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BACKGROUND: Cyclin E1 (CCNE1) is a potential predictive marker and therapeutic target in tubo-ovarian high-grade serous carcinoma (HGSC). Smaller studies have revealed unfavorable associations for CCNE1 amplification and CCNE1 overexpression with survival, but to date no large-scale, histotype-specific validation has been performed. The hypothesis was that high-level amplification of CCNE1 and CCNE1 overexpression, as well as a combination of the two, are linked to shorter overall survival in HGSC. METHODS: Within the Ovarian Tumor Tissue Analysis consortium, amplification status and protein level in 3029 HGSC cases and mRNA expression in 2419 samples were investigated. RESULTS: High-level amplification (>8 copies by chromogenic in situ hybridization) was found in 8.6% of HGSC and overexpression (>60% with at least 5% demonstrating strong intensity by immunohistochemistry) was found in 22.4%. CCNE1 high-level amplification and overexpression both were linked to shorter overall survival in multivariate survival analysis adjusted for age and stage, with hazard stratification by study (hazard ratio [HR], 1.26; 95% CI, 1.08-1.47, p = .034, and HR, 1.18; 95% CI, 1.05-1.32, p = .015, respectively). This was also true for cases with combined high-level amplification/overexpression (HR, 1.26; 95% CI, 1.09-1.47, p = .033). CCNE1 mRNA expression was not associated with overall survival (HR, 1.00 per 1-SD increase; 95% CI, 0.94-1.06; p = .58). CCNE1 high-level amplification is mutually exclusive with the presence of germline BRCA1/2 pathogenic variants and shows an inverse association to RB1 loss. CONCLUSION: This study provides large-scale validation that CCNE1 high-level amplification is associated with shorter survival, supporting its utility as a prognostic biomarker in HGSC. ispartof: CANCER vol:129 issue:5 pages:697-713 ispartof: location:United States status: published

Background Recently, we showed a >60% difference in 5-year survival for patients with tubo-ovarian high-grade serous carcinoma (HGSC) when stratified by a 101-gene mRNA expression prognostic signature. Given the varied patient outcomes, this study aimed to translate prognostic mRNA markers into protein expression assays by immunohistochemistry and validate their survival association in HGSC. Methods Two prognostic genes, FOXJ1 and GMNN, were selected based on high-quality antibodies, correlation with protein expression and variation in immunohistochemical scores in a preliminary cohort (n = 134 and n = 80, respectively). Six thousand four hundred and thirty-four (FOXJ1) and 5470 (GMNN) formalin-fixed, paraffin-embedded ovarian neoplasms (4634 and 4185 HGSC, respectively) represented on tissue microarrays from the Ovarian Tumor Tissue Analysis consortium underwent immunohistochemical staining and scoring, then univariate and multivariate survival analysis. Results Consistent with mRNA, FOXJ1 protein expression exhibited a linear, increasing association with improved overall survival in HGSC patients. Women with >50% expression had the most favourable outcomes (HR = 0.78, 95% CI 0.67–0.91, p 35% GMNN expression showed a trend for better outcomes, though this was not significant. Conclusion We provide foundational evidence for the prognostic value of FOXJ1 in HGSC, validating the prior mRNA-based prognostic association by immunohistochemistry.

PURPOSE Tubo-ovarian cancer (TOC) is a sentinel cancer for BRCA1 and BRCA2 pathogenic variants (PVs). Identification of a PV in the first member of a family at increased genetic risk (the proband) provides opportunities for cancer prevention in other at-risk family members. Although Australian testing rates are now high, PVs in patients with TOC whose diagnosis predated revised testing guidelines might have been missed. We assessed the feasibility of detecting PVs in this population to enable genetic risk reduction in relatives. PATIENTS AND METHODS In this pilot study, deceased probands were ascertained from research cohort studies, identification by a relative, and gynecologic oncology clinics. DNA was extracted from archival tissue or stored blood for panel sequencing of 10 risk-associated genes. Testing of deceased probands ascertained through clinic records was performed with a consent waiver. RESULTS We identified 85 PVs in 84 of 787 (11%) probands. Familial contacts of 39 of 60 (65%) deceased probands with an identified recipient (60 of 84; 71%) have received a written notification of results, with follow-up verbal contact made in 85% (33 of 39). A minority of families (n = 4) were already aware of the PV. For many (29 of 33; 88%), the genetic result provided new information and referral to a genetic service was accepted in most cases (66%; 19 of 29). Those who declined referral (4 of 29) were all male next of kin whose family member had died more than 10 years before. CONCLUSION We overcame ethical and logistic challenges to demonstrate that retrospective genetic testing to identify PVs in previously untested deceased probands with TOC is feasible. Understanding reasons for a family member's decision to accept or decline a referral will be important for guiding future TRACEBACK projects.

