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Background
Chemoresistance assay testing is an in-vitro diagnostic technique intended to identify drugs to which a patient's tumor is resistant. Chemoresistant assays are based on the same principles as the chemosensitivity assay. Both assays are performed to evaluate whether tumor growth is inhibited by a known chemotherapy drug or, more commonly, a panel of drugs. However, chemoresistance assays focus on negative predictive accuracy (the ability of the assay to identify ineffective agents) rather than positive predictive accuracy (the ability of the assay to identify agents capable of inducing a clinical response). Weisenthal and Kern (1991) reported that the negative predictive value (NPV) for in-vitro drug response assays was generally 90 % to 99 % while the positive predictive value (PPV) was 50 % to 70 %. Thus, in-vitro drug testing is purportedly more reliable for identifying drugs that are more likely to be ineffective (resistant) as opposed to identifying drugs that would be effective (sensitive). By eliminating ineffective drugs from treatment regimens, it is hoped that chemoresistance assay testing will spare the patient unnecessary toxicity, correlate a patient's response to chemotherapy and/or patient survival and tailor treatment to individual patients.
Extreme drug resistance (EDR) assays were designed to provide a very high NPV (greater than 99 %), with the intent to deselect ineffective chemotherapies. After successful culture, tumor cells obtained from fresh biopsy specimens are labeled with tritiated thymidine. The level of uptake is tracked after exposure to chemotherapy concentrations that approximate the peak level achieved clinically. Extreme resistance is identified when thymidine incorporation is inhibited in the presence of the drug by less than 1 standard deviation of the median cell inhibition measured for several hundred refence tumor samples. In this test, a positive resistance to a specific drug should prevent the selection of “ineffective” drugs for the human tumor cells in the individual patient.
A slightly different form of tumor cell resistance assay testing consists of exposing tumor cells to anti-neoplastic agents and performing at least 1 or more of the following 3 cell death endpoint tests to determine tumor cell survival: (i) differential cell staining and microscopic counting; (ii) microplating and use of coloring reagent MTT (dimethyl thiazol-diphenyltetrzolium thiazolyl blue); and (iii) measuring ATP content.
Mehta et al (2001) reported the results of EDR testing on breast tumor tissue (n = 103). Extreme drug resistance assay scores of 2 for low, 1 for intermediate, or 0 for EDR were determined for each agent tested. In-vitro EDR scores for 4-hydroxycyclophosphamide (4HC) and doxorubicin were summed for patients treated with AC, or for 4HC and 5-FU for patients treated with CMF. Treatment selection was blinded to assay results. The authors reported that median time to progression was significantly shorter for patients with extreme or intermediate in-vitro resistance (n = 55, 48 months), compared to patients with low in-vitro resistance, (n = 41, 100 months, p = 0.022). Patients demonstrating extreme to intermediate drug resistance showed poorer survival than the low resistance group (49.5 months versus not reached, median follow-up 48 months, p = 0.011). Compared to EDR scores of 4, summed EDR scores of 0 to 1 and summed EDR scores of 2 to 3 were associated with a relative risk of death of 3.09 (95 % confidence interval [CI]: 1.05 to 9.06, Cox proportional hazards model, p = 0.040) and 2.35 (95 % CI: 1.07 to 5.15, Cox proportional hazards model, p = 0.033), respectively. The authors concluded that EDR testing identified patients with individual patterns of drug resistance prior to therapy and that summed EDR scores were significantly associated with time to tumor progression and overall survival.
Haroun et al (2002) described a drug resistance profile (extreme, intermediate, or low) based on statistical comparison to a historical database of brain tumor specimens tested against the same panel of chemotherapeutic agents. The authors stated that through continued analysis and compilation of data from multiple institutions, chemoresistance profiles could assist future investigators with the development of rationale clinical trials and treatment regimens for patients with brain tumors.
In a retrospective study of EDR testing on newly diagnosed advanced ovarian cancer patients, Holloway et al (2002) stated that patients with ovarian tumors demonstrating in- vitro drug resistance to platinum were at significantly increased risk for progression and death when treated with standard platinum-based regimens. Median progression free survival was 6 months for tumors exhibiting extreme resistance to platinum (n = 17) compared to 24 months for tumors exhibiting low resistance to platinum (n = 62).
