High-grade Serous Ovarian Carcinoma Cases Research Articles

Evaluation of the prognostic potential of histopathological subtyping in high-grade serous ovarian carcinoma.

High-grade serous ovarian carcinoma (HGSOC) can be categorized into four gene expression-based subtypes, with supposedly distinct prognoses and treatment responses. Murakami et al. translated these gene expression-based subtypes into the histopathological mesenchymal, immunoreactive, solid and proliferative, and papilloglandular subtypes, showing differences in survival outcomes. Miyagawa et al. refined these criteria to improve the interobserver concordance. The current retrospective study evaluated the interobserver variability and the prognostic differences between the histopathologic subtypes using the criteria of both Murakami et al. and Miyagawa et al. in 208 HGSOC cases. The mesenchymal subtype was considered first, followed by the immunoreactive subtype. Non-conforming cases were categorized as solid and proliferative or papilloglandular. The mesenchymal subtype was identified in 122 patients (58.7%) for both criteria. Using the criteria of Murakami et al., 10 cases (4.8%) were immunoreactive, 26 (12.5%) solid and proliferative, and 50 (24%) papilloglandular, with a concordance rate of 62.5% (κ = 0.34, p < .001). Using the Miyagawa et al. criteria, 23 cases (11%) were immunoreactive, 20 (9.6%) solid and proliferative, and 43 (20.7%) papilloglandular. No survival differences were observed between the subtypes. The fair reproducibility of the histopathological subtype classification of HGSOC and the lack of survival differences among these subtypes indicate the need for further refinement of the criteria and exploration of their correlation with overall survival outcomes before clinical application.

Genetic characteristics of platinum-sensitive ovarian clear cell carcinoma.

Most ovarian clear cell carcinomas are resistant to platinum-based chemotherapy, while a small subset shows a positive response. The aim of this study was to clarify the clinical, pathological and genetic characteristics of platinum-sensitive ovarian clear cell carcinomas. The study included 53 patients with stage III-IV ovarian clear cell carcinoma who had residual tumours after primary surgery and received platinum-based therapy between 2009 and 2018. A retrospective examination of platinum sensitivity was performed using the criterion of ≥6months from the last day of first-line platinum therapy until recurrence/progression. Cases determined to be platinum-sensitive were subjected to immunohistochemical staining, genomic analyses using target sequencing (i.e. NCC Oncopanel) and homologous recombination deficiency (myChoice® HRD Plus) assays. Of the 53 stage III-IV ovarian clear cell carcinoma cases, 11 (21%) were platinum-sensitive. These cases showed better progression-free and overall survival than platinum-resistant cases (hazard ratio=0.16, P<0.001). Among the seven sensitive cases whose tumour tissues were available for molecular profiling, five were pure ovarian clear cell carcinoma based on pathological and genetic features, whereas the remaining two cases were re-diagnosed as high-grade serous ovarian carcinoma. The pure ovarian clear cell carcinomas lacked BRCA1 and BRCA2 mutations, consistent with the absence of the homologous recombination deficiency phenotype, whereas two cases (40%) had ATM mutations. By contrast, the two high-grade serous ovarian carcinoma cases had BRCA1 or BRCA2 mutations associated with the homologous recombination deficiency phenotype. The subset of platinum-sensitive ovarian clear cell carcinomas includes a majority with pure ovarian clear cell carcinoma features that lack the homologous recombination deficiency phenotype.

Molecular Genetic Characteristics of FANCI, a Proposed New Ovarian Cancer Predisposing Gene.

