Tumor Markers
Number: 0352
Table Of Contents
PolicyApplicable CPT / HCPCS / ICD-10 Codes
Background
References
Policy
Scope of Policy
This Clinical Policy Bulletin addresses tumor markers, including somatic (acquired) mutations, in oncology.
For criteria related to germline (inherited) mutations, see CPB 0140 - Genetic Testing.
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Medical Necessity
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Aetna considers any of the following tumor markers for the stated indication medically necessary (unless otherwise stated):
- 1p19q codeletion molecular cytogenetic analysis for astrocytomas and gliomas;
- 5-hydroxyindoleacetic acid (5-HIAA) for neuroendocrine tumors;
- Afirma Thyroid FNA analysis for assessing fine needle aspiration samples from thyroid nodules that are indeterminate; experimental for other indications. Repeat testing is considered experimental, investigational, or unproven;
- ALK expression for pancreatic adenocarcinoma, pediatric Hodgkin's lymphoma, inflammatory myofibroblastic tumor (IMT) with ALK translocation, breast implant-associated ALCL, peripheral T-cell lymphoma, and uterine sarcoma;
- ALK gene fusion as a molecular biomarker in non-small cell lung cancer;
- ALK gene rearrangement for diffuse large B cell lymphoma, anaplastic thyroid carcinoma, primary cutaneous CD30+ T-cell lymphoproliferative disorders, post-transplant lymphoproliferative disorder, and non-small cell lung cancer;
- Alpha fetoprotein (AFP) for testing for hepatocellular carcinoma in hepatitis B carriers, or for persons with cirrhosis and one or more of the following risk factors: alcohol use; alpha-1 antitrypsin deficiency; Asian female at least 50 years of age; Asian male at least 40 years of age; family history of HCC; genetic hemochromatosis; hepatitis C; nonalcoholic steatohepatitis; and stage 4 primary biliary cirrhosis;
- Alpha fetoprotein (AFP) for the following indications: hepatocellular carcinoma; mediastinal mass; ovarian cancer; pelvic mass; testicular cancer; testicular mass; thymic carcinoma; and thymoma;
- Alpha fetoprotein (AFP): serial measurements to diagnose germ cell tumors in members with adenocarcinoma, or carcinoma not otherwise specified, involving mediastinal nodes; or the diagnosis and monitoring of hepatocellular carcinoma (e.g., before considering liver transplantation);
- Androgen receptor splice variant 7 (AR-V7) in circulating tumor cells to select therapy in metastatic castrate-resistant prostate cancer after progression on abiraterone or enzalutamide;
- APC for familial adenomatous polyposis when criteria are met in CPB 0140 - Genetic Testing; and for desmoid fibromatosis; experimental for other indications;
- BCL2 and BCL6 for the diagnosis of non-Hodgkin’s lymphoma and Castleman's disease;
- BCR/ABL for acute lymphocytic leukemia (ALL), acute myeloid leukemia (AML), B-cell lymphoblastic lymphoma, chronic myelogenous leukemia (CML), and suspected myeloproliferative neoplasm; experimental, investigational, or unproven for other indications;
- Beta-2 microglobulin (B2M) for multiple myeloma, non-Hodgkin's lymphoma and Waldenström's macroglobulinemia/ lymphoplasmacytic lymphoma;
- BIRC3 and MALT1 for gastric MALT lymphoma, non-gastric MALT lymphoma, nodal marginal zone lymphoma, and splenic marginal zone lymphoma;
- BRAF V600 mutation for indeterminate thyroid nodules, hairy cell leukemia; gastrointestinal stromal tumors; colorectal cancer, Lynch syndrome; non-small cell lung cancer; thyroid carcinoma; infiltrative glioma, pancreatic adenocarcinoma, and melanoma (see CPB 0715 - Pharmacogenomic and Pharmacodynamic Testing); or Lynch syndrome for persons meeting criteria in CPB 0140 - Genetic Testing; and colorectal cancer if KRAS nonmutated; experimental for other indications;
- Breast Cancer Index (BCI) Footnote2** to assess necessity of adjuvant chemotherapy or adjuvant endocrine therapy in females or males with recently diagnosed breast tumors, where all of the following criteria are met:
- Breast cancer is nonmetastatic (node negative) or with 1-3 involved ipsilateral axillary lymph nodes; and
- Breast tumor is estrogen receptor and/or progesterone receptor positive; and
- Breast tumor is HER2 receptor negative; and
- Adjuvant therapy is not precluded due to any other factor (e.g., advanced age and/or significant co-morbidities); and
- Member and physician (prior to testing) have discussed the potential results of the test and agree to use the results to guide therapy;
BCI is also considered medically necessary for persons with HER2-negative breast cancer with 0-3 positive nodes who received 5 years of endocrine therapy without recurrence to guide decisions about extended endocrine therapy.
- BTK (Bruton's tyrosine kinase) for chronic lymphocytic leukemia/small lymphocytic lymphoma;
- CA 15-3: Serial measurements of CA 15-3 (also known as CA 27-29 or Truquant RIA) in following the course of treatment in women diagnosed with breast cancer, especially advanced metastatic breast cancer (an increasing CA 15-3 level may suggest treatment failure);
- CA 19-9 for the following indications:
- To monitor the clinical response to therapy or detect early recurrence of disease in members with known gastric cancer, pancreatic cancer, gallbladder cancer, cholangiocarcinoma, ovarian cancer, small bowel adenocarcinoma, or adenocarcinoma of the ampulla of Vater; or
- To rule out cholangiocarcinoma in persons with primary sclerosing cholangitis undergoing liver transplantation; or
- For evaluation of jaundice, abnormal liver function tests (LFTs) or hepatobiliary obstruction/abnormality on abdominal imaging; or
- As a tumor marker for mucinous appendiceal carcinoma;
- CALB2 (calretinin) expression for lung cancer and occult primary;
- CALCA (calcitonin) expression for medullary thyroid cancer or for adenocarcinoma or anaplastic/undifferentiated tumors of the head and neck;
- CALR (calreticulin) for chronic myeloid leukemia (chronic phase, adult), myelodysplastic syndrome, or myeloproliferative neoplasms;
- Cancer antigen 125 (CA 125) levels for any of the following:
- As a preoperative diagnostic aid in women with ovarian masses that are suspected to be malignant, such that arrangements can be made for intraoperative availability of a gynecological oncologist if the CA 125 is increased; or
- As a screening test for ovarian cancer when there is a family history of hereditary ovarian cancer syndrome (a pattern of clusters of ovarian cancer within two or more generations), where testing is performed concurrently with transvaginal ultrasound and prophylactic salpingo-oophorectomy has not been performed. For this indication, screening is considered medically necessary every six months beginning at 30 years of age or 10 years before the earliest age of the first diagnosis of ovarian cancer in the family; or
- Diagnosis of ovarian cancer in women with new symptoms (bloating, pelvic or abdominal pain, difficulty eating or feeling full quickly, or urinary frequency and urgency) that have persisted for three or more weeks, where the clinician has performed a pelvic and rectal examination and suspects ovarian cancer; or
- In members with adenocarcinoma of unknown primary, to rule out ovarian cancer; or
- In members with known ovarian cancer, as an aid in the monitoring of disease, response to treatment, detection of recurrent disease, or assessing value of performing second-look surgery;
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Carcinoembryonic antigen (CEA) for any of the following:
- As a preoperative prognostic indicator in members with known colorectal carcinoma or mucinous appendiceal carcinoma when it will assist in staging and surgical treatment planning; or
- Pancreatic cyst fluid CEA for distinguishing mucinous from non-mucinous malignant pancreatic cysts; or
- To detect asymptomatic recurrence of colorectal cancer after surgical and/or medical treatment for the diagnosis of colorectal cancer (not as a screening test for colorectal cancer); or
- To monitor response to treatment for metastatic colorectal cancer; or
- For cholangiocarcinoma, gallbladder cancer, lung cancer, medullary thyroid cancer, metastatic breast cancer, mucinous ovarian cancer, and occult primary; or
- For evaluation of jaundice, abnormal liver function tests (LFTs) or for obstruction/abnormality of the bile duct on liver imaging;
- CBFB for acute myeloid leukemia;
- CCND1 (cyclin D1) for B-cell lymphomas, primary cutaneous B-cell lymphomas, chronic lymphocytic leukemia/small lymphocytic lymphoma, and hairy cell leukemia;
- CD 20, for determining eligibility for anti-CD20 treatment (rituximab) (see CPB 0314 - Rituximab);
- CD 25, for determining eligibility for denileukin diftitox (Ontak) treatment;
- CD 31 immunostaining, for diagnosis of angiosarcoma;
- CD 33, for lymphoblastic lymphoma and for determining eligibility for anti-CD33 (gemtuzumab, Mylotarg) treatment;
- CD 52, for post-transplant lymphoproliferative disorder, T-cell prolymphocytic leukemia, and for determining eligibility for anti-CD52 (alemtuzumab, Campath) treatment;
- CD117 (c-kit), for acute myeloid leukemia, cutaneous melanoma, gastrointestinal stromal tumors and systemic mastocytosis;
- CHGA (Chromogranin A) expression for neuroendocrine tumors, non-small cell lung cancer, small cell lung cancer, Merkel cell carcinoma and occult primary;
- Copy number alterations molecular testing for pediatric diffuse high-grade glioma;
- DecipherFootnote3*** for the following indications:
- Post biopsy in men with NCCN very-low-risk, low-risk, and favorable intermediate-risk prostate cancer who have a greater than 10 year life expectancy who have not received treatment for prostate cancer and are candidates for active surveillance or definitive therapy; or
- Post biopsy in men with intermediate-risk prostate cancer when deciding whether to add androgen-deprivation therapy to radiation; or
- Men with an undetectable PSA after prostatectomy for prostate cancer, to determine adjuvant versus salvage radiation therapy or to determine whether to initiate systemic therapies;
- DecisionDx-UM (Castle Biosciences, Phoenix, AZ) for risk stratification of persons with localized uveal melanoma;
- EndoPredict (also known as 12-gene score)Footnote2** to assess necessity of adjuvant chemotherapy in females or males with recently diagnosed breast tumors, where all of the following criteria are met:
- Breast cancer is nonmetastatic (node negative) or with 1-3 involved ipsilateral axillary lymph nodes; and
- Breast tumor is estrogen receptor positive; and
- Breast tumor is HER2 receptor negative; and
- Adjuvant chemotherapy is not precluded due to any other factor (e.g., advanced age and/or significant co-morbidities); and
- Member and physician (prior to testing) have discussed the potential results of the test and agree to use the results to guide therapy;
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EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit) for the workup of the following:
- myelodysplastic syndrome (MDS), and
- myeloproliferative neoplasms (MPN) to evaluate for higher-risk mutations associated with disease progression in members with primary myelofibrosis (PMF);
Aetna considers EZH2 experimental, investigational, or unproven for all other indications including diffuse large B-cell lymphomas;
- FIP1L1-PDGFRA fusion oncogene for systemic mastocytosis with peripheral blood eosinophilia;
- FIP1L1-PDGFRA gene rearrangements for myeloid/lymphoid neoplasms with peripheral blood eosinophilia and tyrosine kinase fusion genes;
- FLT3 gene mutation testing for acute lymphoblastic leukemia, acute myeloid leukemia (AML), myelodysplastic syndromes, myeloproliferative neoplasms, and myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase fusion genes;
- Human chorionic gonadotropin (HCG), serial measurement to diagnose germ cell tumors in members with adenocarcinoma, or carcinoma not otherwise specified, involving mediastinal nodes, or to monitor treatment in members with known trophoblastic tumors (invasive hydatidiform moles and choriocarcinomas) and germinal cell tumors (teratocarcinoma and embryonal cell carcinoma) of the ovaries or testes, or to monitor for relapse after remission is achieved;
- Human chorionic gonadotropin, beta (beta-HCG) for mediastinal mass; ovarian cancer; pelvic mass; testicular mass; testicular cancer; thymoma; or thymic carcinoma;
- Human epidermal growth factor receptor 2 (HER2) (ERBB2) evaluation in biliary tract, bladder, breast, cervical, colorectal, esophageal, esophageal gastric junction, gastric, non-small cell lung cancer (NSCLC), ovarian/fallopian tube, and salivary gland tumors. See CPB 0313 - Trastuzumab (Herceptin and biosimilars), Trastuzumab and Hyaluronidase-oysk (Herceptin Hylecta);
- Human papillomavirus (HPV) tumor testing (p16) for the workup of head and neck cancer (including oropharynx cancer) or occult primary cancers;
- IGH@ (Immunoglobulin heavy chain locus), gene rearrangement analysis to detect abnormal clonal population(s) in non-Hodgkin’s lymphomas, chronic lymphocytic leukemia, hairy cell leukemia, and post-transplant lymphoproliferative disorder;
- IGK@ (Immunoglobulin kappa light chain locus), gene rearrangement analysis, evaluation to detect abnormal clonal population(s) for non-Hodgkin’s lymphoma, systemic light chain amyloidosis;
- INHA (inhibin) expression for ovarian cancer or pelvic mass;
- Isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) gene mutation for AML, chondrosarcomas, myelodysplastic syndromes, myeloproliferative neoplasms, or gliomas and glioblastomas;
- KRAS for metastatic colorectal cancer, myelodysplastic syndromes, non-small cell lung cancer, pancreatic adenocarcinoma, and uterine sarcoma;
- Lactate dehydrogenase (LDH) for acute lymphoblastic leukemia (ALL), bone cancer, kidney cancer, kidney mass, lung cancer, multiple myeloma, non-Hodgkin's lymphoma, pelvic mass, ovarian cancer, testicular cancer, or testicular mass;
- Liquid biopsy (up to 50 genes) (e.g., Resolution ctDx Lung, InVisionFirst-Lung) for persons with non-small cell lung cancer who are not medically fit for invasive sampling, or there is insufficient tissue for molecular analysis and follow-up tissue-based analysis will be done if an oncogenic driver is not identified; large liquid biopsy panels (greater than 50 genes) are considered experimental, investigational, or unproven for non-small cell lung cancer; for Guardant360CDx non-small cell lung cancer and FoundationOne Liquid CDx for non-small cell lung cancer and prostate cancer (see CPB 0715 - Pharmacogenetic and Pharmacodynamic Testing);
- MammaprintFootnote2** to assess necessity of adjuvant chemotherapy in females or males with recently diagnosed breast tumors, where all of the following criteria are met:
- Breast cancer is nonmetastatic (node negativeFootnote1*) or with 1-3 involved ipsilateral axillary lymph nodes; and
- Breast tumor is estrogen receptor positive or progesterone receptor positive; and
- Breast tumor is HER2 receptor negative (Rationale: adjuvant chemotherapy with trastuzumab (Herceptin) is considered to be medically necessary regardless of Mammaprint score for HER2 receptor positive lesions); and
- Member is determined to be at "high clinical risk" of recurrence using Adjuvant! Online (see page 20 of MINDACT study supplement for definitions of high clinical risk); and
- Adjuvant chemotherapy is not precluded due to any other factor (e.g., advanced age and/or significant co-morbidities); and
- Member and physician (prior to testing) have discussed the potential results of the test and agree to use the results to guide therapy;
- Measurement of estrogen receptors (ESR1) for breast cancer, endometrial carcinoma, non-small cell lung cancer, occult primary, ovarian cancer, or uterine sarcoma;
- Measurement of progesterone receptors (PGR) for breast cancer, non-small cell lung cancer, occult primary, or uterine sarcoma;
- Microsatellite instability (MSI) molecular testing for any of the following indications:
- Adrenal gland tumor (including adrenocortical carcinoma)
- Biliary tract cancers (i.e., extrahepatic cholangiocarcinoma, gallbladder cancer, intrahepatic cholangiocarinoma)
- Bone cancer (i.e., chondrosarcoma, chordoma, Ewing sarcoma, osteosarcoma)
- Breast cancer (invasive)
- Cervical cancer
- Colon cancer (including appendiceal adenocarcinoma)
- Esophageal and esophagogastric junction cancers
- Gastric cancer
- Head and neck cancer (including salivary gland tumors)
- Lynch syndrome
- Neuroendocrine (i.e., extrapulmonary poorly differentiated neuroendocrine carcinoma / large or small cell carcinoma / mixed neuroendocrine-non-neuroendocrine neoplasm)
- Occult primary
- Ovarian cancer / fallopian tube cancer / primary peritoneal cancer (including epithelial ovarian cancer, and less common ovarian cancers [e.g., grade 1 endometrioid carcinoma])
- Penile cancer
- Prostate cancer
- Rectal cancer
- Small bowel adenocarcinoma
- Testicular Cancer (including nonseminoma, seminoma)
- Thyroid carcinoma (i.e., anaplastic, follicular, oncocytic, papillary)
- Upper genitourinary tract (GU) tract tumors
- Uterine neoplasms (i.e., endometrial carcinoma, uterine sarcoma)
- Vulvar cancer - squamous cell carcinoma;
- Mismatch repair (MSI/dMMR) (MLH1, MSH2, MSH6, PMS2) tumor testing (somatic mutations) for breast cancer, ovarian cancer, colorectal cancer, small bowel adenocarcinoma, esophageal cancer, esophagogastric junction cancer, gastric cancer, pancreatic cancer, cholangiocarcinoma, gallbladder cancer, pancreatic adenocarcinoma, cervical cancer, uterine cancer, prostate cancer, testicular cancer, penile cancer, myelodysplastic syndromes, Ewing sarcoma, and occult primary; for medical necessity of screening of germline mutations for HNPCC/Lynch Syndrome with MLH1, MSH2, MSH6, see CPB 0140 - Genetic Testing;
- MLH1 tumor promoter hypermethylation for endometrial cancer;
- MPL (myeloproliferative leukemia protein) for chronic myeloid leukemia (chronic phase, adult), myelodysplastic syndromes, or myeloproliferative neoplasms;
- Murine double minute 2 (MDM2) for uterine sarcoma and soft tissue sarcoma;
- Mycosis fungoides, diagnosis: polymerase chain reaction (PCR) for T-cell receptor gamma chain gene rearrangement as an adjunct to the histopathologic diagnosis of mycosis fungoides;
- MYD88 (myeloid differentiation primary response 88) to differentiate Waldenstrom's macroglobinemia (WM) versus marginal zone lymphoma (MZL) if plasmacytic differentiation present for gastric MALT lymphoma, nodal marginal zone lymphoma, nongastric MALT lymphoma, and splenic marginal zone lymphoma; and for multiple myeloma;
- Myeloperoxidase (MPO) immunostaining, CEBPA mutation, and KIT mutation for diagnosis of acute myeloid leukemia;
- MyMRD NGS Panel for comprehensive prognostic assessment in individuals with acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS);
- Next generation sequencing of tumor DNA (e.g., ClonoSeq) to detect or quantify minimal residual disease in persons with multiple myeloma or acute lymphocytic leukemia;
- NPM1 in acute myeloid leukemia (AML), chronic myeloid leukemia (chronic phase, adult), myelodysplastic syndromes, or myeloproliferative neoplasms; experimental for other indications;
- NRAS for colorectal cancer, myelodysplastic syndrome, or blastic plasmacytoid dendritic cell neoplasm (BPDCN);
- NTRK for all solid tumors;
- Oncotype Dx Breast (also known as 21 gene RT-PCR test) to assess necessity of adjuvant chemotherapy in females or males with recently diagnosed breast tumors, where all of the following criteria are met:
- Breast cancer is nonmetastatic (node negativeFootnote1*) or with 1-3 involved ipsilateral axillary lymph nodes; and
- Breast tumor is estrogen receptor positive; and
- Breast tumor is HER2 receptor negative or breast tumor is HER2 receptor positive and less than 1 cm in diameter. (Rationale: adjuvant chemotherapy with trastuzumab (Herceptin) is considered to be medically necessary regardless of an Oncotype Dx Breast score for HER2 receptor positive lesions 1 cm or more in diameter); and
- Adjuvant chemotherapy is not precluded due to any other factor (e.g., advanced age and/or significant co-morbidities); and
- Member and physician (prior to testing) have discussed the potential results of the test and agree to use the results to guide therapy (i.e., member will forgo adjuvant chemotherapy if Oncotype Dx Breast score is low);
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Oncotype DX ProstateFootnote3*** for the following indications post biopsy:
- Men with NCCN very-low-risk, low-risk, and favorable intermediate-risk prostate cancer who have greater than 10 year life expectancy and who have not received treatment for prostate cancer and are candidates for active surveillance or definitive therapy; or
- Men with intermediate-risk prostate cancer when deciding whether to add androgen-deprivation therapy to radiation;
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PAM50 Risk of Recurrence (ROR) Score (also known as Prosigna Breast Cancer Prognostic Gene Signature Assay)Footnote2** to assess necessity of adjuvant chemotherapy in females or males with recently diagnosed breast tumors, where all of the following criteria are met:
- Breast cancer is nonmetastatic (node negative); and
- Breast tumor is estrogen receptor positive; and
- Breast tumor is HER2 receptor negative; and
- Adjuvant chemotherapy is not precluded due to any other factor (e.g., advanced age and/or significant co-morbidities); and
- Member and physician (prior to testing) have discussed the potential results of the test and agree to use the results to guide therapy;
- PDGFRA for gastrointestinal stromal tumors (GIST) and for pediatric acute lymphoblastic leukemia (see also entry above for FIP1L1-PDGFRA gene rearrangements and fusions);
- PDGFRB testing for myelodysplastic syndromes (MDS), dermatofibrosarcoma protuberans, acute lymphoblastic leukemia, and for myeloid/lymphoid neoplasms with peripheral blood eosinophilia and tyrosine kinase fusion genes;
- Phosphatidylinositol-4,5-bisphosphonate 3-kinase, catalytic subunit alpha polypeptide gene (PIK3CA) for breast cancer and uterine sarcoma;
- Placental alkaline phosphatase (PLAP), to diagnose germ cell seminoma and non-seminoma germ cell tumors in unknown primary cancers;
- PLCG2 (phospholipase C gamma 2) for chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL);
- PML/RARA for acute promyelocytic leukemia; experimental for all other indications;
- Predicting response to EGFR-targeting tyrosine kinase inhibitors in non-small cell lung cancer (NSCLC); KRAS mutation testing to predict non-response to treatment of anal adenocarcinoma, metastatic colorectal cancer, NSCLC, and small bowel adenocarcinoma; or ROS-1 to predict response to treatment of NSCLC, see CPB 0715 - Pharmacogenetic and Pharmacodynamic Testing
- ProlarisFootnote3*** for the following indications post-biopsy:
- Men with NCCN very-low-risk, low-risk, and favorable intermediate-risk prostate cancer who have greater than 10 year life expectancy and who have not received treatment for prostate cancer and are candidates for active surveillance or definitive therapy; or
- Men with intermediate-risk prostate cancer when deciding whether to add androgen-deprivation therapy to radiation;
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ProMarkFootnote3*** for the following indications post-biopsy:
- Men with NCCN very-low-risk, low-risk men, and favorable intermediate risk prostate cancer who have greater than 10 year life expectancy and who have not received treatment for prostate cancer and are candidates for active surveillance or definitive therapy; or
- Men with intermediate-risk prostate cancer when deciding whether to add androgen-deprivation therapy to radiation;
- Prostate-specific antigen (PSA) for prostate cancer screening (see CPB 0521 - Prostate Cancer Screening), staging, monitoring response to therapy, and detecting disease recurrence;
- PTEN for uterine sarcoma and for persons meeting Cowden syndrome testing criteria in CPB 0140 - Genetic Testing; experimental for all other indications;
- Quest Diagnostics Thyroid Cancer Mutation Panel for assessing fine needle aspiration samples from thyroid nodules that are indeterminate; experimental for other indications. Repeat testing is considered experimental, investigational, or unproven;
- RUNX1 for acute myeloid leukemia, myelodysplastic syndrome, and systemic mastocytosis;
- SF3B1 (splicing factor 3b subunit 1) for chronic myeloid leukemia (chronic phase, adult), myelodysplastic syndromes, myeloproliferative neoplasms, or uveal melanoma;
- SRSF2 (serine and arginine rich splicing factor 2) for chronic myeloid leukemia (chronic phase, adult), myelodysplastic syndromes, myeloproliferative neoplasms, or systemic mastocytosis;
- Steroid hormone receptor status in both pre-menopausal and post-menopausal members to identify individuals most likely to benefit from endocrine forms of adjuvant therapy and therapy for recurrent or metastatic breast cancer;
- Targeted hematologic genomic sequencing panel (5-50 genes) for acute lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, myelodysplastic syndromes (MDS) and myeloproliferative neoplasms (MPN) (e.g., MedFusion myeloid malignancy analysis panel). Repeating a hematologic malignancy genomic sequencing panel within 60 days of prior panel testing for the same indication is considered not medically necessary;
- Targeted solid organ genomic sequencing panel (5-50 genes) for colorectal cancer, cutaneous melanoma, pancreatic cancer, prostate cancer and non-small cell lung cancer (including Oncomine Dx Target Test (Thermo Fisher Scientific, Carlsbad, CA)). Repeating a solid organ malignancy genomic sequencing panel within 60 days of prior panel testing for the same indication is considered not medically necessary;
- T-cell receptor gene rearrangements (TRA@, TRB@, TRD@, TRG@) for T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, nasal type extranodal NK/T-cell lymphoma, hepatosplenic gamma-delta T-cell lymphoma, peripheral T-cell lymphoma, primary cutaneous CD30+ T-cell lymphoproliferative disorders, myelodysplastic syndromes, Castleman's disease, mycosis fungoides/Sezary syndrome and myeloid/lymphoid neoplasms with eosinophilia and tyrosine kinase fusion genes;
- TERT (telomerase reverse transcriptase) medically necessary for the workup of:
- Gliomas (i.e., infiltrative supratentorial astrocytoma/oligodendroglioma, anaplastic gliomas/glioblastoma), and
- Myelodysplastic syndrome (MDS).
Aetna considers TERT experimental, investigational, or unproven for all other indications including thyroid carcinoma.
- ThyGeNEXT Thyroid Oncogene Panel (formerly e.g., ThyGenX, miRInform thyroid test) and ThyraMIR microRNA Classifier for assessing fine needle aspiration samples from thyroid nodules that are indeterminate; experimental for other indications; repeat testing is considered experimental, investigational, or unproven;
- Thymidine kinase for chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL);
- Thyroglobulin antibodies for thyroid cancer;
- Thyroglobulin (TG) expression for thyroid cancer, occult primary, and adenocarcinoma or anaplastic/undifferentiated tumors of the head and neck
- Thyroid transcription factor-1 (TTF-1) for lung cancer or neuroendocrine tumors;
- Thyroseq for assessing fine needle aspiration samples from thyroid nodules that are indeterminate; experimental for other indications. Repeat testing is considered experimental, investigational, or unproven;
- TP53 for acute myeloid leukemia; adult medulloblastoma; chronic lymphocytic leukemia/small lymphocytic lymphoma; chronic myeloid leukemia (chronic phase, adult); endometrial carcinoma; malignant peritoneal or pleural mesothelioma; mantle cell lymphoma; myelodysplastic syndromes; myeloproliferative neoplasms; occult primary; pediatric acute lymphoblastic leukemia; peripheral T-cell lymphomas; splenic marginal zone lymphoma; uterine sarcoma; or well-differentiated, grade 3 neuroendocrine tumors;
- Tumor mutation burden (TMB) molecular testing for testicular cancer (nonseminoma, seminoma);
- U2AF1 (U2 small nuclear RNA auxiliary factor 1) for blastic plasmacytoid dendritic cell neoplasm (BPDCN), chronic myeloid leukemia (chronic phase, adult), myelodysplastic syndromes, or myeloproliferative neoplasms;
- Urokinase plasminogen activator (uPA) and plasminogen activator inhibitor 1 (PAI-1)Footnote2** to assess necessity of adjuvant chemotherapy in females or males with recently diagnosed breast tumors, where all of the following criteria are met:
- Breast cancer is nonmetastatic (node negative); and
- Breast tumor is estrogen receptor positive; and
- Breast tumor is HER2 receptor negative; and
- Adjuvant chemotherapy is not precluded due to any other factor (e.g., advanced age and/or significant co-morbidities); and
- Member and physician (prior to testing) have discussed the potential results of the test and agree to use the results to guide therapy;
In addition, urokinase plasminogen activator (uPA) and plasminogen activator inhibitor 1 (PAI-1) is considered medically necessary for the determination of prognosis in persons with newly diagnosed, node negative breast cancer;
- Vascular endothelial growth factor (VEGF) expression for Castleman's disease;
- Veristrat proteomic testing for members with advanced NSCLC, whose tumors were without EGFR and anaplastic lymphoma kinase (ALK) mutations, who had progressed after at least one chemotherapy regimen), and for whom erlotinib was considered an appropriate treatment;
- WT-1 gene expression for desmoplastic round cell tumors, ovarian clear cell carcinomas, non-small cell lung cancer and occult primary;
- ZAP-70, for assessing prognosis and need for aggressive therapy in persons with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL);
- ZRSR2 (zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2) for chronic myeloid leukemia (chronic phase, adult) or myelodysplastic syndromes.
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Aetna considers somatic genomic testing for Janus Kinase 2 (JAK2) mutations in persons with chronic myeloproliferative disorders (CMPDs) medically necessary for the following indications:
- Qualitative assessment of JAK2-V617F sequence variant using methods with detection thresholds of up to 5% for initial diagnostic assessment of adult members presenting with symptoms of CMPD;
- Diagnostic assessment of polycythemia vera in adults; and
- Differential diagnosis of essential thrombocytosis and primary myelofibrosis from reactive conditions in adults.
Aetna considers somatic genomic testing for Janus Kinase 2 (JAK2) mutations in persons with chronic myeloproliferative disorders (CMPDs) experimental, investigational, or unproven for any other indication including:
- Diagnostic assessment of myeloproliferative disorders in children;
- Quantitative assessment of JAK2-V617F allele burden subsequent to qualitative detection of JAK2-V617F.
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Aetna considers the use of fluorescence immunocytology (e.g., ImmunoCyt/uCyt) medically necessary as an adjunct to cystoscopy or cytology in the monitoring of persons with bladder cancer.
Aetna considers the ImmunoCyte/uCyt immunohistochemistry test experimental, investigational, or unproven in the evaluation of hematuria, diagnosing bladder cancer, or for screening for bladder cancer in asymptomatic persons.
- Aetna considers matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS or MASS-FIX) and immunoprecipitation for detection and isotyping of immunoglobulin paraprotein (M-protein) medically necessary for the evaluation and management of plasma cell dyscrasias.
- Aetna considers urinary biomarkers (e.g., bladder tumor antigen (BTA) (e.g., BTA Stat and BTA TRAK), nuclear matrix protein (NMP22) test, the fibrin/fibrinogen degradation products (Aura-Tek FDfP) test, or fluorescence in situ hybridization (FISH) (e.g., Pathnostics Bladder FISH test, UroVysion Bladder Cancer test medically necessary in any of the following conditions:
- Follow-up of treatment for bladder cancer; or
- Monitoring for eradication of bladder cancer; or
- Recurrences after eradication.
Aetna considers the BTA Stat test, the NMP22 test, the Aura-Tek FDP test, or the UroVysion fluorescent in situ hybridization (FISH) test experimental, investigational, or unproven for screening of bladder cancer, evaluation of hematuria, and diagnosing bladder cancer in symptomatic individuals, and all other indications.
Footnote1* Either standard node dissection negative by hematoxylin and eosin (H&E) staining or sentinel node negative by H&E staining (if sentinel node is negative by H&E, but immunoassay is positive, then still considered node negative for this purpose). In addition, women with isolated tumor cells in lymph nodes (micrometastases) are considered node negative.
More than one Oncotype Dx test may be medically necessary for persons with breast cancer who have two or more histologically distinct tumors that meet medical necessity criteria. Repeat Oncotype Dx testing or testing of multiple tumor sites in the same person has no proven value for other indications. Oncotype Dx is considered experimental, investigational, or unproven for ductal carcinoma in situ (OncotypeDx DCIS), colon cancer (OncotypeDx Colon), and all other indications other than breast cancer and prostate cancer.
Footnote2** Aetna considers use of more than one type of test to determine necessity of adjuvant therapy in breast cancer (Oncotype Dx Breast, Breast Cancer Index, EndoPredict, PAM50, Mammaprint, or uPA and PAI-1) experimental, investigational, or unproven.
Footnote3*** Aetna considers repeat testing or use of more than one type of test to assess risk of prostate cancer progression (Oncotype Dx Prostate, Decipher, Prolaris, or ProMark) experimental, investigational, or unproven.
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Experimental, Investigational, or Unproven
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Aetna considers each of the following experimental, investigational, or unproven. The peer-reviewed medical literature does not support these tests as having sufficient sensitivity or specificity necessary to define their clinical role:
- 3D Predict Ovarian Doublet Panel
- 3D Predict Ovarian PARP Panel
- 4Kscore
- Afirma Xpression Atlas
- AFP for the diagnosis of trophoblastic tumors and oncologic indications other than those listed in Section I
- AMBLor Melanoma Prognostic Test
- ArteraAI Prostate Test
- Assaying for loss of heterozygosity (LOH) on the long arm of chromosome 18 (18q) or deleted in colon cancer (DCC) protein (18q-LOH/DCC) for colorectal cancer
- Augusta Hematology Optical Genome Mapping
- Auria for breast cancer screening
- Avantect Pancreatic Cancer test
- Aventa FusionPlus
- BBDRisk Dx
- Biodesix BDX-XL2, Nodify CDT, Nodify Lung, or Nodify XL2 test for distinguishing benign from malignant lung nodules
- Biomarker Translation (BT) test for breast cancer and other indications
- BioSpeciFx, including Comprehensive Tumor Profiling for any indication
- BostonGene Tumor Portrait Test
- BRAF and EGFR for esophageal carcinoma
- Breast Cancer Gene Expression Ratio (HOXB13:IL17BR)
- BreastSentry
- BTG Early Detection of Pancreatic Cancer
- CA 125 for all other indications including use as a screening test for colorectal cancer or ovarian cancer (other than as indicated in Section I) or for differential diagnosis of members with symptoms of colonic disease
- CA 19-9 for all other indications not listed in Section I. including breast, colorectal, esophageal, gastro-esophageal, liver, or uterine cancer; ovarian cyst, NUT midline carcinoma of the nasal cavity, prediction of prognosis or treatment effect in persons with bladder (urothelial) cancer, screening persons with primary sclerosing cholangitis without signs or symptoms of cholangiocarcinoma; or screening persons with primary sclerosing cholangitis for development of cholangiocarcinoma
- Carcinoembryonic antigen cell adhesion molecule 6 (CEACAM6) (e.g., Benign Diagnostics Risk Test) for breast atypical hyperplasia and for predicting the risk of breast cancer
- Carcinoembryonic antigen cellular adhesion molecule-7 (CEACAM-7) expression as a predictive marker for rectal cancer recurrence
- Caris Molecular Intelligence/Caris Target Now Molecular Profiling Test
- Castle Biosciences myPath Melanoma (formerly Myriad myPath Melanoma)
- CDH1 for ovarian cancer
- CDX2 as a prognostic biomarker for colon cancer
- CEA used for all other indications not noted in Section I including any of the following:
- As a screening test for colorectal cancer; or
- As a sole determinant to treat a colorectal cancer member with adjuvant therapy or systemic therapy for presumed metastatic disease; or
- For diagnosis of esophageal carcinoma; or
- For screening, diagnosis, staging or routine surveillance of gastric cancer
- Circulating cell-free nucleic acids in colorectal cancer
- Circulating tumor cell (CTC) assays for all indications, including, but not limited to metastatic breast, colorectal, melanoma, and prostate cancers. Below includes CTC assays considered experimental, investigational, or unproven (not an all-inclusive list):
- CellMax Life
- CELLSEARCH Circulation Multiple Myeloma Cell (CMMC)
- CELLSEARCH HER2 Circulating Tumor Cell (CTC-HER2)
- FirstSightCRC
- Circulating tumor DNA (ctDNA) (also referred to as a liquid biopsy) for any indication (other than small panels, less than 50 genes, for non-small cell lung cancer), including, but not limited to, colorectal cancer, melanoma, ovarian cancer or prostate cancer. Note: for EGFR liquid biopsy for non-small cell lung cancer (e.g., cobas EGFR Mutation Test v2), PIK3CA testing (therascreen PIK3CA RGQ PCR Kit) for breast cancer, ESR1 gene mutations (e.g., Guardant360 CDx assay) for breast cancer, and for other ctDNA/liquid biopsy testing in predicting response in members undergoing immunotherapy or targeted treatment, see CPB 0715 - Pharmacogenetic and Pharmacodynamic Testing. Below includes ctDNA/liquid biopsy tests considered experimental, investigational, or unproven (not an all-inclusive list):
- CancerIntercept
- Colvera
- DefineMBC Epic Sciences ctDNA metastatic breast cancer panel
- GeneStrat
- FoundationACT
- FoundationOne Liquid
- Guardant Reveal minimal residual disease (MRD) assessment and monitoring in breast, colorectal, and lung cancers
- Guardant360
- HPV-SEQ for monitoring disease burden in HPV-related cancers
- LiquidHALLMARK
- Neolab Prostate;
- CK5, CK14, p63, and Racemase P504S testing for prostate cancer
- c-Met expression for predicting prognosis in persons with advanced NSCLC and colorectal cancer, and other indications
- Cyfra21-1 (a cytokeratin 19 fragment), p53, squamous cell carcinoma antigen (SCC-Ag) and vascular endothelial growth factor C (VEGF-C) for diagnosis of esophageal carcinoma
- Cofilin (CFL1) as a prognostic and drug resistance marker in non-small cell lung cancer
- ColonSentry test for screening of colorectal cancer
- ColoPrint, CIMP, LINE-1 hypomethylation, and Immune cells for colon cancer
- Colorectal Cancer DSA (Almac Diagnostics, Craigavon, UK)
- ColoScape Test
- ConfirmMDx for prostate cancer
- Cxbladder tests (e.g., Cxbladder Triage, Cxbladder Detect+) for bladder cancer
- Cyclin D1 and FADD (Fas-associated protein with death domain) for head and neck squamous cell carcinoma
- CyPath Lung
- DAWN IO Melanoma
- DCIS Recurrence Score
- DCISionRT
- Decipher Bladder
- DecisionDx DiffDx-Melanoma (Castle Biosciences, Phoenix, AZ)
- DecisionDx-Melanoma (Castle Biosciences, Phoenix, AZ)
- DecisionDx-SCC (Castle Biosciences, Phoenix, AZ)
- Des-gamma-carboxy prothrombin (DCP) (also known as "prothrombin produced by vitamin K absence or antagonism II" [PIVKA II]) for diagnosing and monitoring hepatocellular carcinoma (HCC) and other indications
- DetermaRx
- DiviTum TKa test
- EarlyCDT-Lung test
- EarlyTect Bladder Cancer Detection (EarlyTect BCD)
- EGFR gene expression analysis for transitional (urothelial) cell cancer
- EGFRVIII for glioblastoma multiforme
- EML4-ALK as a diagnostic tool for stage IV non-small-cell lung cancer
- Endeavor Comprehensive Genomic Profiling
- Envisia Genomic Classifier
- Excision repair cross-complementation group 1 protein (ERCC1) for persons with NSCLC, colon or with gastric cancer who are being considered for treatment with platinum-based chemotherapy, and other indications
- ExoDx Prostate/ExosomeDx Prostate (IntelliScore)
- Fibrin/fibrinogen degradation products (FDP) test (e.g., DR-70 or Onko-Sure) for colorectal cancer
- FoundationOne, FoundationOne CDx and FoundationOne Heme (except where FoundationOne CDx is used as a companion diagnostic test for somatic/tumor BRCA testing, see CPB 0227 - BRCA Testing, Prophylactic Mastectomy, and Prophylactic Oophorectomy and CPB 0715 - Pharmacogenetic and Pharmacodynamic Testing)
- Galectin-3 for breast cancer, myelodysplastic syndrome, osteosarcoma, ovarian cancer, pancreatic cancer, and prostate cancer
- Gene hypermethylation for prostate cancer
- GeneKey (GeneKey Corp., Boston, MA)
- GeneSearch Breast Lymph Node (BLN) assay
- Glutathione-S-transferase P1 (GSTP1) for screening, detection and management of prostate cancer
- Grail Galleri Test
- Guanylyl cyclase c (GCC or GUCY2C) (e.g., Previstage GCC Colorectal Cancer State Test) for colorectal cancer
- Guardant360 TissueNext
- HelioLiver Test
- HeproDx
- HER2 testing of appendiceal cancer
- HERmark testing for breast cancer and other indications
- HMGB1 and RAGE in cutaneous malignancy (e.g., basal cell carcinoma, melanoma, and squamous cell carcinoma)
- Human epididymis protein 4 (HE4) (e.g., Elecsys HE4 assay) for endometrial cancer, ovarian cancer, or evaluation of pelvic mass, including to assist in the determination of referral for surgery to a gynecologic oncologist or general surgery, and for other indications
- IHC4 (e.g., NexCourse IHC4 by AQUA Technology) for breast cancer
- IMMray PanCan-d for detecting pancreatic ductal adenocarcinoma
- Immunoassay using magnetic nanosensor for diagnosis of lung cancer
- Immunoscore for estimating risk of recurrence or determining adjuvant therapy in persons with colon cancer
- Insight DX Breast Cancer Profile
- Insight TNBCtype
- Invitae PCM MRD Monitoring test
- Invitae PCM Tissue Profiling and MRD Baseline Assay
- IsoPSA
- Ki67 for breast cancer
- Ki-67 in upper tract urinary carcinoma
- Lectin-reactive alpha-fetoprotein (AFP-L3) for liver cancer
- Long non-coding RNA in gallbladder cancer
- LungLB and LungLife AI
- LungOI
- Lymph2CX and Lymph3Cx Lymphoma Molecular Classification Assay to distinguish between primary mediastinal B-cell lymphoma (PMBCL) and diffuse large B-cell lymphoma (DLBCL)
- Mammostrat
- Mass spectrometry-based proteomic profiling for indeterminate pulmonary nodules
- MatePair targeted rearrangements (whole genome next-generation sequencing) for hematolymphoid neoplasia and solid organ neoplasia
- Mayo Clinic Laboratories Urinary Steroid Profile for the management of adrenal malignancies
- MelaNodal Predict for the management of cutaneous melanoma
- Merkel SmT Oncoprotein Antibody Titer
- Merkel Virus VP1 Capsid Antibody
- MI Cancer Seek
- Microarray-based gene expression profile testing using the MyPRS test for multiple myeloma
- Micro-RNAs (miRNAs) miRview mets and miRview mets2 (Rosetta Genomics Laboratories, Philadelphia, PA; Rosetta Genomics Ltd., Rehovot, Israel)
- M-inSight Patient Definition Assay
- M-inSight Patient Follow-Up Assessment
- miR-31now
- miR Sentinel Prostate Cancer Test
- Molecular Intelligence Services, including MI Profile and MI Profile X (formerly Target Now Molecualr Profiling Test, including Target Now Select and Target Now Comprehensive)
- Molecular subtyping profile (e.g., BluePrint) for breast cancer
- mRNA gene expression profiling for cutaneous melanoma
- mRNA sequence analysis
- MSK-IMPACT
- MUC1 in gastric cancer
- Mucin 4 expression as a predictor of survival in colorectal cancer
- Mucin 5AC (MUC5AC) as serum marker for biliary tract cancer
- My Prognostic Risk Signature (MyPRS) (Signal Genetics LLC, New York, NY)
- MyAML Next Generation Sequencing Panel
- MyProstateScore (formerly Mi-Prostate Score [MiPS]), an assay of TMPRSS2:ERG (T2:ERG) gene fusion, post-DRE urine expression of PCA3, and serum PSA (KLK3)
- MyProstateScore 2.0
- NantHealth GPS Cancer Panels
- NavDx for surveillance of cancer recurrence in HPV-associated oropharyngeal cancer
- NETest
- NF1, RET, and SDHB for ovarian cancer
- NRAS mutation for selecting persons with metastatic colorectal cancer who may benefit from anti-VEGF antibody bevacizumab; to predict disease prognosis and select persons with melanoma who may benefit from tyrosine kinase inhibitor therapies, and other indications
- OmniSeq Advance DNA and RNA sequencing (OmniSeq and LabCorp)
- OncInsights (Intervention Insights, Grand Rapids, MI)
- OncobiotaLUNG
- Oncomap ExTra (formerly known as Oncotype MAP)
- OncoOmicDx Targeted Proteomic Assay
- OncoSignal test for analysis of solid tumors
- OncoTarget/OncoTreat
- Oncotype MAP PanCancer Tissue Test
- OncoVantage
- Oncuria Detect, Oncuria Monitor and Oncuria Predict for bladder cancer and all other indications
- OVA1/Overa test
- OvaCheck test
- OvaSure
- OvaWatch
- PancreaSeq Genomic Classifier
- PanGIA Prostate for determining if an individual should undergo a prostate biopsy
- Pathwork Tissue of Origin Test/ResponseDx Tissue of Origin Test
- Percepta Bronchial Genomic Classifier
- PGDx elio tissue complete (Personal Genome Diagnostics, Inc.) for tumor mutation profiling
- Pharmaco-oncologic AlgorithmicTreatment Ranking Service
- Phosphatidylinositol-4,5-bisphosphonate 3-kinase, catalytic subunit alpha polypeptide gene (PIK3CA) for predicting disease prognosis and selecting individuals with metastatic colorectal cancer who are being considered for treatment with EGFR antagonists cetuximab and panitumumab, and indications other than breast cancer and uterine sarcoma
- PLCG2 (phospholipase C gamma 2) for all indications other than chronic lymphocytic leukemia (CLL)
- Praxis Somatic Combined Whole Genome Sequencing and Optical Genome Mapping
- Praxis Somatic Optical Genome Mapping
- Praxis Somatic Transcriptome
- Praxis Somatic Whole Genome Sequencing
- PreciseDx Breast Cancer Test
- PreOvar test for the KRAS-variant to determine ovarian cancer risk
- ProOnc TumorSourceDx test (Prometheus Laboratories, San Diego, CA) to identify tissue or origin for metastatic tumor
- PROphet NSCLC test
- Prostate core mitotic test
- Prostate Px and Post-Op Px for predicting recurence of prostate cancer
- Prostate Cancer Risk Panel (FISH analysis by Mayo Clinic)
- Proveri prostate cancer assay (PPCA)
- PSA for screening women with breast cancer or for differentiating benign from malignant breast masses
- PTEN gene expression for non-small cell lung cancer
- RadTox cfDNA test
- Ras oncogenes (except KRAS, NRAS and BRAF)
- ResponseDx Colon
- Ribonucleotide reductase subunit M1 (RRM1) for persons with NSCLC who are being considered for treatment with gemcitabine-based chemotherapy, and other indications
- RNA gene expression profiling for hematolymphoid disorder or neoplasm
- RNA gene expression for solid organ neoplasm
- ROMA (Risk of Ovarian Malignancy Algorithm) for ovarian cancer
- Rotterdam Signature 76-gene panel
- Salivary metatranscriptome analysis for oral cancers (i.e., mRNA CancerDetect)
- SelectMDx for prostate cancer
- Sentinel Prostate Test for prostate cancer screening and determining the risk level of the disease
- Serum amyloid A as a biomarker for endometrial endometrioid carcinoma to monitor disease recurrence and target response to therapy
- Signatera for carcinoid lung cancer
- Signatera molecular residual disease (MRD) assay for:
- alveolar soft tissue sarcoma
- breast cancer
- colorectal cancer
- cutaneous melanoma
- gastric adenocarcinoma
- ovarian sex cord stromal tumor
- pancreatic cancer
- prostate cancer
- renal cell carcinoma, and
- uterine cancer
- Solid Tumor Expanded Panel (Quest)
- Strata Select
- TargetPrint gene expression test for evaluation of estrogen receptor, progesterone receptor, and HER2receptor status in breast cancer
- Tempus Tumor Origin (TO) testing
- The 41-gene signature assay
- Theros CancerType ID (bioTheranostics Inc., San Diego, CA)
- Thymidylate synthase
- Thyroid GuidePx
- TMPRSS fusion genes for prostate cancer
- Topographic genotyping (Pancragen (formerly PathFinderTG))
- Total (whole) gene sequencing for cancer
- TP53 mutation analysis for ovarian cancer
- UriFind Blood Cancer Assay for bladder cancer
- UroAmp MRD for bladder cancer
- UroCor cytology panels (DD23 and P53) for bladder cancer
- Vascular Endothelial Growth Factor (VEGF) except for Castleman's disease
- Vascular endothelial growth factor receptor 2 (VEGFR2) expression for identifying persons with colorectal cancer that is likely to respond to VEGF inhibition, and other indications
- Whole exome sequencing (somatic mutations) (e.g., EXaCT-1 Whole Exome Testing) for cancer.
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Any of the following circulating tumor markers are also considered experimental, investigational, or unproven for screening asymptomatic subjects for cancer, diagnosis, staging, routine surveillance of cancer and monitoring the response to treatment (also see CPB 0715 - Pharmacogenetic and Pharmacodynamic Testing):
a2-PAG CA-SCC MAM-6 TAG12 AMACR Cathepsin-D, Cathepsin-L Motility-related protein (MRP) TAG72 Cyclin E (fragments or whole length) Multidrug resistance glycoprotein (Mdr1) TAG72.3 BCM DU-PAN-2 TAG72.5 CA195 Early prostate cancer antigen (EPCA) NSE TATI CA242 Guanylyl cyclase C (Previstage GCC molecular test) Thrombospondin-1 (THBS-1) CA50 Hepsin PCA3 (DD3) / UpM3 Thymosin B15 CA549 Human kallikrein 2 (HK2) PNA/ELLA TNF-a CA72-4 LASA Prostate stem cell antigen (PSCA) Topoisomerase II Alpha (TOP2A) CAM17-1 LPA SCC TPA CAM26 M 26 SLEX Thymosin B15 CAM29 M 29 SPAN-1 Nuclear Matrix Protein 66 (NMP66) CAR-3 MSA SLX Anti-malignin antibody screen (AMAS) test CYFRA21-1 MCA ST-439
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Related Policies
- CPB 0140 - Genetic Testing
- CPB 0227 - BRCA Testing, Prophylactic Mastectomy, and Prophylactic Oophorectomy
- CPB 0245 - Tumor Chemosensitivity Assays
- CPB 0313 - Trastuzumab (Herceptin and biosimilars), Trastuzumab and Hyaluronidase-oysk (Herceptin Hylecta)
- CPB 0314 - Rituximab
- CPB 0319 - RET Proto-Oncogene Testing
- CPB 0521 - Prostate Cancer Screening
- CPB 0715 - Pharmacogenetic and Pharmacodynamic Testing
- CPB 0758 - Tumor Chemoresistance Assays
Background
A tumor marker is a substance such as a protein, antigen or hormone in the body that may indicate the presence of cancer. Generally, these markers are specific to certain types of cancer and can be detected in blood, urine and tissue samples. The body may produce the marker in response to cancer or the tumor itself may produce the marker. The detection of tumor markers may be used to determine a diagnosis or as an indicator of disease (cancer) progression. It can also be used to document clinical response to treatment. Tumor markers include, but may not be limited to, alpha- fetoprotein (AFP), CA 15-3/CA 27.29, CA 19-9, CA-125, carcinoembryonic antigen (CEA) and prostate-specific antigen (PSA).
Tumor markers are normally produced in low quantities by cells in the body. Detection of a higher-than-normal serum level by radioimmunoassay or immunohistochemical techniques usually indicates the presence of a certain type of cancer. Currently, the main use of tumor markers is to assess a cancer's response to treatment and to check for recurrence. In some types of cancer, tumor marker levels may reflect the extent or stage of the disease and can be useful in predicting how well the disease will respond to treatment. A decrease or return to normal in the level of a tumor marker may indicate that the cancer has responded favorably to therapy. If the tumor marker level rises, it may indicate that the cancer is spreading. Finally, measurements of tumor marker levels may be used after treatment has ended as a part of follow-up care to check for recurrence.
- tumor marker levels can be elevated in people with benign conditions;
- tumor marker levels are not elevated in every person with cancer, especially in the early stages of the disease; and
- many tumor markers are not specific to a particular type of cancer; and
- the level of a tumor marker can be elevated by more than one type of cancer.
Examples of Tumor Markers Include
- 5-Hydroxyindoleacetic acid (5-HIAA) – the main metabolite of serotonin, used as a marker in the evaluation of carcinoid tumors;
- Beta-2-Microglobulin (B2M) – A protein found on the surface of many cells. High levels of B2M are an indicator of certain kinds of cancer, including chronic lymphocytic leukemia, non-Hodgkin's lymphoma and multiple myeloma or kidney disease;
- Beta Human Chorionic Gonadotropin (beta HCG) – A type of tumor marker that may be found in higher than normal amounts in individuals with some types of cancer;
- Calcitonin – Hormone secreted by the thyroid that lowers blood calcium;
- Calretinin – A calcium-binding protein that is used as a marker in the evaluation of lung cancer and other diseases.
- Chromogranin A – A protein found inside neuroendocrine cells, which releases chromogranin A and other hormones into the blood. Chromogranin A may be found in higher than normal amounts in individuals with certain neuroendocrine tumors, small cell lung cancer, prostate cancer and other conditions
- Guanylyl cyclase c (GCC) – An enzyme that may be expressed only in the cells that line the intestine from the duodenum to the rectum.
- Inhibin – One of two hormones (designated inhibin-A and inhibin-B) secreted by the gonads (by Sertoli cells in the male and the granulosa cells in the female) and inhibits the production of follicle-stimulating hormone (FSH) by the pituitary gland;
- Lactate Dehydrogenase (LDH) – Marker used to monitor treatment of testicular cancer;
- Mucin-1 (MUC-1) – Carbohydrate antigen elevated in individuals with tumors of the breast, ovary, lung and prostate as well as other disorders;
- Napsin A – Protein used as a marker in the evaluation of lung cancer;
- Prealbumin – Marker of nutritional status and a sensitive indicator of protein synthesis. Also referred to as transthyretin;
- Prostate Specific Antigen (PSA) – Substance produced by the prostate gland. Levels of PSA in the blood often increase in men with prostate cancer.
- Thyroglobulin – Protein found in the thyroid gland. Some thyroglobulin can be found in the blood and this amount may be measured after thyroid surgery to determine whether thyroid cancer has recurred;
- Thyroid Transcription Factor-1 (TTF-1) – A protein that is used as a tumor marker in the evaluation of lung cancer;
- Transferrin – A protein in blood plasma that carries iron derived from food intake to the liver, spleen and bone marrow.
Tumors may be evaluated with histology, which involves examination of the structure, especially the microscopic structure, of organic tissues. Methods of detecting tumor markers include, but are not limited to: Fluorescence in Situ Hybridization (FISH) – Laboratory technique used to detect small deletions or rearrangements in chromosomes. Immunohistochemical (IHC) Analysis – Laboratory process of detecting an organism in tissues with antibodies.
Gene mutation testing can purportedly be used to find somatic mutations in cancerous cells that are not inherited. Some examples of genes that may have somatic mutations include: IDH1 and IDH2 genes (associated with acute myeloid leukemia [AML], gliomas and chondrosarcomas); NPM1 and FLT3 genes (associated with AML).
Individualized molecular tumor profiling is a laboratory method of testing a panel of tumor markers, which may include genetic as well as biochemical markers, to establish a personalized molecular profile of a tumor to recommend treatment options.
Mass spectrometry based proteomic profiling (eg, Veristrat, Xpresys Lung) is a multivariate serum protein test that uses mass spectrometry and proprietary algorithms to analyze proteins in an individual’s serum. The Xpresys is no longer on the market.
Next-generation sequence (NGS) tests use select genes to purportedly identify molecular growth drivers for improved risk stratification and targeted therapies. Examples include: FoundationOne and OncoVantage for solid tumor cancers; FoundationOne Heme for hematological cancers and sarcomas; and ThyGenX for indeterminate thyroid nodules.
Liquid biopsy refers to serum testing for DNA fragments that are shed by cancer cells and released into the bloodstream. This method is purportedly used for screening, diagnosis and/or monitoring of cancer cells that may otherwise require a tissue sample.
Multianalyte assays with algorithmic analyses (MAAAs) are laboratory measurements that use a mathematical formula to analyze multiple markers that may be associated with a particular disease state and are designed to evaluate disease activity or an individual’s risk for disease. The laboratory performs an algorithmic analysis using the results of the assays and sometimes other information, such as sex and age and converts the information into a numeric score, which is conveyed on a laboratory report. Generally, MAAAs are exclusive to a single laboratory which owns the algorithm. MAAAs have been proposed for the evaluation of pelvic masses, including assisting in the determination of referral for surgery to a gynecologic oncologist or to a general surgeon.
Topographic genotyping (eg, PathFinderTG) is a test that examines a panel of 15 to 20 genetic markers in tissue biopsy or other tissue specimens to purportedly aid in the determination of indeterminate or equivocal cancer diagnoses.
An ASCO Provisional Opinion on somatic genomic testing in patients with metastatic or advanced cancer (Chakravarty, et al., 2022) states: "Repeat genomic testing may be performed for patients with acquired resistance on targeted therapies, especially when known acquired resistance mechanisms may affect the choice of next-line therapy. Repeat testing may also assist in identifying new targets in tumors after progression or after prolonged stable disease on targeted therapies."
AFP
Alpha-fetoprotein (AFP) is a protein that is normally elevated in pregnant women since it is produced by the fetus; however, AFP is not usually found in the blood of adults. In men and in women who are not pregnant, an elevated level of AFP may indicate liver, ovarian or testicular cancer.
Alpha-fetoprotein is normally produced by a developing fetus. Alpha fetoprotein levels begin to decrease soon after birth and are usually undetectable in the blood of healthy adults, except during pregnancy. According to accepted guidelines, an elevated level of AFP strongly suggests the presence of either primary liver cancer or germ cell cancer of the ovary or testicle. As AFP is an established test for the diagnosis and monitoring of hepatoma, it is used as a screening tool to rule out the presence of a liver neoplasm before considering liver transplantation. This is especially pertinent in cases (e.g., cirrhosis) where there is an increased risk of developing a primary liver tumor.
Elevated serum AFP levels are most closely associated with nonseminomatous testicular cancer and hepatocellular cancer (Chin, 2006). The rate of clearance from serum after treatment is an indicator of the effectiveness of therapy. Conversely, the growth rate of progressive disease can be monitored by serially measuring serum AFP concentrations over time.
B15
Hutchinson et al (2005) stated that in tissue-based assays, thymosin beta15 (B15) has been shown to correlate with prostate cancer and with recurrence of malignancy. To be clinically effective, it must be shown that thymosin B15 is released by the tumor into body fluids in detectable concentrations. These researchers developed a quantitative assay that can measure clinically relevant levels of thymosin B15 in human urine. Sixteen antibodies were raised against recombinant thymosin B15 and/or peptide conjugates. One antibody, having stable characteristics over the wide range of pH and salt concentrations found in urine and minimal cross-reactivity with other beta thymosins, was used to develop a competitive enzyme-linked immunosorbent assay (ELISA). Urinary thymosin B15 concentration was determined for control groups; normal (n = 52), prostate intraepithelial neoplasia (PIN, n = 36), and patients with prostate cancer; untreated (n = 7) with subsequent biochemical failure, radiation therapy (n = 17) at risk of biochemical recurrence. The operating range of the competition ELISA fell between 2.5 and 625 ng/ml. Recoveries exceeded 75%, and the intra- and inter-assay coefficients of variability were 3.3% and 12.9%, respectively. No cross-reactivity with other urine proteins was observed. A stable thymosin B15 signal was recovered from urine specimens stored at -20 degrees C for up to 1 year. At a threshold of 40 (ng/dl)/microg protein/mg creatinine), the assay had a sensitivity of 58% and a specificity of 94%. Relative to the control groups, thymosin B15 levels were greater than this threshold in a significant fraction of patients with prostate cancer (p < 0.001), including 5 of the 7 patients who later experienced PSA recurrence. The authors concluded that an ELISA that is able to detect thymosin B15 at clinically relevant concentrations in urine from patients with prostate cancer has been established. They noted that the assay will provide a tool for future clinical studies to validate urinary thymosin B15 as a predictive marker for recurrent prostate cancer.
Bcl-2
Bcl-2 (B-cell CLL/lymphoma 2; BCL2) is a proto-oncogene whose protein product, bcl-2, suppresses programmed cell death (apoptosis), resulting in prolonged cellular survival without increasing cellular proliferation. Dysregulation of programmed cell death mechanisms plays a role in the pathogenesis and progression of cancer as well as in the responses of tumors to therapeutic interventions. Many members of the Bcl-2 family of apoptosis-related genes have been found to be differentially expressed in various malignancies (Reed, 1997).
Salgia (2008) reviewed the evidence for detection of Bcl-2 in lung cancer. The author observed that Bcl-2 over-expression has been reported in 22 to 56% of lung cancers with a higher expression in squamous cell carcinoma as compared to adenocarcinoma histology. The author concluded, however, that the association of Bcl-2 expression and prognosis in non-small cell lung cancer is unclear. Multiple reports have demonstrated that Bcl-2-positive lung cancers are associated with a superior prognosis compared to those that are Bcl-2 negative. However, other studies have failed to demonstrate any survival impact with bcl-2 positivity, while over-expression has also been associated with a poorer outcome. A meta-analysis that included 28 studies examining the prognostic influence of Bcl-2 in non-small cell lung cancer concluded that over-expression of Bcl-2 was associated with a significantly better prognosis in surgically resected (hazard ratio 0.5, 95% CI 0.39-0.65).
Compton (2008) recently reviewed the evidence on the Bcl-2 oncogene and other tumor markers in colon cancer. Compton explained that Bcl-2 is a gene related to apoptosis/cell suicide. Bcl-2 over-expression leading to inhibition of cell death signaling has been observed as a relatively early event in colorectal cancer development. The author concluded that the independent influence of the Bcl-2 oncogene on prognosis remains unproven, and explained that the variability in assay methodology, conflicting results from various studies examining the same factor, and the prevalence of multiple small studies that lack statistically robust, multivariate analyses all contribute to the lack of conclusive data. Compton concluded that before the Bcl-2 oncogene and certain other tumor markers can be incorporated into clinically meaningful prognostic stratification systems, "more studies are required using multivariate analysis, well-characterized patient populations, reproducible and current methodology, and standardized reagents."
BTK (Brution's Typrosine Kinase) and PLCG2 (Phospholipase C Gamma 2)
The National Comprehensive Cancer Network (NCCN) guidelines for "Chronic lymphocytic/smalllymphocytic lymphoma" (v.2.2019) states that testing for BTK and PLCG2 mutations may be useful in patients receiving ibrutinib and suspected of having progression; however, BTK and PLCG2 mutation status alone is not an indication to change treatment. Testing for mutations as screening for resistance is not currently recommended.
Lampson and Brown (2018) state that BTK and PLCG2 mutations are found in approximately 80% of CLL patients with acquired resistance to ibrutinib; however, it remains unclear if these mutations are solely associated with disease relapse or is the direct cause. The authors reviewed the properties of both CLL and ibrutinib that complicate attempts to definitively conclude whether BTK/PLCG2 mutations are passengers or drivers of ibrutinib-resistant disease. The authors concluded that while BTK/PLCG2 mutations have characteristics suggesting that these mutations can drive ibrutinib resistance, a definitive answer remains formally unproven until specific inhibition of such mutations is shown to cause regression of ibrutinib-resistant CLL. Furthermore, data suggest that alternative mechanisms of resistance do exist in some patients. The authors further conclude that multiple unanswered questions remain regarding resistance to ibrutinib in CLL, requiring a need for further exploration. Testing the efficacy of drugs that can inhibit the BTK C481S mutation in patients with ibrutinib-resistant disease is warranted.
CA-125
Cancer antigen 125 (CA-125) is a test that evaluates ovarian cancer treatment. CA-125 is a protein that is found more in ovarian cancer cells than in other cells. CA-125 is expressed by >80 percent of non-mucinous ovarian epithelial neoplasms (Chin et al, 2006). Approximately half of women with metastatic ovarian cancer have an elevated CA-125 level.
The Gynecologic Cancer Foundation, the Society of Gynecologic Oncologists, and the American Cancer Society have issued a consensus statement to promote early detection of ovarian cancer, which recommends that women who have symptoms, including bloating, pelvic or abdominal pain, difficulty eating or feeling full quickly, and urinary frequency and urgency, are urged to see a gynecologist if symptoms are new and persist for more than three weeks (ACS, 2007; SGO, 2007). Ovarian cancer is among the deadliest types of cancer because diagnosis usually comes very late, after the cancer has spread. If the cancer is found and surgically removed before it spreads outside the ovary, the five year survival rate is 93%. However, only 19% of cases are detected early enough for that kind of successful intervention. It is estimated that 22,430 new cases and 15,280 deaths will be reported in 2007 (ACS, 2007). The consensus statement recommendations are based on studies that show the above symptoms appeared in women with ovarian cancer more than in other women (Goff, et al., 2004; Daly & Ozols, 2004). The recommendations acknowledge that there is not consensus on what physicians should do when patients present with these symptoms. According to a consensus statement issued by the Gynecologic Cancer Foundation, pelvic and rectal examination in women with the symptoms is one first step. If there is a suspicion of cancer, the next step may be a transvaginal ultrasound to check the ovaries for abnormal growths, enlargement, or telltale pockets of fluid that can indicate cancer. Testing for CA-125 levels should also be considered.
There is no evidence available that measurement of CA-125 can be effectively used for widespread screening to reduce mortality from ovarian cancer, nor that the use of this test would result in decreased rather than increased morbidity and mortality. According to the available literature, not all women with elevated CA 125 levels have ovarian cancer. CA 125 levels may also be elevated by cancers of the uterus, cervix, pancreas, liver, colon, breast, lung, and digestive tract. Non-cancerous conditions that can cause elevated CA 125 levels include endometriosis, pelvic inflammatory disease, peritonitis, pancreatitis, liver disease, and any condition that inflames the pleura. Menstruation and pregnancy can also cause an increase in CA 125. However, according to the available literature, changes in CA 125 levels can be effectively used in the management of treatment for ovarian cancer. In women with ovarian cancer being treated with chemotherapy, the literature suggests a falling CA 125 level generally indicates that the cancer is responding to treatment and increased survival is expected. Increasing CA 125 levels during or after treatment, on the other hand, may suggest that the cancer is not responding to therapy or that residual cancer remains. According to the available literature, failure of the CA 125 level to return to normal after three cycles of chemotherapy indicates residual tumor, early treatment failure and decreased survival. Under accepted guidelines, CA 125 levels can also be used to monitor patients for recurrence of ovarian cancer. Although an elevated CA 125 level is highly correlated with the presence of ovarian cancer, the literature suggests a normal value does not exclude residual or recurrent disease.
Aetna's preventive services guidelines are based on the recommendations of leading primary care medical professional organizations and federal public health agencies. None of these organizations recommend routine screening of average-risk, asymptomatic women with serum CA-125 levels for ovarian cancer. These organizations have concluded that serum CA-125 levels are not sufficiently sensitive or specific for use as a screening test for ovarian cancer, and that the harms of such screening outweigh the benefits.
The American College of Obstetricians and Gynecologists (2002) has stated that "[u]nfortunately, there is no screening test for ovarian cancer that has proved effective in screening low-risk asymptomatic women. Measurement of CA 125 levels and completion of pelvic ultrasonography (both abdominal and transvaginal) have been the two tests most thoroughly evaluated.... Data suggest that currently available tests do not appear to be beneficial for screening low-risk, asymptomatic women because their sensitivity, specificity, positive predictive value, and negative predictive value have all been modest at best. Because of the low incidence of disease, reported to be approximately one case per 2,500 women per year, it has been estimated that a test with even 100% sensitivity and 99% specificity would have a positive predictive value of only 4.8%, meaning 20 of 21 women undergoing surgery would not have primary ovarian cancer. Unfortunately, no test available approaches this level of sensitivity or specificity."