HR pathway mutations

Supplementary methods

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

Revised Supplementary Data, containing Supplementary Methods, Supplementary Figures, Supplementary Tables Supplementary Figure S1. Outline of cohort selection and analyses. Supplementary Figure S2. Clinical response and therapy course of 96 patients with exceptional responses to chemotherapy. Supplementary Figure S3. Distribution and type of TP53 mutations. Supplementary Figure S4. RB1 protein expression altered by genomic inactivation. Supplementary Figure S5. Characterization of CD8 and Ki-67 in tumors according to homologous recombination mutation status. Supplementary Table S2 Immunohistochemical analysis: primary antibodies and staining conditions Supplementary Table S3 Homologous recombination and DNA repair panel Supplementary Table S6 Comparison of molecular alteration prevalence between clinical subgroups Supplementary Table S7 Patient characteristics of tissue microarray cohort

Low-grade serous ovarian carcinomas (LGSC) are associated with a poor response to chemotherapy and are molecularly characterized by RAS pathway activation. Using exome and whole genome sequencing, we identified recurrent mutations in the protein translational regulator EIF1AX and in NF1, USP9X, KRAS, BRAF, and NRAS. RAS pathway mutations were mutually exclusive; however, we found significant co-occurrence of mutations in NRAS and EIF1AX. Missense EIF1AX mutations were clustered at the N-terminus of the protein in a region associated with its role in ensuring translational initiation fidelity. Coexpression of mutant NRAS and EIF1AX proteins promoted proliferation and clonogenic survival in LGSC cells, providing the first example of co-occurring, growth-promoting mutational events in ovarian cancer. Cancer Res; 77(16); 4268–78. ©2017 AACR.

Supplemental Material - Group Authorship from EIF1AX and NRAS Mutations Co-occur and Cooperate in Low-Grade Serous Ovarian Carcinomas

Supplemental materials and methods

Supplementary Table S1. Patient characteristics; Supplementary Table S2. Ki67 proliferation index; Supplementary Table S3. WES and WGS performance statistics; Supplementary Table S4. Amplicon sequencing primers; Supplementary Table S5. High confidence variants from exome and whole genome sequencing; Supplementary Table S6. Summary of SNVs and indels by sample; Supplementary Table S7. Significantly mutated genes (MuSiC output); Supplementary Table S8. Somatic variants in recurrently mutated genes; Supplementary Table S9. Validation cohort variants; Supplementary Table S10. TP53 variants in validation cohort; Supplementary Table S11. EIF1AX mutations reported in cancer

Purpose:Although ovarian clear cell carcinomas (OCCC) are commonly resistant to platinum-based chemotherapy, good clinical outcomes are observed in a subset of patients. The explanation for this is unknown but may be due to misclassification of high-grade serous ovarian cancer (HGSOC) as OCCC or mixed histology.Experimental Design:To discover potential biomarkers of survival benefit following platinum-based chemotherapy, we ascertained a cohort of 68 Japanese and Australian patients in whom progression-free survival (PFS) and overall survival (OS) could be assessed. We performed IHC reclassification of tumors, and targeted sequencing and immunohistochemistry of known driver genes. Exome sequencing was performed in 10 patients who had either unusually long survival (N = 5) or had a very short time to progression (N = 5).Results:The majority of mixed OCCC (N = 6, 85.7%) and a small proportion of pure OCCC (N = 3, 4.9%) were reclassified as likely HGSOC. However, the PFS and OS of patients with misclassified samples were similar to that of patients with pathologically validated OCCC. Absent HNF1B expression was significantly correlated with longer PFS and OS (P = 0.0194 and 0.0395, respectively). Mutations in ARID1A, PIK3CA, PPP2R1A, and TP53 were frequent, but did not explain length of PFS and OS. An exploratory exome analysis of patients with favorable and unfavorable outcomes did not identify novel outcome-associated driver mutations.Conclusions:Survival benefit following chemotherapy in OCCC was not associated with pathological misclassification of tumor histotype. HNF1B loss may help identify the subset of patients with OCCC with a more favorable outcome.

