Jillian Hung

Novel Molecular Subtypes of Serous and Endometrioid Ovarian Cancer Linked to Clinical Outcome

Purpose: The study aim to identify novel molecular subtypes of ovarian cancer by gene expression profiling with linkage to clinical and pathologic features. Experimental Design: Microarray gene expression profiling was done on 285 serous and endometrioid tumors of the ovary, peritoneum, and fallopian tube. K-means clustering was applied to identify robust molecular subtypes. Statistical analysis identified differentially expressed genes, pathways, and gene ontologies. Laser capture microdissection, pathology review, and immunohistochemistry validated the array-based findings. Patient survival within k-means groups was evaluated using Cox proportional hazards models. Class prediction validated k-means groups in an independent dataset. A semisupervised survival analysis of the array data was used to compare against unsupervised clustering results. Results: Optimal clustering of array data identified six molecular subtypes. Two subtypes represented predominantly serous low malignant potential and low-grade endometrioid subtypes, respectively. The remaining four subtypes represented higher grade and advanced stage cancers of serous and endometrioid morphology. A novel subtype of high-grade serous cancers reflected a mesenchymal cell type, characterized by overexpression of N-cadherin and P-cadherin and low expression of differentiation markers, including CA125 and MUC1. A poor prognosis subtype was defined by a reactive stroma gene expression signature, correlating with extensive desmoplasia in such samples. A similar poor prognosis signature could be found using a semisupervised analysis. Each subtype displayed distinct levels and patterns of immune cell infiltration. Class prediction identified similar subtypes in an independent ovarian dataset with similar prognostic trends. Conclusion: Gene expression profiling identified molecular subtypes of ovarian cancer of biological and clinical importance.

Whole–genome characterization of chemoresistant ovarian cancer

Patients with high-grade serous ovarian cancer (HGSC) have experienced little improvement in overall survival, and standard treatment has not advanced beyond platinum-based combination chemotherapy, during the past 30 years. To understand the drivers of clinical phenotypes better, here we use whole-genome sequencing of tumour and germline DNA samples from 92 patients with primary refractory, resistant, sensitive and matched acquired resistant disease. We show that gene breakage commonly inactivates the tumour suppressors RB1, NF1, RAD51B and PTEN in HGSC, and contributes to acquired chemotherapy resistance. CCNE1 amplification was common in primary resistant and refractory disease. We observed several molecular events associated with acquired resistance, including multiple independent reversions of germline BRCA1 or BRCA2 mutations in individual patients, loss of BRCA1 promoter methylation, an alteration in molecular subtype, and recurrent promoter fusion associated with overexpression of the drug efflux pump MDR1.

Genomic Classification of Serous Ovarian Cancer with Adjacent Borderline Differentiates RAS Pathway and TP53-Mutant Tumors and Identifies NRAS as an Oncogenic Driver

Purpose: Low-grade serous ovarian carcinomas (LGSC) are Ras pathway-mutated, TP53 wild-type, and frequently associated with borderline tumors. Patients with LGSCs respond poorly to platinum-based chemotherapy and may benefit from pathway-targeted agents. High-grade serous carcinomas (HGSC) are TP53-mutated and are thought to be rarely associated with borderline tumors. We sought to determine whether borderline histology associated with grade 2 or 3 carcinoma was an indicator of Ras mutation, and we explored the molecular relationship between coexisting invasive and borderline histologies. Experimental Design: We reviewed >1,200 patients and identified 102 serous carcinomas with adjacent borderline regions for analyses, including candidate mutation screening, copy number, and gene expression profiling. Results: We found a similar frequency of low, moderate, and high-grade carcinomas with coexisting borderline histology. BRAF/KRAS alterations were common in LGSC; however, we also found recurrent NRAS mutations. Whereas borderline tumors harbored BRAF/KRAS mutations, NRAS mutations were restricted to carcinomas, representing the first example of a Ras oncogene with an obligatory association with invasive serous cancer. Coexisting borderline and invasive components showed nearly identical genomic profiles. Grade 2 cases with coexisting borderline included tumors with molecular features of LGSC, whereas others were typical of HGSC. However, all grade 3 carcinomas with coexisting borderline histology were molecularly indistinguishable from typical HGSC. Conclusion: Our findings suggest that NRAS is an oncogenic driver in serous ovarian tumors. We demonstrate that borderline histology is an unreliable predictor of Ras pathway aberration and underscore an important role for molecular classification in identifying patients that may benefit from targeted agents. Clin Cancer Res; 20(24); 6618–30. ©2014 AACR.