The National Cancer Institute (2004) has stated: "There is insufficient evidence to establish that screening for ovarian cancer with serum markers such as CA 125 levels, transvaginal ultrasound, or pelvic examinations would result in a decrease in mortality from ovarian cancer. A serious potential harm is the false-positive test result, which may lead to anxiety and invasive diagnostic procedures. There is good evidence that screening for ovarian cancer with the tests above would result in more diagnostic laparoscopies and laparotomies than new ovarian cancers found. Unnecessary oophorectomies may also result."
The U.S. Preventive Services Task Force (2004) recommends against routine screening with serum CA-125 level for ovarian cancer. The Task Force concluded that the potential harms of such screening outweigh the potential benefits.
CA 15-3
Cancer antigen 15-3 (CA 15-3) is a serum cancer antigen that has been used in the management of patients with breast cancer. According to the available literature, its low detection rate in early stage disease indicates that CA 15-3 cannot be used to screen or diagnose patients with breast cancer. It has been widely used to monitor the effectiveness of treatment for metastatic cancer. Elevated serum CA 15-3 concentrations are found in 5 percent of stage I, 29 percent of stage II, 32 percent of stage III and 95 percent of stage IV carcinoma of the breast (Chin, et al, 2006). Most (96 percent) patients with a CA 15-3 increase of greater than 25 percent have disease progression. Most (nearly 100 percent) patients with a CA 15-3 decrease of greater than 50 percent are responding to treatment.
Cancers of the ovary, lung, and prostate may also raise CA 15-3 levels. The literature indicates elevated levels of CA 15-3 may be associated with non-cancerous conditions, such as benign breast or ovarian disease, endometriosis, pelvic inflammatory disease, and hepatitis.
Similar to the CA 15-3 antigen, CA 27-29 is found in the blood of most breast cancer patients. The literature indicates CA 27-29 levels may be used in conjunction with other procedures (such as mammograms and measurements of other tumor marker levels) to check for recurrence in women previously treated for stage II and stage III breast cancer. CA 27-29 levels can also be elevated by cancers of the colon, stomach, kidney, lung, ovary, pancreas, uterus, and liver. First trimester pregnancy, endometriosis, ovarian cysts, benign breast disease, kidney disease, and liver disease are non-cancerous conditions that can also elevate CA 27-29 levels.
Elevated CA 27.29 levels are primarily associated with metastatic breast cancer, where it can be used to monitor the course of disease, response to treatment, and detect disease recurrence (Chin, et al., 2006). Elevated serum CA 27.29 concentrations are found in 95 percent of stage IV breast cancer. In addition, CA 27.29 has been found to be elevated in lung (43 percent), pancreas (47 percent), ovarian (56 percent), and liver (55 percent) cancer.
CA 19-9
Cancer antigen 19-9 (CA 19-9) is a mucin-glycoprotein first identified from a human colorectal carcinoma cell line and is present in epithelial tissue of the stomach, gall bladder, pancreas and prostate (Chin, et al., 2006). Concentrations are increased in patients with pancreatic, gastric, and colon cancer as well as in some nonmalignant conditions. Increasing levels generally indicate disease progression, whereas decreasing levels suggest therapeutic response.
Initially found in colorectal cancer patients, CA 19-9 has also been identified in patients with pancreatic, stomach, hepatocellular cancer, and bile duct cancer. In those who have pancreatic cancer, the literature indicates higher levels of CA 19-9 tend to be associated with more advanced disease. Although the sensitivity of the CA 19-9 level in patients with pancreatic cancer is relatively high, the specificity is lowered by elevations that occur in patients with benign pancreatic or liver disease. Non-cancerous conditions that may elevate CA 19-9 levels include gallstones, pancreatitis, cirrhosis of the liver, and cholecystitis. Although excellent correlations have been reported between CA 19-9 measurements and relapse in patients with pancreatic cancer who are followed after surgical resection, no patient has been salvaged by the earlier diagnosis of relapse, a fact that reflects the lack of effective therapy.
Guidelines from the National Comprehensive Cancer Network (NCCN, 2010) state that measurement of CA 19-9 should be considered in evaluating patients with intrahepatic or extrahepatic cholangiocarcinoma and gallbladder cancer. The guidelines note that CA 19-9 is often elevated in persons with cholangiocarcinoma or gallbladder cancer, although this marker is not specific for these cancers. Nehls, et al. (2004) considered CA19-9 as one of the several new potential tumor markers for the diagnosis of cholangiocarcinoma. Levy, et al. (2005) aimed to characterize the test properties of CA 19-9 and of a change in CA 19-9 over time in predicting cholangiocarcinoma in patients with primary sclerosing cholangitis. Charts of 208 patients were reviewed. Fourteen patients had cholangiocarcinoma. Median CA 19-9 was higher with cholangiocarcinoma (15 versus 290 U/ml, p < 0.0001). A cutoff of 129 U/ml provided: sensitivity 78.6%, specificity 98.5%, adjusted positive predictive value 56.6% and negative predictive value 99.4%. The median change over time was 664 U/ml in cholangiocarcinoma compared to 6.7 U/ml in primary sclerosing cholangitis alone (p < 0.0001). A cutoff of 63.2 U/ml for change in CA 19-9 provided: sensitivity 90%, specificity 98% and positive predictive value 42%.
CA 19-9 is produced by adenocarcinomas of the pancreas, stomach, gall-bladder, colon, ovary, and lung, and it is shed into the circulation. Although numerous studies have addressed the potential utility of CA 19-9 in adenocarcinoma of the colon and rectum, the sensitivity of CA 19-9 was always less than that of the CEA test for all stages of disease. Its use for screening asymptomatic populations has been hampered by a false-positive rate of 15% to 30% in patients with non-neoplastic diseases of the pancreas, liver, and biliary tract. Only a few studies have addressed the use of CA 19-9 in monitoring patients' post-primary therapy. Significant postsurgical decreases are observed for CA 19-9, but these decreases have not been correlated with survival or disease-free interval. In monitoring response to treatment, decreases in CEA have been found to more accurately reflect response to therapy than did decreases of CA 19-9. Progressive increases of the marker may signal disease progression in 25% of the patients who express the CA 19-9 marker, but this monitoring provides only a minimal lead time of 1 to 3 months. Monitoring with CA 19-9 has not been shown to improve the management of patients with colorectal cancer. The serum CA 19-9 level does not add significant information to that provided by CEA, which is currently regarded as the marker of choice for this neoplasm.
Sinakos and colleagues (2011) evaluated the long-term outcomes in Mayo Clinic patients presenting with primary sclerosing cholangitis (PSC) between 2000 and 2010 (n= 73) for incidence of cholangiocarcinoma (CCA). The results showed initial levels of CA 19-9 in patients without CCA were significantly lower than those from patients with CCA (p < 0.0001). No factors known to affect CA 19-9 levels were identified in 33% of the patients without CCA; endoscopic treatment and recurrent bacterial cholangitis were associated with levels of CA 19-9 in 26% and 22% of these patients, respectively.
Juntermanns (2011) prospectively analyzed a bile duct tumor database and retrieved records of 238 patients who underwent surgery between 1999 and 2008. Their findings included that pre-operative CA19-9 serum levels did not show a statically reliable differentiation between benign or malignant dignity. The authors concluded that current diagnostics cannot differentiate malignant from benign tumor masses in the hepatic hilum with required reliability. The authors further concluded that administration of CIK cells, thymus factor, IL-2 and IFN-alpha after AHSCT could improve the immunologic function of patients, and TH1/TH2 ratio may virtually reflect the immune status of patients, but that more information is required to make prognostic assessments of immune reconstruction and the long-term survival rate.
Sarbia et al (1993) investigated 69 adenocarcinomas of the esophagogastric junction and found high rates of antigen expression were found for the "intestinal" markers CA 19-9 (between 55.5% and 100%) and BW 494 (between 42.9 and 86.7%). The authors concluded that these data, in combination with CK-20 expression, PGII, and 2B5 indicate that the distribution of adenocarcinomas with gastric and.or intestinal differentiation at the esophagogastric junction forms a continuum with out clear-cut borders. This study has not been replicated and NCCN guidelines for Esophageal and Esophagogastric Junction Cancers does not include recommendations for CA 19-9 testing for these indications (NCCN, 2011).
The American Society of Clinical Oncology (ASCO)'s update of recommendations for the use of tumor markers in gastrointestinal cancer (Gershon, et al., 2006) stated that for pancreatic cancer, CA 19-9 can be measured every 1 to 3 months for patients with locally advanced or metastatic disease receiving active therapy.
Mucinous carcinoma of the appendix is a rare entity most commonly associated with primary tumors of the appendix and colon, and for which spread is generally confined to the abdominal cavity (Andreopoulou et al, 2007). Imaging assessment of these mucinous lesions is difficult, and recent studies have explored the use of tumor markers as clinical tools in evaluation of mucinous carcinoma of the appendix.
Carmignani et al (2004) evaluated patients with synchronous systemic and intraperitoneal dissemination of appendix cancer treated with cytoreductive surgery and perioperative regional chemotherapy with a mean follow up time of 42.6 months. Results of this study indicated that patients with elevated CEA and CA 19-9 levels had a shorter median survival time (p=0.0083 and p = 0.0193, respectively). In a subsequent study, Carmingnani et al (2004) prospectively recorded tumor markers CEA and CA19-9 within 1 week prior to definitive treatment. The investigators found CEA elevated in 56% of 532 patients and CA19-9 elevated in 67.1% of those patients. They reported that "although the absolute level of tumor marker did not correlate with prognosis, a normal value indicated an improved survival." Their findings included an elevated CEA in 35.2% of 110 patients determined to have recurrent disease and an elevated CA 19-9 in 62.9%, while 68.2% of patients had at least one of the tumor markers elevated.
Current guidelines indicate that for liver transplantation for primary sclerosing cholangitis, stringent efforts should be made to detect superadded cholangiocarcinoma, including measurement of CA 19-9 (Devlin & O'Grady, 1999).
Carmignani et al (2004a) conducted a study to report the role of combined treatments, including cytoreductive surgery and perioperative regional chemotherapy, in patients with synchronous systemic and intraperitoneal dissemination of appendix cancer. Study subjects were treated with cytoreductive surgery and perioperative regional chemotherapy and statistical analysis of variables utilized survival as an end point and included demographic characteristics, prior surgical score (PSS), tumor marker levels, peritoneal cancer index (PCI), and completeness of cytoreduction (CC). With a mean follow-up of 42.6 months, median survival time (MST) for 15 patients was 28 months and 5-year survival rate was 29.4 %. Female patients had a longer MST than male patients (p = 0.0199) and survival was better in patients with PSS 0 and 1 (p = 0.0277). Patients with elevated CEA and CA 19-9 levels had a shorter MST (p = 0.0083 and p = 0.0193, respectively) while PCI and CC comparisons did not show significant differences. The morbidity rate (n = 2) was 13.3 % and the mortality (n = 2) rate was also 13.3 %. The authors concluded that "acceptable morbidity and mortality and a 29.4 % 5-year survival rate allows cytoreductive surgery and regional chemotherapy to be considered as a treatment option for selected patients with synchronous systemic and intraperitoneal dissemination of appendix cancer."
Carmignani et al (2004b) in a further publication regarding gastrointestinal cancer, stated that carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA 19-9) tumor markers have found selected clinical application. The authors remarked that the use of these tumor markers in mucinous epithelial tumors of the appendix has not been previously determined. Thus, the authors conducted a study in which, in patients with peritoneal dissemination of a mucinous epithelial malignancy of the appendix, tumor markers CEA and CA 19-9 were prospectively recorded preoperatively within 1 week prior to definitive treatment and if the appendiceal tumor recurred, the tumor marker was determined. The primary endpoint was the accuracy of these two tumor markers in the management of this disease for these two specific clinical situations. CEA was elevated in 56 % of 532 patients and CA 19-9 was elevated in 67.1 % of these patients. Although the absolute level of tumor marker did not correlate with prognosis, a normal value indicated an improved survival. CEA was elevated in 35.2 % of 110 patients determined to have recurrent disease and CA 19-9 was elevated in 62.9 %. At least one of the tumor markers was elevated in 68.2 % of patients. An elevated CEA tumor marker at the time of recurrence indicated a reduced prognosis and both CEA and CA 19-9 tumor markers were elevated in a majority of these patients. This should be a valuable diagnostic tool previously underutilized in this group of patients. These tumor markers were also of benefit in the assessment of prognosis in that a normal level indicated an improved prognosis. At the time of a reoperative procedure, CEA and CA 19-9 tumor markers gave information regarding the progression of disease and have practical value in the management of epithelial appendiceal malignancy with peritoneal dissemination.
Andreopoulou et al (2007) stated that mucinous carcinoma of the appendix is a rare entity with a distinct natural history that poses diagnostic and therapeutic challenges and that mucinous peritoneal carcinomatosis is most commonly associated with primary tumors of the appendix and colon. The authors stated that usually the spread remains confined to the abdominal cavity and that imaging assessment of these mucinous lesions is difficult, while tumor markers (CEA and CA19.9) may be surrogates for extent of disease.
Recruitment for large scale studies given the rare nature of mucinous appendiceal carcinoma would be challenging. However, available evidence does illustrate a benefit to use of CA 19-9 in patients with mucinous appendiceal carcinoma.
National Comprehensive Cancer Network’s clinical practice guideline on “Hepatobiliary cancers” (Version 1.2021) states that CEA and Ca 19-9 are baseline tests, and should not be performed to confirm diagnosis of gallbladder cancer, or cholangiocarcinoma (extra-hepatic or intra-hepatic).
An UpToDate review on “Tumors of the nasal cavity” (Dagan et al, 2021) does not mention CA 19-9.
Furthermore, National Comprehensive Cancer Network’s Biomarkers Compendium (2021) does not list NUT midline carcinoma tumor of the nasal cavity to be associated with CA 19-9 expression.
Cathepsins
This enzyme plays a critical role in protein catabolism and tissue remodeling (Chin, et al., 2006). Over-expression is associated with non-ductal carcinoma and metastasis at the time of breast cancer diagnosis. High levels may have clinical significance in predicting decreased metastasis-free survival and decreased overall survival in women with node-negative breast cancer.
Svatek et al (2008) examined the role of urinary cathepsin B and L in the detection of bladder urothelial cell carcinoma. These investigators concluded that urinary cathepsin L is an independent predictor of bladder cancer presence and invasiveness in patients with a history of urothelial carcinoma of the bladder. They stated that further evaluation of this marker is necessary before its use as an adjunct to cystoscopy for urothelial carcinoma of the bladder.
CD 20
CD 20 is used to determine eligibility for rituximab (Rituxan; anti-CD20) treatment in patients with B-cell non-Hodgkin's lymphomas (NHL) (Chin, et al., 2006). Rituximab is a genetically engineered, chimeric murine/human monoclonal antibody directed against the CD20 antigen found on the surface of normal and malignant B-cell lymphocytes. Since non-Hodgkin's Lymphoma (NHL) subtypes may differ in their response to rituximab, determination of drug sensitivity is important for choosing therapy.
CD 25
CD 25 is used to determine eligibility for denileukin diftitox treatment in patients with persistent or recurrent CTCL (Chin, et al., 2006). Denileukin diftitox (Ontak) is a cutaneous T-cell lymphoma (CTCL) therapy that targets the high-affinity interleukin-2 (IL-2) receptor. The IL-2 receptor may exist in a low-affinity form (CD25), an intermediate-affinity form (CD122/CD132), and a high-affinity form (CD25/CD122/CD132). Patients whose malignant cells express the CD25 component of the IL-2 receptor may respond to Ontak therapy.
CD 31
Compton (2008) reviewed the evidence for intratumor microvessel density (MVD) and antibodies against CD31 in colorectal cancer. The author explained that intratumoral MVD is a reflection of tumor-induced angiogenesis. Microvessel density has been independently associated with shorter survival in some, but not all studies. A meta-analysis of all studies relating MVD expression to prognosis concluded that at least some of the variability could be explained by the different methods of MVD assessment. The author noted that there was a significant inverse correlation between immunohistochemical expression and survival when MVD was assessed using antibodies against CD31 or CD34, but not factor VIII. The author concluded, however, that there is a need for evaluation of MVD in large studies of prognostic factors using multivariate analysis; however, standard guidelines for staining, evaluation, and interpretation of MVD are lacking.
In a review, Hayes (2008) reviewed the evidence for assessing angiogenesis factors in breast cancer. The author noted that, in an early report, MVD count (as indicated by IHC staining for endothelial cells, such as factor VIII-related antigen or CD31) was a statistically significant independent predictor of both disease-free and overall survival in women with both node-negative and node-positive breast cancer. The author noted, however, that subsequent data are conflicting, with some studies confirming and others refuting the initial findings. The author stated that, "As with many of the other tumor marker studies, evaluation of angiogenesis is complicated by technical variation, reader inconsistency, and potential interaction with therapy."
Burgdorf (2006) reviewed the use of CD31 in acquired progressive lymphangioma. The author stated that special staining techniques reveal that the cells are variably positive for CD31, but that the staining patterns are too variable to be of diagnostic importance.
Some authorities have stated that CD31 staining may be useful for diagnosing angiosarcomas (Schwartz, 2008; Carsi and Sim, 2008; Fernandez and Schwartz, 2007; McMains and Gourin, 2007). CD31 immunostaining can help confirm that the tumor originates from blood vessels.
CD 33
CD 33 is used to determine eligibility for gemtuzumab (Mylotarg, anti-CD33) treatment in patients with acute myeloid leukemia (Chen, et al., 2006). Gemtuzumab consists of a recombinant, humanized IgG kappa antibody conjugated to a cytotoxic anti-tumor antibiotic, calicheamicin, which binds specifically to the CD33 antigen. This antigen is found on the surface of leukemic blasts and immature normal cells of myelomonocytic lineage, but not in normal hematopoietic stem cells.
CD 52
CD 52 is used to determine eligibility for alemtuzumab (Campath, anti-CD52) treatment in patients with chronic lymphocytic leukemia (Chen, et al., 2006). CD52 is an antigen that can be expressed at high density on the surface of malignant CLL cells. Alemtuzumab is a humanized antibody targeted against CD52 and its binding is necessary for cell death and therapeutic response.
CD 117, c-kit
CD 117 is used to determine eligibility for treatment with imatinib mesylate in patients with c-kit-positive gastrointestinal stromal tumors (GISTs) (Chen, et al., 2006). The glycoprotein c-kit (CD117) is a member of the receptor tyrosine kinase subclass III family and has been implicated in a number of malignancies. Imatinib mesylate, a tyrosine kinase inhibitor, is effective in treating GISTs and other tumors that express c-kit.
CEA
Carcinoembryonic antigen (CEA) is a normal cell product that is over-expressed by adenocarcinomas, primarily of the colon, rectum, breast, and lung. It is normally found in small amounts in the blood of most healthy people, but may become elevated in people who have cancer or some benign conditions.
CEA is an oncofetal glycoprotein present in the gastrointestinal tract and body fluids of the embryo and fetus (Chin, et al., 2006). It is also present in certain adult gastrointestinal cells, including the mucosal cells of the colorectum, and small amounts are present in blood. Blood levels are often elevated in patients with disseminated cancers and in some patients with nonmalignant disease.
According to the available literature, the primary use of CEA is in monitoring colorectal cancer, especially when the disease has metastasized. CEA is also used after treatment to check for recurrence of colorectal cancer. However, the literature indicates a wide variety of other cancers can produce elevated levels of this tumor marker, including melanoma; lymphoma; and cancers of the breast, lung, pancreas, stomach, cervix, bladder, kidney, thyroid, liver, and ovary. Elevated CEA levels can also occur in patients with non-cancerous conditions, including inflammatory bowel disease, pancreatitis, and liver disease.
The American Society of Clinical Oncology (ASCO)'s update of recommendations for the use of tumor markers in gastrointestinal cancer (Gershon, et al., 2006) stated that post-operative CEA levels should be performed every 3 months for stage II and III disease for at least 3 years if the patient is a potential candidate for surgery or chemotherapy of metastatic disease.
ER, PR
Estrogen receptor (ER) and progesterone receptor (PR) predicts response to hormone therapy for women with advanced breast cancer and those receiving adjuvant treatment, and prognosticates the aggressiveness of a tumor (Chin, 2006).
The estrogen receptor and progesterone receptor are intracellular receptors that are measured directly in tumor tissue. These receptors are polypeptides that bind their respective hormones, translocate to the nucleus, and induce specific gene expression. Breast cancers are dependent upon estrogen and/or progesterone for growth and this effect is mediated through ERs and progesterone receptors (ER/PR) (Chin, et al., 2006). Both receptors may be over-expressed in malignant breast tissue. Most oncologists have used the estrogen receptor and also the progesterone receptor not only to predict the probability of response to hormonal therapy at the time of metastatic disease, but also to predict the likelihood of recurrent disease, and to predict the need for adjuvant hormonal therapy or chemotherapy. Although these latter uses for estrogen and progesterone receptors are commonly accepted by most oncologists, the data on which these conclusions are based are controversial.
ERCC1
Yu and colleagues (2012) stated that the excision repair cross-complementation group 1 (ERCC1) plays an essential role in DNA repair and has been linked to resistance to platinum-based anticancer drugs among advanced NSCLC patients. These investigators examined if ERCC1 Asn118Asn and C8092A genetic variants are associated with treatment response of platinum chemotherapy. They performed a meta-analysis using 10 eligible cohort studies (including 11 datasets) with a total of 1,252 NSCLC patients to summarize the existing data on the association between the ERCC1 Asn118Asn and C8092A polymorphisms and response to platinum regiments. Odds ratio or hazard ratio with 95 % CI were calculated to estimate the correlation. These researchers found that neither ERCC1 C8092A polymorphism nor Asn118Asn variant is associated with different response of platinum-based treatment among advanced NSCLC patients. Additionally, these 2 genetic variants are not related to treatment response in either Caucasian patients or Asian patients. The authors concluded that the findings of this meta-analysis indicated that the ERCC1 Asn118Asn and C8092A polymorphisms may not be good prognostic biomarkers for platinum-based chemotherapy in patients with stage III-IV NSCLC.
Wang et al (2012) performed a meta-analysis by using 20 eligible studies to examine polymorphisms of ERCC1, GSTs, TS and MTHFR in predicting clinical outcomes (response rate, OS and toxicity) of gastric cancer (GC) patients treated with platinum/5-Fu-based chemotherapy. The association was measured using random/fixed effect odds ratios (ORs) or hazard ratios (HRs) combined with their 95 % CIs according to the studies' heterogeneity. Statistical analysis was performed with the software STATA 9.0 package. No significant association was found between response rate and genetic polymorphism in TS, MTHFR, ERCC1, GSTM1 and GSTP1. However, response rate was higher in GSTT1 (+) genotype compared with GSTT1 (-) genotype (T-/T+: OR = 0.67, 95 % CI: 0.47 to 0.97). With regard to long-term outcomes, these researchers observed a significant longer OS in TS 3R/3R [(2R2R+2R3R)/3R3R: HR = 1.29, 95 % CI: 1.02 to 1.64] and GSTP1 GG/GA [(GG+AG)/AA: HR = 0.51, 95 % CI: 0.39 to 0.67] genotypes. In addition, significant association was demonstrated between toxicity and genetic polymorphism in TS, MTHFR and GSTP1 in included studies. The authors concluded that polymorphisms of ERCC1, GSTs, TS and MTHFR were closely associated with clinical outcomes of GC patients treated with platinum/5-Fu-based chemotherapy. Moreover, they state that studies with large sample size using the method of multi-variant analyses may help us to give more persuasive data on the putative association in future.
In a meta-analysis, Gong and colleagues (2012) examined if RRM1 expression is associated with the clinical outcome of gemcitabine-containing regimen in advanced NSCLC. An electronic search was conducted using the databases PubMed, Medline, EMBASE, Cochrane library and CNKI, from inception to May, 2011. A systemic review of the studies on the association between RRM1 expression in advanced NSCLC and clinical outcome of gemcitabine-containing regimen was performed. Pooled odds ratios (OR) for the response rate, weighted median survival and time to progression were calculated using the software Revman 5.0. The search strategy identified 18 eligible studies (n = 1,243). Response rate to gemcitabine-containing regimen was significantly higher in patients with low/negative RRM1 (OR = 0.31, 95 % CI: 0.21 to 0.45, p < 0.00001). Non-small cell lung cancer SCLC patients with low/negative RRM1 who were treated with gemicitabine-containing regimen survived 3.94 months longer (95 % CI: 2.15 to 5.73, p < 0.0001) and had longer time to progression for 2.64 months (95 % CI: 0.39 to 4.89, p = 0.02) than those with high/positive RRM1. The authors concluded that low/negative RRM1 expression in advanced NSCLC was associated with higher response rate to gemcitabine-containing regimen and better prognosis. Moreover, they stated that large phase III randomized trials are needed to identify whether RRM1 detection is clinically valuable for predicting the prognosis and sensitivity to gemcitabine-containing regimen in advanced NSCLC.
Friboulet et al (2013) stated that the ERCC1 protein is a potential prognostic biomarker of the effectiveness of cisplatin-based chemotherapy in NSCLC. Although several ongoing trials are evaluating the level of expression of ERCC1, no consensus has been reached regarding a method for evaluation.
Besse et al (2013) noted that somatic ERCC1 and ribonucleotide reductase M1 (RRM1) expression levels have been extensively explored as markers of DNA repair capacity in tumor cells. Although low ERCC1 and/or RRM1 expression is generally associated with sensitivity to platinum, the results published in retrospective and prospective studies are not always consistent. These researchers examined the function of these 2 biomarkers as well as the tools available for their assessment and the associated technical issues. Their prognostic and predictive values were summarized and considered in terms of customizing systemic therapy according to biomarker (ERCC1 and RRM1) expression levels. The authors discussed why the use of both markers should at this point be restricted to clinical research.
EZH2 (Ehancer of Zeste 2 Polycomb Repressive Complex 2 Subunit)
The National Comprehensive Cancer Center (NCCN) Biomarkers Compendium (2019) for "EZH2" includes the following category 2A recommendations:
- Myelodysplastic syndromes (MDS) for somatic mutation of EZH2 for cytopenia(s), suspect myelodysplasia. For initial evaluation, consider genetic testing for somatic mutations (i.e., acquired mutations) in genes associated with MDS.
- Myeloproliferative neoplasms (MPN) - additional molecular testing using multi-gene NGS panel should be considered to evaluate for higher-risk mutations associated with disease progression in patients with primary myelofibrosis (PMF). Next-generation sequencing (NGS) remains a research tool in many situations. However, it may be useful to establish clonality in selected circumstances (e.g., "triple negative" non-mutated JAK2, MPL, and CALR. Identification of "higher-risk" mutations may be helpful in the decision-making regarding allogeneic HCT for patients with PMF.
The NCCN guidelines on "B-cell lymphomas" (v.1.2019) does not provide a recommendation for EZH2 testing. Thus, NCCN does not provide a recommendation for diffuse large B-cell lymphoma (DLBC).
Intlekofer et al (2018) state that there is an unmet need to develop genomic biomarker-driven therapeutics to improve outcomes for patients with diffuse large B-cell lymphoma (DLBCL), which currently has a relapse rate of over 30%. The authors sought to define the genomic landscape of DLBCL by using formalin-fixed paraffin-embedded (FFPE) biopsy specimens in order to help underline genomic alterations that characterize DLBCL. Archived FFPE biopsy specimens from 1989 to 2012 were reviewed on 198 patients with DLBCL. Samples were sequenced using the FoundationOne-Heme platform that uses DNA sequencing to interrogate the entire coding sequence of 406 genes, selected introns of 31 genes involved in rearrangements, and utilizes RNA sequencing to interrogate 265 genes known to be somatically altered in human hematologic malignancies. Of 219 FFPE DLBCL samples attempted, 214 were successfully sequenced. The median number of genomic alterations (Gas) per case was 6, with 97% of patients harboring at least one alteration. The most commonly identified single nucleotide variants (SNVs) were in KMT2D (MLL2; 31%, n = 62), TP53 (24%, n = 48), MYD88 (18%, n = 36), CREBBP (18%, n = 35), and B2M (Beta-2-microglobulin; 17%; n = 33). A cluster of BCL2trans and KMT2Dmut corresponded with a GCB subtype and with high rates of TP53mut, EZH2mut, and TNFRSF14mut (p = 0.002). Of note, the largest cluster of 80 patients (40%) did not have a distinct genomic signature. The authors further observed an enrichment in MYD88mut, ETV6mut, and PRDM1mut among non-GCB and EZH2mut among GCB tumors; however, these did not remain significant after correction for FDR. In 41% (n = 81) there was a GA targeted by a non-FDA-approved drug with compelling clinical evidence either in DLBCL (level 3A; 33%, n = 66; mostly histone deacetylase and EZH2 inhibitors in CREBBPmut, EP300mut, and EZH2mut) or in another indication (level 3B; 8%, n = 15). The authors note that prior studies reported EZH2 mutations frequencies as high as 24%, whereas they found EZH2mut in 11% of their cohort, a difference that would have major implications for designing a trial with sequencing-based selection of patients for treatment with EZH2 inhibitors. The authors concluded that despite an accumulating body of research into the genomic landscape of DLBCL, very few GAs have been found to be associated with treatment refractoriness or disease relapse. The authors report that their study confirms prior associations between TP53mut and survival. Though marginally significant, CDKN2A/Bdel and B2Mmut were also found to be associated with shorter OS. As larger sequencing cohorts are assembled, future studies will continue to refine the association between GAs and treatment outcomes.
FIP1L1-PDGFRA Fusion Oncogene
Patnaik et al (2007) noted that systemic mastocytosis is characterized by abnormal growth and accumulation of neoplastic mast cells in various organs. The clinical presentation is varied and may include skin rash, symptoms related to release of mast cell mediators, and/or organopathy from involvement of bone, liver, spleen, bowel, or bone marrow. These investigators reviewed pathogenesis, disease classification, clinical features, diagnosis, and treatment of mast cell disorders; they examined pertinent literature emerging during the last 20 years in the field of mast cell disorders. The authors concluded that the cornerstone of diagnosis is careful bone marrow histologic examination with appropriate immunohistochemical studies. Ancillary tests such as mast cell immunophenotyping, cytogenetic/molecular studies, and serum tryptase levels assist in confirming the diagnosis. Patients with cutaneous disease or with low systemic mast cell burden are generally managed symptomatically. In the patients requiring mast cell cytoreductive therapy, treatment decisions are increasingly being guided by results of molecular studies. Most patients carry the kit D816V mutation and are predicted to be resistant to imatinib mesylate (Gleevec) therapy. In contrast, patients carrying the FIP1L1-PDGFRA mutation achieve complete responses with low-dose imatinib therapy. Other therapeutic options include use of interferon-alpha, chemotherapy (2-chlorodeoxyadenosine), or novel small molecule tyrosine kinase inhibitors currently in clinical trials.
Tefferi et al (2008) stated that current classification and diagnosis of systemic mastocytosis, and its distinction from other myeloid malignancies associated with bone marrow mastocytosis, remain challenging for both clinicians and hematopathologists. In its upcoming revision, due out in 2008, the World Health Organization (WHO) classification system for myeloid malignancies considers mast cell disease as a myeloproliferative neoplasm and systemic mastocytosis as a subcategory of mast cell disease with bone marrow involvement. At the same time, the WHO document distinguished the usually KIT-mutated systemic mastocytosis from myeloid neoplasms associated with bone marrow mastocytosis and PDGFR mutations (e.g., FIP1L1-PDGFRA, PRKG2-PDGFRB). The latter are often associated with eosinophilia or basophilia and sensitive to treatment with imatinib. WHO-defined systemic mastocytosis is sometimes associated with a clonally-related second myeloid neoplasm, which is not surprising considering its origin as a stem cell disease with multi-lineage clonal involvement. Conversely, an otherwise well-defined myeloid malignancy, such as myelodysplastic syndrome or a non-mast cell disease myeloproliferative neoplasm, might harbor neoplastic mast cells. The authors’ approach to diagnosis in systemic mastocytosis starts with bone marrow examination with tryptase staining and mast cell CD25 immunophenotyping. The former enhances morphologic and the latter immunophenotypic distinction between normal (round and CD25-negative) and abnormal (spindle-shaped and CD25-positive) mast cells. Bone marrow examination also allows detection of a 2nd hematologic neoplasm, if present. In addition, in the presence of blood eosinophilia, these investigators screened for FIP1L1-PDGFRA, using either FISH or RT-PCR. By contrast, they relied on conventional cytogenetics to identify cases of bone marrow mastocytosis associated with a PDGFRB re-arrangement (i.e., chromosomal translocations involving 5q31-32). In general, the authors considered mutation screening for KITD816V and measurement of serum tryptase or urinary histamine metabolites as being complementary for the diagnosis of mast cell disease. It is to be noted that the likelihood of detecting a KIT mutation is significantly higher with the use of both highly sensitive PCR-based assay and mast cell-enriched test samples.
An UpToDate review on “Advanced systemic mastocytosis: Management and prognosis” (Gotlib, 2021) states that “Imatinib is generally effective only for unmutated KIT or KIT mutations outside of exon 17. Case reports have reported sensitivity to imatinib for SM with mutations in exons 8 to 10 of KIT: F522C (transmembrane mutation), germline K509I mutation, deletion of codon 419 in exon 8, and p.A502_Y503dup exon 9 mutation. It is important to recognize that many previously reported responses to imatinib were likely to be rare KIT mutations that are sensitive to imatinib or misdiagnoses (e.g., FIP1L1-PDGFRA-positive myeloid/lymphoid neoplasms with eosinophilia that can also exhibit an increase in bone marrow MC numbers and elevated serum tryptase levels)”.
Furthermore, National Comprehensive Cancer Network’s clinical practice guideline on “Systemic mastocytosis” (Version 1.2020) provides the following information:
- Screen for FIP1L1-PDGFRA if eosinophilia is present
- Useful in certain circumstances: Imatinib (only if KIT D816V mutation negative or unknown or if eosinophilia is present with FIP1L1-PDGFRA fusion gene. (In cases with a primarily interstitial pattern of mast cells, peripheral blood eosinophilia, and negativity of KIT D816V mutation, then the FIP1L1-PDGFRA fusion gene should be tested). The FIP1L1-PDGFRA fusion oncogene should be tested in patients with eosinophilia in peripheral blood who do not have the KIT D816V mutation.
HCG
Human chorionic gonadotropin (HCG) is normally produced in increasing quantities by the placenta during pregnancy. Accepted guidelines provide that HCG levels can be used to screen for choriocarcinoma in women who are at high risk for the disease, and to monitor the treatment of trophoblastic disease. The literature states that elevated HCG levels may also indicate the presence of cancers of the testis, ovary, liver, stomach, pancreas, and lung.
Accepted guidelines provide that alpha fetoprotein (AFP) and b-HCG measurements are valuable for determining prognosis and monitoring therapy in patients with non-seminomatous germ cell cancer. Because of the low incidence of elevated AFP and b-HCG levels in early-stage cancer, the literature suggests these markers have no value in screening for testicular cancer. However, the specificity of these markers is such that when determined simultaneously, at least one marker will be positive in 85% of patients with active cancer. The value of AFP and b-HCG as markers is enhanced by a low frequency of false-positive results and by the chemoresponsiveness of testicular cancer. The literature states that only rarely do patients with other types of cancer have elevated levels of AFP. Non-cancerous conditions that can cause elevated AFP levels include benign liver conditions, such as cirrhosis or hepatitis, ataxia telangiectasia, Wiscott-Aldrich syndrome, and pregnancy.
HE4
Human Epididymis Protein 4 (HE4) is a secreted glycoprotein that is being studied as a potential marker for ovarian cancer.
A variety of other tumor markers have been investigated for early detection of ovarian cancer as well as different combinations of tumor markers complementary to CA 125 that could potentially offer greater sensitivity and specificity than CA 125 alone. Preliminary studies on HE4 (human epididymis protein 4), a marker for ovarian cancer, reported similar sensitivity to CA 125 when comparing ovarian cancer cases to healthy controls, and a higher sensitivity when comparing ovarian cancer cases to benign gynecologic disease (Hellstrom, et al., 2003 & 2008; Moore, et al., 2008;) However, an assessment on genomic tests for ovarian cancer prepared by Duke University for the Agency for Healthcare Research and Quality (AHRQ, 2006) stated, "Although research remains promising, adaptation of genomic tests into clinical practice must await appropriately designed and powered studies in relevant clinical settings." Further studies are needed to determine if HE4 significantly adds to the sensitivity of CA 125 while maintaining a high specificity.
National Comprehensive Cancer Network (NCCN) guidelines (2016) state that data show that HE4 and several other markers do not increase early enough to be useful in detecting early-stage ovarian cancer.
Her-2/neu
Estrogen and progestin receptors are important prognostic markers in breast cancer, and the higher the percentage of overall cells positive as well as the greater the intensity, the better the prognosis. Estrogen and progesterone receptor positivity in breast cancer cells is an indication the patient may be a good candidate for hormone therapy. HER-2/neu is an oncogene. Its gene product, a protein, is over-expressed in approximately 20 to 30% of breast cancers. The over-expressed protein is present in unusually high concentration on the surface of some malignant breast cancer cells, causing these cells to rapidly proliferate. It is important because these tumors are susceptible to treatment with Herceptin (trastuzumab), which specifically binds to this over-expressed protein. Herceptin blocks these protein receptors, inhibiting continued replication and tumor growth. HER2/neu may also be expressed in ovarian, gastric, colorectal, endometrial, lung, bladder, prostate, and salivary gland (Chen, et al., 2006).
HER-2/neu is an oncogene encoding a growth factor receptor related to epidermal growth factor receptor (EGFR) and is amplified in approximately 25-30 percent of node-positive breast cancers (Chin, et al. 2006). Overexpression of HER-2/neu is associated with decreased disease-free and overall survival. Over-expression of HER-2/neu may be used to identify patients who may be may benefit from trastuzumab (Herceptin™ ) and/or high dose chemotherapy. Trastuzumab is a humanized monoclonal antibody targeting the HER 2/neu (c-erbB-2) oncogene.
Her-2 has been used to: assess prognosis of stage II, node positive breast cancer patients; predict disease-free and overall survival in patients with stage II, node positive breast cancer treated with adjuvant cyclophosphamide, doxorubicin, 5-fluorouracil chemotherapy; and determine patient eligibility for Herceptin treatment (Chen, et al., 2006). The College of American Pathologists (CAP) recommends FISH as an optimal method for HER2/neu testing; therefore, positive IHC results are usually confirmed by FISH testing.
There are additional tests that may be used in breast cancer cases, such as DNA ploidy, Ki-67 or other proliferation markers. However, most authorities believe that HER-2/neu, estrogen and progesterone receptor status are the most important to evaluate first. The other tests do not have therapeutic implications and, when compared with grade and stage of the disease, are not independently significant with respect to prognosis.
Harris et al (2007) updated ASCO's recommendations for the use of tumor marker tests in the prevention, screening, treatment, and surveillance of breast cancer. Thirteen categories of breast tumor markers were considered, 6 of which were new for the guideline. The following categories showed evidence of clinical utility and were recommended for use in practice: CA 15-3, CA 27.29, CEA, estrogen receptor, progesterone receptor, human epidermal growth factor receptor 2, urokinase plasminogen activator, plasminogen activator inhibitor 1, and certain multi-parameter gene expression assays. Not all applications for these markers were supported, however. The following categories demonstrated insufficient evidence to support routine use in clinical practice: DNA/ploidy by flow cytometry, p53, cathepsin D, cyclin E (fragments or whole length), proteomics, certain multi-parameter assays, detection of bone marrow micrometastases, and circulating tumor cells (e.g., CellSearch assay). These guidelines found present data insufficient to recommend measurement of Ki67, cyclin D, cyclin E, p27, p21, thymidine kinase, topoisomerase II, or other markers of proliferation to assign patients to prognostic groups. The guidelines also found insufficient data to recommend assessment of bone marrow micrometastases for management of patients with breast cancer.
Guidelines from the American Society for Clinical Oncology (2016) recommend against the use of soluble HER2 levels to guide selection of type of adjuvant therapy in breast cancer. This is a moderate-strength recommendation based upon low-quality evidence. The guidelines also recommend against the use of HER2 gene coamplification to guide adjuvant chemotherapy selection in breast cancer.
IgVh Mutation Status
Chronic lymphocytic leukemia (CLL) patients can be divided into two basic groups on the basis of the mutational status of the immunoglobulin heavy-chain variable-region (IgVH) gene in leukemic cells (Chin, 2006). Patients with IgVH mutations have longer survival than those without IgVH mutation. Thus, mutation analysis may be useful for planning management strategies.
Kappa / Lambda Light Chain
Elevated serum levels of monoclonal free light chains are associated with malignant plasma cell proliferation (e.g., multiple myeloma), primary amyloidosis, and light chain deposition disease (Chen et al, 2006). The appearance of higher levels of free light chains in the urine may be indicative of kidney disease or malignant lymphoproliferative disease such as multiple myeloma. These tests have been used for the detection of multiple myeloma.
Ki67
There is a strong correlation between proliferation rate and clinical outcome in a variety of tumor types and measurement of cell proliferative activity is an important prognostic marker (Chen, et al., 2006). This marker correlates with flow cytometric S-phase.
There is insufficient evidence for Ki67. NCCN guidelines on breast cancer (2015) state: "The measurement of nuclear antigen, Ki-67 by IHC, gives an estimate of the tumor cells in the proliferative phase (G1, G2 and M phases) of the cell cycle. Studies have demonstrated the prognostic value of Ki-67 as a biomarker and its usefulness in predicting response and clinical outcome. One small study suggests that measurement of Ki-67 after short-term exposure to endocrine treatment may be useful to select patients resistant to endocrine therapy and those who may benefit from additional interventions. However, these data require larger analytic and clinical validation. In addition, standardization of tissue handling and processing is required to improve the reliability and value of Ki-67 testing. At this time, there is no conclusive evidence that Ki-67 alone, especially baseline Ki-67 as an individual biomarker, helps to select the type of endocrine therapy for an individual patient. Therefore, the NCCN Breast Cancer Panel does not currently recommend assessment of Ki-67."
The p16/KI-67 Dual Stain test (CINtec PLUS) claims to detect virally induced oncogenic molecular changes in the cell through the immune cytochemical double staining of the tumor suppressor gene p16INK4a and the proliferation marker Ki-67 and thereby to improve the triage of women with equivocal cytological results (Kisser, et al., 2014). The Ludwig Boltzmann Institut conducted a systematic review of studies assessing utlity of the p16/Ki-67 Dual Stain test in the triage of equivocal or mild to moderate dysplasia results in cervical cancer screening. The authors of the assessment stated that they could not identify any studies assessing clinical outcomes such as mortality or morbidity and only one high quality study assessing diagnostic accuracy of the test: the evaluation of the clinical utility of the test was therefore not possible (Kisser, et al., 2014). Consequently the test was not recommended for inclusion in the benefits catalogue of public health insurances.
Guidelines from the American Society for Clinical Oncology (2016) state: "Protein encoded by the MKI67 gene labeling index by IHC should not be used to guide choice on adjuvant chemotherapy." This is a moderate-strength recommendation based upon intermediate-quality evidence.
KRAS
The ras proto-oncogenes are normal cellular components, which are thought to be important for transduction of signals required for proliferation and differentiation. The ras oncogene family has 3 members: H-ras, K-ras, and N-ras. Ras gene mutations can be found in a variety of tumor types, although the incidence varies greatly. The highest incidences are found in adenocarcinomas of the pancreas (90 %), colon (50 %), and lung (30 %); thyroid tumors (50 %), and myeloid leukemia (30 %).
Investigators have established an association between some genotypes of K-ras (KRAS) oncogenes and response to treatment with cetuximab or panitumumab (Lievre et al, 2006 and 2008; Di Fiore et al, 2007; Gonçalves et al, 2008; De Roock et al, 2008). Patients whose tumors express specific forms of the KRAS gene exhibit considerably decreased responses to cetuximab and panitumumab. It has been theorized that cetuximab and panitumumab do not target epidermal growth factor receptor (EGFR) associated with these specific KRAS mutations and thus are unable to block their activation. It has been suggested that KRAS genotype be considered as a selection factor for cancer patients who are candidates for treatment with cetuximab or panitumumab.
Karapetis and colleagues (2008) stated that treatment with cetuximab improves overall survival (OS) and progression-free survival (PFS) and preserves the quality of life in patients with colorectal cancer that has not responded to chemotherapy. The mutation status of the K-ras gene in the tumor may affect the response to cetuximab and have treatment-independent prognostic value. These investigators analyzed tumor samples, obtained from 394 of 572 patients (68.9 %) with colorectal cancer who were randomly assigned to receive cetuximab plus best supportive care or best supportive care alone, to look for activating mutations in exon 2 of the K-ras gene. They evaluated if the mutation status of the K-ras gene was associated with survival in the cetuximab and supportive-care groups. Of the tumors evaluated for K-ras mutations, 42.3 % had at least one mutation in exon 2 of the gene. The effectiveness of cetuximab was significantly associated with K-ras mutation status (p = 0.01 and p < 0.001 for the interaction of K-ras mutation status with OS and PFS, respectively). In patients with wild-type K-ras tumors, treatment with cetuximab as compared with supportive care alone significantly improved OS (median of 9.5 versus 4.8 months; hazard ratio for death, 0.55; 95 % confidence interval [CI], 0.41 to 0.74; p < 0.001) and PFS (median of 3.7 months versus 1.9 months; hazard ratio for progression or death, 0.40; 95 % CI, 0.30 to 0.54; p < 0.001). Among patients with mutated K-ras tumors, there was no significant difference between those who were treated with cetuximab and those who received supportive care alone with respect to OS (hazard ratio, 0.98; p = 0.89) or PFS (hazard ratio, 0.99; p = 0.96). In the group of patients receiving best supportive care alone, the mutation status of the K-ras gene was not significantly associated with OS (hazard ratio for death, 1.01; p = 0.97). The authors concluded that patients with a colorectal tumor bearing mutated K-ras did not benefit from cetuximab, whereas patients with a tumor bearing wild-type K-ras did benefit from cetuximab. The mutation status of the K-ras gene had no influence on survival among patients treated with best supportive care alone.
The ASCO's provisional clinical opinion on testing for KRAS gene mutations in patients with metastatic colorectal carcinoma to predict response to anti-EGFR monoclonal antibody therapy (Allegra et al, 2009) stated that based on systematic reviews of the relevant literature, all patients with metastatic colorectal carcinoma who are candidates for anti-EGFR antibody therapy should have their tumor tested for KRAS mutations in a CLIA-accredited laboratory. If KRAS mutation in codon 12 or 13 is detected, then patients with metastatic colorectal carcinoma should not receive anti-EGFR antibody therapy as part of their treatment.
The KRAS oncogene mutation tests are intended to aid in the formulation of treatment decisions for patients who may be candidates for treatment of metastatic epithelial cancers with anti-EGFR therapies such as cetuximab or panitumumab. Several tests for KRAS mutation are currently available in the United States; however, at this time, no KRAS genotype test kits have been approved by the FDA.
At the 2008 Annual Meeting of the American Society of Clinical Oncology (ASCO), data on 540 patients with metastatic colorectal cancer in the randomized, phase III CRYSTAL trial were presented. Among 192 patients with KRAS mutations, there was no improvement in overall responses or PFS from the addition of cetuximab to standard chemotherapy. In the patients with normal KRAS, the 1-year PFS rate was 43 % for patients receiving cetuximab versus 25 % for those receiving only standard chemotherapy, and the overall response rate was 59 % versus 43 %, respectively (van Cutsem, 2008). Also at the 2008 ASCO meeting, data from 233 metastatic colorectal cancer patients were presented that confirmed the correlation of KRAS status with patient response to anti-EGFR therapy. No benefit was found after addition of cetuximab to standard chemotherapy with FOLFOX (the combination of fluorouracil, leucovorin, and oxaliplatin) in patients with a mutated KRAS; however, addition of cetuximab to FOLFOX increased both response rate and PFS in patients with a wild-type (i.e., un-mutated) KRAS gene (Bokemeyer, 2008). Response to panitumumab was correlated to KRAS status in a published phase III trial. A total of 427 patients with metastatic colorectal cancer received either panitumumab or best supportive care. Panitumumab exhibited a 17% response rate among patients with normal KRAS, but 0% response among patients with KRAS mutations (Amado, 2008).
A meta-analysis of results from 8 studies involving 817 patients with colorectal cancer found that the presence of KRAS mutation predicted lack of response to treatment with anti-EGFR monoclonal antibodies (e.g., panitumumab or cetuximab), whether as stand-alone therapy or in combination with chemotherapy (Linardou et al, 2008). This analysis also provided empirical evidence that k-RAS mutations are highly specific negative predictors of response (de-novo resistance) to single-agent EGFR tyrosine-kinase inhibitors in advanced non-small cell lung cancer; and similarly to anti-EGFR monoclonal antibodies alone or in combination with chemotherapy in patients with metastatic colorectal cancer.
The Blue Cross and Blue Shield Association (BCBSA, 2008) Technology Evaluation Center Medical Advisory Panel concluded that use of KRAS mutation analysis meets TEC criteria to predict non-response to anti-EGFR monoclonal antibodies cetuximab and panitumumab to treat metastatic colorectal cancer. The TEC assessment found that the evidence is sufficient to conclude that patients with mutated KRAS tumors in the setting of metastatic colorectal cancer do not respond to anti-EGFR monoclonal antibody therapy. The assessment explained that the data show that the clinical benefit of using EGFR inhibitors in treating metastatic colorectal cancer, either as monotherapy or in combination with other treatment regimens, is not seen in patients with KRAS-mutated tumors. The assessment found: "This data supports knowing a patient's tumor mutation status before consideration of use of an EGFR inhibitor in the treatment regimen. Identifying patients whose tumors express mutated KRAS will avoid exposing patients to ineffective drugs, avoid exposure to unnecessary drug toxicities, and expedite the use of the best available alternative therapy."
Colorectal cancer guidelines from the National Comprehensive Cancer Network (NCCN, 2010) recommend consideration of reflex BRAF testing in patients with wild type KRAS. The NCCN guidelines explain that several small studies suggest that patients with wild-type KRAS and a BRAF mutation are unlikely to respond to anti-EGFR therapies such as cetuximab and panitumumab. The guidelines explain that patients with a known BRAF mutation are unlikely to respond to anti-EGFR antibodies, although the data are somewhat inconsistent. Studies demonstrate that in patients with metastatic colorectal cancer, about 8 percent have mutations in the BRAF gene. Testing for the BRAF V600E mutation is performed by PCR amplification and direct DNA sequence analysis.
Ratner et al (2010) stated that ovarian cancer (OC) is the single most deadly form of women's cancer, typically presenting as an advanced disease at diagnosis in part due to a lack of known risk factors or genetic markers of risk. The KRAS oncogene and altered levels of the microRNA (miRNA) let-7 are associated with an increased risk of developing solid tumors. In this study, these researchers investigated a hypothesized association between an increased risk of OC and a variant allele of KRAS at rs61764370, referred to as the KRAS-variant, which disrupts a let-7 miRNA binding site in this oncogene. Specimens obtained were tested for the presence of the KRAS-variant from non-selected OC patients in 3 independent cohorts, 2 independent ovarian case-control studies, and OC patients with hereditary breast and ovarian cancer syndrome (HBOC) as well as their family members. The results indicated that the KRAS-variant is associated with more than 25 % of non-selected OC cases. Furthermore, these researchers found that it is a marker for a significant increased risk of developing OC, as confirmed by 2 independent case-control analyses. Lastly, they determined that the KRAS-variant was present in 61 % of HBOC patients without BRCA1 or BRCA2 mutations, previously considered uninformative, as well as in their family members with cancer. These findings supported the hypothesis that the KRAS-variant is a genetic marker for increased risk of developing OC, and they suggested that the KRAS-variant may be a new genetic marker of cancer risk for HBOC families without other known genetic abnormalities.
Hollestelle et al (2011) noted that recently, a variant allele in the 3'UTR of the KRAS gene (rs61764370 T>G) was shown to be associated with an increased risk for developing non-small cell lung cancer, as well as OC, and was most enriched in OC patients from HBOC families. This functional variant has been shown to disrupt a let-7 miRNA binding site leading to increased expression of KRAS in vitro. In the current study, these investigators genotyped this KRAS-variant in breast cancer index cases from 268 BRCA1 families, 89 BRCA2 families, 685 non-BRCA1/BRCA2 families, and 797 geographically matched controls. The allele frequency of the KRAS-variant was found to be increased among patients with breast cancer from BRCA1, but not BRCA2 or non-BRCA1/BRCA2 families as compared to controls. As BRCA1 carriers mostly develop ER-negative breast cancers, these researchers also examined the variant allele frequency among indexes from non-BRCA1/BRCA2 families with ER-negative breast cancer. The prevalence of the KRAS-variant was, however, not significantly increased as compared to controls, suggesting that the variant allele not just simply associates with ER-negative breast cancer. Subsequent expansion of the number of BRCA1 carriers with breast cancer by including other family members in addition to the index cases resulted in loss of significance for the association between the variant allele and mutant BRCA1 breast cancer. In this same cohort, the KRAS-variant did not appear to modify breast cancer risk for BRCA1 carriers. More importantly, results from the current study suggested that KRAS-variant frequencies might be increased among BRCA1 carriers, but solid proof requires confirmation in a larger cohort of BRCA1 carriers.
Therascreen KRAS RGQ PCR Kit (Qiagen) is intended to detect 7 mutations in codons 12 and 13 of the KRAS gene (Raman, et al., 2013). The kit utilizes two technologies — ARMS and Scorpions — for detection of mutations in real-time PCR. The therascreen KRAS RGQ PCR kit is being developed as a companion diagnostic to aid clinicians, through detection of KRAS mutations, in the identification of patients with metastatic colorectal cancer (mCRC) who are more likely to benefit from cetuximab.
PreOvar™ tests (Mira Dx) for the KRAS-variant, and will help identify ovarian cancer patients whose female relatives should also be evaluated for the KRAS-variant (Raman, et al., 2013). PreOvar™ may also help assess the relative risk of developing ovarian cancer for women who have a family history of ovarian cancer without a living proband (ancestor with the disease). The KRAS-Variant is present in 6-10% of the general population and 25% of non-selected women with epithelial ovarian cancer. Additionally, the KRAS-variant was identified in over 60% of Hereditary Breast and Ovarian Cancer (HBOC) patients that were previously classified as "uninformative," or negative for other known genetic markers of ovarian cancer risk. The test determines if KRAS-variant may put someone at increased risk for developing ovarian cancer.
The Evaluation of Genomic Applications in Practice and Prevention (EGAPP) Working Group (EWG) (2013) found that, for patients with metastatic colorectal cancer (mCRC) who are being considered for treatment with cetuximab or panitumumab, there is convincing evidence to recommend clinical use of KRAS mutation analysis to determine which patients are KRAS mutation positive and therefore unlikely to benefit from these agents before initiation of therapy. The level of certainty of the evidence was deemed high, and the magnitude of net health benefit from avoiding potentially ineffective and harmful treatment, along with promoting more immediate access to what could be the next most effective treatment, is at least moderate.
The EWG found insufficient evidence to recommend for or against BRAF V600E testing for the same clinical scenario (EGAPP, 2013). The level of certainty for BRAF V600E testing to guide antiepidermal growth factor receptor (EGFR) therapy was deemed low. The EWG encourages further studies of the potential value of testing in patients with mCRC who were found to have tumors that are wild type (mutation negative) for KRAS to predict responsiveness to therapy.
LASA
LASA is a complex marker that measures the amount of sialic acid in serum and can be elevated in serum from patients with any number of different neoplasms. Elevations in blood LASA levels have been reported in patients with mammary (63 percent), gastroenteric (65 percent), pulmonary (79 percent), and ovarian (94 percent) neoplasms as well as those with leukemia (86 percent), lymphoma (87 percent), melanoma (84 percent), sarcoma (97 percent), and Hodgkin disease (91 percent). As a result, this assay may not have high specificity or sensitivity necessary for cancer detection (Chen, et al., 2006). This serum cancer marker has not been widely accepted for use in the detection or prognosis of colorectal carcinoma. There is no practical information concerning outcome and the use of LASA in the medical literature. Although several articles describe the use of LASA in the diagnosis of colorectal cancer and its association with tumor-node-metastasis (TNM) stage, it has been shown that patients with colorectal polyps and colorectal carcinoma both have elevated LASA levels, and that the levels returned to baseline after removal of either polyps or carcinomas.
mdr1
In a review on multidrug resistance in acute leukemia, List and Spier (1992) explained that the mdr1 gene or its glycoprotein product, P-glycoprotein, is detected with high frequency in secondary acute myeloid leukemia (AML) and poor-risk subsets of acute lymphoblastic leukemia. Investigations of mdr1 regulation in normal hematopoietic elements have shown a pattern that corresponds to its regulation in acute leukemia, explaining the linkage of mdr1 to specific cellular phenotypes. Therapeutic trials are now in progress to test the ability of various MDR-reversal agents to restore chemotherapy sensitivity in high-risk acute leukemias.
In a phase III multi-center randomized study to determine whether quinine would improve the survival of adult patients with de novo AML, Soary et al (2003) reported that neither mdr1 gene or P-glycoprotein expression influenced clinical outcome.
A phase I/II study of the MDR modulator Valspodar (PSC 833, Novartis Pharma) combined with daunorubicin and cytarabine in patients with relapsed and primary refractory acute myeloid leukemia (Gruber et al, 2003) reported that P-glycoprotein did not give an obvious improvement to the treatment results.
MRP-1
Motility-related protein (MRP-1) is a glycoprotein with a sequence identical to that of CD9, a white blood cell differentiation antigen. The level of MRP-1/CD9 expression has been found in investigational studies to inhibit cell motility and low MRP-1/CD9 expression may be associated with the metastatic potential of breast cancer (Miyake et al, 1995). CD9 immuno-expression is also being investigated as a potential new predictor of tumor behavior in patients with squamous cell carcinoma of the head and neck (Mhawech et al, 2004) as well as other tumors (e.g., urothelial bladder carcinoma, colon cancer, lung cancer); however, prospective studies are needed to determine the clinical role of MRP-1/CD9 expression in tumors.
MYD88 (Myeloid Differentiation Primary Response 88)
NCCN Biomarkers Compendium (2019) for "MYD88" includes the following category 2A recommendations:
- Gastric MALT lymphoma - Useful under certain circumstances, such as molecular analysis to detect antigen receptor gene rearrangements; MYD88 mutation status to differentiate Waldenstrom’s macroglobinemia (WM) versus marginal zone lymphomas (MZL) if plasmacytic differentiation present
- Nodal marginal zone lymphoma and nongastric MALT lymphoma - Molecular analysis to detect antigen receptor gene rearrangements; MYD88 mutation status to differentiate WM versus MZL if plasmacytic differentiation present; PCR for t(11;18)
- Splenic marginal zone lymphoma - Useful under certain circumstances, such as molecular analysis to detect antigen receptor gene rearrangements; MYD88 mutation status to differentiate WM versus MZL if plasmacytic differentiation present; BRAF mutation status to differentiate MZL from HCL by IHC or sequencing; PCR for t(11;18).
NSE
Neuron-specific enolase (NSE) has been detected in patients with neuroblastoma, small cell lung cancer, Wilms' tumor, melanoma, and cancers of the thyroid, kidney, testicle, and pancreas. However, studies of NSE as a tumor marker have concentrated primarily on patients with neuroblastoma and small cell lung cancer. According to the available literature, measurement of NSE level in patients with these diseases cannot be correlated to the extent of the disease, the patient's prognosis, or the patient's response to treatment because of the poor sensitivity of this marker.
p53
p53 is a tumor suppressor gene on the short arm of chromosome 17 that encodes a protein that is important in the regulation of cell division. Although the full role of p53 in the normal and neoplastic cell is unknown, there is evidence that the gene product is important in preventing the division of cells containing damaged DNA. p53 gene deletion or mutation is a frequent event along with other molecular abnormalities in colorectal carcinogenesis. The literature on p53 abnormality and prognosis in colorectal cancer suffers from a paucity of reported data and the use of a variety of techniques in assay and statistical analysis in the small numbers of cases analyzed. For these reasons, the literature generally does not recommend p53 analysis as a routine approach to assisting in the management of patients with colorectal cancer.
Guidelines from the American Society for Clinical Oncology (2016) recommend against the use of p53 to guide adjuvant chemotherapy in breast cancer. This is a moderate-strength recommendation based upon intermediate-quality evidence.
PCA3
Prostate cancer antigen 3 (PCA3, also known as DD3) is a gene that has been found to be highly overexpressed in prostate cancer. This gene has been investigated as a potential diagnostic marker for prostate cancer. However, there are no published clinical outcome studies of the effectiveness of the PCA3 gene in screening, diagnosis or management of prostate cancer.
Prostate cancer antigen 3 (PCA3) (Progensa, Gene-Probe, Inc.) encodes a prostate-specific mRNA. It is one of the most prostate cancer-specific genes identified, with over-expression in about 95% of cancers tested. The PCA3 urine assay is an amplified nucleic acid assay, which uses transcription-mediated amplification (TMA) to quantify PCA3 and PSA mRNA in prostate cells found in urine samples. The PCA3 score is calculated as the ratio between PCA3 and PSA mRNA. The main target population of this non-invasive test is men with raised PSA but a negative prostate biopsy. Other target groups include men with a slightly raised PSA, as well as men with signs and symptoms suggestive of prostate cancer.
van Gils and colleagues (2007) stated that PCA3 is a promising prostate cancer marker. These investigators performed a multi-center study to validate the diagnostic performance of the PCA3 urine test established in an earlier single-institution study. The first voided urine after digital rectal examination (DRE) was collected from a total of 583 men with serum PSA levels between 3 and 15 ng/ml who were to undergo prostate biopsies. These researchers determined the PCA3 score in these samples and correlated the results with the results of the prostate biopsies. A total of 534 men (92 %) had an informative sample. The area under the receiver-operating characteristic curve, a measure of the diagnostic accuracy of a test, was 0.66 for the PCA3 urine test and 0.57 for serum PSA. The sensitivity for the PCA3 urine test was 65 %, the specificity was 66 % (versus 47 % for serum PSA), and the negative predictive value was 80 %. The authors concluded that the findings of this multi-center study validated the diagnostic performance of the PCA3 urine test in the largest group studied thus far using a PCA3 gene-based test.
Marks and associates (2007) examined the potential utility of the investigational PCA3 urine assay to predict the repeat biopsy outcome. Urine was collected after DRE (3 strokes per lobe) from 233 men with serum PSA levels persistently 2.5 ng/ml or greater and at least one previous negative biopsy. The PCA3 scores were determined using a highly sensitive quantitative assay with TMA. The ability of the PCA3 score to predict the biopsy outcome was assessed and compared with the serum PSA levels. The RNA yield was adequate for analysis in the urine samples from 226 of 233 men (i.e., the informative specimen rate was 97 %). Repeat biopsy revealed prostate cancer in 60 (27 %) of the 226 remaining subjects. Receiver operating characteristic curve analysis yielded an area under the curve of 0.68 for the PCA3 score. In contrast, the area under the curve for serum PSA was 0.52. Using a PCA3 score cutoff of 35, the assay sensitivity was 58 % and specificity 72 %, with an odds ratio of 3.6. At PCA3 scores of less than 5, only 12 % of men had prostate cancer on repeat biopsy; at PCA3 scores of greater than 100, the risk of positive biopsy was 50 %. The authors concluded that in men undergoing repeat prostate biopsy to rule out cancer, the urinary PCA3 score was superior to serum PSA determination for predicting the biopsy outcome. The high specificity and informative rate suggest that the PCA3 assay could have an important role in prostate cancer diagnosis.
Groskopf et al (2007) reported that the PCA3 score is independent of prostate volume and was highly correlated with the risk of positive biopsy. The PCA3 test was performed on 529 men scheduled for prostate biopsy. Overall, the PCA3 score had a sensitivity of 54% and a specificity of 74%. A PCA3 score of less than 5 was associated with a 14% risk of positive biopsy, while a PCA3 score of greater than 100 was associated with a 69% risk of positive biopsy.
Haese et al (2007) presented preliminary results from a European multicenter study of PCA3. Enrolled patients had a PSA level of less than or equal to 2.5 ng/mL, had 1 or 2 previous negative biopsies, and were scheduled for repeat biopsy. The specificity of the PCA3 score (cutoff 35) was found to be 78%, and the sensitivity was 67%. Patients with a PCA3 score of greater than or equal to 35 had a 33% probability of a positive repeat biopsy, compared to a 6% probability for those with a PCA3 score of less than 35.
In a review on biomarkers for prostate cancer detection, Parekh, et al. (2007) stated that prostate stem cell antigen, alpha-methyl coenzyme-A racemase, PCA3, early prostate cancer antigen, hepsin and human kallikrein 2 are promising markers that are currently undergoing validation.
An assessment by the BlueCross BlueShield Association Technology Evaluation Center (BCBSA, 2008) found that, in general, PCA3 assay results to date are preliminary; interpretation of results has not been standardized and clinical utility studies of decision-making for initial biopsy, repeat biopsy or treatment have not been reported.
Tosoian et al (2010) evaluated the relationship between PCA3 and prostate biopsy results in men in a surveillance program. Urine specimens were obtained from 294 men with prostate cancer enrolled in the Johns Hopkins surveillance program. The follow-up protocol included semi-annual free and total PSA measurements, digital rectal examination and annual surveillance prostate biopsy. Cox proportional hazards regression was used to evaluate the association between PCA3 results and progression on surveillance biopsy (defined as Gleason pattern 4 or 5, more than 2 positive biopsy cores or more than 50% involvement of any core with cancer). Patients with progression on biopsy (12.9%) had a mean PCA3 score similar to that of those without progression (60.0 versus 50.8, p = 0.131). Receiver operating characteristics analysis suggested that PCA3 alone could not be used to identify men with progression on biopsy (area under the curve = 0.589, 95% CI 0.496 to 0.683, p = 0.076). After adjustment for age and date of diagnosis PCA3 was not significantly associated with progression on biopsy (p = 0.15). The authors concluded that in men with low risk prostate cancer who were carefully selected for surveillance the PCA3 score was not significantly associated with short-term biopsy progression. They stated that further analysis is necessary to assess the usefulness of PCA3 in combination with other biomarkers or in selected subsets of patients undergoing surveillance.
While there are studies examining the positive and negative predictive values of the PCA3 urine assay, there is currently a lack of evidence of the effect of this test on management of individuals with or suspected of prostate cancer. The PCA3 urine assay shows promise as a prostate cancer diagnostic tool, however, more research is needed to ascertain the clinical value of this assay for screening and diagnostic purposes.
An assessment of PCA3 prepared for the Agency for Healthcare Research and Quality (2013) concluded: "For diagnostic accuracy, there was a low strength of evidence that PCA3 had better diagnostic accuracy for positive biopsy results than tPSA elevations, but insufficient evidence that this led to improved intermediate or long-term health outcomes. For all other settings, comparators, and outcomes, there was insufficient evidence."
The Evaluation of Genomic Applications in Practice and Prevention (EGAPP) Working Group (2013) found insufficient evidence to recommend prostate cancer antigen 3 (PCA3) testing to inform decisions for when to re-biopsy previously biopsy-negative patients for prostate cancer or to inform decisions to conduct initial biopsies for prostate cancer in at-risk men (e.g., previous elevated prostate-specific antigen test or suspicious digital rectal examination). The EGAPP Working Group found insufficient evidence to recommend PCA3 testing in men with cancer-positive biopsies to determine if the disease is indolent or aggressive in order to develop an optimal treatment plan. The EGAPP Working Group concluded that, based on the available evidence, the overall certainty of clinical validity to predict the diagnosis of prostate cancer using PCA3 is deemed "low." The EGAPP Working Group discouraged clinical use for diagnosis unless further evidence supports improved clinical validity. The EGAPP Working Group also found that, based on the available evidence, the overall certainty of net health benefit is deemed "low." The EGAPP Working Group discourages clinical use unless further evidence supports improved clinical outcomes.
Guidelines from the European Association of Urology (2015) state that "[b]iological markers, include urine markers such as PCA3, the TMPRSS2: ERG fusion gene or PSA isoforms such as the Phi index, appear promising as does genomics on the tissue sample itself. However, further study data will be needed before such markers can be used in standard clinical practice."