FANCI was recently identified as a new candidate ovarian cancer (OC)-predisposing gene from the genetic analysis of carriers of FANCI c.1813C>T; p.L605F in OC families. Here, we aimed to investigate the molecular genetic characteristics of FANCI, as they have not been described in the context of cancer. We first investigated the germline genetic landscape of two sisters with OC from the discovery FANCI c.1813C>T; p.L605F family (F1528) to re-affirm the plausibility of this candidate. As we did not find other conclusive candidates, we then performed a candidate gene approach to identify other candidate variants in genes involved in the FANCI protein interactome in OC families negative for pathogenic variants in BRCA1, BRCA2, BRIP1, RAD51C, RAD51D, and FANCI, which identified four candidate variants. We then investigated FANCI in high-grade serous ovarian carcinoma (HGSC) from FANCI c.1813C>T carriers and found evidence of loss of the wild-type allele in tumour DNA from some of these cases. The somatic genetic landscape of OC tumours from FANCI c.1813C>T carriers was investigated for mutations in selected genes, copy number alterations, and mutational signatures, which determined that the profiles of tumours from carriers were characteristic of features exhibited by HGSC cases. As other OC-predisposing genes such as BRCA1 and BRCA2 are known to increase the risk of other cancers including breast cancer, we investigated the carrier frequency of germline FANCI c.1813C>T in various cancer types and found overall more carriers among cancer cases compared to cancer-free controls (p = 0.007). In these different tumour types, we also identified a spectrum of somatic variants in FANCI that were not restricted to any specific region within the gene. Collectively, these findings expand on the characteristics described for OC cases carrying FANCI c.1813C>T; p.L605F and suggest the possible involvement of FANCI in other cancer types at the germline and/or somatic level.

Immunoexpression of p53 and p16 in Low and High-grade Serous Ovarian Cancer: A Cross-sectional Study

Introduction: The most common malignancy of the ovary is serous carcinoma, which can be classified as either Low-grade Serous Ovarian Carcinoma (LGSOC) or High-grade Serous Ovarian Carcinoma (HGSOC) and originates from the surface epithelium. However, the overall prognosis for both cancers is very poor. Immunohistochemical analysis of p53 and p16 expression is commonly used to detect mutations. Diffuse and strong mutations (mutant type) are almost always observed in cases of HGSOC, while focal expression (wild type) suggests the absence of mutations in HGSOC. LGSOCs are characterised by a low number or absence of genetic mutations. Aim: To investigate the association between p53 and p16 expression in different grades and stages of Serous Ovarian Carcinoma (SOC). Materials and Methods: This observational cross-sectional descriptive study was conducted on 62 patients diagnosed with ovarian serous carcinoma. The study focused on examining the expressions of p53 and p16 using Immunohistochemistry (IHC) in the Department of Pathology, Nil Ratan Sircar Medical College and Hospital, Kolkata, West Bengal, India, over a period of one and a half years (February 2021 to July 2022). The study parameters included clinical features, histological findings, staging, The Federation of Gynaecology and Obstetrics (FIGO), grading, p53 and p16 expression by IHC in all cases, and the association between p53 and p16 expression with the grade and stage of the cancer. Statistical analysis was performed using Analysis of Variance (ANOVA) and Statistical Package for Social Sciences (SPSS) software. A p-value of less than 0.05 was considered significant. Results: A total of 62 cases were included in the study, with 55 cases (88.70%) classified as LGSOC and 7 cases (11.29%) as HGSOC. The mean age for LGSOC was 53.5 years, while for HGSOC it was 54 years. Among the HGSOC cases (n=55), 45 cases (81.80%) showed diffuse positive results (mutant type) for p53. In contrast, there was no diffuse p53 expression in LGSOC cases (n=7), with 5 cases (71.40%) showing focal positivity (wild type). The p-value for comparing p53 expression in both cases was significant (&lt;0.00001). As for p16 expression, among the HGSOC cases (n=55), 31 cases (56.40%) showed diffuse positivity (mutant type), while among the LGSOC cases (n=7), most of the cases, 5 cases (71.40%), showed focal positivity (wild type). The p-value for comparing p16 expression in both cases was significant (&lt;0.003794). Conclusion: In conclusion, p53 along with p16 are good markers for grading SOC, and p53 is highly effective in differentiating HGSOC from LGSOC based on the positivity pattern (diffuse and strong positive for high-grade/mutant type, and focal positive for low-grade cancers). Thus, p53 has become an attractive target for the development of moleculetargeted therapies for this disease.

