|
Background
Tumor markers are substances 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. However, in many cases the literature states that measurements of tumor marker levels alone are insufficient to diagnose cancer for the following reasons: (1) tumor marker levels can be elevated in people with benign conditions; (2) tumor marker levels are not elevated in every person with cancer, especially in the early stages of the disease; and (3) many tumor markers are not specific to a particular type of cancer; and (4) the level of a tumor marker can be elevated by more than one type of cancer. Elevated levels of Prostate-Specific Antigen (PSA) may be found in the blood of men with benign prostate conditions, such as prostatitis and benign prostatic hyperplasia (BPH), or with a malignant growth in the prostate. 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 annual 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. 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. 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. 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. 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. 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. 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 relatively new 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. 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. 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. 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. 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. 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. 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. 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. 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 quite controversial. 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. 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. 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. 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. 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 & 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 EGFR receptors 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. 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 - 75% of treated TCC recur. Furthermore, 10 – 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. 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. 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. 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. 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). 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, Bast & 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.” 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 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 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. 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). The Oncotype Dx has also been promoted for use in colorectal cancer. However, there is a lack of evidence establishing the clinical utility of this test in colorectal cancer. 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. Furthermore, a major clinical study is now underway to determine if the test really helps patients avoid needless chemotherapy. The European study will recruit 6,000 patients with early-stage breast cancer. 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 (Gunvén, et al, 2010; 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. In reviewing gene expression profiling as a guide for the management of early stage breast cancer, the California Technology Assessment Forum (CTAF, 2007) stated that the Oncotype Dx recurrence score meets CTAF Technology Assessment Criteria 1 through 5 for safety, effectiveness and improvement in health outcomes when used with other tools to inform the decision to use chemotherapy in patients recently diagnosed with invasive breast cancer meeting the following criteria: On the other hand, the CTAF stated that the use of other forms of gene expression profiling, including the 70-gene prognostic signature (Mammaprint), do not meet CTAF Technology Assessment Criteria 3 through 5 for safety, effectiveness and improvement in health outcomes. A draft CTAF reassessment of the Mammaprint is currently under review. Furthermore, the ASCO's 2006 update of the breast cancer follow-up and management guidelines in the adjuvant setting stated that the use of CBCs, chemistry panels, bone scans, chest radiographs, liver ultrasounds, computed tomography scans, [18F]fluorodeoxyglucose-positron emission tomography scanning, magnetic resonance imaging, or tumor markers (CEA, CA 15-3, and CA 27.29) is not recommended for routine breast cancer follow-up in an otherwise asymptomatic patient with no specific findings on clinical examination. It stated that careful history taking, physical examination, and regular mammography are appropriate for detecting breast cancer recurrence. Currently, there is a lack of evidence that the use of Mammaprint would influence the decisions that women and their physicians make with regard to adjuvant therapy. The clinical value of this test has yet to be established. A report prepared by the Evaluation of Genomic Applications in Practice and Prevention (EGAPP) Working Group evaluated the evidence for three gene expression assays on the market; Oncotype Dx, MammaPrint and the Breast Cancer Profiling (BCP or H/I ratio) test, and for gene expression signatures underlying the assays. The report found that, for clinical validity, studies differed according to whether they examined the actual test that is currently being offered to patients or the underlying gene signature. The report found that almost all of the Oncotype Dx evidence was for the marketed test, the strongest validation study being from one arm of a randomized controlled trial (NSABP-14) with a clinically homogeneous population. This study showed that the test, added in a clinically meaningful manner to standard prognostic indices. The MammaPrint signature and test itself was examined in studies with clinically heterogeneous populations (e.g., mix of ER positivity and tamoxifen treatment) and showed a clinically relevant separation of patients into risk categories, but it was not clear exactly how many predictions would be shifted across decision thresholds if this were used in combination with traditional indices.The BCP test itself was examined in one study, and the signature was tested in a variety of formulations in several studies. The report identified one randomized controlled trial that provided high quality retrospective evidence of the clinical utility of Oncotype Dx to predict chemotherapy treatment benefit, but evidence for clinical utility was not found for MammaPrint or the H/I ratio. The report concluded that Oncotype Dx is furthest along the validation pathway, with strong retrospective evidence that it predicts distant spread and chemotherapy benefit to a clinically relevant extent over standard predictors, in a well-defined clinical subgroup with clear treatment implications. The report concluded that the evidence for clinical implications of using MammaPrint was not as clear as with Oncotype Dx, and the ability to predict chemotherapy benefit does not yet exist. The report concluded that the H/I ratio test requires further validation. For all tests, the relationship of predicted to observed risk in different populations still needs further study, as does their incremental contribution, optimal implementation, and relevance to patients on current therapies. The EGAPP Working Group (2009) reported that they found adequate evidence regarding the association of the Oncotype Dx Recurrence Score with disease recurrence and adequate evidence for response to chemotherapy. The EWG found adequate evidence to characterize the association of MammaPrint with future metastases, but inadequate evidence to assess the added value to standard risk stratification, and could not determine the population to which the test would best apply. The evidence was inadequate to characterize the clinical validity of the Quest H:I Test. The EGAPP Working Group reported that they found no evidence regarding the clinical utility of the MammaPrint and Quest H:I Ratio tests, and inadequate evidence regarding Oncotype Dx. The EGAPP Working Group noted that these technologies have potential for both benefit and harm. 