A Cancer Care Ontario Guideline on prostate cancer surveillance (Morash, et al., 2015), which has been endorsed by the American Society for Clinical Oncology (2016), did not include PCA3 level in their recommendation because evidence of PCA3 to predict disease reclassification in prostate cancer was lacking.
National Institute for Health and Care Excellence (NICE)’s clinical practice guideline on "Diagnosing prostate cancer: PROGENSA PCA3 assay and Prostate Health Index" (2015) stated that " The PROGENSA PCA3 assay and the Prostate Health Index are not recommended for use in people having investigations for suspected prostate cancer, who have had a negative or inconclusive transrectal ultrasound prostate biopsy". The assessment cited studies finding that adding the PCA3 score to clinical assessment and MRI had very little effect on the size of the reported area under the curve, with minimal change in derived sensitivity and specificity for clinical assessment with MRI compared with clinical assessment using MRI and the PCA3 assay.
In a Lancet review of prostate cancer, Attard, et al. (2016) stated that "[s]everal studies have so far proven inconclusive as to whether PCA3 is useful to selectively detect aggressive prostate cancers."
PDGFRB Testing
The National Comprehensive Cancer Network’s Biomarkers Compendium (2016) recommends the following for PDGFRB testing:
Myelodysplastic Syndromes (MDS): Helpful in some clinical situations: Evaluate CMML patients for 5q31-33 translocations and/or PDGFR beta gene rearrangements. (Category of Evidence: 2A).
Non-Melanoma Skin Cancers - Dermatofibrosarcoma Protuberans (DFSP): Tumors lacking the t(17;22) translocation may not respond to imatinib. Molecular analysis of a tumor using cytogenetics may be useful prior to the institution of imatinib therapy. (Category of Evidence: 2A).
PSA
Prostate Specific Antigen (PSA) is a substance produced by the prostate gland. Levels of PSA in the blood often increase in men with prostate cancer. Elevated levels of Prostate-Specific Antigen (PSA) may also be found in the blood of men with benign prostate conditions, such as prostatitis and benign prostatic hyperplasia (BPH). While PSA does not allow distinction between benign prostate conditions and cancer, an elevated PSA level may indicate that other tests are necessary to determine whether cancer is present. PSA levels have been shown to be useful in monitoring the effectiveness of prostate cancer treatment, and in checking for recurrence after treatment has ended. Use of PSA for screening remains very controversial. Although researchers are in the process of studying the value of PSA along with digital rectal exams for routine screening of men ages 55 to 74 for prostate cancer; and the literature does not show at this time whether using PSA to screen for prostate cancer actually does reduce the number of deaths caused by this cancer. The American Cancer Society recommends clinicians and patients consider screening with PSA and digital rectal exam for African American men and men with familial tendency age 40 or older and all men age 50 or older.
Cancer Care Ontario guidelines on active surveillance of prostate cancer (Morash, et al., 2015) state that the active surveillance protocol should include the following tests: PSA test every 3 to 6 months; digital rectal examination every year, and a 12- to 14-core confirmatory transrectal ultrasound (TRUS) biopsy (including anterior directed cores) within 6 to 12 months, then serial biopsy a minimum of every 3 to 5 years thereafter. The guidelines state that "[c]urrent evidence shows that PSA kinetics does not reliably predict disease stability or reclassification to higher risk state. There was conflicting evidence whether PSA is a good predictor of disease progression or reclassification. Differences were also found in the ability of different measures of PSA, such as PSA velocity, PSA density, and PSA doubling time for predicting progression or reclassification. PSA monitoring is considered a necessary component of an AS protocol, but a rising PSA may be best viewed as a trigger for reappraisal (e.g., MRI, repeat biopsy) rather than a trigger for intervention."
Thrombospondin-1
Thrombospondin-1 (THBS-1), an angiogenesis inhibitor, has been identified as a potential monitoring marker in gynecologic malignancies. In a randomized phase III study on the co-expression of angiogenic markers and their associations with prognosis in advanced epithelial ovarian cancer, Secord, et al. (2007) reported that high THBS-1 may be an independent predictor of worse progression-free and overall survival in women with advanced-stage EOC. However, the authors stated, "A larger prospective study is warranted for validation of these findings."
Thymidylate Synthase
Thymidylate synthase is a DNA synthesis related gene. According to Compton (2008), the prognostic value of this promising and potentially clinically applicable molecular marker has been studied in colorectal cancer. Compton found that the independent influence of this marker on prognosis remains unproven. Compton explained that "[v]ariability in assay methodology, conflicting results from various studies examining the same factor, and the prevalence of multiple small studies that lack statistically robust, multivariate analyses all contribute to the lack of conclusive data." Compton concluded that before this marker can be incorporated into clinically meaningful prognostic stratification systems, more studies are required using multivariate analysis, well-characterized patient populations, reproducible and current methodology, and standardized reagents.
In a special report on pharmacogenomics of cancer, the BlueCross and BlueShield Association's Technology Evaluation Center (TEC) (2007) described the results of a meta-analysis on thymidylate synthase protein expression and survival in colorectal cancer that stated low thymidylate synthase expression was significantly associated with better survival, but heterogeneity and possible bias prevented firm conclusions.
Guidelines from the American Society for Colon and Rectal Surgeons (2004) stated: "In the future, DNA analysis and the intratumoral expression of specific chemical substances", including thymidylate synthase, "may be used routinely to further assess prognosis or response to therapy." In addition, Shankaran et al (2008) stated in a review on the role of molecular markers in predicting response to therapy in patients with colorectal cancer, "Although to date no molecular characteristics have emerged as consistent predictors of response to therapy, retrospective studies have investigated the role of a variety of biomarkers, including microsatellite instability, loss of heterozygosity of 18q, type II transforming growth factor beta receptor, thymidylate synthase, epidermal growth factor receptor, and Kirsten-ras (KRAS)."
TOP2A
Topoisonmerase II alpha is a protein encoded by the TOP2A gene and is proposed as a predictive and prognostic marker for breast cancer. It is also proposed as an aid in predicting response to anthracycline therapy in breast cancer. Two types of tests are available for topoisonmerase II alpha: topoisomerase II alpha protein expression testing by immunohistochemistry (IHC); and TOP2A gene amplification testing by FISH (eg, TOP2A FISH pharmDx Assay).
The topoisomerase II alpha gene (TOP2A) is located adjacent to the HER-2 oncogene at the chromosome location 17q12-q21 and is either amplified or deleted (with equal frequency) in a great majority of HER-2 amplified primary breast tumors and also in tumors without HER-2 amplification. Recent experimental as well as numerous, large, multi-center trials suggest that amplification (and/or deletion) of TOP2A may account for both sensitivity or resistance to commonly used cytotoxic drugs (e.g., anthracyclines) depending on the specific genetic defect at the TOP2A locus. An analysis of TOP2A aberrations in the Danish Breast Cancer Cooperative Group trial 89D (Nielsen, et al., 2008) suggested a differential benefit of adjuvant chemotherapy in patients with primary breast cancer, favoring treatment with epirubicin in patients with TOP2A amplifications, and perhaps deletions; however, the authors concluded that, "Additional studies are needed to clarify the exact importance of TOP2A deletions on outcome, but deletions have proven to be associated with a very poor prognosis."
The National Comprehensive Cancer Network (NCCN, 2008) guideline on breast cancer does not address the use of TOP2A testing. Guidelines from the American Society for Clinical Oncology (2016) state: "The clinician should not use TOP2A gene amplification or TOP2A protein expression by IHC to guide adjuvant chemotherapy selection.: This is a moderate-strength recommendation based upon high quality evidence. The guidelines also recommend against the use of TOP2A gene coamplification to guide adjuvant chemotherapy selection.
TSP-1
Ghoneim et al (2008) explained that thrombospondin-1 (TSP-1) is a member of a family of five structurally related extracellular glycoproteins that plays a major role in cell-matrix and cell to cell interactions. Due to its multifunctional nature and its ability to bind to a variety of cell surface receptors and matrix proteins, TSP-1 has been identified as a potential regulator of angiogenesis and tumor progression. Data collected by Secord, et al. (2007) suggested that high THBS-1 levels may be an independent predictor of worse progression-free and overall survival in women with advanced-stage epithelial ovarian cancer. However, a phase II clinical trail (Garcia, et al., 2008) of bevacizumab and low-dose metronomic oral cyclophosphamide in recurrent ovarian cancer reported that levels of TSP-1 were not associated with clinical outcome.
uPA
The serine protease urokinase-type plasminogen activator (uPA) and its primary inhibitor, plasminogen activator inhibitor-1 (PAI-1), have shown promise for risk assessment and prediction of therapeutic response in primary breast cancer (Chin, et al., 2006). High levels of uPA or PAI-1 in primary tumor tissue are associated with an aggressive disease course and poor prognosis in both node-positive and node-negative breast cancer.
A report by the Belgian Healthcare Knowledge Centre (KCE) (San Miguel, et al., 2015) found no studies reporting on the impact of uPA/PAI-1 on clinical management (clinical utility).
Guidelines from the American Society for Clinical Oncology (2016) state: "If a patient has ER/PgR-positive, HER2-negative (node-negative) breast cancer, the clinician may use urokinase plasminogen activator and plasminogen activator inhibitor type 1 to guide decisions on adjuvant systemic therapy." This is a weak recommendation based upon high-quality evidence. The ASCO guidelines recommend the use of urokinase plasminogen activator and plasminogen activator inhibitor type 1 to guide decisions on adjuvant systemic therapy in patients with HER2-positive breast cancer or TN breast cancer.
Zap-70
Zeta-chain-associated protein kinase 70, which is used as a prognostic marker in (CLL).
Zap-70 is indicated to assess prognosis and need for aggressive therapy in patients with chronic lymphocytic leukemia (CLL) (Chin, et al., 2006). ZAP-70 is a 70-kD member of the Syk family of protein tyrosine kinases. It is expressed primarily in T-cells and natural killer (NK) cells and is critical for signal transduction following T-cell receptor engagement. In CLL B-cells, elevated ZAP-70 expression appears to predict the need for therapy as effectively as IgVH mutation status. Although ZAP-70 expression is strongly correlated with IgVH mutation status, the combination of the two markers may provide greater prognostic value than either marker alone. Positive ZAP-70 results predict an aggressive disease course.
4K Score
4Kscore Test measures the blood plasma levels of four different prostate-derived kallikrein proteins [Total PSA, Free PSA, Intact PSA and Human Kallikrein2 (hK2)] and combines results in an algorithm with age, DRE (nodules, no nodules) and prior biopsy results. The result is purportedly an individual’s specific probability for finding a high-grade, Gleason score 7 or higher prostate cancer upon biopsy.
Parekh et al (2015) performed the first prospective evaluation of the 4Kscore in predicting Gleason ≥7 PCa in the USA. The investigators prospectively enrolled 1012 men scheduled for prostate biopsy, regardless of prostate-specific antigen level or clinical findings, from 26 US urology centers between October 2013 and April 2014. The primary outcome was Gleason ≥7 PCa on prostate biopsy. The area under the receiver operating characteristic curve, risk calibration, and decision curve analysis (DCA) were determined, along with comparisons of probability cutoffs for reducing the number of biopsies and their impact on delaying diagnosis. Gleason ≥7 PCa was found in 231 (23%) of the 1012 patients. The investigators stated that the 4Kscore showed excellent calibration and demonstrated higher discrimination (area under the curve [AUC] 0.82) and net benefit compared to a modified Prostate Cancer Prevention Trial Risk Calculator 2.0 model and standard of care (biopsy for all men) according to DCA. A possible reduction of 30-58% in the number biopsies was identified with delayed diagnosis in only 1.3-4.7% of Gleason ≥7 PCa cases, depending on the threshold used for biopsy. Pathological assessment was performed according to the standard of care at each site without centralized review.
Stattin et al (2015) conducted a case-control study nested within a population-based cohort. PSA and three additional kallikreins (4KScore) were measured in cryopreserved blood from a population-based cohort in Västerbotten, Sweden. Of 40,379 men providing blood at ages 40, 50, and 60 years from 1986 to 2009, 12,542 men were followed for >15 yr. From this cohort, the Swedish Cancer Registry identified 1423 incident PCa cases, 235 with distant metastasis. Most metastatic cases occurred in men with PSA in the top quartile at age 50 yr (69%) or 60 yr (74%), whereas 20-yr risk of metastasis for men with PSA below median was low (≤0.6%). The investigators reported that, among men with PSA >2 ng/ml, a prespecified model based on four kallikrein markers significantly enhanced the prediction of metastasis compared with PSA alone. About half of all men with PSA >2 ng/ml were defined as low risk by this model and had a ≤1% 15-yr risk of metastasis. The authors concluded that, for men in their fifties, screening should focus on those in the top 10% to 25% of PSA values because the majority of subsequent cases of distant metastasis are found among these men. Testing of four kallikrein markers in men with an elevated PSA could aid biopsy decision making.
Voigt et al (2014) conducted a systematic review and meta-analysis to examine the aggregated results from published studies of the Kallikrein Panel. The results of the meta-analysis were used to model the Kallikrein Panel's effect on healthcare costs. The authors reported that meta-analysis demonstrates a statistically significant improvement of 8-10% in predictive accuracy. The authors estimated that 48% to 56% of current prostate biopsies could be avoided and that use of the Kallikrein Panel could result in annual US savings approaching $1 billion.
Konety et al (2015) conducted a clinical utility study to assess the influence of the 4Kscore Test on the decision to perform prostate biopsies in men referred to urologists for abnormal PSA and/or DRE results. The study population included 611 patients seen by 35 academic and community urologists in the United States. Urologists ordered the 4Kscore Test as part of their assessment of men referred for abnormal PSA and/or DRE test results. Results for the patients were stratified into low risk (< 7.5%), intermediate risk (7.5%-19.9%), and high risk (≥ 20%) for aggressive prostate cancer. The investigators reported that the 4Kscore Test results influenced biopsy decisions in 88.7% of the men. Performing the 4Kscore Test resulted in a 64.6% reduction in prostate biopsies in patients; the actual percentage of cases not proceeding to biopsy were 94.0%, 52.9%, and 19.0% for men who had low-, intermediate-, and high-risk 4Kscore Test results, respectively. A higher 4Kscore Test was associated with greater likelihood of having a prostate biopsy (P < 0.001). The investigators reported that, among the 171 patients who had a biopsy, the 4Kscore risk category is strongly associated with biopsy pathology.
Lin et al (2016) sought to evaluate the utility of the 4Kscore in predicting the presence of high-grade cancer in men on active surveillance. Plasma collected before the first and subsequent surveillance biopsies was assessed for 718 men prospectively enrolled in the multi-institutional Canary PASS trial. Biopsy data were split 2:1 into training and test sets. The investigators developed statistical models that included clinical information and either the 4Kpanel or serum prostate-specific antigen (PSA). The endpoint was reclassification to Gleason ≥7. The investigators used receiver operating characteristic (ROC) curve analyses and area under the curve (AUC) to assess discriminatory capacity, and decision curve analysis (DCA) to report clinical net benefit. Significant predictors for reclassification were 4Kpanel (odds ratio [OR] 1.54, 95% confidence interval [CI] 1.31-1.81) or PSA (OR 2.11, 95% CI 1.53-2.91), ≥20% cores positive (OR 2.10, 95% CI 1.33-3.32), two or more prior negative biopsies (OR 0.19, 95% CI 0.04-0.85), prostate volume (OR 0.47, 95% CI 0.31-0.70), and body mass index (OR 1.09, 95% CI 1.04-1.14). ROC curve analysis comparing 4K and base models indicated that the 4Kpanel improved accuracy for predicting reclassification (AUC 0.78 vs 0.74) at the first surveillance biopsy. Both models performed comparably for prediction of reclassification at subsequent biopsies (AUC 0.75 vs 0.76). In DCA, both models showed higher net benefit compared to biopsy-all and biopsy-none strategies. Limitations include the single cohort nature of the study and the small numbers; results should be validated in another cohort before clinical use.
Guidelines from the National Comprehensive Cancer Network (NCCN, 2016) lists the 4Kscore nonpreferentially among a number of tests (i.e., the percent free PSA and the Prostate Health Index (PHI)) that can be considered for patients prior to biopsy and among several tests (i.e., percent free PSA, PHI, PCA3 and ConfirmMDx) for those with prior negative biopsy for men thought to be at higher risk for clinically significant prostate cancer. The NCCN guidelines state that the 4Kscore cannot be recommended over other tests (i.e., the percent free PSA, the Prostate Health Index (PHI), The NCCN guidelines explain that head-to-head comparisons have been performed in Europe for some of these tests, performed individually or in combinations in the intial or repeat biopsy settings, but sample sizes were small and results varied. The NCCN guidelines stated that the optimal order of biomarker tests and imaging is unknown, and that it remains unclear how to interpret multiple tests in individual patients, especially when results are contradictory. The panel states that it is important for patients and their urologists to understand, however, that no cutoff threshold has been established for the 4KScore.
Recommendations from Memorial Sloan Kettering (Vickers, et al., 2016) state that in biopsy-naive men with PSA ≥3 ng/mL, prostate MRI is the strongest independent predictor of clinically significant prostate cancer, but "[a]s evidence continues to build, we believe that prostate MRI may emerge as a valuable tool to reduce overdiagnosis of PCa, most likely in concert with newer biomarkers, such as the Prostate Health Index, the 4Kscore, and single nucleotide polymorphism panels.
A 2016 MolDx assessment of the 4KScore concluded that "the intended use population has been inadequately validated; the 4Kscore model has continuously changed; the model has been recurrently tested on potentially inappropriate patients (PSA > 10) and patients with inadequate biopsy sampling; it is unclear how much the hK2 and possibly intact PSA contribute to the model; the value of the 4Kscore model/algorithm is fraught with statistical hypothesis and not prospective outcomes or concordance in a defined patient population likely to be considered for biopsy (eg: PSA 3-10 ng/mL); assumptions are made that no harm will come to following young men with unknown low grade prostate cancer (not on AS); there is significant difficulty equating the model used in the Swedish study to the presently proposed formula; and the incidence of clinically diagnosable prostate cancer in patients with low risk by the model/algorithm at 10 years is very concerning."
Anceschi et al (2019) stated that in recent years, several biomarkers alternative to standard prostate specific antigen (PSA) for PCa diagnosis have become available. In a systematic review, these researchers examined current knowledge about alternative serum and urinary biomarkers for the diagnosis of PCa. A research was conducted in Medline, restricted to English language articles published between December 2014 and June 2018 with the aim to update previously published series on PCa biomarkers. The preferred reporting items for systematic reviews and meta-analyses (PRISMA) criteria were used for selecting studies with the lowest risk of bias. Emerging role and actual controversies on serum and urine alternative biomarkers to standard PSA for PCa diagnosis, staging and prognosis assessment, such as prostate health index (PHI), PCA3, ConfirmMDx, Aberrant PSA glycosylation, MiPS, miRNAs were critically presented in the current review. The authors concluded that although the use of several biomarkers has been recommended or questioned by different international guidelines, larger prospective randomized studies are still necessary to validate their efficacy in PCa detection, discrimination, prognosis and treatment effectiveness. To-date, only PHI and 4Kscore have shown clinical relevance for discriminating more aggressive PCa. Furthermore, a new grading classification based on molecular features relevant for PCa risk-stratification and tailoring treatment is still needed.
Kim et al (2019) noted that prostate cancer (CaP) is the most common cancer diagnosed among men in the United States and the 5th most common cancer among men in Korea. Unfortunately, the early stages of CaP may have no symptoms. Therefore, early detection is very important and physicians managing voiding dysfunction must have awareness regarding CaP. The traditional tests used for early detection of CaP are the prostate-specific antigen (PSA) blood test and digital rectal examination (DRE). However, a high PSA level is not specific for CaP. Benign prostatic hyperplasia (BPH), prostatitis, urinary tract infection (UTI), and urinary retention can all cause a high PSA level. Thus, no test shows sufficient accuracy to truly be useful for screening men for CaP. A prostate biopsy is the only method that yields a definitive diagnosis of CaP; however, this test is invasive and uncomfortable. Recently, new biomarkers for CaP detection have been proposed to improve the accuracy of the PSA test. These investigators summarized their knowledge of various new biomarkers, including PSA-associated biomarkers (the prostate health index and 4Kscore), molecular biomarkers (PCA3, TMPRSS2: ERG fusion gene, and various miRNAs), and proteomics-associated biomarkers, and the ways in which they may improve the detection rate of CaP. The authors concluded that until now, there has been many efforts to predict early stage CaP such as PSA associated markers, various molecular markers, miRNA markers, and protein markers. Unfortunately, the follow-up validation studies are lack due to several reasons. Thus, future studies of CaP biomarkers need to focus on combinations of molecular biomarkers and clinical variables, rather than on biomarkers alone.
Marzouk et al (2019) stated that recent years have seen the development of biomarkers and imaging technologies designed to improve the specificity of PSA. Widespread implementation of imaging technologies, such as mp-MRI raises considerable logistical challenges. These researchers evaluated a biopsy strategy that utilizes selective mp-MRI as a follow-up test to biomarkers to improve the detection of significant PCa. They developed a conceptual approach based on the risk calculated from the 4Kscore using results from the U.S. prospective validation study, multiplied by the likelihood ratio of mp-MRI from the PROMIS trial. The primary outcome was Gleason grade greater than or equal to 7 (grade group greater than or equal to 2) cancer on biopsy. Using decision curve analysis, the net benefit was determined for this model and compared with the use of the 4Kscore and mp-MRI independently at various thresholds for biopsy. For a cut-point of 7.5 % risk of high-grade disease, patients with less than 5 % risk from a blood marker would not have risk of significant PCa sufficiently increased by a positive mp-MRI to warrant biopsy; comparably, patients with a risk of greater than 23 % would not have risk sufficiently reduced by a negative imaging study to forgo biopsy. From the 4Kscore validation study, 46 % of men considered for biopsy in the U.S. have risks 5 % to 23 %. Net benefit was highest for the combined strategy, followed by 4Kscore alone. The authors concluded that selective mp-MRI in men with intermediate scores on a secondary blood test resulted in a biopsy strategy that was more scalable than mp-MRI for all men with elevated PSA. These researchers stated that prospective validation is needed to examine if the predicted properties of combined blood and imaging testing are empirically confirmed.
Falagario et al (2020) stated that the 2019 European Association of Urology guidelines recommended mp-MRI for biopsy-naïve patients with clinical suspicion of PCa and avoiding biopsy in patients with negative mp-MRI and low clinical suspicion. However, consensus on the optimal definition of low clinical suspicion is lacking. These researchers evaluated 266 biopsy-naïve patients who underwent mp-MRI, the 4Kscore test, and prostate biopsy to define the best strategy to avoid unnecessary testing and biopsies. The European Randomized Study of Screening for Prostate Cancer risk calculator (ERSPC-RC) and PSA density (PSAd) were also considered. For men with Prostate Imaging-Reporting and Data System v2.0 (PI-RADS) 1⿿2 lesions, the highest negative predictive value (NPV) was observed for those with low or intermediate 4Kscore risk (96.9 % and 97.1 %), PSAd < 0.10 ng/ml/cm3 (98.7 %), and ERSPC-RC less than 2 % (98.7 %). For men with PI-RADS 3⿿5 lesions the lowest positive predictive value (PPV) was observed for those with low 4Kscore risk (0 %), PSAd less than 0.10 ng/ml/cm3 (13.2 %), and ERSPC-RC of less than 2 % (12.3 %). The best biopsy strategy was an initial 4Kscore followed by mp-MRI if the 4Kscore was greater than 7.5 % and a subsequent biopsy if the mp-MRI was positive (PI-RADS 3⿿5) or the 4Kscore was 18 %. This would result in missing 2.7 % (2/74) of clinically significant PCs (csPCs) and avoiding 34.2 % of biopsies. Initial mp-MRI followed by biopsy for negative mp-MRI (PI-RADS 1⿿2) if the 4Kscore was 18 % or PSAd was 0.10 ng/ml/cm3 resulted in a similar percentage of csPC missed (2.7 % [2/74] and 1.3 % [1/74]) but slightly fewer biopsies avoided (25.2 % and 28.1 %). Physicians should consider clinical risk screening tools when ordering and interpreting mp-MRI results to avoid unnecessary testing and diagnostic errors. The authors stated that performing the 4Kscore test in conjunction with mp-MRI for men with a clinical suspicion of prostate cancer may help to reduce unnecessary biopsies. These researchers stated that this study was limited by its small sample size and its retrospective nature; prospective validation of these findings is needed before their implementation in clinical practice.
An UpToDate review on “Screening for prostate cancer” (Hoffman, 2021) states that “Referral for urologic evaluation will not necessarily result in a prostate biopsy. Other tests (e.g., free to total PSA ratio [f/T PSA], PCA3, 4Kscore test, and/or magnetic resonance imaging [MRI]) may be done by the urologist to help determine the likelihood that the PSA is elevated due to prostate cancer, the PSA may be followed over time, or a biopsy may be performed. Relevant considerations include the patient's health status, clinical likelihood for harboring significant disease, and personal wishes”.
Auria
Auria (Namida Lab, Inc.) is a home labororaty breast cancer screening test that evaluates for S100A8 and S100A9 biomarkers by ELISA method on tear fluid. The sample strip is a thin piece of filter paper that is commonly used to test for dry eye. The results are interpretated using an algorithm and reported as a risk score.
Auria is intended for women ages 30 and over. Auria is not a replacement for screening mammograms. Auria is not intended for women with an unevaluated palpable mass or area of concern in their breast tissue. It is also not intended for women who no longer have breast tissue. Moreover, this test is intended for informational and educational use only, and is not intended to be used for diagnostic purposes.
Avantect Pancreatic Cancer Test
The Avantect test (ClearNote Health) is a cell-free DNA (cfDNA)-based blood test that combines whole-genome sequencing with 5-hydroxymethylcytosine (5hmC) profiling in a single assay to evaluate persons at high risk for pancreatic cancer. The test incorporates machine learning and a bioinformatic algorithm to detect the presence of cancer. The results are reported as detected or not detected.
Currently, there is insufficient evidence in the peer-reviewed literature to support the sensitivity or specificity of this test.
Aventa FusionPlus
Aventa FusionPlus (Aventa Genomics, LLC) is a next-generation sequencing test that detects gene fusions, translocations, and other rearrangements across 361 genes from formalin-fixed, paraffin embedded (FFPE) tumor tissue. The test uses 3D genomics, which involves exploring the 3-dimensional organization of DNA in the nucleus to reveal insights into the genome’s sequence, structure, and regulatory landscape. The test is indicated for patients with driver-negative solid tumors (e.g., RAS-wt NSCLC and PDAC, unresolved sarcomas, any other driver-negative solid tumor). The aim of the test is to see all clinically relevant variants to enable physicians to identify druggable targets, better understand prognosis, and resolve diagnostic dilemmas (Aventa, 2024).
There is insufficient evidence in published peer-reviewed literature to support the clinical value of Aventa FusionPlus.
Bladder Cancer: BTA-stat, NMP22, Urovysion, ImmunoCyt
In the United States, bladder malignancy is the 4th commonest cancer in men and the 8th commonest in women. Patients usually present with urinary tract symptoms (e.g., gross or microscopic hematuria or irritative voiding symptoms such as frequency, dysuria, and urgency). Evaluations of these patients usually entail voided-urine cytology, cystoscopy, and upper urinary tract imaging such as intravenous pyelography, renal sonography, or retrograde pyelography. Most newly diagnosed bladder cancers are superficial (i.e., not invading beyond the lamina propria on histological examination), and are known as transitional cell carcinoma (TCC). These superficial bladder cancers are usually managed by transurethral resection. However, the literature shows that approximately 50 to 75 % of treated TCC recur. Furthermore, 10 to 15 % of TCC progress to muscle-invasive bladder cancer. According to the literature, the prevalence of recurrence after initial treatment as well as the natural history of TCC necessitates long-term follow-up. Following treatment, accepted guidelines provide that patients who have previously been diagnosed with TCC should usually undergo urine cytology/cystoscopy every 3 months in the 1st year, every 6 months in the 2nd year, and once-yearly afterwards.
Currently, urine cytology with confirmatory cystoscopy represents the cornerstone for the identification of bladder tumors. However, the subjectivity and low sensitivity of cytology led to the development of several urine-based tests as adjuncts to cytology/cystoscopy for the diagnosis and follow-up of patients with TCC. These tests include the BTA Stat test (Bard Diagnostic, Redmond, WA), the NMP22 test (Matritech, Newton, MA), the Aura-Tek FDP test (PerImmune, Rockville, MD), and the Vysis UroVysion FISH Test (Vysis, Inc., Downers Grove, IL). They are usually objective, qualitative (BTA Stat and Aura-Tek FDP), or quantitative (NMP22, UroVysion), and have higher sensitivity than cytology, but some have lower specificity. So far, no single bladder tumor marker has emerged as the generally accepted test of choice, and none has been established as a screening tool for bladder malignancy.
Urine-based markers, such as proteins with increased cancer cell expression or chromosomal abnormalities in the urine, may be detected using a variety of laboratory methods to aid in the management of bladder cancer. The following markers/tests are currently available:
- Bladder tumor antigen (BTA) (eg, BTA stat and BTA TRAK)
- Fluorescence immunocytology (eg, ImmunoCyt/uCyt+)
- Fluorescence in situ hybridization (FISH) (eg, UroVysion)
- mRNA quantification by RT-qPRC testing (eg, Cxbladder)
- Nuclear matrix protein 22 (NMP22) (eg, NMP22 BladderChek and Matritech NMP22 Test).
Urine-based markers have a role in the detection of bladder cancer recurrence in individuals with a history of bladder cancer and are used adjunctively with urinary cytology and cystoscopy. These tests have also been proposed for bladder cancer screening, diagnosis of bladder cancer in individuals symptomatic of bladder cancer and for the evaluation of hematuria.
The UroVysion Bladder Cancer Kit (UroVysion Kit) (Baycare Laboratories) is designed to detect aneuploidy for chromosomes 3, 7, 17, and loss of the 9p21 locus via fluorescence in situ hybridization (FISH) in urine specimens from persons with hematuria suspected of having bladder cancer (Raman, et al., 2013). FISH analysis is used in conjunction with cystoscopy to monitor for recurrence among those with previously diagnosed bladder cancer. FISH analysis is a surveillance tool in established primary and secondary bladder adenocarcinoma.
The ImmunoCyt is an immunocytochemistry assay for the detection of tumor cells shed in the urine of patients previously diagnosed with bladder cancer (Chen, et al., 2006). This test is intended to augment the sensitivity of cytology for the detection of tumor cells in the urine of individuals previously diagnosed with bladder cancer. The test has been used for detection of tumor cells in the urine of individuals previously diagnosed with bladder cancer, and for use in conjunction with cytoscopy as an aid in the management of bladder cancer.
Although urine cytology has been shown to be less accurate than urinary biomarker tests, familiarity with the method as well as ease of performance justify the continued routine use of the former by primary care physicians, especially in patients who have no history of bladder malignancy. The urine-based biomarker tests have been shown to be accurate in detecting low-grade bladder tumors. In particular, these tests may be of help in deciding the need for further diagnostic assessment of patients with a history of bladder cancer and negative results on urine cytology. For example, elevated levels of urinary bladder tumor markers in patients with a history of TCC may warrant earlier, rather than delayed, cystoscopic examination. On the other hand, consideration may be given to lengthening the intervals between cystoscopic investigations when values of these tumor markers are normal.
An assessment by the Adelaide Health Technology Assessment (Mundy & Hiller, 2009) concluded that the NMP BladderCheck and UroVysion FISH assay, designed for the detection of bladder cancer in high risk patients, have poor sensitivity and poor positive predictive values. The assessment recommended that these assays not be used in asymptomatic patients. The assessment suggested, however, that these tests may be useful in the monitoring of patients with transitional cell carcinoma between cytoscopies. The AHTA recommended that this technology not be assessed further.
An assessment prepared for the Agency for Healthcare Research and Quality (Meleth, et al., 2014) found: "Although UroVysion is marketed as a diagnostic rather than a prognostic test, limited evidence from two small studies (total N=168) rated as low or medium risk of bias supported associations between test result and prognosis for risk of recurrence. We found no studies that directly assessed the impact of a test of interest on both physician decision-making and downstream health outcomes to establish clinical utility. We attempted to construct an indirect chain of evidence to answer the overarching question, but we were unable to do so. Even in the cases where the tests seemed to add value in determining prognosis (i.e., evidence of clinical validity), we found no evidence that using the test was related to improved outcomes for patients."
The American Urologic Association’s guideline on "Diagnosis, evaluation and follow-up of asymptomatic microhematuria (AMH) in adults" (Davis et al, 2012) stated that "The use of urine cytology and urine markers (Nuclear Matrix Protein 22 [NMP22], bladder tumor antigen [BTA]-stat, and UroVysion fluorescence in situ hybridization assay [FISH]) is not recommended as a part of the routine evaluation of the asymptomatic microhematuria patient".
Chou et al (2015) systematically reviewed the evidence on the accuracy of urinary biomarkers for diagnosis of bladder cancer in adults who have signs or symptoms of the disease or are undergoing surveillance for recurrent disease. Data sources included Ovid MEDLINE (January 1990 through June 2015), Cochrane Central Register of Controlled Trials, Cochrane Database of Systematic Reviews, and reference lists. A total of 57 studies that evaluated the diagnostic accuracy of quantitative or qualitative nuclear matrix protein 22 (NMP22), qualitative or quantitative bladder tumor antigen (BTA), FISH, fluorescent immunohistochemistry (ImmunoCyt [Scimedx]), and Cxbladder (Pacific Edge Diagnostics USA) using cystoscopy and histopathology as the reference standard met inclusion criteria; case-control studies were excluded. Dual extraction and quality assessment of individual studies were carried out; overall strength of evidence (SOE) was also assessed. Across biomarkers, sensitivities ranged from 0.57 to 0.82 and specificities ranged from 0.74 to 0.88. Positive likelihood ratios ranged from 2.52 to 5.53, and negative likelihood ratios ranged from 0.21 to 0.48 (moderate SOE for quantitative NMP22, qualitative BTA, FISH, and ImmunoCyt; low SOE for others). For some biomarkers, sensitivity was higher for initial diagnosis of bladder cancer than for diagnosis of recurrence. Sensitivity increased with higher tumor stage or grade. Studies that directly compared the accuracy of quantitative NMP22 and qualitative BTA found no differences in diagnostic accuracy (moderate SOE); head-to-head studies of other biomarkers were limited. Urinary biomarkers plus cytologic evaluation were more sensitive than biomarkers alone but missed about 10 % of bladder cancer cases. The authors concluded that urinary biomarkers miss a substantial proportion of patients with bladder cancer and are subject to false-positive results in others; accuracy is poor for low-stage and low-grade tumors. They stated that research is needed to understand how the use of these biomarkers with other diagnostic tests affect the use of cystoscopy and clinical outcomes.
In an editorial that accompanied the afore-mentioned study, Abbosh and Plimack (2015) stated that "Until urinary biomarkers become available that are sufficiently accurate to supplant the current recommended detection algorithms in biomarker-negative patients, they will not be a cost-effective addition to strategies to detect bladder cancer".
In summary, urine-based bladder tumor marker tests have been shown to be useful as an adjunct to urine cytology and cystoscopy in monitoring for recurrences of bladder cancer, but according to the available literature should not be used as a screening tool for bladder malignancy. The U.S. Preventive Services Task Force (USPSTF, 2004) has concluded that the potential harms of screening for bladder cancer using available tests, such as microscopic urinalysis, urine dipstick, urine cytology, or such new tests as bladder tumor antigen (BTA) or nuclear matrix protein (NMP22) immunoassay, outweigh any potential benefits.
BluePrint
Molecular subtyping profile or BluePrint is proposed for the evaluation of an individual’s prognosis when diagnosed with breast cancer. The multigene profile classifies breast cancer into basal type, luminal type and ERBB type (HER2/neu positive) molecular subclasses to stratify an individual’s risk to purportedly assist with treatment decisions.
Agendia BluePrint has an 80-gene profile that classifies breast cancer into molecular subtypes (Raman, et al., 2013). The profile separates tumors into Basal-type, Luminal-type and ERBB2-type subgroups by measuring the functionality of downstream genes for each of these molecular pathways to inform the physician of the potential effect of adjuvant therapy.
Krijgsman et al (2012) noted that classification of breast cancer into molecular subtypes maybe important for the proper selection of therapy, as tumors with seemingly similar histopathological features can have strikingly different clinical outcomes. Herein, these researchers reported the development of a molecular subtyping profile (BluePrint), which enables rationalization in patient selection for either chemotherapy or endocrine therapy prescription. An 80-Gene Molecular Subtyping Profile (BluePrint) was developed using 200 breast cancer patient specimens and confirmed on 4 independent validation cohorts (n = 784). Additionally, the profile was tested as a predictor of chemotherapy response in 133 breast cancer patients, treated with T/FAC neoadjuvant chemotherapy. BluePrint classification of a patient cohort that was treated with neoadjuvant chemotherapy (n = 133) shows improved distribution of pathological Complete Response (pCR), among molecular subgroups compared with local pathology: 56 % of the patients had a pCR in the Basal-type subgroup, 3 % in the MammaPrint low-risk, luminal-type subgroup, 11 % in the MammaPrint high-risk, luminal-type subgroup, and 50 % in the HER2-type subgroup. The group of genes identifying luminal-type breast cancer is highly enriched for genes having an Estrogen Receptor binding site proximal to the promoter-region, suggesting that these genes are direct targets of the Estrogen Receptor. Implementation of this profile may improve the clinical management of breast cancer patients, by enabling the selection of patients who are most likely to benefit from either chemotherapy or from endocrine therapy.
An assessment by the National Institute for Health Research (Ward, et al., 2013) found the evidence for Blueprint was limited. Because of the limited available data identified for this test, the NIHR was unable to draw firm conclusions about its analytical validity, clinical validity (prognostic ability) and clinical utility. The report stated that further evidence on the prognostic and predictive ability of this test was required.
A report by the Belgian Healthcare Knowledge Centre (KCE) (San Miguel, et al., 2015) found that limited evidence for the prognostic ability (clinical validity) of BluePrint. The KCE found insufficient evidence on the impact of BluePrint on clinical management (clinical utility).
Furthermore, there is no information regarding BluePrint/molecular subtyping from NCCN’s clinical practice guideline on "Breast cancer" (Version 2.2013).
Breast Cancer Gene Expression Ratio / Breast Cancer Index
The Breast Cancer Gene Expression Ratio (HOXB13:IL17BR, also known as H/I) (AviaraDx, Inc., Carlsbad, CA) is intended to predict the risk of disease recurrence in women with estrogen receptor (ER)-positive, lymph node-negative breast cancer. The Breast Cancer Gene Expression Ratio is based on the ratio of the expression of two genes: the homeobox gene-B13 (HOXB13) and the interleukin- 17B receptor gene (IL17BR). In breast cancers that are more likely to recur, the HOXB13 gene tends to be over-expressed, while the IL-17BR gene tends to be under-expressed.
Ma et al (2004) reported on the early validation of the HOXB13:IL17BR gene expression ratio. The investigators generated gene expression profiles of hormone receptor-positive primary breast cancers in a set of 60 patients treated with adjuvant tamoxifen monotherapy. An expression signature predictive of disease-free survival was reduced to a two-gene ratio, HOXB13 versus IL17BR, which outperformed existing biomarkers. The investigators concluded that ectopic expression of HOXB13 in MCF10A breast epithelial cells enhances motility and invasion in vitro, and its expression is increased in both preinvasive and invasive primary breast cancer. The investigators suggested that HOXB13:IL17BR expression ratio may be useful for identifying patients appropriate for alternative therapeutic regimens in early-stage breast cancer.
In an 852-patient retrospective study, Ma, et al (2006) found that the HOXB13:IL17BR ratio (H:I expression ratio) independently predicted breast cancer recurrence in patients with ER-positive, lymph-node negative cancer. The H:I expression ratio was found to be predictive in patients who received tamoxifen therapy as well as in those who did not. Expression of HOXB13, IL17BR, CHDH, estrogen receptor (ER) and progesterone receptor (PR) were quantified by real-time polymerase chain reaction (PCR) in 852 formalin-fixed, paraffin-embedded primary breast cancers from 566 untreated and 286 tamoxifen-treated breast cancer patients. Gene expression and clinical variables were analyzed for association with relapse-free survival (RFS) by Cox proportional hazards regression models. The investigators reported that, in the entire cohort, expression of HOXB13 was associated with shorter RFS (p = .008), and expression of IL17BR and CHDH was associated with longer RFS (p < 0.0001 for IL17BR and p = 0.0002 for CHDH). In ER-positive patients, the HOXB13:IL17BR index predicted clinical outcome independently of treatment, but more strongly in node-negative patients. In multivariate analysis of the ER-positive node-negative subgroup including age, PR status, tumor size, S phase fraction, and tamoxifen treatment, the two-gene index remained a significant predictor of RFS (hazard ratio [HR] = 3.9; 95 % CI:1.5 to 10.3; p = .007).
The value of the Breast Cancer Gene Expression Ratio was also evaluated in a study by Goetz et al (2006). That study found that a high H:I expression ratio is associated with an increased rate of relapse and mortality in ER-positive, lymph node-negative cancer patients treated with surgery and tamoxifen. Goetz et al (2006) examined the association between the ratio of the HOXB13 to IL17BR expression and the clinical outcomes of relapse and survival in women with ER-positive breast cancer enrolled onto a North Central Cancer Treatment Group adjuvant tamoxifen trial (NCCTG 89-30-52). Tumor blocks were obtained from 211 of 256 eligible patients, and quantitative reverse transcription-PCR profiles for HOXB13 and IL-17BR were obtained from 206 patients. In the node-positive cohort (n = 86), the HOXB13/IL-17BR ratio was not associated with relapse or survival. In contrast, in the node-negative cohort (n = 130), a high HOXB13/IL-17BR ratio was associated with significantly worse RFS [HR, 1.98; p = 0.031], disease-free survival (DFS) (HR, 2.03; p = 0.015), and OS (HR, 2.4; p = 0.014), independent of standard prognostic markers.
The Blue Cross and Blue Shield Association Technology Evaluation Center (BCBSA, 2007) announced that its Medical Advisory Panel (MAP) concluded that the use of the Breast Cancer Gene Expression Ratio gene expression profiling does not meet the TEC criteria.
The Evaluation of Genomic Applications in Practice and Prevention (EGAPP) Working Group (2009) found insufficient evidence to make a recommendation for or against the use of the H:I ratio test to improve outcomes in defined populations of women with breast cancer. EGAPP concluded that the evidence is insufficient to assess the balance of benefits and harms of the proposed uses of this test. The EWG encouraged further development and evaluation of these technologies.
In a systematic review on gene expression profiling assays in early-stage breast cancer, Marchionni, et al. (2008) summarized evidence on the validity and utility of 3 gene expression-based prognostic breast cancer tests: Oncotype Dx, MammaPrint, and H/I. The authors concluded that gene expression technologies show great promise to improve predictions of prognosis and treatment benefit for women with early-stage breast cancer. However, more information is needed on the extent of improvement in prediction, characteristics of women in whom the tests should be used, and how best to incorporate test results into decision making about breast cancer treatment.
Guidelines from the American Society for Clinical Oncology (Harris, et al., 2007) found that, in newly diagnosed patients with node-negative, estrogen-receptor positive breast cancer, the Oncotype Dx assay can be used to predict the risk of recurrence in patients treated with tamoxifen. The ASCO guidelines concluded that Oncotype Dx may be used to identify patients who are predicted to obtain the most therapeutic benefit from adjuvant tamoxifen and may not require adjuvant chemotherapy. The ASCO guidelines found, in addition, that patients with high recurrence scores appear to achieve relatively more benefit from adjuvant chemotherapy than from tamoxifen. ASCO found that there are insufficient data at present to comment on whether these conclusions generalize to hormonal therapies other than tamoxifen, or whether this assay applies to other chemotherapy regimens. Guidelines from the American Society for Clinical Oncology (Harris, et al., 2007) concluded that the precise clinical utility and appropriate application for other multiparameter assays, such as the MammaPrint assay, the Rotterdam Signature, and the Breast Cancer Gene Expression Ratio are under investigation. ASCO also found insufficient data to recommend use of proteomic patterns for management of patients with breast cancer.
Sgori et al (2013) found that, in the absence of extended letrozole therapy, high H/I identifies a subgroup of ER-positive patients disease-free after 5 years of tamoxifen who are at risk for late recurrence. The investigators also found that, when extended endocrine therapy with letrozole is prescribed, high H/I predicts benefit from therapy and a decreased probability of late disease recurrence. Sgori, et al. conducted a prospective-retrospective, nested case-control design of 83 recurrences matched to 166 nonrecurrences from letrozole- and placebo-treated patients within MA.17 trial. Expression of H/I within primary tumors was determined by reverse-transcription polymerase chain reaction with a prespecified cutpoint. The investigators determined the predictive ability of H/I for ascertaining benefit from letrozole using multivariable conditional logistic regression including standard clinicopathological factors as covariates. All statistical tests were two-sided. The investigators reported that high H/I was statistically significantly associated with a decrease in late recurrence in patients receiving extended letrozole therapy (odds ratio [OR] = 0.35; 95% confidence interval [CI] = 0.16 to 0.75; P = .007). In an adjusted model with standard clinicopathological factors, high H/I remained statistically significantly associated with patient benefit from letrozole (OR = 0.33; 95% CI = 0.15 to 0.73; P = .006). Reduction in the absolute risk of recurrence at 5 years was 16.5% for patients with high H/I (P = .007). The interaction between H/I and letrozole treatment was statistically significant (P = .03).
BioTheranostics Breast Cancer Index (BCI) is a prognostic biomarker that provides quantitative assessment of the likelihood of distant recurrence in patients diagnosed with estrogen receptor-positive, lymph node-negative breast cancer (Raman, et al., 2013). In development and validation studies, BCI stratified about 50% of tamoxifen treated ER+, node-negative breast cancer patients into a low risk group for 10-year distant recurrence. BCI is a molecular assay developed from the combination of two indices: HOXB13:IL17BR and five cell cycle-associate gene index (BUB1B, CENPA, NEK2, RACGAP1, RRM2) that assesses tumor grade. The test is performed on a formalin-fixed, paraffin-embedded (FFPE) tissue block.
Ma et al (2008) reported on the development and early validation of a five-gene reverse transcription PCR assay for molecular grade index (MGI) that has subsequently been incorporated into BCI and is suitable for analyzing routine formalin-fixed paraffin-embedded clinical samples. The investigators found that the combination of MGI and HOXB13:IL17BR outperformed either alone and identifies a subgroup (approximately 30%) of early stage estrogen receptor-positive breast cancer patients with very poor outcome despite endocrine therapy. From their previously published list of genes whose expression correlates with both tumor grade and tumor stage progression, the investigators selected five cell cycle-related genes to build MGI and evaluated MGI in two publicly available microarray data sets totaling 410 patients. Using two additional cohorts (n =323), the investigators developed a real-time reverse transcription PCR assay for MGI, validated its prognostic utility, and examined its interaction with HOXB13:IL17BR. The investigators reported that MGI performed consistently as a strong prognostic factor and was comparable with a more complex 97-gene genomic grade index in multiple data sets. In patients treated with endocrine therapy, MGI and HOXB13:IL17BR modified each other's prognostic performance. High MGI was associated with significantly worse outcome only in combination with high HOXB13:IL17BR, and likewise, high HOXB13:IL17BR was significantly associated with poor outcome only in combination with high MGI.
Jerevall et al (2011) reported on the development of the Breast Cancer Index, a dichotomous index combining two gene expression assays, HOXB13:IL17BR (H:I) and molecular grade index (MGI), to assess risk of recurrence in breast cancer patients. The study objective was to demonstrate the prognostic utility of the combined index in early-stage breast cancer. In a blinded retrospective analysis of 588 ER-positive tamoxifen-treated and untreated breast cancer patients from the randomized prospective Stockholm trial which was conducted during 1976 to 1990, H:I and MGI were measured using real-time RT-PCR. Association with patient outcome was evaluated by Kaplan-Meier analysis and Cox proportional hazard regression. A continuous risk index was developed using Cox modelling. The investigators found that the dichotomous H:I+MGI was significantly associated with distant recurrence and breast cancer death. The greater than 50% of tamoxifen-treated patients categorized as low-risk had less than 3% 10-year distant recurrence risk. A continuous risk model (Breast Cancer Index (BCI)) was developed with the tamoxifen-treated group and the prognostic performance tested in the untreated group was 53% of patients categorized as low risk with an 8.3% 10-year distant recurrence risk.
Jankowitz et al (2011) reported on a study to validate the prognostic performance of BCI in estrogen-receptor positive, lymph node negative breast cancer patients. The investigators found that, in this characteristically low-risk cohort, BCI classified high versus low-risk groups with about a five-fold difference in 10-year risk of distant recurrence and breast cancer-specific death. The investigators identified tumor samples from 265 estrogen-receptor positive, lymph-node negative tamoxifen-treated patients from a single academic institution's cancer research registry. They performed the BCI assay and assigned scores based on a predetermined risk model. The investigators assessed risk by BCI and Adjuvant Online! (AO) and correlated these to clinical outcomes in the patient cohort. The investigators found that BCI was a significant predictor of outcome in this cohort of estrogen-receptor positive, lymph-node negative patients (median age: 56-y; median follow-up: 10.3-y), treated with adjuvant tamoxifen alone or tamoxifen with chemotherapy (32%). BCI categorized 55%, 21%, and 24% of patients as low, intermediate and high-risk, respectively. The 10-year rates of distant recurrence were 6.6%, 12.1% and 31.9% and of breast cancer-specific mortality were 3.8%, 3.6% and 22.1% in low, intermediate, and high-risk groups, respectively. In a multivariate analysis including clinicopathological factors, BCI was a significant predictor of distant recurrence (HR for 5-unit increase = 5.32 [CI 2.18-13.01; P = 0.0002]) and breast cancer-specific mortality (HR for a 5-unit increase = 9.60 [CI 3.20-28.80; P < 0.0001]). AO was significantly associated with risk of recurrence. In a separate multivariate analysis, both BCI and AO were significantly predictive of outcome. In a time-dependent (10-year) ROC curve accuracy analysis of recurrence risk, the addition of BCI and AO increased predictive accuracy in all patients from 66% (AO only) to 76% (AO+BCI) and in tamoxifen-only treated patients from 65% to 81%. The authors concluded that BCI and AO are independent predictors with BCI having additive utility beyond standard of care parameters that are encompassed in AO. The authors acknowledge that this study is limited by the fact that it was a retrospective, single-institution study and that results may have been biased on the basis of specimen availability and patterns of referral to the tertiary academic center.
Mathieu et al (2012) assessed the performance of BCI to predict chemosensitivity based on pathological complete response (pCR) and breast conservation surgery (BCS). The authors performed the BCI assay on tumor samples from 150 breast cancer patients from a single institution treated with neoadjuvant chemotherapy. The authors used logistical regression and c-index to assess predictive strength and additive accuracy of BCI beyond clinicopathologic factors. BCI classified 42% of patients as low, 35% as intermediate and 23% as high risk. Low BCI risk group had 98.4% negative predictive value (NPV) for pCR and 86% NPV for BCS. High versus low BCI group had a 34 and 5.8 greater likelihood of achieving pCR and BCS, respectively (P=0.0055; P=0.0022). BCI increased c-index for pCR (0.875-0.924; p=0.017) and BCS prediction (0.788-0.843; p=0.027) beyond clinicopathologic factors. The authors concluded that BCI significantly predicted pCR and BCS beyond clinicopathologic factors. High NPVs indicate that BCI could be a useful tool to identify breast cancer patients who are not eligible for neoadjuvant chemotherapy. The authors concluded that "these results suggest that BCI could be used to assess both chemosensitivity and eligibility for BCS." The authors stated that an important limitation of this study is that, in this retrospective analysis, patients were not selected based on ER or HER2 expression for the indications of neoadjuvant chemotherapy. The authors explained that this could have increased the predictive strength of BCI given that this biomarker was initially developed and validated in ER + node-negative patients
Zhang et al (2013) examined the prognostic performance of BCI for prediction of early (0-5 years) and late (more than 5 years) risk of distant recurrence in patients with estrogen receptor-positive (ER(+)), lymph node-negative (LN(-)) tumors. The BCI model was validated by retrospective analyses of tumor samples from tamoxifen-treated patients from a randomized prospective trial (Stockholm TAM, n = 317) and a multi-institutional cohort (n = 358). Within the Stockholm TAM cohort, BCI risk groups stratified the majority (approximately 65%) of patients as low risk with less than 3% distant recurrence rate for 0 to 5 years and 5 to 10 years. In the multi-institutional cohort, which had larger tumors, 55% of patients were classified as BCI low risk with less than 5% distant recurrence rate for 0 to 5 years and 5 to 10 years. Zhang and colleagues found that, for both cohorts, continuous BCI was the most significant prognostic factor beyond standard clinicopathologic factors for 0 to 5 years and more than five years. The authors concluded that the prognostic sustainability of BCI to assess early- and late-distant recurrence risk at diagnosis has clinical use for decisions of chemotherapy at diagnosis and for decisions for extended adjuvant endocrine therapy beyond five years.
Sgori et al (2013) compared the prognostic ability of the BCI assay, the Oncotype DX Breast, and IHC4 for both early and late recurrence in patients with estrogen-receptor-positive, node-negative (N0) disease who took part in the Arimidex, Tamoxifen, Alone or in Combination (ATAC) clinical trial. In this prospective comparison study, Sgori and colleagues obtained archival tumor blocks from the TransATAC tissue bank from all postmenopausal patients with estrogen-receptor-positive breast cancer from whom the Oncotype DX and IHC4 values had already been derived. The investigators did BCI analysis in matched samples with sufficient residual RNA using two BCI models -- cubic (BCI-C) and linear (BCI-L)-using previously validated cutoffs. The prospectively-defined primary study objective was to evaluate overall (0–10y) prognostic performance of the BCI-C model for DR in ER+ N0 patients. Secondary objectives were: 1) assessment of the prognostic performance of the BCI-L model and its components, H/I and MGI, for overall (0–10y), early (0–5y) and late (5–10y) DR; 2) comparative performance of BCI-L versus the Oncotype DX RS and IHC4. To assess the ability of the biomarkers to predict recurrence beyond standard clinicopathological variables, the investigators calculated the change in the likelihood-ratio from Cox proportional hazards models. Suitable tissue was available from 665 patients with estrogen-receptor-positive, N0 breast cancer for BCI analysis. The primary analysis showed significant differences in risk of distant recurrence over 10 years in the categorical BCI-C risk groups (p<0·0001) with 6·8% (95% CI 4·4-10·0) of patients in the low-risk group, 17·3% (12·0-24·7) in the intermediate group, and 22·2% (15·3-31·5) in the high-risk group having distant recurrence. BCI-C analyzed as a continuous variable was not significantly associated with overall (0–10y) risk of DR when adjusted for CTS (inter-quartile HR=1·39; 95% CI, 0·99 to 3·70; LR-Δχ2=3·70; P=0·054). Comparison of the prognostic performance of BCI-L to BCI-C indicated that unlike BCI-C, BCI-L was a significant predictor of risk of recurrence as a continuous variable, and the HR after adjustment with CTS was 2·19 versus 4.86 between high- and low-risk groups for BCI-C and BCI-L, respectively. Thus, all subsequent analyses were performed utilizing BCI-L. The secondary analysis showed that BCI-L was a much stronger predictor for overall (0-10 year) distant recurrence compared with BCI-C (interquartile HR 2·30 [95% CI 1·62-3·27]; likelihood ratio (LR)-Δχ(2)=22·69; p<0·0001). When compared with BCI-L, the Oncotype Dx breast score was less predictive (HR 1·48 [95% CI 1·22-1·78]; LR-Δχ(2)=13·68; p=0·0002) and IHC4 was similar (HR 1·69 [95% CI 1·51-2·56]; LR-Δχ(2)=22·83; p<0·0001). All further analyses were done with the BCI-L model. In a multivariable analysis, all assays had significant prognostic ability for early distant recurrence (BCI-L HR 2·77 [95% CI 1·63-4·70], LR-Δχ(2)=15·42, p<0·0001; Oncotype Dx Breast score HR 1·80 [1·42-2·29], LR-Δχ(2)=18·48, p<0·0001; IHC4 HR 2·90 [2·01-4·18], LR-Δχ(2)=29·14, p<0·0001); however, only BCI-L was significant for late distant recurrence (BCI-L HR 1·95 [95% CI 1·22-3·14], LR-Δχ(2)=7·97, p=0·0048; 21-gene recurrence score HR 1·13 [0·82-1·56], LR-Δχ(2)=0·48, p=0·47; IHC4 HR 1·30 [0·88-1·94], LR-Δχ(2)=1·59, p=0·20). The authors concluded that BCI-L was the only significant prognostic test for risk of both early and late distant recurrence and identified two risk populations for each timeframe. BCI-L could help to identify patients at high risk for late distant recurrence who might benefit from extended endocrine or other therapy. An important limitation is that the evaluation of BCI-L was a secondary objective of this study; the primary objective was evaluation of BCI-C.
An editorial (Ignatiadis, 2013) accompanying the study by Sgroi, et al. stated that the BCI test is "ready for prime time" in treatment decision making for post-menopausal, estrogen-receptor positive women who have undergone 5 years of hormonal therapy. The editorial noted that there are other molecular diagnostic assays that also have been shown to predict late recurrence. For support, the editorial cited a study by Sestak, et al. (2013), which found that, in the last follow-up phase, Clinical Treatment Score (CTS) added most prognostic information for distant recurrence in years 5 to 10 for breast cancer patients in the ATAC trial. Sestak, et al. reported that, in a multivariate model that incorporated CTS, PAM50 provided the strongest additional prognostic factor in the 5 to 10 year followup phase, followed by BCI, and with IHC4 and RS adding the least prognostic information.
A manufacturer funded study (Gustavsen, et al., 2014) reported on a model that found BCI to be cost saving from a third-party payer perspective, based upon assumptions about the impact of BCI on adjuvant chemotherapy use, extended endocrine therapy use, and endocrine therapy compliance. The authors developed two economic models to project the cost-effectiveness of BCI in a hypothetical population of patients with estrogen-receptor positive, lymph-node negative breast cancer compared with standard clinicopathologic diagnostic modalities. The authors modeled costs associated with adjuvant chemotherapy, toxicity, followup, endocrine therapy, and recurrence over 10 years. The models examined cost utility compared with standard practice when used at diagnosis and in patients disease-free at 5 years post diagnosis. The authors reported that use of BCI was projected to be cost saving in both models. In the newly diagnosed population, net cost savings were $3803 per patient tested. In the 5 years post diagnosis population, BCI was projected to yield a net cost savings of $1803 per patient tested. The authors reported that sensitivity analyses demonstrated that BCI was cost saving across a wide range of clinically relevant input assumptions.
Preliminary data suggest that molecular approaches including gene expression platforms such as BCI may add to classical clinical parameters including tumor size and node status at diagnosis, but further research is needed (Smith, et al., 2014; Bianchini & Gianni, 2013; Ignatiadis and Sotiriou, 2013). The clinical utility of BCI and other molecular diagnostics in predicting late recurrence has yet to be established (Foukakis and Bergh, 2015). It also remains to be established which of several molecular diagnostic tests in development are the most appropriate for detecting late recurrence (Sestak & Kuzick, 2015).
An assessment by the National Institute for Health Research (Ward, et al., 2013) found that, based on the limited available data, no firm conclusions can be drawn about the analytical validity, clinical validity (prognostic ability) and clinical utility of the Breast Cancer Index. The assessment stated that further evidence on the prognostic and predictive ability of this test is required. An assessment by IETS (2013) and a consensus statement (Azim, et al., 2013) reached similar conclusions.
An assessment by the BlueCross BlueShield Association (2015) concluded that the evidence is insufficient to permit conclusions about the Breast Cancer Index on health outcomes. Although evidence supports the association of risk classes defined by the Breast Cancer Index and recurrence and survival outcomes, it remains to be shown whether the Breast Cancer Index adds incremental prognostic information to standard clinical risk classifiers.
An assessment by the Belgian Healthcare Knowledge Centre (KCE) (San Miguel, et al.,2015) found that the evidence for the H/I ratio assay is limited to studies supporting the prognostic ability (clinical validity) of the test. They found insufficient evidence for the impact of the H/I ratio assay on clinical management (clinical utility).
A review published in the ASCO Educational Book (Smith, et al., 2014) reviewed the BCI and other currently available molecular diagnostics for selecting and determining the optimal duration of endocrine adjuvant therapy in women with early stage estrogen receptor positive breast cancer: "Further research into applying molecular features and gene expression scores to standard clinico-pathologic criteria for tailoring extended endocrine therapy is now a high priority.... An important research challenge is now to identify which patients are likely to benefit from this type of long-term therapy. Preliminary data suggest that molecular approaches including gene expression platforms such as ROR may add to classical clinical parameters including tumor size and node status at diagnosis."
A Palmetto Medicare Local Coverage Determination (LCD) allows coverage of the Breast Cancer Index in certain post-menopausal women with estrogen-receptor positive breast cancer, reasoning that the data defined benefit of the BCI test appears to be when a woman is having significant side effects or has other concerns regarding adjuvant tamoxifen therapy and is opposed to taking more than 5 years of tamoxifen or starting on an AI (letrazole) after tamoxifen (CMS, 2014). The LCD noted, however, that, there is an increase in recurrence risk with increasing BCI score such that, "at the 95% confidence interval (CI), the risk in some individuals categorized in the BCI-low group could be as high as 20%. Due to the data complexity, there is a significant possibility that a physician might consider all BCI-L patients at negligible risk, and thus not consider extended hormone therapy and consequently lead women from the NCCN recommended interventions. Given the low toxicity and low cost of extended therapy, the false sense of security could deny many women from lifesaving therapy."
There is a lack of consensus among guidelines regarding the value of molecular assays in determining whether longer durations of adjuvant endocrine therapy beyond 5 years are clinically indicated. Guidelines from the American Society for Clinical Oncology (Burstein, et al., 2014) on adjuvant endocrine therapy for hormone-receptor positive breast cancer state: "Well-established clinical factors including tumor size; nodal status; ER, PgR, and HER2 biomarkers; and molecular diagnostic assays serve as prognostic factors for breast cancer recurrence. However, there are no robust specific clinical or biomarker measures that selectively predict early versus late recurrence, nor predict whether tamoxifen or AI therapy would be appropriate treatment, nor determine whether longer durations of adjuvant endocrine therapy are clinically indicated." The National Comprehensive Cancer Network guidelines for breast cancer version 2, 2015 states: "Multiple other multi-gene mor multi-gene expression assay systems have been developed. These systems are generally based upon small, retrospective studies, and the Panel believes that none are currently sufficiently validated to warrant inclusion in the guideline." The St. Gallen guideline panel (Coates, et al., 2015) found that Oncotype DX, MammaPrint, PAM-50 ROR score, EndoPredict and the Breast Cancer Index were all considered usefully prognostic for years 1-5, but only the Oncotype Dx commanded a majority in favor of its value in predicting the usefulness of chemotherapy. The Panel agreed that the PAM50 ROR score was clearly prognostic beyond five years, and that the Mammaprint was not prognostic beyond 5 years. The Panel was divided about the prognostic value of the Breast Cancer Index, the Oncotype DX, and EndoPredict in this time period. ESMO guidelines (Senkus, et al., 2013) state: "Molecular signatures for ER-positive breast cancer such as OncotypeDx, EndoPredict, Breast Cancer Index or for all types of breast cancer (pNO-1) such as MammaPrint and Genomic Grade Index are commercially available, but none of them have proven robust clinical utility so far. In some cases of difficult decision, such as grade 2 ER-positive HER-2 negative and node-negative breast cancer, MammaPrint and Oncotype DX may be used in conjunction with all clinicopathological factors, to help in treatment decision-making."
Guidelines from the American Society for Clinical Oncology (2016) state: "If a patient has ER/PgR-positive, HER2-negative (node-negative) breast cancer, the clinician may use the Breast Cancer Index to guide decisions on adjuvant systemic therapy." This is a moderate strength recommendation based upon intermediate quality evidence. ASCO guidelines recommend use of the Breast Cancer Index to guide decisions on adjuvant systemic therapy in patients with ER/PgR=positive, HER2-negative (node-positive) breast cancer. The guidelines also recommend against the use of the Breast Cancer Index in HER2-positive breast cancer or TN breast cancer. The guidelines also recommended against the use of The Breast Cancer Index to guide decisions on extended endocrine therapy for patients with ER/PgR-positive, HER-2 negative (node-negative) breast cancer who have had 5 years of endocrine therapy without evidence of recurrence.
BT Test
Provista Life Sciences (Phoenix, AZ) has developed a laboratory test called the Biomarker Translation Test, or the BT Test, which is a test score based on a multi-protein biomarker analysis (i.e., IL-2, -6,-8,-12, TNFa, EGF, FGF, HGF, VEGF) and medical profile of an individual's risk factors for breast cancer. It is intended to be used as an adjunctive test along with other breast cancer detection modalities, however, there are no published studies of the effectiveness of this test.
BTG Early Detection of Pancreatic Cancer
BT-Reveal Early Pancreatic Cancer Test (Breakthrough Genomics), also referred to as BTG Early Detection of Pancreatic Cancer test, uses a plasma specimen to evaluate circulating tumor cell–free DNA (cfDNA) for 59 biomarkers to help detect early pancreatic cancer in patients at high risk for developing the disease. The test is intended for use as a screening tool for at-risk individuals. A positive result does not guarantee the individual has cancer. For all positive results, additional follow-up imaging and other confirmatory tests are recommended.
The peer-reviewed publications discussed in the manufacturer’s website includes literature on the underlying technology used in the development of the test; however, none reflect the specific performance metrics of the test or the initial clinical data that was submitted to the FDA as part of the test’s designation as a ‘Breakthrough Device’.
Circulating Tumor Cells (e.g., CellMax Life and FirstSightCRC) for Screening of Colorectal Cancer
Yang and colleagues (2017) noted that CTCs have been accepted as a prognostic marker in patients with mCRC (International Union for Cancer Control [UICC] stage IV). However, the prognostic value of CTCs in patients with non-mCRC (Union for International Cancer Control [UICC] stage I to III) still remains in dispute. These researchers carried out a meta-analysis to examine the prognostic significance of CTCs detected by the RT-PCR method in patients diagnosed with non-mCRC patients. A comprehensive literature search for relevant articles was performed in the Embase, PubMed, Ovid, Web of Science, Cochrane library and Google Scholar databases. The studies were selected according to pre-determined inclusion/exclusion criteria. Using the random-effects model of Stata software, version12.0 (2011) (Stata Corp, College Station, TX), to conduct the meta-analysis, and the HR, RR and their 95 % CIs were regarded as the effect measures. Subgroup analyses and meta-regression were also conducted to clarify the heterogeneity. A total of 12 eligible studies, containing 2,363 patients with non-mCRC, were suitable for final analyses. The results showed that the OS (HR = 3.07, 95 % CI: 2.05 to 4.624, p < 0.001; I2 = 55.7 %, p = 0.008) and DFS (HR = 2.58, 95 % CI: 2.00 to 3.32, p < 0.001; I2 = 34.0 %, p = 0.085) were poorer in patients with CTC-positive, regardless of the sampling time, adjuvant therapy and TNM stage. CTC-positive was also significantly associated with regional lymph nodes (RLNs) metastasis (RR = 1.62, 95 % CI: 1.17 to 2.23, p = 0.003; I2 = 74.6 %, p < 0.001), depth of infiltration (RR = 1.41, 95 % CI: 1.03 to 1.92, p = 0.03; I2 = 38.3 %, p = 0.136), vascular invasion (RR = 1.66, 95 % CI: 1.17 to 2.36, p = 0.004; I2 = 46.0 %, p = 0.135), tumor grade (RR = 1.19, 95 % CI: 1.02 to 1.40, p = 0.029; I2 = 0 %, p = 0.821) and TNM stage (I, II versus III) (RR = 0.76, 95 % CI: 0.71 to 0.81, p < 0.001; I2 = 0 %, p = 0.717). However, there was no significant relationship between CTC-positive and tumor size (RR = 1.08, 95 % CI: 0.94 to 1.24, p = 0.30; I2 = 0 %, p = 0.528). The authors concluded that detection of CTCs by RT-PCR method had prognostic value for non-mCRC patients, and CTC-positive was associated with poor prognosis and poor clinicopathological prognostic factors. However, the prognostic value of CTCs supported the use of CTCs as an indicator of metastatic disease prior to the current classification of mCRC meaning it is detectable by CT/MRI. This study did not address the use of CTC for screening of CRC.
