Peter A. Fasching

Patterns of Associations with Epidemiologic Factors by High-Grade Serous Ovarian Cancer Gene Expression Subtypes

Abstract Background: Ovarian high-grade serous carcinomas (HGSC) comprise four distinct molecular subtypes based on mRNA expression patterns, with differential survival. Understanding risk factor associations is important to elucidate the etiology of HGSC. We investigated associations between different epidemiologic risk factors and HGSC molecular subtypes. Methods: We pooled data from 11 case–control studies with epidemiologic and tumor gene expression data from custom NanoString CodeSets developed through a collaboration within the Ovarian Tumor Tissue Analysis consortium. The PrOTYPE-validated NanoString-based 55-gene classifier was used to assign HGSC gene expression subtypes. We examined associations between epidemiologic factors and HGSC subtypes in 2,070 cases and 16,633 controls using multivariable-adjusted polytomous regression models. Results: Among the 2,070 HGSC cases, 556 (27%) were classified as C1.MES, 340 (16%) as C5.PRO, 538 (26%) as C2.IMM, and 636 (31%) as C4.DIF. The key factors, including oral contraceptive use, parity, breastfeeding, and family history of ovarian cancer, were similarly associated with all subtypes. Heterogeneity was observed for several factors. Former smoking [OR = 1.25; 95% confidence interval (CI) = 1.03, 1.51] and genital powder use (OR = 1.42; 95% CI = 1.08, 1.86) were uniquely associated with C2.IMM. History of endometriosis was associated with C5.PRO (OR = 1.46; 95% CI = 0.98, 2.16) and C4.DIF (OR = 1.27; 95% CI = 0.94, 1.71) only. Family history of breast cancer (OR = 1.44; 95% CI = 1.16, 1.78) and current smoking (OR = 1.40; 95% CI = 1.11, 1.76) were associated with C4.DIF only. Conclusions: This study observed heterogeneous associations of epidemiologic and modifiable factors with HGSC molecular subtypes. Impact: The different patterns of associations may provide key information about the etiology of the four subtypes.

Surrogate End Points for Overall Survival in Neoadjuvant Randomized Clinical Trials for Early Breast Cancer

PURPOSE To assess trial-level surrogacy value for overall survival (OS) of the pathologic complete response (pCR) and invasive disease-free survival (iDFS) in randomized clinical trials (RCTs) for early breast cancer (BC). METHODS Individual patient data of neoadjuvant RCTs with available data on pCR, iDFS, and OS were included in the analysis. We used the coefficient of determination R 2 from weighted linear regression models to quantify the association between treatment effects on OS and on the surrogate end points. RESULTS Eleven RCTs, for a total of 15 treatment comparisons and 12,247 patients, were included in the analysis. There was a weak association between hazard ratios (HRs) for OS and odds ratio of pCR overall ( R 2 , 0.07; 95% CI, 0.00 to 0.48), as well as in all the subgroups explored. Overall, the R 2 for the association between HR OS and HR iDFS was 0.46 (95% CI, 0.08 to 0.71), which is just below the cutoff of 0.5 for moderate surrogacy. In the majority of subgroups explored, the R 2 ranged from 0.5 to <0.7, while in hormone receptor–/human epidermal growth factor receptor 2– subtype, histologic grade 1-2 tumors, and lobular tumors, surrogacy was strong (ie, R 2 ≥0.7). The surrogacy value of iDFS for OS was affected by follow-up (FUP) length: R 2 substantially increased up to 36 months of FUP, with little further improvement after 48 months of FUP. CONCLUSION iDFS with sufficient FUP is an acceptable surrogate end point to confidently anticipate final OS results of neoadjuvant RCTs for early BC. This recommendation holds true across many subgroups, with the notable exception of HR+ disease. There is definite need to reassess whether OS is the optimal end point for treatment efficacy measurement in HR+ early BC.