Supplementary Figure S1. Representative hematoxylin and eosin stained sections from sequenced LGSC; Supplementary Figure S2. Age distribution analysis of patients from the Australian Ovarian Cancer Study. Includes patients with advanced stage, serous epithelial ovarian cancer (n = 684); Supplementary Figure S3. RAS pathway mutations in LGSC; Supplementary Figure S4. Verification of EIF1AX mutations; Supplementary Figure S5. Characterization of the AOCS2 cell line; Supplementary Figure S6. Functional effect of EIF1AX mutations and gene suppression in a LGSC cell line; Supplementary Figure S7. (a) EIF1AX and NRAS function in the mTOR and RAS/ERK signaling pathways to regulate protein translation, cell proliferation and cell survival

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PDF file - 119K, Supp Table S1. Regions of differential copy number change between primary tumor samples and most distant abdominal deposit Supp Table S2. Top 50 pathways altered between primary tumors and abdominal deposits Supp Table S3. Individual patient chemotherapy regimens for paired primary and relapse ovarian tumors Supp Table S4. BRCA mutation details for mutation positive cases Supp Table S5. Regions of discrete copy number amplification Supp Table S6. Regions of discrete copy number deletion

TP53 mutations are implicated in the progression of mucinous borderline tumors (MBOT) to mucinous ovarian carcinomas (MOC). Optimized immunohistochemistry (IHC) for TP53 has been established as a proxy for the TP53 mutation status in other ovarian tumor types. We aimed to confirm the ability of TP53 IHC to predict TP53 mutation status in ovarian mucinous tumors and to evaluate the association of TP53 mutation status with survival among patients with MBOT and MOC. Tumor tissue from an initial cohort of 113 women with MBOT/MOC was stained with optimized IHC for TP53 using tissue microarrays (75.2%) or full sections (24.8%) and interpreted using established criteria as normal or abnormal (overexpression, complete absence, or cytoplasmic). Cases were considered concordant if abnormal IHC staining predicted deleterious TP53 mutations. Discordant tissue microarray cases were re-evaluated on full sections and interpretational criteria were refined. The initial cohort was expanded to a total of 165 MBOT and 424 MOC for the examination of the association of survival with TP53 mutation status, assessed either by TP53 IHC and/or sequencing. Initially, 82/113 (72.6%) cases were concordant using the established criteria. Refined criteria for overexpression to account for intratumoral heterogeneity and terminal differentiation improved concordance to 93.8% (106/113). In the expanded cohort, 19.4% (32/165) of MBOT showed evidence for TP53 mutation and this was associated with a higher risk of recurrence, disease-specific death, and all-cause mortality (overall survival: HR = 4.6, 95% CI 1.5-14.3, p = 0.0087). Within MOC, 61.1% (259/424) harbored a TP53 mutation, but this was not associated with survival (overall survival, p = 0.77). TP53 IHC is an accurate proxy for TP53 mutation status with refined interpretation criteria accounting for intratumoral heterogeneity and terminal differentiation in ovarian mucinous tumors. TP53 mutation status is an important biomarker to identify MBOT with a higher risk of mortality.