Lopresti and associates (2019) stated that CTCs represent an easy, repeatable and representative access to information regarding solid tumors. However, their detection remains difficult because of their paucity, their short half-life, and the lack of reliable surface biomarkers. Flow cytometry (FC) is a fast, sensitive and affordable technique, ideal for rare cells detection. Adapted to CTCs detection (i.e., extremely rare cells), most FC-based techniques require a time-consuming pre-enrichment step, followed by a 2-hours staining procedure, impeding on the efficiency of CTCs detection. These researchers overcame these caveats and reduced the procedure to less than 1 hour, with minimal manipulation. First, cells were simultaneously fixed, permeabilized, then stained. Second, using low-speed FC acquisition conditions and 2 discriminators (cell size and pan-cytokeratin expression), these investigators suppressed the pre-enrichment step. Applied to blood from donors with or without known malignant diseases, this protocol ensured a high recovery of the cells of interest independently of their epithelial-mesenchymal plasticity and could predict which samples were derived from cancer donors. The authors concluded that this proof-of-concept study laid the bases of a sensitive tool to detect CTCs from a small amount of blood upstream of in-depth analyses (colorectal cancer was one of the key words in this study).
Baek and co-workers (2019) noted that CTCs in the blood have been used as diagnostic markers in patients with CRC. In a prospective study, these researchers evaluated a CTC detection system based on cell size to examine CTCs and their potential as early diagnostic and prognostic biomarkers for CRC. From 2014 to 2015, a total of 88 patients with newly diagnosed CRC, who were scheduled for surgery, and 31 healthy volunteers were enrolled and followed-up in Pusan National University Hospital; CTCs were enriched using a centrifugal microfluidic system with a new fluid-assisted separation technique (FAST) and detected by cytomorphological evaluation using fluorescence microscopy. Two or more CTCs were detected using FAST in 74 patients and 3 healthy volunteers. The number of CTCs in the CRC group was significantly higher than that in the healthy volunteers (p < 0.001). When a ROC curve was created to differentiate patients with CRC from healthy volunteers, the sensitivity and specificity were almost optimized when the critical CTC value was 5/7.5 ml of blood. When this value was used, the sensitivity and specificity in differentiating patients with CRC from the healthy controls were 75 % and 100 %, respectively. In patients with CRC with greater than or equal to 5 CTCs, vascular invasion was frequently identified (p = 0.035). All patients with stage IV were positive for CTCs. Patients with greater than or equal to 5 CTCs showed a trend toward poor OS and PFS. The authors concluded that this study demonstrated promising results with the use of FAST-based CTC detection for the early diagnosis and prognosis of CRC. This study did not address the use of CTC for screening of CRC.
UpToDate reviews on "Screening for colorectal cancer: Strategies in patients at average risk" (Doubeni, 2019) and "Screening for colorectal cancer in patients with a family history of colorectal cancer or advanced polyp" (Ramsey and Grady, 2019) do not mention measurement of circulating tumor cells as a screening tool.
Furthermore, National Comprehensive Cancer Network’s clinical practice guidelines on "Colon cancer" (Version 2.2019) and "Rectal cancer" (Version 2.2019) do not mention measurement of circulating tumor cells as a screening tool.
Circulating Tumor Cells (e.g., CELLSEARCH)
Circulating tumor cell (CTC) test, CELLSEARCH, is a blood test that has been proposed as a method to determine prognosis, evaluate progression and assess treatment response in individuals with metastatic breast, colorectal and prostate cancers. CTC assays were developed to detect cells that break away from tumors and enter the blood stream.
The CellSearch™ Epithelial Cell Kit, along with the CellSpotter™ Analyzer (Veridex, LLC, Warren, NJ) is a device designed to automate the detection and enumeration of circulating tumor cells (CTCs) of epithelial origin (CD45-, EpCAM+, and cytokeratins 8, 18+ and/or 19+) in whole blood in patients with advanced breast cancer (Ellery, et al., 2010; Raman, et al., 2011). It is intended for use in adjunctively monitoring and predicting cancer disease progression and response to therapy.
The CellSearch Epithelial Cell Kit received FDA 510(k) clearance on January 21, 2004. The FDA concluded that the device is substantially equivalent to immunomagnetic circulating cancer cell selection and enumeration systems. These devices consist of biological probes, fluorochromes and other reagents, preservation and preparation devices and semi-automated analytical instruments to select and count circulating cancer cells in a prepared sample of whole blood.
The CellSearch Epithelial Cell Kit quantifies CTCs by marking cancerous cells with tiny, protein-coated magnetic balls in whole blood. These cells are stained with fluorescent markers for identification and then dispensed into a cartridge for analysis where a strong magnetic field is applied to the mixture causing the magnetically marked cells to move to the cartridge surface. The cartridge is then analyzed by the CellSpotter Analyzer. A medical professional rechecks the CTCs and the CellSpotter Analyzer tallies the final CTC count.
In a prospective, multicenter study, Cristofanilli et al (2004) used the CellSearch System on 177 patients with measurable metastatic breast cancer for levels of CTCs both before the patients started a new line of therapy and at follow-up. The progression of the disease or the response to treatment was determined with the use of standard imaging studies at the participating centers every nine to twelve weeks. Outcomes were assessed according to levels of CTCs at baseline, before the patients started a new therapy. In the first test, patients with 5 or more CTCs per 7.5 ml of blood compared to a group with fewer than 5 CTCs had a shorter median progression-free survival (2.7 months vs. 7.0 months) and shorter overall survival (10.1 months vs. greater than 18 months). At the follow-up visit, approximately three to four weeks after the initiation of therapy, the percentage of patients with more than 5 CTC was reduced from 49 percent to 30 percent, suggesting a benefit from therapy. The difference in progression-free survival between the two groups remained consistent (2.1 months for women with 5 or more CTCs vs. 7 months for women with less than 5 CTCs). Overall, survival in the women with more than 5 CTCs was 8.2 months compared to greater than 18 months in the cohort with less than 5 CTCs. Cristofanilli concluded that the number of CTCs before treatment was an independent predictor of progression-free survival and overall survival in patients with metastatic breast cancer. However, Cristofanilli also concluded that the results may not be valid for patients who do not have measurable disease or for those starting a new regimen of hormone therapy, immunotherapy, or both. He states, "The prognostic implications of an elevated level of circulating tumor cells for patients with metastatic disease who are starting a new treatment may be an opportunity to stratify these patients in investigational studies". Furthermore, the study did not address whether patients with an elevated number of circulating tumor cells might benefit from other therapies. Thus, this minimally invasive assay requires further evaluation as a prognostic marker of disease progression and response to therapy.
The clinical application of quantifying CTCs in the peripheral blood of breast cancer patients remains unclear. Published data in the peer-reviewed medical literature are needed to determine how such measurements would guide treatment decisions and whether these decisions would result in beneficial patient outcomes (Kahn, et al., 2004; Abeloff, et al., 2004). An assessment of CellSearch by AETSA (2006) concluded "In the current stage of development of this technology, there is no evidence that it provides any advantage over existing technology for CTC identification or indeed any additional clinical use." Guidelines from the American Society for Clinical Oncology (Harris, et al., 2007) found: "The measurement of circulating tumor cells (CTCs) should not be used to make the diagnosis of breast cancer or to influence any treatment decisions in patients with breast cancer. Similarly, the use of the recently U.S. Food and Drug Administration (FDA)-cleared test for CTC (CellSearch Assay) in patients with metastatic breast cancer cannot be recommended until further validation confirms the clinical value of this test."
An assessment by the Canadian Agency for Drugs and Technologies in Health (CADTH, 2012) found that studies indicate that measurement of CTCs using the CellSearch system could be used as prognostic factors for progression of the disease and the potential treatment of patients with ovarian cancer. No economic studies were identified, therefore the cost-effectiveness of the CellSearch system could not be summarized.
Although studies relate circulating tumor cells to prognostic indicators (see, e.g., Cohen, et al., 2008; De Giorgi, et al., 2009), there are a lack of published prospective clinical studies demonstrating that measurement of CTCs alters management such that clinical outcomes are improved. Such clinical outcome studies are currently ongoing. Current guidelines from the National Comprehensive Cancer Network (NCCN) make no recommendations for use of circulating tumor cells.
Guidelines from the American Society for Clinical Oncology (2016) state: "The clinician should not use circulating tumor cells to guide decisions on adjuvant systemic therapy." This is a strong recommendation based upon intermediate-quality evidence.
Scher et al (2015) noted that clinical trials in castration-resistant prostate cancer (CRPC) need new clinical end-points that are valid surrogates for survival. These researchers evaluated circulating tumor cell (CTC) enumeration as a surrogate outcome measure. Examining CTCs alone and in combination with other biomarkers as a surrogate for OS was a secondary objective of COU-AA-301, a multi-national, randomized, double-blind phase III trial of abiraterone acetate plus prednisone versus prednisone alone in patients with metastatic CRPC previously treated with docetaxel. The biomarkers were measured at baseline and 4, 8, and 12 weeks, with 12 weeks being the primary measure of interest. The Prentice criteria were applied to test candidate biomarkers as surrogates for OS at the individual-patient level. A biomarker panel using CTC count and lactate dehydrogenase (LDH) level was shown to satisfy the 4 Prentice criteria for individual-level surrogacy; 12-week surrogate biomarker data were available for 711 patients. The abiraterone acetate plus prednisone and prednisone-alone groups demonstrated a significant survival difference (p = 0.034); surrogate distribution at 12 weeks differed by treatment (p < 0.001); the discriminatory power of the surrogate to predict mortality was high (weighted c-index, 0.81); and adding the surrogate to the model eliminated the treatment effect on survival. Overall, 2-year survival of patients with CTCs less than 5 (low risk) versus patients with CTCs greater than or equal to 5 cells/7.5 ml of blood and LDH greater than 250 U/L (high risk) at 12 weeks was 46 % and 2 %, respectively. The authors concluded that a biomarker panel containing CTC number and LDH level was shown to be a surrogate for survival at the individual-patient level in this trial of abiraterone acetate plus prednisone versus prednisone alone for patients with metastatic CRPC. They stated that independent phase III clinical trials are needed to validate these findings.
An assessment from the Institut National d’Excellence en Santé et Services Sociaux (INESSS) (Arsenault & Le Blanc, 2016) concluded: "Based on the scientific literature identified, the use of CellSearch tests as a predictive and prognostic biomarker in patients with early-stage breast cancer is not justified. The evidence is insufficient for establishing a concrete association between the presence of CTCs pre- and posttreatment and patient survival. In the case of patients with metastatic breast cancer, the examination of the scientific literature suggests that CTC enumeration prior to treatment could be a prognostic biomarker for patient survival. Despite the prognostic value of CTC enumeration, based on studies, its clinical utility has yet to be confirmed. For now, CellSearch tests should not be used outside the context of a clinical study. Further studies are needed to determine if the CellSearch test could play a clinically significant role in managing breast cancer patients."
The CELLSEARCH Circulating Multiple Myeloma Test (Menarini Silicon Biosystems, Inc) analyzes peripheral blood for circulating multiple myeloma cells (CMMCs). The test consists of circulating plasma cell immunologic selection, identification, morphological characterization, and enumeration of plasma cells based on differential CD138, CD38, CD19, and CD45 protein biomarker expression.
The CELLSEARCH HER2 Circulating Tumor Cell (CTC-HER2) Test (Menarini Silicon Biosystems, Inc) analyzes peripheral blood for HER2 status of the circulating tumor cells. The test consists of circulating tumor cell selection, identification, morphological characterization, detection and enumeration based on differential EpCAM, cytokeratins 8, 18, and 19, and CD45 protein biomarkers, and quantification of HER2 protein biomarker–expressing cells.
Cxbladder
Cxbladder is a suite of non-invasive genomic urine tests designed to help rule out urothelial bladder cancer in patients experiencing hematuria and to monitor for recurrent disease in those who have been treated for non-mucle invasive bladder cancer.
O'Sullivan and colleagues (2012) examined if the RNA assay uRNA® and its derivative Cxbladder® have greater sensitivity for the detection of bladder cancer than cytology, NMP22™ BladderChek™ and NMP22™ ELISA, and whether they are useful in risk stratification. A total of 485 patients presenting with gross hematuria but without a history of urothelial cancer were recruited prospectively from 11 urology clinics in Australasia. Voided urine samples were obtained before cystoscopy. The sensitivity and specificity of the RNA tests were compared to cytology and the NMP22 assays using cystoscopy as the reference. The ability of Cxbladder to distinguish between low grade, stage Ta urothelial carcinoma and more advanced urothelial carcinoma was also determined. uRNA detected 41 of 66 urothelial carcinoma cases (62.1 % sensitivity, 95 % confidence interval [CI]: 49.3 to 73.8) compared with NMP22 ELISA (50.0 %, 95 % CI: 37.4 to 62.6), BladderChek (37.9 %, 95 % CI: 26.2 to 50.7) and cytology (56.1 %, 95 % CI: 43.8 to 68.3). Cxbladder, which was developed on the study data, detected 82 %, including 97 % of the high grade tumors and 100 % of tumors stage 1 or greater. The cut-offs for uRNA and Cxbladder were pre-specified to give a specificity of 85 %. The specificity of cytology was 94.5 % (95 % CI: 91.9 to 96.5), NMP22 ELISA 88.0 %, (95 % CI: 84.6 to 91.0) and BladderChek 96.4 % (95 % CI: 94.2 to 98.0). Cxbladder distinguished between low-grade Ta tumors and other detected urothelial carcinoma with a sensitivity of 91 % and a specificity of 90 %. The authors concluded that uRNA and Cxbladder showed improved sensitivity for the detection of urothelial carcinoma compared to the NMP22 assays. Stratification with Cxbladder provides a potential method to prioritize patients for the management of waiting lists.
An UpToDate review on "Clinical presentation, diagnosis, and staging of bladder cancer" (Lotan and Choueiri, 2013) does not mention the use of mRNA biomarkers/PCR testing as a management tool for bladder cancer. Furthermore, NCCN’s clinical practice guideline on "Bladder cancer" (Version 1.2014) does not mention the use of mRNA biomarkers/PCR testing as a management tool for bladder cancer.
An assessment of urinary biomarkers for diagnosis of bladder cancer prepared for the Agency for Healthcare Research and Quality (Chou, et al., 2016) identified only one study of Cxbladder meeting inclusion criteria, graded as moderate quality, with an overall strength of evidence of "low".
Cxbladder Triage and the Cxbladder Detect+ are urine-based tests designed to rule out the presence of bladder cancer in low-risk patients with hematuria. The test uses an algorithm to incorporate clinical risk factor markers (age, sex, smoking history and macrohematuria frequency) and genetic information (mRNA), using gene-expression profiling by real-time quantitative PCR of 5 biomarker genes (MDK, HOXA13, CDC2 [CDK1], IGFBP5, and CXCR2), to determine a risk score for having urothelial carcinoma. The Cxbladder Detect+ includes urinary analysis of 6 single nucleotide polymorphisms for the FGFR3 and TERT genes, in addition to the current 5 mRNA biomarkers and clinical risk factors.
Lotan et al (2017) noted that patients with urothelial carcinoma (UC) undergo rigorous surveillance for recurrence. Non-invasive urine tests are not currently recommended by guideline panels owing to insufficient clinical benefit. In a prospective study, these researchers compared the performance of the Cxbladder Monitor test to other commonly available urine markers and cytology for surveillance of patients with UC. A total of 1,036 urine samples were collected from 803 patients undergoing surveillance for UC. Of these, 1,016 samples were directly assessed using cytology, NMP22 Bladderchek and NMP22 enzyme-linked immunosorbent assay (ELISA), and the clinically validated Cxbladder Monitor test. An exploratory analysis was also performed comparing data from 157 samples where UroVysion fluorescence in-situ hybridization (FISH) analysis was performed locally. The sensitivity of Cxbladder Monitor (0.91) significantly out-performed cytology (0.22), NMP22 ELISA (0.26), and NMP22 BladderChek (0.11). The negative predictive value (NPV) of Cxbladder Monitor was also superior at 0.96 compared with cytology (0.87), NMP22 ELISA (0.87), and NMP22 BladderChek (0.86). All false-negative results (n = 14) observed using Cxbladder Monitor were also negative for cytology, NMP22 ELISA, and NMP22 BladderChek. In the more limited set, UroVysion FISH also had inferior sensitivity (0.33) and NPV (0.92). The authors concluded that the Cxbladder Monitor test significantly out-performed current Food and Drug Administration (FDA)-approved urine-based monitoring tests, as well as cytology, in a large representative population undergoing surveillance for recurrent UC. This supported using Cxbladder Monitor as a confirmatory negative adjunct to cystoscopy or to justify postponing cystoscopic investigations in patients with a low-risk of recurrence.
The authors noted that the performance characteristics of the comparator FDA-approved non-invasive urine tests performed centrally by independent laboratories as part of the study were lower than those reported previously. Cytology results obtained as part of standard of care (SOC) and used in the patient evaluation was also collected and referred to as local cytology. A comparison of local versus central cytology confirmed the validity of study cytology results. Central and local cytology review was comparable for sensitivity with an overall agreement of greater than 90 %, suggesting that locally and centrally obtained results were comparable and not subject to any significant confounding factors or bias. However, differences in outcomes across locations for UroVysion FISH results was a potential limitation of the analyses performed in this study, as this test was not performed as part of central pathologic review. Although Cxbladder Monitor compared favorably to FISH across the available data, the broad confidence intervals (CIs) were indicative of the relatively low sample size of this analysis. Overall, this test showed a change in the use of urine biomarker diagnostic tests to concentrate specifically on performance metrics (sensitivity and NPV) that added significant utility to the surveillance clinical pathway by ruling out recurrent UC. These investigators noted that although a negative result is highly correlated with the absence of disease, a result that is not negative only suggests that a patient should continue with physician-prescribed procedures and does not require additional work-up, nor is a non-negative result indicative of future recurrence.
Konety et al (2019) stated that Cxbladder diagnostic tests combine genomic information from urinary mRNA with phenotypic information to either rule out low-risk individuals or identify patients at a high risk of UC. In a retrospective study, these researchers examined the performance of Cxbladder and urinary cytology, and Cxbladder's adjudication of atypical cytology and equivocal cystoscopy. They analyzed pooled data from 3 prospective Cxbladder clinical trials and 1 real-world clinical study. Physicians were blinded to Cxbladder results, and Cxbladder providers were blinded to clinical results. This trial analyzed diverse urology practices in the U.S., Australia, and New Zealand. A total of 1,784 consecutive, prospectively recruited patients with hematuria or previously diagnosed UC provided 852 samples with both local cytology and Cxbladder results; 153 had atypical cytologies and 14 had both atypical cytology and equivocal cystoscopy. Outcome measures included NPV and proportion of tumors missed for Cxbladder and local cytology, as well as evaluation of Cxbladder for adjudicating atypical cytology and equivocal cystoscopy. Cxbladder ruled out 35 % of patients and NPV 97 % (95 % CI: 94 % to 98 %) compared with 93 % (95 % CI: 91 % to 94 %) for cytology; Cxbladder missed 8.5 % and cytology missed 63 % of tumors. UC was diagnosed in 26/153 cases of atypical cytology (17 %). Cxbladder correctly adjudicated all these patients including those with both atypical cytology and equivocal cystoscopy; these patients had a positive Cxbladder result and were diagnosed with UC by pathology. The incidence of patients with both atypical cytology and equivocal cystoscopy was low. The authors concluded that Cxbladder correctly adjudicated all patients diagnosed with UC among those with atypical cytology and equivocal cystoscopy, and out-performed cytology for accurately identifying patients who did not have UC.
The authors stated that drawbacks of this trial included the fact that cytology interpretation was not centralized, rather a deliberate choice to better reflect real-world clinical conditions. Furthermore, although the proportion of atypical cytologies was within the representative range, there were a limited number of atypical cytologies with equivocal cystoscopies.
Laukhtina et al (2021) conducted a systematic review and network meta-analysis (NMA) on the diagnostic accuracy of novel urinary biomarker tests (UBTs) in non-nuscle-invasive bladder cancer (NMIBC). PubMed, Web of Science, and Scopus were searched up to April 2021 to identify studies addressing the diagnostic values of UBTs: Xpert bladder cancer, Adxbladder, Bladder EpiCheck, Uromonitor and Cxbladder Monitor, and Triage and Detect. The primary endpoint was to assess the pooled diagnostic values for disease recurrence in NMIBC patients using a DTA meta-analysis and to compare them with cytology using an NMA. The secondary endpoints were the diagnostic values for high-grade (HG) recurrence as well as for the initial detection of bladder cancer. Twenty-one studies, comprising 7330 patients, were included in the quantitative synthesis. In most of the studies, there was an unclear risk of bias. For NMIBC surveillance, novel UBTs demonstrated promising pooled diagnostic values with sensitivities up to 93%, specificities up to 84%, positive predictive values up to 67%, and negative predictive value up to 99%. Pooled estimates for the diagnosis of HG recurrence were similar to those for the diagnosis of any-grade recurrence. The analysis of the number of cystoscopies potentially avoided during the follow-up of 1000 patients showed that UBTs might be efficient in reducing the number of avoidable interventions with up to 740 cystoscopies. The NMA revealed that diagnostic values (except specificity) of the novel UBTs were significantly higher than those of cytology for the detection of NMIBC recurrence. There were too little data on UBTs in the primary diagnosis setting to allow a statistical analysis. The authors concluded that their analyses support high diagnostic accuracy of the studied novel UBTs, supporting their utility in the NMIBC surveillance setting. All of these might potentially help prevent unnecessary cystoscopies safely. There are not enough data to reliably assess their use in the primary diagnostic setting. These results have to be confirmed in a larger cohort as well as in head-to-head comparative studies.
Lotan et al (2023) developed enhanced Cxbladder tests that incorporate DNA analysis of 6 single nucleotide polymorphisms for the FGFR3 and TERT genes, in addition to the current 5 mRNA biomarkers and clinical risk factors. Two multicenter, prospective studies were undertaken in: (i) U.S. patients with gross hematuria aged 18 years or older, and (ii) Singaporean patients with gross hematuria or microhematuria who were older than 21 years. All patients provided a midstream urine sample and underwent cystoscopy. Samples were retrospectively analyzed using enhanced Cxbladder-Triage (risk stratifies patients), enhanced Cxbladder-Detect (risk stratifies patients and detects positive patients), and the combination enhanced Cxbladder-Triage × Cxbladder-Detect. The authors found that in the pooled cohort (N=804; gross hematuria: n=484, microhematuria: n=320), enhanced Cxbladder-Detect had a sensitivity of 97% (95% CI 89%-100%), specificity of 90% (95% CI 88%-92%), and negative predictive value of 99.7% (95% CI 99%-100%) for detection of urothelial carcinoma. Overall, 83% of patients were enhanced Cxbladder-Detect-negative (ie, needed no further work-up). Of 133 enhanced Cxbladder-Detect-positive patients, 59 had a confirmed tumor, of which 19 were low-grade noninvasive papillary carcinoma or papillary urothelial neoplasm of low malignant potential. In total, 40 tumors were high-grade Ta, T1-T4, Tis, including concomitant carcinoma in situ. Of the 74 patients with normal cystoscopy, 41 were positive by single nucleotide polymorphism analysis. Enhanced Cxbladder-Triage and enhanced Cxbladder-Detect had significantly better specificity than the first-generation Cxbladder tests (P < .001). The authors acknowledged limitations to their study, such as pooling data from 2 different study, and that results may have been affected by referral bias, as the study relied on referrals from primary care to urology. Data were also subject to potential selection bias due to patient eligibility, particularly among those presenting with microhematuria. Lastly, some of the urine samples and patients included in the current analytical validation study were also used during the development of the Cxb+ tests. Thus, external validation is needed and ongoing, particularly with regard to the choice of test thresholds. The authors concluded that their study in ethnically diverse patients with hematuria showed the analytical validity of the enhanced Cxbladder tests. Furthermore, addition of detection of DNA SNPs from FGFR3 and TERT enhances the performance of Cxbladder tests, and is analytically validated in patients with microhematuria or gross hematuria, providing accurate risk stratification and guidance on which patients require further evaluation by cystoscopy.
Li et al (2023) noted that bladder cancer surveillance is associated with high costs and patient burden. CxMonitor (CxM) allows patients to skip their scheduled surveillance cystoscopy if CxM-negative indicating a low probability of cancer presence. In a prospective, multi-center study, these researchers presented outcomes from of CxM to reduce surveillance frequency during the coronavirus pandemic. Eligible patients due for cystoscopy from March to June 2020 were offered CxM and skipped their scheduled cystoscopy if CxM-negative. CxM-positive patients came for immediate cystoscopy. The primary outcome was safety of CxM-based management, assessed by frequency of skipped cystoscopies and detection of cancer at immediate or next cystoscopy. Patients were surveyed on satisfaction and costs. During the study period, a total of 92 patients received CxM and did not differ in demographics nor history of smoking/radiation between sites; 9 of 24 (37.5 %) CxM-positive patients had 1 T0, 2 Ta, 2 Tis, 2 T2, and 1 Upper tract urothelial carcinoma (UTUC) on immediate cystoscopy and subsequent evaluation; 66 CxM-negative patients skipped cystoscopy, and none had findings on follow-up cystoscopy requiring biopsy – 6 of these patients did not attend follow-up, 4 elected to undergo additional CxM instead of cystoscopy, 2 stopped surveillance, and 2 died of unrelated causes. CxM-negative and CxM-positive patients did not differ in demographics, cancer history, initial tumor grade/stage, AUA risk group, or number of prior recurrences. Median satisfaction (5/5, inter-quartile range [IQR] 4 to 5) and costs (26/33, 78.8 % no out-of-pocket costs) were favorable. The authors concluded that CxM safely reduced frequency of surveillance cystoscopy in real-world settings and appeared acceptable to patients as an at-home test. Moreover, these researchers stated that additional validation and cost-effectiveness analyses are needed.
The authors stated that this study had several drawbacks. First, it primarily reflected a small series (n = 92) that may or may not be generalizable to other settings with a limited follow-up period (next scheduled cystoscopy up to 12 months after scheduled cystoscopy). Thus, these findings only supported skipping 1 cystoscopy with re-surveillance necessary on alternative visits. Second, there was also variation in implementation across sites as repeat CxM instead of follow-up cystoscopy was carried out for some CxM-negative patients at Michigan due to patient preference. Third, some subjects were lost to follow-up due to events such as change of address, missed appointments, or death from co-morbidities. These drawbacks reflected the reality of differences in care between institutions and the complexity of real-world practice. Fourth, this trial had limited enrollment of patients from under-represented groups despite CxM being offered to all eligible patients; this likely reflected existing social inequities in access to bladder cancer care.
Soorojebally et al (2023) noted that bladder cancer detection and follow-up is based on cystoscopy and/or cytology, but it remains imperfect and invasive. Current research focuses on diagnostic biomarkers that could improve bladder cancer detection and follow-up by discriminating patients at risk of aggressive cancer who need confirmatory TURBT (Transurethral Resection of Bladder Tumor) from patients at no risk of aggressive cancer who could be spared from useless explorations. These investigators carried out a systematic review of data on the clinical validity and clinical use of 11 urinary biomarkers (VisioCyt, XpertBladder, BTA stat, BTA TRAK, NMP22 BC, NMP22 BladderChek Test, ImmunoCyt/uCyt1+, UroVysion Bladder Cancer Kit, Cxbladder, ADXBLADDER, Urodiag) for bladder cancer diagnosis and for non-muscle invasive bladder cancer (NMIBC) follow-up. All available studies on the 11 biomarkers published between May 2010 and March 2021 and present in Medline were reviewed. The main endpoints were clinical performance for bladder cancer detection, recurrence or progression during NMIBC monitoring, and additional value compared to cytology and/or cystoscopy. Most studies on urinary biomarkers had a prospective design and high-level of evidence; however, their results should be interpreted with caution given the heterogeneity among studies. Most of the biomarkers under study displayed higher detection sensitivity compared with cytology, but lower specificity. Some biomarkers may have clinical use for NMIBC surveillance in patients with negative or equivocal cystoscopy or negative or atypical urinary cytology findings, and also for recurrence prediction. The authors concluded that urinary biomarkers might have a complementary place in bladder cancer diagnosis and NMIBC surveillance; however, their clinical benefit remains to be confirmed.
Maas et al (2023) stated that urinary biomarkers to detect bladder cancer have been the subject of research for decades. The idea that urine -- being in continuous contact with tumor tissue -- should provide a vector of tumor information remains an attractive concept. Research on this topic has resulted in a complex landscape of many different urine markers with varying degrees of clinical validation. These markers range from cell-based assays to proteins, transcriptomic markers and genomic signatures, with a clear trend towards multiplex assays. Unfortunately, the number of different urine markers and the efforts in research and development of clinical grade assays are not reflected in the use of these markers in clinical practice, which is currently limited. Numerous prospective trials are in progress with the objective of increasing the quality of evidence regarding urinary biomarkers in bladder cancer to achieve guideline implementation. The current research landscape suggests a division of testing approaches. Some efforts are directed towards addressing the limitations of current assays to improve the performance of urinary biomarkers for a straightforward detection of bladder cancer. Furthermore, comprehensive genetic analyses are emerging based on advances in next-generation sequencing (NGS) and are expected to substantially affect the potential application of urinary biomarkers in bladder cancer.
Furthermore, National Comprehensive Cancer Network’s clinical practice guideline on “Bladder Cancer” (Version 1.2024) does not mention Cxbladder/urinary biomarker as a management tool.
CyPath Lung
CyPath Lung (Precision Pathology Services and bioAffinity Technologies Inc.) assay uses flow cytometry to evaluate a self–collected sputum specimen for the level of 5 markers (meso-tetra [4- carboxyphenyl] porphyrin [TCPP], CD206, CD66b, CD3, CD19) indicative of lung cancer. The test includes an algorithmic analysis of the findings and reports the likelihood that the patient has lung cancer.
Lemieux et al (2023) conducted an observational cohort study to identify differential characteristics (using CyPath Lung test) between sputum samples taken from healthy participants, participants at high risk for lung cancer who are free of the disease, and participants with confirmed lung cancer. The healthy cohort was defined as a current non-smoker who has smoked less than 5 pack-years in his or her lifetime, and if smoked, quit more than 15 years ago, and has no known lung disease. High-risk cohort was defined as individuals aged 55 to 74 years who is a current smoker with a smoking history of at least 30 pack-years or current non-smoker who has a smoking history of at least 30 pack-years and quit smoking within the past 15 years. The cancer cohort was defined as individuals who have been diagnosed by a physician as highly suspect for having lung cancer, but has not yet undergone a biopsy nor received therapy, and after providing a sputum sample is confirmed to have lung cancer by biopsy. The study included single cell suspensions prepared from induced sputum samples that were collected over three consecutive days and labeled with a viability dye to exclude dead cells, antibodies to distinguish cell types, and a porphyrin to label cancer-associated cells. The labeled cell suspension was run on a flow cytometer and the data collected. An analysis pipeline combining automated flow cytometry data processing with machine learning was developed to distinguish cancer from non-cancer samples from 150 patients at high risk of whom 28 had lung cancer. Flow data and patient features were evaluated to identify predictors of lung cancer. Random training and test sets were chosen to evaluate predictive variables iteratively until a robust model was identified. The final model was tested on a second, independent group of 32 samples, including six samples from patients diagnosed with lung cancer. The authors stated that the automated analysis combined with machine learning resulted in a predictive model that achieved an area under the ROC curve (AUC) of 0.89 (95% CI 0.83-0.89). The sensitivity and specificity were 82% and 88%, respectively, and the negative and positive predictive values 96% and 61%, respectively. Importantly, the test was 92% sensitive and 87% specific in cases when nodules were < 20 mm (AUC of 0.94; 95% CI 0.89-0.99). Testing of the model on an independent second set of samples showed an AUC of 0.85 (95% CI 0.71-0.98) with an 83% sensitivity, 77% specificity, 95% negative predictive value and 45% positive predictive value. The model is robust to differences in sample processing and disease state. The authors concluded that the CyPath Lung correctly classifies samples as cancer or non-cancer with high accuracy, including from participants at different disease stages and with nodules less than 20 mm in diameter. This test is intended for use after lung cancer screening to improve early-stage lung cancer diagnosis.
The National Comprehensive Cancer Network (NCCN) Biomarkers Compendium and NCCN clinical practice guidelines on “Lung cancer screening” (Version 2.2024) do not include this type of testing method or specimen type for evaluating risk of lung cancer.
DefineMBC
The Epic Sciences circulating tumor DNA (ctDNA) metastatic breast cancer panel, also known as DefineMBC, is a blood-based liquid biopsy that incorporates both cell-based and cell-free analysis to provide tumor histology and comprehensive molecular profiling of biomarkers when tissue biopsy is not available.
DefineMBC is designed for use in patients with breast cancer when metastatic disease is suspected or has previously been confirmed. The test work by analyzing all nucleated cells and ctDNA from a whole blood sample, which is then incorporated into a comprehensive clinical report that includes the following:
- Detection of circulating tumor cells (CTCs). DefineMBC combines immunofluorescence (IF) imaging, proprietary machine learning algorithms, and individual cell retrieval to identify CTCs.
- Assessment of protein expression (HER2, ER) on CTCs
- Determination of ERBB2 copy number alterations (CNA) within individual CTCs
- ctDNA analysis for identification of single nucleotide variants (SNVs), indels, fusions, and CNAs from a targeted 56-gene next-generation sequencing (NGS) panel
- The calculation of microsatellite instability (MSI) and blood tumor mutational burden (bTMB).
The clinical use of ctDNA in metastatic breast cancer is not yet included in the National Comprehensive Cancer Network (NCCN) guidelines for breast cancer for disease assessment and monitoring. Further, NCCN Biomarkers Compendium does not provide a recommendation for evaluating 56 or more genes in patients with invasive breast cancer (NCCN, 2024).
DiviTum TKa Test
DiviTum TKa (Biovica Inc.) is a blood-based biomarker test that monitors and predicts treatment response in hormone receptor-positive metastatic breast cancer. The test measures thymidine kinase activity (TKa) which reflects cell proliferation. The test includes an algorithmic analysis that generates an activity score to help monitor changes in tumor cell proliferation in response to therapy.
Bergqvist and colleagues (2023) state that serum thymidine kinase activity (TKa) levels, an indicator of cell-proliferation, is a potential biomarker for monitoring endocrine therapy (ET) and predicting metastatic breast cancer (MBC) outcome. The authors examined data on progression within 30/60 days post sampling, with a new, FDA approved version of DiviTum TKa highlighting differences versus a Research Use Only version. The evaluation included 1,546 serum samples from 454 patients, collected at baseline and at 4 subsequent timepoints during treatment. A predefined cut-off tested the ability to predict disease progression. A new measuring unit, DuA (DiviTum® unit of Activity) was adopted. The authors found that a DiviTum TKa score less than 250 DuA provides a much lower risk of progression within 30/60 days after blood draw, the negative predictive value (NPV) was 96.7% and 93.5%, respectively. Patients less than 250 DuA experienced significantly longer progression-free survival and overall survival, demonstrated at baseline and for all time intervals. The authors concluded that DiviTum TKa provides clinically meaningful information for patients with HR+ MBC, and that low TKa levels provide such a high NPV for rapid progression that such patients might forego additional therapy added to single agent ET.
The National Comprehensive Cancer Network Clinical Practice Guidelines on “Breast cancer” (version 2.2024) does not discuss the utility of serum thymidine kinase activity for management of MBC.
EarlyTect Bladder Cancer Detection (EarlyTect BCD)
EarlyTect Bladder Cancer Detection (EarlyTect BCD; Promis Diagnostics, Inc.) is a non-invasive urine test that uses methylated PENK DNA detection by linear target enrichment-quantitative methylation-specific real-time PCR (LTE-qMSP) to check the likelihood of bladder cancer.
Bang et al (2024) evaluated the validity of EarlyTect BCD, a streamlined PENK methylation test in urine DNA, for detecting bladder cancer in patients with hematuria. This study was comprised of two clinical studies: a retrospective case-control study as the training set (n = 105) and a prospective study as the validation set (n = 238), which was composed of patients who were scheduled for cystoscopy between April 2020 and December 2023 in the Republic of Korea and the United States. All patients had hematuria before enrolling in the study. Voided urine samples were collected before cystoscopy. In the US cohort, 41 patients were additionally enrolled to collect bladder wash samples during cystoscopy regardless of urine samples. The test integrates two steps, linear target enrichment and quantitative methylation-specific PCR within a single closed tube. The detection limitation of the test was approximately two genome copies of methylated PENK per milliliter of urine. In the retrospective training set (n = 105), an optimal cutoff value was determined to distinguish BC from non-BC, resulting in a sensitivity of 87.3% and a specificity of 95.2%. In the prospective validation set (n = 210, 122 Korean and 88 American patients), the overall sensitivity for detecting all stages of BC was 81.0%, with a specificity of 91.5% and an area under the curve value of 0.889. There was no significant difference between the two groups. The test achieved a sensitivity of 100% in detecting high-grade Ta and higher stages of BC. The negative predictive value of the test was 97.7%, and the positive predictive value was 51.5%. The authors concluded that their findings demonstrate that EarlyTect BCD is a highly effective noninvasive diagnostic tool for identifying BC among patients with hematuria. The authors acknowledged study limitations. The study included a limited number of patients in the US group and incomplete information regarding the stage and grade of patients with BC. Despite these constraints, the study holds substantial value in highlighting the noninvasive diagnostic potential of the EarlyTect BCD, aiding in early BC detection in patients presenting with hematuria, which warrants a well-designed large-scale multicenter prospective study to use the test in practice. The authors state that the study's findings underline the substantial capability of the EarlyTect BCD in accurately identifying bladder cancer in patients with hematuria, potentially lowering the requirement of excessive diagnostic cystoscopy in cases with a negative test result.
Endeavor Comprehensive Genomic Profiling
Endeavor Comprehensive Genomic Profiling (PathGroup) uses the elio tissue complete next-generation sequencing (NGS) assay (Personal Genome Diagnostics) to analyze 505 genes for all therapeutically actionable, solid tumor companion diagnostic biomarkers for relevant variants such as single nucleotide variants (SNVs), insertions and deletions, select translocations, select amplifications, tumor mutational burden (TMB) and microsatellite instability (MSI).
There is insufficient evidence in the published peer-reviewed literature for the Endeavor to support the sensitivity or specificity of this test. For information on the elio tissue complete NGS assay, see "PGDx elio Tissue Complete for Tumor Mutation Profiling".
EXaCT-1
EXaCT-1 (Weill Cornell Medicine Clinical Genomics Laboratory) is a clinical genomic test that examines genomic mutation of all genes in each patient's cancer cells. The test involves HaloPlex (Agilent) PCR target enrichment and next-generation sequencing to identify point mutations, copy-number alterations, and indels (insertion and deletion mutations). In addition to mutations associated with specific treatments, the test identifies “potentially relevant” mutations. The EXaCT-1 test is intended to help select appropriate targeted therapies.
Rennert and colleagues (2016) described the Exome Cancer Test v1.0 (EXaCT-1) whole-exome sequencing (WES)-based test for precision cancer care, which uses HaloPlex (Agilent) target enrichment followed by next-generation sequencing (Illumina) of tumor and matched constitutional control DNA. The authors presented a detailed clinical development and validation pipeline suitable for simultaneous detection of somatic point/indel mutations and copy-number alterations (CNAs). A computational framework for data analysis, reporting and sign-out was also presented. For the validation, the authors tested EXaCT-1 on 57 tumors covering 5 distinct clinically relevant mutations. Results demonstrated elevated and uniform coverage compatible with clinical testing as well as complete concordance in variant quality metrics between formalin-fixed paraffin embedded and fresh-frozen tumors. Extensive sensitivity studies identified limits of detection threshold for point/indel mutations and CNAs. Prospective analysis of 337 cancer cases revealed mutations in clinically relevant genes in 82% of tumors, demonstrating that EXaCT-1 is an accurate and sensitive method for identifying actionable mutations, with reasonable costs and time, greatly expanding its utility for advanced cancer care. However, the authors acknowledged some limitations. "Focused sequencing for most of the targeted NGS panels achieves coverage at 500–1,000×, whereas total coverage for WES assays is only 100× or less. The technology also does not cover each and every exon. A small number of exons, such as those buried in stretches of repeats out towards the chromosome tips, or in duplicated regions are not covered. In our hands, ~1% of the HaloPlex exome is poorly covered with <10 reads per target base, likely due to low mapability.16 Furthermore, approximately one-fifth (~23% on average) of the captured regions in clinically relevant genes did not achieve the required minimum depth of coverage for accurate negative-mutation calls. This means that it is in some cases difficult to completely exclude the presence of low abundance, near-detection threshold mutations in our assay. This limitation is most acute for tumour suppressor genes, where deleterious mutations could potentially occur anywhere along the entire length of the coding region. In other words, it may be difficult to accurately report such genes as not mutated even when no mutated reads are found." Moreover, the authors note that 0.41% of the exons in their analysis had no coverage at all, stating that this is possibly owing to coverage gap affected by the location of restriction enzyme sites used for fragmentation and differences in the designed and actual library insert size. "Finally, despite the increased demand and proven utility of WES, routine whole exome is still associated with many challenges including the data generation and interpretation, and manipulation and storage of the data, increasing the costs of the testing and requiring highly trained health-care professionals as well as special solutions for data management such as cloud storage facilities."
Gene Expression Profiling for Cancer of Unknown Primary
Carcinoma of unknown primary (CUP) is a biopsy-proven metastatic solid tumor with no primary tumor identified and represents approximately 2% to 4% of all cancer cases. The diagnosis of CUP is made following inconclusive results from standard tests (e.g., biopsy, immunochemistry and other blood work, chest x-rays, and occult blood stool test). The absence of a known primary tumor presents challenges to the selection of appropriate treatment strategies. As a result, patients have a poor prognosis, and fewer than 25% survive 1 year from the time of diagnosis. A variety of tissue-biopsy testing techniques currently are used to determine the origin of the CUP, including immunochemistry; histological examination of specimens stained with eosin and hematoxylin, and electron microscopy. These techniques definitively identify the type of carcinoma in less than 20% to 30% of CUP.
Gene expression profiling is a technique used to identify the genetic makeup of a tissue sample by characterizing the patterns of mRNA transcribed, or "expressed", by its DNA. Specific patterns of gene expression, reflected in unique configurations of mRNA, are associated with different tumor types. By comparing the gene expression profile (GEP) of an unknown tumor to the profiles of known primary cancers ("referent" profiles), it may be possible to determine the type of tumor from which the CUP originated.
In July 2008, the FDA cleared for marketing the Pathwork Tissue of Origin test (Pathwork Diagnostics, Sunnyvale, CA), a gene expression profiling test that uses microarray processing to determine the type of cancer cells present in a tumor of unknown origin. The test uses the PathChip (Affymetrix Inc., Santa Clara, C), a custom-designed gene expression array, to measure the expression from 1,668 probe sets to quantify the similarity of tumor specimens to 15 common malignant tumor types, including: bladder, breast, colorectal, gastric, germ cell, hepatocellular, kidney, non-small cell lung, non-Hodgkin's lymphoma, melanoma, ovarian, pancreatic, prostate, soft tissue sarcoma, and thyroid. The degree of correspondence between the tissue sample's GEP and a referent profile is quantified and expressed as a probability-based score.
A multi-center, clinical validation study reported on comparisons of diagnoses based on GEP from 477 banked tissue samples of undifferentiated and poorly differentiated metastases versus standard of care pathology based diagnoses. Comparison of the GEP based diagnoses versus pathology based diagnoses yielded an 89 % agreement and the concurrence was greater than 92 % for 8 out of 15 types of primary tumors. The overall accuracy of the test was approximately 95 % and 98 % for positive and negative determinations, respectively (Monzon et al, 2007).
Gene expression profiling is a promising technology in the management of CUP; however, there is insufficient evidence of its clinical utility compared to that achieved by expert pathologists using current standards of practice. A draft clinical guideline on metastatic malignant disease of unknown origin by the National Institute for Clinical Excellence (NICE, 2010) recommends against using gene expression profiling (e.g., Pathwork TOT, CupPrint, Theros CancerTypeID, miRview Mets) to identify primary tumors in patients with CUP. The guideline explained that currently there is no evidence that gene-expression based profiling improves the management or changes the outcomes for patients with CUP. Guidelines on occult primary from the National Comprehensive Cancer Network (NCCN, 2010) state that, while gene expression profiling looks promising, "prospective clinical trials are necessary to confirm whether this approach can be used in choosing treatment options which would improve the prognosis of patients with occult primary cancers."
An assessment by the Andalusian Agency for Health Technology Assessment (AETSA, 2012) of microRNAs as a diagnostic tool for lung cancer found only two studies assessing the analytical validity of miRview in patients with non-small cell lung cancer. The sensitivity of the miRNA for the detection of carcinoma was between 96% and 100% and the specificity was between 90% and 100%. The area under the ROC curve was close to unity and the positive and negative probability ratios showed a high diagnostic accuracy (9.6 and 0.04, respectively). The assessment stated that, although the quality of the studies was moderate to high, the sensitivity of the diagnostic test may be overestimated as it is a case-control design.
A technology assessment prepared for the Agency for Healthcare Research and Quality (Meleth et al, 2013) found that the clinical accuracy of the PathworkDx, miRview, and CancerTypeID are similar, ranging from 85 percent to 88 percent, and that the evidence that the tests contribute to identifying a tumor of unknown origin was moderate. The assessment concluded that we do not have sufficient evidence to assess the effect of the tests on treatment decisions and outcomes. The assessment noted that most studies of these tests were funded wholly or partially by the manufacturers of these tests, and that the most urgent need in the literature is to have the clinical utility of the tests evaluated by research groups that have no evidence conflict of interest.
Monzon et al (2009) stated that malignancies found in unexpected locations or with poorly differentiated morphologies can pose a significant challenge for tissue of origin determination. Current histologic and imaging techniques fail to yield definitive identification of the tissue of origin in a significant number of cases. The aim of this study was to validate a predefined 1,550-gene expression profile for this purpose. Four institutions processed 547 frozen specimens representing 15 tissues of origin using oligonucleotide microarrays were used in this study. Half of the specimens were metastatic tumors, with the remainder being poorly differentiated and undifferentiated primary cancers chosen to resemble those that present as a clinical challenge. In this blinded multi-center validation study the 1,550-gene expression profile was highly informative in tissue determination. The study found overall sensitivity (positive percent agreement with reference diagnosis) of 87.8 % (95 % CI: 84.7 % to 90.4 %) and overall specificity (negative percent agreement with reference diagnosis) of 99.4 % (95 % CI: 98.3 % to 99.9 %). Performance within the subgroup of metastatic tumors (n = 258) was found to be slightly lower than that of the poorly differentiated and undifferentiated primary tumor subgroup, 84.5 % and 90.7 %, respectively (p = 0.04). Differences between individual laboratories were not statistically significant. The authors concluded that this study represents the first adequately sized, multi-center validation of a gene-expression profile for tissue of origin determination restricted to poorly differentiated and undifferentiated primary cancers and metastatic tumors. These results indicate that this profile should be a valuable addition or alternative to currently available diagnostic methods for the evaluation of uncertain primary cancers.
Monzon and Koen (2010) stated that tumors of uncertain or unknown origin are estimated to constitute 3 % to 5 % of all metastatic cancer cases. Patients with these types of tumors show worse outcomes when compared to patients in which a primary tumor is identified. New molecular tests that identify molecular signatures of a tissue of origin have become available. The authors reviewed the literature on existing molecular approaches to the diagnosis of metastatic tumors of uncertain origin and discuss the current status and future developments in this area. Published peer-reviewed literature, available information from medical organizations (NCCN), and other publicly available information from tissue-of-origin test providers and/or manufacturers were used in this review. The authors concluded that molecular tests for tissue-of-origin determination in metastatic tumors are available and have the potential to significantly impact patient management. However, available validation data indicate that not all tests have shown adequate performance characteristics for clinical use. Pathologists and oncologists should carefully evaluate claims for accuracy and clinical utility for tissue-of-origin tests before using test results in patient management. The personalized medicine revolution includes the use of molecular tools for identification/confirmation of the site of origin for metastatic tumors, and in the future, this strategy might also be used to determine specific therapeutic approaches.
Anderson and Weiss (2010) noted that pathologists use various panels of IHC stains to identify the site of tissue of origin for metastatic tumors, particularly poorly or undifferentiated cancers of unknown or uncertain origin. Although clinicians believe that immunostains contribute greatly to determining the probable primary site among 3 or more possibilities, objective evidence has not been convincingly presented. This meta-analysis reviews the objective evidence supporting this practice and summarizes the performance reported in 5 studies published between 1993 and 2007. A literature search was conducted to identify IHC performance studies published since 1990 that were masked, included more than 3 tissues types, and used more than 50 specimens. The 5 studies found in this search were separated into 2 subgroups for analysis: those, which included only metastatic tumors (n = 368 specimens) and the blended studies, which combined primary tumors and metastases (n = 289 specimens). The meta-analysis found that IHCs provided the correct tissue identification for 82.3 % (95 % CI: 77.4 % to 86.3 %) of the blended primary and metastatic samples and 65.6 % (95 % CI: 60.1 % to 70.7 %) of metastatic cancers. This difference is both clinically and statistically significant. The authors concluded that this literature review confirms that there is still an unmet medical need in identification of the primary site of metastatic tumors. It establishes minimum performance requirements for any new diagnostic test intended to aid the pathologist and oncologist in tissue of origin determination.
GeneSearch BLN
The presence of breast tumor cells in axillary lymph nodes is a key prognostic indicator in breast cancer. During surgery to remove breast tumors, patients often undergo biopsy of the sentinel (i.e., first) node(s) that receive lymphatic fluid from the breast. Excised sentinel lymph nodes are currently evaluated post-operatively by formalin-fixed paraffin-embedded Hematoxylin and Eosin (H&E) histology and IHC. GeneSearch™ Breast Lymph Node (BLN) assay (Veridex, LLC, Warren, NJ) is a novel method to examine the extracted sentinel lymph nodes for metastases and can provide information during surgery within 30 to 40 minutes from the time the sentinel node is removed, potentially avoiding a second operation for some patients. The GeneSearch BLN assay received FDA pre-market approval on July 16, 2007 as a qualitative in vitro diagnostic test for the rapid detection of metastases larger than 0.2 mm in nodal tissue removed from sentinel lymph node biopsies of breast cancer patients. GeneSearch BLN assay uses real time reverse transcriptase polymerase chain reaction (RT-PCR) to detect the gene expression markers, mammaglobin (MG) and cytokeratin 10 (CI19), which are abundant in breast tissue but scarce in lymph node cells. In the clinical trial conducted by Veridex, which was submitted to the FDA, the sensitivity of the GeneSearch BLN Assay was reported to be 87.6 % and the specificity was 94.2 % (Julian et al, 2008). According to the product labeling, "The GeneSearch™ Breast Lymph Node (BLN) assay may be used in conjunction with sentinel lymph node biopsy for a patient who has been counseled on use of this test and has been informed of its performance. False positive results may be associated with increased morbidity. False negative and inconclusive test results may be associated with delayed axillary node dissection. Clinical studies so far are inconclusive about a benefit from treatment based on findings of breast cancer micro-metastases in sentinel lymph nodes."
Blumencranz et al (2007) compared the GeneSearch BLN assay with results from conventional histologic evaluation from 416 patients at 11 clinical sites and reported that the GeneSearch BLN assay detected 98 % of metastases greater than 2 mm in size and 57 % of metastasis less than 0.2 mm. False positives were reported in 4 % of the cases. However, there were several limitations of this study, including the lack of a description of patient recruitment, inadequate descriptions of several analyses performed, substantial variations in test performance across sites, and ad hoc comparison of the assay to other intra-operative techniques.
Viale et al (2008) analyzed 293 lymph nodes from 293 patients utilizing the GeneSearch BLN assay. Using histopathology as the reference standard, the authors reported that the BLN assay correctly identified 51 of 52 macro-metastatic and 5 of 20 micro-metastatic sentinel lymph nodes (SLNs), with a sensitivity of 98.1 % to detect metastases larger than 2 mm, 94.7 % for metastases larger than 1 mm, and 77.8 % for metastases larger than 0.2 mm. The overall concordance with histopathology was 90.8 %, with a specificity of 95.0 %, a positive predictive value of 83.6 %, and a negative predictive value of 92.9 %. When the results were evaluated according to the occurrence of additional metastases to non-SLN in patients with histologically positive SLNs, the assay was positive in 33 (91.7 %) of the 36 patients with additional metastases and in 22 (66.6 %) of the 33 patients without further echelon involvement. The authors concluded that the sensitivity of the GeneSearch BLN assay is comparable to that of the histopathologic examination of the entire SLN by serial sectioning at 1.5 to 2 mm.
Although treatment for metastases larger than 2.0 mm is widely accepted as beneficial, clinical studies have not yet provided data for a consensus on benefit from treatment based on very small breast cancer metastases (between 0.2 mm and 2.0 mm) in SLNs. False positive results may be associated with increased morbidity, usually due to effects of axillary node dissection surgery. Patients who undergo axillary lymph node dissection (ALND) have significantly higher rates of increased swelling in the upper arm and forearm (lymphedema), pain, numbness, and motion restriction about the shoulder when compared with patients who undergo only sentinel lymph node dissection (SLND). False negative and inconclusive test results may be associated with delayed axillary node dissection. Clinical studies so far are inconclusive regarding a benefit from treatment based on findings of breast cancer micro-metastases in SLNs. Preliminary data suggest that the GeneSearch BLN assay has high specificity and moderate sensitivity when only macro-metastases are included in the analysis. The clinical significance of micro-metastases is still being debated in the literature, thus, the failure of the GeneSearch BLN assay to perform adequately in the detection of micro-metastases is of unknown significance.
A systematic evidence review by the BlueCross BlueShield Association Technology Evaluation Center (BCBSA, 2007) determined that the use of the GeneSearch BLN assay to detect sentinel node metastases in early stage breast cancer does not meet the TEC criteria. The assessment stated, "There are several operational issues that add difficulty to the use of the GeneSearch assay, including the need for fresh specimens (rather than putting them in formalin for permanent fixation), the learning curve involved in reducing both the percentage of invalid results (from about 15% initially to 4 - 8% for more experienced technicians) and the time to perform the test compared to alternative intra-operative techniques (which take less than 15 minutes)." Furthermore, the assessment stated "The GeneSearch assay also provides less information for staging than other intra-operative procedures, since it cannot distinguish between micro- and macro-metases. Nor can it indicate the location of the metastasis (inside or outside the node). Post-operative histology is therefore required in all cases. It is less crucial when frozen section histology is performed, since pathologists can judge the size of the metastasis and its location from this test, although distortion is possible. To summarize, the data available is inadequate to assess the clinical utility of the GeneSearch assay compared to either post-operative histology alone or to the alternative intra-operative tests such as imprint cytology and frozen section histology. In addition, the balance of benefits versus harms may require higher specificity to avoid unnecessary ALNDs and their sequelae, whereas the GeneSearch design emphasizes sensitivity."
A report by Adelaide Health Technology Assessment stated that, if the GeneSearch BLN Assay is to play a role in reducing the mortality of breast cancer patients, it will be through more accurate diagnosis of breast cancer metastasis during SNB (Ellery, et al., 2010). The report noted, however, that, as yet there are no data to indicate whether SNB itself lowers the mortality rate among breast cancer patients. Hence, it is unclear whether the GeneSearch BLN Assay would have any indirect effect on breast cancer mortality until further investigation into SNB concludes.
- determine the assay turn-around-time from the time of node removal to the report of the assay result to the surgeon and
- determine whether the assay result was or was not received in time to make an intra-operative decision and
- collect data in relation to other surgical procedures during the sentinel lymph node dissection/breast surgery to determine if the assay turn-around-time resulted in longer surgery time.
Guardant Reveal
Guardant Reveal is a blood-based liquid biopsy test that detects circulating tumor DNA (ctDNA) for minimal residual disease (MRD) assessment in early-stage colorectal, breast, and lung cancers. In addition to detection of MRD, Guardant Reveal is marketed to monitor recurrence in previously diagnosed patients.
Hashimoto et al (2023) state that the presence of ctDNA in plasma after surgery may signify the presence of MRD in various cancers. Thus, the authors launched a study protocol for a multi-institutional prospective observational study of ctDNA for MRD detection in conjunction with a randomized, controlled phase III trial (JCOG1801) evaluating the efficacy of preoperative chemoradiotherapy (pre-CRT) compared with up-front surgery for locally recurrent rectal cancer (LRRC). Blood samples will be obtained according to a pre-established protocol schedule in JCOG1801, thus ensuring reliable results for clinical application. The analysis of plasma samples will be performed using Guardant Reveal. The authors state that this study has the potential to offer insights into the utility of ctDNA as a prognostic factor or to predict treatment response in LRRC patients. Limitations of this study include its observational design and lack of robust statistical power due to the relatively small sample size.
Slater et al (2023) state that ctDNA to detect MRD is emerging as a biomarker to predict recurrence in patients with curatively treated early stage colorectal cancer (CRC). ctDNA risk stratifies patients to guide adjuvant treatment decisions. Thus, the authors are conducting the UK’s first multicenter, prospective, randomized study to determine whether a de-escalation strategy using ctDNA to guide adjuvant chemotherapy (ACT) decisions is non-inferior to standard of care (SOC) chemotherapy, as measured by 3-year disease free survival (DFS) in patients with resected CRC with no evidence of MRD (ctDNA negative post-operatively). In doing so the authors state that they may be able to spare patients unnecessary chemotherapy and associated toxicity and achieve significant cost savings for the National Health Service (NHS). The study opened with the Guardant Reveal plasma-only ctDNA assay in August 2022.
The National Comprehensive Cancer Network Biomarkers Compendium (NCCN, 2024) does not provide a recommendation for ctDNA for MRD testing in colorector, breast or lung cancer.
Guardant360
The Guardant360 panel analyzes cell-free circulating tumor DNA (liquid biopsy) for 73 genes associated with a wide variety of solid tumors.
Noting that data on the influence of hybrid capture (HC)-based NGS on treatment are limited, Rozenblum, et al. (2017) investigated its impact on treatment decisions and clinical outcomes in a series of patients at a cancer center. This retrospective study included patients with advanced lung cancer on whom HC-based NGS with broad gene panels was performed between November 2011 and October 2015. HC-based NGS was performed upon the recommendation of the treating physician, mostly on the basis of young age and smoking history. The results of standard molecular testing for EGFR mutations and ALK rearrangements were negative before HC-based NGS in 80.2% (81 of 101) and 70.3% (71 of 101) of the patients, respectively. Upfront HC-based NGS was performed on 15 patients because of very little biopsy material. HC-based NGS was performed off-site on tumor samples with FoundationOne (Foundation Medicine, Inc., Cambridge, MA) (n = 82) or on blood samples using a liquid biopsy approach with Guardant360 if the tissue sample had been exhausted (n= 18). The study focused on gene analyses (GAs) with potential clinical relevance. Initial analysis (level 1) included GAs associated with U.S. Food and Drug Administration–approved anticancer therapies (including off-label drugs) for all cancer types. A subsequent analysis (level 2) included GAs with appropriate evidence-based targeted agents with antidriver activity in lung cancer, as recommended by the National Comprehensive Cancer Network (NCCN) guidelines for NSCLC. GAs associated with investigational treatments were not included in the current analysis. Demographic and clinicopathologic characteristics, treatments, and outcome data were collected. A total of 101 patients were included (median age 63 years [53% females, 45% never-smokers, and 85% with adenocarcinoma]). HC-based NGS was performed upfront and after EGFR/ALK testing yielded negative or inconclusive results in 15% and 85% of patients, respectively. In 51.5% of patients, HC-based NGS was performed before first-line therapy, and in 48.5%, it was performed after treatment failure. HC-based NGS identified clinically actionable genomic alterations in 50% of patients, most frequently in EGFR (18%), Ret proto-oncogene (RET) (9%), ALK (8%), Mesenchymal-epithelial transition factor (MET) receptor tyrosine kinase gene (6%), and erb-b2 receptor tyrosine kinase 2 gene (ERBB2) (5%). In 15 patients, it identified EGFR/ALK aberrations after negative results of prior standard testing. Treatment strategy was changed for 43 patients (42.6%). The overall response rate in these patients was 65% (complete response 14.7%, partial response 50%). Median survival was not reached. Immunotherapy was administered in 33 patients, mostly without an actionable driver, with a presenting disease control rate of 32%, and with an association with tumor mutation burden. The authors noted a number of limitations of this study, including its retrospective nature, its small sample size, and its being a single-center study. In addition, the high percentage of never-smokers, the preponderance of female patients, and the relatively young median age of the patient group represented a selection bias with a high pretest probability for the existence of driver mutation. The authors noted that the results of large prospective trials such as the UK National Lung Matrix Trial and the National Cancer Institute's Molecular Analysis for Therapy Choice Program are thus eagerly anticipated.
Kim et al (2017) reported on an interim analysis of an open-label prospective, clinical trial of ctDNA in patients with metastatic NSCLC, gastric cancer (GC), and other cancers. The investigators reported that somatic alterations were detected in 59 patients with GC (78%), and 25 patients (33%) had targetable alterations (ERBB2, n = 11;MET, n = 5; FGFR2, n = 3; PIK3CA, n = 6). In NSCLC, 62 patients (85%) had somatic alterations, and 34 (47%) had targetable alterations (EGFR, n = 29; ALK, n = 2; RET, n = 1; ERBB2, n = 2). A subgroup of subjects (10 with GC and 17 with NSCLC) who had tissue for confirmation of ctDNA findings were treated with targeted therapy. The investigators reported that response rate and disease control rate were 67% and 100%, respectively, in GC and 87% and 100%, respectively, in NSCLC. The authors noted that this is the first prospective study to examine the clinical utility of comprehensive ctDNA genomic testing to guide matched therapy selection. The authors stated that, because this study was not randomized, its primary limitation is the potential for selection bias to enroll patients more likely to benefit. In addition, the cohort is heterogeneous, including patients at varying lines of therapy and with various concomitant treatments, which limits conclusions in this interim analysis. Not all patients with targetable alterations could receive matched therapy because of the various requirements of the multiple parallel matched therapy substudy protocols, performance status, or loss to follow-up. The authors stated that the final analysis will help to address the modest sample size of this interim analysis as well as report on progression-free survival. The authors stated that future studies should examine ctDNAguided matched therapy outcomes in more racially diverse cohorts.
Noting that there is a paucity of data on the concordance between plasma cell-free circulating tumor DNA (ctDNA) and tissue-based genomic testing, Villaflor, et al. (2016) reported on a descriptive study of subjects with NSCLC undergoing analysis of ctDNA using Guardant360 next-generation sequencing assay at a single institution. The authors stated that this study is the first clinic-based series of NSCLC patients assessing outcomes of targeted therapies using a commercially available ctDNA assay. Of the 90 patients submitted for ctDNA analysis as part of clinical care, 68 had provided informed consent for inclusion in this study. Thirty-eight samples from the 68 subjects were tested using the 54-gene ctDNA panel while the remaining 31 samples were analyzed on the 68-gene ctDNA panel. Of note, the 54-gene panel did not include ALK, RET or ROS1 fusions. Tissue-based testing was performed on 44 subjects using 9 different testing platforms. Demographic, clinicopathologic information and results from tissue and plasma-based genomic testing were reviewed for each subject. The majority of patients had a diagnosis of lung adenocarcinoma (n = 55, 81%), with the remainder lung squamous cell carcinoma (n = 12, 17.7%) and other lung cancers (n = 1, 1.3%). Over 80% of patients had detectable ctDNA. Thirty-one patients had matched tissue and blood samples; the reason for lack of tissue results for the remaining 37 patients was not routinely documented. In cases with detectable ctDNA and completed tissue analysis, an EGFR activating was found in both tissue and blood in 5 paired samples, and in tissue only in 2 samples (71% concordance). The time between biopsy and blood draw ranged from 0 days to 7 years, with an average of 8.8 months and median of 1.4 years between biopsy and blood draw. The investigators found no correlation between concordance and timing of blood draw versus tissue biopsy. A total of 9 subjects with paired tissue and blood samples had an EGFR driver mutation identified in plasma and tissue (n = 5), plasma only (n = 1) or tissue only (n = 3). Eight of these individuals were treated with erlotinib or afatinib at first or second line. Two patients were still responding to therapy at the time of data analysis. Of the 6 remaining patients, the median progression-free survival was 11.5 months (range 7.5 months–29 months; 95% CI–5.7–28.7). The investigators stated that these data suggest that biopsy-free ctDNA analysis is a viable first choice when the diagnostic tissue biopsy is insufficient for genotyping or at the time of progression when a repeated invasive tissue biopsy is not possible/preferred. The authors noted however, that the numbers in this series are modest and further research in larger prospective cohorts is needed.
Thompson et al (2016) evaluated the feasibility of using cell-free circulating tumor DNA (ctDNA) NGS as a complement or alternative to tissue NGS in a single-center observational study. A total of 112 plasma samples obtained from a consecutive study of 102 prospectively enrolled patients with advanced NSCLC were subjected to ultra-deep sequencing of 68 or 70 genes and matched with tissue samples, when possible. The investigators detected 275 alterations in 45 genes, and at least one alteration in the ctDNA for 86 of 102 patients (84%), with EGFR variants being most common. ctDNA NGS detected 50 driver and 12 resistance mutations, and mutations in 22 additional genes for which experimental therapies, including clinical trials, are available. Although ctDNA NGS was completed for 102 consecutive patients, tissue sequencing was only successful for 50 patients (49%). The overall concordance for all variants covered and detected by both platforms was 60%. When wild-type calls, that is, genes for which no variants were detected, are considered, the overall concordance was 97.5%. Actionable EGFR mutations were detected in 24 tissue and 19 ctDNA samples, yielding concordance of 79%, with a shorter time interval between tissue and blood collection associated with increased concordance (P = 0.038). ctDNA sequencing identified eight patients harboring a resistance mutation who developed progressive disease while on targeted therapy, and for whom tissue sequencing was not possible.
Schwaederle et al (2016) extracted plasma from 171 patients with a variety of cancers and analyzed the plasma for ctDNA (54 genes and copy number variants (CNVs) in three genes (EGFR, ERBB2 and MET)). The most represented cancers were lung (23%), breast (23%), and glioblastoma (19%). Ninety-nine patients (58%) had at least one detectable alteration, where actionability was defined as an alteration that was either the direct target or a pathway component that could be targeted by at least one FDA approved or investigational drug in a clinical trial. The most frequent alterations were TP53 (29.8%), followed by EGFR (17.5%), MET (10.5%), PIK3CA (7%), and NOTCH1 (5.8%). In contrast, of 222 healthy volunteers, only one had an aberration (TP53). Ninety patients with non-brain tumors had a discernible aberration (65% of 138 patients; in 70% of non-brain tumor patients with an alteration, the anomaly was potentially actionable). Nine of 33 patients (27%) with glioblastoma had an alteration (6/33 (18%) potentially actionable). Overall, sixty-nine patients had potentially actionable alterations (40% of total; 69.7% of patients (69/99) with alterations); 68 patients (40% of total; 69% of patients with alterations), by an FDA-approved drug. In summary, 65% of diverse cancers (as well as 27% of glioblastomas) had detectable ctDNA aberration(s), with the majority theoretically actionable by an approved agent. The authors noted a number of study limitations. First, this study included a limited number of patients in each histology. Second, clinical annotation was not available since the database was de-identified. Third, the definition of "actionable" and the level of evidence needed for such a determination is a matter of debate and in constant evolution. Fourth, the use of tissue-based next generation sequencing as a comparison to establish clinical utility was not accessible for this group of de-identified patients. Finally, whether or not the patients would have responded to these drugs could not be addressed in this study, and will require further investigation.