Dose-Response Association of CD8+ Tumor-Infiltrating Lymphocytes and Survival Time in High-Grade Serous Ovarian Cancer.

Importance Cytotoxic CD8+ tumor-infiltrating lymphocytes (TILs) participate in immune control of epithelial ovarian cancer; however, little is known about prognostic patterns of CD8+ TILs by histotype and in relation to other clinical factors. Objective To define the prognostic role of CD8+ TILs in epithelial ovarian cancer. Design, Setting, and Participants This was a multicenter observational, prospective survival cohort study of the Ovarian Tumor Tissue Analysis Consortium. More than 5500 patients, including 3196 with high-grade serous ovarian carcinomas (HGSOCs), were followed prospectively for over 24 650 person-years. Exposures Following immunohistochemical analysis, CD8+ TILs were identified within the epithelial components of tumor islets. Patients were grouped based on the estimated number of CD8+ TILs per high-powered field: negative (none), low (1-2), moderate (3-19), and high (≥20). CD8+ TILs in a subset of patients were also assessed in a quantitative, uncategorized manner, and the functional form of associations with survival was assessed using penalized B-splines. Main Outcomes and Measures Overall survival time. Results The final sample included 5577 women; mean age at diagnosis was 58.4 years (median, 58.2 years). Among the 5 major invasive histotypes, HGSOCs showed the most infiltration. CD8+ TILs in HGSOCs were significantly associated with longer overall survival; median survival was 2.8 years for patients with no CD8+ TILs and 3.0 years, 3.8 years, and 5.1 years for patients with low, moderate, or high levels of CD8+ TILs, respectively (P value for trend = 4.2 × 10−16). A survival benefit was also observed among women with endometrioid and mucinous carcinomas, but not for those with the other histotypes. Among HGSOCs, CD8+ TILs were favorable regardless of extent of residual disease following cytoreduction, known standard treatment, and germline BRCA1 pathogenic mutation, but were not prognostic for BRCA2 mutation carriers. Evaluation of uncategorized CD8+ TIL counts showed a near-log-linear functional form. Conclusions and Relevance This study demonstrates the histotype-specific nature of immune infiltration and provides definitive evidence for a dose-response relationship between CD8+ TILs and HGSOC survival. That the extent of infiltration is prognostic, not merely its presence or absence, suggests that understanding factors that drive infiltration will be the key to unraveling outcome heterogeneity in this cancer.

Homologous Recombination DNA Repair Pathway Disruption and Retinoblastoma Protein Loss Are Associated with Exceptional Survival in High-Grade Serous Ovarian Cancer.

Purpose: Women with epithelial ovarian cancer generally have a poor prognosis; however, a subset of patients has an unexpected dramatic and durable response to treatment. We sought to identify clinical, pathological, and molecular determinants of exceptional survival in women with high-grade serous cancer (HGSC), a disease associated with the majority of ovarian cancer deaths. Experimental Design: We evaluated the histories of 2,283 ovarian cancer patients and, after applying stringent clinical and pathological selection criteria, identified 96 with HGSC that represented significant outliers in terms of treatment response and overall survival. Patient samples were characterized immunohistochemically and by genome sequencing. Results: Different patterns of clinical response were seen: long progression-free survival (Long-PFS), multiple objective responses to chemotherapy (Multiple Responder), and/or greater than 10-year overall survival (Long-Term Survivors). Pathogenic germline and somatic mutations in genes involved in homologous recombination (HR) repair were enriched in all three groups relative to a population-based series. However, 29% of 10-year survivors lacked an identifiable HR pathway alteration, and tumors from these patients had increased Ki-67 staining. CD8+ tumor-infiltrating lymphocytes were more commonly present in Long-Term Survivors. RB1 loss was associated with long progression-free and overall survival. HR deficiency and RB1 loss were correlated, and co-occurrence was significantly associated with prolonged survival. Conclusions: There was diversity in the clinical trajectory of exceptional survivors associated with multiple molecular determinants of exceptional outcome in HGSC patients. Concurrent HR deficiency and RB1 loss were associated with favorable outcomes, suggesting that co-occurrence of specific mutations might mediate durable responses in such patients. Clin Cancer Res; 24(3); 569–80. ©2017 AACR. See related commentary by Peng and Mills, p. 508

Serous ovarian and primary peritoneal cancers: A comparative analysis of clinico-pathological features, molecular subtypes and treatment outcome

