Rama Raghavan

Genome-Wide Study of Response to Platinum, Taxane, and Combination Therapy in Ovarian Cancer: In vitro Phenotypes, Inherited Variation, and Disease Recurrence

Background: The standard treatment for epithelial ovarian cancer (EOC) patients with advanced disease is carboplatin-paclitaxel combination therapy following initial debulking surgery, yet there is wide inter-patient variation in clinical response. We sought to identify pharmacogenomic markers related to carboplatin-paclitaxel therapy. Methods: The lymphoblastoid cell lines, derived from 74 invasive EOC patients seen at the Mayo Clinic, were treated with increasing concentrations of carboplatin and/or paclitaxel and assessed for in vitro drug response using MTT viability and caspase3/7 apoptosis assays. Drug response phenotypes IC50 (effective dose at which 50% of cells are viable) and EC50 (dose resulting in 50% induction of caspase 3/7 activity) were estimated for each patient to paclitaxel and carboplatin (alone and in combination). For each of the six drug response phenotypes, a genome-wide association study was conducted. Results: Statistical analysis found paclitaxel in vitro drug response phenotypes to be moderately associated with time to EOC recurrence (p = 0.008 IC50; p = 0.058 EC50). Although no pharmacogenomic associations were significant at p < 5 × 10−8, seven genomic loci were associated with drug response at p < 10−6, including at 4q21.21 for carboplatin, 4p16.1 and 5q23.2 for paclitaxel, and 3q24, 10q, 1q44, and 13q21 for combination therapy. Nearby genes of interest include FRAS1, MGC32805, SNCAIP, SLC9A9, TIAL1, ZNF731P, and PCDH20. Conclusions: These results suggest the existence of genetic loci associated with response to platinum-taxane therapies. Further research is needed to understand the mechanism by which these loci may impact EOC clinical response to this commonly used regimen.

Assessment of multifactor gene-environment interactions and ovarian cancer risk: candidate genes, obesity, and hormone-related risk factors

Background: Many epithelial ovarian cancer (EOC) risk factors relate to hormone exposure and elevated estrogen levels are associated with obesity in postmenopausal women. Therefore, we hypothesized that gene–environment interactions related to hormone-related risk factors could differ between obese and non-obese women. Methods: We considered interactions between 11,441 SNPs within 80 candidate genes related to hormone biosynthesis and metabolism and insulin-like growth factors with six hormone-related factors (oral contraceptive use, parity, endometriosis, tubal ligation, hormone replacement therapy, and estrogen use) and assessed whether these interactions differed between obese and non-obese women. Interactions were assessed using logistic regression models and data from 14 case–control studies (6,247 cases; 10,379 controls). Histotype-specific analyses were also completed. Results: SNPs in the following candidate genes showed notable interaction: IGF1R (rs41497346, estrogen plus progesterone hormone therapy, histology = all, P = 4.9 × 10−6) and ESR1 (rs12661437, endometriosis, histology = all, P = 1.5 × 10−5). The most notable obesity–gene–hormone risk factor interaction was within INSR (rs113759408, parity, histology = endometrioid, P = 8.8 × 10−6). Conclusions: We have demonstrated the feasibility of assessing multifactor interactions in large genetic epidemiology studies. Follow-up studies are necessary to assess the robustness of our findings for ESR1, CYP11A1, IGF1R, CYP11B1, INSR, and IGFBP2. Future work is needed to develop powerful statistical methods able to detect these complex interactions. Impact: Assessment of multifactor interaction is feasible, and, here, suggests that the relationship between genetic variants within candidate genes and hormone-related risk factors may vary EOC susceptibility. Cancer Epidemiol Biomarkers Prev; 25(5); 780–90. ©2016 AACR.

Bayesian accrual prediction for interim review of clinical studies: open source R package and smartphone application

BackgroundSubject recruitment for medical research is challenging. Slow patient accrual leads to increased costs and delays in treatment advances. Researchers need reliable tools to manage and predict the accrual rate. The previously developed Bayesian method integrates researchers’ experience on former trials and data from an ongoing study, providing a reliable prediction of accrual rate for clinical studies.MethodsIn this paper, we present a user-friendly graphical user interface program developed in R. A closed-form solution for the total subjects that can be recruited within a fixed time is derived. We also present a built-in Android system using Java for web browsers and mobile devices.ResultsUsing the accrual software, we re-evaluated the Veteran Affairs Cooperative Studies Program 558— ROBOTICS study. The application of the software in monitoring and management of recruitment is illustrated for different stages of the trial.ConclusionsThis developed accrual software provides a more convenient platform for estimation and prediction of the accrual process.