Germline variants of homology‐directed repair or mismatch repair genes in cervical cancer

AbstractWhile cervical cancer is associated with a persistent human papillomavirus (HPV) infection, the progression to cancer is influenced by genomic risk factors that have remained largely obscure. Pathogenic variants in genes of the homology‐directed repair (HDR) or mismatch repair (MMR) are known to predispose to diverse tumour entities including breast and ovarian cancer (HDR) or colon and endometrial cancer (MMR). We here investigate the spectrum of HDR and MMR germline variants in cervical cancer, with particular focus on the HPV status and histological subgroups. We performed targeted next‐generation sequencing for 5 MMR genes and 12 HDR genes on 728 German patients with cervical dysplasia or invasive cancer. In total, 4% of our patients carried a pathogenic germline variant, based on ClinVar classifications and additional ESM1b and AlphaMissense predictions. These included 15 patients with truncating variants in HDR genes (BARD1, BRCA1, BRCA2, BRIP1, FANCM, RAD51D and SLX4). MMR‐related gene variants were less prevalent and mainly of the missense type. While MMR‐related gene variants tended to associate with adenocarcinomas, HDR gene variants were commonly observed in squamous cancers. While one patient with HPV‐negative cancer carried a pathogenic MMR gene variant (in MSH6), the HDR germline variants were found in patients with HPV‐positive cancers and tended to associate with HPV18. Taken together, our study supports a potentially risk‐modifying role of MMR and HDR germline variants in cervical cancer but no association with HPV‐negative status. These variants may be exploitable in future therapeutic managements.

Real-world utilization of aromatase inhibitors, tamoxifen, and ovarian function suppression in premenopausal patients with early hormone receptor-positive, HER2-negative breast cancer with increased recurrence risk

Mendelian randomization analyses suggest a role for cholesterol in the development of endometrial cancer

AbstractBlood lipids have been associated with the development of a range of cancers, including breast, lung and colorectal cancer. For endometrial cancer, observational studies have reported inconsistent associations between blood lipids and cancer risk. To reduce biases from unmeasured confounding, we performed a bidirectional, two‐sample Mendelian randomization analysis to investigate the relationship between levels of three blood lipids (low‐density lipoprotein [LDL] and high‐density lipoprotein [HDL] cholesterol, and triglycerides) and endometrial cancer risk. Genetic variants associated with each of these blood lipid levels (P < 5 × 10−8) were identified as instrumental variables, and assessed using genome‐wide association study data from the Endometrial Cancer Association Consortium (12 906 cases and 108 979 controls) and the Global Lipids Genetic Consortium (n = 188 578). Mendelian randomization analyses found genetically raised LDL cholesterol levels to be associated with lower risks of endometrial cancer of all histologies combined, and of endometrioid and non‐endometrioid subtypes. Conversely, higher genetically predicted HDL cholesterol levels were associated with increased risk of non‐endometrioid endometrial cancer. After accounting for the potential confounding role of obesity (as measured by genetic variants associated with body mass index), the association between genetically predicted increased LDL cholesterol levels and lower endometrial cancer risk remained significant, especially for non‐endometrioid endometrial cancer. There was no evidence to support a role for triglycerides in endometrial cancer development. Our study supports a role for LDL and HDL cholesterol in the development of non‐endometrioid endometrial cancer. Further studies are required to understand the mechanisms underlying these findings.

Genome‐Wide Association Analyses of HPV16 and HPV18 Seropositivity Identify Susceptibility Loci for Cervical Cancer

ABSTRACTInfection by high‐risk human papillomavirus is known to exacerbate cervical cancer development. The host immune response is crucial in disease regression. Large‐scale genetic association studies for cervical cancer have identified few susceptibility variants, mainly at the human leukocyte antigen locus on chromosome 6. We hypothesized that the host immune response modifies cervical cancer risk and performed three genome‐wide association analyses for HPV16, HPV18 and HPV16/18 seropositivity in 7814, 7924, and 7924 samples from the UK Biobank, followed by validation genotyping in the German Cervigen case‐control series of cervical cancer and dysplasia. In GWAS analyses, we identified two loci associated with HPV16 seropositivity (6p21.32 and 15q26.2), two loci associated with HPV18 seropositivity (5q31.2 and 14q24.3), and one locus for HPV16 and/or HPV18 seropositivity (at 6p21.32). MAGMA gene‐based analysis identified HLA‐DQA1 and HLA‐DQB1 as genome‐wide significant (GWS) genes. In validation genotyping, the genome‐wide significant lead variant at 6p21.32, rs9272293 associated with overall cervical disease (OR = 0.86, p = 0.004, 95% CI = 0.78–0.95, n = 3710) and HPV16 positive invasive cancer (OR = 0.73, p = 0.005, 95% CI = 0.59–0.91, n = 1431). This variant was found to be a robust eQTL for HLA‐DRB1, HLA‐DQB1‐AS1, C4B, HLA‐DRB5, HLA‐DRB6, HLA‐DQB1, and HLA‐DPB1 in a series of cervical epithelial tissue samples. We additionally genotyped twenty‐four HPV seropositivity variants below the GWS threshold out of which eleven variants were found to be associated with cervical disease in our cohort, suggesting that further seropositivity variants may determine cervical disease outcome. Our study identifies novel genomic risk loci that associate with HPV type‐specific cervical cancer and dysplasia risk and provides evidence for candidate genes at one of the risk loci.