Liang et al (2016) performed a retrospective chart review of 100 patients with stage 4 or high-risk stage 3 breast cancer. Of the 100 patients included in this study, 29 had a tissue analysis done during the course of treatment. Only the specific genomic alterations tested in both the cell-free DNA (cfDNA) and tissue DNA were included in this analysis. Of the 29 patients with tissue analysis, 6 had no evidence of disease at the time of cfDNA analysis and were excluded from the comparative analysis of genomic alterations found between cfDNA and tissue DNA. A total of 55 single nucleotide variants (SNVs) and 4 copy number variants (CNVs) were evaluated for both cfDNA and tissue DNA from the 23 remaining patients. The degree of agreement between genomic alterations found in tumor DNA (tDNA) and cfDNA was determined by Cohen's Kappa. Clinical disease progression was compared to mutant allele frequency using a 2-sided Fisher's exact test. The presence of mutations and mutant allele frequency was correlated with PFS using a Cox proportional hazards model and a log-rank test. The most commonly found genomic alterations were mutations in TP53 and PIK3CA, and amplification of EGFR and ERBB2. PIK3CA mutation and ERBB2 amplification demonstrated robust agreement between tDNA and cfDNA (Cohen's kappa = 0.64 and 0.77, respectively). TP53 mutation and EGFR amplification demonstrated poor agreement between tDNA and cfDNA (Cohen's kappa = 0.18 and 0.33, respectively). The directional changes of TP53 and PIK3CA mutant allele frequency were closely associated with response to therapy (p = 0.002). The investigators stated that the presence of TP53 mutation (p = 0.0004) and PIK3CA mutant allele frequency [p = 0.01, HR 1.074 (95 % CI: 1.018 to 1.134)] was excellent predictors of PFS. The authors concluded that identification of selected cancer-specific genomic alterations from cfDNA may be a non-invasive way to monitor disease progression, predict PFS, and offer targeted therapy. They noted that this study was limited by its small sample size and the inherent nature of retrospective data collection of existing genomic information.
Aggarwal et al (2019) noted that the clinical implications of adding plasma-based circulating tumor DNA next-generation sequencing (NGS) to tissue NGS for targetable mutation detection in non-small cell lung cancer (NSCLC) have not been formally assessed. In a prospective, cohort study, these researchers examined if plasma NGS testing was associated with improved mutation detection and enhanced delivery of personalized therapy in a real-world clinical setting. This trial enrolled 323 patients with metastatic NSCLC who had plasma testing ordered as part of routine clinical management. Plasma NGS was performed using a 73-gene commercial platform. Patients were enrolled at the Hospital of the University of Pennsylvania from April 1, 2016, through January 2, 2018. The database was locked for follow-up and analyses on January 2, 2018, with a median follow-up of 7 months (range of 1 to 21 months). The number of patients with targetable alterations detected with plasma and tissue NGS; the association between the allele fractions (AFs) of mutations detected in tissue and plasma; and the association of response rate with the plasma AF of the targeted mutations. Among the 323 patients with NSCLC (60.1 % women; median age of 65 years [range of 33 to 93]), therapeutically targetable mutations were detected in EGFR, ALK, MET, BRCA1, ROS1, RET, ERBB2, or BRAF for 113 (35.0 %) overall; 94 patients (29.1 %) had plasma testing only at the discretion of the treating physician or patient preference. Among the 94 patients with plasma testing alone, 31 (33.0 %) had a therapeutically targetable mutation detected, thus obviating the need for an invasive biopsy. Among the remaining 229 patients who had concurrent plasma and tissue NGS or were unable to have tissue NGS, a therapeutically targetable mutation was detected in tissue alone for 47 patients (20.5 %), whereas the addition of plasma testing increased this number to 82 (35.8 %); 36 of 42 patients (85.7 %) who received a targeted therapy based on the plasma result achieved a complete response (CR) or a partial response (PR) or stable disease (SD). The plasma-based targeted mutation AF had no correlation with depth of Response Evaluation Criteria in Solid Tumors (RECIST) response (r = -0.121; p = 0.45). The authors concluded that given the ease of obtaining plasma-based genotyping and the success observed with such a non-invasive approach, these findings argued for incorporation of plasma-based genotyping into routine clinical management of patients with NSCLC.
The authors stated that this study had several drawbacks. This single-center study was conducted among physicians who were comfortable ordering and interpreting plasma NGS tests. This user bias probably enriched for patients who had plasma NGS only and were likely to have targetable mutations. A sizeable proportion of patients underwent testing after progression to detect resistance mutations, which likely increased the frequency of patients with EGFR T790M. Moreover, this study only considered plasma NGS testing at a single point. The clinical utility of longitudinal plasma NGS-based monitoring is an area of active study in this group.
In an editorial that accompanied the afore-mentioned study by Aggarwal et al (2019), Gyawali and West (2018) noted that "Putting aside the question of whether and when NGS is appropriate, what does the study by Aggarwal and colleagues demonstrate for the role of plasma vs tissue NGS? We cannot conclude from this work that plasma testing should obviate the need for tissue NGS in most patients, since 29 % of the patients with a therapeutically targetable mutation and who had undergone NGS testing from both plasma and tissue had the mutation detected in tissue only. But the study compellingly demonstrates that plasma NGS can obviate the need for tissue NGS in patients in whom plasma testing demonstrates a mutation, given the response and disease control rate among patients who had therapeutically targetable mutations identified from plasma. The relatively high rate of molecular marker detection from plasma also offers a strong option for patients for whom tissue is not available and challenging to obtain. These results, combined with the patient satisfaction with the relative ease of providing blood rather than a solid tissue sample, suggest a clinical strategy of pursuing plasma NGS first, then tissue NGS if plasma NGS cannot detect relevant mutations. Another driver of plasma NGS is the cost-effectiveness of liquid biopsy over tissue biopsy, as suggested in the Statement Paper by the International Association for the Study of Lung Cancer5; however, data to support this claim are still lacking. In the context of initial workup of advanced non-squamous NSCLC, for which many therapeutically targetable mutations are potentially present, broader clinical use of NGS from one source or another seems reasonable, based on cost and time and tissue efficiency. However, this may not hold true in other contexts in which the relevant targets are very limited, such asT790Min acquired resistance on an earlier-generation EGFR TKI, or are extremely infrequent and/or are not clinically relevant, as in squamous NSCLC, acquired resistance to various other driver mutations, and many other cancer settings. In summary, mounting data now support a role for plasma NGS as a helpful tool to supplement or even obviate the need for often scarce and difficult-to-obtain tissue for NGS testing, but this should not circumvent the central question of whether NGS testing will improve clinical outcomes and thus whether it should be performed at all. Next-generation sequencing should not be presumed to be the right tool for every job … A shotgun approach may be appropriate if there is a sufficient chance of hitting a target suspected to be there, but we do not know exactly where; however, there are more accurate and precise weapons if we have a better idea where the true target is. If not, and if there is little reason to expect the existence of a real target, merely having a readily available shotgun should not lead us to shoot blindly in the dark without acknowledging that we may do unexpected damage".
Odegaard et al (2018) stated that liquid biopsies are powerful tools that enable non-invasive genotyping of advanced solid tumors; however, comprehensive, structured validation studies employing validated orthogonal comparator methods are lacking. These researchers analytically and clinically validated a circulating cell-free tumor DNA sequencing test for comprehensive tumor genotyping and demonstrated its clinical feasibility. Analytical validation was conducted according to established principles and guidelines. Blood-to-blood clinical validation comprised blinded external comparison to clinical digital droplet PCR across 222 consecutive biomarker-positive clinical samples. Blood-to-tissue clinical validation comprised comparison of Digital Sequencing calls to those documented in the medical record of 543 consecutive lung cancer patients. Clinical experience was reported from 10,593 consecutive clinical samples. Digital sequencing technology enabled variant detection down to 0.02 % to 0.04 % allelic fraction/2.12 copies with less than or equal to 0.3 %/2.24 to 2.76 copies 95 % limits of detection while maintaining high specificity (prevalence-adjusted positive predictive value (PPV) greater than 98 %). Clinical validation using orthogonal plasma- and tissue-based clinical genotyping across more than 750 patients demonstrated high accuracy and specificity (positive percent agreement (PPAs) and negative percent agreement (NPAs) greater than 99 % and PPVs 92 to 100 %). Clinical use in 10,593 advanced adult solid tumor patients demonstrated high feasibility (greater than 99.6 % technical success rate) and clinical sensitivity (85.9 %), with high potential actionability (16.7 % with FDA-approved on-label treatment options; 72.0 % with treatment or trial recommendations), particularly in non-small cell lung cancer where 34.5 % of patient samples comprised a directly targetable standard-of-care biomarker. The authors concluded that high concordance with orthogonal clinical plasma- and tissue-based genotyping methods supported the clinical accuracy of digital sequencing across all 4 types of targetable genomic alterations. Digital sequencing’s clinical applicability is further supported by high rates of technical success and biomarker target discovery.
McCoach et al (2018) stated that patients with advanced NSCLC whose tumors harbor anaplastic lymphoma kinase (ALK) gene fusions benefit from treatment with ALK inhibitors (ALKi). Analysis of cell-free circulating tumor DNA (cfDNA) may provide a non-invasive way to identify ALK fusions and actionable resistance mechanisms without an invasive biopsy. The Guardant360 (G360; Guardant Health) de-identified database of NSCLC cases was queried to identify 88 consecutive patients with 96 plasma-detected ALK fusions. G360 is a clinical cfDNA NGS test that detects point mutations, select copy number gains, fusions, insertions, and deletions in plasma. Identified fusion partners included EML4 (85.4 %), STRN (6 %), and KCNQ, KLC1, KIF5B, PPM1B, and TGF (totaling 8.3 %); 42 ALK-positive patients had no history of targeted therapy (cohort 1), with tissue ALK molecular testing attempted in 21 (5 negative, 5 positive, and 11 tissue insufficient). Follow-up of 3 of the 5 tissue-negative patients showed responses to ALKi; 31 patients were tested at known or presumed ALKi progression (cohort 2); 16 samples (53% ) contained 1 to 3 ALK resistance mutations. In 13 patients, clinical status was unknown (cohort 3), and no resistance mutations or bypass pathways were identified. In 6 patients with known EGFR-activating mutations, an ALK fusion was identified on progression (cohort 4; 4 STRN, 1 EML4; 1 both STRN and EML4); 5 harbored EGFR T790M. The authors concluded that in this cohort of cfDNA-detected ALK fusions, these researchers demonstrated that comprehensive cfDNA NGS provided a non-invasive means of detecting targetable alterations and characterizing resistance mechanisms on progression.
The authors stated that this study had several drawbacks. First, this was a retrospective analysis reliant on clinical information provided on sample submission. Thus, complete treatment history and clinical follow-up was not available (and cannot be verified) for all patients. This included patient demographic information, type and length of prior therapies, local tissue testing modality, and prior molecular testing results both at diagnosis and progression re-biopsy. Further, there were limitations to the cfDNA platform including the identification of multiple sub-clonal populations, which may not be clinically relevant to resistance. Additionally, given G360 is a clinical cfDNA assay, only ALK fusion events that occurred with partners with known biologic significance were reported. Finally, in this study these researchers identified 6 patients in cohort 2 whose ALK fusion were not identified by cfDNA, instead they were identified by the presence of the ALK resistance mutation. This reflected the complexity of fusion proteins and the fact that ALK has numerous fusion variants that may hinder identification by small fragment cfDNA analyses. Additionally, these investigators were unable to estimate the true false negative rate of cfDNA in detecting ALK fusions given the database search parameters.
Laufer-Geva et al (2018) stated that NGS of cfDNA enables non-invasive genomic analysis of NSCLC patients. Although plasma-detected genomic alterations (GAs) have been shown to predict targeted therapy response, evidence of durability of response is lacking or limited to small cohorts as is the impact of cfDNA NGS results on clinical decisions. This retrospective study of stage IIIB/IV NSCLC patients between the years 2014 and 2017 in Israel used cfDNA NGS (Guardant360) to identify targetable GAs. These researchers consecutively tested 116 NSCLC patients, 41.4 % before 1st-line therapy (group A), 34.5 % upon progression on chemotherapy or immunotherapy (group B1), and 24.1 % upon progression on EGFR tyrosine kinase inhibitors (group B2). Targetable GAs were found in 31 % of group A (15 of 48 patients), 32.5 % in group B1 (13 of 40 patients) and 71 % in group B2 (20 of 28 patients). Treatment decision was changed to targeted therapy in 23 % (11 of 48 patients), 25 % (10 of 40 patients) and 32 % (9 of 28 patients), respectively (total cohort 26 %; 30/116). Objective response rate (Response Evaluation Criteria in Solid Tumors [RECIST]) was 43 % (12 of 28 patients) including 1 CR, PR in 39 % (11 of 28 patients), SD in 32 % (9 of 28 patients), and progressive disease in 25 % (7 of 28 patients). Disease control rate was 75 % for 5 months median treatment duration. The authors concluded that comprehensive cfDNA testing impacted clinical decisions in 1/4 to 1/3 of initial and subsequent lines of treatment in advanced NSCLC patients. This retrospective study extended previous reports by showing that responses based on cfDNA were durable and change treatment decisions at initial presentation and at progression.
The authors stated that limitations of this study included its retrospective nature, although the response and disease control rate (DCR) were consistent with 2 previous prospective studies. Also, more than 50 % of the at-progression patients were in the 3rd-line of treatment or higher, where response rates would be expected to be lower than published studies of 2nd-line targeted therapies. Response rate, survival, and duration of treatment in this study population, which was enriched for plasma-positive patients with limited or failed tumor tissue genotyping, may introduce theoretical selection bias as cfDNA may be more likely to be positive in patients with aggressively growing metastases whereas single lesion biopsy-based genotyping is indifferent to whether disease is indolent or aggressive. If true, however, then the plasma-based treatment results become more, rather than less, compelling. Nonetheless, the clinical outcomes reported in this study reflected the real-life impact. Because patients were not randomized, and cfDNA testing ordered on those with a higher pre-test probability of mutation (female, non-smoking, etc.) the prevalence of GAs here may be higher than a cohort enrolled by randomization.
Lam and associates (2019) stated that major guidelines do not recommend routine molecular profiling of lung squamous-cell carcinoma (LUSC) because the prevalence of actionable alterations is thought to be low. Increased utilization of next-generation sequencing (NGS), particularly with cfDNA, facilitates re-evaluation of this premise. These investigators retrospectively evaluated the prevalence of actionable alterations in 2 distinct LUSC cohorts totaling 492 patients. A total of 410 consecutive patients with stage 3B or 4 LUSC were tested with a targeted cfDNA NGS assay, and 82 patients with LUSC of any stage were tested with a tissue NGS cancer panel. In the overall cohort, 467 patients (94.9 %) had a diagnosis of LUSC, and 25 patients (5.1 %) had mixed histology with a squamous component. A total of 10.5 % of the LUSC subgroup had somatic alterations with therapeutic relevance, including in EGFR (2.8 %), ALK/ROS1 (1.3 %), BRAF (1.5 %), and MET amplification or exon 14 skipping (5.1 %); 16 % of patients with mixed histology had an actionable alteration. In the LUSC subgroup, 3 evaluable patients were treated with targeted therapy for an actionable alteration; all of them experienced partial response. The authors concluded that in this large, real-world LUSC cohort, they observed a clinically significant prevalence of actionable alterations. These researchers stated that further evaluation of the genomic landscape in this setting is needed to potentially identify under-appreciated treatment options.
Leighl and colleagues (2019) stated that complete and timely tissue genotyping is challenging, leading to significant numbers of patients with newly diagnosed metastatic NSCLC (mNSCLC) being under-genotyped for all 8 genomic biomarkers recommended by professional guidelines. These researchers attempted to demonstrate non-inferiority of comprehensive cfDNA relative to physician discretion SOC tissue genotyping to identify guideline-recommended biomarkers in patients with mNSCLC. Prospectively enrolled patients with previously untreated mNSCLC undergoing physician discretion SOC tissue genotyping submitted a pre-treatment blood sample for comprehensive cfDNA analysis (Guardant360). Among 282 patients, physician discretion SOC tissue genotyping identified a guideline-recommended biomarker in 60 patients versus 77 cfDNA identified patients (21.3 % versus 27.3 %; p < 0.0001 for non-inferiority). In tissue-positive patients, the biomarker was identified alone (12/60) or concordant with cfDNA (48/60), an 80 % cfDNA clinical sensitivity for any guideline-recommended biomarker. For FDA-approved targets (EGFR, ALK, ROS1, BRAF) concordance was greater than 98.2 % with 100 % PPV for cfDNA versus tissue (34/34 EGFR, ALK, or BRAF positive patients). Utilizing cfDNA in addition to tissue increased detection by 48 %, from 60 to 89 patients, including those with negative, not assessed, or insufficient tissue results. cfDNA median turnaround time was significantly faster than tissue (9 versus 15 days; p < 0.0001). Guideline-complete genotyping was significantly more likely (268 versus 51; p < 0.0001). The authors concluded that a comprehensive, sensitive, and specific cfDNA test used in patients with newly diagnosed mNSCLC successfully identified guideline recommended biomarkers at a rate at least as high as SOC tissue testing and returned these results significantly faster and for a significantly higher proportion of the population. Moreover, cfDNA-detected guideline recommended biomarkers were invariably present in tissue, when tissue was successfully tested, reinforcing that cfDNA genotyping results may be used in clinical management in the same way tissue genotyping results are currently used. Lastly, when modeled together, these results suggested that initial biomarker assessment using cfDNA rather than tissue ("blood first"), reserving tissue for PD-L1 IHC and reflex testing when cfDNA is negative for any known oncogenic driver mutations, improved biomarker discovery rate, turn-around time, and increased the number of patients with newly diagnosed mNSCLC who receive guideline complete biomarker testing.
The authors stated that 1 main drawback of this study was that, while cfDNA testing utilized a single platform, tissue genomic assessment was not standardized but was instead left to physician’s discretion SOC, which included a variety of methodologies, including PCR, FISH, IHC, and/or NGS. As only 18 % of patients successfully underwent comprehensive tissue genomic profiling, many alterations that were identified in cfDNA alone were in fact a result of incomplete tissue genotyping due to methodology choice and/or tissue testing failure as opposed to analytical discordance between the tests. As part of the study design, providers were specifically instructed to not make any changes to their SOC tissue genotyping practices, however, these investigators could not rule out the possibility that the receipt of a cfDNA clinical result may have influenced the decision to pursue further tissue genotyping in instances of sequential testing. Moreover, these findings may not apply to other cfDNA tests that are less sensitive or less comprehensive. They stated that while this limited certain comparisons, this design was critical to the fundamental question addressed by this study, whether a well-validated cfDNA test can match or even improve upon SOC tissue methods.
An accompanying commentary (Meador and Oxnard, 2019) noted that the sensitivity of cfDNA genotyping can be low in patients with lower metastatic burden, likely due to reduced shed of tumor DNA into the plasma. The commentators stated that this insensitivity of cfDNA sequencing must be acknowledged as a significant barrier to its application. "Ultimately, negative cfDNA sequencing may be better thanno genotyping at all, but it is not sufficient to rule out the presence of targetable driver mutations given the impaired sensitivity of these assays and unknown rate of tumor shed in any given patient."
Willis et al (2019) sought to analytically validate microsatellite instability (MSI) testing using Guardant360 according to established guidelines and clinically validate it using 1,145 cfDNA samples for which tissue MSI status based on standard-of-care tissue testing was available. The landscape of cfDNA-based MSI across solid tumor types was investigated in a cohort of 28,459 clinical plasma samples. Clinical outcomes for 16 patients with cfDNA MSI-H gastric cancer treated with immunotherapy were evaluated. In evaluable patients, cfDNA testing accurately detected 87% (71/82) of tissue MSI-H and 99.5% of tissue microsatellite stable (863/867) for an overall accuracy of 98.4% (934/949) and a positive predictive value of 95% (71/75). Concordance of cfDNA MSI with tissue PCR and next-generation sequencing was significantly higher than IHC. Prevalence of cfDNA MSI for major cancer types was consistent with those reported for tissue. Finally, robust clinical activity of immunotherapy treatment was seen in patients with advanced gastric cancer positive for MSI by cfDNA, with 63% (10/16) of patients achieving complete or partial remission with sustained clinical benefit. Limitations included the small number of subjects for which clinical outcomes were evaluated.
National Comprehensive Cancer Network’s clinical practice guideline on "Non-small cell lung cancer" (Version 3.2019) states that "The panel feels that cell-free/circulating tumor DNA testing should not be used in lieu of tissue diagnosis. Standard and guidelines for cell-free DNA (cfDNA)/circulating tumor DNA testing for genetic alterations have not been established, there is up to a 30 % false-negative rate, and alterations can be detected that are not related to the tumor (e.g., clonal hematopoiesis of indeterminate potential [CHIP] … However, cfDNA testing can be used in specific circumstances if the patient is not medically fit for invasive tissue sampling, or there is insufficient tissue for molecular analysis and follow-up tissue-based analysis will be done if an oncogenic driver is not identified. Given the previous caveats, careful consideration is required to determine whether cfDNA findings reflect a true driver mutation or an unrelated finding". Since the Guardant360 includes a panel of 68 genes, and only about 5 of which are actionable. The clinical value of the of the entire gene panel of Guardant360 has not been established.
Turner and colleagues (2020) stated that ctDNA testing might provide a current assessment of the genomic profile of advanced cancer, without the need to repeat tumor biopsy. In an open-label, multi-center, multi-cohort, phase-IIa platform trial, these researchers examined the accuracy of ctDNA testing in advanced breast cancer and the ability of ctDNA testing to select patients for mutation-directed therapy. This study was carried out in 18 United Kingdom hospitals. Participants were women (aged greater than or equal to 18 years) with histologically confirmed advanced breast cancer and an ECOG performance status of 0 to 2. Patients had completed at least 1 previous line of treatment for advanced breast cancer or relapsed within 12 months of neoadjuvant or adjuvant chemotherapy. Patients were recruited into 4 parallel treatment cohorts matched to mutations identified in ctDNA: cohort A comprised patients with ESR1 mutations (treated with IM extended-dose fulvestrant 500 mg); cohort B comprised patients with HER2 mutations (treated with oral neratinib 240 mg, and if estrogen receptor-positive with IM standard-dose fulvestrant); cohort C comprised patients with AKT1 mutations and estrogen receptor-positive cancer (treated with oral capivasertib 400 mg plus IM standard-dose fulvestrant); and cohort D comprised patients with AKT1 mutations and estrogen receptor-negative cancer or PTEN mutation (treated with oral capivasertib 480 mg). Each cohort had a primary endpoint of confirmed ORR. For cohort A, 13 or more responses among 78 evaluable patients were needed to infer activity and 3 or more among 16 were needed for cohorts B, C, and D. Recruitment to all cohorts was complete and long-term follow-up is ongoing. Between December 21, 2016, and April 26, 2019, a total of 1,051 patients registered for the study, with ctDNA results available for 1,034 patients. Agreement between ctDNA digital PCR and targeted sequencing was 96% to 99 % (n = 800, kappa 0.89 to 0.93). Sensitivity of digital PCR ctDNA testing for mutations identified in tissue sequencing was 93 % (95 % CI: 83 to 98) overall and 98 % (87 to 100) with contemporaneous biopsies. In all cohorts, combined median follow-up was 14.4 months (IQR 7.0 to 23.7). Cohorts B and C met or exceeded the target number of responses, with 5 (25 % [95 % CI: 9 to 49]) of 20 patients in cohort B and 4 (22 % [6 to 48]) of 18 patients in cohort C having a response. Cohorts A and D did not reach the target number of responses, with 6 (8 % [95 % CI: 3 to 17]) of 74 in cohort A and 2 (11 % [1 to 33]) of 19 patients in cohort D having a response. The most common grade 3 to 4 AEs were raised gamma-glutamyltransferase (13 [16 %] of 80 patients; cohort A); diarrhea (4 [25 %] of 20; cohort B); fatigue (4 [22 %] of 18; cohort C); and rash (5 [26 %] of 19; cohort D); 17 serious adverse reactions occurred in 11 patients, and there was 1 treatment-related death caused by grade 4 dyspnea (in cohort C). The authors concluded that ctDNA testing offered accurate, rapid genotyping that enabled the selection of mutation-directed therapies for patients with breast cancer, with sufficient clinical validity for adoption into routine clinical practice. These researchers stated that these findings demonstrated clinically relevant activity of targeted therapies against rare HER2 and AKT1 mutations, confirming these mutations could be targetable for breast cancer treatment.
The authors stated that this study had several drawbacks. Inclusion of relatively heavily pre-treated patients might reduce activity of the targeted drugs, especially in cohort A, and future ctDNA selection trials might benefit from more restrictive entry criteria. The study was designed to examine the activity of therapies against specific genomic events; however, it did not target PIK3CA mutations, and as a result relatively few of the patients registered to the trial had a response to therapy (17 [1.6 %] of 1,051 patients). However, mutation-directed therapy with alpelisib is now approved to target PIK3CA mutations, and this study showed the clinical validity of using ctDNA to direct therapy. Cohort D was designed as a basket cohort from the outset, to examine the activity of capivasertib against different AKT pathway activating mutations. Only cohort D allowed entry of patients with previous tissue sequencing results, as it was anticipated that ctDNA testing alone might not recruit sufficient patients. Although these researchers identified low activity of capivasertib in PTEN-mutant cancers when used as a single agent, AKT inhibition in combination with paclitaxel chemotherapy might be effective in PTEN mutant cancers. Capivasertib plus fulvestrant might be effective in endocrine-resistant estrogen receptor-positive breast cancer without mutation selection, as shown in the FAKTION trial. It was not possible to robustly compare plasma MATCH with FAKTION, as patients enrolled in plasma MATCH had more previous lines of treatment, and AKT1 mutations were not assessed and would be few in number in FAKTION.
In a retrospective, single-center study, Alvarez et al (2020) reported their experience of cfDNA testing at the time of diagnosis and how this intervention could help avoid further invasive interventions, how it could be used to determine initiation of therapy, and how variation allele frequency of the somatic alteration affects response to subsequent treatment. This trial included patients with advanced NSCLC who had cfDNA from plasma tested using the Guardant360 panel, which identifies somatic genomic alterations by massive parallel sequencing of target genes. An institutional Clinical Laboratory Improvement Amendments tissue panel using FISH (for MET, RET, ROS1, and ALK) and NGS for selected genes was employed for tissue analysis. Actionable mutations are those with FDA-approved targeted therapies (EGFR, ALK, ROS, BRAF, NTRK fusions) or therapies soon to be approved (RET fusions and MET amplifications; or MET exon 14 skipping mutation). A total of 163 blood samples from 143 patients were evaluated, 82 at diagnosis and 81 at disease progression. A total of 94 cases had tissue and cfDNA testing performed within 12 weeks of each other; 76 (81 %) of 94 cases were concordant, of which 22 cases were concordantly positive and 54 concordantly negative; 18 (19 %) of 94 cases were discordant, of which 11 had negative blood and positive tissue results, and 7 had positive blood and negative tissue results. cfDNA testing had a sensitivity of 67 % (95 % CI: 51 % to 83 %), specificity of 89 % (95 % CI: 81 % to 97 %), NPV of 83 % (95 % CI: 74 % to 92 %), and PPV of 76 % (95 % CI: 60 % to 91 %); 19 (21 %) of 82 cfDNA samples analyzed at diagnosis had actionable mutations identified (4 EGFR exon 19 deletion, 2 EGFR exon 21 L858R, 2 EGFR L861Q, 1 L861R, 4 EML4-ALK fusion, 2 CD74-ROS1 fusion, 2 MET exon 14 skipping mutation, 2 KIF5B-RET fusion). Of the 82 patients with cfDNA testing performed at the time of diagnosis, 8 patients (10 %) initiated targeted therapy on the basis of cfDNA results only, with 6 patients experiencing PR, 1 patient CR, and 1 patient SD. The response rate for patients who initiated targeted therapies on the basis of cfDNA only at diagnosis was 88 %. Variant allele frequency had no impact on response. The authors concluded that initiation of targeted therapy for advanced NSCLC was feasible based only on identification of actionable mutations by cfDNA testing in 9 % of the cases for which tissue diagnosis could not be obtained. Actionable targets were identified by cfDNA in 20 % of the samples sent at diagnosis. A substantial number of patients benefited from cfDNA testing at initial diagnosis because it identified actionable mutations that led to appropriate targeted treatments. These researchers stated that cfDNA testing results are being incorporated and accepted in many clinical trials for patient enrollment, which also represents an opportunity to expand patient access to innovative treatments when obtaining further tissue for expanded molecular testing is challenging. Progressive disease remains a challenge, and cfDNA testing results can provide some insight into the mechanism of resistance to tyrosine kinase inhibitors that in some instances tissue re-biopsy molecular testing might not reflect. It should also be noted that cfDNA variation allele frequency does not predict depth of response to targeted therapies.
In a retrospective data review, Dvir et al (2021) presented their real-world data on the use of liquid biopsies in the routine management of NCSLC patients. These investigators carried out a review of 279 consecutive patients with NSCLC in the community setting, who had liquid biopsies performed between 2014 and 2019 as part of routine clinical management. Over a period of 5 years, a total of 337 liquid biopsy samples, taken from 279 patients were sent for plasma NGS testing. The median age at diagnosis was 73 years (range of 36to 93, SD 10.4), 141 (51 %) were men and 138 (49 %) were women. The majority were White or Caucasian (80 % versus 8 % Black or African American versus 12 % multi-racial or unknown race) and had a history of smoking (79 %). Excluding synonymous mutations and variants of unknown significance, 254 AAs were detected in 106 patients. Commonly detected AAs were EGFR (n = 127, 50%), KRAS (n = 61, 24%), BRAF (n = 24, 9.5%), and MET (n = 23, 9%). Tissue NGS detected actionable aberrations in 45 patients, with EGFR (n = 28, 57 %) and KRAS (n = 10, 20 %) being the most common actionable aberrations. Concordance agreement between plasma and tissue NGS modalities was detected in 39 of 45 (86.7 %) patients and was demonstrated most commonly in EGFR (n = 25) and KRAS (n = 11). In 44 of 106 (41.5 %) of patients, for whom tissue NGS was not carried out, additional precision treatment was guided by the actionable aberrations detected through liquid biopsy. The authors concluded that integration of liquid biopsy into the routine management of patients with NSCLC demonstrated actionable aberrations detection in 44 additional patients, which comprised a 42 % increase in actionable aberrations detection rate, when tissue NGS was not performed. Moreover, these researchers stated that more powered studies are needed to examine if incremental benefit exists between tissue NGS and liquid biopsy; these findings cautiously showed a role for the use of liquid biopsy as part of routine clinical management.
The authors stated that this study was limited by its retrospective nature. The information regarding tissue NGS and mortality was captured via review of the electronic medical records, which may be incomplete. The liquid biopsy information was obtained from the Guardant360 database. Detection bias may be introduced if providers chose to order liquid biopsies in selected cases depending on specific characteristics (e.g., insurance coverage and non-smoking status). Evaluating whether clinicians had access to the genomic NGS data and offered patients informed therapy was not routinely documented, limiting the understanding of clinician decision-making with broad-based genomic-sequencing results.
In a prospective study, Palmero et al (2021) examined comprehensive NGS of cfDNA compared with SOC tissue-based testing to identify guideline-recommended alterations in advanced NSCLC (aNSCLC). Patients with treatment-naive aNSCLC were tested using a well-validated NGS cfDNA panel, and results were compared with SOC tissue testing. The primary objective was non-inferiority of cfDNA versus tissue analysis for the detection of 2 guideline-recommended biomarkers (EGFR and ALK) and an additional 6 actionable biomarkers. Secondary analyses included tissue versus cfDNA biomarker discovery, ORR, PFS to targeted therapy, and PPV of cfDNA. The primary objective was met with cfDNA identifying actionable mutations in 46 patients versus 48 by tissue (p < 0.05). In total, 0/186 patients were genotyped for all 8 biomarkers with tissue, compared with 90.8% using cfDNA. Targetable alterations or KRAS were identified in 80.7 % when cfDNA was used first versus 57.1 % when tissue was used first; PPV for cfDNA-detected EGFR was 100.0 % (25/25); ORR and PFS in patients receiving targeted therapy based on tissue or cfDNA were similar to those previously reported. The authors concluded that this study confirmed a previous report that comprehensive cfDNA testing was non-inferior to SOC tissue testing in detecting aNSCLC-recommended biomarkers. Furthermore, cfDNA-based 1st-line therapy produced outcomes similar to tissue-based testing, demonstrating the clinical utility of comprehensive cfDNA genotyping as the initial genotyping modality in patients with treatment-naive aNSCLC when tissue was insufficient or when all actionable biomarkers could not be rapidly assessed.
The authors stated that this study had several drawbacks. First, the lack of a standardized tissue-based testing algorithm precluded direct comparison of comprehensive cfDNA versus tissue testing performance. Physicians used the tissue assays available to them as per their institutional SOC, as this study was designed to specifically address the critical question of what impact the addition of comprehensive cfDNA-based testing might have on real-world patient care and was not intended to be a head-to-head comparison of tissue and cfDNA NGS testing. In this study, only 2 patients had comprehensive NGS tissue testing. Second, although multi-center, this study was limited to Spain and may not reflect results in other patient populations or healthcare systems, although several studies with very similar results have been reported in the U.S. and elsewhere in the world. Third, as this study was powered for a primary endpoint of non-inferiority, only a small number of patients received targeted therapy for any individual biomarker and were available for central RECIST assessment of response. Fourth, these researchers did not examine the correlation of clinicopathologic features with cfDNA detection rates and blood-tissue concordance. This topic has been addressed elsewhere and could be included in future studies. Finally, the findings of this study were only applicable to liquid biopsies that, like the current assay, perform comprehensive genomic profiling as defined by the MolDx program.
Cui et al (2022) noted that genomic sequencing is necessary for 1st-line advanced NSCLC (aNSCLC) treatment decision-making. While tissue NGS is standard, however, tissue quantity, quality, and time-to-results remains problematic. These researchers compared upfront cfDNA NGS clinical utility against routine tissue testing in patients with aNSCLC. cfDNA-NGS was carried out in consecutive, newly identified aNSCLC patients between December 2019 to October 2021 alongside routine tissue genotyping. Variants were interpreted using AMP/ASCO/CAP guidelines. The primary endpoint was tier-1 variants detected on cfDNA-NGS. cfDNA-NGS results were compared to tissue results. Of 311 patients, 282 (91 %) had an informative cfDNA-NGS test; 118 (38 %) patients had a tier-1 variant identified by cfDNA-NGS. Of 243 patients with paired tissue-cfDNA tests, 122 (50 %) tissue tests were informative; 85 (35 %) tissue tests identified a tier-1 variant. cfDNA-NGS detected 39 additional tier-1 variants compared to tissue alone, increasing the tier-1 detection rate by 46 % (from 85 to 124). The sensitivity of cfDNA-NGS relative to tissue was 75 % (25 % tissue tier-1 variants were not detected on cfDNA-NGS); 33 % of cfDNA tier-1 variants were not identified on tissue tests. Median time from request-to-report was shorter for cfDNA-NGS versus tissue (8 days versus 22 days; p < 0.0001). A total of 245 (79 %) patients received 1st-line systemic-therapy: 49 (20 %) with cfDNA-NGS results alone. Median time from sampling-to-commencement of 1st-line treatment was shorter for cfDNA-NGS blood draw versus 1st tissue biopsy (16 days versus 35 days; p < 0.0001). The authors concluded that given the ability of cfDNA-NGS to rapidly detect clinically relevant genomic variants, a plasma-first, tissue-next (if no relevant variants are detected on plasma) testing approach could improve the speed and accuracy of therapeutic decision-making and should be considered a key strategy to increase adequacy and timeliness of target identification and treatment for all patients with aNSCLC.
The authors stated that this trial was carried out at a single U.K. academic cancer center, a drawback of this study. Patients were referred from a variety of diagnostic services with different tissue molecular ordering methods and timelines to the authors’ center. Thus, to minimize unquantifiable bias, the tissue test turn-around time was calculated from the date of biopsy rather than the date of tissue molecular test request as this could not be accurately ascertained. In addition, in this real-world study, the tissue testing methods were heterogenous, reflecting their standard referral pathways and use of sequential hierarchical single-gene testing at some sites could have biased the comparisons between cfDNA-NGS and tissue testing times. Nevertheless, these comparisons did reflect real-world testing scenarios and were, therefore, clinically relevant. Moreover, these findings were based on a healthcare model of government-funded genotype testing, identifying the potential challenges and benefits of implementing cfDNA-NGS in this setting.
Garcia-Pardo et al (2023) stated that clinical management of patients with newly diagnosed advanced NSCLC requires molecular testing of genetic alterations to guide 1st-line treatment. Next generation sequencing in tumor tissue is recommended by clinical guidelines for diagnosis and molecular profiling for advanced non-squamous NSCLC. However, despite tissue-NGS is becoming more widely used, access to NGS testing is still limited in many parts of the world due to a lack of resources and infrastructure. Furthermore, tissue NGS in NSCLC can be challenging due to the need of invasive, limited-tissue content tumor biopsies, which are both time- and resource-consuming. Inadequate or delayed tumor tissue genotyping is one of the major barriers to access to targeted therapy. Detection of plasma ctDNA is becoming widely adopted as complementary to tissue tumor genotyping in NSCLC. Plasma-based molecular profiling is less invasive, faster, requires fewer resources, and it is non-inferior to tumor tissue genotyping in NSCLC. In a retrospective study, these investigators reported their experience using plasma-based NGS in patients with advanced NSCLC. They analyzed the frequency of oncogenic drivers detected, the proportion of patients treated with genotype-directed therapy, and survival outcomes. These researchers analyzed the findings of 109 patients with advanced NSCLC who underwent either Foundation One Liquid (n = 91) or Guardant360 (n = 18) as part of molecular pre-screening for clinical trials. They noted that plasma-based NGS can potentially identify additional driver alterations in patients with NSCLC compared to single-gene testing for EGFR, ALK and ROS1; this can be relevant when there is limited tissue for molecular profiling or if tissue biopsy is not feasible. The identification of driver alterations in plasma aids NSCLC patients to be treated with matched targeted therapy, which eventually might lead to better outcomes. The authors stated that further studies examining the cost-effectiveness of complementary liquid biopsy in addition to tissue genotyping are needed, as well as novel strategies such as a tissue-sparing, plasma-first approach. Moreover, they stated that timely and equitable access to targeted therapies will also be critical to translate research in precision medicine into improved patient care.
The authors stated that this study had several drawbacks. First, this was a retrospective study, and the sample size was relatively small. Second, not all patients with advanced NSCLC referred the authors’ center were systematically enrolled in the molecular pre-screening studies, potentially leading to selection bias; this may account, in part, for the high rate of cases without full-valid tissue genotyping. Third, although not intended, this trial compared plasma-based NGS to single-gene testing for EGFR, ALK, and ROS1 only, due to access constraints to broad molecular testing in Spain; tissue-based NGS is considered the SOC for molecular profiling in NSCLC, and any kind of NGS (either plasma or tissue-based) is preferred to single-gene testing for EGFR, ALK and ROS1.
Powell et al (2024) stated that although immune checkpoint inhibitor immunotherapies are contraindicated as 1st-line treatment of advanced NSCLC in patients with ALK re-arrangement and EGFR mutation, many receive them. These researchers examined the association between optimal 1st-line treatment in this population and clinical outcomes. Claims and genomic data from patients with advanced or metastatic NSCLC were extracted from a nationally representative GuardantINFORM data-set. Patients who had their 1st claim mentioning advanced or metastatic NSCLC between March 2019 and February 2020 and had ALK rearrangement or EGFR mutation detected by CGP were included in this study. Patients were classified as having received optimal or suboptimal 1st-line treatment. Claims were reviewed to determine real-world time to next treatment, real-world time to discontinuation, and health services utilization (emergency department, in-patient, and out-patient) in the 12 months following 1st-line treatment initiation. Survival analyses were carried out using Kaplan-Meier plots and Cox proportional hazard models. Health services utilization was compared between the groups using t-tests and negative binomial models. Of the 359 patients included, 280 (78.0 %) received optimal 1st-line treatment. Optimally treated patients had longer median real-world time to next treatment (11.2 versus 4.4 months; p < 0.01) and real-world time to discontinuation (10.4 versus 1.9 months; p < 0.01). The optimal group had significantly fewer emergency department presentations (0.76 versus 1.27; p < 0.01) and out-patient visits (22.9 versus 42.7; p < 0.01) than the suboptimal group but did not significantly differ in in-patient utilization. Adjusted utilization analysis yielded similar findings. The authors concluded that patients with NSCLC who received optimal treatment, as determined by CGP using NGS-based circulating tumor DNA testing (Guardant360), had significantly superior clinical and utilization outcomes, reinforcing existing guidelines recommending profiling at the onset of treatment.
Guardant360 TissueNext
The Guardant360 TissueNext (Guardant Health, Inc.) is a next-generation sequencing (NGS)-based pan-cancer tissue test for patients with advanced solid tumor. The test is used to help oncologists identify actionable biomarkers for therapeutic management. The test applies targeted genomic sequence and DNA analysis of 84 or more genes and includes interrogation for sequence variants, gene copy number amplifications, gene rearrangements, microsatellite instability (MSI) and tumor mutational burden (TMB) from formalin-fixed paraffin-embedded (FFPE) tumor tissue.
HPV-SEQ Test
HPV-SEQ (Sysmex Inostics, Inc.) is a a quantitative method for circulating tumor DNA (ctDNA) detection indicated for treatment monitoring of disease burden in HPV-related cancers. Specifically, it uses next-generation sequencing (NGS) based quantification of 8 DNA targets, cell free HPV 16 and 18 DNA from plasma, to detect minimal residual disease (MRD).
In a proof-of-principle study, Leung and colleagues (2021) asked whether a next-generation sequencing approach, HPV sequencing (HPV-seq), could provide quantitative and qualitative assessment of HPV ctDNA in low disease burden settings. The authors conducted preclinical technical validation studies on HPV-seq and applied it retrospectively to a prospective multicenter cohort of patients with locally advanced cervix cancer (NCT02388698) and a cohort of patients with oropharynx cancer. HPV-seq results were compared with digital polymerase chain reaction (dPCR). The primary outcome was progression-free survival (PFS) according to end-of-treatment HPV ctDNA detectability. The authors found that HPV-seq achieved reproducible detection of HPV DNA at levels less than 0.6 copies in cell line data. HPV-seq and dPCR results for patients were highly correlated (R2 = 0.95, P = 1.9 × 10–29) with HPV-seq detecting ctDNA at levels down to 0.03 copies/mL plasma in dPCR-negative posttreatment samples. Detectable HPV ctDNA at end-of-treatment was associated with inferior PFS with 100% sensitivity and 67% specificity for recurrence. Accurate HPV genotyping was successful from 100% of pretreatment samples. HPV ctDNA fragment sizes were consistently shorter than non–cancer-derived cell-free DNA (cfDNA) fragments, and stereotyped cfDNA fragmentomic patterns were observed across HPV genomes. The authors concluded that HPV-seq is a quantitative method for ctDNA detection that outperforms dPCR and reveals qualitative information about ctDNA. They state that their findings in this proof-of-principle study could have implications for treatment monitoring of disease burden in HPV-related cancers; however, future prospective studies are needed to confirm that patients with undetectable HPV ctDNA following chemoradiotherapy have exceptionally high cure rates.
Sanz-Garcia et al (2024) state that up to 30% of patients with locally advanced head and neck squamous cell carcinoma (LA-HNSCC) relapse. Molecular residual disease (MRD) detection using multiple assays after definitive therapy has not been reported. In this study, the authors included patients with LA-HNSCC (stage III Human Papilloma virus (HPV)-positive, III-IVB HPV-negative) treated with curative intent. Plasma was collected pre-treatment, at 4–6 weeks (FU1) and 8-12 weeks (FU2) post-treatment. Circulating tumor DNA (ctDNA) was analyzed using a tumor-informed (RaDaR®) and a tumor-naïve (CAPP-seq) assay. HPV DNA was measured using HPV-sequencing (HPV-seq) and digital PCR (dPCR). A total of 86 plasma samples from 32 patients were analyzed; all patients with at least 1 follow-up sample. Most patients were stage III HPV-positive (50%) and received chemoradiation (78%). No patients had radiological residual disease at FU2. With a median follow-up of 25 months, there were 7 clinical relapses. ctDNA at baseline was detected in 15/17 (88%) by RaDaR and was not associated with recurrence free survival (RFS). Two patients relapsed within a year after definitive therapy and showed MRD at FU2 using RaDaR; detection of ctDNA during follow-up was associated with shorter RFS (p < 0.001). ctDNA detection by CAPP-seq pre-treatment and during follow-up was not associated with RFS (p = 0.09). HPV DNA using HPV-seq or dPCR during follow-up was associated with shorter RFS (p < 0.001). Sensitivity and specificity for MRD at FU2 using RaDaR was 40% and 100% versus 20 and 90.5% using CAPP-seq. Sensitivity and specificity for MRD during follow-up using HPV-seq was 100% and 91.7% versus 50% and 100% using dPCR. The authors concluded that HPV DNA and ctDNA can be detected in LA-HNSCC before definitive therapy. The RaDaR assay but not CAPP-seq may detect MRD in patients who relapse within 1 year. HPV-seq may be more sensitive than dPCR for MRD detection. The authors acknowledged study limitations. Despite this being a prospective study, the plasma analysis was performed after all patients were recruited. However, research personnel performing the plasma analysis were blinded to clinical outcome. Second, the number of recruited patients and plasma samples are lower than expected, partially due to limitations to recruitment and plasma biobanking imposed by COVID restrictions. Moreover, all tests could not be performed in all samples, reducing the validity of our conclusions when making comparisons across the assays. They note that the small sample size in their study limits the potential applicability of their findings without further validation in larger studies. Finally, this study was not powered to evaluate the potential value of ctDNA kinetics in the immediate post-treatment setting. The authors state that further validation of the results is ongoing in a prospective multi-centric investigator-initiated study (MERIDIAN, NCT05414032), which includes a cancer interception strategy by randomizing patients who have MRD at FU2 to a novel bispecific checkpoint inhibitor (AZD2936) or observation. This study will aim to complete recruitment by the end of 2025 with an expected readout in 2026.
Invitae PCM MRD Monitoring
Invitae PCM MRD Monitoring (Invitae Corporation) test evaluates a patient blood specimen for circulating tumor DNA (ctDNA) related to the patient’s tumor profile identified in the Invitae PCM Tissue Profiling and MRD Baseline Assay. The test is used to help clinicians identify the presence of cancer cells following treatment (minimal residual disease, or MRD) and to monitor patients for cancer recurrence. This assay uses exome sequencing of a patient's tumor and blood specimens to identify tumor-specific variants for inclusion in the patient-specific panel. This panel is subsequently used to detect ctDNA in the patient’s peripheral blood using high throughput next generation sequencing (NGS).
Currently, there is insufficient evidence in the peer-reviewed literature to support the sensitivity or specificity of this test.
Invitae PCM Tissue Profiling and MRD Baseline Assay
Invitae PCM Tissue Profiling and MRD Baseline Assay (Invitae Corporation) uses targeted next-generation sequencing (NGS) to analyze circulating tumor DNA (ctDNA) in a non-invasive blood sample to identify patient-specific somatic mutations for subsequent minimal residual disease (MRD) evaluation, which is the level of cancer cells in the blood following treatment. This test is indicated for baseline testing. Results for ctDNA status are reported as “Detected”, “Not Detected”, or “Results Are Unavailable”.
Zhao et al (2023) state that emerging evidence suggest there is clinical utility for highly sensitive molecular assays for detecting plasma-based circulating tumor DNA (ctDNA) for monitoring MRD and recurrent disease, providing prognostic information, and monitoring therapy responses in patients with solid tumors. The authors point out that the Invitae Personalized Cancer Monitoring assay uses a patient-specific, tumor-informed variant signature identified through whole exome sequencing (WES) to detect ctDNA in peripheral blood of patients with solid tumors. The authors analytically validated the assay's tumor WES and ctDNA detection components using 250 unique human specimens and nine commercial reference samples that generated 1349 WES and cell-free DNA (cfDNA)-derived libraries. Cell-free DNA (cfDNA) is fragmented DNA shed by cells in the body into the bloodstream and other body fluids. The cfDNA released from tumors is specifically referred to as ctDNA and comprises only a fraction of total cfDNA. A comparison of tumor and germline WES was used to identify patient-specific tumor variant signatures and generate patient-specific panels, followed by targeted NGS of plasma-derived cfDNA using the patient-specific panels with anchored multiplex polymerase chain reaction (PCR) chemistry leveraging unique molecular identifiers. The authors found that WES resulted in overall sensitivity of 99.8% and specificity of > 99.9%. Patient-specific panels were successfully designed for all 63 samples (100%) with ≥ 20% tumor content and 24 (80%) of 30 samples with ≥ 10% tumor content. Limit of blank studies using 30 histologically normal, formalin-fixed paraffin-embedded (FFPE) specimens resulted in 100% expected panel design failure. The ctDNA detection component demonstrated specificity of > 99.9% and sensitivity of 96.3% for a combination of 10 ng of cfDNA input, 0.008% allele frequency, 50 variants on the patient-specific panels, and a baseline threshold. Limit of detection ranged from 0.008% allele frequency when utilizing 60 ng of cfDNA input with 18-50 variants in the patient-specific panels (> 99.9% sensitivity) with a baseline threshold, to 0.05% allele frequency when using 10 ng of cfDNA input with an 18-variant panel with a monitoring threshold (> 99.9% sensitivity). The authors concluded that the Invitae Personalized Cancer Monitoring assay, featuring a flexible patient-specific panel design with 18-50 variants, demonstrated high sensitivity and specificity for detecting ctDNA at variant allele frequencies as low as 0.008%. Moreover, this assay may support patient prognostic stratification, provide real-time data on therapy responses, and enable early detection of residual/recurrent disease. The authors acknowledged limitations of ctDNA assays, such as they are prone to biological limitations. The amount of ctDNA in the blood depends on many factors, including tumor type, burden, and stage of disease, and may vary over the course of treatment. Particular cancer types, such as colorectal cancer, and higher stage tumors have higher shed rates, and early-stage tumors may have low shed rates that make ctDNA fractions at or below the lower limits of detection (LOD) of a ctDNA assay. Circulating tumor DNA detection can also be affected by low fractions of ctDNA owing to other factors such as a patient’s body mass index (BMI), which can result in ctDNA fractions below an assay’s LOD. As the clinical utility of ctDNA testing matures, ctDNA analysis and clinical reporting will also evolve. The authors state that as with any emerging field, it will be essential to develop clinical practice guidelines along with education and training of clinical personnel for the application and interpretation of ctDNA testing, and that harmonization of reported results between different laboratories offering this type of test and recommendations on essential reported values will also be critical.
LiquidHALLMARK
LiquidHALLMARK (Lucence Health Inc.) test uses next–generation sequencing (NGS) from plasma to profile ctDNA mutations in 80 genes, fusions in 10 genes, insertions/deletions, copy number alterations, and microsatellite instability (MSI). As an add-on option, LiquidHALLMARK analyses 36 ctRNA targets for actionable and emerging fusions. LiquidHALLMARK targets genes that are commonly associated with 15 cancers, including lung, breast and colon cancer. The test reports any identified mutations along with a guide to possible clinically actionable treatment options. Per Lucence Health, ctRNA results are currently investigational – results provided for informational, non-diagnostic purposes only.
Poh et al (2022) reported on the analytical and clinical validation of LiquidHALLMARK, an amplicon-based NGS liquid biopsy assay which interrogates 80 cancer-related genes for SNVs, INDELs, CNAs, and gene fusions, as well as additional biomarkers including oncogenic viruses (EBV and HBV) and MSI. The authors examined a total of 1592 samples submitted to their laboratory between January 2018 and May 2021. While the assay is intended for advanced cancer patients to aid clinical therapeutic decision making, 4.5% (n = 71) of the samples were comprised of either screening cases in healthy individuals (n = 61), or suspected cancer cases (n = 10). Additionally, samples from patients with localized tumors constituted 8.7%. The authors found that overall, 73.6% (1120/1521) of cancer samples harbored at least one detectable genetic variant (ctDNA positive), including 40.6% of samples from localized tumors and 78.5% of samples from metastatic tumors. The most commonly altered genes among the ctDNA positive samples were TP53, KRAS, PIK3CA, APC, SMAD4, and PTEN. EGFR alterations were detected in 36.1% of all ctDNA positive samples and in 62.8% of the lung cancer samples. The assay demonstrated a high sensitivity for detecting point mutations (99.38%) and insertions/deletions (95.83%) at a 0.1% variant allele frequency (VAF), and gene fusions at a 0.5% VAF with a sensitivity of 91.67%. Specificity in non-cancer samples exceeded 99.9999%. Clinical application indicated that 74.8% of cancer samples tested ctDNA-positive, with significant representation from lung cancer patients. Among ctDNA-positive lung cancers, 72.5% harbored at least one biomarker with a guideline-approved drug indication. The authors concluded that these results establish the high sensitivity, specificity, accuracy, and precision of the LiquidHALLMARK assay and supports its clinical application for blood-based genomic testing. The assay has some limitations. Although patient-matched tumor tissue and plasma samples were obtained, these samples were not temporally matched, and the treatment status of patients between collection of tumor tissue and blood is unknown. This potentially underestimates the true positive percent agreement of the assay and represents a limitation of the tissue concordance studies performed. In addition to high sensitivity, the assay exhibits uniformly high sequencing depth across the targeted regions of genome coverage, recovering a median 69.6% of unique input DNA molecules (range 42.0–98.4%), with 98.7% of calls having a unique coverage of >1000X. Moreover, its lower sensitivity for gene fusions compared to point mutations and deletions may affect its performance in certain cancer types where thee fusions are prevalent diagnostic markers. Further, the external validation with the cobas EGFR Mutation Test v2 showed a reduced concordance of 84.00%, indicating potential variability in performance with different reference standards or in clinical settings. Overall, the LiquidHALLMARK assay provides a platform for ctDNA analysis with broad genomic coverage; however, it has specific areas where performance may be less optimal, particularly in detecting gene fusions and certain complex genomic alterations at low frequencies. Additional studies to demonstrate clinical validity and utility are ongoing.
Ravi and colleagues (2022) evaluated serial plasma collections from 39 patients with advanced urothelial carcinoma (aUC) receiving immune checkpoint (ICI) therapy and profiled ctDNA using a novel and sensitive amplicon-based NGS assay, LiquidHALLMARK. At least one genomic alteration was detected in the ctDNA samples of 37 (95%) patients pre-therapy and 39 (100%) patients post-therapy. There was a median of three unique genomic alterations per patient in both the pre- and post-immune checkpoint inhibitor therapy samples. The most common genomic alterations were in TP53 (54% pre- and post-therapy), TERT (49% pre-therapy and 59% post-therapy, respectively) and BRCA1/BRCA2 (33% pretherapy and 33% post-therapy, respectively). At the time of the post-therapy sample, 9 of 36 evaluable patients (25%) had a complete or partial response to treatment. Among these 9 patients, 7 (78%) demonstrated clearance of one or more genomic alterations per ctDNA. Four patients had clearance of TP53 variants. Further, patients in whom clearance of TP53 variants was seen during ICI therapy had a higher likelihood of response compared to those in whom TP53 variants remained or emerged during therapy (50% versus 12.5%; p = .046). The authors state that while these findings are hypothesis-generating and require validation and evaluation in other settings (chemotherapy, antibody-drug conjugates), noninvasive serial evaluation of ctDNA may assist in monitoring response to therapy and guide the development of rational therapeutic combinations with ICI.
LungOI
The LungOI (Imagene AI Ltd.) test performs an augmentative algorithmic analysis using artificial intelligence (AI) and bioinformatic databases to evaluate digitized pathology slides from formalin–fixed paraffin–embedded (FFPE) lung tumor tissue stained with hematoxylin and eosin (H&E). LungOI evaluates the slide for 8 genes (ALK, BRAF, EGFR, ERBB2, MET, NTRK1-3, RET, ROS1), and KRAS G12C and PD-L1, which are then reported as positive or negative for each biomarker.
Currently, there is insufficient evidence in the peer-reviewed literature to support the sensitivity or specificity of this test.
MammaPrint
MammaPrint a 70-gene profile that classifies breast cancer into Low Risk or High Risk of recurrence, by measuring genes representative of all the pathways of cancer metastases which were selected for their predictive relationship to 10-year recurrence probability (Raman, et al., 2013). MammaPrint is indicated for women who have stage I or II breast cancer, are lymph node positive or negative, are ER-positive or negative and tumor size of less than five centimeters. MammaPrint determines if the patient is a candidate for chemotherapy.
In February 2007, the Food and Drug Administration (FDA) approved Mammaprint (Agendia, Amsterdam), a DNA microarray-based test used to predict whether women with early breast cancer might face the disease again. The test measures the activity of 70 genes, providing information about the likelihood that cancer will recur. It measures each of these genes in a sample of a woman's breast-cancer tumor and then uses a specific formula to produce a score that determines if the patient is deemed low-risk or high-risk for metastasis. In clinical trials, 1 in 4 women found to be at high risk by Mammaprint had recurrence of their cancer within 5 years. However, there are questions regarding the accuracy of this test. The positive predictive values at 5 and 10 years were 23 % and 29 %, respectively, while the corresponding negative predictive values were 95 % and 90 %, respectively.
Mammaprint was tested on 307 patients under the age of 61 years who underwent surgery for stage I or stage II breast cancer, and who have tumor size equal to or less than 5 cm, and lymph node-negative. The study found that Mammaprint more than doubled physicians' ability to predict breast cancer recurrence.
Cardoso et al (2016) conducted a study to evaluate the clinical utility of the 70-gene signature test (MammaPrint). The study was exerpted from a phase III randomized trial. In this study, of 6693 enrolled women with early stage breast cancer, women with low clinical and genomic risk did not receive chemotherapy whereas those at high risk did receive chemotherapy. All study subjects had their genomic risk evaluated using MammaPrint. The authors noted that "the primary goal was to assess whether, among patients with high-risk clinical features and a low-risk-gene-expression profile who did not receive chemotherapy, the lower boundary of the 95% confidence interval for the rate of 5-year survival without distant metastasis would be 92% (i.e. the noninferiority boundary, or higher). The number of women found to be at high clinical risk and low genomic risk was 1550. In this group, the 5 year survival rate without distant metastases was 94.7% among those not receiving chemotherapy. The authors concluded that among women with early-stage-breast cancer who were at high clinical risk and low genomic risk for recurrence, the receipt of chemotherapy on the basis of the 60 gene signature led to a 5-year survival rate without distant metastasis that was 1.5 percentage points lower than the rate with chemotherapy.
A comment by Hudis and Dickler (2016) stated that it can be challenging to convince practitioners that chemotherapy is not need in an otherwise healthy younger population. They further noted that the primary aim of the study on one study of a 70-gene signature test was to "declare non-inferiority against a predefined benchmark of a 5 year metastasis-free survival rate in just one cohort: patients with a high clinical risk for whom a discordant low genomic risk led to the omission of otherwise standard chemotherapy." They concluded that although for select patients providers may wish to use the MammaPrint, the actions they will take as a result of this testing will be variable and may over time change as a result of further study.
The study by Cardoso et al (2016) was a 5-year median follow-up results of the MINDACT trial, which is to follow subjects for 10 years. The authors noted that follow-up is ongoing to determine whether their findings remain valid for longer-term outcome. These investigators noted that "In the critical group of patients at high clinical risk and low genomic risk, the use of adjuvant chemotherapy led to a trend toward a higher rate of the 5-year outcome than that with no chemotherapy, which included a rate of survival without distant metastasis that was 1.5 percentage points higher, a rate of disease-free survival that was 2.8 percentage points higher, and a rate of overall survival that was 1.4 percentage points higher with chemotherapy than with no chemotherapy in the intention-to-treat population and a rate of survival without distant metastasis that was 1.9 percentage points higher, a rate of disease-free survival that was 3 percentage points higher, and a rate of overall survival that was 1.5 percentage points higher with chemotherapy than with no chemotherapy in the per-protocol population. The study was not powered to assess the statistical significance of these differences. Some 50 % of the study patients were defined as being at low clinical risk. In this group, we did not find any meaningful difference in the 5-year rate of survival without distant metastasis between patients at high genomic risk who received chemotherapy and those who did not receive chemotherapy. On the basis of these data, the results for the 70-gene signature do not provide evidence for making recommendations regarding chemotherapy for patients at low clinical risk".
In an editorial that accompanied the afore-mentioned study, Hudis and Dickler (2016) stated that "a difference of 1.5 percentage points, if real, might mean more to one patient than to another. Thus, the stated difference does not precisely exclude a benefit that clinicians and patients might find meaningful. An adequately powered randomization or a higher threshold for 5-year metastasis-free survival might have provided a more convincing result but would have raised other major challenges for the investigators".
A focused update by the American Society for Clinical Oncology (ASCO) (Kopp, et al., 2017) states that If a patient has hormone receptor–positive, human epidermal growth factor receptor 2 (HER2)–negative, node-negative breast cancer, the MammaPrint assay may be used in those with high clinical risk to inform decisions on withholding adjuvant systemic chemotherapy due to its ability to identify a good-prognosis population with potentially limited chemotherapy benefit. The guidelines state that, if a patient has hormone receptor–positive, HER2-negative, node-positive breast cancer, the MammaPrint assay may be used in patients with one to three positive nodes and a high clinical risk to inform decisions on withholding adjuvant systemic chemotherapy. However, such patients should be informed that a benefit from chemotherapy cannot be excluded, particularly in patients with greater than one involved lymph node. The guideline update was based upon an assessment of data on clinical utility from the MINDACT trial plus other published literature.
Mammostrat
Mammostrat (Clarient) is a novel test for estimating the risk for recurrence in hormone-receptor positive, early stage breast cancer that is independent of proliferation and grade (Raman, et al., 2013). Five biomarkers are combined with a defined mathematical algorithm resulting in a risk index. Mammostrat is clinically validated and has been studied on more than 4,500 total patients in numerous independent cohorts that include the NSABP B14 and B20 trials. Clinicians and patients are faced with difficult choices as to whether to add toxic adjuvant chemotherapy in addition to standard endocrine treatment. Mammostrat may help clinicians understand the inherent aggressiveness of the tumor and the likelihood of tumor recurrence.
The Mammostrat is a prognostic immunohistochemistry (IHC) test that measures the risk of breast cancer recurrence in post-menopausal, node-negative, estrogen receptor-expressing breast cancer patients who will receive hormonal therapy and are considering adjuvant chemotherapy. The test analyzes five monoclonal antibody biomarkers and applies a diagnostic algorithm to assess whether patients have a high, moderate, or low risk of recurrence after they have had their breast cancer tumor surgically removed and have been treated with tamoxifen.
Bartlett et al (2010) tested the efficacy of the Mammostrat in a mixed population of cases treated in a single center with breast-conserving surgery and long-term follow-up. Tissue microarrays from a consecutive series of 1,812 women managed by wide local excision and post-operative radiotherapy were collected. Of 1,390 cases stained, 197 received no adjuvant hormonal or chemotherapy, 1,044 received tamoxifen only, and 149 received a combination of hormonal therapy and chemotherapy. Median age at diagnosis was 57 years, 71% were post-menopausal, 23.9% were node-positive and median tumor size was 1.5 cm. Samples were stained using triplicate 0.6 mm2 tissue microarray cores, and positivity for p53, HTF9C, CEACAM5, NDRG1 and SLC7A5 was assessed. Each case was assigned a Mammostrat risk score, and distant recurrence-free survival (DRFS), relapse-free survival (RFS) and overall survival (OS) were analyzed by marker positivity and risk score. Increased Mammostrat scores were significantly associated with reduced DRFS, RFS and OS in ER-positive breast cancer (p < 0.00001). In multivariate analyses the risk score was independent of conventional risk factors for DRFS, RFS and OS (p < 0.05). In node-negative, tamoxifen-treated patients, 10-year recurrence rates were 7.6 +/- 1.5% in the low-risk group versus 20.0 +/- 4.4% in the high-risk group. Further, exploratory analyses revealed associations with outcome in both ER-negative and un-treated patients. The authors concluded that the Mammostrat can act as an independent prognostic tool for ER-positive, tamoxifen-treated breast cancer and the results of the study revealed a possible association with outcome regardless of node status and ER-negative tumors.
There is insufficient evidence to determine whether the Mammostrat test is better than conventional risk assessment tools in predicting the recurrence of breast cancer. Furthermore, neither NCCN or ASCO have incorporated the test into their guidelines as a management tool. Guidance from the National Institute for Health and Clinical Excellence (NICE, 2013) states that the Mammostrat is "only recommended for use in research in people with ER+, LN− and HER2− early breast cancer, to collect evidence about potentially important clinical outcomes and to determine the ability of the tests to predict the benefit of chemotherapy ... The tests are not recommended for general use in these people because of uncertainty about their overall clinical benefit and consequently their cost effectiveness."
An assessment by the Belgian Healthcare Knowledge Centre (KCE) (San Miguel, et al., 2015) found that the evidence for Mammostrat is mainly limited to studies supporting the prognostic ability (clinical validity) of the test. The KCE stated that these studies include a large sample size and appear to be of reasonable quality. The KCE cited one study reporting on clinical utility in terms of the predictive ability of the test by risk group. "However, further evidence is required."
Guidelines from the American Society for Clinical Oncology (2016) state: "If a patient has ER/PgR-positive, HER2-negative (node-positive or node-negative) breast cancer, the clinician should not use the five-protein assay (Mammostrat; Clarient, a GE Healthcare company, Aliso Viejo, CA) to guide decisions on adjuvant systemic therapy." This is a moderate strength recommendation based upon intermediate-quality evidence. The ASCO guidelines recommend against the use of Mammostrat to guide decisions on adjuvant systemic therapy for patients with HER2-positive or TN breast cancer.
MelaNodal Predict
The MelaNodal Predict (Quest Diagnostics and based on technology developed by SkylineDx) assay is a quantitative reverse transcription polymerase chain reaction (qRT-PCR) assay based upon the CP-GEP (clinicopathologic and gene expression profile) model that combines the gene expression of 8 target genes (MLANA, GDF15, CXCL8, LOXL4, TGFBR1, ITGB3, PLAT, SERPINE2) and two control genes (RLP0 and ACTB) with the clinicopathological features of patient age and Breslow depth to predict a cutaneous melanoma patient's risk of sentinel lymph node metastasis from formalin-fixed, paraffin-embedded primary tumor tissue obtained from those who are being considered for the sentinel lymph node biopsy procedure (SLNB).
Bellomo et al (2020) noted that more than 80 % of patients who undergo SLNB have no nodal metastasis. In a retrospective study, these researchers described a model that combines clinicopathologic and molecular variables to identify patients with thin and intermediate thickness melanomas who may forgo the SLNB due to their low risk of nodal metastasis. Genes with functional roles in melanoma metastasis were discovered by analysis of NGS data and case control studies. These investigators then used PCR to quantify gene expression in diagnostic biopsy tissue across a prospectively designed archival cohort of 754 consecutive thin and intermediate thickness primary cutaneous melanomas. Outcome of interest was SLNB metastasis within 90 days of melanoma diagnosis. A penalized maximum likelihood estimation algorithm was used to train logistic regression models in a repeated cross validation scheme to predict the presence of SLN metastasis from molecular, clinical, and histologic variables. Expression of genes with roles in epithelial-to-mesenchymal transition (glia derived nexin, growth differentiation factor 15, integrin β3, interleukin 8, lysyl oxidase homolog 4, TGFβ receptor type 1 and tissue-type plasminogen activator) and melanosome function (melanoma antigen recognized by T cells 1) were associated with SLN metastasis. The predictive ability of a model that only considered clinicopathologic or gene expression variables (CP-GEP) was out-performed by a model that included molecular variables in combination with the clinicopathologic predictors Breslow thickness and patient age; AUC, 0.82; 95 % CI: 0.78 to 0.86; SLN biopsy reduction rate of 42 % at a NPV of 96 %. The authors concluded that a combined model including CP-GEP improved the identification of melanoma patients who may forgo the SLNB due to their low risk of nodal metastasis. Moreover, these researchers stated that additional investigation is ongoing to externally validate these findings.