In a retrospective study, Loizzi et al (2003) reported that recurrent ovarian cancer patients (n = 50) with EDR directed therapy had an overall response rate of 65 % compared with 35 % in patients who were treated empirically. The overall and progression-free median survival were 38 and 15 months in the EDR group compared with 21 and 7 months in the control group, respectively.
In a prospective phase II blinded study of 48 patients with recurrent malignant glioma, Parker et al (2004) evaluated the predictive reliability of an EDR assay to identify clinical resistance to irinotecan (CPT-11) using tumor biopsies obtained from patients immediately prior to their first dose of CPT-11 therapy. In-vitro tumor response to SN38 (bioactive species of CPT-11 used in the EDR assay) determined prior to treatment was correlated with objective response, time to tumor progression (TTP) and survival following the administration of CPT-11. SN38 activity was tested in 19 of 29 tumors, with 15 of 18 assay results evaluable for correlation with clinical outcomes. In-vitro drug resistance was classified as either extreme, intermediate (IDR), or low (LDR). Median TTP for IDR/LDR cases was 3 months versus 6 weeks for EDR cases. A 13-week median survival for EDR cases was significantly shorter compared to 38 weeks for IDR/LDR cases (p = 0.029). At follow-up, 2 of 3 survivors were patients who had tumors IDR/LDR to SN38.
Cloven et al (2004) reported the results of EDR testing to epithelial ovarian cancer (n = 5,195) and found EDR to cisplatin (10 %), carboplatin (16 %), cyclophosphamide (16 %), doxorubicin (40 %), gemcitabine (21 %), paclitaxel (22 %), and topotecan (13 %). The investigators also reported differences of EDR among histologic subtypes of epithelial ovarian cancer.
Fruehauf et al (2006) described in-vitro drug response profiles on biopsy specimens from patients with malignant gliomas (n = 478). Samples were tested for drug resistance to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), cisplatin, dacarbazine, paclitaxel, vincristine, and irinotecan. Biomarkers associated with drug resistance were detected by imunohistochemistry, including multi-drug resistance gene-1, glutathione S-transferase pi (GSTP1), O(6)-methylguanine-DNA methyltransferase (MGMT), and mutant p53. In-vitro drug resistance in malignant gliomas was independent of prior therapy. High-grade glioblastomas showed a lower level of EDR than low-grade astrocytomas to cisplatin (11 % versus 27 %), temozolomide (14 % versus 27 %), irinotecan (33 % versus 53 %), and BCNU (29 % versus 38 %). A substantial percentage of brain tumors overexpressed biomarkers associated with drug resistance, including MGMT (67 %), GSTP1 (49 %), and mutant p53 (41 %). MGMT and GSTP1 over-expression was independently associated with in-vitro resistance to BCNU, whereas coexpression of these 2 markers was associated with the greatest degree of BCNU resistance.
d'Amato et al (2006) reported EDR or IDR to non-small cell lung cancer specimens (n = 3,042) to carboplatin (68 %), cisplatin (63 %), doxorubicin (75 %), etoposide (63 %), gemcitabine (72 %), navelbine (42 %), paclitaxel (40 %), taxotere (52 %), and topotecan (31 %). In a follow-up study, d'Amato et al (2007) reported resistance to multiple-agent chemotherapy to non-small cell lung cancer specimens (n = 4,571) to carboplatin-paclitaxel (30 %), cisplatin-navelbline (24 %), cisplatin-gemcitabine (42 %), and cisplatin-docetaxel (27 %).
In a retrospective analysis comparing the differences of EDR testing between invasive micropapillary/low-grade serous ovarian carcinoma (n = 13) and high-grade serous ovarian carcinoma (n = 31), patients with recurrent invasive micropapillary/low-grade serous ovarian carcinoma were more likely to manifest EDR to standard chemotherapy agents. Compared to high-grade serous ovarian carcinoma, invasive micropapillary/low-grade serous ovarian carcinoma were more likely to manifest EDR to the drugs paclitaxel (69 % versus 14 %, p < 0.001), carboplatin (50 % versus 17 %, p = 0.05), cyclophosphamide (40 % versus 23 %, p = 0.41), gemcitabine (36 % versus 19 %, p = 0.40), and cisplatin (33 % versus 28 %, p = 0.72) and less likely to be resistant to etoposide (0 % versus 44 %, p = 0.007), doxorubicin (8 % versus 45 %, p = 0.03), and topotecan (8 % versus 21 %, p = 0.65) (Santillan et al, 2007).