TP53 mutations in Pap test DNA are a potential biomarker of ovarian cancer risk in high-risk women (226)

<b>Objectives:</b> The use of Pap tests as a molecular diagnostic for high-grade serous ovarian carcinoma (HGSOC) is a promising concept, but studies to detect tumor DNA report limited sensitivity. <i>TP53</i> somatic mutations from clonal expansions in non-neoplastic tissue can be detected in Pap tests and blood and could potentially be utilized for cancer risk assessment. We aimed to determine whether <i>TP53</i> somatic mutations in blood and Pap test DNA are associated with HGSOC in women with and without germline <i>BRCA1</i> or <i>BRCA2 (BRCA)</i> mutations. <b>Methods:</b> Peripheral blood and Pap samples were collected from 30 women with HGSOC (10 with a germline <i>BRCA</i> mutation) and 40 women without cancer (20 with a germline <i>BRCA</i> mutation) who underwent gynecologic surgery at the University of Washington. Genomic DNA from buffy coats and Pap cell pellets was used for ultra-deep sequencing of the <i>TP53</i> coding region (mean depth ~3300x) using duplex sequencing for error correction. Mutations were characterized based on pathogenicity and presence in the UMD <i>TP53</i> Cancer Database. Mutation frequencies (total coding mutations divided by nucleotides sequenced) were compared between groups using t-tests. Fitted linear regression models were used to explore age-related trends, and logistic regression models were used to predict cancer with adjustments for age. <b>Results:</b> Total 436 coding <i>TP53</i> mutations were identified in DNA from Pap tests, and 262 mutations were identified in the DNA from blood. Pap tests demonstrated an overall higher <i>TP53</i> mutation frequency in patients with HGSOC than in those without cancer (2.0x10^-6 vs 1.4x10^-6, p=0.03), but this effect was driven by and limited to patients with <i>BRCA</i> mutations (2.4x10^-6 vs 1.4x10^-6, p=0.01). Women with <i>BRCA</i> mutations and HGSOC also demonstrated a greater frequency of mutations affecting the DNA-binding domain, predicted to be pathogenic, and frequently found in cancer (referred to as ‘cancerlike') (all p<0.05). Cancer-like mutations increased with age in Pap test samples from women without cancer (p<0.01, R²=0.23) but not in patients with HGSOC. Age-adjusted logistic regression confirmed that cancer-like <i>TP53</i> mutation frequency was associated with HGSOC in patients with germline <i>BRCA</i> mutations (OR: 4.2, 95% CI: 1.2-14.3). There were no significant differences between HGSOC and non-cancer cases when examining <i>TP53</i> mutations in blood samples. Among 22 cases with known <i>TP53</i> tumor mutations, the tumor mutation was identified in six Pap samples (27%) and zero blood samples. <b>Conclusions:</b> The identification of HGSOC-specific mutations in Pap tests, even with highly accurate DNA sequencing methods, has limited sensitivity for cancer detection. However, Pap tests can detect abundant low-frequency cancer-like <i>TP53</i> mutations at a higher rate in women with germline <i>BRCA</i> mutations and HGSOC compared to women without cancer. Somatic <i>TP53</i> mutation frequency using age-related thresholds should be further explored as a biomarker for ovarian cancer risk in high-risk women.

Evaluation of TP53 mutations in Pap test and blood DNA as novel biomarkers of ovarian cancer risk

