Abstract SSY01-04: Discovery and validation of genome-wide genetic signatures of chemotherapy susceptibility: A translational model

Background: The paradigm of individualized drug therapy based on an individual9s unique genetic make-up is especially desirable in the field of hematology/oncology. If clinicians could better predict which individuals are likely to respond to chemotherapy and who is at the greatest risk of suffering chemotherapy-related toxicities, this could greatly improve the overall care of cancer patients. Evidence suggests that germline genetic differences between individuals explain a significant portion of observed treatment susceptibility heterogeneity. We chose two widely-used chemotherapy drugs in which to further investigate these genetic factors. Capecitabine is an ideal agent in which to investigate germline susceptibility loci since it has been previously shown that 26-65% of the cytotoxic effects of capecitabine9s metabolite, 5-FU, are genetically heritable (1), and because candidate genes (such as DPYD) have been previously identified as potentially important factors in 5-FU-related toxicity. Perhaps more importantly, capecitabine is often used as monotherapy which allows clear attribution of any toxicities and response to the drug– an important feature of pharmacogenetic inquiry. As the second drug, we chose cisplatin because the pharmacogenetics of the platinum agents has been an area of particular scientific activity since discovery of the role of the ERCC1 gene. Additionally, susceptibility to the cytotoxic effects of cisplatin is a heritable trait, with up to 47% of cisplatin toxicity susceptibility being under appreciable genetic influence (2). Yet, despite the active investigation into the pharmacogenetics of both of these agents, there has been limited clinical applicability of the findings, and the proposed candidate genes have been found to have relatively modest, if any, effect sizes. We believe that part of this limitation is likely due to the fact that rarely do single genes entirely explain an individual9s chemotherapeutic susceptibility risk, because chemotherapy sensitivity is most likely a multi-genic trait. To account for this, we developed an experimental model which permits elucidation of genetic determinants of chemotherapy susceptibility via a genome-wide approach. This approach allows simultaneous pre-clinical discovery of multiple single nucleotide polymorphisms (SNPs) and genes involved in drug susceptibility with no bias towards well-studied genes. After identification of these novel candidates, clinical replication of their importance is essential, and this project describes our bench-to-bedside translational model for validation of these pharmacogenomic findings in relevant cancer care clinical settings. Hypothesis: Germline genetic polymorphisms are critical determinants of chemotherapy-related toxicity, and possibly also efficacy. Methods: For both chosen chemotherapy agents, we employed an experimental model using EBV-transformed B-lymphoblastoid cell lines (LCLs) from healthy individuals of different ethnic backgrounds to identify genetic determinants of chemotherapy-related susceptibility via an unbiased genome-wide approach. The LCLs are from the International HapMap collection, in which, for all individuals, rich genotypic information is already known. LCLs from these HapMap cell lines are treated in vitro with each chemotherapeutic agent, and individual cytotoxicity “susceptibility” phenotypes are identified which are then included in a genome-wide association study (GWAS) with the known genotypes. This powerful statistical analysis allows identification of a small list of potential SNPs which represent a “genetic signature” that may govern sensitivity to the given chemotherapeutic agent. These pre-clinical LCL GWAS studies have now been performed for both capecitabine and cisplatin. However, clinical replication of these findings was needed. To accomplish this, we designed two clinical trials– one for patients receiving cisplatin, and one for patients receiving capecitabine– to test these novel genetic variants (along with previously-identified candidate-gene SNPs) in carefully-selected clinical settings with well-described and important clinical phenotypes as the relevant endpoints. Results: For each drug, the pre-clinical LCL models produced a workable set of SNPs (approximately 25 unique SNP candidates per drug) and their related genes (approximately the same number) as outputs of various GWAS analyses. Where possible, SNPs were then replicated in independent HapMap populations which were separate from the discovery sets. The SNP candidates for each drug