Based on a review of the current literature, the American Society of Clinical Oncology (ASCO) Working Group found insufficient evidence to support the use of any chemotherapy sensitivity and resistance assays (CSRAs) in oncological practice. Specifically, the ASCO Work Group found limitations in the literature that included small sample sizes and a lack of prospective studies. For technically challenging CSRAs that require colony formation (e.g., the human tumor cloning assay), and for surgical procedures including the sub-renal capsule assay, the success rate of the CSRAs is modest. Furthermore, preparation of the assay may involve complex laboratory work, limiting a broad application of the technology to routine clinical practice. Because the in-vitro analytic strategy has potential importance, participation in clinical trials evaluating these technologies remains a priority (Schrag et al, 2004).
Chemoresistance assays, using measurements of extreme drug resistance, have been shown to have a high negative predictive value for identifying those drugs which are ineffective. Although advocates of chemoresistance assays may argue that the relevant outcome is avoidance of the toxicity and morbidity of an ineffective drug, it is impossible to separate this limited outcome from the final outcome of improved survival based on the selection of potentially more effective chemotherapy. For example, when one drug is eliminated on the basis of chemoresistance assays, it is implied that the remaining drugs, which did not show chemoresistance, are potentially more effective. In this way, by a process of elimination, chemoresistance results dictate the positive selection of potentially effective drugs. However, even the avoidance of potentially unnecessary toxicity is not a straightforward outcome. If one drug is eliminated from a regimen on the basis of chemoresistance, in most cases another drug with its associated toxicity will be substituted.
Proponents of the EDR assays believe the ability to predict clinical failure with 99 % accuracy is the compelling feature of the EDR assay (Mehta, 2001; Holloway, 2002; Loizzi, 2003; Parker, 2004) while others (Schrag, 2004) find no convincing evidence that chemoresistant assays should be integrated into routine oncology care until further randomized controlled testing is done.
Guidelines from the National Comprehensive Cancer Network (2011) stated that chemosensitivity and chemoresistance assays are used in some centers for decisions related to future chemotherapy in situations where there are multiple equivalent chemotherapy options available; "the current level of evidence is not sufficient to supplant current standard of care chemotherapy."
Burstein et al (2011) updated the ASCO Technology Assessment guidelines on CSRAs published in 2004. An Update Working Group reviewed data published between December 1, 2003, and May 31, 2010. Medline and the Cochrane Library were searched yielding 11,313 new articles. The limits for "human and English" were used, and then standard ASCO search strings for randomized controlled trials (RCTs), meta-analyses, guidelines, and reviews were added, yielding 1,298 articles for abstract review. Of these, only 21 articles met pre-defined inclusion criteria and underwent full text review, and 5 reports of RCTs were included for data extraction. Review of the literature does not identify any CSRAs for which the evidence base is sufficient to support use in oncology practice. The authors concluded that the use of CSRAs to select chemotherapeutic agents for individual patients is not recommended outside of the clinical trial setting. They noted that oncologists should make chemotherapy treatment recommendations based on published reports of clinical trials and a patient's health status and treatment preferences. Because the in-vitro analytic strategy has potential importance, participation in clinical trials evaluating these technologies remains a priority.