<h3>Objectives:</h3> The use of Pap tests as a molecular diagnostic for high-grade serous ovarian carcinoma (HGSC) is a promising concept, but studies to detect tumor DNA report limited sensitivity. ‘Cancer-like' somatic mutations from clonal expansions in non-neoplastic tissue can be detected in Pap tests and blood, and could potentially be utilized for cancer risk-assessment. We aimed to determine whether <i>TP53</i> somatic mutations in blood and Pap test DNA are associated with HGSC or with inherited ovarian cancer susceptibility. <h3>Methods:</h3> Peripheral blood and Pap samples were collected from 26 women with HGSC (10 with germline <i>BRCA1</i> or <i>BRCA2</i> [<i>BRCA</i>] mutations) and 35 women without cancer (17 with germline <i>BRCA</i> mutations), all of whom underwent gynecologic surgery at a single academic institution. Blood samples alone were available for an additional four women. DNA was extracted from the blood buffy coat layer and Pap samples. The coding region of <i>TP53</i> was deeply sequenced (mean depth ~3800x) using duplex sequencing, an ultra-accurate error-correction sequencing approach. Mutations frequently found in the UMD <i>TP53</i> Cancer Database were termed ‘cancer-like'. Mutation frequencies (total coding mutations divided by nucleotides sequenced, MF) were compared between groups using t-tests, restricted to those between the ages of 40 and 60 for improved age-matching. Age-related trends were explored with fitted linear regression models. <h3>Results:</h3> 572 coding-region <i>TP53</i> mutations were identified in DNA from Pap tests from 26 women with HGSC (300 mutations) and 35 women without cancer (272 mutations). 287 mutations were identified in the DNA from blood buffy coat from 29 women with HGSC (161 mutations) and 36 without cancer (226 mutations). DNA from Pap tests demonstrated a higher MF in patients with HGSC than those without cancer (2.5x10^-6 vs. 1.6x10^-6, p=0.03). Cancer-like MF was also significantly higher in patients with HGSC (8.8x10^-7 vs. 5.0x10^-7, p=0.01). There were no significant differences in MF amongst cases without cancer with or without germline <i>BRCA</i> mutations, or amongst cases without cancer in those with <i>BRCA1</i> vs <i>BRCA2</i> germline mutations. Cancer-like mutations increased with age in Pap test samples from women without cancer (p<0.01, R2 0.34) but not in patients with HGSC (p=0.58, R2 0.01). In DNA from blood, there were no significant differences in average MF between HGSC and non-cancer cases nor between <i>BRCA</i> wildtype and mutant cases. No age-related correlations were found. Among twelve cases where the tumor mutation was known, duplex sequencing identified the mutation in five Pap samples (42%) and zero blood samples. <h3>Conclusions:</h3> Identifying the tumor-specific mutation in Pap tests, even with highly accurate DNA sequencing methods, does not reliably detect ovarian cancer. However, Pap tests carry abundant low frequency cancer-like <i>TP53</i> mutations, which are found in higher rates in women with HGSC than without. In patients without cancer, these <i>TP53</i> mutations increase linearly with age. Somatic <i>TP53</i> mutation frequency using age-related thresholds should be further explored as a biomarker for ovarian cancer risk.

Oncogenic B-Myb Is Associated With Deregulation of the DREAM-Mediated Cell Cycle Gene Expression Program in High Grade Serous Ovarian Carcinoma Clinical Tumor Samples.

Cell cycle control drives cancer progression and treatment response in high grade serous ovarian carcinoma (HGSOC). MYBL2 (encoding B-Myb), an oncogene with prognostic significance in several cancers, is highly expressed in most HGSOC cases; however, the clinical significance of B-Myb in this disease has not been well-characterized. B-Myb is associated with cell proliferation through formation of the MMB (Myb and MuvB core) protein complex required for transcription of mitotic genes. High B-Myb expression disrupts the formation of another transcriptional cell cycle regulatory complex involving the MuvB core, DREAM (DP, RB-like, E2F, and MuvB), in human cell lines. DREAM coordinates cell cycle dependent gene expression by repressing over 800 cell cycle genes in G0/G1. Here, we take a bioinformatics approach to further evaluate the effect of B-Myb expression on DREAM target genes in HGSOC and validate our cellular model with clinical specimens. We show that MYBL2 is highly expressed in HGSOC and correlates with expression of DREAM and MMB target genes in both The Cancer Genome Atlas (TCGA) as well as independent analyses of HGSOC primary tumors (N = 52). High B-Myb expression was also associated with poor overall survival in the TCGA cohort and analysis by a DREAM target gene expression signature yielded a negative impact on survival. Together, our data support the conclusion that high expression of MYBL2 is associated with deregulation of DREAM/MMB-mediated cell cycle gene expression programs in HGSOC and may serve as a prognostic factor independent of its cell cycle role. This provides rationale for further, larger scale studies aimed to determine the clinical predictive value of the B-Myb gene expression signature for treatment response as well as patient outcomes.