Characterization of fusion genes in common and rare epithelial ovarian cancer histologic subtypes

Gene fusions play a critical role in some cancers and can serve as important clinical targets. In epithelial ovarian cancer (EOC), the contribution of fusions, especially by histological type, is unclear. We therefore screened for recurrent fusions in a histologically diverse panel of 220 EOCs using RNA sequencing. The Pipeline for RNA-Sequencing Data Analysis (PRADA) was used to identify fusions and allow for comparison with The Cancer Genome Atlas (TCGA) tumors. Associations between fusions and clinical prognosis were evaluated using Cox proportional hazards regression models. Nine recurrent fusions, defined as occurring in two or more tumors, were observed. CRHR1-KANSL1 was the most frequently identified fusion, identified in 6 tumors (2.7% of all tumors). This fusion was not associated with survival; other recurrent fusions were too rare to warrant survival analyses. One recurrent in-frame fusion, UBAP1-TGM7, was unique to clear cell (CC) EOC tumors (in 10%, or 2 of 20 CC tumors). We found some evidence that CC tumors harbor more fusions on average than any other EOC histological type, including high-grade serous (HGS) tumors. CC tumors harbored a mean of 7.4 fusions (standard deviation [sd] = 7.4, N = 20), compared to HGS EOC tumors mean of 2.0 fusions (sd = 3.3, N = 141). Few fusion genes were detected in endometrioid tumors (mean = 0.24, sd = 0.74, N = 55) or mucinous tumors (mean = 0.25, sd = 0.5, N = 4) tumors. To conclude, we identify one fusion at 10% frequency in the CC EOC subtype, but find little evidence for common (> 5% frequency) recurrent fusion genes in EOC overall, or in HGS subtype-specific EOC tumors.

Transcriptomic Characterization of Endometrioid, Clear Cell, and High-Grade Serous Epithelial Ovarian Carcinoma

Background: Endometrioid carcinoma (EC) and clear cell carcinoma (CC) histotypes of epithelial ovarian cancer are understudied compared with the more common high-grade serous carcinomas (HGSC). We therefore sought to characterize EC and CC transcriptomes in relation to HGSC. Methods: Following bioinformatics processing and gene abundance normalization, differential expression analysis of RNA sequence data collected on fresh-frozen tumors was completed with nonparametric statistical analysis methods (55 ECs, 19 CCs, 112 HGSCs). Association of gene expression with progression-free survival (PFS) was completed with Cox proportional hazards models. Eight additional multi-histotype expression array datasets (N = 852 patients) were used for replication. Results: In the discovery set, tumors generally clustered together by histotype. Thirty-two protein-coding genes were differentially expressed across histotype (P < 1 × 10−10) and showed similar associations in replication datasets, including MAP2K6, KIAA1324, CDH1, ENTPD5, LAMB1, and DRAM1. Nine genes associated with PFS (P < 0.0001) showed similar associations in replication datasets. In particular, we observed shorter PFS time for CC and EC patients with high gene expression for CCNB2, CORO2A, CSNK1G1, FRMD8, LIN54, LINC00664, PDK1, and PEX6, whereas, the converse was observed for HGSC patients. Conclusions: The results suggest important histotype differences that may aid in the development of treatment options, particularly those for patients with EC or CC. Impact: We present replicated findings on transcriptomic differences and how they relate to clinical outcome for two of the rarer ovarian cancer histotypes of EC and CC, along with comparison with the common histotype of HGSC. Cancer Epidemiol Biomarkers Prev; 27(9); 1101–9. ©2018 AACR.

Developing a genetic signature to predict drug response in ovarian cancer

There is a lack of personalized treatment options for women with recurrent platinum-resistant ovarian cancer. Outside of bevacizumab and a group of poly ADP-ribose polymerase inhibitors, few options are available to women that relapse. We propose that efficacious drug combinations can be determined via molecular characterization of ovarian tumors along with pre-established pharmacogenomic profiles of repurposed compounds. To that end, we selectively performed multiple two-drug combination treatments in ovarian cancer cell lines that included reactive oxygen species inducers and HSP90 inhibitors. This allowed us to select cell lines that exhibit disparate phenotypes of proliferative inhibition to a specific drug combination of auranofin and AUY922. We profiled altered mechanistic responses from these agents in both reactive oxygen species and HSP90 pathways, as well as investigated PRKCI and lncRNA expression in ovarian cancer cell line models. Generation of dual multi-gene panels implicated in resistance or sensitivity to this drug combination was produced using RNA sequencing data and the validity of the resistant signature was examined using high-density RT-qPCR. Finally, data mining for the prevalence of these signatures in a large-scale clinical study alluded to the prevalence of resistant genes in ovarian tumor biology. Our results demonstrate that high-throughput viability screens paired with reliable in silico data can promote the discovery of effective, personalized therapeutic options for a currently untreatable disease.

An ensemble-based Cox proportional hazards regression framework for predicting survival in metastatic castration-resistant prostate cancer (mCRPC) patients