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. 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. 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 < .0001 for IL17BR and p = .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 = 3.9; 95% confidence interval 1.5 to 10.3; p = .007). The clinical 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 [hazard ratio (HR), 1.98; p = 0.031], 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 is currently preparing an evaluation of the H:I ratio test. 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. 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 diagnostic tool. 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 prevalences 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 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. 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." 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. 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. Current 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)." 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. PCA3 (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 the recently discovered 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 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. 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. 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". Sutcliffe, et al. (2009) provided an evidence-based perspective on the prognostic value of novel markers in localized prostate cancer and identified the best prognostic model including the 3 classical markers and investigated if models incorporating novel markers are better. Eight electronic bibliographic databases were searched. The reference lists of relevant articles were checked and various health services research-related resources consulted via the internet. The search was restricted to publications from 1970 onwards in the English language. Selected studies were assessed, data extracted using a standard template, and quality assessed using an adaptation of published criteria. Because of the heterogeneity regarding populations, outcomes and study type, meta-analyses were not undertaken and the results are presented in tabulated format with a narrative synthesis of the results. A total of 30 papers met the inclusion criteria, of which 28 reported on prognostic novel markers and 5 on prognostic models. A total of 21 novel markers were identified from the 28 novel marker studies. There was considerable variability in the results reported, the quality of the studies was generally poor and there was a shortage of studies in some categories. The marker with the strongest evidence for its prognostic significance was PSA velocity (or doubling time). There was a particularly strong association between PSA velocity and prostate cancer death in both clinical and pathological models. In the clinical model the hazard ratio for death from prostate cancer was 9.8 (95 % CI 2.8 to 34.3, p < 0.001) in men with an annual PSA velocity of more than 2 ng/ml versus an annual PSA velocity of 2 ng/ml or less; similarly, the hazard ratio was 12.8 (95 % CI 3.7 to 43.7, p < 0.001) in the pathological model. The quality of the prognostic model studies was adequate and overall better than the quality of the prognostic marker studies. Two issues were poorly dealt with in most or all of the prognostic model studies: (i) inclusion of established markers, and (ii) 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". 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. 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." 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) and the American Society for Clinical Oncology (ASCO) make no recommendation for use of circulating tumor cells. 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. 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. The 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. 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) 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.
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, CA), 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, 2009) 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." 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." Thus, there is insufficient evidence to make a conclusion about the effectiveness of the GeneSearch BLN assay. The FDA is requiring the manufacturer to conduct two post-approval studies. The primary objective of the first study is to estimate the positive predictive concordance between the GeneSearch BLN assay and histology as routinely practiced and the objectives of the second clinical study are (i) determine the assay turn-around-time from the time of node removal to the report of the assay result to the surgeon and (ii) determine whether the assay result was or was not received in time to make an intra-operative decision and (iii) 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. 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. 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 aptosis/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." 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 & Sim, 2008; Fernandez & Schwartz, 2007; McMains & Gourin, 2007). CD31 immunostaining can help confirm that the tumor originates from blood vessels. 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. Glioblastomas are among the most aggressive of all known human tumors. The median survival times remain in the 12 to15-month range despite aggressive surgery, radiation, and chemotherapy. Through molecular and genetic profiling efforts, underlying mechanisms of resistance to these therapies are becoming better understood. Resistance to alkylating agents via direct DNA repair by O(6)-methylguanine methyltransferase (MGMT) has been investigated as a barrier to successful treatment. Assessment of MGMT status could help identify gliomas patients more likely to respond to chemotherapy or to benefit from MGMT depletion strategies. Strategies to overcome MGMT-mediated chemoresistance are being actively investigated. In a review, Hegi, et al. (2008) stated that one strategy to overcome MGMT-mediated chemoresistance includes treatment with nontoxic pseudo-substrate inhibitors of MGMT, such as O(6)-benzylguanine, or RNA interference-mediated gene silencing of MGMT. However, the author reported that systemic application of MGMT inhibitors is limited by an increase in