Kinome capture sequencing of high-grade serous ovarian carcinoma reveals novel mutations in the JAK3 gene.

High-grade serous ovarian carcinoma (HGSOC) remains the deadliest form of epithelial ovarian cancer and despite major efforts little improvement in overall survival has been achieved. Identification of recurring “driver” genetic lesions has the potential to enable design of novel therapies for cancer. Here, we report on a study to find such new therapeutic targets for HGSOC using exome-capture sequencing approach targeting all kinase genes in 127 patient samples. Consistent with previous reports, the most frequently mutated gene was TP53 (97% mutation frequency) followed by BRCA1 (10% mutation frequency). The average mutation frequency of the kinase genes mutated from our panel was 1.5%. Intriguingly, after BRCA1, JAK3 was the most frequently mutated gene (4% mutation frequency). We tested the transforming properties of JAK3 mutants using the Ba/F3 cell-based in vitro functional assay and identified a novel gain-of-function mutation in the kinase domain of JAK3 (p.T1022I). Importantly, p.T1022I JAK3 mutants displayed higher sensitivity to the JAK3-selective inhibitor Tofacitinib compared to controls. For independent validation, we re-sequenced the entire JAK3 coding sequence using tagged amplicon sequencing (TAm-Seq) in 463 HGSOCs resulting in an overall somatic mutation frequency of 1%. TAm-Seq screening of CDK12 in the same population revealed a 7% mutation frequency.Our data confirms that the frequency of mutations in kinase genes in HGSOC is low and provides accurate estimates for the frequency of JAK3 and CDK12 mutations in a large well characterized cohort. Although p.T1022I JAK3 mutations are rare, our functional validation shows that if detected they should be considered as potentially actionable for therapy. The observation of CDK12 mutations in 7% of HGSOC cases provides a strong rationale for routine somatic testing, although more functional and clinical characterization is required to understand which nonsynonymous mutations alterations are associated with homologous recombination deficiency.

Abstract A49: Homologous recombination deficiency status-based classification of high-grade serous ovarian carcinoma

Abstract Background: Homologous recombination repair (HRR) pathway deficiency (HRD) is deeply involved in the tumorigenesis and progression of high-grade serous ovarian carcinoma (HGSOC) as well as in the sensitivity to platinum chemotherapy drugs. Until now, the proposed cutoff value for the HRD score, determined by calculating genomic scar signature scores associated with HRD, namely, the loss of heterozygosity (LOH), telomeric allelic imbalance (TAI), and large-scale state transitions (LST), has been ≥ 42 based on a combined dataset of HGSOC and breast cancer. In this study, we propose a rational method to classify HGSOC based on HRD status and investigated its clinical significance. Methods: We obtained data from The Cancer Genome Atlas (TCGA) on HGSOC and determined the HRD score as the sum of LOH, TAI, and LST scores. We then investigated the relationships among the score, genetic alterations in HRR-related genes, and the clinical data. Results: BRCA1/2 mutations were enriched in the group with HRD scores ≥ 63. BRCA1/2 mutations represented 38% of the cases with HRD scores ≥ 63 (49/128), 10% with HRD scores between 42 and 62 (12/118), and 10% with HRD scores 41 and below (5/50); no enrichment of BRCA1/2 mutation cases with HRD scores from 42 to 62 was observed compared with those with scores ≤ 41. Compared with the groups with scores ≤ 62, this group had a good prognosis (p &amp;lt; 0.0001); we thus considered HRD scores ≥ 63 to be the best cutoff point for identifying HRD cases in HGSOC. Among the other HRR pathway gene mutations, only CHEK1 homozygous deletions and PTEN homozygous deletions were associated with high HRD scores (p = 0.0038 and p = 0.035, respectively). In terms of promoter methylation, BRCA1 methylations and RAD51C methylations were associated with high HRD scores (p &amp;lt; 0.0001 and p = 0.029, respectively). Classification of HGSOC cases by the HRD status revealed a better prognosis for HRD cases caused by genetic alterations (HRD-G) than those caused by epigenetic changes (HRD-E), those caused by undetermined reasons (HRD-U), and non-HRD cases (p &amp;lt; 0.0001). A combined analysis of HRD status and residual tumor after primary debulking surgery revealed that, among cases without macroscopic residual tumors, 11 of 12 HRD-G cases survived after the median observation period of 6.6 years, showing remarkably higher survival rates than HRD-E, HRD-U, and non-HRD cases (p = 0.0059). Conclusion: HRD score ≥ 63 is an optimal cut-off to define HRD in HGSOC. HGSOC can be classified into subtypes with different prognoses according to HRD status. This classification could be useful for personalized HGSOC treatment. Citation Format: Noriomi Matsumura, Hisamitsu Takaya, Shiro Takamatsu, Hidekatsu Nakai. Homologous recombination deficiency status-based classification of high-grade serous ovarian carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A49.