From March through August 2015, nearly 60 teams from around the world participated in the Prostate Cancer Dream Challenge (PCDC). Participating teams were faced with the task of developing prediction models for patient survival and treatment discontinuation using baseline clinical variables collected on metastatic castrate-resistant prostate cancer (mCRPC) patients in the comparator arm of four phase III clinical trials. In total, over 2,000 mCRPC patients treated with first-line docetaxel comprised the training and testing data sets used in this challenge. In this paper we describe: (a) the sub-challenges comprising the PCDC, (b) the statistical metrics used to benchmark prediction performance, (c) our analytical approach, and finally (d) our team’s overall performance in this challenge. Specifically, we discuss our curated, ad-hoc, feature selection (CAFS) strategy for identifying clinically important risk-predictors, the ensemble-based Cox proportional hazards regression framework used in our final submission, and the adaptation of our modeling framework based on the results from the intermittent leaderboard rounds. Strong predictors of patient survival were successfully identified utilizing our model building approach. Several of the identified predictors were new features created by our team via strategically merging collections of weak predictors. In each of the three intermittent leaderboard rounds, our prediction models scored among the top four models across all participating teams and our final submission ranked 9 th place overall with an integrated area under the curve (iAUC) of 0.7711 computed in an independent test set. While the prediction performance of teams placing between 2 nd- 10 th (iAUC: 0.7710-0.7789) was better than the current gold-standard prediction model for prostate cancer survival, the top-performing team, FIMM-UTU significantly outperformed all other contestants with an iAUC of 0.7915. In summary, our ensemble-based Cox regression framework with CAFS resulted in strong overall performance for predicting prostate cancer survival and represents a promising approach for future prediction problems.

Abstract 1833: Transcriptome characterization of high grade serous and endometrioid epithelial ovarian cancer tumors

Background: Little transcriptomic research has compared epithelial ovarian cancer (EOC) histological subtypes. We set out to characterize the transcriptomes of high-grade serous carcinomas (HGSC) and endometrioid carcinomas (EC), which make up around 70% and 20% of EOC tumors, respectively, and have some histopathological similarities. Methods: Fresh frozen tumors from EOC patients seen at the Mayo Clinic (30 EC and 62 HGSC) were used. 1ug RNA riboZero was used for library preparation using the Illumina TruSeq kit and sequenced on a HiSeq 2000 machine. Reads were aligned using TopHat2 followed by quantification of abundances using RSEM and differential expression analysis with edgeR. We analyzed transcriptomes, conducted pathway analyses, and summarized key candidate gene sets. Expressed SNVs (eSNVs) from the RNA-seq data were determined using GATK and RVboost. Results: The analysis found 699 genes with FDR Discussion: Using one of the largest sets of identically processed fresh-frozen EOC tumors, some patterns emerged among the numerous EC v HGSC transcriptomic differences. TPH1, up-expressed in EC, is regulated by SOX4 which was also up-regulated in EC. Two sets of genes related to Kallikreins serine proteases were differentially expressed, including KLK2 which is known to regulate EGFR and pro-inflammatory cytokines and is regulated by MYC. Lastly, TRAF3IP2 encodes for a protein involved in regulating cytokines through members of the NFKB pathway. Conclusions: These findings suggest important biological insights into one of the rarer EOC histologies and may aid in the development of targeted treatment options. Research is on-going to incorporate additional features (e.g., DNA methylation, copy number) into a “systems biology” framework to better understand the molecular differences between EOC histologies. Citation Format: Brooke L. Fridley, Junqiang Dai, Rama Raghavan, Chen Wang, Pengcheng Lu, Stacey Winham, Madalene Earp, Kate Lawrenson, Simon A. Gayther, Kimberly R. Kalli, Ellen L. Goode. Transcriptome characterization of high grade serous and endometrioid epithelial ovarian cancer tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1833.

Abstract 4853: A bioinformatics approach to drug discovery: patient based connectivity mapping

Epithelial ovarian cancer (EOC) is the fifth leading cause of cancer death among women in the United States (5% of cancer deaths). The standard treatment for patients with advanced EOC is initial debulking surgery followed by carboplatin-paclitaxel combination chemotherapy. Unfortunately, even with modern chemotherapy, most patients relapse and die with the five-year overall survival around 45%. In addition, those patients that initially respond to taxane-platinum therapy eventually develop platinum-resistant tumors and relapse. Thus, finding novel therapeutics for treating EOC is essential. One approach that has been used widely in cancer drug discovery is Connectivity Mapping (CMAP) using gene expression data and drug phenotype data assessed on cancer cell lines (CCL) (i.e. “sensitive” or “resistant” based on dose-response drug data). However, recent publications have highlighted some issues with the use of these standard CCL sets that are often used in these drug screening studies. Therefore, instead of CMAP analysis based on gene expression signatures developed on drug response data collected on a small set of EOC cell lines, we took a novel CMAP approach based on information collected on EOC patients and clinical endpoints. That is, we determined the gene expression signature (e.g., set of genes) that were associated with time to recurrence in data collected on EOC patients within TCGA (n = 518) and a Mayo Clinic study (n = 474). The set of genes associated with time to recurrence (with and without adjustment for additional clinical covariates) was completed separately for TCGA and Mayo Clinic studies, restricting all analyses to EOC tumor of high-grade serous histology. Each of these 4 sets of genes, where genes with hazard ratios (HR) > 1 coded as “positively” associated and genes with HR Note: This abstract was not presented at the meeting. Citation Format: Brooke L. Fridley, Rama Raghavan, Gottfried E. Konecny, Chen Wang, Ellen L. Goode, Harsh B. Pathak, Stephen Hyter. A bioinformatics approach to drug discovery: patient based connectivity mapping. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4853. doi:10.1158/1538-7445.AM2015-4853