hematologic toxicity. Another strategy, the author explained, is to deplete MGMT activity in tumor tissue using a dose-dense temozolomide schedule. The author stated that these alternative schedules are well tolerated; however, it remains unclear whether they are more effective than the standard dosing regimen or whether they effectively deplete MGMT activity in tumor tissue. The author noted that not all patients with glioblastoma having MGMT promoter methylation respond to alkylating agents, and even those who respond will inevitably experience relapse. Data from a retrospective study reported by Cancer Care Ontario (2006) suggested that newly diagnosed malignant glioma patients who had undergone surgery and external beam radiotherapy had a greater benefit from temozolomide treatment if their tumors had MGMT promoter methylation versus patients with unmethylated MGMT tumors. However, it should be noted that these studies were performed retrospectively and prospective validation is required before MGMT methylation can be used for clinical stratification purposes. Idbaih, et al. (2007) reviewed recent studies on molecular markers including MGMT in the treatment of glioma and found evidence that MGMT inactivation is a prognostic marker and predictor of chemosensitivity in gliomas. The author stated, "Although such markers remain to be formally validated by ongoing and planned prospective trials, it is likely that they will soon become essential for optimizing treatment decisions." 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. 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. 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. 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). 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 the disease-free survival (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. 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 & Liebman, 1993;). Aoyagi, et al.(1996) and Weitz & 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. 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 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. Noon, et al. (2010) stated that renal cell carcinoma (RCC) is the most common type of kidney cancer and follows an unpredictable disease course. These researchers reviewed 2 critical genes associated with disease progression -- p53 and murine double minute 2 (MDM2) -- and provided a comprehensive summary and critical analysis of the literature regarding these genes in RCC. Information was compiled by searching the PubMed database for articles that were published or e-published up to April 1, 2009. Search terms included renal cancer, renal cell carcinoma, p53, and MDM2. Full articles and any supplementary data were examined; and, when appropriate, references were checked for additional material. All studies that described assessment of p53 and/or MDM2 in renal cancer were included. The authors concluded that increased p53 expression, but not p53 mutation, is associated with reduced overall survival/more rapid disease progression in RCC. There also was evidence that MDM2 up-regulation is associated with decreased disease-specific survival. Two features of RCC stood out as unusual and will require further investigation: (i)increased p53 expression is tightly linked with increased MDM2 expression; and (ii) 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. 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. 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. In an editorial on the OVA1 Test, Muller (2010) stated that the role of OVA1 in a generalized practice setting and cost-benefit ratio has yet to be determined. Furthermore, the ACOG Committee Opinion on the role of the obstetrician-gynecologist in the early detection of epithelial ovarian cancer (2011) noted that the clinical utility of the OVA1 Test "is not yet established". 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. 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. An assessment by the BlueCross BlueShield Association Technology Evaluation Center (2010) concluded that tumor-cell epidermal growth factor receptor (EGFR) mutation analysis to predict response to erlotinib (Tarceva) in patients with advanced non-small cell lung cancer (NSCLC) meets the Blue Cross and Blue Shield Association Technology Evaluation Center (TEC) criteria. Furthermore, guidelines from the National Comprehensive Cancer Network (NCCN, 2010) recommend EGFR testing for the following histologic subtypes of NSCLC: (i) adenocarcinoma, (ii) large cell, and (iii) NSCLC not otherwise specified. Epidermal growth factor receptor testing is not recommended for squamous cell carcinoma. 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. 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. 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. 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. 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 (2010) 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. 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. 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. 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. 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. Evidence to support the use of Caris Target Now molecular profiling is limited to a single pilot study. The 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 greater than or equal 1.3 (95% confidence interval 17% to 38%). Therefore, the null hypothesis (that less than or equal to 15% of this patient population would have a PFS ratio of greater than or equal to 1.3) was rejected. The authors concluded that, in 27% of patients, the molecular profiling approach resulted in a longer PFS on an molecular profiling-suggested regimen than on the regimen on which the patient had just experienced progression. An accompanying editorial (Doroshow, 2010) noted that the trial had a number of significant limitations, including uncertainty surrounding the achievement of time to progression (the study’s primary endpoint), and a lack of a randomized design for this trial. Glossary of Terms: | a2-PAG | Pregnancy-associated alpha2 glycoprotein | | BCM | Breast cancer mucin | | BTA | Bladder tumor antigen | | CA19-9 | Cancer antigen 19-9 | | CA50 | Cancer antigen 50 | | CA72-4 | Cancer antigen 72-4 | | CA195 | Cancer antigen 195 | | CA242 | Cancer antigen 242 | | CA549 | Cancer antigen 549 | | CA-SCC | Squamous cell carcinoma | | CAM17-1 | Monoclonal antimucin antibody 17-1 | | CAM26 | Monoclonal antimucin antibody 26 | | CAM29 | Monoclonal antimucin antibody 29 | | CAR3 | Antigenic determinant recognized by monoclonal antibody AR3 | | DU-PAN-2 | Sialylated carbohydrate antigen DU-PAN-2 | | FDP | Fibrin/fibrinogen degradation products | | GCC | Guanylyl cyclase C | | MCA | Mucin-like carcinoma-associated antigen | | NMP22 | Nuclear matrix protein22 | | NSE | Neuron-specific enolase | | PLAP | Placental alkaline phosphatase | | PNA-ELLA | Peanut lectin-bonding assay | | SLEX | Sialylated Lewis X-antigen | | SLX | Sialylated SSEA-1 antigen | | SPAN-1 | Sialylated carbonated antigen SPAN-1 | | ST-439 | Sialylated carbonated antigen ST-439 | | TAG12 | Tumor-associated glycoprotein 12 | | TAG72 | Tumor-associated glycoprotein 72 | | TAG72.3 | Tumor-associated glycoprotein 72.3 | | TATI | Tumor-associated trypsin inhibitor | | TNF-a | Tumor necrosis factor alpha | | TPA | Tissue polypeptide antigen |
|
Close Window