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

Objective Mucinous ovarian carcinoma (MOC) is an uncommon ovarian cancer histotype that responds poorly to conventional chemotherapy regimens. Although long overall survival outcomes can occur with early detection and optimal surgical resection, recurrent and advanced disease are associated with extremely poor survival. There are no current guidelines specifically for the systemic management of recurrent MOC. We analyzed data from a large cohort of women with MOC to evaluate the potential for clinical utility from a range of systemic agents. Methods We analyzed gene copy number (n = 191) and DNA sequencing data (n = 184) from primary MOC to evaluate signatures of mismatch repair deficiency and homologous recombination deficiency, and other genetic events. Immunohistochemistry data were collated for ER, CK7, CK20, CDX2, HER2, PAX8 and p16 (n = 117–166). Results Molecular aberrations noted in MOC that suggest a match with current targeted therapies include amplification of ERBB2 (26.7%) and BRAF mutation (9%). Observed genetic events that suggest potential efficacy for agents currently in clinical trials include: KRAS/NRAS mutations (66%), TP53 missense mutation (49%), RNF43 mutation (11%), ARID1A mutation (10%), and PIK3CA/PTEN mutation (9%). Therapies exploiting homologous recombination deficiency (HRD) may not be effective in MOC, as only 1/191 had a high HRD score. Mismatch repair deficiency was similarly rare (1/184). Conclusions Although genetically diverse, MOC has several potential therapeutic targets. Importantly, the lack of response to platinum-based therapy observed clinically corresponds to the lack of a genomic signature associated with HRD, and MOC are thus also unlikely to respond to PARP inhibition.

Mucinous ovarian carcinoma (MOC) is a unique subtype of ovarian cancer with an uncertain etiology, including whether it genuinely arises at the ovary or is metastatic disease from other organs. In addition, the molecular drivers of invasive progression, high-grade and metastatic disease are poorly defined. We perform genetic analysis of MOC across all histological grades, including benign and borderline mucinous ovarian tumors, and compare these to tumors from other potential extra-ovarian sites of origin. Here we show that MOC is distinct from tumors from other sites and supports a progressive model of evolution from borderline precursors to high-grade invasive MOC. Key drivers of progression identified are TP53 mutation and copy number aberrations, including a notable amplicon on 9p13. High copy number aberration burden is associated with worse prognosis in MOC. Our data conclusively demonstrate that MOC arise from benign and borderline precursors at the ovary and are not extra-ovarian metastases., Whether mucinous ovarian carcinoma (MOC) arises from cells at the ovary or from metastases from other primary sites is an unanswered question. Here, Cheasley et al perform a genetic analysis of the disease, showing that MOC arises at the ovary.

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

Low-grade serous ovarian carcinomas (LGSC) are associated with a poor response to chemotherapy and are molecularly characterized by RAS pathway activation. Using exome and whole genome sequencing, we identified recurrent mutations in the protein translational regulator EIF1AX and in NF1, USP9X, KRAS, BRAF, and NRAS. RAS pathway mutations were mutually exclusive; however, we found significant co-occurrence of mutations in NRAS and EIF1AX. Missense EIF1AX mutations were clustered at the N-terminus of the protein in a region associated with its role in ensuring translational initiation fidelity. Coexpression of mutant NRAS and EIF1AX proteins promoted proliferation and clonogenic survival in LGSC cells, providing the first example of co-occurring, growth-promoting mutational events in ovarian cancer. Cancer Res; 77(16); 4268–78. ©2017 AACR.

ABCB1 encodes Multidrug Resistance protein (MDR1), an ATP-binding cassette member involved in the cellular efflux of chemotherapeutic drugs. Here we report that ovarian and breast samples from chemotherapy treated patients are positive for multiple transcriptional fusions involving ABCB1, placing it under the control of a strong promoter while leaving its open reading frame intact. We identified 15 different transcriptional fusion partners involving ABCB1, as well as patients with multiple distinct fusion events. The partner gene selected depended on its structure, promoter strength, and chromosomal proximity to ABCB1. Fusion positivity was strongly associated with the number of lines of MDR1-substrate chemotherapy given. MDR1 inhibition in a fusion positive ovarian cancer cell line increased sensitivity to paclitaxel more than 50-fold. Convergent evolution of ABCB1 fusion is therefore frequent in chemotherapy resistant recurrent ovarian cancer. As most currently approved PARP inhibitors (PARPi) are MDR1 substrates, prior chemotherapy may precondition resistance to PARPi., ABCB1 encodes Multidrug Resistance Protein which promotes efflux of chemotherapeutic and targeted agents. Here, in breast and ovarian cancer the authors identify multiple transcriptional fusion partners involving ABCB1 that are associated with treatment failure and previous treatment regimens.