The authors stated that a limitation of the simultaneous selection of CP variables and genes by their feature selection algorithm was the absence of established variables easily recognizable by clinicians, such as ulceration, in the CP-GEP model. They noted that this study was also limited by referral bias and variations in pathologic assessment. The exclusion of patients with ambiguous less than 0.1 mm metastasis from model development could have influenced the results. Moreover, these researchers excluded T4 lesions (i.e., melanoma with a Breslow thickness of less than 4 mm) because the pre-test probability of regional metastasis for these patients was very high. For example, 21 (70 %) of 30 patients with T4 lesions in their cohort presented with regional metastasis, which was well above the recommend threshold for recommending SLNB. Clinicians may not want to forgo SLNB for an a priori high-risk T4 melanoma, even if a molecular classifier was available. Finally, eligibility of patients with T1 melanoma was determined by the Mayo Clinic institutional practice guidelines for recommending SLNB, which select for higher-risk patients, such as those of less than 40 years of age and with T1b melanoma. Indiscriminate inclusion of low-risk patients could have biased test performance calculations.
Yousaf et al (2021) stated that about 85 % of melanoma patients who undergo a SLNB are node-negative. Melanoma incidence is highest in patients 65 years or older; however, their SLNB positivity rate is lower than in younger patients. CP-GEP identifies primary cutaneous melanoma (CM) patients who may safely forgo SLNB due to their low risk for nodal metastasis. These investigators attempted to validate CP-GEP in a U.S. melanoma patient cohort. A cohort of 208 adult patients with primary CM from the Mayo Clinic and West Virginia University was used. Patients were stratified according to their risk for nodal metastasis: CP-GEP High Risk and CP-GEP Low Risk. The main performance measures were SLNB reduction rate (RR) and NPV. SLNB positivity rate for the entire cohort was 21 %. Most patients had a T1b (34 %) or T2a (31 %) melanoma. In the T1-T2 group (153 patients), CP-GEP achieved an SLNB RR of 41.8 % (95 % CI: 33.9 to 50.1) at an NPV of 93.8 % (95 % CI: 84.8 to 98.3). Subgroup analysis showed similar performance in T1-T2 patients 65 years or older of age (51 patients; SLNB positivity rate, 9.8 %): SLNB RR of 43.1 % (95 % CI: 29.3 to 57.8) at an NPV of 95.5 % (95 % CI: 77.2 to 99.9). The authors confirmed the potential of CP-GEP to reduce negative SLNB in all relevant age groups. These researchers stated that these findings were especially relevant to patients 65 years or older, where surgery is often elective. They stated that CP‐GEP (Merlin Assay) may provide a promising tool to reduce SLNB procedures by guiding doctors and patients in their clinical decision‐making.
Mulder et al (2021) noted that the CP-GEP model was developed to accurately identify patients with T1-T3 primary cutaneous melanoma at low risk for nodal metastasis. These researchers attempted to validate the CP-GEP model in an independent Dutch cohort of patients with melanoma. Patients (aged 18 years or older) with primary cutaneous melanoma who underwent SLNB between 2007 and 2017 at the Erasmus Medical Centre Cancer Institute were eligible. The CP-GEP model combines clinicopathological features (age and Breslow thickness) with the expression of eight target genes involved in melanoma metastasis (ITGB3, PLAT, SERPINE2, GDF15, TGFBR1, LOXL4, CXCL8 and MLANA). Using the pathology result of SLNB as the gold standard, performance measures of the CP-GEP model were calculated, resulting in CP-GEP high risk or low risk for nodal metastasis. A total of 210 patients were included in the study. Most patients presented with T2 (n = 94, 45 %) or T3 (n = 70, 33 %) melanoma. Of all patients, 27 % (n = 56) had a positive SLNB, with nodal metastasis in 0 %, 30 %, 54 % and 16 % of patients with T1, T2, T3 and T4 melanoma, respectively. Overall, the CP-GEP model had a NPV of 90.5 % (95 % CI: 77.9 to 96.2), with an NPV of 100 % (95 % CI: 72.2 to 100) in T1, 89.3 % (95 % CI: 72.8 to 96.3) in T2 and 75.0 % (95 % CI: 30.1 to 95.4) in T3 melanomas. The CP-GEP indicated high risk in all T4 melanomas. The authors concluded that the CP-GEP model was a non-invasive and validated tool that accurately identified patients with primary cutaneous melanoma at low risk for nodal metastasis. In this validation cohort, the CP-GEP model has shown the potential to reduce SLNB procedures in patients with melanoma. These researchers stated that the CP‐GEP model is a promising tool for patient care with a low implementation threshold, which may reduce the number of SLN‐negative procedures, and can guide doctors and patients in their clinical decision‐making for SLNB.
These researchers stated that the low number of T1 melanomas (n = 11, 5 % of the validation cohort) and lack of nodal metastasis in this group could be interpreted as a limitation of the study, but was rather a result of adequate de-selection (based on current clinical guidelines) for SLNB of these patients. However, 10 (out of 11) patients with a T1 melanoma could have safely forgone SLNB and 1 patient would have undergone SLNB without having SLN metastasis, if the CP‐GEP model outcome had been used. Another challenge was the presence of too little tumor material of the FFPE primary melanoma, which occurred mainly in thin melanomas (i.e., T1). In addition, the inclusion of T1 melanomas without SLN metastasis may have resulted in a higher NPV. On the other hand, the percentage of T1 melanomas was significantly higher in the development cohort (n = 192, 25 %), of which 6 patients had a positive SLNB. Moreover, NPV was high in both the development and validation cohorts (96 % and 90.5%, respectively). Because the algorithm has been bridged to a different platform (QuantstudioDx), this study did not directly validate the development platform (Fluidigm). To validate the CP‐GEP algorithm, both discovery and bridging have been done in a stringent document‐controlled product development environment and all acceptance criteria, coefficients and the cut‐off value were pre-defined. Moreover, these investigators stated that to demonstrate the added value of using the CP‐GEP model in clinical practice, more validation data are needed.
Johansson et al (2022) noted that in patients with cutaneous melanoma, SLNB serves as an important technique to examine disease stage and to guide adjuvant systemic therapy. A model using CP-GEP (Merlin Assay) has recently been introduced to identify patients that may safely forgo SLNB. These investigators presented data from an independent validation cohort of the CP-GEP model in Swedish patients. Archival histological material (primary melanoma tissue) from a prospectively collected cohort of 421 consecutive patients with pT1-T4 melanoma undergoing SLNB between 2006 and 2014 was analyzed using the CP-GEP model. CP-GEP combines Breslow thickness and patient age with the expression levels of 8 genes from the primary melanoma. Stratification is based on their risk for nodal metastasis: CP-GEP Low Risk or CP-GEP High Risk. The SLNB positivity rate was 13 %. Of 421 primary melanomas, the CP-GEP model identified 86 patients as having a low risk for nodal metastasis. In patients with pT1-2 melanomas, the SLNB reduction rate was 35.4 % (95 % CI: 29.4 to 41.8) with a NPV of 96.5 % (95 % CI: 90.0 to 99.3). Among patients with pT1-3 melanomas, CP-GEP suggested a SLNB reduction rate of 24.0 % (95 % CI: 19.7 to 28.8) and a NPV of 96.5 % (95 % CI: 90.1 to 99.3). Only 1 of 118 pT3 tumors was classified as CP-GEP Low Risk, and all pT4 tumors were classified as being high risk for nodal metastasis. The authors concluded that the findings of this study showed that CP-GEP could identify patients with a low risk for nodal metastasis. Patients with pT1-2 melanomas had the highest clinical benefit from using the test, where 35 % of the patients could forgo a SLNB procedure.
Stassen et al (2023) stated that SLNB is recommended for patients with >pT1b cutaneous melanoma, and should be considered and discussed with patients diagnosed with pT1b cutaneous melanoma for the purpose of staging, prognostication and determining eligibility for adjuvant therapy. Previously, the CP-GEP model was developed to identify patients who can forgo SLNB because of a low risk for sentinel node metastasis. In a prospective, multi-center study, these researchers examined the clinical use and implementation of the CP-GEP model. Both test performance and feasibility for clinical implementation were assessed in 260 patients with T1-T4 melanoma. The CP-GEP model showed an overall NPV of 96.7 % and PPV of 23.7 %, with a potential SLNB reduction rate of 42.2 % in patients with T1-T3 melanoma. With a median time of 16 days from initiation to return of test results, there was sufficient time left before the SLNB was performed. The authors concluded that based on these outcomes, the model may support clinical decision-making to identify patients who can forgo SLNB in clinical practice.
The authors stated that one potential drawback of this study was that it was carried out in 4 dedicated melanoma centers, which may result in a relatively homogeneous study population. However, it is important to note that the population included in this study was likely to exhibit similarities with the broader population eligible for SLNB. This similarity can be attributed to the composition of the participating centers, which encompass both academic as well as teaching hospitals, with some academic centers also serving as regional hospitals. As a consequence, the characteristics and diversity of the study population are expected to be representative and comparable to the general population eligible for SLNB. Moreover, these researchers stated that if the model is incorporated into clinical practice, it has the potential to significantly reduce SLNBs, as the reduction rate is robust and significant when performed in the appropriate patient population (i.e., pT2). Thus, although not actively examined in this study, the incorporation of the CP-GEP model can be beneficial to patients for different reasons. First, after shared decision-making, patients with a low-risk can forgo SLNB, decreasing the risk of complications associated with the SLNB, such as lymphedema and infection. Second, the reduction in surgeries may result in a decrease in healthcare costs, and allow healthcare resources to be allocated to other departments, so that capacity issues may be addressed.
Amaral et al (2023) noted that patients with cutaneous melanoma stage I/IIA disease are currently not eligible for adjuvant therapy, despite their risk for relapses and death. Ina retrospective study, these investigators attempted to validate the ability of a model combining CP-GEP to identify patients at high risk for disease recurrence in stage I/II and subgroup stage I/IIA. A total of 543 patients with stage I/II primary cutaneous melanoma diagnosed between 2000 and 2017 were analyzed; all patients received SLNB. Analysis was carried out for a separate group of 80 patients who did not undergo SLNB. CP-GEP stratified 424 stage I/IIA patients (78 % of the cohort) according to their risk for recurrence, with 5-year relapse-free survival (RFS) rates of 77.8 % and 93 % for CP-GEP high risk (195 patients) and low risk (229 patients), respectively, and HR of 3.53 (p < 0.001). In patients who did not receive SLNB biopsy, CP-GEP captured 6 out of 7 relapses. The authors concluded that CP-GEP may help better select stage I/IIA melanoma patients at high risk for disease recurrence and should get access to adjuvant therapy. CP-GEP also showed value in patients who did not receive SLNB biopsy, capturing 6 out of 7 relapses; thus, showing the potential to replace SLNB and stratify patients based on their risk for disease recurrence more accurately. These researchers stated that a drawback of this trial was its retrospective nature and the fact that data came from only 1 center. In addition, a longer follow-up time for the subgroup that did not undergo SLNB would have been desired.
In a review on “Debating sentinel lymph node biopsy for melanoma in the modern adjuvant era”, Zhang et al (2023) noted that “While both gene expression profiling of the primary lesion and circulating tumor DNA assays appear to be promising, neither has demonstrated adequate prospective validation to be broadly recommended”.
Sun et al (2024) stated that cutaneous melanoma is becoming more prevalent in the U.S. and has the highest mortality among cutaneous malignancies. The majority of melanomas are diagnosed at an early stage and, as such, survival is generally favorable. However, there remains prognostic uncertainty among subsets of early- and intermediate-stage melanoma patients, some of whom go on to develop advanced disease while others remain disease-free. Melanoma GEP has evolved with the notion to aid in bridging this gap and identify higher- or lower-risk patients to better tailor treatment and surveillance protocols. These tests seek to prognosticate melanomas independently of established AJCC 8 cancer staging and clinicopathologic features (sex, age, primary tumor location, thickness, ulceration, mitotic rate, lympho-vascular invasion, micro-satellites, and/or SLNB status). While there is a significant opportunity to improve the accuracy of melanoma prognostication and diagnosis, it is equally important to understand the current landscape of molecular profiling for melanoma treatment. The authors concluded that specialty society guidelines currently do not recommend molecular testing outside of clinical trials for melanoma clinical decision-making, citing insufficient high-quality evidence in guiding indications for the testing and interpretation of results.
Furthermore, National Comprehensive Cancer Network’s clinical practice guideline on “Melanoma: Cutaneous” (Version 1.2024) states that “Gene expression profiling for melanoma could be an enormously valuable contribution to understanding the biology of the disease. However, the difficulty of embracing gene expression profiling as an independent predictor of outcome is illustrated by the inconsistency of results across studies aimed at defining the most predictive gene sets for melanoma. Comparison of the gene signatures identified in these studies show minimal overlap in specific genes thought to be predictive of outcome. The identification and validation of a prognostic gene expression profile is a complicated multi-step and often multi-study process, and there are many ways in which specifics of study design and methodology can impact the end result. The lack of overlap in gene signatures identified as prognostic for melanoma is likely due to substantial differences in study design and methodology. Efforts to develop gene expression profiling prognostic assays for other types of cancer have also resulted in limited or partial overlap in the “gene signature” identified by different studies”.
miR Sentinel Prostate Cancer Test
The miR Sentinel Prostate Cancer Test (miR Scientific) is a platform of individual assays for both prostate cancer screening and determining the risk level of the disease. The miR Sentinel Prostate Cancer Test applies exosome-based analysis of 442 small noncoding RNAs (sncRNAs) by quantitative reverse transcription polymerase chain reaction (RT-qPCR) from a urine sample. The test is powered by an algorithm-based miR platform with results being reported as molecular evidence of no-, low-, intermediate- or high-risk of prostate cancer.
Wu and associates (2020) stated that exosomes are defined as small membranous vesicles. After RNA content was discovered in exosomes, they emerged as a novel approach for the treatment and diagnosis of cancer. Long non-coding RNAs (lncRNA), a kind of specific RNA transcript, have been reported to function as tumor growth, metastasis, invasion, and prognosis by regulating the tumor micro-environment in exosomes. These researchers examined the potential diagnostic of exosomal lncRNA in solid tumors. They carried out a meta-analysis from January 2000 to October 2019 and identified publications in the English language; all relevant English literature from the Web of Science, Embase, and PubMed data-bases through October 1, 2019 were searched. The articles were strictly screened by these investigators’ criteria and critiqued using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. There were 28 studies with 19 articles (4,017 subjects) identified, including studies on BC, CRC, cholangiocarcinoma, esophageal SCC, gastric cancer, HCC, laryngeal SCC, NSCLC, and PCa. A meta-analysis showed that the combined value of sensitivity in 29 studies was 0.74 (95 % CI: 0.7 to 0.78), and the combined value of specificity in the studies was 0.81 (95 % CI: 0.78 to 0.83); suggesting the high diagnostic efficacy of liquid exosomes in cancer patients. It is statistically insignificant in terms of sex, ethnicity, and year. The diagnostic power of urinary system tumors was found to be higher than that of digestive system tumors by several subgroup analyses. The authors performed a meta-analysis and literature review of 28 studies that included 4,017 patients with 10 malignant cancer types. They stated that mechanistically, this study demonstrated that lncRNAs in exosomes could be a promising bio-indicator for the diagnosis and prognosis of solid tumors. These investigators hoped that their findings would encourage more researchers to examine the prognostic and diagnostic role of lncRNA in exosomes as well as examine the underlying biomechanisms in different cancers.
Wang and colleagues (2020) noted that this is the 1st report of the development and performance of a platform that interrogates small non-coding RNAs (sncRNA) isolated from urinary exosomes. The Sentinel PCa Test classifies patients with PCa from subjects with no evidence of PCa, the miR Sentinel CS Test stratifies patients with PCa between those with low risk PCa (Grade Group 1) from those with intermediate and high risk disease (Grade Group 2-5), and the miR Sentinel HG Test stratifies patients with PCa between those with low and favorable intermediate risk PCa (Grade Group 1 or 2) and those with high risk (Grade Group 3-5) disease. sncRNAs were extracted from urinary exosomes of 235 subjects and interrogated on miR 4.0 microarrays. Using proprietary selection and classification algorithms, informative sncRNAs were selected to customize an interrogation OpenArray platform that forms the basis of the tests. The tests were validated using a case-control sample of 1,436 subjects. The performance of the miR Sentinel PCa Test demonstrated a sensitivity of 94 % and specificity of 92 %. The Sentinel CS Test demonstrated a sensitivity of 93 % and specificity of 90 % for prediction of the presence of Grade Group 2 or greater cancer, and the Sentinel HG Test demonstrated a sensitivity of 94 % and specificity of 96 % for the prediction of the presence of Grade Group 3 or greater cancer. The authors concluded that the Sentinel PCa, CS and HG Tests demonstrated high levels of sensitivity and specificity, highlighting the utility of interrogation of urinary exosomal sncRNAs for non-invasively diagnosing and classifying PCa with high precision.
These researchers stated that discordance between the Sentinel test results and the core biopsy outcomes may reflect pathological miss of higher grade cancer or a true test mis-classification. Given the known false-negative rate of core needle biopsies, these investigators estimated the apparent false-positive rate of the Sentinel PC is 6 % to 12 % based on the 95 % CI. This compares favorably to the 50 % to 60 % false-positive rate reported for systematic transrectal ultrasound-guided core needle biopsies and 30 % to 40 % false-positive rates reported for various MRI-targeted biopsies. The combined apparent false-positive and negative rates of the Sentinel HG Test with biopsy outcome is around 10 %. This performance is within the confidence limits of the well-established rate of mis-attribution of grade resulting from systematic biopsies. Therefore, it is plausible that in this case the apparent false-positive cases resulting from the Sentinel HG Test may in fact be those who harbor higher grade cancer missed on the systematic biopsy. An alternative explanation is that some of these represent actual false-positive test results. To further examine this issue, these researchers are currently conducting a large retrospective study comparing the Sentinel Scores with radical prostatectomy pathology.
In an editorial that accompanied the afore-mention study, Helfand (2020) stated that "Overall, the results appear to be promising new PCa biomarkers for each of these tests. However, some caution should be made with any novel test, including the requirement to validate the results in other independent cohorts and racially diverse groups. In addition, because of inherent sampling errors with biopsy results, further comparison to final surgical pathology should be made. Finally, research should be devoted to further characterize the small noncoding RNAs as they may provide additional insights and/or therapeutic targets into the biology of PCa".
Per the National Comprehensive Cancer Network Clinical Practice Guidelines for “Prostate cancer early detection” (Version 2.2024), the MiR Sentinel Prostate Cancer Test awaits further validation, especially in the group of patients with negative DREs and PSAs in the range where most such tests are used (ie, 2.5–10.0 ng/mL)”.
M-Protein Detection and Isotyping for Plasma Cell Dyscrasias
An M protein is a monoclonal immunoglobulin (antibody) found in unusually large amounts in the blood or urine of people with multiple myeloma and other types of plasma cell tumors. Testing classically uses electrophoretic techniques, supplemented with additional tests for protein quantification and methodologies to determine whether the protein arises from a single clone (i.e., monoclonal). The initial evaluation usually includes a serum protein electrophoresis (SPEP), serum immunofixation, routine urinalysis, 24-hour urine protein electrophoresis (UPEP), and urine immunofixation (Murray, 2024). Another widely utilized assay is the serum free light chain (sFLC) assay. Moreover, mass spectrometry is considered a useful alternative to confirm the existence of an M-protein after a positive finding by SPEP (Murray et al, 2021).
Two mass spectrometry methods have emerged in the literature. Both methods start with immune-enrichment of patient immunoglobulins (Igs) but differ on the analytical target used to detect the M-protein. One method utilizes Ig trypsin digestion and detection of peptides specific to the M-protein CDR15–18. This method has been termed the “clonotypic peptide” approach. However, more clinical data is still needed to determine if the clonotypic approach translates into disease-free and overall survival benefits in persons with multiple myeloma (Murray et al, 2021).
The second method utilizes total free light chain (LC) mass distributions from Igs which have been chemically reduced and denatured into heavy and light chain components. This is a more simpler and practical approach for M-protein detection, which relies on scanning the overall mass distribution of denatured intact Ig LCs. This method was adapted to matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS), which eliminated the chromatography step and reduced analytical time from 20 min to 10 s. Eventually, the MALDI-TOF method was modified to include immuno-enrichments to conclusively identify the M-protein isotype in a similar fashion to immunofixation electrophoresis (IFE). The intact LC MALDI-TOF MS assay, coined as Mass-Fix, has been found to be comparable to IFE in terms of assay turnaround time and ease of interpretation with the added benefit of reduced labor. These features make the intact LC method more economically attractive in comparison to the clonotypic method. Commercial efforts are being made to automate the technique and provide a high-throughput method which would be widely accessible for routine laboratory implementation" (Murray et al, 2021).
The International Myeloma Working Group (IMWG) Mass Spectrometry Committee endorses detection of M-proteins by MS (intact MALDI-TOF method) as an alternative to IFE for clinical practice and clinical trials. The group also endorses MS for distinguishing residual M-protein from therapeutic monoclonal antibodies for clinical practice, and for accurate interpretation and determination of complete response in clinical trials (Murray et al, 2021).
Li et al (2022) developed a MALDI-TOF Mass spectrometry-based method for the screening test of M-proteins in human serum. The study evaluated 212 samples including 110 electrophoresis positive samples (62 SPE positive M-proteins and 48 M-proteins detectable by IFE only) and 102 IFE negative serum samples. In addition, the utility of current MALDI-TOF MS-based method for M-protein level monitoring was tested in a cohort of eight multiple myeloma (MM) patients whose serum samples including the diagnostic and plus 5 available posttreatment samples from a serum bank in a large referral hospital. The authors report that all 62 patients with SPE positive results could be identified by the current MALDI-TOF MS method. In contrast, additional 43 SPE-/IFE+ and 7 cases with SPE-/IFE- patient were detected as positive with their method, indicating a higher analytical sensitivity. Compared to the IFE positive/negative results, the overall sensitivity and specificity of the MDT-MALDI assay were determined as 95% and 93%, respectively. The authors concluded that compared to the front-line electrophoresis technologies, the current assay demonstrated with high analytical performance and throughput, more rapid, convenient and economical. The current method could be a new choice for the diagnosis and disease monitoring of plasma cell dyscrasias.
Keren et al (2022) developed evidence-based guideline on laboratory detection and initial diagnosis of monoclonal gammopathies. A total of 60 articles were included for qualitative analysis and potential data extraction, and 25 studies provided data that informed the recommendations. Based on the literature review, the panel made a strong recommendation for confirming a SPEP abnormality suspicious for presence of an M protein with additional testing by sIFE or alternative method with similar sensitivity. "Though not widely available, a new technique involving immunoenrichment followed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MASS-FIX) has been shown to be a highly sensitive, specific, and cost-effective method comparable to sIFE to detect and identify M proteins)".
Mehra et al (2023) state several studies have demonstrated the analytical sensitivity of MALDI-TOF mass spectrometry (MALDI-TOF MS) by immunoenrichment for M-protein analysis.
M-inSight Patient Definition Assay and M-inSight Patient Follow-Up Assessment
M-inSight Patient Definition Assay (Corgenix Clinical Laboratory) is a blood serum test that uses liquid chromatography with tandem mass spectrometry (LC–MS/MS) to identify specific protein pieces, known as clonotypic peptides, that come from cells in a patient with multiple myeloma (MM). The results are reported as baseline presence or absence of detectable clonotypic peptides.
M-inSight Patient Follow-Up Assessment (Corgenix Clinical Laboratory) is a blood serum test that uses LC–MS/MS to quantify specific clonotypic peptides from cells in a patient with multiple myeloma. The test includes comparison with the separately reported baseline test, M–inSight Patient Definition Assay, and determines the abundance of monoclonal protein (M–protein).
The M-inSight platform is a personalized, serum-based, targeted mass spectrometry assay for minimal residual disease (MRD) monitoring in multiple myeloma. M-inSight tracks patient-specific clonotypic peptides from the M-protein secreted by the tumor cells. Ultra-sensitivity is reached by analyzing unique peptides derived from the variable region of the M-protein (clonotypic peptides) corresponding to patient specific V(D)J-gene rearrangements and somatically hypermutated gene sequences. The M-inSight platform works in 2 steps: Patient Definition Assay - M-protein is sequenced from serum, then the best clonotypic peptides are selected from this sequence. This is a one-time process; and Patient Follow-up Assessment - M-protein quantitation is obtained over time measuring clonotypic peptides in serum.
M-inSight uses a combination of 3 dimensions to reach the highest sensitivity: (i) high resolution separation by using nano ultra high performance liquid chromatography (nUPLC), (ii) high resolution mass analyzer with the Orbitrap technology to accurately quantify at the lowest level, and (iii) unique clonotypic peptides targeting which avoids the interference with the polyclonal background and therapeutic monoclonal antibody.
Conventional M-protein analysis includes serum protein electrophoresis (SPEP) and immunofixation. Zajec et al (2018) developed a targeted mass-spectrometry (MS) assay (liquid chromatography MS) to detect M-protein in serum of an MM patient in the presence of therapeutic mAb’s. The authors stated that their developed mass-spectrometry assay can circumvent repeated bone marrow aspirations, enable simultaneous absolute quantification of M-protein and therapeutic monoclonal antibodies, and is more than two orders of magnitude more sensitive than conventional M-protein diagnostics. Their Technical Note purports the feasibility of the approach; however, it needs to be validated on longitudinally collected samples from a cohort of multiple myeloma patients.
More clinical data is still needed to determine if the MRD positivity by the clonotypic approach translates into disease-free and overall survival benefits. While most studies utilizing the mass spectrometry clonotypic method detect the presence of the M-protein in samples from patients who are bone marrow MRD-negative, the clinical implications of this will require more time to elucidate (Murray et al, 2021).
Myeloproliferative Neoplasms and Myelodysplastic Syndromes
Myeloproliferative neoplasms and myelodysplastic syndromes are both blood cell diseases and both carry an increased risk of transformation into acute myelogenous leukemia (AML). Myelodysplastic syndromes (MDSs) refer to a heterogeneous group of myeloid disorders characterized by varying reductions in the production of red blood cells, platelets, and mature granulocytes that may also exhibit functional (i.e., qualitative) defects.
Conversely, Myeloproliferative neoplasms (MPN) refer to a group of heterogenous disorders characterized by overproduction of one or more types of blood cells. MPNs include polycythemia vera, essential thrombocythemia, chronic myeloid leukemia, primary myelofibrosis, chronic neutrophilic leukemia, and other less well defined entities such as chronic eosinophilic leukemia, not otherwise categorized.
A third category, Myelodysplastic/myeloproliferative neoplasms (MDS/MPN), include disorders that manifest both dysplastic and proliferative features. These include chronic myelomonocytic leukemia, juvenile myelomonocytic leukemia, atypical CML (aCML, BCR-ABL1 negative), MDS/MPN with ring sideroblasts and thrombocytosis, and unclassifiable MDS/MPN.
MyProstateScore (Mi-Prostate Score [MiPS]) and MyProstateScore 2.0
The MiPS assay is a multiplex analysis of TMPRSS2:ERG (T2:ERG) gene fusion, post-DRE urine expression of PCA3, and serum PSA (KLK3). The MiPS assay tests for the presence of two prostate cancer biomarkers: a piece of RNA made from the PCA3 gene, found to be overactive in 95 percent of all prostate cancers, and another RNA marker that is found only when TMPRSS2 and ERG abnormally fuse. TMPRSS2:ERG, or T2-ERG, is a strong indicator of prostate cancer. The MiPS test is not meant to be used alone as a prostate cancer screening tool, nor is it intended to replace PSA. The Mi-Prostate Score test is designed to provide additional information for patients who have undergone PSA testing. The performance in men who have not undergone PSA testing is unknown.
Salami et al (2013) sought to develop a clinical algorithm combining serum PSA with detection of TMPRSS2:ERG fusion and PCA3 in urine collected after digital rectal exam (post-DRE urine) to predict prostate cancer on subsequent biopsy. Post-DRE urine was collected in 48 consecutive patients before prostate biopsy at 2 centers; quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect PCA3 and TMPRSS2:ERG fusion transcript expression. Serum PSA was measured by clinical assay. The performance of TMPRSS2:ERG fusion, PCA3, and serum PSA as biomarkers predicting prostate cancer at biopsy was measured; a clinically practical algorithm combining serum PSA with TMPRSS2:ERG and PCA3 in post-DRE urine to predict prostate cancer was developed. Post-DRE urine sediment provided informative RNA in 45 patients; prostate cancer was present on subsequent biopsy in 15. TMPRSS2:ERG in post-DRE urine was associated with prostate cancer (OR = 12.02; P < 0.001). PCA3 had the highest sensitivity in predicting prostate cancer diagnosis (93%), whereas TMPRSS2:ERG had the highest specificity (87%). TMPRSS2:ERG had the greatest discriminatory value in predicting prostate cancer (AUC = 0.77 compared with 0.65 for PCA3 and 0.72 for serum PSA alone). Combining serum PSA, PCA3, and TMPRSS2:ERG in a multivariable algorithm optimized for clinical utility improved cancer prediction (AUC = 0.88; specificity = 90% at 80% sensitivity). The authors concluded that a clinical algorithm specifying biopsy for all patients with PSA ≥ 10 ng/ml, while restricting biopsy among those with PSA <10 ng/ml to only those with detectable PCA3 or TMPRSS2:ERG in post-DRE urine, performed better than the individual biomarkers alone in predicting prostate cancer.
Tomlins et al (2016) state TMPRSS2:ERG (T2:ERG) and prostate cancer antigen 3 (PCA3) are the most advanced urine-based prostate cancer (PCa) early detection biomarkers. The authors aimed to validate logistic regression models, termed Mi-Prostate Score (MiPS), that incorporate serum prostate-specific antigen (PSA; or the multivariate Prostate Cancer Prevention Trial risk calculator version 1.0 [PCPTrc]) and urine T2:ERG and PCA3 scores for predicting PCa and high-grade PCa on biopsy. T2:ERG and PCA3 scores were generated using clinical-grade transcription-mediated amplification assays. Pretrained MiPS models were applied to a validation cohort of whole urine samples prospectively collected after digital rectal examination from 1244 men presenting for biopsy. Area under the curve (AUC) was used to compare the performance of serum PSA (or the PCPTrc) alone and MiPS models. Decision curve analysis (DCA) was used to assess clinical benefit. Among informative validation cohort samples (n=1225 [98%], 80% from patients presenting for initial biopsy), models incorporating T2:ERG had significantly greater AUC than PSA (or PCPTrc) for predicting PCa (PSA: 0.693 vs 0.585; PCPTrc: 0.718 vs 0.639; both p<0.001) or high-grade (Gleason score >6) PCa on biopsy (PSA: 0.729 vs 0.651, p<0.001; PCPTrc: 0.754 vs 0.707, p=0.006). MiPS models incorporating T2:ERG score had significantly greater AUC (all p<0.001) than models incorporating only PCA3 plus PSA (or PCPTrc or high-grade cancer PCPTrc [PCPThg]). DCA demonstrated net benefit of the MiPS_PCPTrc (or MiPS_PCPThg) model compared with the PCPTrc (or PCPThg) across relevant threshold probabilities. The authors concluded that incorporating urine TMPRSS2:ERG (T2:ERG) and PCA3 scores improves the performance of serum PSA (or PCPTrc) for predicting PCa and high-grade PCa on biopsy. The authors noted that limitations of this study, which included included the use of PCPTrc_v1 and PCPThg_v1, rather than updated version 2 (v2) risk calculators, because the MiPS models were locked for subsequent validation studies prior to PCPT_v2 risk calculator development. Of note, PCPTrc_v2 and PCPThg_v2 were poorly calibrated in the validation cohort, with no significant difference in AUCs compared with version 1 (PCPThg_v1 showed significantly increased AUC compared to PCPThg_v2). In addition, the authors observed greater improvement for predicting all cancers, compared with high-grade cancer only, when incorporating T2:ERG plus PCA3 scores. Although overdiagnosis of low-grade cancer drives overtreatment, whether these models show utility in identifying the subset of patients with low-grade cancer who harbor undiagnosed higher grade cancer ( approximately 20–40%) or can be combined with novel imaging or tissue-based prognostic tests should be investigated. Of note, tissue and urine assessment of PCA3 and/or T2:ERG have been variably associated with significant disease and progression, supporting the need for additional investigation in these settings. Last, the validation cohort consisted of men without cancer undergoing biopsy based on current standard of care (i.e., elevated serum PSA), so conclusions regarding performance in men on active surveillance or screening populations is unknown.
Sanda et al (2017) stated potential survival benefits from treating aggressive (Gleason score, ≥7) early-stage prostate cancer are undermined by harms from unnecessary prostate biopsy and overdiagnosis of indolent disease. The objective of their study was to evaluate the a priori primary hypothesis that combined measurement of PCA3 and TMPRSS2:ERG (T2:ERG) RNA in the urine after digital rectal examination would improve specificity over measurement of prostate-specific antigen alone for detecting cancer with Gleason score of 7 or higher. As a secondary objective, to evaluate the potential effect of such urine RNA testing on health care costs. They conducted a prospective, multicenter diagnostic evaluation and validation in academic and community-based ambulatory urology clinics. Participants were a referred sample of men presenting for first-time prostate biopsy without preexisting prostate cancer: 516 eligible participants from among 748 prospective cohort participants in the developmental cohort and 561 eligible participants from 928 in the validation cohort. The interventions included: Urinary PCA3 and T2:ERG RNA measurement before prostate biopsy. The main outcome measure was the presence of prostate cancer having Gleason score of 7 or higher on prostate biopsy. Pathology testing was blinded to urine assay results. In the developmental cohort, a multiplex decision algorithm was constructed using urine RNA assays to optimize specificity while maintaining 95% sensitivity for predicting aggressive prostate cancer at initial biopsy. Findings were validated in a separate multicenter cohort via prespecified analysis, blinded per prospective-specimen-collection, retrospective-blinded-evaluation (PRoBE) criteria. Cost effects of the urinary testing strategy were evaluated by modeling observed biopsy results and previously reported treatment outcomes. Among the 516 men in the developmental cohort (mean age, 62 years; range, 33-85 years) combining testing of urinary T2:ERG and PCA3 at thresholds that preserved 95% sensitivity for detecting aggressive prostate cancer improved specificity from 18% to 39%. Among the 561 men in the validation cohort (mean age, 62 years; range, 27-86 years), analysis confirmed improvement in specificity (from 17% to 33%; lower bound of 1-sided 95% CI, 0.73%; prespecified 1-sided P = .04), while high sensitivity (93%) was preserved for aggressive prostate cancer detection. Forty-two percent of unnecessary prostate biopsies would have been averted by using the urine assay results to select men for biopsy. Cost analysis suggested that this urinary testing algorithm to restrict prostate biopsy has greater potential cost-benefit in younger men. The authors concluded that combined urinary testing for T2:ERG and PCA3 can avert unnecessary biopsy while retaining robust sensitivity for detecting aggressive prostate cancer with consequent potential health care cost savings.
The NCCN Prostate Cancer Early Detection Guidelines (v.2.2019) states that rearrangements of the ERG gene are found in approximately half of prostate cancers and early studies suggested that the combination of TMPRSS2:ERG (T2:ERG) gene fusion and PCA3 improved the prediction of prostate cancer on biopsy (Tomlinson 2016; Sanda 2017). Based on these early results, the NCCN panel considers MiPS to be investigational at present time, but will review additional information as it becomes available.
Newcomb et al (2019) stated for men on active surveillance for prostate cancer, biomarkers may improve prediction of reclassification to higher grade or volume cancer. This study examined the association of urinary PCA3 and TMPRSS2:ERG (T2:ERG) with biopsy-based reclassification. Urine was collected at baseline, 6, 12, and 24 months in the multi-institutional Canary Prostate Active Surveillance Study (PASS), and PCA3 and T2:ERG levels were quantitated. Reclassification was an increase in Gleason score or ratio of biopsy cores with cancer to ≥34%. The association of biomarker scores, adjusted for common clinical variables, with short- and long-term reclassification was evaluated. Discriminatory capacity of models with clinical variables alone or with biomarkers was assessed using receiver operating characteristic (ROC) curves and decision curve analysis (DCA). Seven hundred and eighty-two men contributed 2069 urine specimens. After adjusting for PSA, prostate size, and ratio of biopsy cores with cancer, PCA3 but not T2:ERG was associated with short-term reclassification at the first surveillance biopsy (OR = 1.3; 95% CI 1.0-1.7, p = 0.02). The addition of PCA3 to a model with clinical variables improved area under the curve from 0.743 to 0.753 and increased net benefit minimally. After adjusting for clinical variables, neither marker nor marker kinetics was associated with time to reclassification in subsequent biopsies. The authors stated that studies evaluating the use of PCA3 in active surveillance have been limited and sample sizes have been small. In the current study, which is the largest study to date of PCA3 in men using active surveillance, after adjustment for clinical variables available after cancer diagnosis, the authors found a significant association of PCA3 with reclassification at the sBx1 (adjusted OR = 1.3, p = 0.02), but not for subsequent biopsies (adjusted OR = 1.01, p = 0.96). Although they found no association between T2:ERG and biopsy reclassification, some studies have suggested improved performance when PCA3 and T2:ERG are used in combination or combined into a MiPS score for the initial diagnosis of PCa. Thus, the authors combined PCA3 and T2:ERG into a MIPS score, but found little or no improvement over PCA3 alone. The authors concluded that PCA3 but not T2:ERG was associated with cancer reclassification in the first surveillance biopsy but has negligible improvement over clinical variables alone in ROC or DCA analyses. Neither marker was associated with reclassification in subsequent biopsies.
Lebastchi et al (2020) evaluated the association of the MyProstateScore (MPS) urine test on the decision to undergo biopsy in men referred for prostate biopsy in urology practice. MPS testing was offered as an alternative to immediate biopsy in men referred to the University of Michigan for prostate biopsy from October 2013 through October 2016. The primary endpoint was the decision to perform biopsy. The proportion of patients undergoing biopsy was compared to predicted risk scores from the Prostate Cancer Prevention Trial risk calculator (PCPTrc). Analyses were stratified by the use of multiparametric magnetic resonance imaging (mpMRI). The associations of PCPTrc, MPS, and mpMRI with the decision to undergo biopsy were explored in a multivariable logistic regression model. Of 248 patients, 134 (54%) proceeded to prostate biopsy. MPS was significantly higher in biopsied patients (29 vs14, p < .001). The use of biopsy was strongly associated with MPS, with biopsy rates of 26%, 38%, 58%, 90%, and 85% in the first through fifth quintiles, respectively (p < .001). MPS association with biopsy persisted upon stratification by mpMRI. On multivariable analysis, MPS was strongly associated with the decision to undergo biopsy when modeled as both a continuous (odds ratio [OR] 1.05, 95%; confidence interval [CI] 1.04-1.08; <.001) and binary (OR 7.76, 95%; CI 4.14-14.5; p < .001) variable. The authors concluded that 46% of patients undergoing clinical MPS testing as an alternative to immediate prostate biopsy were able to avoid biopsy. Increasing MPS was strongly associated with biopsy rates. The authors report these findings were robust to use of mpMRI. The authors acknowledge limitations to their study.
First, these data reflect a study period in which MPS thresholds for clinical use were not proposed or validated, and MPS results were provided across a range from 0 to 100. Thus, there was no objective measure of a “negative” or “low-risk” MPS, and the decision to pursue biopsy was ultimately based on discretion. Therefore, the association of MPS with the primary outcome was based on numerical categorization (i.e., quintiles). Second, the study was single-arm with no comparison cohort without MPS testing. Third, while measures of the proportion biopsied imply a baseline 100% biopsy rate in the absence of MPS or mpMRI, it is likely that other considerations could have reduced use of biopsy in the absence of MPS. Finally, the pathology of patients who did not undergo biopsy is unknown, thus the current study should not be considered a robust assessment of clinical validity. As this was not the primary objective, it is nonetheless notable that the limited assessment of validity performed herein was consistent with existing data.
Mi-Prostate (MiPS) was renamed MyProstate score in 2021.
The National Comprehensive Cancer Network Clinical Practice Guidelines on “Prostate cancer early detection” (Version 2.2024) states that MyProstateScore (MPS) can be considered for patients prior to biopsy and for those with prior negative biopsy who are thought to be at higher risk for clinically significant prostate cancer. However, the extent of validation of this test across diverse populations is variable, and it is not yet known how such a test could be applied in optimal combination with MRI.
MyProstateScore 2.0 (Lynx DX) is a next-generation prostate cancer risk assessment test that evaluates the activity (gene expression) of 18 genes (includes novel gene fusion, T2:ERG) from a first-catch urine specimen collected post-digital rectal examination (post-DRE). An algorithmic analysis of the findings reports a probability score for the likelihood that the patient has clinically significant prostate cancer grade Group 2 or higher (Grade Group ≥2 or Gleason score ≥7).
Tosoian and colleagues (2024) discussed the development and validation of an 18-gene urine test for high-grade prostate cancer. Their objective was to develop a multiplex urinary panel for high-grade prostate cancer (PCa) and validate its external performance relative to current guideline-endorsed biomarkers. They hypothesized that augmenting the prior generation of cancer-associated biomarkers with novel molecules selectively expressed by high-grade, aggressive cancers would improve testing accuracy. The original MyProstateScore (MPS) test incorporates prostate cancer antigen 3 (PCA3) and TMPRSS2:ERG gene fusion expression with serum PSA level to estimate risk of high-grade cancers. To derive a gene panel for high-grade cancers, the authors performed differential expression analysis of 58 724 genetic targets in multi-institutional RNA sequencing data, which identified 54 markers of PCa, including 17 markers uniquely overexpressed by high-grade cancers. Gene expression and clinical factors were modeled in a new urinary test for high-grade PCa (MyProstateScore 2.0 [MPS2]). Optimal models were developed in parallel without prostate volume (MPS2) and with prostate volume (MPS2+). The locked models underwent blinded external validation in a prospective National Cancer Institute trial cohort. Data were collected from January 2008 to December 2020, and data were analyzed from November 2022 to November 2023. Multiple biomarker tests were assessed in the validation cohort, including serum PSA alone, the Prostate Cancer Prevention Trial risk calculator, and the Prostate Health Index (PHI) as well as derived multiplex 2-gene and 3-gene models, the original 2-gene MPS test, and the 18-gene MPS2 models. Under a testing approach with 95% sensitivity for PCa of GG 2 or greater, measures of diagnostic accuracy and clinical consequences of testing were calculated. Cancers of GG 3 or greater were assessed secondarily. Of 761 men included in the development cohort, the median (IQR) age was 63 (58-68) years, and the median (IQR) PSA level was 5.6 (4.6-7.2) ng/mL; of 743 men included in the validation cohort, the median (IQR) age was 62 (57-68) years, and the median (IQR) PSA level was 5.6 (4.1-8.0) ng/mL. In the validation cohort, 151 (20.3%) had high-grade PCa on biopsy. Area under the receiver operating characteristic curve values were 0.60 using PSA alone, 0.66 using the risk calculator, 0.77 using PHI, 0.76 using the derived multiplex 2-gene model, 0.72 using the derived multiplex 3-gene model, and 0.74 using the original MPS model compared with 0.81 using the MPS2 model and 0.82 using the MPS2+ model. At 95% sensitivity, the MPS2 model would have reduced unnecessary biopsies performed in the initial biopsy population (range for other tests, 15% to 30%; range for MPS2, 35% to 42%) and repeat biopsy population (range for other tests, 9% to 21%; range for MPS2, 46% to 51%). Across pertinent subgroups, the MPS2 models had negative predictive values of 95% to 99% for cancers of GG 2 or greater and of 99% for cancers of GG 3 or greater. The authors state that in this study, a new 18-gene PCa test had higher diagnostic accuracy for high-grade PCa relative to existing biomarker tests, and that these data support use in patients with elevated PSA levels to reduce the potential harms of screening while preserving its long-term benefits. The authors acknowledged limitations to their study. For one, there was limited racial diversity in the study population. Second, the reference standard was systematic biopsy, which is subject to undersampling that could increase NPV and decrease positive predictive value relative to surgical pathology. Repeated model development in patients with more definitive pathologic data (eg, radical prostatectomy), and prostatectomy-derived MPS2 models did not differ substantially. Although the derived multiplex models capture the components of other commercially available tests, these models should not be interpreted as equivalent to the commercial assays, just as no conclusions can be drawn regarding biomarkers not assessed. This study population was not suitable for comparing biomarkers with mpMRI, which remains a critical knowledge gap. The authors are currently conducting a prospective multicenter trial for this assessment. Regardless, the externally validated performance of MPS2 supports its effectiveness in accurately ruling out the need for mpMRI and biopsy altogether; however, additional studies are needed to corroborate these data and confirm the observed positive impact of MPS2 testing on longer-term outcomes.
Wei and colleagues (2023a, 2023b) discuss considerations for a prostate biopsy in the 2023 American Urological Association and Society of Urologic Oncology (AUA/SUO) guidelines. The authors state that “while there are a plethora of serum, urine, tissue, and imaging biomarkers to assess the likelihood of high-grade prostate cancer, there is little knowledge on comparative effectiveness, how they may complement or supplement each other, and how various stepwise algorithms perform. Considerable research is required to achieve the goal of a highly effective, practical, scalable, and widely available approach”. “In a recent study, MPS was shown to be significantly associated with GG2+ cancer across all PI-RADS scores inclusive of PI-RADS 3 lesions. Pending future prospective validation studies, biomarkers may augment mpMRI for identifying patients for prostate biopsy especially in patients with negative or equivocal mpMRI findings but with ongoing suspicion for GG2+ cancer.” The PSA blood test remains the first-line screening test of choice based on randomized trials of PSA-based screening showing reductions in metastasis and prostate cancer death.
In a AUA/SUO (2023) guideline statement, a conditional recommendation with Grade C evidence (low level of certainty) states that clinicians may use adjunctive urine or serum markers when further risk stratification would influence the decision regarding whether to proceed with biopsy. Wei et al acknowledge that it is debatable which of the newer biomarkers (alone or in combination) is best, as comparative studies are sparse.
The National Comprehensive Cancer Network Biomarkers Compendium (NCCN, 2024) for “Prostate cancer early detection” and “Prostate cancer” do not include recommendations for the second generation MyProstateScore 2.0 test.
NavDx for Surveillance of Cancer Recurrence in HPV-Associated Oropharyngeal Cancer
In a prospective, multi-center study, Chera et al (2019) identified a profile of circulating tumor human papilloma virus (HPV) DNA (ctHPVDNA) clearance kinetics that is associated with disease control following chemoradiotherapy (CRT) for HPV-associated oropharyngeal squamous cell carcinoma (OPSCC). This trial was carried out in 103 patients with p16-positive OPSCC; M0 disease; and receipt of definitive CRT. Blood specimens were collected at baseline, weekly during CRT, and at follow-up visits. Optimized multi-analyte digital PCR assays were used to quantify ctHPVDNA (types 16/18/31/33/35) in plasma. A control cohort of 55 healthy volunteers and 60 patients with non-HPV-associated malignancy was also analyzed. Baseline plasma ctHPVDNA had high specificity (97 %) and high sensitivity (89 %) for detecting newly diagnosed HPV-associated OPSCC. Pre-treatment ctHPV16DNA copy number correlated with disease burden, tumor HPV copy number, and HPV integration status. These investigators defined a ctHPV16DNA favorable clearance profile as having high baseline copy number (greater than 200 copies/ml) and greater than 95 % clearance of ctHPV16DNA by day 28 of CRT; 19 of 67 evaluable patients had a ctHPV16DNA favorable clearance profile, and none had persistent or recurrent regional disease after CRT. In contrast, patients with adverse clinical risk factors (T4 or greater than 10 total pack years [TPY]) and an unfavorable ctHPV16DNA clearance profile had a 35 % actuarial rate of persistent or recurrent regional disease after CRT (p = 0.0049). The authors concluded that a rapid clearance profile of ctHPVDNA may predict likelihood of disease control in patients with HPV-associated OPSCC patients treated with definitive CRT and may be useful in selecting patients for de-intensified therapy. These researchers stated that future studies assessing ctHPVDNA as an integral biomarker to guide treatment de-intensification are needed; and may facilitate personalized treatment decisions based on tumor biology in addition to clinical risk factors. Finally, prospective evaluation of ctHPVDNA as a biomarker in other HPV-associated malignancies (e.g., cervical and anal cancers) should be evaluated.
The authors stated that although this study included over 100 patients, it was under-powered due to low occurrence of disease persistence/recurrence and limited follow-up. Despite these limitations, these investigators observed an early trend in worse regional DFS (RDFS) in patients with greater than 10 TPY and unfavorable ctHPVDNA kinetics. Validation of these findings using a clinical-grade test in independent and larger patient cohorts is needed to confirm these findings.
Chera et al (2020) noted that plasma ctHPVDNA is a sensitive and specific biomarker of HPV-associated OPSCC. In a prospective, clinical trial, these investigators examined if longitudinal monitoring of ctHPVDNA during post-treatment surveillance could accurately detect clinical disease recurrence. This study was carried out in patients with non-metastatic HPV-associated (p16-positive) OPSCC. All patients were treated with curative-intent CRT. Patients underwent a 3-month post-CRT positron emission tomography/computed tomography (PET/CT) scan and were thereafter clinically evaluated every 2 to 4 months (years 1 to 2), then every 6 months (years 3 to 5). Chest imaging was carried out every 6 months. Blood specimens were collected every 6 to 9 months for analysis of plasma ctHPVDNA using a multi-analyte digital PCR assay. The primary endpoint was to estimate the NPV and PPV of ctHPVDNA surveillance. A total of 115 patients were enrolled, and 1,006 blood samples were analyzed. After a median follow-up time of 23 months (range of 6.1 to 54.7 months), 15 patients (13 %) developed disease recurrence; 87 patients had undetectable ctHPVDNA at all post-treatment time-points, and none developed recurrence (NPV, 100 %; 95 % CI: 96 % to 100 %); 28 patients developed a positive ctHPVDNA during post-treatment surveillance, 15 of whom were diagnosed with biopsy-proven recurrence; 16 patients had 2 consecutively positive ctHPVDNA blood tests, 15 of whom developed biopsy-proven recurrence. Two consecutively positive ctHPVDNA blood tests had a PPV of 94 % (95 % CI: 70 % to 99 %). Median lead time between ctHPVDNA positivity and biopsy-proven recurrence was 3.9 months (range of 0.37 to 12.9 months). The authors concluded that post-treatment surveillance of cancer recurrence with plasma ctHPVDNA monitoring has exceptional NPV (100 %) and PPV (94 %) when using 2 consecutively positive ctHPVDNA tests as the criterion for positivity. Patients who have undetectable ctHPVDNA during clinical follow-up are unlikely to have recurrent disease and may be spared routine radiographic and in-office nasopharyngo-laryngoscopic surveillance. These researchers stated that future studies should examine if ctHPVDNA-based monitoring may help to reduce the financial toxicity of cancer survivorship and whether earlier detection of cancer relapse improves post-recurrence survival outcomes.
The authors stated that whether earlier detection of disease recurrence may positively impact survival outcomes in HPV-associated OPSCC remains an open question. ctHPVDNA-based monitoring may result in a higher rate of identifying oligo-recurrence and potentially a higher rate of salvage with surgery or radiotherapy. Therefore, clinical trials of early intervention after ctHPVDNA-based detection are needed. The findings described in this study were specific for the optimized multi-analyte ctHPVDNA assay and may not apply to alternative ctHPVDNA assays. In addition, detection of 2 consecutively positive ctHPVDNA tests to improve the PPV was not prospectively defined and warrants further validation. Consistent with NCCN guidelines, there is no “gold standard” for surveillance imaging. Therefore, these researchers opted to use a more stringent criterion of biopsy-proven recurrence to define a change in disease status. A shorter interval between ctHPVDNA testing (e.g., every 3 months) would more precisely define the extent of earlier detection. Several heterogenous clinical factors (intensity of treatment, tobacco pack-years, use of chemotherapy) may have impacted the pattern and frequency of disease recurrence in this study. Although this trial showed feasibility of ctHPVDNA-based surveillance for both distant and local-regional recurrences, more research is needed to examine potential differences in detection efficiency.
Misawa et al (2020) noted that HPV-associated oropharyngeal cancer (OPC) is an independent tumor type with regard to cellular, biological, and clinical features. The use of non-invasive biomarkers such as ctDNA may be relevant in early diagnosis and eventually improve the outcomes of patients with HNSCC. Genome-wide discovery using RNA sequencing and reduced representation bi-sulfite sequencing yielded 21 candidates for methylation-targeted genes. A verification study (252 HNSCC patients) using quantitative methylation-specific PCR (Q-MSP) identified 10 genes (ATP2A1, CALML5, DNAJC5G, GNMT, GPT, LY6D, LYNX1, MAL, MGC16275, and MRGPRF) that showed a significant increase recurrence in methylation groups with OPC. Further investigation on ctDNA using Q-MSP in HPV-associated OPC showed that 3 genes (CALML5, DNAJC5G, and LY6D) had a high predictive ability as emerging biomarkers for a validation set, each capable of discriminating between the plasma of the patients from healthy individuals. Among the 42 ctDNA samples, methylated CALML5, DNAJC5G, and LY6D were observed in 31 (73.8 %), 19 (45.2 %), and 19 (45.2 %) samples, respectively. Among pre-treatment ctDNA samples, methylated CALML5, DNAJC5G, and LY6D were observed in 8/8 (100 %), 7/8 (87.5 %), and 7/8 (87.5 %) samples, respectively. Methylated CALML5, DNAJC5G, and LY6D were found in 2/8 (25.0 %), 0/8 (0 %), and 1/8 (12.5 %) of the final samples in the series, respectively. The authors presented the relationship between the methylation status of 3 specific genes and cancer recurrence for risk classification of HPV-associated OPC cases. These researchers concluded that ctDNA analysis has the potential to aid in determining patient prognosis and real-time surveillance for disease recurrences and could serve as an alternative method of screening for HPV-associated OPC.
The authors stated that currently the lack of an effective OPC screening program is because there are no identified OPC precursor lesions. The HPV-associated carcinogenesis initially arises in tonsillar crypts, which may be the most likely reason why the HPV prevalence in tonsillar cells is higher. In the future, these researchers think that highly sensitive and tissue-specific ctDNA biomarkers of HPV-associated OPC will be discovered, allowing early detection. This study, entailing human specimens and high-throughput profiling platforms, may be susceptible to measurement bias from various sources; accordingly, the use of methylation markers in clinical practice requires further testing in prospective studies with larger HNSCC cohorts. Finally, these efforts will lead to the identification of new oncological biomarkers for early detection and outcome prediction, which is a pre-requisite for realizing the advantages of precision medicine. This trial demonstrated the use of using parallel serial assessment of ctDNA methylation in the treatment evaluation of HPV-associated OPC.
Wuerdemann et al (2020) noted that g lobal incidences of OPSCC are rising due to an association with high-risk HPV. Although there is an improved OS of HPV-related OPSCC; up to 25 % of the patients develop recurrent or distant metastatic disease with a fatal outcomes. Biomarkers to monitor this disease are not established. In a meta-analysis, these investigators examined the role of cell-free HPV DNA in liquid biopsy (LB) as a biomarker for HPV-related OPSCC. PubMed, Livivo, and Cochrane Library databases were searched from inception to August, 2020. All studies were analyzed by Meta-DiSc 1.4 and Stata 16.0 statistical software. A total of 16 studies were considered for systematic review, whereas 11 studies met inclusion criteria for meta-analysis, respectively. Pooled sensitivity of cfHPV-DNA at first diagnosis and during follow-up was 0.81 (95 % CI: 0.78 to 0.84) and 0.73 (95 % CI: 0.57 to 0.86), while pooled specificity was 0.98 (95 % CI: 0.96 to 0.99) and 1 (95 % CI: 0.99 to 1.00). The DOR at first diagnosis was 200.60 (95 % CI: 93.31 to 431.22) and 300.31 (95 % CI: 60.94 to 1479.88) during follow-up. The AUC of SROC was 0.99 at first diagnosis, and 1.00 during follow-up, respectively. The authors concluded that cfHPV-DNA in the blood of patients with HPV-related OPSCC presented a potential biomarker with high specificity at first diagnosis and during follow-up. Testing for cfHPV-DNA proved to be a promising application of liquid biopsy for early detection of primary OPSCC in high-risk groups such as immune-deficient patients. Moreover, these researchers stated that heterogeneity of sensitivity and NLR could be explained by different specimens and methods used for the detection of cfHPV-DNA, as well as variability in the estimation of HPV status in the primary. They noted that although publication bias was ruled out, the findings of this analyses suggested that additional studies with larger sample sizes and homogeneous study protocols are needed to increase sensitivity and to further examine proof diagnostic accuracy in patients with HPV-related OPSCC.
The authors stated that the drawbacks of this meta-analysis were the relatively small number of studies included with somewhat low patient and control numbers. In addition, these investigators only included studies written in the English language, which could yield selection bias for the language and populations studied. Furthermore, there was a high variability throughout study settings and material and methods used for the detection of cfHPV-DNA, which restricted the liability of conclusions drawn. These researchers stated that although publication bias was not significant and subgroup analysis was carried out to examine the cause of heterogeneity, the inclusion of only a few factors left the risk of not taking other relevant ones into account. Once a higher number of studies is available, a more thorough evaluation of the cause of heterogeneity will be possible to strengthen the role of cfHPV-DNA as a biomarker in HPV-related OPSCC.
Berger et al (2022) stated that despite generally favorable outcomes, 15 % to 25 % of patients with HPV-driven OPSCC will have recurrence. Current post-treatment surveillance practices rely on physical examinations and imaging and are inconsistently applied. In a retrospective, clinical case-series study, these researchers examined circulating tumor tissue modified viral (TTMV)-HPV DNA obtained during routine post-treatment surveillance among a large population of real-world patients. This trial included 1,076 consecutive patients across 108 U.S. sites who were 3 or more months post-treatment for HPV-driven OPSCC and who had 1 or more TTMV-HPV DNA tests (NavDx, Naveris Laboratories) obtained during surveillance between February 6, 2020, and June 29, 2021. Test results were compared with subsequent clinical evaluations. Circulating TTMV-HPV DNA was positive in 80 of 1,076 (7.4 %) patients, with follow-up available on all. At 1st positive surveillance testing, 21 of 80 (26 %) patients had known recurrence while 59 of 80 (74 %) patients were not known to have recurrent disease. Among these 59 patients, 55 (93 %) subsequently had a confirmed recurrence, 2 patients had clinically suspicious lesions, and 2 had clinically "no evidence of disease" (NED) at last follow-up. To-date, the overall PPV of TTMV-HPV DNA testing for recurrent disease was 95 % (n = 76/80). Furthermore, the point-in-time NPV was 95 % (n = 1,198/1,256). The authors concluded that these findings highlighted the clinical potential for circulating TTMV-HPV DNA testing in routine practice. As a surveillance tool, TTMV-HPV DNA positivity was the 1st indication of recurrence in the majority of cases, pre-dating identification by routine clinical and imaging exams. These researchers stated that these findings may inform future clinical and guideline-endorsed strategies for HPV-driven malignancy surveillance.
The authors stated that this study had several drawbacks. First, the majority of patients (78 %) had a single surveillance test result obtained during the study period with more than half (55 %) greater than 12 months from completion of therapy. Sequential values obtained in this cohort over time would be of interest, as would baseline or pre-treatment and on-treatment test results. The vast majority of positive or detectable cases reflected HPV subtype 16 disease (93 %); therefore, further investigation is needed to validate these findings in non-HPV16 high-risk subtypes, although these investigators expect similar findings. Second, as this was a cross-sectional analysis of the NPV of the test, whether a negative TTMV-HPV DNA result remains predictive of the absence of recurrence 3, 6, or 12 months after a negative blood test is currently unknown. Further follow-up is needed to clarify this point and help further refine the role of TTMV-HPV DNA testing in the surveillance setting. Third, the length of time a negative test remains predictive is the subject of continued study as the cohort ages.
An accompanying commentary (Colevas, 2022) stated that "Lack of prospectively planned follow-up and minimal characterization of the patient population studied complicate interpretation of circulating human papillomavirus (HPV) DNA as a prognostic biomarker for patients with HPV-associated oropharyngeal carcinoma treated with curative intent. Controlled clinical trials with embedded biomarkers will be necessary to optimize the utility of circulating tumor HPV DNA assays".
Rettig et al (2022) stated that circulating tumor tissue-modified viral (TTMV) HPV DNA is a dynamic, clinically relevant biomarker for HPV-positive oropharyngeal squamous cell carcinoma (SCC). Reasons for its wide pre-treatment inter-patient variability are not well understood. In a cross-sectional study, these researchers characterized clinicopathologic factors associated with TTMV HPV DNA. This study included patients evaluated for HPV-positive oropharyngeal SCC at Dana-Farber Cancer Institute (Boston, MA) between December 2019 and January 2022 and who were undergoing curative-intent treatment. Clinicopathologic characteristics included demographic variables, tumor and nodal staging, HPV genotype, and imaging findings. Main outcome measures entailed pre-treatment circulating TTMV HPV DNA from 5 genotypes (16, 18, 31, 33, and 35) assessed using a commercially available digital droplet polymerase chain reaction (PCR)-based assay, considered as either detectable/undetectable or a continuous score (fragments/ml). Among 110 included patients, 96 were men (87 %) and 104 were White (95 %), with a mean (SD) age of 62.2 (9.4) years. Circulating TTMV HPV DNA was detected in 98 patients (89 %), with a median (inter-quartile range [IQR]) score of 315 (47 to 2,686) fragments/ml (range of 0 to 60,061 fragments/ml). Most detectable TTMV HPV DNA was genotype 16 (n = 86 [88 %]), while 12 patients (12 %) harbored other genotypes. Circulating TTMV HPV DNA detection was most strongly associated with clinical N stage. Although few patients had clinical stage N0 disease, only 4 of these 11 patients (36 %) had detectable DNA compared with 94 of 99 patients (95 %) with clinical stage N1 to N3 disease (proportion difference, 59 %; 95 % confidence interval [CI]: 30 % to 87 %). Among patients with undetectable TTMV HPV DNA, more than half (7 of 12 [58 %]) had clinical stage N0 disease. The TTMV HPV DNA prevalence and score increased with progressively higher clinical nodal stage, diameter of largest lymph node, and higher nodal maximum standardized uptake value on positron emission tomography/computed tomography (PET/CT). In multi-variable analysis, clinical nodal stage and nodal maximum standardized uptake value were each strongly associated with TTMV HPV DNA score. Among 27 surgically treated patients, more patients with than without lympho-vascular invasion had detectable TTMV HPV DNA (12 of 12 [100 %] versus 9 of 15 [60 %]). The authors concluded that in this cross-sectional study, circulating TTMV HPV DNA was statistically significantly associated with nodal disease at HPV-positive OPSCC diagnosis. The few patients with undetectable levels had predominantly clinical stage N0 disease, suggesting assay sensitivity for diagnostic purposes may be lower among patients without cervical lymphadenopathy. These researchers stated that mechanisms underlying this association, and the use of this biomarker for surveillance of patients with undetectable baseline values, warrant further investigation.
Gunning et al (2023) noted that the blood test analyzes tumor tissue modified viral (TTMV)-HPV DNA to provide a reliable means of detecting and monitoring HPV-driven cancers. The test has been clinically validated in a large number of independent studies and has been integrated into clinical practice by over 1,000 healthcare providers at over 400 medical sites in the U.S. This Clinical Laboratory Improvement Amendments (CLIA), high complexity laboratory developed test, has also been accredited by the College of American Pathologists (CAP) and the New York State Department of Health. These investigators reported a detailed analytical validation of the NavDx assay, including sample stability, specificity as measured by limits of blank (LOBs), and sensitivity illustrated via limits of detection and quantitation (LODs and LOQs). LOBs were 0 to 0.32 copies/μL, LODs were 0 to 1.10 copies/μL, and LOQs were less than 1.20 to 4.11 copies/μL, demonstrating the high sensitivity and specificity of data provided by NavDx. In-depth evaluations including accuracy and intra- and inter-assay precision studies were shown to be well within acceptable ranges. Regression analysis revealed a high degree of correlation between expected and effective concentrations, demonstrating excellent linearity (R2 = 1) across a broad range of analyte concentrations. The authors concluded that these findings showed that NavDx accurately and reproducibly detects circulating TTMV-HPV DNA, which has been shown to aid in the diagnosis and surveillance of HPV-driven cancers. It should be noted that all authors are employed by Naveris, Inc. and may hold equity or stock options in Naveris, Inc. S.K. is listed as an inventor on patents.
Hanna et al (2023) noted that HPV is causally linked to OPSCC. Consensus guidelines recommend clinical examinations and imaging in decreasing frequency as part of post-treatment surveillance for recurrence. Plasma TTMV-HPV DNA testing has emerged as a biomarker that could inform disease status during surveillance. In a retrospective, observational, cohort study, these researchers determined the NPV of TTMV-HPV DNA for recurrence when matched to physician-reported clinical outcome data (median follow-up time: 27.9 months; range of 4.5 to 154 months). This trial entailed 543 patients who completed curative-intent therapy for HPV-associated OPSCC between February 2020 and January 2022 at 8 U.S. cancer care institutions. The cohort included mostly men with a median age of 61 who had loco-regionally advanced disease. HPV status was determined by p16 positivity in 87 % of patients, with a positive HPV PCR/ISH among 55 %; while pre-treatment TTMV-HPV DNA status was unknown for most (79 %) patients. Patients had a mean of 2.6 tests and almost 50 % had 3 or more TTMV-HPV DNA results during surveillance. The per-test and per-patient sensitivity of the assay was 92.5 % (95 % CI: 87.5 to 97.5) and 87.3 % (95 % CI: 79.1 to 95.5), respectively. The NPV for the assay was 99.4 % (95 % CI: 98.9 to 99.8) and 98.4 % (95 % CI: 97.3 to 99.5), respectively. The authors concluded that TTMV-HPV DNA surveillance testing yielded few false negative results and few missed recurrences. These data could inform decisions on when to pursue reimaging following 1st disease re-staging and could inform future surveillance practice. Moreover, these researchers stated that further investigation of how pre-treatment TTMV-HPV DNA status would impact sensitivity for recurrence is needed.
The authors stated that the findings of this study further clarified the potential clinical utility of TTMV-HPV DNA as a blood-based tumor biomarker that can be incorporated into clinical practice for HPV OPSCC disease-related surveillance. Moreover, these investigators acknowledged the drawbacks of retrospective, observational studies; however, these real-world data reflected an important geographically diverse, multi-institutional cohort with a large sample representative of the broader HPV-positive OPSCC population across the U.S.
Zhang et al (2023) noted that plasma HPV circulating tumor DNA (ctDNA) analyses provided a promising, minimally invasive approach to examine tumor burden in patients with HPV-related oropharynx SCC (OPSCC). The authors stated that this case demonstrated the utility of using plasma ctDNA testing for early diagnosis of oligometastatic disease, which was able to be treated with surgical resection. Moreover, these researchers stated that further prospective studies are needed to guide evidence-based surveillance guidelines for patients with HPV-related OPSCC.