Development and Validation of the Gene Expression Predictor of High-grade Serous Ovarian Carcinoma Molecular SubTYPE (PrOTYPE)

Abstract Purpose: Gene expression–based molecular subtypes of high-grade serous tubo-ovarian cancer (HGSOC), demonstrated across multiple studies, may provide improved stratification for molecularly targeted trials. However, evaluation of clinical utility has been hindered by nonstandardized methods, which are not applicable in a clinical setting. We sought to generate a clinical grade minimal gene set assay for classification of individual tumor specimens into HGSOC subtypes and confirm previously reported subtype-associated features. Experimental Design: Adopting two independent approaches, we derived and internally validated algorithms for subtype prediction using published gene expression data from 1,650 tumors. We applied resulting models to NanoString data on 3,829 HGSOCs from the Ovarian Tumor Tissue Analysis consortium. We further developed, confirmed, and validated a reduced, minimal gene set predictor, with methods suitable for a single-patient setting. Results: Gene expression data were used to derive the predictor of high-grade serous ovarian carcinoma molecular subtype (PrOTYPE) assay. We established a de facto standard as a consensus of two parallel approaches. PrOTYPE subtypes are significantly associated with age, stage, residual disease, tumor-infiltrating lymphocytes, and outcome. The locked-down clinical grade PrOTYPE test includes a model with 55 genes that predicted gene expression subtype with >95% accuracy that was maintained in all analytic and biological validations. Conclusions: We validated the PrOTYPE assay following the Institute of Medicine guidelines for the development of omics-based tests. This fully defined and locked-down clinical grade assay will enable trial design with molecular subtype stratification and allow for objective assessment of the predictive value of HGSOC molecular subtypes in precision medicine applications. See related commentary by McMullen et al., p. 5271

Identification of a Locus Near ULK1 Associated With Progression-Free Survival in Ovarian Cancer

Abstract Background: Many loci have been found to be associated with risk of epithelial ovarian cancer (EOC). However, although there is considerable variation in progression-free survival (PFS), no loci have been found to be associated with outcome at genome-wide levels of significance. Methods: We carried out a genome-wide association study (GWAS) of PFS in 2,352 women with EOC who had undergone cytoreductive surgery and standard carboplatin/paclitaxel chemotherapy. Results: We found seven SNPs at 12q24.33 associated with PFS (P < 5 × 10–8), the top SNP being rs10794418 (HR = 1.24; 95% CI, 1.15–1.34; P = 1.47 × 10–8). High expression of a nearby gene, ULK1, is associated with shorter PFS in EOC, and with poor prognosis in other cancers. SNP rs10794418 is also associated with expression of ULK1 in ovarian tumors, with the allele associated with shorter PFS being associated with higher expression, and chromatin interactions were detected between the ULK1 promoter and associated SNPs in serous and endometrioid EOC cell lines. ULK1 knockout ovarian cancer cell lines showed significantly increased sensitivity to carboplatin in vitro. Conclusions: The locus at 12q24.33 represents one of the first genome-wide significant loci for survival for any cancer. ULK1 is a plausible candidate for the target of this association. Impact: This finding provides insight into genetic markers associated with EOC outcome and potential treatment options. See related commentary by Peres and Monteiro, p. 1604

Association of genomic variants at the human leukocyte antigen locus with cervical cancer risk, HPV status and gene expression levels