Purpose: Although ovarian clear cell carcinomas (OCCC) are commonly resistant to platinum-based chemotherapy, good clinical outcomes are observed in a subset of patients. The explanation for this is unknown but may be due to misclassification of high-grade serous ovarian cancer (HGSOC) as OCCC or mixed histology. Experimental Design: To discover potential biomarkers of survival benefit following platinum-based chemotherapy, we ascertained a cohort of 68 Japanese and Australian patients in whom progression-free survival (PFS) and overall survival (OS) could be assessed. We performed IHC reclassification of tumors, and targeted sequencing and immunohistochemistry of known driver genes. Exome sequencing was performed in 10 patients who had either unusually long survival (N = 5) or had a very short time to progression (N = 5). Results: The majority of mixed OCCC (N = 6, 85.7%) and a small proportion of pure OCCC (N = 3, 4.9%) were reclassified as likely HGSOC. However, the PFS and OS of patients with misclassified samples were similar to that of patients with pathologically validated OCCC. Absent HNF1B expression was significantly correlated with longer PFS and OS (P = 0.0194 and 0.0395, respectively). Mutations in ARID1A, PIK3CA, PPP2R1A, and TP53 were frequent, but did not explain length of PFS and OS. An exploratory exome analysis of patients with favorable and unfavorable outcomes did not identify novel outcome-associated driver mutations. Conclusions: Survival benefit following chemotherapy in OCCC was not associated with pathological misclassification of tumor histotype. HNF1B loss may help identify the subset of patients with OCCC with a more favorable outcome.

Purpose: The majority of patients with high-grade serous ovarian cancer (HGSC) develop progressive disease following primary treatment, with a five-year survival rate of ~30%. However, a subset of patients have an extraordinary response to treatment and ~15% survive more than ten years (long-term survivors). The Multidisciplinary Ovarian Cancer Outcomes Group (MOCOG) aims to uncover factors that influence long-term survival of HGSC patients. Here, we investigated the genomic and immunologic determinants of exceptional survival of this deadly disease. Experimental Design: Patient characteristics and clinical histories were evaluated to identify patients diagnosed with advanced stage (Stage IIIC/IV) and histopathologically confirmed HGSC with greater than 10-year overall survival. Whole-genome sequencing (WGS) was performed on primary tumors (median 78x coverage) and germline samples (median 39x coverage) of 55 long-term survivors. Primary tumor samples were also characterised by RNA sequencing, DNA methylation profiling and immunohistochemistry. Results: A total 38 (69%) of long-term surviving patients had residual disease following surgery, suggestive of highly chemo-sensitive disease. Most patients (41, 75%) were alive at last follow-up and 26 (47%) were progression-free. Somatic mutation burden was higher in primary tumors of long-term survivors relative to controls (316 unselected HGSC patients in The Cancer Genome Atlas). Genome-wide mutational signatures were predominantly Signature 3 (associated with homologous recombination deficiency), Signature 1 (age related) and Signatures 5, 8 and 16 (unknown etiology). Inactivation of the tumor suppressor RB1 by structural rearrangements or homozygous deletion was frequent in long-term survivors, with 33% of tumors showing associated loss of RB1 protein expression by immunohistochemistry compared to 13% of unselected HGSC controls (n=207; P = 0.001). Staining of adjacent tumor tissue revealed that RB1 loss was associated with increased numbers of PD-1+ tumor-infiltrating lymphocytes (P = 0.015) and MHC class I on tumor cells (P = 0.002). In an independent HGSC cohort (n=847), RB1 protein loss was associated with prolonged survival (HR: 0.75, P < 0.001) compared to patients with RB1 positive tumors. Furthermore, co-occurrence of germline mutations in BRCA1 or BRCA2 and RB1 loss was associated with a significantly longer overall survival compared to patients with retained RB1 protein expression and no germline BRCA mutation (HR: 0.44, P < 0.001). Conclusions: This study delineates the full landscape of genomic alterations in HGSC of long-term survivors. Our findings indicate that specific mutations might be associated with enhanced host immune responses and long-term survival. Citation Format: Dale W. Garsed, Ahwan Pandey, Sian Fereday, Kathryn Alsop, Maartje C. Wouters, Flurina Saner, Jessica A. Beach, Katy Milne, Catherine J. Kennedy, Joy Hendley, Nadia Traficante, Celeste L. Pearce, Malcolm C. Pike, Multidisciplinary Ovarian Cancer Outcomes Group, Susan J. Ramus, Martin Köbel, Brad H. Nelson, Ellen L. Goode, Anna deFazio, David D. Bowtell. The genomic landscape of high-grade serous ovarian cancer in long-term survivors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2722.