Ferrandino et al (2023) stated that there is growing interest in the use of circulating plasma tumor human papillomavirus (HPV) DNA for diagnosis and surveillance of patients with HPV-associated oropharyngeal squamous cell carcinoma (OPSCC). Recent advances in the assays, combining the identification of circulating HPV tumor DNA and tumor DNA fragment analysis (tumor tissue-modified viral [TTMV]-HPV DNA), have been shown to be highly accurate. However, use of these newer techniques has been limited to small cohort studies and clinical trials. In a retrospective, observational study, these researchers attempted to establish the effectiveness of plasma TTMV-HPV DNA testing in the diagnosis and surveillance of HPV-associated OPSCC in a contemporary clinical setting. This trial included patients with OPSCC who underwent TTMV-HPV DNA testing between April 2020 and September 2022 during the course of routine clinical care. For the diagnosis cohort, patients with at least 1 TTMV-HPV DNA measurement before initiation of primary therapy were included. Patients were included in the surveillance cohort if they had at least 1 TTMV-HPV DNA test carried out after completion of definitive or salvage therapy. Main outcomes and measures included per-test performance metrics, including sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV), for TTMV-HPV DNA testing. Of 399 patients included in the analysis, 163 were in the diagnostic cohort (median [inter-quartile range (IQR)] age, 63 [56 to 68.5] years; 142 [87.1 %] men), and 290 were in the surveillance cohort (median [IQR] age, 63 [57 to 70] years; 237 [81.7 %] men). Of the 163 patients in the diagnostic cohort, 152 (93.3 %) had HPV-associated OPSCC; while 11 (6.7 %) had HPV-negative OPSCC. The TTMV-HPV DNA sensitivity in pre-treatment diagnosis was 91.5 % (95 % CI: 85.8 % to 95.4 % [139 of 152 tests]), and the specificity was 100 % (95 % CI: 71.5 % to 100 % [11 of 11 tests]). In the surveillance cohort, 591 tests conducted in 290 patients were evaluated. A total of 23 patients had molecularly confirmed pathologic recurrences. The TTMV-HPV DNA test demonstrated sensitivity of 88.4 % (95 % CI: 74.9 % to 96.1 % [38 of 43 tests]) and specificity of 100 % (95 % CI: 99.3 % to 100 % [548 of 548 tests]) in detecting the recurrences; PPV was 100 % (95 % CI: 90.7 % to 100 % [38 of 38 tests]), and NPV was 99.1 % (95 % CI: 97.9 % to 99.7 % [548 of 553 tests]). The median (range) lead time from positive TTMV-HPV DNA test to pathologic confirmation was 47 (0 to 507) days. The authors concluded that the findings of this cohort study showed that when evaluated in a clinical setting, the TTMV-HPV DNA assay showed 100 % specificity in both diagnosis and surveillance. However, the sensitivity was 91.5 % for the diagnosis cohort and 88.4 % for the surveillance cohort, signifying that nearly 1 in 10 negative tests among patients with HPV-associated OPSCC was a false negative. These investigators stated that additional research is needed to validate the assay's performance and, if validated, then further research into the implementation of this assay into standard clinical practice guidelines will be required. They stated that prospective studies are needed to determine he optimal timing of testing and how results should inform treatment decisions.
The authors stated that this study had several drawbacks. First, the diagnostic cohort was subject to considerable ascertainment bias in that the testing was applied to patients with known or suspected OPSCC. While the sensitivity was very good (91.5 %), this trial was not powered or designed to examine its use as a screening tool in the general population. Second, with respect to surveillance, this was a retrospective cohort study in which the majority of patients did not have a pre-treatment TTMV-HPV DNA measurement, and as such, these researchers could not exclude the possibility that a false negative may be the result of the test being negative at baseline as opposed to below the level of detection. Third, the definition of a false negative recurrence using a 3-month time frame may be overly conservative and may have artificially lowered the calculated sensitivity of this test by classifying a patient who was truly disease-free at that point in time as a false negative. As such, the authors believed that prospective work with scheduled TTMV-HPV DNA testing alongside standard-of-care (SOC) imaging/clinical examinations will be critical for further elucidating the true sensitivity and appropriate testing interval for this test. Fourth, there was no standardized protocol for incorporating testing into the surveillance; thus, it was difficult to make accurate conclusions regarding lead time detection, as TTMV-HPV testing may have been prompted in some cases by clinical or radiographic findings. Fifth, while it was presumed that an earlier detection of disease may result in improved outcomes, it was beyond the scope of this study to make conclusions regarding how incorporation of TTMV-HPV DNA testing into practice may affect the overall survival (OS) as well as the financial and psychological health of patients.
In an invited commentary on the afore-mentioned study by Ferrandino et al (2023), Lango (2023) noted that Ferrandino and colleagues used data that were collected during the course of routine clinical care and at the discretion of the treating clinician. On average, patients had 1 to 2 TTMV-HPV DNA tests following treatment (591 tests in 290 patients), and it did not appear that testing was obtained according to a schedule. In addition, it was unclear if standardized surveillance imaging was used, which might have affected the time at which recurrences were clinically detected. Lango stated that the appropriate interval for surveillance testing remains unclear. Testing every 3 months in the 1st year, 4 in the next, and every 6 months subsequently -- intervals frequently employed for surveillance visits with or without imaging -- appeared reasonable. Baseline, pre-treatment testing is preferred but not mandatory if HPV-related disease is confirmed. Ferrandino and co-workers stated that a prospective clinical validation study is needed, Lango agreed with this sentiment, and noted that the use of this technology shows remarkable promise to transform the ability to identify and follow patients with HPV-related disease. The author stated that testing is likely to be increasingly used in routine clinical care, as it is commercially available. It is incumbent on researchers to establish evidence for strong and detailed surveillance guidelines to share among the cancer community.
Roof et al (2024) noted that clinical and imaging examinations often provide indeterminate findings during cancer surveillance, which can result in over-treatment and cause psychological as well as financial harm to the patient. In a retrospective study, these researchers examined the need to enhance diagnostic precision and decision-making in the management of HPV-associated OPC. This trial examined the use of TTMV-HPV DNA to resolve indeterminate disease status following definitive treatment for HPV-associated OPC. Participants included patients treated for HPV-associated OPC at 8 U.S. centers, and who received 1 or more TTMV-HPV DNA tests during post-treatment surveillance between February 2020 and January 2022. Among 543 patients, 210 patients (38.7 %; 210/543) experienced 1 or more clinically indeterminate findings (CIFs) during surveillance, with 503 CIFs recorded. Of those patients with an "indeterminate" disease status at a point during surveillance, 79 were associated with contemporaneous TTMV-HPV DNA testing. TTMV-HPV DNA testing showed high accuracy (97.5 %; 77/79) in correctly determining recurrence status. Patients whose disease status was "indeterminate" at the time of a positive TTMV-HPV DNA test were clinically confirmed to recur faster than those whose disease status was "no evidence of disease". Only 3 % of patients (17/543) experienced indeterminate TTMV-HPV DNA tests during surveillance. Discordance between TTMV-HPV DNA tests and clinical results was minimal, with only 0.6 % (3/543) of patients showing positive tests without recurrence. The authors concluded that these findings supported the use of circulating TTMV-HPV DNA in resolving indeterminate disease status and informing the subsequent clinical course.
The authors stated that this study had several drawbacks. First, TTMV-HPV DNA testing was carried out at the discretion of treating clinicians. Second, the lack of a central review of imaging may reduce the consistency of these findings, although the distributed nature of the reviews more likely reflected daily practice. In addition, given the retrospective nature of the study, clinical uncertainty may be influenced by knowledge of the disease outcome, although this would likely result in fewer reported CIFs. Third, while this study revealed the potential for earlier detection of recurrence with TTMV-HPV DNA surveillance, it remained unclear if earlier detection could result in improved oncologic or survival outcomes, although opportunities to treat oligometastatic and low tumor burden disease may result in significant decreases in treatment-associated morbidity and increases in patient’s QOL; thus further research is needed to examine the clinical benefit of earlier detection of recurrence, which must be balanced with the healthcare costs associated with surveillance in general. Fourth, since not all patients received pre-treatment TTMV-HPV DNA testing, not at all tumors were tested for HPV via PCR or ISH, and not all tumors were HPV genotyped, there was a risk of including patients in this analysis whose tumors possessed HPV genotypes not covered by the assay or patients who had p16-positive but HPV-negative tumors. The inclusion of such patients could have increased the variability of the presented findings.
In a “Letter to the Editor” on the afore-mentioned study by Roof et al (2024), Daungsupawong and Wiwanitkit (2024) stated that the study’s drawbacks could be attributed to the cohort’s retrospective design and limited sample size. Moreover, the study did not identify the specific types of unclear results that patients experienced, which may have limited the generalizability of the results. In addition, neither the cost-effectiveness of TTMV-HPV DNA testing nor its potential implications for patients with OPC associated with HPV were addressed in the study. They stated that larger prospective studies are needed to confirm the usefulness of TTMV-HPV DNA testing in this patient population and studies examining the effect of TTMV-HPV DNA testing on patient outcomes, such as survival rates and QOL, may be the future paths for study. In addition, TTMV-HPV DNA testing’s cost-effectiveness and comparison to alternative surveillance tactics may offer important information for therapeutic decision-making regarding the treatment of HPV-associated OPC.
Furthermore, National Comprehensive Cancer Network’s clinical practice guideline on “Head and neck cancers” (Version 3.2024) does not mention circulating tumor DNA as a management tool.
OncotypeDx Breast
Oncotype Dx (Genomic Health, Inc., Redwood City, CA) is a diagnostic laboratory-developed assay that quantifies the likelihood of breast cancer recurrence in women with newly diagnosed, stage I or II, node negative, estrogen receptor positive breast cancer, who will be treated with tamoxifen. The assay analyzes the expression of a panel of 21 genes, and is intended for use in conjunction with other conventional methods of breast cancer analysis. Together with staging, grading, and other tumor marker analyses, Oncotype Dx is intended to provide greater insight into the likelihood of systemic disease recurrence. Clinical studies have evaluated the prognostic significance of the Oncotype Dx multigene assay in breast cancer (Paik et al, 2004; Esteva et al, 2003).
Oncotype Dx analyses the patterns of 21 genes is being applied as a quantification tool for likelihood of breast cancer recurrence within 10 years of newly diagnosed, stage I or II, lymph node-negative, hormone receptor-positive breast cancer in women who will be treated with tamoxifen (Raman, et a., 2013). Oncotype is being applied as a quantification tool for likelihood of breast cancer recurrence in 10 years in women with newly diagnosed breast cancer. It is also intended to assist in making decisions regarding adjuvant chemotherapy based on recurrence likelihood.
There currently is a lack of evidence from prospective clinical studies of the impact of this test on the management of women with breast cancer demonstrating improvements in clinical outcomes (Lopez, et al., 2010; Romeo, et al., 2010; Tiwana, et al., 2013; IETS, 2013), Bast and Hortobagyi (2004) commented that "[b]efore use of the recurrence score [from the Oncotype Dx multigene assay] is applied to general patient care, however, additional studies are needed." The National Cancer Institute is sponsoring a prospective, randomized controlled clinical study, the TAILORx study, using the Oncotype Dx assay to help identify a group of patients with a mid-range risk of recurrence to determine whether treating patients with hormonal therapy only is equivalent to treating them with hormonal therapy in combination with adjuvant chemotherapy.
However, there is indirect evidence of the clinical utility of the Oncotype Dx. Paik et al (2006) used banked tumor samples from previous clinical studies of tamoxifen and adjuvant chemotherapy in early breast cancer to assess the performance of the Oncotype Dx multigene assay in predicting response to adjuvant chemotherapy. These investigators examined tumor samples from subjects enrolled in the National Surgical Adjuvant Breast and Bowel Project (NSABP) B20 trial to determine whether there is a correlation between the recurrence score (RS) determined by Oncotype Dx in tumor samples and subsequent response to adjuvant chemotherapy. A total of 651 patients were assessable (227 randomly assigned to tamoxifen and 424 randomly assigned to tamoxifen plus chemotherapy). The test for interaction between chemotherapy treatment and RS was statistically significant (p = 0.038). Patients with high-RS (RS greater than or equal to 31) tumors (ie, high risk of recurrence) had a large benefit from chemotherapy (relative risk, 0.26; 95% confidence interval 0.13 to 0.53; absolute decrease in 10-year distant recurrence rate: mean, 27.6%; standard error, 8.0%). Patients with low-RS (less than 18) tumors derived minimal, if any, benefit from chemotherapy treatment (relative risk, 1.31; 95% confidence interval, 0.46 to 3.78; absolute decrease in distant recurrence rate at 10 years: mean, -1.1%; standard error, 2.2%). The investigators found that patients with intermediate-RS tumors did not appear to have a large benefit, but the investigators concluded that the uncertainty in the estimate cannot exclude a clinically important benefit.
One limitation of the study by Paik et al (2006) is that the NASBP B20 trial was conducted before the advent of important advances in breast cancer chemotherapy, including the introduction of trastuzumab (Herceptin), which has been demonstrated to improve overall and disease-free survival in breast cancer patients with HER2 positive tumors. Current guidelines recommend the use of trastuzumab adjuvant chemotherapy in women with metastatic HER2 positive breast cancer, and women with HER2 positive nonmetastatic breast cancers 1 cm or more in diameter. Thus, the Oncotype Dx score would not influence the decision to use adjuvant trastuzumab in women with HER2 positive tumors 1 cm or more in diameter.
Commenting on an early report of this study by Paik et al, of the Oncotype Dx presented in abstract form, the BlueCross BlueShield Association Technology Evaluation Center assessment stated that "additional studies in different populations are needed to confirm whether risk prediction is sufficiently accurate for physicians and patients to choose with confidence whether to withhold adjuvant chemotherapy."
An international consensus group (Azim, et al., 2013) found the available evidence on the analytical and clinical validity of Oncotype Dx Breast to be convincing. However, neither the Oncotype Dx or none of the other genomic tests the evaluated demonstrated robust evidence of clinical utility: they stated that it was not clear from the current evidence that modifying treatment decisions based on the results of a given genomic test could result in improving clinical outcome.
The selection criteria for the TailorRx prospective trial of OncotypeDx state that candidates should have negative axillary nodes as determined by a sentinel lymph node biopsy and/or axillary dissection as defined by the American Joint Committee on Cancer 6th Edition Staging System (NCI, 2009). The American Joint Committee on Cancer (AJCC) 6th Edition criteria redefined isolated tumor cells as node negative (the prior version of the criteria, AJCC 5th Edition, classified isolated tumor cells as node positive). "Isolated tumor cells (single cells or cell deposits) will now be defined as tumor cell deposits no larger than 0.2 mm in diameter that may or may not (but usually do not) show histologic evidence of malignant activity. Pending further information, isolated tumor cells will be classified as node-negative, because it is believed that the unknown benefits of providing treatment for these small lesions would not outweigh the morbidity caused by the treatment itself." (Singletary, et al., 2002). However, the banked tumor samples used in the study by Paik, et al. (2006) to validate the OncotypeDx were classified based on AJCC 5th Ed. criteria. In addition, there is new evidence demonstrating that women with isolated tumor cells are at a significantly increased risk of breast cancer. Investigators from the Netherlands found an association between isolated tumor cells and micrometastases in regional lymph nodes and clinical outcome of breast cancer (de Boer, et al., 2009). These investigators identified all patients in the Netherlands who underwent a sentinel-node biopsy for breast cancer before 2006 and had breast cancer with favorable primary-tumor characteristics and isolated tumor cells or micrometastases in the regional lymph nodes. Patients with node-negative disease were randomly selected from the years 2000 and 2001. The primary end point was disease-free survival. The investigators identified 856 patients with node-negative disease who had not received systemic adjuvant therapy (the node-negative, no-adjuvant-therapy cohort), 856 patients with isolated tumor cells or micrometastases who had not received systemic adjuvant therapy (the node-positive, no-adjuvant-therapy cohort), and 995 patients with isolated tumor cells or micrometastases who had received such treatment (the node-positive, adjuvant-therapy cohort). The median follow-up was 5.1 years. The adjusted hazard ratio for disease events among patients with isolated tumor cells who did not receive systemic therapy, as compared with women with node-negative disease, was 1.50 (95% confidence interval [CI], 1.15 to 1.94); among patients with micrometastases, the adjusted hazard ratio was 1.56 (95% CI, 1.15 to 2.13). Among patients with isolated tumor cells or micrometastases, the adjusted hazard ratio was 0.57 (95% CI, 0.45 to 0.73) in the node-positive, adjuvant-therapy cohort, as compared with the node-positive, no-adjuvant-therapy cohort. The investigators concluded that isolated tumor cells or micrometastases in regional lymph nodes were associated with a reduced 5-year rate of disease-free survival among women with favorable early-stage breast cancer who did not receive adjuvant therapy. In patients with isolated tumor cells or micrometastases who received adjuvant therapy, disease-free survival was improved.
The Medical Advisory Panel of the BlueCross BlueShield Association Technology Evaluation Center (BCBCA, 2014) concluded that use of Oncotype DX to determine recurrence risk for deciding whether to undergo adjuvant chemotherapy in women with unilateral, nonfixed, hormone receptor‒positive, lymph node‒negative breast cancer who will receive hormonal therapy meets the Blue Cross and Blue Shield Association Technology Evaluation Center (TEC) criteria. A technology assessment by the BlueCross BlueShield Association (2014) stated: "Technical performance of the assay is well documented and is unlikely to be a major source of variability; rather, tissue sampling is likely the greatest source of variability. Retrospective epidemiologic analyses indicated strong, independent associations between Oncotype DX recurrence score (RS) result and distant disease recurrence or death from breast cancer. The evidence identified a subset of conventionally classified, high-risk patients who are at sufficiently low risk of recurrence by Oncotype DX that they might reasonably decide that the harms (toxicity) of chemotherapy outweigh the very small absolute benefit. Two studies of the original validation data, in which conventionally classified patients were reclassified by Oncotype DX result, indicated that the test provides significant recurrence risk information in addition to conventional criteria for individual patient risk classification. Additional evidence indicated that Oncotype DX results are significantly associated with breast cancer death in a community-based patient population, and that RS high-risk patients benefit from chemotherapy, whereas benefits for other RS categories were not statistically significant. Thus, the evidence was judged sufficient to permit conclusions regarding probable health outcomes."
The Oncotype Dx has also been promoted for use in women with node-positive, ER-positive breast cancer. An assessment by the BlueCross BlueShield Association (2010) concluded that it has not yet been demonstrated whether use of the Oncotype Dx for selecting adjuvant chemotherapy in patients with lymph-node-positive breast cancer improves health outcomes. The report explained that the evidence for not selecting chemotherapy for women with low RS values is based on low event rates and wide confidence intervals that include the possibility of benefit from chemotherapy. Because the data allow for a possible benefit of chemotherapy in patients with low RS results, it is unknown if health outcomes would be improved, the same, or worse, if chemotherapy was withheld in these women. The report stated that, due to the lack of clear and sufficient information, there is a need for a second, confirmatory study. The report stated that the Fred Hutchinson Cancer Research Center will conduct a nationwide, NCI-sponsored, Phase III clinical trial to determine the predictive ability of the Oncotype Dx to identify which patients with lymph-node-positive breast cancer will benefit from chemotherapy.
The clinical evidence base for OncotypeDX is considered to be the most robust. There was some evidence on the impact of the test on decision-making and to support the case that OncotypeDX predicts chemotherapy benefit; however, few studies were UK based and limitations in relation to study design were identified. OncotypeDX has a more robust evidence base, but further evidence on its impact on decision-making in the UK and the predictive ability of the test in an estrogen receptor positive (ER+), lymph node negative (LN-), human epidermal growth factor receptor 2 negative (HER2-) population receiving current drug regimens is needed.
Guidelines from the National Comprehensive Cancer Network (NCCN, 2015) state that "the 21-gene RT-PCR assay recurrence score can be considered in select patients with 1-3 involved ipsilateral axillary lymph nodes to guide the addition of combination chemotherapy to standard hormone therapy. A retrospective analysis of a prospective randomized trial suggests that the test is predictive in this group similar to its performance in node-negative disease." The NCCN guidelines (2015) explained: "Unplanned, retrospective subset analysis from a single randomized clinical trial in post-menopausal, ALN-positive, ER-positive breast cancer found that the 21-gene RT-PCR assay may provide predictive information for chemotherapy in addition to tamoxifen [citing Albain, et al., 2010]. Patients with a high score in the study benefited from chemotherapy, whereas patients with a low score did not appear to benefit from the addition of chemotherapy regardless of the number of positive lymph nodes. Patient selection for assay use remains controversial." "The RxPONDER trial will confirm the SWOG-8814 trial data for women with ER-positive, node-positive disease treated with endocrine therapy with or without chemotherapy based on risk scores."
Guidance from the National Institute for Health and Care Excellence (2013) stated: "Oncotype DX is recommended as an option for guiding adjuvant chemotherapy decisions for people with estrogen ER+, LN- and HER2- early breast cancer if: The person is assessed as being at intermediate risk; and information on the biological features of the cancer provided by Oncotype DX is likely to help in predicting the course of the disease and would therefore help when making the decision about prescribing chemotherapy; and the manufacturer provides Oncotype DX to National Health Service (NHS) organisations according to the confidential arrangement agreed with the National Institute for Health and Care Excellence (NICE). NICE encourages further data collection on the use of Oncotype DX in the NHS."
An assessment by the Belgian Healthcare Knowledge Center (KCE) (San Miguel, et al., 2015) concluded that "the evidence for Oncotype DX is more robust than the evidence for other tests." The KCE Report noted, however, that important evidence gaps are still present. The KCE review mostly identified studies supporting the prognostic ability (clinical validity) of the test. The KCE judged these studies to be of moderate to high quality. The KCE found no prospective studies reporting on the impact of Oncotype DX on long-term outcomes such as overall survival, while four studies indicated that Oncotype DX leads to changes in decision making. The KCE identified two studies on the predictive benefit of the test, one for lymph node patients. The KCE reported also noted that the first evidence relating to improvements in quality of life and reductions in patient anxiety as a result of using the test has been reported, but this is based on small patient numbers and further evidence is required.
Guidelines from the American Society for Clinical Oncology (2016) state: "If a patient has ER/PgR-positive, HER2-negative (node-negative) breast cancer, the clinician may use the 21-gene recurrence score (RS; Oncotype DX; Genomic Health, Redwood City, CA) to guide decisions on adjuvant systemic chemotherapy." This is a strong recommendation based upon high quality evidence. The ASCO guidelines recommend against OncotypeDx Breast to guide decisions on adjuvant systemic chemotherapy for patients with ER/PgR-positive, HER2-negative (node-positive) breast cancer. The guidelines also recommend against the use of OncotypeDx Breast in women with HER2-positive breast cancer or TN breast cancer. The guidelines recommended against the use of OncotypeDx Breast to guide decisions on extended endocrine therapy for patients with ER/PgR-positive, HER-2 negative (node-negative) breast cancer who have had 5 years of endocrine therapy without evidence of recurrence.
Acceptance of 21-gene recurrence score assays as tools for clinical decision making in women or men with early stage breast cancer is controversial due to the lack of prospective validation studies, nevertheless, 2007 guidelines from an expert panel convened by ASCO on tumor markers in breast cancer concluded that multiparameter gene expression analysis (i.e., Oncotype Dx assay) can be used to predict the risk of recurrence in women with newly diagnosed, node-negative, ER-positive breast cancer. Although it is reasonable to consider the use of a 21-gene recurrence score assay in males, none of the data generated to date have been in men with breast cancer (Gradishar, 2010).
A 2009 abstract that looked at cases of male breast cancer (BC) with Oncotype Dx, concluded, "This large genomic study of male BC reveals a heterogeneous biology as measured by the standardized quantitative oncotype Dx breast cancer assay, similar to that observed in female BC. Some differences, which may reflect the differences in hormone biology between males and females, were noted and deserve further study." (Shak et al, 2009).
OvaChek
The OvaCheck™ (Correlogic Systems, Inc.) is a proteomic analysis of blood for the early detection of ovarian cancer. A similar test, which involved a different molecular pattern, was the subject of a 2002 study of 216 women with ovarian cancer. That study showed that the proteomic test had a specificity of 100% and a sensitivity of 95%, with a positive predictive value of 94% (Petricoin, et al., 2002). While this study showed that a proteomic test detected ovarian cancers even where CA-125 levels were normal, this study included only women who had been detected with ovarian cancer by other means. There is inadequate evidence that this test will be effective for screening women with undetected ovarian cancer.
In addition, there is concern, given the low prevalence of ovarian cancer, that this test is not sufficiently specific for use in screening. The National Cancer Institute explains that even an ovarian cancer test with a specificity of 99% means that 1% of those who did not have cancer would test positive, which is "far too high a rate for commercial use" (NCI, 2004). For a rare disease such as ovarian cancer, which has an approximate prevalence of 1 in 2,500 in the general population, a 99% specificity and 100% sensitivity translates into 25 women falsely identified for every one true cancer found.
The OvaCheck™ test employs electrospray ionization (ESI) type of mass spectrometry using highly diluted denatured blood samples. This method differs from a matrix-assisted laser desorption ionization (MALDI) analysis of undiluted native sera samples that was used in the Lancet study and is currently under investigation by the National Cancer Institute and Food and Drug Administration (NCI, 2004). The NCI notes that "[t]he class of molecules analyzed by these two approaches, and thus the molecules that constitute the diagnostic patterns, would be expected to be entirely different." Neither the NCI nor FDA has been involved in the design or validation of OvaCheck™ methodology.
As the Ovacheck test is performed as a "home-brewed" test by two national laboratories instead of as a commercially available kit, FDA approval of the OvaCheck test may not be required. The Society for Gynecologic Oncologists (SGO, 2004) has reviewed the literature regarding OvaCheck and concluded that "more research is needed to validate the test’s effectiveness before offering it to the public." Similarly, the American College of Obstetricians and Gynecologists (2004) has stated that "more research is needed to validate the test's effectiveness before recommending it to the public."
An assessment of the Ovacheck test and other genomic tests for ovarian cancer prepared for the Agency for Healthcare Research and Quality by the Duke Evidence-Based Practice Center (Myers, et al., 2006) reached the following conclusions: "Genomic test sensitivity/specificity estimates are limited by small sample sizes, spectrum bias, and unrealistically large prevalence of ovarian cancer; in particular, estimates of positive predictive values derived from most of the studies are substantially higher than would be expected in most screening or diagnostic settings. We found no evidence relevant to the question of the impact of genomic tests on health outcomes in asymptomatic women. Although there is a relatively large literature on the association of test results and various clinical outcomes, the clinical utility of changing management based on these results has not been evaluated." Specifically regarding Ovacheck and other proteomic tests for ovarian cancer, the assessment found that, "[a] lthough all studies reported good discrimination for the particular protein profile studied, there were several recurrent issues that limit the ability to draw inferences about potential clinical applicability," in particular technical issues with the assays themselves, variations in analytic methods used among studies, and an unrealistically high prevalence of ovarian cancer in the datasets compared to what would be expected in a normal screening population.
OvaSure
OvaSure is an ovarian cancer screening test that entails the use of 6 biomarkers (leptin, prolactin, osteopontin, insulin-like growth factor II, macrophage inhibitory factor and CA-125) to assess the presence of early stage ovarian cancer in high-risk women. Visintin et al (2008) characterized and validated the OvaSure for discriminating between disease-free and ovarian cancer patients. These researchers analyzed 362 healthy controls and 156 newly diagnosed ovarian cancer patients. Concentrations of leptin, prolactin, osteopontin, insulin-like growth factor II, macrophage inhibitory factor, and CA-125 were determined using a multiplex, bead-based, immunoassay system. All 6 markers were evaluated in a training set (181 samples from the control group and 113 samples from ovarian cancer patients) and a test set (181 sample control group and 43 ovarian cancer). Multiplex and ELISA exhibited the same pattern of expression for all the biomarkers. None of the biomarkers by themselves was good enough to differentiate healthy versus cancer cells. However, the combination of the 6 markers provided a better differentiation than CA-125. Four models with less than 2% classification error in training sets all had significant improvement (sensitivity 84 % to 98% at specificity 95%) over CA-125 (sensitivity 72% at specificity 95%) in the test set. The chosen model correctly classified 221 out of 224 specimens in the test set, with a classification accuracy of 98.7%. The authors noted that the OvaSure is the first blood biomarker test with a sensitivity of 95.3% and a specificity of 99.4% for the detection of ovarian cancer. Six markers provided a significant improvement over CA-125 alone for ovarian cancer detection. Validation was performed with a blinded cohort. They stated that this novel multiplex platform has the potential for efficient screening in patients who are at high risk for ovarian cancer.
However, the Society of Gynecologic Oncologists (SGO, 2008) released an opinion regarding OvaSure, which stated that additional research is needed before the test should be offered to women outside the context of a research study. Moreover, SGO stated that it will "await the results of further clinical validation of OvaSure with great interest".
Furthermore, according to the FDA’s web site, the FDA sent the Laboratory Corporation of America a warning letter stating that it is illegally marketing OvaSure to detect ovarian cancer. According to the FDA warning letter, their review indicates that this product is a device under section 201(h) of the Food, Drug, and Cosmetic Act (FDCA or Act), 21 U.S.C. 321(h), because it is intended for use in the diagnosis of disease or other conditions, or in the cure, treatment, prevention, or mitigation of disease. The Act requires that manufacturers of devices that are not exempt obtain marketing approval or clearance for their products from the FDA before they may offer them for sale. This helps protect the public health by ensuring that new devices are shown to be both safe and effective or substantially equivalent to other devices already legally marketed in this country for which approval is not required. According to the FDA warning letter, no such determination has been made for OvaSure.
NCCN Guidelines Panel Members (NCCN, 2016) believe that the OvaSure screening test should not be used to detect ovarian cancer. The NCCN guidelines explain that the OvaSure test uses 6 biomarkers, including leptin, prolactin, osteopontin, insulin-like growth factor II, macrophae inhibitory factor, and CA-125.
PancraGen (formerly PathFinderTG - Pancreas)
PathFinderTG (RedPath Integrated Pathology, Pittsburgh, PA), also known as topographic genotyping, is described by the manufacturer as a quantitative genetic mutational analysis platform for resolving "indeterminate, atypical, suspicious, equivocal and non-diagnostic specimen" diagnoses from pathology specimens (RedPath, 2007). The manufacturer states that PathFinder TG "focuses on acquired mutational damage rather than inherited genetic predisposition for certain diseases, although there are certain NIH recommended inherited conditions for which we do test." The manufacturer states that the temporal sequence of acquired mutational damage revealed by the PathFinderTG test is an earlier demonstration of tumor biological aggressiveness than current staging systems that rely on the depth of invasion already achieved by the tumor. Most available published evidence for topographic genotyping focuses on retrospective analyses of pathology specimens examining correlations of test results with tumor characteristics (e.g., Saad et al, 2008; Lin et al, 2008; Finkelstein et al, 2003; Pollack et al, 2001; Riberio et al, 1998; Kounelis et al, 1998; Finkelstein et al, 1998; Holst et al, 1998; Jones et al, 1997; Holst et al, 1997; Pricolo et al, 1997; Przygodzki et al, 1997; Finkelstein et al, 1996; Kanbour-shakir et al, 1996; Ribeiro et al, 1996; Pryzgodzki et al, 1996; Safatle-Ribeiro et al, 1996; Papadaki et al, 1996; Przygodzki et al, 1996; Pricolo et al, 1996; Finkelstein et al, 1994). There are no prospective clinical outcome studies on the use of topographic genotyping in guiding patient management. Current evidence-based guidelines from leading medical professional organizations and public health agencies do not include recommendations for topographic genotyping. In a review on molecular analysis of pancreatic cyst fluid, Shen and colleagues (2009) stated that a large study with validation of PathFinderTG molecular testing of pancreatic fluid will be needed before a firm conclusion can be drawn.
A systematic evidence review of the PathFinderTG prepared for the Agency for Healthcare Research and Quality (Trikalinos, et al., 2010) reviewed evidence available at that time, and found that most studies on loss-of-heterozygosity based topographic genotyping with PathfinderTG were excluded because they only described the molecular profile of different tumors, without assessing the ability of the method to help make diagnosis, prognosis or treatment guidance. The review found no studies that directly measured whether using loss-of-heterozygosity based topographic genotyping with PathfinderTG improves patient-relevant clinical outcomes. The review reported that eligible studies on the diagnostic and prognostic ability of loss-of-heterozygosity based topographic genotyping with PathfinderTG were small in sample sizes and had overt methodological limitations. The review reported that important characteristics of their designs were not clearly reported. The report noted that loss-of-heterozygosity based topographic genotyping with PathfinderTG is claimed to be particularly useful in cases where conventional pathology is unable to provide a conclusive diagnosis. However, the included studies were not designed to address this question. Therefore, it is unclear if the findings of the reviewed studies are directly applicable to patients with the same cancers but with inconclusive diagnosis.
A subsequent study by Panarelli et al (2012) comparing PathFinderTG to cytological examination, finding concordance in 35 percent of cases. The authors concluded that the PathfinderTG panel may aid the classification of pancreatic lesions, but is often inaccurate and should not replace cytologic evaluation of these lesions.
The manufacturer has announced that the PathginderTG - Pancreas has been rebranded Pancragen.
Al Haddad et al (2015) reported on a multicenter retrospective chart review study to determine the diagnostic accuracy of integrated molecular pathology (Pancragen) for pancreatic adenocarcinoma, and the utility of IMP testing under current guideline recommendations for managing pancreatic cysts. The authors found that Pancragen more accurately determined the malignant potential of pancreatic cysts than a Sendai 2012 guideline management criteria model. Patients who had undergone previous Pancragen testing as prescribed by their physician and for whom clinical outcomes were available from retrospective record review were included (n = 492). Performance was determined by correlation between clinical outcome and previous Pancragen diagnosis ("benign"/"statistically indolent" vs. "statistically higher risk [SHR]"/"aggressive") or an International Consensus Guideline (Sendai 2012) criteria model for "surveillance" vs. "surgery." The Cox proportional hazards model determined hazard ratios for malignancy. Benign and statistically indolent Pancragen diagnoses had a 97 % probability of benign follow-up for up to 7 years and 8 months from initial Pancragen testing. SHR and aggressive diagnoses had relative hazard ratios for malignancy of 30.8 and 76.3, respectively (both P < 0.0001). Sendai surveillance criteria had a 97 % probability of benign follow-up for up to 7 years and 8 months, but for surgical criteria the hazard ratio was only 9.0 (P < 0.0001). In patients who met Sendai surgical criteria, benign and statistically indolent Pancragen diagnoses had a > 93 % probability of benign follow-up, with relative hazard ratios for SHR and aggressive IMP diagnoses of 16.1 and 50.2, respectively (both P < 0.0001). The authors concluded that Pancragen may improve patient management by justifying more relaxed observation in patients meeting Sendai surveillance criteria.
Loren et al (2016) used registry data to determine if initial adjunctive Pancragen testing influenced future real-world pancreatic cyst management decisions for intervention or surveillance relative to 2012 International Consensus Guideline (ICG) recommendations, and if this benefitted patient outcomes. Using data from the National Pancreatic Cyst Registry, the investigators evaluated associations between real-world decisions (intervention vs. surveillance), ICG model recommendations (surgery vs. surveillance) and Pancragen diagnoses (high-risk vs. low-risk) using 2 × 2 tables. The investigators used Kaplan Meier and hazard ratio analyses to assess time to malignancy. Odds ratios (OR) for surgery decision were determined using logistic regression. Of 491 patients, 206 received clinical intervention at follow-up (183 surgery, 4 chemotherapy, 19 presumed by malignant cytology). Overall, 13 % (66/491) of patients had a malignant outcome and 87 % (425/491) had a benign outcome at 2.9 years' follow-up. When ICG and Pancragen were concordant for surveillance/surgery recommendations, 83 % and 88 % actually underwent surveillance or surgery, respectively. However, when discordant, Pancragen diagnoses were predictive of real-world decisions, with 88 % of patients having an intervention when ICG recommended surveillance but Pancragen indicated high risk, and 55 % undergoing surveillance when ICG recommended surgery but Pancragen indicated low risk. These Pancragen-associated management decisions benefitted patient outcomes in these subgroups, as 57 % had malignant and 99 % had benign outcomes at a median 2.9 years' follow-up. Pancragen was also more predictive of real-world decisions than ICG by multivariate analysis: OR 11.4 (95 % CI 6.0 - 23.7) versus 3.7 (2.4 - 5.8), respectively.
Kowalski et al (2016) examined the utility of integrated molecular pathology (IMP) in managing surveillance of pancreatic cysts based on outcomes and analysis of false negatives (FNs) from a previously published cohort (n=492). In endoscopic ultrasound with fine-needle aspiration (EUS-FNA) of cyst fluid lacking malignant cytology, IMP demonstrated better risk stratification for malignancy at approximately 3 years’ follow-up than International Consensus Guideline (Fukuoka) 2012 management recommendations in such cases. The investigators reviewed patient outcomes and clinical features of Fukuoka and IMP FN cases. Practical guidance for appropriate surveillance intervals and surgery decisions using IMP were derived from follow-up data, considering EUS-FNA sampling limitations and high-risk clinical circumstances observed. Surveillance intervals for patients based on IMP predictive value were compared with those of Fukuoka. Outcomes at follow-up for IMP low-risk diagnoses supported surveillance every 2 to 3 years, independent of cyst size, when EUS-FNA sampling limitations or high-risk clinical circumstances were absent. In 10 of 11 patients with FN IMP diagnoses (2% of cohort), EUS-FNA sampling limitations existed; Fukuoka identified high risk in 9 of 11 cases. In 4 of 6 FN cases by Fukuoka (1% of cohort), IMP identified high risk. Overall, 55% of cases had possible sampling limitations and 37% had high-risk clinical circumstances. Outcomes support more cautious management in such cases when using IMP.
An American Gastroenterological Association Technical Review (Scheiman et al, 2014) stated: "Testing for molecular alterations in pancreatic cyst fluid is currently available and reimbursed by Medicare under certain circumstances. Case series have confirmed malignant cysts have greater number and quality of molecular alterations, but no study has been properly designed to identify how the test performs in predicting outcome with regard need to surgery, surveillance or predicts interventions leading to improved survival. This adjunct to fine-needle aspiration (FNA) may provide value in distinct clinical circumstances, such as confirmation of a serous lesion due to a lack of KRAS or GNAS mutation in a macrocystic serous cystadenoma, but its routine use is not supported at the present time."
A guideline from the American Society for Gastrointestinal Endoscopy (Muthusamy, et al., 2016) stated: "A more recent study demonstrated that integrated molecular analysis of cyst fluid (ie, combining molecular analysis with results of imaging and clinical features) was able to better characterize the malignant potential of pancreatic cysts compared to consensus guidelines for the management of mucinous cysts [citing Al Haddad, et al., 2015]. ... Molecular analysis (which requires only 200 mL of fluid) may be most useful in small cysts with nondiagnostic cytology, equivocal cyst fluid CEA results, or when insufficient fluid is present for CEA testing [citing Al Haddad, et al., 2014]. However, additional research is needed to determine the precise role molecular analysis of cyst fluid will play in evaluating pancreatic cystic lesions."
Guidelines published in April 2015 by the American Gastroenterological Association (Vege, et al., 2015) have no recommendations for use of topographic genotyping for evaluating pancreatic cysts. Other guidelines (NCCN, 2015; Vege, et al., 2015; Del Chiaro, et al., 2013; Sahani, et al., 2013; Tanaka, et al., 2012) have no firm recommendations for topographic genotyping for assessing indeterminate pancreatic cysts.
The International consensus guidelines for "The management intraductal papillary mucinous neoplasm (IPMN) and mucinous cystic neoplasm (MCN) of the pancreas" (Tanaka et al, 2012) stated that endoscopic ultrasound (EUS) is recommended for all cysts with worrisome features or for cysts greater than 3 cm without these features. Endoscopic US confirmation of a mural nodule, any features of main duct involvement (intraductal mucin or thickened main duct wall), or suspicious or positive cytology for malignancy is an indication for surgical resection. Cysts with high-risk stigmata should be resected in patients medically fit for surgery, although EUS is optional. Endoscopic US can be considered in smaller cysts without worrisome features but is not required. Endoscopic US analysis should include at least cytology, amylase level, and CEA. The guidelines stated that elevated CEA is a marker that distinguishes mucinous from non-mucinous cysts, but not benign from malignant cysts.
Khalid et al (2004) noted that brush cytology of biliary strictures to diagnose pancreaticobiliary malignancy suffers from poor sensitivity. These researchers attempted to improve the diagnostic yield of pancreaticobiliary brush cytology through analysis of tumor suppressor gene linked microsatellite marker loss of heterozygosity (LOH) and k-ras codon 12 mutation detection. A total of 26 patients with biliary strictures underwent endoscopic retrograde cholangiography with brush cytology. A panel of 12 polymorphic microsatellite markers linked to 6 tumor suppressor genes was developed. Genomic DNA from cell clusters acquired from brush cytology specimens and micro-dissected surgical malignant and normal tissue underwent polymerase chain amplification reaction (PCR); PCR products were compared for LOH and k-ras codon 12 mutations. A total of 17 patients were confirmed to have pancreaticobiliary adenocarcinoma; 9 patients had benign strictures (8 proven surgically, 1 by follow-up). Cytomorphological interpretation was positive for malignancy (n = 8), indeterminate (n = 10), and negative for malignancy (n = 8). Selected malignant appearing cytological cell clusters and micro-dissected histological samples from cancer showed abundant LOH characteristic of malignancy while brushings from 9 cases without cancer carried no LOH (p < 0.001); LOH and k-ras mutations profile of the cytological specimens was almost always concordant with the tissue samples. Presence of k-ras mutation predicted malignancy of pancreatic origin (p < 0.001). The authors concluded that LOH and k-ras codon 12 mutation analysis of PCR amplified DNA from biliary brush cytology discriminated reactive from malignant cells, with 100 % sensitivity, specificity, and accuracy. Minor variations in LOH in brushings and in different sites within the same tumor likely reflect intra-tumoral mutational heterogeneity during clonal expansion of pre- and neoplastic lineages.
Nodit et al (2006) noted that the clinical course of pancreatic endocrine tumor (PET) varies depending on tumor aggressiveness, disease extent, and possibly accumulated molecular alterations. Endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) results, although accurate in diagnosing PET, correlated poorly with PET outcome. The role of detecting key molecular abnormalities in predicting PET behavior and clinical outcome from EUS-FNA material remains unknown. In this study, patients with confirmed PET who underwent EUS-FNA during a 32-month period were included. Patient demographics and clinical data were recorded and follow-up information was obtained by contacting their physician to evaluate disease progression. Representative tumor cells were micro-dissected from the FNA material. DNA was harvested and amplified, targeting a panel of 17 polymorphic microsatellite markers on chromosomes 1p, 3p, 5q, 9p, 10q, 11q, 17p, 17q, 21q, and 22q. The polymerase chain reaction (PCR) products were subjected to fluorescent capillary gel electrophoresis to detect microsatellite loss. The fractional allelic loss (FAL) was calculated. A total of 25 patients were studied; 13 were classified histologically as benign PET limited to the pancreas and 12 as malignant PET (invasive or metastatic). The mean FAL in the benign and malignant PET was 0.03 and 0.37 (p < 0.0001), respectively. In addition, the mean FAL was significantly greater in those with disease progression as compared with patients with stable disease (0.45 versus 0.09, respectively, p < 0.0001). The authors concluded that micro-satellite loss analysis of EUS-FNA material from PET can be performed reliably and an FAL value of more than 0.2 is associated with disease progression . These researchers stated that this technique may have value in the pre-operative assessment and risk stratification of patients with PET.
The authors stated that the small sample size and limited follow-up period were drawbacks of this study, which needed replication in larger prospective studies with longer follow-up periods. The impact of individual micro-satellite markers on the PET clinical course also required further study. In this study certain microsatellites (3p26, 5q23, 17q23, and 21q23) were lost only in malignant PETs, but with the small number of specimens studied the significance of this was unclear. Interestingly, both malignant PETs with a single allelic loss (5q23 and 17q23) each involved micro-satellites not lost in the benign PET.
- selected cells micro-dissected from cytology slides; and
- corresponding cell-free DNA (cfDNA) in residual supernatant fluid.
From 43 patients with brushings of bile or pancreatic duct strictures, DNA was extracted from micro-dissected cells and 1 to 2 ml of cytocentrifugation supernatant fluid. Mutational analysis targeted 17 genomic sites associated with pancreaticobiliary cancer, including sequencing for KRAS point mutation and LOH analysis of micro-satellites located at 1p, 3p, 5q, 9p, 10q, 17p, 17q, 21q, and 22q. Mutations were found in 25/28 patients with malignancy, and no mutations were found in 5/5 patients with benign surgical results. The cell-free supernatant fluid generally contained higher levels and quality of DNA, resulting in increased detection of mutations in most patients. KRAS mutations only occurred in patients with pancreatic cancer; MP of supernatant fluid specimens resulted in high sensitivity and specificity for malignancy, improving the detection of malignancy over cytology alone. The authors concluded that In this study they had shown that neoplastic free DNA was present in the extracellular compartment even when a particular cytology sample lacked sufficient cellularity to afford a definitive diagnosis. Most importantly, the cell-free supernatant, available as a residual specimen after cytocentrifugation, should be regarded as a potentially valuable source of information due to its content of adequate amounts of free DNA for robust mutational analysis with the capacity to address issues related to sampling variation and to detect neoplasia at an early stage of development.
The authors stated that this study had several limitations. First, the total number of test samples was relatively small and the results shown here require confirmation with additional specimens. In particular, the addition of more confirmed negative specimens would strengthen the findings around sensitivity. Second, this study was restricted to the use of Saccomanno’s fixation which, though commonly used, was not the only fixative used in cytology practice. While each fixative merits individual testing with respect to its capacity to deliver adequate levels of representative, intact supernatant DNA for MP, it was reasonable to expect favorable results with other methods of sample preparation as most cytology fixatives were alcohol-based and not expected to induce significant DNA degradation. Indeed, additional unpublished work in the authors’ laboratory involving testing of other supernatant fluids indicated that most common cytology fixatives yielded amplifiable DNA (with the notable exception of CytoRich Red). This was consistent with their prior experience in genotyping micro-dissected cytology slides, in which most slides yielded amplifiable DNA regardless of the cytology fixative used. While none of the supernatant specimens evaluated in this study failed to provide adequate DNA for MP, it was expected that a small proportion of markedly hypocellular specimens, likely from non-neoplastic states, would fail to meet the lower limit of DNA quantity for analysis. It should be noted that in this study, when micro-dissection alone was used, 2/18 cases proved to be false negative for mutation detection (1 cholangiocarcinoma and 1 pancreatic adenocarcinoma). While no false negative malignant stricture cases were seen in cohort 2A where both micro-dissection and supernatant fluid analysis were utilized, in 2 patients, the supernatant fluid manifested a lesser extent of mutational change than that present in the corresponding micro-dissected stained cytology cells. These findings emphasized that the sampling variation and other limitations may nevertheless be present in individual cases limiting or preventing the detection of cancer. It remained essential to integrate all of the information including clinical and imaging findings to optimize individual patient diagnosis.
Finkelstein et al (2014) noted that diagnosis of fine-needle aspirations of pancreatic solid masses is complicated by many factors that keep its false-negative rate high. These researchers’ novel approach analyzes cell-free cytocentrifugation supernatant, currently a discarded portion of the specimen. Supernatant and cytology slides were collected from 25 patients: 11 cases with confirmed outcome [5 positive (adenocarcinoma) and 6 negative (inflammatory states)], plus 14 without confirmed outcomes. Slides were micro-dissected, DNA was extracted from micro-dissections and corresponding supernatants, and all were analyzed for KRAS point mutation and loss of heterozygosity. Notably, higher levels of free DNA were found in supernatants than in corresponding micro-dissected cells. Supernatants contained sufficient DNA for mutational profiling even when samples contained few to no cells. Mutations were present in 5/5 malignancies and no mutations were present in inflammatory states. The authors concluded that these findings supported using supernatant for mutational genotyping when diagnostic confirmation is needed for pancreatic solid masses. These researchers stated that the data presented suggested that supernatant fluid should be regarded as a valuable source of information that may address many diagnostic issues and may serve as a useful, complimentary tool for pathologists when microscopic examination is suboptimal.
The authors stated that several limitations of this molecular analysis of cytocentrifugation supernatant were recognized. The total number of test samples was not large, and the promising results shown here need to be evaluated with a greater number of specimens. In addition, this study was restricted to the use of Saccomanno’s fixation. Ideally, each commonly used fixative should be individually tested for its capacity to deliver adequate levels of representative supernatant DNA for mutational profiling. It is reasonable, however, to expect favorable results with other methods of sample preparation since most cytology fixatives are alcohol based and are not expected to induce significant DNA degradation. Consistently, prior work has shown that cytology specimens based on micro-dissected stained cytology cells, are especially suitable for mutational analysis.
Deftereos et al (2014) stated that FNA of pancreatic solid masses can be significantly impacted by sampling variation. Molecular analysis of tumor DNA can be an aid for more definitive diagnosis. These investigators evaluated how molecular analysis of the cell-free cytocentrifugation supernatant DNA can help reduce sampling variability and increase diagnostic yield. A total of 23-FNA smears from pancreatic solid masses were performed. Remaining aspirates were rinsed for preparation of cytocentrifuged slides or cell blocks. DNA was extracted from supernatant fluid and assessed for DNA quantity spectrophotometrically and for amplifiability by quantitative PCR (qPCR). Supernatants with adequate DNA were analyzed for mutations using PCR/capillary electrophoresis for a broad panel of markers (KRAS point mutation by sequencing, micro-satellite fragment analysis for loss of heterozygosity (LOH) of 16 markers at 1p, 3p, 5q, 9p, 10q, 17p, 17q, 21q, and 22q). In selected cases, micro-dissection of stained cytology smears and/or cytocentrifugation cellular slides were analyzed and compared. In all, 5/23 samples cytologically confirmed as adenocarcinoma showed detectable mutations both in the micro-dissected slide-based cytology cells and in the cytocentrifugation supernatant. While most mutations detected were present in both micro-dissected slides and supernatant fluid specimens, the latter showed additional mutations supporting greater sensitivity for detecting relevant DNA damage. Clonality for individual marker mutations was higher in the supernatant fluid than in micro-dissected cells. Cytocentrifugation supernatant fluid contains levels of amplifiable DNA suitable for mutation detection and characterization. The finding of additional detectable mutations at higher clonality indicated that supernatant fluid may be enriched with tumor DNA. The authors concluded that the findings of this study suggested how the supernatant fluid can be utilized as a source of molecular information and could become a powerful addition to standard cytology evaluation. Mutational profiling of DNA in normally discarded supernatant fluid may help resolve occasional diagnostic challenges and may serve as a useful, complementary tool for cytopathologists when microscopic examination yielded no conclusive diagnosis or when a specimen is suboptimal.
Malhotra et al (2014) aimed to better understand the supporting role that MP of DNA from micro-dissected cytology slides and supernatant specimens may play in the diagnosis of malignancy in FNA and biliary brushing specimens from patients with pancreaticobiliary masses. Cytology results were examined in a total of 30 patients with associated surgical (n = 10) or clinical (n = 20) outcomes; MP of DNA from micro-dissected cytology slides and from discarded supernatant fluid was analyzed in 26 patients with atypical, negative or indeterminate cytology. Cytology correctly diagnosed aggressive disease in 4 patients. Cytological diagnoses for the remaining 26 were as follows: 16 negative (9 false negative), 9 atypical, 1 indeterminate. MP correctly determined aggressive disease in 1 false negative cytology case and confirmed a negative cytology diagnosis in 7 of 7 cases of non-aggressive disease. Of the 9 atypical cytology cases, MP correctly diagnosed 7 as positive and 1 as negative for aggressive disease. One specimen that was indeterminate by cytology was correctly diagnosed as non-aggressive by MP. When first-line malignant (positive) cytology results were combined with positive 2nd-line MP results, 12/21 cases of aggressive disease were identified, compared to 4/21 cases identified by positive cytology alone. The authors concluded that when 1st-line cytology results were uncertain (atypical), questionable (negative), or not possible (non-diagnostic/indeterminate), MP provided additional information regarding the presence of aggressive disease. When used in conjunction with 1st-line cytology, MP increased detection of aggressive disease without compromising specificity in patients that were difficult to diagnose by cytology alone.
The authors stated that this study had several drawbacks including a small sample size that limited their ability to calculate the diagnostic performance of MP in pancreatic masses and associated biliary strictures. Although MP allowed these researchers to detect additional cases of aggressive disease, even when cytology and MP results were combined into one overall diagnosis, 9 cases of malignancy were missed. These falsely negative results were likely due to a combination of the less than perfect sensitivity of both tests as well as to sampling limitations related to FNA and brushing techniques. These investigators noted that despite such limitations, these promising findings do provide support for future larger scale studies, with the addition of supernatant analysis providing an opportunity to overcome some of these limitations.
Gonda et al (2017) stated that it is a challenge to detect malignancies in biliary strictures. Various sampling methods are available to increase diagnostic yield, but these require additional procedure time and expertise. These investigators evaluated the combined accuracy of fluorescence in situ hybridization (FISH) and PCR-based DNA MP of specimens collected using standard brush techniques. These researchers performed a prospective study of 107 consecutive patients treated for biliary strictures by endoscopic retrograde cholangiopancreatography from June 2012 through June 2014. They carried out routine cytology and FISH analyses on cells collected by standard brush techniques, and analyzed supernatants for point mutations in KRAS and LOH mutations in tumor-suppressor genes at 10 loci (MP analysis was performed at Interpace Diagnostics). Strictures were determined to be non-malignant based on repeat image analysis or laboratory test results 12 months after the procedure. Malignant strictures were identified based on subsequent biopsy or cytology analyses, pathology analyses of samples collected during surgery, or death from biliary malignancy. These researchers determined the sensitivity and specificity with which FISH and MP analyses detected malignancies using the exact binomial test. The final analysis included 100 patients; 41 % had biliary malignancies. Cytology analysis identified patients with malignancies with 32 % sensitivity and 100 % specificity. Addition of FISH or MP results to cytology results increased the sensitivity of detection to 51 % (p < 0.01) without reducing specificity. The combination of cytology, MP, and FISH analyses detected malignancies with 73 % sensitivity (p < 0.001); FISH identified an additional 9 of the 28 malignancies not detected by cytology analysis, and MP identified an additional 8 malignancies; FISH and MP together identified 17 of the 28 malignancies not detected by cytology analysis. The authors concluded that these findings supported the use of both FISH testing and PCR-based MP of tumor-suppressor gene LOH and KRAS in evaluation of cytology-negative or indeterminate biliary strictures; MP allowed for increased diagnostic yield from each individual brush, given that normally discarded, cell-free supernatant material that contains DNA can be analyzed. Based on these findings, these researchers suggested using either FISH or MP as a 2nd-line diagnostic modality to 1st-line cytology. They stated that MP may be best prioritized to scenarios of low cellularity. Any case that is negative or indeterminate by 2 testing modalities should undergo a 3rd to increase the probability of detecting possible malignancy. To do so, normally discarded supernatant fluid should be retained for MP testing during the standard cytology cytocentrifugation preparation of cells for cytology. These researchers stated that additional studies may help to better understand the reflex order of sequential testing and the impact of this reflex on health economics.
The authors stated that this study had several drawbacks that may have impacted generalized conclusions. A somewhat higher benign stricture rate was noted in their cases than in other prior series. There also were relatively few primary sclerosing cholangitis (PSC) patients included in this study. Prior studies have shown that there is a significant aneuploidy rate associated with pre-malignant lesions seen in PSC. Because of this, specificity of FISH for malignancy was expected to be lower in a cohort of PSC patients than the authors reported in their cohort. Less was known about detection of KRAS mutations in the progression of PSC to cholangiocarcinoma. However, based on this study cohort and prior studies, these findings likely were not generalizable to the PSC population.
Khosravi et al (2018) stated that indeterminate cytology occurs in a significant number of patients with solid pancreaticobiliary lesion that undergo EUS-FNA or endoscopic retrograde cholangiopancreatography (ERCP) and can incur further expensive testing and inappropriate surgical intervention. Mutation profiling improves diagnostic accuracy and yield but the impact on clinical management is uncertain. These researchers determined the performance of MP in clinical practice and its impact on management in solid pancreaticobiliary patients with indeterminate cytology. Solid pancreaticobiliary patients with non-diagnostic, benign, atypical or suspicious cytology who had past MP testing were included. Mutation profiling examined KRAS mutation and a tumor suppressor gene associated loss of heterozygosity mutation panel covering 10 genomic loci. Two endo-sonographers made management recommendations without and then with MP results, indicating their level of confidence. Mutation profiling improved diagnostic accuracy in 232 patients with indeterminate cytology. Among patients with non-diagnostic cytology, low-risk MP provided high specificity and negative predictive value (NPV) for the absence of malignancy while high-risk MP identified malignancies otherwise undetected. Mutation profiling increased clinician confidence in management recommendations and resulted in more conservative management in 10 % of patients. Mutation profiling increased the rate of benign disease in patients recommended for conservative management (84 % to 92 %, p < 0.05) and the rate of malignant disease in patients recommended for aggressive treatment (53 % to 71 %, p < 0.05). The authors concluded that MP improved diagnostic accuracy and significantly impacted management decisions. Low-risk MP results increased recommendations for conservative management and increased the rate of benign outcomes those patients, helping to avoid unnecessary aggressive interventions and improve patient outcomes. These researchers stated that their study was limited by its retrospective nature. Moreover, they noted that although high-risk MP results were able to help confirm the presence of malignancy in cases in which cytology indicated a high suspicion of malignancy, low-risk results could not effectively exclude the possibility of malignancy in such cases.
Kushnir et al (2019) noted that routine cytology of biliary stricture brushings obtained during ERCP has suboptimal sensitivity for malignancy. These investigators compared the individual and combined ability of cytology, FISH analysis and PCR-based MP to detect malignancy in standard biliary brushings. They performed a prospective study of patients undergoing ERCP using histology or 1 year follow-up to determine patient outcomes; MP was performed on free-DNA from biliary brushing specimens using normally discarded supernatant fluid. MP examined KRAS point mutations and tumor suppressor gene associated LOH mutations at 10 genomic loci; FISH examined chromosome specific gains or losses. A total of 101 patients were included in final analysis and 69 % had malignancy. Cytology had 26 % sensitivity and 100 % specificity for malignancy. Using either FISH or MP in combination with cytology increased sensitivity to 44 % and 56 %, respectively. The combination of all 3 tests (cytology, FISH, and MP) had the highest sensitivity for malignancy (66 %). There was no difference in the specificity of cytology, FISH or MP testing when examined alone or in combination; MP improved diagnostic yield of each procedure from 22 % to 100 %; FISH improved yield to 90 %; MP detected 21 malignancies beyond that identified by cytology; FISH detected an additional 13. The combination of FISH and MP testing detected an additional 28 malignancies. The authors concluded that both MP and FISH are complimentary molecular tests that could significantly increase detection of biliary malignancies when used in combination with routine cytology of standard biliary brush specimens.
Previstage GCC
Guanylyl Cyclase C (GCC or GUCY2C) (Diagnocure) a gene coding for a protein found in cells, lining the intestine from the duodenum to the rectum (Raman, et al., 2013). It is involved in water transport, crypt morphology and suppression of tumorigenesis. It is not normally found in tissue in other parts of the body, and therefore, GCC detected outside of the intestine, indicates presence of colorectal cancer metastases. Early studies have indicated that the presence of GCC in the blood may be an early indicator of micrometastases that would otherwise escape detection by the current standard methods of monitoring. Earlier detection provides an opportunity for more immediate treatment or surgical intervention to potentially improve patient outcomes and survival rates. This is a diagnostic test for recurrence by identification of micrometastasis in the blood.
Guanyl cyclase C (GCC) is a receptor protein normally expressed in high concentrations on the luminal surface of the gastrointestinal epithelium. Expression of GCC persists on mucosal cells that have undergone malignant transformation. Thus, GCC has potential use as a marker to determine spread of colorectal cancer to lymph nodes. A retrospective study of 21 patients post surgical resection of colorectal cancer found that all 11 of 21 patients who were free of cancer for 5 years or more were negative for GCC in lymph nodes, whereas all 10 of 21 patients whose cancer returned within 3 years of surgery were positive for GCC. However, the value of the GCC marker test in the management of colorectal cancer needs to be evaluated in prospective clinical outcome studies. A large prospective study is currently being conducted to compare standard histological examination of lymph nodes to the GCC marker test.
Previstage™ Guanylyl Cyclase C (GCC or GUCY2C) (Diagnocure) is a gene coding for a protein found in cells, lining the intestine from the duodenum to the rectum (Raman, et al., 2013). It is involved in water transport, crypt morphology and suppression of tumorigenesis. It is not normally found in tissue in other parts of the body, and therefore, GCC detected outside of the intestine, indicates presence of colorectal cancer metastases. GCC mRNA has shown to be highly accurate in detecting the spread and recurrence of colorectal cancer, respectively in lymph nodes and blood, thereby representing a significant improvement over traditional detection methods. Previstage is a predictive test for risk stratification of recurrence and prognostic marker for recurrence.
PROphet NSCLC Test
The PROphet NSCLC test (Oncohost, Inc.) is a plasma-based proteomic analysis tool used to guide first-line immunotherapy decision-making in patients with advanced, unresectable non-small cell lung cancer (NSCLC). From a single blood sample, the test identifies expression patterns in a panel of approximately 7,000 proteins and assigns a score reflecting the clinical benefit probability (progression-free survival greater than 12 months) from PD-1/PD-L1 inhibitor immunotherapy-based treatment plans. The score is reported as positive or negative for each biomarker.
Ben et al (2024) examined the analytical validity of the PROphet test, which is based on the SomaScan platform both experimentally and computationally, in treatment decision-making guidance for patients with metastatic NSCLC. The experimental part involved proteomic measurements obtained using the SomaScan assay, an aptamer-based proteomics platform (SomaLogic Inc.). The computational part was a model generated on a cohort of NSCLC patients. Plasma samples were obtained as part of a clinical study (PROPHETIC; NCT04056247) and were collected either before treatment commencement or before the second treatment. The authors found that experimental precision analysis displayed a median coefficient of variation (CV) of 3.9 % and 4.7 % for intra-plate and inter-plate examination, respectively, and the median accuracy rate between sites was 88 %. Computational precision (defined as the extent to which the algorithm output for a given sample is consistent between multiple runs) exhibited a high accuracy rate, with 93 % of samples displaying complete concordance in results. A cross-platform comparison between SomaScan and other proteomics platforms yielded a median Spearman's rank correlation coefficient of 0.51, affirming the consistency and reliability of the SomaScan platform as used under the PROphet test. The authors concluded that their study presents a robust framework for evaluating the analytical validity of a platform that combines an experimental assay with subsequent computational algorithms, and that, when applied to the PROphet test, strong analytical performance of the test was demonstrated.
PROPHETIC is a prospective, multicenter, international study to develop an algorithm that predicts patient treatment outcomes. The algorithm serves as a treatment decision-making tool for physicians. Investigators also aim to identify the metabolic pathways that could lead to better therapeutic options. The study will enroll approximately 10,000 patients worldwide. Each will participate in the study for up to 5 years.
The National Comprehensive Cancer Network does not mention the use of plasma-based proteomic testing for evaluating therapy for patients with advanced, unresectable NSCLC (NCCN, 2024).
ProstatePx
Donovan et al (2008) from Aureon, the manufacturer of Prostate Px, reported on the development and validation of their systems pathology model for predicting prostate cancer recurrence after prostatectomy. The clinical utility of defining high risk for failure after radical prostatectomy is to decide whether patients require closer follow-up than average or whether adjuvant radiotherapy, hormone therapy, or chemotherapy would be of benefit. In this analysis, the concordance index for the systems pathology approach used by Aureon was 0.83, but was only slightly better than a 10-variable model that used only the usual clinical parameters, with a concordance index of 0.80. The corresponding hazard ratios for clinical failure were 6.37 for the 10-variable clinical model, and 9.11 for the systems pathology approach. In an accompanying editorial, Klein, et al. (2009) questioned the clinical significance of these differences. They noted that "[a]lthough the difference in concordance indices was statistically significant, the question is whether there is sufficient clinical relevance to justify the extra effort, expense, and clinical expertise needed for the systems approach ... In contemporary clinical practice, a patient with a hazard ratio of 6.37 generated by the model using easily derived, routinely reported clinical and pathological parameters is just as likely to be a candidate for closer monitoring or adjuvant therapy than one with a hazard ratio of 9.11 generated by the systems approach".
- inclusion of established markers, and
- consideration of the possible biases from study attrition. Given the heterogeneity of the models, they can not be considered comparable.
Only 2 models did not include a novel marker, and 1 of these included several demographical and co-morbidity variables to predict all-cause mortality. Only 2 models reported a measure of model performance, the C-statistic, and for neither was it calculated in an external data set. It was not possible to assess whether the models that included novel markers performed better than those without. This review highlighted the poor quality and heterogeneity of studies, which render much of the results inconclusive. It also pinpointed the small proportion of models reported in the literature that are based on patient cohorts with a mean or median follow-up of at least 5 years, thus making long-term predictions unreliable. Prostate-specific antigen velocity, however, stood out in terms of the strength of the evidence supporting its prognostic value and the relatively high hazard ratios. There is great interest in PSA velocity as a monitoring tool for active surveillance but there is as yet no consensus on how it should be used and, in particular, what threshold should indicate the need for radical treatment.
In an editorial on clinically relevant prognostic markers for prostate cancer, Gelmann and Henshall (2009) stated that "[u]ntil we have sufficiently discriminating markers to inform treatment decisions, the problem of whom to treat will continue to grow exponentially as the number of cases of screening-detected low-risk cancer increases".
Rotterdam Signature 76-Gene Panel
The Rotterdam Signature test (Veridex) is a 76-gene expression assay (Raman, 2013). Sixty genes are intended to evaluate estrogen-receptor positive samples and 16 genes to evaluate estrogen-receptor negative samples. In a validation study that tested the signature on samples from 148 women, 50 fell into the low-risk group and 98 into the high-risk group. The test had 88% specificity and 39% sensitivity for the low-risk group, with a hazard ratio for distant relapse within 5 years of 5.74 comparing the high-risk group to the low-risk group. The Rotterdam Signature identifies women at high and low risk of disease recurrence.
The Rotterdam Signature 76-gene panel (Veridex, LLC) is a multivariate index assay that is intended to assist in assessing a patient’s risk of systemic recurrence of cancer following successful initial treatment of localized node-negative breast cancer with surgery and tamoxifen alone. This multigene assay is intended for use in lymph-node negative breast cancer patients. The Rotterdam Signature panel uses microarray processing to measure cellular concentrations of mRNA in fresh tissue samples. The Rotterdam Signature panel uses the Human Genome U133a GeneChip (Affymetrix, Inc.) to identify patients that have gene expression signatures associated with either a low or high risk of developing metastatic disease. A multicenter study investigated the ability of the Rotterdam 76-gene signature to identify patients at risk of distant metastases within 5 and 10 years of first diagnosis, using frozen tissue samples from 180 patients with node-negative breast cancer who had not received systemic chemotherapy (Foekens, et al., 2006). The Rotterdam 76-gene signature correctly identified 27 out of 30 cases of relapse within 5 years (90% sensitivity) and 75 out of 150 patients who did not relapse (50% specificity). An earlier summary of the same study (Foekens, et al., 2005) reported a hazard ratio for distant metastasis-free survival comparing favorable versus unfavorable signature = 7.41 (95% confidence interval 2.63-20.9); p = 8.5 x 10-6). The hazard ratio of overall survival comparing favorable versus unfavorable signature = 5.45 (95% confidence interval 1.62-18.3); p = .002. There are no published studies that have assessed the clinical utility of the Rotterdam 76-gene signature by monitoring the long-term outcomes of the patients selected and not selected for chemotherapy on the basis of assay results.
Strata Select
Strata Oncology, Inc. (Ann Arbor, MI), a next-generation precision oncology company, developed Strat Select, a molecular profiling test for patients with advanced cancer which features the Immunotherapy Response Score, a novel multivariate predictive biomarker algorithm for PD-1/PD-L1 checkpoint inhibitor immunotherapy benefit. The Immunology Response Score integrates pertinent biological factors (TMB, PD-L1, PD-1, and tumor microenvironment components) into a simple score to predict immunotherapy benefit across all solid tumors. Strata Select analyzes DNA and RNA across 437 genes to provide guidance for genomic-alteration targeted therapies. The targeted RNA sequencing enables analysis of 950 fusion isoforms involving 59 primary driver genes (Strata Oncology, 2023).
Currently, there is insufficient evidence in the peer-reviewed literature to support the sensitivity or specificity of this test.