AbstractThe human leukocyte antigen (HLA) locus on chromosome 6 has been reported to be associated with cervical cancer. We investigated two independent single‐nucleotide polymorphisms in a large case‐control series of cervical dysplasia and carcinoma that has been newly established by the German Cervigen Consortium, comprising a total of 2481 cases and 1556 healthy females. We find significant associations for both variants, rs9272117 at HLA‐DQA1 and rs2844511 at MICA and HCP5, with cervical disease. Both variants showed evidence of association with invasive cervical cancer (rs9272117: OR 0.89, 95% CI 0.79‐0.99, P = .036; rs2844511: OR 1.17, 95% CI 1.04‐1.31, P = .008) and with high‐grade dysplasia (rs9272117: OR 0.78, 95% CI 0.70‐0.87, P = 7.1 × 10−6; rs2844511: OR 1.13, 95% CI 1.01‐1.26, P = .035), as well as in a combined analysis of both groups (rs9272117: OR 0.83, 95% CI 0.75‐0.91, P = 6.9 × 10−5; rs2844511: OR 1.14, 95% CI 1.04‐1.26, P = .005). Variant rs2844511, but not rs9272117, also showed modest evidence of association with low‐grade dysplasia (OR 1.26, 95% CI 1.04‐1.54, P = .019). In case‐only analyses, rs2844511 tended to predict HPV status (P = .044) and rs9272117 tended to associate with HPV16 (P = .022). RNA studies in cervical samples showed a significant correlation in the transcript levels of MICA, HCP5 and HLA‐DQA1, suggesting extensive co‐regulation. All three genes were upregulated in HPV16‐positive samples. In stratified analyses, rs9272117 was associated with HLA‐DQA1 levels, specifically in HPV‐positive samples, while rs2844511 was associated with MICA and HCP5 levels. The risk allele of rs2844511 was required for correlations between MICA or HCP5 with HLA‐DQA1. Altogether, our results support 6p21.32‐33 as the first consistent cervical cancer susceptibility locus and provide evidence for a link between genetic risk variants, HPV16 status and transcript levels of HLA‐DQA1, HCP5 and MICA, which may contribute to tumor immune evasion.

Concurrent RB1 Loss and BRCA Deficiency Predicts Enhanced Immunologic Response and Long-term Survival in Tubo-ovarian High-grade Serous Carcinoma

Abstract Purpose: The purpose of this study was to evaluate RB1 expression and survival across ovarian carcinoma histotypes and how co-occurrence of BRCA1 or BRCA2 (BRCA) alterations and RB1 loss influences survival in tubo-ovarian high-grade serous carcinoma (HGSC). Experimental Design: RB1 protein expression was classified by immunohistochemistry in ovarian carcinomas of 7,436 patients from the Ovarian Tumor Tissue Analysis consortium. We examined RB1 expression and germline BRCA status in a subset of 1,134 HGSC, and related genotype to overall survival (OS), tumor-infiltrating CD8+ lymphocytes, and transcriptomic subtypes. Using CRISPR-Cas9, we deleted RB1 in HGSC cells with and without BRCA1 alterations to model co-loss with treatment response. We performed whole-genome and transcriptome data analyses on 126 patients with primary HGSC to characterize tumors with concurrent BRCA deficiency and RB1 loss. Results: RB1 loss was associated with longer OS in HGSC but with poorer prognosis in endometrioid ovarian carcinoma. Patients with HGSC harboring both RB1 loss and pathogenic germline BRCA variants had superior OS compared with patients with either alteration alone, and their median OS was three times longer than those without pathogenic BRCA variants and retained RB1 expression (9.3 vs. 3.1 years). Enhanced sensitivity to cisplatin and paclitaxel was seen in BRCA1-altered cells with RB1 knockout. Combined RB1 loss and BRCA deficiency correlated with transcriptional markers of enhanced IFN response, cell-cycle deregulation, and reduced epithelial–mesenchymal transition. CD8+ lymphocytes were most prevalent in BRCA-deficient HGSC with co-loss of RB1. Conclusions: Co-occurrence of RB1 loss and BRCA deficiency was associated with exceptionally long survival in patients with HGSC, potentially due to better treatment response and immune stimulation.