High-grade serous cancer (HGSC), the most common subtype of ovarian cancer, often becomes resistant to chemotherapy, leading to poor patient outcomes. Intratumoral heterogeneity occurs in nearly all solid cancers, including ovarian cancer, contributing to the development of resistance mechanisms. In this study, we examined the spatial and temporal genomic variation in HGSC using high-resolution single-nucleotide polymorphism arrays. Multiple metastatic lesions from individual patients were analyzed along with 22 paired pretreatment and posttreatment samples. We documented regions of differential DNA copy number between multiple tumor biopsies that correlated with altered expression of genes involved in cell polarity and adhesion. In the paired primary and relapse cohort, we observed a greater degree of genomic change in tumors from patients that were initially sensitive to chemotherapy and had longer progression-free interval compared with tumors from patients that were resistant to primary chemotherapy. Notably, deletion or downregulation of the lipid transporter LRP1B emerged as a significant correlate of acquired resistance in our analysis. Functional studies showed that reducing LRP1B expression was sufficient to reduce the sensitivity of HGSC cell lines to liposomal doxorubicin, but not to doxorubicin, whereas LRP1B overexpression was sufficient to increase sensitivity to liposomal doxorubicin. Together, our findings underscore the large degree of variation in DNA copy number in spatially and temporally separated tumors in HGSC patients, and they define LRP1B as a potential contributor to the emergence of chemotherapy resistance in these patients. Cancer Res; 72(16); 4060–73. ©2012 AACR.

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.

Cyclin D1 binds and regulates the activity of cyclin-dependent kinases (CDKs) 4 and 6. Phosphorylation of the retinoblastoma protein by cyclin D1.CDK4/6 complexes during the G(1) phase of the cell cycle promotes entry into S phase. Cyclin D1 protein is ubiquitinated and degraded by the 26 S proteasome. Previous studies have demonstrated that cyclin D1 ubiquitination is dependent on its phosphorylation by glycogen synthase kinase 3beta (GSK-3beta) on threonine 286 and that this phosphorylation event is greatly enhanced by binding to CDK4 (Diehl, J. A., Cheng, M. G., Roussel, M. F., and Sherr, C. J. (1998) Genes Dev. 12, 3499-3511). We now report an additional pathway for the ubiquitination of free cyclin D1 (unbound to CDKs). We show that, when unbound to CDK4, a cyclin D1-T286A mutant is ubiquitinated. Further, we show that a mutant of cyclin D1 that cannot bind to CDK4 (cyclin D1-KE) is also ubiquitinated in vivo. Our results demonstrate that free cyclin D1 is ubiquitinated independently of its phosphorylation on threonine 286 by GSK-3beta, suggesting that, as has been shown for cyclin E, distinct pathways of ubiquitination lead to the degradation of free and CDK-bound cyclin D1. The pathway responsible for ubiquitination of free cyclin D1 may be important in limiting the effects of cyclin D1 overexpression in a variety of cancers.