Symphony
Symphony (Agendia) provides complete tumor profiling and is used to support therapeutic choices for breast cancer (Raman, 2013). SYMPHONY includes four assays to support breast cancer treatment decisions: MammaPrint® determines the risk of recurrence. BluePrint™ determines molecular subtypes and TargetPrint® determines estrogen receptor (ER), progesterone receptor (PR), and HER2 status. TheraPrint™ identifies alternative types of therapy for metastatic disease. SYMPHONY provides genomic information assisting with therapeutic decisions even for cases that have been otherwise classified as indeterminate, such as grade 2, small tumors, HER2 and/or lymph node positive. MammaPrint® determines if the patient is a candidate for chemotherapy. TargetPrint® determines if the patient is a candidate for hormonal therapy. BluePrint® provides information on the sub-classification of the tumor which guides the choice of therapies and combinations of therapies. TheraPrint® identifies alternative types of therapy for metastatic disease.
TargetPrint
TargetPrint®, ER/PR/HER2 Expression Assay (Agendia) is a microarray-based gene expression test which offers a quantitative assessment of the patient’s level of estrogen receptor (ER), progesterone receptor (PR) and HER2/neu overexpression within her breast cancer (Raman, et al., 2013). TargetPrint is offered in conjunction with MammaPrint to provide the physician an even more complete basis for treatment decisions. TargetPrint delivers an added benefit to the diagnostic process. Immunohistochemistry provides a semi-quantitative positive or negative result, whereas the gene expression result provided by TargetPrint allows physicians to integrate the absolute level of ER, PR and HER2 gene expression into treatment planning. TargetPrint determines if the patient is a candidate for hormonal therapy.
TargetPrint is a microarray-based gene expression test which offers a quantitative assessment of the patient’s level of estrogen receptor (ER), progesterone receptor (PR) and HER2/neu overexpression in breast cancer. The manufacturer states that TargetPrint is offered in conjunction with MammaPrint gene expression profiling to provide the physician an even more complete basis for treatment decisions. The manufacturer states that, as compared to Immunohistochemistry (IHC), TargetPrint provides additional information. Whereas IHC provides a semi-quantitative positive or negative result, the gene expression result provided by TargetPrint provides data on the absolute level of ER, PR and HER2 gene expression. Published information on the TargetPrint is limited to studies examining its correlation with measurements of ER, PR, and HER2 receptors (Gunven et al, 2011; Gevensleben et al, 2010; Roepman et al, 2009). There is a lack of evidence from published prospective clinical studies that demonstrates that quantification of ER, PR, and HER2 gene expression by TargetPrint alters management such that clinical outcomes are improved.
UriFind Blood Cancer Assay
UriFind (AnchorDx) is a non-invasive quantitative real-time PCR (qPCR) assay that has been designed for detecting two DNA methylation biomarkers in urine specimens from patients suspected of having bladder cancer.
In November 2022, AnchorDx announced that they have enrolled the first patient in a multicenter, prospective clinical trial that will assess the performance of the UriFind assay in more than 1,000 targeted patients. In July 2021, the assay was granted a Breakthrough Device Designation (BTD) by the U.S. FDA. Results of this clinical trial are aimed towards meeting the requirements for an application for an FDA Premarketing Approval (PMA). The trial is expected to include about 10 sites of Urology clinics and 3 CAP/CLIA laboratories.
UroAmp MRD
UroAmp MRD (Convergent Genomics) is a non-invasive genomic urine test indicated for detecting bladder cancer or predicting its recurrence before clinical signs or symptoms appear. The test interrogates 60 urothelial cancer genes while broadly measuring changes across the wole genome. Moreover, it uses an algorithm reported as minimal residual disease (MRD) status positive or negative and quantitative disease burden, a measure of a patient’s disease burden compared to bladder cancer patients previously tested with UroAmp. UroAmp MRD is purported to help monitor disease progression, recurrence, and response to therapeutic interventions.
In a prospectively designed and accrued cohort study, Rac et al (2024) examined the utility of urinary comprehensive genomic profiling (uCGP) for predicting recurrence risk following transurethral resection of bladder tumor (TURBT) and evaluating longitudinal IVT response. Urine was collected before and after intravesical therapy (IVT) instillation and uCGP testing was done using the UroAmp™ platform. UroAmp uses next‐generation sequencing to detect six classes of mutations. Five of these—single‐nucleotide variants (SNVs), small insertion‐deletions (INDELs), targeted gene‐level copy‐number variants (CNVs), microsatellite instability (MSI), and copy‐neutral loss of heterozygosity (LOH)—are assayed across a 60‐gene panel, while the sixth mutation type is whole‐genome aneuploidy. UroAmp is a tumor‐naïve test. Detected mutations are annotated using publicly available data sources, including dbSNP, 1000 Genomes Project, The Cancer Genome Atlas, Sanger/COSMIC, and AARC Project GENIE. Mutation profiles serve as input features to machine‐learned algorithms disease and molecular grade prediction. UroAmp risk algorithms are calculated independent of any clinical features. The investigators found that baseline uCGP following TURBT identified patients with high (61%) and low (39%) recurrence risk. At 24 months, recurrence‐free survival (RFS) was 100% for low‐risk and 45% for high‐risk patients with a hazard ratio (HR) of 9.3. Longitudinal uCGP classified patients as minimal residual disease (MRD) Negative, IVT Responder, or IVT Refractory with 24‐month RFS of 100%, 50%, and 32%, respectively. Compared with MRD Negative patients, IVT Refractory patients had a HR of 10.5. Collectively, uCGP enables noninvasive risk assessment of patients following TURBT and induction IVT. uCGP could inform surveillance cystoscopy schedules and identify high‐risk patients in need of additional therapy. The investigators acknowledged study limitations, with the most significant being the cohort size. This study was powered for general assessment of MRD detection and its longitudinal relationship with recurrence. However, it was not powered to evaluate progression, which is a critical consideration but would require larger sample size and follow‐up. The limited cohort size also restricted the investigators’ ability to assess individual molecular relationships.
VEGF
Tumour angiogenesis is associated with invasiveness and the metastatic potential of various cancers. Vascular endothelial growth factor (VEGF), the most potent and specific angiogenic factor identified to date, regulates normal and pathologic angiogenesis. An evidence report from Cancer Care Ontario (Welch et al, 2008) on the use of the VEGF inhibitor bevacizumab in colorectal cancer explained that the increased expression of VEGF has been correlated with metastasis, recurrence, and poor prognosis in many cancers, including colorectal cancer. Guidelines from the National Institute for Health and Clinical Excellence (NICE, 2007) explained that bevacizumab (Avastin) is a recombinant humanised monoclonal IgG1 antibody that acts as an angiogenesis inhibitor. It targets the biological activity of VEGF, which stimulates new blood vessel formation in the tumour. However, neither the FDA approved labeling of bevacizumab or evidence-based guidelines recommend measurement of VEGF to diagnose colorectal cancer or to select patients for treatment. In a special report on pharmacogenomics of cancer, the BlueCross and BlueShield Association's Technology Evaluation Center (TEC) (2007) stated that pre-treatment VEGF levels do not appear to be predictive of response to anti-angiogenic therapy.
Shin and colleagues (2013) evaluated inhibitory effects of bevacizumab on VEGF signaling and tumor growth in-vitro and in-vivo, and assessed phosphorylation of VEGF receptor 2 (VEGFR2) and downstream signaling in endothelial cells as pharmacodynamic markers using phospho-flow cytometry. These researchers also validated markers in patients with mCRC treated with bevacizumab-based chemotherapy. In in-vitro studies, bevacizumab inhibited proliferation of human umbilical vein endothelial cells in association with reduced VEGF signaling. Notably, bevacizumab inhibited VEGF-induced phosphorylation of VEGFR-2, Akt, and extra-cellular signal-regulated kinase (ERK). In-vivo, treatment with bevacizumab inhibited growth of xenografted tumors and attenuated VEGF-induced phosphorylation of Akt and ERK. The median percentages of VEGFR2 + pAkt + and VEGFR2 + pERK + cells, determined by phospho-flow cytometry, were approximately 3-fold higher in mCRC patients than in healthy controls. Bevacizumab treatment decreased VEGFR2 + pAkt + cells in 18 of 24 patients on day 3. The authors concluded that bevacizumab combined with chemotherapy decreased the number of VEGFR2 + pAkt + cells, reflecting impaired VEGFR2 signaling. Together, these data suggested that changes in the proportion of circulating VEGFR2 + pAkt + cells may be a potential pharmacodynamic marker of the effectiveness of anti-angiogenic agents, and could prove valuable in determining drug dosage and administration schedule.
PLAP
The National Comprehensive Cancer Network's guideline on occult primary tumors includes placental alkaline phosphatase (PLAP) as a useful marker to assist in identifying germ cell seminoma and non-seminoma germ cell tumors in unknown primary cancer (NCCN, 2009).
MPO
Myeloperoxidase (MPO), a blood protein, is a major component of azurophilic granules of neutrophils. Myeloperoxidase analysis has been used to distinguish between the immature cells in acute myeloblastic leukemia (cells stain positive) and those in acute lymphoblastic leukemia (cells stain negative). The National Comprehensive Cancer Network guidelines on acute myeloid leukemia (AML) include MPO analysis in the classification of AML (NCCN, 2008).
Matsuo et al (2003) examined the prognostic factor of the percentage of MPO-positive blast cells for AML. Cytochemical analysis of MPO was performed in 491 patients who were registered to the Japan Adult Leukemia Study Group (AML92 study). Patients were divided into two groups using the percentage of MPO-positive blast (high [ > or = 50%] and low [< 50%]). Complete remission rates were 85.4% in the former and 64.1% in the latter (p = 0.001). The OS and DFS were significantly better in the high MPO group (48.3 versus 18.7% for OS, and 36.3 versus 20.1% for DFS, p < 0.001, respectively). Multi-variate analysis showed that both karyotype and the percentage of MPO-positive blast cells were equally important prognostic factors. The high MPO group still showed a better survival even when restricted to the intermediate chromosomal risk group or the patients with normal karyotype (p < 0.001). The OS of patients with normal karyotype in the high MPO group was almost equal to that of the favorable chromosomal risk group. The authors concluded that the percentage of MPO-positive blast cells is a simple and highly significant prognostic factor for AML patients, and especially useful to stratify patients with normal karyotype.
DCP
The most commonly used marker for hepatocellular carcinoma (HCC) is the AFP level. Des-gamma-carboxy prothrombin (DCP) (also known as "prothrombin produced by vitamin K absence or antagonism II" [PIVKA II]) has also shown promise in the diagnosis of HCC (Toyoda et al, 2006; Ikoma et al, 2002; Nomura et al, 1996; Liebman et al, 1984). In one series of 76 patients with HCC, this marker was elevated in 69 patients with a mean serum concentration of 900 mcg/L. Much lower mean values were seen in patients with chronic active hepatitis, metastatic disease to the liver, and normal subjects (10 and 42 mcg/L and undetectable, respectively) (Liebman et al, 1984). Elevations in serum levels of DCP are less frequent in tumors less than 3 cm in size (Nakamura et al, 2006; Weitz and Liebman, 1993;). Aoyagi et al (1996) as well as Weitz and Liebman (1993) reported that abnormal prothrombin levels do not correlate well with serum AFP.
Toyoda at al (2006) measured AFP, lens culinaris agglutinin A-reactive fraction of AFP (AFP-L3), and DCP for the evaluation of tumor progression and prognosis of patients with HCC (n = 685) at the time of initial diagnosis. Positivity for AFP > 20 ng/dL, AFP-L3 > 10% of total AFP, and/or DCP > 40 mAU/mL was determined. In addition, tumor markers were measured after treatment of HCC. Of the 685 patients, 337 (55.8%) were positive for AFP, 206 (34.1%) were positive for AFP-L3, and 371 (54.2%) were positive for DCP. In a comparison of patients positive for only 1 tumor marker, patients positive for AFP-L3 alone had a greater number of tumors, whereas patients positive for DCP alone had larger tumors and a higher prevalence of portal vein invasion. When patients were compared according to the number of tumor markers present, the number of markers present clearly reflected the extent of HCC and patient outcomes. The number of markers present significantly decreased after treatment. The authors concluded that tumor markers AFP-L3 and DCP appeared to represent different features of tumor progression in patients with HCC and that the number of tumor markers present could be useful for the evaluation of tumor progression, prediction of patient outcome, and treatment efficacy.
The National Comprehensive Cancer Network's guideline on HCC (NCCN, 2008) does not include measurement of DCP among the surveillance test options for HCC. According to NCCN guidelines, proposed surveillance for the early detection of HCC among high-risk populations (e.g., chronic hepatitis C virus-infected patients) includes liver ultrasonography every 3 to 6 months and evaluation of alkaline phosphatase, albumin, and AFP. The guidelines stated, "It is not yet clear if early detection of hepatocellular cancer with routine screening improves the percentage of patients detected with disease at a potentially curative stage, but high-risk chronic hepatitis C virus - infected patients should be considered for ongoing recurrent screening until these issues have been resolved. The level of des-gamma-carboxy-prothrombin protein induced by vitamin K absence (PIVKA-II) is also increased in many patients with hepatocellular carcinoma. However, as is true with AFP, PIVKA-II may be elevated in patients with chronic hepatitis." Furthermore, according to Sherman (2008), DCP has not been adequately studied as a screening test for HCC and cannot be recommended at this time.
NMP66
Researchers at Matritech (Newton, MA) have detected the presence of nuclear matrix protein (NMP) in the blood of women at the early stage of breast cancer, which is absent in the blood of healthy women, as well as those with fibroadenoma. NMP66 has been selected as a marker for further development and clinical trials of a test for use in the detection and monitoring of women with, or at risk for, breast cancer have been initiated (Wright and McGechan, 2003). However, there are no published studies on the effectiveness of NMP66 testing at this time.
HERmark
HERmark Breast Cancer Assay ( biosciences monogram) is used to help determine prognosis and therapeutic choices for metastatic breast cancer (Raman, et al., 2013). Clinical practice guidelines recommend determining HER2 status in patients with all invasive breast cancer, but caution that current HER2 testing methods such as central immunohistochemistry and Fluorescence in situ Hybridization test may be inaccurate in approximately 20% of cases. According to the HERmark Web site, their method precisely quantifies HER2 total protein and HER2 homodimer levels in formalin-fixed, paraffin-embedded tissue sections and outperformed Fluorescence in situ Hybridization at determining patient outcomes in patients with metastatic breast cancer.
HERmark testing has been proposed for a number of indications, including use to predict response to trastuzumab in the treatment of metastatic breast cancer. Monogram, the manufacturer of the HERmark test, claims that the test can provide a more precise and quantitative measurement of the HER2 gene than IHC and fluorescent in-situ hybridization (FISH) tests. The HERmark provides a quantitative measurement of HER2 total protein and HER2 homodimer levels, while conventional methods are an indirect measure of the HER2 gene, the manufacturer claims. The HERmark test will be offered as a CLIA-validated assay through Monogram's CAP-certified clinical laboratory. Other proposed indications for HERmark include determining the prognosis for breast cancer, and predicting treatment results in cancers other than breast cancer (e.g., ovarian prostate, head and neck, etc.). There are no current recommendations from leading medical professional organizations for the use of HERmark testing for breast cancer.
Yardley et al (2015) compared quantitative HER2 expression by the HERmark Breast Cancer Assay (HERmark) with routine HER2 testing by immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH), and correlated HER2 results with overall survival (OS) of breast cancer patients in a multicenter Collaborative Biomarker Study (CBS). Two hundred and thirty-two formalin-fixed, paraffin-embedded breast cancer tissues and local laboratory HER2 testing results were provided by 11 CBS sites. HERmark assay and central laboratory HER2 IHC retesting were retrospectively performed in a blinded fashion. HER2 results by all testing methods were obtained in 192 cases. HERmark yielded a continuum of total HER2 expression (H2T) ranging from 0.3 to 403 RF/mm2 (approximately 3 logs). The distribution of H2T levels correlated significantly (P<0.0001) with all routine HER2 testing results. The concordance of positive and negative values (equivocal cases excluded) between HERmark and routine HER2 testing was 84% for local IHC, 96% for central IHC, 85% for local FISH, and 84% for local HER2 status. OS analysis revealed a significant correlation of shorter OS with HER2 positivity by local IHC (HR=2.6, P=0.016), central IHC (HR=3.2, P=0.015), and HERmark (HR=5.1, P<0.0001) in this cohort of patients most of whom received no HER2-targeted therapy. The OS curve of discordant low (HER2 positive but H2T low, 10% of all cases) was aligned with concordant negative (HER2 negative and H2T low, HR=1.9, P=0.444), but showed a significantly longer OS than concordant positive (HER2 positive and H2T high, HR=0.31, P=0.024). Conversely, the OS curve of discordant high (HER2 negative but H2T high, 9% of all cases) was aligned with concordant positive (HR=0.41, P=0.105), but showed a significantly shorter OS than concordant negative (HR=41, P<0.0001).
MDM2
- increased p53 expression is tightly linked with increased MDM2 expression; and
- patients who have tumors that display increased p53 and MDM2 expression may have the poorest overall survival.
Because there was no evidence to support the conclusion that p53 mutation is associated with poorer survival, it seemed clear that increased p53 expression in RCC occurs independent of mutation. The authors stated that further investigation of the mechanisms leading to increased p53/MDM2 expression in RCC may lead to improved prognostication and to the identification of novel therapeutic interventions.
OVA1
OVA1 is a blood test used to aid in the evaluation of pelvic masses for the likelihood of malignancy before surgery. OVA1 measures five biomarkers: apolipoprotein A1 (Apo A-1), beta-2 microglobulin (B2M), CA-125 prealbumin and transferrin. The results of these measurements are applied to an algorithm, resulting in a numerical score.
The OVA1 Test (Vermillion Inc. and Quest Diagnostics) is a serum test that is intended to help physicians determine if a woman is at risk for a malignant pelvic mass prior to biopsy or exploratory surgery, when the physician’s independent clinical and radiological evaluation does not indicate malignancy (Mundy, et al., 2010). The OVA1 Test employs an in vitro diagnostic multivariate index (IVDMIA) that combines the results of five immunoassays to produce a numerical score indicating a women's likelihood of malignancy. The OVA1 Test is intended to help physicians assess if a pelvic mass is benign or malignant in order to help determine whether to refer a woman to a gynecologic oncologist for surgery. The OVA1 Test was cleared by the FDA for use in women who meet the following criteria: over age 18, ovarian adnexal mass present for which surgery is planned, and not yet referred to an oncologist. The intended use of the OVA1 Test is an aid to further assess the likelihood that malignancy is present when the physician’s independent clinical and radiological evaluation does not indicate malignancy. According to the product labeling, the OVA1 Test is not intended as a screening or stand-alone diagnostic assay. There is a lack of evidence in the peer-reviewed published medical literature on the OVA1 Test.
Ueland et al (2011) sought to compare the effectiveness of physician assessment with the OVA1 multivariate index assay in identifying high-risk ovarian tumors. The multivariate index assay was evaluated in women scheduled for surgery for an ovarian tumor in a prospective, multi-institutional trial involving 27 primary- care and specialty sites throughout the United States. Preoperative serum was collected, and results for the multivariate index assay, physician assessment, and CA 125 were correlated with surgical pathology. Physician assessment was documented by each physician before surgery. CA 125 cutoffs were chosen in accordance with the referral guidelines of the American College of Obstetricians and Gynecologists. The study enrolled 590 women, with 524 evaluable for the multivariate index assay and CA 125, and 516 for physician assessment. Fifty-three percent were enrolled by nongynecologic oncologists. There were 161 malignancies and 363 benign ovarian tumors. Physician assessment plus the multivariate index assay correctly identified malignancies missed by physician assessment in 70% of nongynecologic oncologists, and 95% of gynecologic oncologists. The multivariate index assay also detected 76% of malignancies missed by CA 125. Physician assessment plus the multivariate index assay identified 86% of malignancies missed by CA 125, including all advanced cancers. The investigators stated that the performance of the multivariate index assay was consistent in early- and late-stage cancers.
Ware Miller et al (2011) sought to estimate the performance of the ACOG referral guidelines for pelvic mass with the OVA1 multivariate index assay. A prospective, multi-institutional trial included 27 primary care and specialty sites throughout the United States. The College guidelines were evaluated in women scheduled for surgery for an ovarian mass. Clinical criteria and blood for biomarkers were collected before surgery. A standard CA 125-II assay was used and the value applied to the multivariate index assay algorithm and the CA 125 analysis. Study results were correlated with surgical pathology. Of the 590 women enrolled with ovarian mass on pelvic imaging, 516 were evaluable. There were 161 malignancies (45 premenopausal and 116 postmenopausal). The College referral criteria had a modest sensitivity in detecting malignancy. Replacing CA 125 with the multivariate index assay increased the sensitivity (77-94%) and negative predictive value (87-93%) while decreasing specificity (68-35%) and positive predictive value (52-40%). Similar trends were noted for premenopausal women and early-stage disease.
Bristow et al (2013) sought to validate the effectiveness of a multivariate index assay in identifying ovarian malignancy compared to clinical assessment and CA125-II, among women undergoing surgery for an adnexal mass after enrollment by non-gynecologic oncology providers. A prospective, multi-institutional trial enrolled female patients scheduled to undergo surgery for an adnexal mass from 27 non-gynecologic oncology practices. Pre-operative serum samples and physician assessment of ovarian cancer risk were correlated with final surgical pathology. A total of 494 subjects were evaluable for multivariate index assay, CA125-II, and clinical impression. Overall, 92 patients (18.6%) had a pelvic malignancy. Primary ovarian cancer was diagnosed in 65 patients (13.2%), with 43.1% having FIGO stage I disease. For all ovarian malignancies, the sensitivity of the multivariate index assay was 95.7% (95%CI=89.3-98.3) when combined with clinical impression. The multivariate index assay correctly predicted ovarian malignancy in 91.4% (95%CI=77.6-97.0) of cases of early-stage disease, compared to 65.7% (95%CI=49.2-79.2) for CA125-II. The multivariate index assay correctly identified 83.3% malignancies missed by clinical impression and 70.8% cases missed by CA125-II. Multivariate index assay was superior in predicting the absence of an ovarian malignancy, with a negative predictive value of 98.1% (95%CI=95.2-99.2). Both clinical impression and CA125-II were more accurate at identifying benign disease. The multivariate index assay correctly predicted benign pathology in 204 patients (50.7%, 95%CI=45.9-55.6) when combined with clinical impression.
Longoria et al (2014) sought to analyze the effectiveness of the OVA1 multivariate index assay (MIA) in identifying early-stage ovarian malignancy compared to clinical assessment, CA 125-II, and modified American Congress of Obstetricians and Gynecologists (ACOG) guidelines among women undergoing surgery for an adnexal mass. Patients were recruited in 2 related prospective, multi-institutional trials involving 44 sites. All women had preoperative imaging and biomarker analysis. Preoperative biomarker values, physician assessment of ovarian cancer risk, and modified ACOG guideline risk stratification were correlated with surgical pathology. A total of 1016 patients were evaluable for MIA, CA 125-II, and clinical assessment. Overall, 86 patients (8.5%) had primary-stage I/II primary ovarian malignancy, with 70.9% having stage I disease and 29.1% having stage II disease. For all early-stage ovarian malignancies, MIA combined with clinical assessment had significantly higher sensitivity (95.3%; 95% confidence interval [CI], 88.6-98.2) compared to clinical assessment alone (68.6%; 95% CI, 58.2-77.4), CA 125-II (62.8%; 95% CI, 52.2-72.3), and modified ACOG guidelines (76.7%; 95% CI, 66.8-84.4) (P < .0001). Among the 515 premenopausal patients, the sensitivity for early-stage ovarian cancer was 89.3% (95% CI, 72.8-96.3) for MIA combined with clinical assessment, 60.7% (95% CI, 42.4-76.4) for clinical assessment alone, 35.7% (95% CI, 20.7-54.2) for CA 125-II, and 78.6% (95% CI, 60.5-89.8) for modified ACOG guidelines. Early-stage ovarian cancer in postmenopausal patients was correctly detected in 98.3% (95% CI, 90.9-99.7) of cases by MIA combined with clinical assessment, compared to 72.4% (95% CI, 59.8-82.2) for clinical assessment alone, 75.9% (95% CI, 63.5-85.0) for CA 125-II, and 75.9% (95% CI, 63.5-85.0) for modified ACOG guidelines.
Bristow et al (2013) assessed the impact on referral patterns of using the OVA1 Multivariate Index Assay, CA125, modified-American College of Obstetricians and Gynecologists referral guidelines, and clinical assessment among patients undergoing surgery for an adnexal mass after initial evaluation by nongynecologic oncologists. Overall, 770 patients were enrolled by nongynecologic oncologists from 2 related, multiinstitutional, prospective trials and analyzed retrospectively. All patients had preoperative imaging and biomarker analysis. The subset of patients enrolled by nongynecologic oncologists was analyzed to determine the projected referral patterns and sensitivity for malignancy based on multivariate index assay (MIA), CA125, modified-American College of Obstetricians and Gynecologists (ACOG) guidelines, and clinical assessment compared with actual practice. The prevalence of malignancy was 21.3% (n = 164). In clinical practice, 462/770 patients (60.0%) were referred to a gynecologic oncologist for surgery. Triage based on CA125 predicted referral of 157/770 patients (20.4%) with sensitivity of 68.3% (95% confidence interval [CI], 60.8-74.9). Triage based on modified-ACOG guidelines would have resulted in referral of 256/770 patients (33.2%) with a sensitivity of 79.3% (95% CI, 72.4-84.8). Clinical assessment predicted referral of 184/763 patients (24.1%) with a sensitivity of 73.2% (95% CI, 65.9-79.4). Risk stratification using multivariate index assay would have resulted in referral of 429/770 (55.7%) patients, with sensitivity of 90.2% (95% CI, 84.7-93.9). MIA demonstrated statistically significant higher sensitivity (P < .0001) and lower specificity (P < .0001) for detecting malignancy compared with clinical assessment, CA125, and modified-ACOG guidelines.
Goodrich et al (2014) investigated the relationship between imaging and the multivariate index assay (MIA) in the prediction of the likelihood of ovarian malignancy before surgery. Subjects were recruited in 2 related prospective, multiinstitutional trials that involved 44 sites across the United States. Women had ovarian imaging, biomarker analysis, and surgery for an adnexal mass. Ovarian tumors were classified as high risk for solid or papillary morphologic condition on imaging study. Biomarker and imaging results were correlated with surgical findings. Of the 1110 women who were enrolled with an adnexal mass on imaging, 1024 cases were evaluable. There were 255 malignant and 769 benign tumors. High-risk findings were present in 46% of 1232 imaging tests and 61% of 1024 MIA tests. The risk of malignancy increased with rising MIA scores; similarly, the likelihood of malignancy was higher for high-risk, compared with low-risk, imaging. Sensitivity and specificity for the prediction of malignancy were 98% (95% CI, 92-99) and 31% (95% CI, 27-34) for ultrasound or MIA; 68% (95% CI, 58-77) and 75% (95% CI, 72-78) for ultrasound and MIA, respectively. For computed tomography scan or MIA, sensitivity was 97% (95% CI, 92-99) and specificity was 22% (95% CI, 16-28); the sensitivity and specificity for computed tomography scan and MIA were 71% (95% CI, 62-79) and 70% (95% CI, 63-76). Only 1.6% of ovarian tumors were malignant when both tests indicated low risk.
An assessment by the BlueCross BlueShield Association Technology Evaluation Center (BCBSA, 2013) stated: "The evidence regarding the effect of OVA1 ...on health outcomes is indirect and based on studies of diagnostic performance of the tests in patients undergoing surgery for adnexal masses. Although the studies show improvements in sensitivity and worsening of specificity with the use of the tests in conjunction with clinical assessment, there are problems in concluding that this results in improved health outcomes. The clinical assessment performed in the studies is not well characterized. Although OVA1 improves sensitivity, specificity declines so much that most patients test positive."
An technology assessment by the ECRI Institute (2015) concluded that the evidence on OVA1 consists of cross-sectional diagnostic accuracy studies. This evidence as reported in article abstracts is unclear as to whether use of OVA1 improves patient-oriented outcomes because none of the studies reported the direct impact of these tests on survival or quality of life. The primary rationale for using these tests is to select the type of surgeon to perform the primary surgery.
Stewart et al (2016) reported on a survey of primary care physicians on how often they refer patients diagnosed with ovarian cancer to gynecological oncologists, finding that a total of 84% of primary care physicians (87% of family/general practitioners, 81% of internists and obstetrician/gynecologists) said they always referred patients to gynecologic oncologists for treatment. Common reasons for not always referring were patient preference or lack of gynecologic oncologists in the practice area. A total of 23% of primary care physicians had heard of the OVA1 test, which helps to determine whether gynecologic oncologist referral is needed. The authors noted that, although referral rates reported here are high, it is not clear whether ovarian cancer patients are actually seeing gynecologic oncologists for care.
Eskander et al (2016) conducted a retrospective chart review of patients who received the OVA1. Twenty-two obstetricians/gynecologists were recruited from a variety of practices and hospitals throughout the United States. A total of 136 patients with elevated-risk assay results were assessed, of whom 122 underwent surgery to remove an adnexal mass. Prior to surgery, 98 (80%) of the patients were referred to a gynecologic oncologist with an additional 11 (9%) having a gynecologic oncologist available if required by intra-operative findings. Primary ovarian cancer was found in 65 (53%) patients, and gynecologic oncologists performed 61 (94%) of the initial surgeries these patients. Similar results were found in premenopausal and postmenopausal patients.
- sensitivity of OVA1;
- sensitivity of mACOG; and
- percentage of patients, not referred to a gynecologic oncologist, who were correctly diagnosed with advanced epithelial ovarian cancer (EOC).
The authors concluded that OVA1 is a more cost-effective triage strategy than mACOG or CA-125. It is expected to increase the percentage of women with ovarian cancer that are referred to gynecologic oncologists, which is shown to improve clinical outcomes. Limitations include the use of assumptions when published data was unavailable, and the use of multiple sources for survival data.
Urban et al (2017) reported that the addition of a patient-reported symptom index (SI), which captures subjective symptoms in an objective manner, improved the sensitivity of the OVA1 multivariate index assay (MIA). The investigators conducted a prospective study of patients seen at a tertiary care medical center. Following consent, patients completed an SI and preoperative serum was collected for an OVA1 multivariate index assay. Results for the SI and OVA1 were correlated with operative findings and surgical pathology. Of 218 patients enrolled, 124 (56.9%) had benign disease and 94 (43.1%) had borderline tumors or carcinomas. Sixty-six patients had a primary ovarian or fallopian tube cancer. The median age of patients enrolled in this study was 54 years (interquartile range, 44-63 years), of whom 148 (67.9%) were postmenopausal. More than a third (36.3%) of patients with benign masses was accurately identified as low risk by MIA and SI. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of the SI relative to primary ovarian cancer was 87.9% (95% CI, 77.9%-93.7%), 70.2% (95% CI, 61.6%-77.5%), 61.1% (95% CI 51.0-70.2%) and 91.6% (95% CI, 84.3%-95.7%), respectively. The sensitivity, specificity, PPV and NPV of CA125 was 75.4% (95% CI, 63.7%-84.2%), 85.7% (95% CI 78.3%-90.9%), 74.2% (62.6%-82.3%) and 86.4% (95% CI, 79.1%-91.5%), respectively. The sensitivity, specificity, PPV and NPV of the MIA were 93.9% (95% CI, 85.4%-97.6%), 55.6 (95% CI 46.9%-64.1%), 53.0% (95% CI 44.0%-61.8% and 94.5% (95% CI, 94.5%-100%), respectively. The overall sensitivity for the combination of MIA plus SI was 100% (66/66; 95% CI, 94.5%-100%), and specificity was 36.3% (45/124; 95% CI, 28.4%-45.0%), with a PPV of 45.5% (37.6% to 53.6%) and a NPV of 100% (95% CI, 92.1%-100%). Limitations of this study noted by the authors include the small sample size and the high prevalence of ovarian malignancies in this population that was largely from a tertiary care center. It should also be noted that the sensitivity and negative predictive value of SI plus CA 125 was 96.9% (95% CI 89.5%-99.2%) and 97.3% (95% CI 90.5%-99.2%), which exceeded that of MIA alone but was somewhat less than MIA plus SI.
Ovarian cancer guidelines from the National Comprehensive Cancer Network (2016) note that the Society of Gynecologic Oncology (SGO), the FDA, and the Mayo Clinic have stated that the OVA1 test should not be used as a screening tool to detect ovarian cancer. The NCCN explains that the OVA1 attempts to preoperatively classify adenixal masses as benign or malignant and suggests that patients can be assessed for who should undergo surgery by an experienced gynecologic oncologist and who can have surgery in the community. "Based upon data documenting an increased survival, NCCN guidelines panel members recommended that all patients should undergo surgery by an experienced gynecologic oncologist (Category 1 recommendation)."
Guidelines on management of adnexal masses from the American College of Obstetricians and Gynecologists (ACOG, 2016) state that the OVA1 multivariate index assay has demonstrated higher sensitivity and negative predictive value compared with clinical impression and CA 125 alone. The guidelines state that serum biomarker panels [OVA1 and ROMA] may be used as an alternative to CA 125 alone in determining the need for referral to or consultation with a gynecological oncologist when an adenexal mass requires surgery. The guidelines state that trials that have evaluated the predictive value of these panels show potential for improved specificity; "[h]owever, comparative research has not yet defined the best testing approach."
- postmenopausal with elevated CA 125 level, ultrasound findings suggestive of malignancy, ascites, a nodular or fixed pelvic mass, or evidence of abdominal or distant metastases;
- premenosausal with very elevated CA 125 level, ultrasound findings suggestive of malignancy, ascites, a nodular or fixed pelvic mass, or evidence of abdominal or distant metastases;
- premenopausal or postmenopausal with an elevated score on a formal risk assessment test such as the multivariate index assay, risk of malignancy index, or the Risk of Ovarian Malignancy algorithm or one of the ultrasound-based scoring systems from the International Ovarian Tumor Analysis group.
The UK National Institute for Health Research Health Technology Assessment Program commissioned an assessment (Westwood, et al., 2016) comparing the Risk of Malignancy Index (RMI) to alternative risk scores for ovarian cancer, including Overa/OVA2 (MIA2G), as well as the ROMA score, IOTA group's simple rules ultrasound classification system (IOTA), and the ADNEX model. The RMI 1 score uses three components (measured serum CA125 levels, ultrasound imaging and menopausal status) to calculate a risk score. The ROMA score uses serum HE4 and serum CA125 levels, along with menopausal status, to generate an individualized estimate of the risk that a person has ovarian cancer. Simple Rules is a morphological scoring system, developed by the International Ovarian Tumour Analysis Group (IOTA), is based on the presence of ultrasound features (described as rules) to characterize an ovarian mass as benign or malignant. The ADNEX model uses nine predictors, three clinical variables [age, serum CA125 and type of referral center (oncology or other)] and six ultrasound variables; iPhone, Android and web applications are available for calculating the ADNEX risk score. The overall objective of the assessment was to summarize the evidence on the clinical effectiveness and cost-effectiveness of using these alternative risk scores to guide referral decisions for women with suspected ovarian cancer in secondary care. In the base-case analysis, the RMI 1 was the least effective and the second least expensive (Westwood, et al., 2018). The IOTA group’s simple ultrasound rules was the least expensive and the second most effective, and thereby dominated the RMI 1. The ADNEX model was the most effective, and compared with the IOTA group’s simple ultrasound rules, resulted in an incremental cost-effectiveness ratio of £15,304 per QALY gained. The remaining risk scores, ROMA and Overa (MIA2G), were dominated (both more costly and less effective) than the IOTA groups simple ultrasound rultes and the ADNEX model. The incremental analysis indicated that, up to thresholds of £15,304 per QALY gained, the IOTA group’s simple ultrasound rules are cost-effective, whereas the IOTA group’s ADNEX model is cost-effective for higher willingness to pay thresholds.
Dunton et al (2019a) stated that based on evidence that African American (AA) women have lower CA125 values than Caucasian (C) women, these investigators examined if this disparity would have an impact on ovarian cancer detection using CA125 and multi-variate index assay (MIA). Serum from 2 prospective trials of 1,029 samples (274 malignancies [250 C/24 AA]) were analyzed for CA125 and MIA results. Clinical performance was calculated. Sensitivity of MIA in Caucasian women was 93.2 %, 74.4 % for CA125 at the ACOG approved cut-off level of 200 U/ml cut-off, and 80.4 % using the 2007, Dearking 67 U/ml cut-off. In AA women, MIA sensitivity was 79.2 %, 33.3 % for CA125 at the ACOG approved cut-off levels and 62.5 % at the 2007, Dearking 67 U/ml cut-off. The authors concluded that these findings supported that CA125 in AA women with adnexal masses had lower sensitivity than MIA no matter what the cut-off value was. Implementation of MIA in evaluation of adnexal masses should increase sensitivity of detection of malignancy compared with CA125, especially in AA women.
These researchers stated that as the number of AA patients in the studies that made up the analysis database for this research was fairly small, they presented data on all malignancies rather than dividing them based on histologic subtype. During their exploratory analysis, these investigators carried out these calculations, but due to the small sample sizes found the confidence intervals (CIs) too wide to draw solid conclusions from. These researchers are in the process of developing research opportunities to add further AA women with primary ovarian malignancies to their specimen repositories in order to confirm the results found in this analysis. Moreover, the authors stated that within the next 10 years, this research could be a stepping-stone toward closing the survivorship gap between Caucasian women and AA women, where ovarian cancer is concerned. Increased use of a more sensitive test such as MIA in minority women, and clinical awareness of the shortcomings of entrenched medical practices like CA125 could potentially increase early detection, which is key for improved survivorship.
Dunton et al (2019b) examined the serum values of risk of ovarian malignancy algorithm (ROMA) and multi-variate index assay (MIA) in subgroups of women who underwent surgery for adnexal masses to determine sensitivity, specificity, and PPV and NPV for the detection of malignancy in different ethnic populations. Serum samples from 2 prospective trials of 1,029 women in which 274 women diagnosed with malignancy were analyzed for ROMA scores and MIA results. Biomarker data were obtained from the previous prospective studies that validated the MIA test. Of these, 250 women were Caucasian (C) and 24 were African-American (AA). Sensitivity, specificity, PPV, NPV, and CIs for pre-operative test results were calculated using DTComPair package of the R programming language. In pre-menopausal women, a ROMA value equal to or greater than 1.14 indicated a high-risk of finding epithelial ovarian cancer. In pre-menopausal women, MIA values greater than 5.0 were associated with a greater risk of malignancy. In post-menopausal women, a ROMA value equal to or greater than 2.99 indicated a high-risk of finding epithelial ovarian cancer. In post-menopausal women, MIA values greater than 4.4 were associated with a greater risk of malignancy. Primary ovarian malignancy was diagnosed in 179 cases (167 C/12 AA) and metastatic disease to the ovary in an additional 27 cases (22 C/5 AA). The authors concluded that these findings showed that ROMA in AA women with adnexal masses exhibited lower sensitivity for the detection of malignancy than did MIA. These investigators stated that implementation of MIA in the evaluation of adnexal masses would increase the sensitivity of the detection of malignancy compared with ROMA, with the most marked results in AA women. Moreover, these researchers stated that this was the 1st study that examined the sensitivity of ROMA and MIA for ovarian malignancy based on ethnic difference. They stated that a drawback of this trial was the small number of African-American women in the 2 prospective studies that were database used for analysis resulting in a lack of statistical evidence of superiority. However, given the biological basis of lower CA 125 in African-American women, the authors felt that these findings were clinically significant to alert practitioners of the possible false negatives of ROMA in African-American women. These investigators were exploring research to add additional African-American women to the database to confirm these findings.
In a retrospective study, Dunton et al (2020) examined the use of Multivariate Index Assay (MIA OVA1) by gynecologists and determined referral practices and surgical decision-making for women with adnexal masses and low-risk MIA OVA1 scores. Information on patients who received an OVA1 test was collected from 22 gynecologic practices via a chart review. Referral patterns were examined for patients with low-risk OVA1 results before 1st surgical intervention. Chart reviews were from a variety of practice and hospital settings representing major geographic regions within the U.S. A total of 282 independent patient charts were reviewed. Low-risk results were found for 146 patients (52 %). Surgery was carried out on 82 (56 %) patients with low-risk scores. The referral rate to specialty care was 21 % (17/82) for low-risk OVA1 patients. A total of 3 low-malignant potential tumors were identified in the low-risk patients, with no cases of invasive malignancy; 86 % of the surgeries carried out on low-risk OVA1 patients were minimally invasive. In 44 % of the low-risk OVA1 patients, no surgical intervention was carried out. The authors concluded that a high proportion of low-risk OVA1 patients were not referred to a gynecologic oncologist before surgery, indicating gynecologists may use MIA OVA1 along with clinical and radiographic findings to appropriately retain patients for their care. This practice is safe and may be cost-saving, with patient satisfaction implications. These researchers stated that these findings may help catalyze the development of prospective studies to better examine the role of OVA1 in the clinical decision-making process and to help with long-term collection of data that can be used to examine outcomes of decisions made on both low-and elevated risk OVA1 tests.
The authors stated that this study had several drawbacks. Because this was a retrospective study, it may be of limited use in causal inference because it was not possible to identify all confounding factors impacting referral and surgical planning. Physicians who chose to participate in the study may have been more likely to have a positive opinion of the test and use it to guide referral. Furthermore, because physicians were allowed to select patients for review there was a possibility of a selection or recall bias for cases that best fit the physician’s ideal of the clinical application in both the elevated and low-risk situations. For instance, in a previous study (Eskander et al, 2016), these investigators showed the malignancy rate of this population to be 64 %, which is much higher than previous studies of intended use populations (Bristow et al, 2013; Ueland et al, 2011).
Dunton et al (2021) noted that ovarian cancer is the deadliest gynecologic cancer, with no recommended screening test to aid in early detection. Cancer antigen 125 (CA125) is a serum biomarker commonly used by clinicians to evaluate pre-operative cancer risk, but it under-performs in pre-menopausal women, early-stage malignancies, and several histologic subtypes. OVA1 is a multi-variate index assay that combines CA125 and 4 other serum proteins to evaluate the malignant risk of an adnexal mass. These researchers examined the performance of OVA1 in a cohort of patients with low-risk serum CA125 values. They analyzed patient data from previous collections (n = 2,305, prevalence = 4.5 %) where CA125 levels were at or below 67 units/milliliter (U/ml) for pre-menopausal women and 35 U/ml for post-menopausal women. These investigators compared the performance of OVA1 to CA125 in classifying the risk of malignancy in this cohort, including sensitivity, specificity, PPV and NPV. The overall sensitivity of OVA1 in patients with a low-risk serum CA125 was 59 % with a false-positive rate of 30 %. OVA1 detected over 50 % of ovarian malignancies in pre-menopausal women despite a low-risk serum CA125. OVA1 also correctly identified 63 % of early-stage cancers missed by CA125. The most common epithelial ovarian cancer subtypes in the study population were mucinous (25 %) and serous (23 %) carcinomas. Despite a low-risk CA125, OVA1 successfully detected 83 % of serous, 58 % of mucinous, and 50 % of clear cell ovarian cancers. The authors concluded that as a standalone test, CA125 missed a significant number of ovarian malignancies that could be detected by OVA1. This was particularly important for pre-menopausal women and early-stage cancers, which have a much better long-term survival than late-stage malignancies. These investigators stated that using OVA1 in the setting of a normal serum CA125 could aid in identifying at-risk ovarian tumors for referral to a gynecologic oncologist, potentially improving OS.
The authors stated that a drawback of this study was the retrospective nature of the data analysis, which was carried out after merging several study databases. Furthermore, the percentage of early-stage ovarian cancer in this study (70 %) was twice that expected in the general population, suggesting a possible sampling bias. However, this shift toward early-stage cancers allowed for a more robust evaluation of test performance in this cohort.
In the largest-of-its-kind study, Reilly et al (2023) examined the use of CA125 and OVA1, commonly employed as ovarian tumor markers for evaluating the risk of malignancy. This study focused on the ability and utility of these tests to reliably predict patients at low risk for ovarian cancer. Clinical utility endpoints were 12-month maintenance of benign mass status, reduction in gynecologic oncologist referral, avoidable surgical intervention, and associated cost savings. This was a retrospective, multi-center review of data from electronic medical records and administrative claims databases. Patients receiving a CA125 or OVA1 test between October 2018 and September 2020 were identified and followed for 12 months using site-specific electronic medical records (EMRs) to examine tumor status and utilization outcomes. Propensity score adjustment was used to control for confounding variables. Payer allowed amounts from Merative MarketScan Research Databases were used to estimate 12-month episode-of-care costs per patient, including surgery and other interventions. Among 290 low-risk OVA1 patients, 99.0 % remained benign for 12 months compared with 97.2 % of 181 low-risk CA125 patients. The OVA1 cohort exhibited 75 % lower odds of surgical intervention in the overall sample of patients (adjusted OR: 0.251, p ≤ 0.0001), and 63 % lower odds of gynecologic oncologist utilization among pre-menopausal women (adjusted OR: 0.37, p = 0.0390) versus CA125. OVA1 showed significant savings in surgical interventions ($2,486, p ≤ 0.0001) and total episode-of-care costs ($2,621, p ≤ 0.0001) versus CA125. The authors concluded that the findings of this study suggested that OVA1 was associated with significant reduction in avoidable surgeries overall and sub-specialty referrals for pre-menopausal patients, as well as substantial cost savings per patient. Moreover, these researchers stated that opportunities for future research include using cost-utility modeling to estimate the long-term impact of using biomarker assays on ovarian tumor treatment costs and quality of life.
The authors stated that there were drawbacks inherent to retrospective chart reviews. Because of the reliance on medical records, data quality is dependent upon accurate documentation and the ability of the medical records reviewer to understand the gathered information. As is true of all “real world” studies, it is not possible to fully control for exogenous factors that could influence the results of the study, such as determination of eligibility to receive an ovarian cancer diagnostic test and which test (OVA1 or CA125) they should receive. Despite both tests using biomarkers to determine the likelihood of ovarian cancer, there may be other clinical factors physicians rely upon to determine which test patients receive. Furthermore, OVA1 is not widely covered across all commercial plans, limiting which physicians can order the test and ultimately which patients can receive it. These investigators examined the impact of the volume imbalance and potential data skewness and determined that a propensity score adjustment methodology was most appropriate to both leverage the considerable treatment cohort volume and maintain the comparator cohort volume. Moreover, the low prevalence of high-risk cases led these researchers to postpone analysis of this subgroup until adequately powered samples can be obtained. In addition, the study’s utilization and economic outcomes were captured from events occurring within the enrolled site’s EMRs, which may have excluded a nominal number of “out-of-network” events. Finally, the surgery metrics did not capture utilization of laparotomies, which the participating sites confirmed was not the standard-of-care and is rarely deployed based on the presence of the preferred minimally invasive surgical approach.
Fritsche and Bullock (2023) stated that patients with adnexal masses suspicious for malignancy benefit from referral to oncology specialists during pre-surgical assessment of the mass. OVA1 is a multi-variate assay using a 5-biomarker panel that offers high overall and early-stage sensitivity; however, OVA1 has a high false-positive rate for benign masses. Overa, a 2nd-generation multi-variate index assay was developed to reduce the false-positive rate. In a retrospective study, these researchers employed Overa as a reflex for OVA1 to increase specificity. OVA1 cut-off scores were established to place patients into 3 categories: low, intermediate, and high cancer risk. Samples with intermediate-risk OVA1 scores were reflexed to the Overa and defined as high or low risk. This protocol was tested with 1,035 prospectively collected serum samples and validated with an independent prospectively collected sample set (n = 207). A total of 359 samples (35 %) had intermediate OVA1 scores. Reflexing these to Overa eliminated 58 % of the false-positives and improved the overall specificity from 50 % to 72 %. This finding was confirmed in the independent dataset, in which the specificity increased from 56 % to 73 %. The authors concluded that reflexing samples with intermediate OVA1 scores significantly lowered the false-positive rate; thus, reducing unnecessary surgical referrals.
The authors stated that a drawback of this study was the retrospective nature of the data. Moreover, these researchers stated that studies are under way to examine the clinical performance and utility of OVA1plus in a population that reflects a “real-world” demographic of patients. It should also be noted that both authors were employed or contracted by Aspira Women's Health Inc. or its subsidiary, Aspira Labs, at the time of contribution. Aspira Women's Health, Inc. provided funding for this study.
Furthermore, an UpToDate review on “Adnexal mass: Role of serum biomarkers in diagnosing epithelial carcinoma of the ovary, fallopian tube, or peritoneum” (Li, 2023) states that “Biomarker panels -- Commercially available biomarker panels, including OVA1, Overa, Risk of Malignancy Algorithm (ROMA), Risk of Malignancy Index (RMI), and the Assessment of Different Neoplasias in the Adnexa (ADNEX) model are used to assess the likelihood of malignancy in patients in whom surgery for an adnexal mass is planned. These tests should not be used alone to decide whether to proceed with surgical exploration for an adnexal mass.
- The individual biomarkers used in each panel vary; test availability and expense may factor into which test is most appropriate for each clinical situation.
- These tests have not been studied for ovarian cancer screening”.
Furthermore, National Comprehensive Cancer Network’s clinical practice guideline on “Ovarian Cancer/Fallopian Tube Cancer/Primary Peritoneal Cancer” (Version 1.2024) states that “There are a number of biomarker tests and prediction algorithms (based on a variety factors, such as symptoms, imaging results, biomarkers, and patient characteristics) that have been developed for assessing the likelihood of malignancy among patients who have an adnexal mass (and have not yet had surgery). It is important to note that these tests are for preoperative assessment only, and none is suitable for ovarian cancer screening prior to detection of an adnexal mass; they are also not for use as stand-alone diagnostic tests. For example, the OVA1 test is a multivariate index assay (MIA) that uses 5 markers (including transthyretin, apolipoprotein A1, transferrin, beta-2 microglobulin, and CA-125) in preoperative serum to assess the likelihood of malignancy in patients with an adnexal mass for which surgery is planned, with the aim of helping community practitioners determine which patients to refer to a gynecologic oncologist for evaluation and surgery. The Society of Gynecologic Oncology (SGO) and the FDA have stated that the OVA1 test should not be used as a screening tool to detect ovarian cancer in patients without any other signs of cancer, or as a stand-alone diagnostic tool. Moreover, based on data documenting an increased survival, the NCCN Guidelines Panel recommends that all patients with suspected ovarian malignancies (especially those with an adnexal mass) should undergo evaluation by an experienced gynecologic oncologist prior to surgery … A number of specific biomarkers and algorithms using multiple biomarker test results have been proposed for preoperatively distinguishing benign from malignant tumors in patients who have an undiagnosed adnexal/pelvic mass. Biomarker tests developed and evaluated in prospective trials comparing preoperative serum levels to postoperative final diagnosis include serum HE4 and CA-125, either alone or combined using the Risk of Ovarian Malignancy Algorithm [ROMA] algorithm; the MIA (brand name OVA1) based on serum levels of 5 markers: transthyretin, apolipoprotein A1, transferrin, beta-2 microglobulin, and CA-125; and the second-generation MIA (MIA2G, branded name OVERA) based on CA-125, transferrin, apolipoprotein A1, follicle-stimulating hormone [FSH], and HE4. The FDA has approved the use of ROMA, OVA1, or OVERA for estimating the risk for ovarian cancer in those with an adnexal mass for which surgery is planned, and have not yet been referred to an oncologist. Although the American Congress of Obstetricians and Gynecologists (ACOG) has suggested that ROMA and OVA1 may be useful for deciding which patients to refer to a gynecologic oncologist, other professional organizations have been non-committal. Not all studies have found that multi-biomarker assays improve all metrics (i.e., sensitivity, specificity, positive predictive value, negative predictive value) for prediction of malignancy compared with other methods (e.g., imaging, single-biomarker tests, symptom index/clinical assessment). Currently, the NCCN Panel does not recommend the use of these biomarker tests for determining the status of an undiagnosed adnexal/pelvic mass”.
ColonSentry
The ColonSentry test (GeneNews, Toronto, Canada) measures the expression of seven genes, which serve as biomarkers to detect colorectal cancer. Interpretation of the status of these seven biomarkers is intended to assist physicians in identifying patients who have an increased current risk. According to the manufacturer, individuals assessed as having an increased current risk of colorectal cancer should consider having a colonoscopy. Individuals assessed as having a decreased current risk of colorectal cancer should discuss with their doctor further screening, including repeating ColonSentry at regular intervals. There is a lack of evidence in the peer-reviewed published medical literature on the effectiveness of colorectal cancer screening with ColonSentry. No current evidence-based guidelines from medical professional organizations or public health agencies recommend ColonSentry for colorectal cancer screening.
Pharmaco-oncologic Algorithmic Treatment Ranking Service
CureMatch, Inc. (San Diego, CA), a leading company in precision medicine support for oncology, developed a therapy matching and scoring service that is a patient-specific, assistive, rules-based algorithm for ranking pharmaco-oncologic treatment options based on the patient's tumor-specific cancer marker information obtained from prior molecular pathology, immunohistochemical, or other pathology results which have been previously interpreted and reported separately (CureMatch, 2023).
Currently, there is insufficient evidence in the peer-reviewed literature to support the sensitivity or specificity of this test.
Prostate Px
Prostate Px (Aureon) uses a prostate cancer patient's biopsy tissue to provide an assessment of disease severity and disease recurrence. Clinical data is integrated with an analysis of each patient’s cancer using tissue histology and molecular biomarkers, such as androgen receptor, associated with disease progression. Although the manufacturer states that the results of the Prostate Px can be used in decision-making, there is a lack of evidence of the clinical utility of this test in altering the management of patients such that clinical outcomes are improved.
Post-Op Px
Post-Op Px is a prognostic test that utilizes a patented systems pathology approach to analyze prostatectomy tissue by combining cellular, molecular and clinical information to provide a thorough and more accurate picture of each patient's individual risk of prostate cancer recurrence. (Aureon, 2010). Donovan et al (2011) evaluated the performance of a systems-based risk assessment tool with standard defined risk groups and the 10 year postoperative normogram for predicting disease progression. The systems model was found to be more accurate than standard risk groups both to predict significant disease progression (p < 0.001) and for predicting prostate-specific antigen recurrence (p < 0.001). However, this study has not been replicated in the peer-reviewed literature.
CEACAM-7
Messick et al (2010) evaluated carcinoembryonic antigen cellular adhesion molecule-7 (CEACAM-7) expression in rectal cancer as a predictive recurrence factor. A single-institution colorectal cancer database and a frozen tissue biobank were queried for rectal cancer patients. CEACAM-7 messenger RNA (mRNA) expression from normal rectal mucosa and rectal cancers was analyzed using quantitative real-time polymerase chain reaction (PCR). Expression-level differences among normal tissue, disease-free survivors, and those that developed recurrence were analyzed. A total of 84 patients were included in the study, which consisted of 37 patients with non-recurrent disease (median follow-up of 170 months), 29 patients with recurrent disease, and 18 patients with stage IV disease. CEACAM-7 expression was decreased 21-fold in rectal cancers compared with normal mucosa (p = 0.002). The expression levels of CEACAM-7 were relatively decreased in tumors that developed recurrence compared with non-recurrence, significantly for stage II patients (14-fold relative decrease, p = 0.002). For stages I-III, disease-free survival segregates were based on relative CEACAM-7 expression values (p = 0.036), specifically for stage II (p = 0.018). The authors concluded that CEACAM-7 expression is significantly decreased in rectal cancer. Expression differences between long-term survivors and those with recurrent disease introduce a potential tumor marker to define a subset of patients who benefit most from adjuvant therapy. Moreover, they stated that additional study and validation are needed before CEACAM-7 can be applied in clinical settings.
CFL1
Castro et al (2010) assessed the potential value of cofilin (CFL1) gene (main member of the invasion/metastasis pathway) as a prognostic and predictive NSCLC biomarker. Meta-analysis of tumor tissue microarray was applied to examine expression of CFL1 in archival lung cancer samples from 111 patients, and its clinicopathologic significance was investigated. The robustness of the finding was validated using another independent data set. Finally, the authors assayed in vitro the role of CFL1 levels in tumor invasiveness and drug resistance using 6 human NSCLC cell lines with different basal degrees of CFL1 gene expression. Cofilin levels in biopsies discriminated between good and bad prognosis at early tumor stages (IA, IB, and IIA/B), where high CFL1 levels are correlated with lower overall survival rate (p < 0.0001). Biomarker performance was further analyzed by IHC, hazard ratio (p < 0.001), and receiver-operating characteristic curve (area = 0.787; p < 0.001). High CFL1 mRNA levels and protein content are positively correlated with cellular invasiveness (determined by Matrigel Invasion Chamber System) and resistance (2-fold increase in drug 50 % growth inhibition dose) against a list of 22 alkylating agents. Hierarchical clustering analysis of the CFL1 gene network had the same robustness for stratified NSCLC patients. The authors concluded that these findings indicated that the CFL1 gene and its functional gene network can be used as prognostic biomarkers for NSCLC and could also guide chemotherapeutic interventions. Moreover, prospective, large-scale, randomized clinical trials are needed to establish the role of CFL1 as a prognostic and drug resistance marker for NSCLC.
EarlyCDT-Lung
The EarlyCDT-Lung (Oncimmune, De Soto, KS) test measures antibodies to 6 tumor-associated antigens: p53, NY-ESO-1, CAGE, GBU4-5, Annexin 1, and SOX2. Elevation of any one of the panel of immuno-biomarkers above a predetermined cut-off value suggests that a tumor might be present. The test is designed to be used in conjunction with diagnostic imaging. High-risk individuals with a positive EarlyCDT-Lung would have additional testing such as a CT scan or the test would be used as a follow-up test for indeterminate lung nodules identified by CT.
Boyle et al (2011) reported the sensitivity and specificity of an autoantibody panel of 6 tumor-related antigens (p53, NY-ESO-1, CAGE, GBU4-5, Annexin 1 and SOX2) in patients with lung cancer. Three cohorts of patients with newly diagnosed lung cancer were identified: group 1 (n = 145), group 2 (n = 241) and group 3 (n = 269). Patients were individually matched by gender, age and smoking history to a control individual with no history of malignant disease. Serum samples were obtained after diagnosis but before any anticancer treatment. Autoantibody levels were measured against the panel of 6 tumor-related antigens (p53, NY-ESO-1, CAGE, GBU4-5, Annexin 1 and SOX2). Assay sensitivity was tested in relation to demographic variables and cancer type/stage. The autoantibody panel demonstrated a sensitivity/specificity of 36 %/91 %, 39 %/89 % and 37 %/90 % in groups 1, 2 and 3, respectively, with good reproducibility. There was no significant difference between different lung cancer stages, indicating that the antigens included covered the different types of lung cancer well. The authors concluded that the assay confirms the value of an autoantibody panel as a diagnostic tool and offers a potential system for monitoring patients at high-risk of lung cancer.
There is insufficient evidence of the effectiveness of the EarlyCDT-Lung as a screening test for the early detection of lung cancer. Systematic screening for lung cancer is not unequivocally recommended by any major professional organization. The USPSTF (2004) concluded that current evidence was insufficient to recommend for, or against, screening for lung cancer. Whether earlier detection of lung cancer will translate to a mortality benefit remains unclear.
E-cad
Deeb et al (2004) stated that E-cadherin (E-cad) and epidermal growth factor receptor (EGFR) are important cell adhesion and signaling pathway mediators. Theyr reported the results of a study which aimed to assess their expression in lung adenocarcinoma (AdC) and squamous cell carcinoma (SCC) and their association with clinicopathologic variables. Two to three cores from 130 resectable lung cancers (stages I-IIIA) were arrayed into three blocks using a Beecher system. Markers expression and coexpression were analyzed against clinicopathologic variables (age, gender, smoking status, performance status, weight loss, histology, grade, stage, and lymph node involvement) and patient survival. For E-cad, 65 cases (55%) were positive (+), 53 (45%) were negative (-); and for EGRF, 43 cases (34%) were (+), and 83 (66%) were (-). There was no significant association between E-cad or EGFR, and any of the clinicopathologic variables except for an association between EGFR(+) and SCC histologic type. Both negative and cytoplasmic staining of E-cad correlated with shorter patient survival with P=0.008 and 0.002, respectively. EGFR expression did not correlate with patient survival, but, patients with E-cad(-)/EGFR(+) phenotype had poorer survival than those with E-cad(+)/EGFR(-) (P=0.026). The authors concluded that lung AdC and SCC may be stratified based on expression of E-cad and EGFR with the E-cad(-)/EGFR(+) expression having a worse disease outcome.
EML4-ALK
Yoshida et al (2011) report that a subset of lung cancers harbors an EML4-ALK (echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase) gene fusion, and they examined 15 lung adenocarcinomas with reverse-transcriptase polymerase chain reaction-proven EML4-ALK fusion transcripts and 30 ALK-negative cases. Positive rearrangement signals (splits or isolated 3' signals) were identified in 13 to 78% (mean ± SD, 41% ± 19%) of tumor cells in the ALK-positive cohort and in 0 to 15% (mean ± SD, 6% ± 4%) of cells in the ALK-negative cohort. Sensitivity was at 93% and specificity at 100%. The only false-negative tumor having only 13% CISH-positive cells displayed predominantly (76%) isolated 5' signals unaccompanied by 3' signals. FISH showed largely similar signal profiles, and the results were completely concordant with CISH.The authors stated that they have successfully introduced CISH for diagnosing EML4-ALK-positive lung adenocarcinoma. This method allows simultaneous visualization of genetics and tumor cytomorphology and facilitates the molecular evaluation and could be applicable in clinical practice to detect lung cancer that may be responsive to ALK inhibitors.
Ellis et al (2011) conducted a systematic review and a consensus meeting of Canadian lung cancer oncologists and pathologists to make recommendations on the use of biomarkers in NSCLC. The articles were reviewed by pairs of oncologists and pathologists to determine eligibility for inclusion. Ten oncologists and pathologists reviewed and summarized the literature at a meeting attended by 37 individuals. The findings included that there is some evidence that histology is prognostic for survival as well as evidence from multiple randomized clinical trials to recommend the following: histologic subtype is predictive of treatment efficacy and for some agents toxicity. Immunohistochemistry testing should be performed on NSCLC specimens that cannot be classified accurately with conventional H&E staining. As EGFR mutations are predictive of benefit from tyrosine kinase inhibitors, diagnostic NSCLC samples should be routinely tested for EGFR-activating mutations. Clinical data on K-RAS mutations are inconsistent, therefore testing is not recommended. There is insufficient evidence to recommend other biomarker testing. No biomarkers to date reliably predict improved efficacy for anti-VEGF therapy. The authors concluded that outine assessment for EML4/ALK mutations is not recommended at present, although emerging data suggest that it may become valuable in the near future.
MUC4
Shanmugan and co-workers (2010) stated that mucin 4 (MUC4) is aberrantly expressed in colorectal adenocarcinomas (CRCs) but its prognostic value is unknown. Archival tissue specimens collected from 132 CRC patients who underwent surgical resection without pre-surgery or pos-tsurgery therapy were evaluated for expression of MUC4 by using a mouse monoclonal antibody and horseradish peroxidase. MUC4 expression levels were correlated with clinicopathologic features and patient survival. Survival was estimated by both uni-variate Kaplan-Meier and multi-variate Cox regression methods. In both normal colonic epithelium and CRCs, MUC4 staining was localized primarily in the cytoplasm. The optimal immunostaining cut-off value (greater than or equal to 75 % positive cells and an immunostaining score greater than or equal to 2.0), which was derived by using the bootstrap method, was used to categorize CRCs into groups of high expression (33 of 132 patients; 25 %) or low expression (99 of 132 patients; 75 %). Patients who had early stage tumors (stages I and II) with high MUC4 expression had a shorter disease-specific survival (log-rank; p = 0.007) than patients who had with low expression. Patients who had advanced-stage CRCs (stages III and IV) did not demonstrate such a difference (log-rank; p = 0.108). Multi-variate regression models that were generated separately for patients with early stage and advanced-stage CRC confirmed that increased expression of MUC4 was an independent indicator of a poor prognosis only for patients who had early stage CRCs (HR 3.77; 95 % CI: 1.46 to 9.73). The authors stated that aftr validating these findings in larger retrospective and prospective studies, a stage-based anayses could establish the utility of MUC4 as a prognostic molecular marker of early stage CRC.
ProOnc TumorSourceDx
ProOnc TumorSourceDx test is designed to identify tissue or origin for metastastic tumor. It identifies 25 possible classes of tissue origin corresponding to 17 distinct tissues and organs. It requires only 48 microRNAs to identify tissue of origin based on microRNA expression levels. However, there is insufficient evidence regarding its clinical value as tumor markers.
SAA
Cocco and associates (2010) examined the expression of serum amyloid A (SAA) in endometrial endometrioid carcinoma and evaluated its potential as a serum biomarker. SAA gene and protein expression levels were evaluated in endometrial endometrioid carcinoma and normal endometrial tissues, by real-time PCR, IHC, and flow cytometry. SAA concentration in 194 serum samples from 50 healthy women, 42 women with benign diseases, and 102 patients including 49 grade 1, 38 grade 2, and 15 grade 3 endometrial endometrioid carcinoma was also studied by a sensitive bead-based immunoassay. SAA gene expression levels were significantly higher in endometrial endometrioid carcinoma when compared with normal endometrial tissues (mean copy number by real-time PCR = 182 versus 1.9; p = 0.001). IHC revealed diffuse cytoplasmic SAA protein staining in poorly differentiated endometrial endometrioid carcinoma tissues. High intra-cellular levels of SAA were identified in primary endometrial endometrioid carcinoma cell lines evaluated by flow cytometry, and SAA was found to be actively secreted in vitro. SAA concentrations (microg/ml) had medians of 6.0 in normal healthy women and 6.0 in patients with benign disease (p = 0.92). In contrast, SAA values in the serum of endometrial endometrioid carcinoma patients had a median of 23.7, significantly higher than those of the healthy group (p = 0.001) and benign group (p = 0.001). Patients harboring G3 endometrial endometrioid carcinoma were found to have SAA concentrations significantly higher than those of G1/G2 patients. The authors concluded that SAA is not only a liver-secreted protein, but is also an endometrial endometrioid carcinoma cell product. SAA is expressed and actively secreted by G3 endometrial endometrioid carcinoma, and it is present in high concentration in the serum of endometrial endometrioid carcinoma patients. SAA may represent a novel biomarker for endometrial endometrioid carcinoma to monitor disease recurrence and response to therapy. They stated that additional studies are needed to validate these findings.
Caris Target Now / Caris Molecular Profiling Service
Molecular Intelligence Services (formerly Target Now Molecular Profiling Test) uses a multi-platform profiling approach including gene sequencing (NGS and Sanger), protein expression analysis (immunohistochemistry) and gene copy number analysis (chromogenic or fluorescence in situ hybridization [FISH]). The test has been used to examine tumor samples for underlying molecular alterations that may yield insights into potentially overlapping and different therapeutic options for individuals with these tumor types.
According to the manufacturer, the Caris Life Sciences molecular profiling test, Caris Target Now, examines the genetic and molecular changes unique to a patient's tumor so that treatment options may be matched to the tumor's molecular profile. The manufacturer states that the Caris Target Now test is performed after a cancer diagnosis has been established and the patient has exhausted standard of care therapies or if questions in therapeutic management exist. Using tumor samples obtained from a biopsy, the tumor is examined to identify biomarkers that may have an influence on therapy. Using this information, Caris Target Now is intended to provide information on the drugs that will be more likely to produce a positive response. The manufacturer states that Caris Target Now can be used with any solid cancer such as lung cancer, breast cancer, and prostate cancer.
There is insufficient evidence to support the use of Caris Target Now molecular profiling. A study (Von Hoff et al, 2010) compared the progression-free survival (PFS) of patients with refractory metastatic cancers using a treatment regimen selected by Caris Target Now molecular profiling of the patient's tumor with the PFS for the most recent regimen on which the patient had experienced progression. The investigators prespecified that a molecular profiling approach would be deemed of clinical benefit for the individual patient who had a PFS ratio (defined as a ratio of PFS on molecular profiling-selected therapy to PFS on prior therapy) of greater than or equal to 1.3. In 86 patients who had molecular profiling attempted, there was a molecular target detected in 84 (98 %). Sixty-six of the 84 patients were treated according to molecular profiling results. Eighteen (27 %) of 66 patients had a PFS ratio of gre