Wang et al (2012) examined if expression of circulating microRNAs (miRNAs) can predict clinical outcome in breast cancer patients treated with adjuvant chemotherapy. Circulating miRNAs in blood serum prior to treatment were determined by quantitative real-time polymerase chain reaction (PCR) in 56 breast cancer patients with invasive ductal carcinoma and pre-operative neoadjuvant chemotherapy. Proliferating cell nuclear antigen (PCNA) immunostaining and TUNEL were performed in surgical samples to determine the effects of chemotherapy on cancer cell proliferation and apoptosis, respectively. Among the miRNAs tested, only miR-125b was significantly associated with therapeutic response, exhibiting higher expression level in non-responsive patients (n = 26, 46 %; p = 0.008). In addition, breast cancers with high miR-125b expression had higher percentage of proliferating cells and lower percentage of apoptotic cells in the corresponding surgical specimens obtained after neoadjuvant chemotherapy. Increased resistance to anti-cancer drug was observed in-vitro in breast cancer cells with ectopic miR-125b expression; conversely, reducing miR-125b level sensitized breast cancer cells to chemotherapy. Moreover, these researchers demonstrated that the E2F3 was a direct target of miR-125b in breast cancer cells. The authors concluded that these findings suggested that circulating miR-125b expression is associated with chemotherapeutic resistance of breast cancer. This finding has important implications in the development of targeted therapeutics for overcoming chemotherapeutic resistance in novel anti-cancer strategies.
Lee and colleagues (2012) examined (i) if immunohistochemistry of multi-drug-resistant (MDR) proteins (MDR1, MRP1, MRP2 and BCRP) in colorectal adenocarcinomas can substitute for histoculture drug resistance assays (HDRA) and (ii) if chemosensitivity as indicated by HDRA and MDR protein expression is related to prognostic parameters in colorectal cancers. Chemosensitivity of cancer tissues to 5-FU, irinotecan and oxaliplatin was assessed by HDRA. Immunohistochemical staining of MDR proteins was quantified by image analysis in 76 colorectal adenocarcinoma patients. Inhibition rates (IRs) of the anti-cancer drugs by HDRA were not related to MDR protein expression. However, the IR of 5-FU was significantly decreased with lymph node metastasis (p = 0.03) and advanced clinical stages (p = 0.047). The IRs of irinotecan and oxaliplatin were not associated with clinicopathological parameters. Immunohistochemically, positive scores for MRP2 and BCRP protein were paradoxically related to lower clinical stages (p = 0.043) and male gender (p = 0.019), respectively. The authors concluded that immunohistochemical staining of MDR proteins can not predict tumor responses to anti-cancer drugs in colorectal cancers. Chemoresistance to 5-FU as indicated by HDRA was highly associated with aggressive prognostic factors.
Ebert and associates (2012) noted that chemotherapy for advanced colorectal cancer (CRC) leads to improved survival; however, predictors of response to systemic treatment are not available. Genomic and epigenetic alterations of the gene encoding transcription factor AP-2 epsilon (TFAP2E) are common in human cancers. The gene encoding dickkopf homolog 4 protein (DKK4) is a potential down-stream target of TFAP2E and has been implicated in chemotherapy resistance. These investigators evaluated the role of TFAP2E and DKK4 as predictors of the response of CRC to chemotherapy. They analyzed the expression, methylation, and function of TFAP2E in CRC cell lines in-vitro and in patients with CRC. They examined an initial cohort of 74 patients, followed by 4 cohorts of patients (a total of 220) undergoing chemotherapy or chemoradiation. TFAP2E was hyper-methylated in 38 of 74 patients (51 %) in the initial cohort. Hyper-methylation was associated with decreased expression of TFAP2E in primary and metastatic CRC specimens and cell lines. Colorectal-cancer cell lines over-expressing DKK4 showed increased chemoresistance to fluorouracil but not irinotecan or oxaliplatin. In the 4 other patient cohorts, TFAP2E hyper-methylation was significantly associated with non-response to chemotherapy (p < 0.001). Conversely, the probability of response among patients with hypo-methylation was approximately 6 times that in the entire population (overall estimated risk ratio, 5.74; 95 % CI: 3.36 to 9.79). Epigenetic alterations of TFAP2E were independent of mutations in key regulatory cancer genes, microsatellite instability, and other genes that affect fluorouracil metabolism. The authors concluded that TFAP2E hyper-methylation is associated with clinical non-responsiveness to chemotherapy in CRC. Functional assays confirm that TFAP2E-dependent resistance is mediated through DKK4. In patients who have CRC with TFAP2E hyper-methylation, targeting of DKK4 may be an option to overcome TFAP2E-mediated drug resistance.
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