Coordinated accumulation of cyclin D1 and D3 is observed in 15% of primary breast cancers and in the breast cancer cell line MCF-7 this simultaneous overexpression is due to a defect in their ubiquitin-mediated proteolysis. The F-box protein Skp2 is a component of an SCF ubiquitin ligase complex and can associate with cyclin D1 and the cdk inhibitor p21 (Zhong-Kang et al., 1998). We extend this observation and show that cyclin D3 can also associate with Skp2 suggesting that cyclins D1, D3 and p21 may share the same SCF complex. In agreement with this hypothesis we report here that in primary breast cancers and in MCF-7 cells where cyclins D1 and D3 are elevated the level of p21 is also elevated. Further, we demonstrate that the turnover of p21 protein is reduced in MCF-7 cells. We show that p21 is active as a cdk inhibitor in this cell line but that the presence of elevated levels of cyclin D3 titrates p21 away from cyclin D1-cdk4/6 complexes and cdk2 complexes resulting in increased kinase activities. Our results suggest that a defect in the SCF complex may occur in 15 – 20% of breast cancers and that the resulting coordinated elevation of cyclins D1 and D3 overcomes the inhibition of cell cycle progression by p21. We propose that in the context of cyclins D1 and D3 overexpression, p21 may promote cell cycle progression.

The three human D-type cyclins, cyclin D1, D2 and D3 share the ability to bind to and activate cdk4 and 6. MMTV-cyclin D1 transgenic mice develop mainly adenocarcinoma, while MMTV-cyclin D2 mice show a lack of alveologenesis during pregnancy and only develop carcinoma at low frequency. The effect of cyclin D3 overexpression in mammary glands remains hitherto unknown. We generated MMTV-cyclin D3 transgenic mice and report here that they develop exclusively squamous cell carcinoma. We show that although cyclin D3 transgene expression was detected early in puberty, postnatal development and mammary gland proliferation were normal in virgin animals. In contrast, multiparous mice develop multiple foci of abnormal growth that correspond to various stages of squamous metaplasia. Therefore, our results support a role for cyclin D3 in squamous differentiation. In addition, we found that p16 expression during involution is abolished, while p27 expression increased in MMTV-cyclin D3 mice, two modifications that have been reported in the other MMTV-D-type cyclin transgenic models. Our observations indicate that despite biochemical redundancy in vitro and in vivo, D-type cyclins promote distinct oncogenic pathways.

Fenretinide has shown promise in the chemoprevention of breast cancer, a tumor type in which the oncogene cyclin D1 is overexpressed frequently. We aimed at determining the effect of cyclin D1 level on the response to fenretinide treatment.Stable clones of T47-D cells were created to overexpress cyclin D1 or a mutant of cyclin D1, injected in nude mice for xenograft formation, and the rate of tumor growth and tumor regression determined.We show here that cells overexpressing cyclin D1 are significantly more sensitive to fenretinide than genetically matched cells that express low levels of cyclin D1, and that fenretinide prevents tumor formation arising from cyclin D1-overexpressing cells. Furthermore, we show that fenretinide is also able to promote the regression of cyclin D1-positive tumors. We also show that cells expressing a mutant of cyclin D1 that cannot bind to cdk4 are also more sensitive to fenretinide.These results suggest that fenretinide may be particularly useful in the treatment of cyclin D1-positive breast cancers, and that the interaction between cyclin D1 and fenretinide is independent of cyclin D1 binding to cdk4.