were also shown to have far more predictive power for the cytotoxicity phenotypes when they were incorporated as haplotype-like sets, referred to as “susceptibility signatures.” For each drug, these genome-wide-derived SNP candidates were then combined with literature-reported lists of other SNPs that had been previously identified as potentially important in candidate gene studies. The composite list of candidate gene-derived SNPs and our novel genome-wide SNPs were then taken forward for testing in a carefully-designed clinical trial for each drug. For capecitabine, a 250 patient, eight institution multi-center trial of patients with breast cancer was designed. This setting was chosen because capecitabine has particular appeal for breast cancer patients with advanced stages of disease because, besides having considerable anti-tumor efficacy, it can be administered as an outpatient and avoids the frequent hospital visits required for administration of other chemotherapies. These quality of life considerations are particular advantages of capecitabine. However, capecitabine is associated with several potentially severe side-effects which counter the palliative goals of the medication and are confounders of effectiveness. The trial allows any adult woman breast cancer patient who is about to be treated with single-agent capecitabine to be eligible. Patients are carefully monitored for three toxicities of interest: diarrhea, hand-foot syndrome, and neutropenia. Toxicity data capture is being studied via traditional means (clinic visit questionnaires) and via a novel patient phone-in toxicity reporting system that captures toxicity during the between-clinic visits. The primary endpoint of the study examines whether the chosen SNPs correlate with development of any or all of the three capecitabine-related toxicities of interest. Secondary endpoints include analysis of whether the genetic determinants of capecitabine toxicity differ for patients who develop “early” toxicity (during cycle 1) versus “late” toxicity (during cycles 2-4). Trial accrual is now ongoing. For cisplatin, a 60 patient, single-institution clinical trial was designed in urothelial cancer of the bladder. Given the importance of the role of cisplatin in urothelial cancer and the controversy surrounding the utility of neoadjuvant cisplatin-based chemotherapy in this disease, we chose the neoadjuvant bladder cancer setting as one in which pharmacogenomic information might be particularly useful for guiding treatment decisions. All bladder cancer patients who are eligible for standard cisplatin-based neoadjuvant chemotherapy are eligible for this trial. Patients will receive standard neoadjuvant chemotherapy and then will proceed to cystectomy. The rate of achievement of pT0 in the cystectomy specimens will be the primary endpoint. Patients with a “cisplatin susceptible” pharmacogenomic signature will be assessed for their rate of pT0 as compared to patients who are predicted to be cisplatin resistant by the pharmacogenomic signature. Accrual to this trial is also ongoing. Conclusion: Prediction of which patients will respond to chemotherapy, and which will experience severe side-effects, is currently often not possible. Our pre-clinical model has allowed the identification of a novel list of SNPs and related genes that, for the first time, offer unbiased variants for clinical testing alongside candidate gene-SNPs as one comprehensive pharmacogenomic signature. We report here the successful implementation of this translational model into current patient testing for two important chemotherapy drugs, capecitabine and cisplatin. Ultimately, validation of pharmacogenetic endpoints in these two clinical trials would provide novel variants which could then be tested in subsequent prospective fashion (e.g., patient dose-targeting by genotype). This will enable the cancer field to move one step closer to the goal of “personalized” medical care, which has the potential to significantly improve the way that cancer patients are treated by minimizing toxicity and maximizing response. References 1. Watters JW, Kraja A, Meucci MA, Province MA, McLeod HL. Genome-wide discovery of loci influencing chemotherapy cytotoxicity. Proc Natl Acad Sci U S A. 2004;101(32):11809-14. 2. Dolan ME, Newbold KG, Nagasubramanian R, Wu X, Ratain MJ, Cook EH Jr, Badner JA. Heritability and linkage analysis of sensitivity to cisplatin-induced cytotoxicity. Cancer Res. 2004;64(12):4353-6. Citation Format: Peter O9Donnell. Discovery and validation of genome-wide genetic signatures of chemotherapy susceptibility: A translational model [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr SSY01-04