OBJECTIVE Primary peritoneal cancer is rare and considered equivalent to stage III/IV ovarian cancer, but questions remain concerning its underlying biology, prognosis and optimal management. METHODS Clinico-pathological and treatment details of primary peritoneal (n=120) and ovarian cancer (n=635) were obtained on women recruited to the Australian Ovarian Cancer Study. Log-rank test was used to compare survival and cox proportional hazards models were fitted to obtain hazard ratios and 95% confidence intervals, both unadjusted and adjusted for age, grade, FIGO stage, residual disease and treatment with neoadjuvant chemotherapy. Molecular subtype was determined by gene expression profiling using published data. RESULTS Compared with advanced serous ovarian cancer, primary peritoneal cancer patients were older (mean age 65.5 vs. 60.2years, p<0.001), more often treated with neoadjuvant chemotherapy (38.4% vs. 11.4%, p<0.001). Gene expression profiling classified a substantially higher proportion of primary peritoneal carcinomas as C1 (mesenchymal, reactive stromal infiltration) subtype (70.6% vs. 32.1%, p=0.029), which was associated with lower complete surgical resection rate. Women with primary peritoneal cancer had significantly shorter progression-free (11.6 vs. 13.6months, p=0.007) and overall survival (31.7 vs. 39.8months, p=0.012). In multivariate analysis, residual disease and neoadjuvant chemotherapy were both independently associated with increased risk of progression and death. CONCLUSIONS Primary peritoneal cancer patients were more frequently treated with neoadjuvant chemotherapy and had inferior survival. Different tumor biology characterized by activated stromal fibrosis in primary peritoneal cancer may underlie the differences in treatment and clinical outcome.

Corrigendum: Whole–genome characterization of chemoresistant ovarian cancer

This corrects the article DOI: 10.1038/nature14410

Primary treatment patterns in women recruited to the Australian Ovarian Cancer Study

Associations between clinical outcome and patient characteristics, such as tumour gene expression, and inherited variation (single nucleotide polymorphisms) can be difficult to reproduce between ovarian cancer cohorts. Sources of variation include the size and composition of patient cohorts and treatment, which has varied over time and can vary between countries. We have undertaken a review of treatment in patients recruited to the Australian Ovarian Cancer Study (AOCS) to determine the primary treatment patterns and levels of consistency across the cohort. Australian clinical practice guidelines for the management of women with epithelial ovarian cancer were published in 2004 [1] and AOCS recruited women with suspected ovarian, fallopian tube or primary peritoneal cancer from 2002-2006. AOCS collected fresh tissue, blood, epidemiological/dietary questionnaires and clinical follow-up data for women aged 18-79 diagnosed with invasive (n=1476) and borderline (n=352) cancer, and material and data are available for research, by application. Clinical data was collected through a national research nurse network at pre-specified intervals: at diagnosis, completion of primary treatment and then at 6-monthly intervals. At the end of Feb 2011 primary treatment data was complete on 99% of eligible cases and the median follow-up time was 5.1 years. Most patients with invasive cancer underwent surgery prior to chemotherapy (n=1190, 82%) and a small proportion had an interval debulk (surgery following ~3 cycles of chemotherapy, 12%) or other procedure (<10%). The vast majority of women with advanced stage cancer (FIGO stage II-IV) received chemotherapy (1116/1167, 96%) and of these, over 99% (1110/1116) received a platin-based regimen. The most common regimens were carboplatin/paclitaxel (77%) and carboplatin alone (9%), most women (86%) received 6-8 cycles and 95% of women who received adjuvant chemotherapy began treatment within 6 weeks of surgery. In conclusion, the majority of women in AOCS were treated according to national guidelines, enabling the selection of cases with uniform treatment for projects investigating associations of genomic and genetic features with clinical outcome.

The Australian Ovarian Cancer Study

Background AOCS commenced in 2000 as a collaborative study between researchers at the Peter MacCallum Cancer Centre (PMCC), Queensland Institute for Medical Research (QIMR), Westmead Institute for Cancer Research (WICR) and University of Melbourne. Patient recruitment ceased on June 30, 2006. AOCS recruited a total of 1834 women with invasive or borderline ovarian cancer, far exceeding the initial target. We have received a total of 1815 completed questionnaires and have collected 1080 fresh tumour tissue samples and 1582 blood samples. Control recruitment is also complete and a total of 1066 control women that did not have ovarian cancer were recruited. Clinical details have been recorded for all AOCS patients, with clinical follow-up done at 6-monthly intervals: we have primary treatment data, including surgery and chemotherapy details on 99% of cases; and 89% of eligible cases have follow-up to five years post-diagnosis.. Thus far only 123 patients (6.5%) have been lost to follow-up, despite that fact that 30-40% of our patients return to regional areas for ongoing treatment.