The mechanism by which all-trans retinoic acid (ATRA) leads to a G(1) arrest of the cell cycle remains unclear. We show here that the decrease in D-type cyclin levels observed following ATRA treatment correlates with an increase in the rate of cyclin D1 ubiquitylation in both T-47D and MCF-7 breast cancer cell lines. However, MCF-7 cells are more resistant to ATRA than T-47D cells indicating that cyclin D1 degradation is not sufficient for ATRA-mediated arrest. We found a striking difference between these cells in that while ATRA induces an elevation in the cdk inhibitor p27 in T-47D cells, this is not observed in the ATRA-resistant MCF-7 cells. Furthermore, we demonstrate that ATRA promotes the ubiquitylation of Skp2, an F-box protein that targets p27 for degradation. Moreover, overexpression of Skp2 in T-47D cells prevents accumulation of p27 and promotes resistance to ATRA. In addition, overexpression of cyclin D1 in T-47D cells also promotes ATRA resistance. We found that the mechanism of ATRA-induced ubiquitylation of cyclin D1 and Skp2 is independent of CUL-1 expression and that ATRA can rescue cyclin D1 degradation in the uterine cell line SK-UT-1, where D-type cyclins are stabilized due to a specific defect in proteolysis. These data suggest that ATRA induces a novel pathway of ubiquitylation and that the degradation of the F-box protein Skp2 is the mechanism underlying p27 accumulation and cyclin E-cdk2 inactivation following ATRA treatment.

D-type cyclins are important cell cycle regulators that promote cellular proliferation in response to growth factors by inactivation of the retinoblastoma protein (Rb). Cyclin D1 has been shown to be overexpressed in several cancer types and to act as an oncogene in breast cancers. As D-type cyclins are rate limiting for progression into S phase, the level at which they accumulate must be carefully regulated. Several mechanisms leading to overexpression of cyclin D1 have been reported including amplification, translocation and stabilization of the mRNA. Here, we present data showing elevated cyclin D1 protein in breast cancer samples in the absence of elevated mRNA level. Further, we found that in these cases, cyclin D3 protein also accumulates and that the coordinate increase in cyclin D1 and D3 occurs in 15% (7/47) of breast cancers. In addition we show that blocking the activity of the 26S proteosome results in the accumulation of cyclin D1 and D3, that both D-type cyclins are ubiquitinated and associate with Cul-1, a component of the SCF ubiquitin ligase complex. Finally, we show that the coordinated elevation of cyclin D1 and D3 is also observed in the breast cell line MCF-7 and demonstrate that the degradation of cyclin D1 and D3 is deficient in this cell line. These results indicate that cyclin D1 and cyclin D3 share a common mechanism of degradation and we propose that the coordinate increase of D-type cyclins observed in primary breast cancers reflects a defect in their proteolysis.

Background: Median overall survival (OS) for women with high-grade serous ovarian cancer (HGSOC) is similar to 4 years, yet survival varies widely between patients. There are no well-established, gene expression signatures associated with prognosis. The aim of this study was to develop a robust prognostic signature for OS in patients with HGSOC. Patients and methods: Expression of 513 genes, selected from a meta-analysis of 1455 tumours and other candidates, was measured using NanoString technology from formalin-fixed paraffin-embedded tumour tissue collected from 3769 women with HGSOC from multiple studies. Elastic net regularization for survival analysis was applied to develop a prognostic model for 5-year OS, trained on 2702 tumours from 15 studies and evaluated on an independent set of 1067 tumours from six studies. Results: Expression levels of 276 genes were associated with OS (false discovery rate < 0.05) in covariate-adjusted single-gene analyses. The top five genes were TAP1, ZFHX4, CXCL9, FBN1 and PTGER3 (P < 0.001). The best performing prognostic signature included 101 genes enriched in pathways with treatment implications. Each gain of one standard deviation in the gene expression score conferred a greater than twofold increase in risk of death [hazard ratio (HR) 2.35, 95% confidence interval (CI) 2.02-2.71; P < 0.001]. Median survival [HR (95% CI)] by gene expression score quintile was 9.5 (8.3 to -), 5.4 (4.6-7.0), 3.8 (3.3-4.6), 3.2 (2.9-3.7) and 2.3 (2.1-2.6) years. Conclusion: The OTTA-SPOT (Ovarian Tumor Tissue Analysis consortium - Stratified Prognosis of Ovarian Tumours) gene expression signature may improve risk stratification in clinical trials by identifying patients who are least likely to achieve 5-year survival. The identified novel genes associated with the outcome may also yield opportunities for the development of targeted therapeutic approaches.

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.