Abstract IA02: DNA repair gene promoter methylation patterns adapt and influence PARP inhibitor response

Abstract PARP inhibitor (PARPi) resistance in high-grade serous ovarian carcinoma (HGSOC) can be acquired as a result of restored homologous recombination (HR) due to secondary or reversion mutations in HR genes, such as BRCA1, BRCA2, and RAD51C, or due to loss of BRCA1 promoter methylation (meBRCA1). We have demonstrated that homozygous meBRCA1 can be lost or reverted to heterozygous methylation following treatment with platinum-based chemotherapy, resulting in HR-competent PARPi-resistant tumors. RAD51C promoter methylation (meRAD51C) is detected in approximately 2% of HGSOC cases and, as with meBRCA1, is associated with gene silencing and HR deficiency. We are exploring PARPi response in meRAD51C preclinical models to determine clinical relevance. Two patient-derived xenograft (PDX) models of HGSOC with RAD51C gene silencing caused by meRAD51C have distinct meRAD51C profiles (measured by methylation-specific high-resolution melt analysis and targeted bisulfite next-generation sequencing), and different responses to PARPi treatment pressure. PDX PH039 loses methylation and regains RAD51C expression after only 2 cycles of PARPi retreatment (niraparib), resulting in PARPi-refractory tumors by cycle 3-4. Illumina EPIC methylation array analysis of PH039 revealed increasing global methylation losses following each round of PARPi treatment. Lack of meRAD51C stability and rapid development of PARPi resistance in PH039 may be due to the high degree of meRAD51C heterogeneity within the tumor favoring selection of pre-existing HR-competent clones under PARPi pressure. In contrast, PDX 183 has a relatively homogeneous and stable meRAD51C profile. We have multiple examples of using unique PDX models to demonstrate various important features of PARPi resistance, including loss of promoter methylation for BRCA1 vs. RAD51C. Thus, meRAD51C confers response to PARPi in HGSOC, but PARPi treatment pressure can cause loss of methylation and drug resistance in some tumors. The contrasting PARPi responses of these PDX provide a platform for the study of meRAD51C stability in vivo and may present therapeutic opportunities to improve meRAD51C durability and PARPi responses in patients. Citation Format: Kasenija Nesic, Rachel M Hurley, Cordelia McGehee, Olga Kondrashova, Maria I. Harrell, Giada V. Zapparoli, Ashan Musafer, Ming E. Wong, John Weroha, Xiaonan Hou, Hu Li, Vivian Negron, Kevin Peterson, Paula Schneider, Elizabeth M. Swisher, Melissa Southey, Alexander Dobrovic, Matthew Wakefield, Scott H. Kaufmann, Clare L. Scott. DNA repair gene promoter methylation patterns adapt and influence PARP inhibitor response [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr IA02.

Neoantigen load and HLA-class I expression identify a subgroup of tumors with a T-cell-inflamed phenotype and favorable prognosis in homologous recombination-proficient high-grade serous ovarian carcinoma

BackgroundThere is increasing evidence for the benefit of poly ADP ribose polymerase (PARP) inhibitors in a subset of high-grade serous ovarian carcinoma (HGSC) patients, especially those with homologous recombination (HR)-deficient...

Immunohistochemical expression of p53 in serous carcinoma of ovary and its correlation with clinicopathological parameters

Immunohistochemical expression of p53 in serous carcinoma of ovary and its correlation with clinicopathological parameters - IJPO- Print ISSN No: - 2394-6784 Online ISSN No:- 2394-6792 Article DOI No:- 10.18231/j.ijpo.2020.007, Indian Journal of Pathology and Oncology-Indian J Pathol Oncol

Homologous recombination deficiency status-based classification of high-grade serous ovarian carcinoma

Homologous recombination repair (HRR) pathway deficiency (HRD) is involved in the tumorigenesis and progression of high-grade serous ovarian carcinoma (HGSOC) as well as in the sensitivity to platinum chemotherapy drugs. In this study, we obtained data from The Cancer Genome Atlas (TCGA) on HGSOC and identified scores for the loss of heterozygosity, telomeric allelic imbalance, and large-scale state transitions, and calculated the HRD score. We then investigated the relationships among the score, genetic/epigenetic alterations in HRR-related genes, and the clinical data. We found that BRCA1/2 mutations were enriched in the group with HRD scores ≥63. Compared with the groups with scores ≤62, this group had a good prognosis; we thus considered HRD scores ≥63 to be the best cutoff point for identifying HRD cases in HGSOC. Classification of HGSOC cases by the HRD status revealed a better prognosis for HRD cases caused by genetic alterations (genetic HRD) than those caused by epigenetic changes and those caused by undetermined reasons (p = 0.0002). Among cases without macroscopic residual tumors after primary debulking surgery, 11 of 12 genetic HRD cases survived after the median observation period of 6.6 years, showing remarkably high survival rates (p = 0.0059). In conclusion, HGSOC can be classified into subtypes with different prognoses according to HRD status. This classification could be useful for personalized HGSOC treatment.

Abstract AP09: COPY-NUMBER SIGNATURES AND MUTATIONAL PROCESSES IN HIGH GRADE SEROUS OVARIAN CARCINOMA

Abstract BACKGROUND: The genomic complexity of profound copy-number aberration has prevented effective molecular stratification of high grade serous ovarian carcinoma (HGSOC). Recent algorithmic advances have enabled interpretation of complex genomic changes by identifying mutational signatures—genomic patterns that are the imprint of mutagenic processes accumulated over the lifetime of a cancer cell. We hypothesized that specific features of copy-number (CN) abnormalities could represent the imprints of distinct mutational processes, and developed methods to identify signatures from copy-number features in HGSOC. METHODS: We derived copy-number signatures from absolute copy number profiles from 253 primary and relapsed HGSOC samples from 132 patients in the BriTROC-1 cohort using low-cost shallow whole-genome sequencing (sWGS; 0.1×). A subset of 56 of these cases had deep whole-genome sequencing (dWGS) performed for mutation analysis and comparison with sWGS data. Independent validation was performed using 112 dWGS HGSOC cases from PCAWG and 415 HGSOC cases with SNP array and whole exome sequence from TCGA. CN signature exposures were correlated with mutation data, SNV signatures, and other measures derived from deep WGS and exome sequencing to identify statistically significant genomic associations using a false discovery rate &amp;lt;0.05. RESULTS: We identified 7 CN signatures that provided a molecular framework to rederive the major defining elements of HGSOC genomes, including defective homologous recombination (HRD), tandem duplication, amplification of CCNE1 and amplification-associated fold-back inversions. Almost all patients with HGSOC demonstrated a mixture of signatures indicative of combinations of mutational processes, including those with early driver events such as BRCA2 mutation (in addition to HRD signatures). High exposure to CN signature 3, characterised by BRCA1/2-related HRD, was associated with improved overall survival. Conversely, high exposure to signature 1, which was characterised by oncogenic RAS signaling (including NF1, KRAS and NRAS mutation), predicted platinum-resistant relapse and poor survival. CONCLUSIONS: HGSOC lacks clinically-relevant patient stratification, which is reflected in poor survival and is a significant barrier to precision medicine. Copy-number signature exposures at diagnosis predict both overall survival and the probability of platinum-resistant relapse. Our results suggest that early TP53 mutation, the ubiquitous initiating event in HGSOC, may permit multiple mutational processes to co-evolve, potentially simultaneously and that additional signature exposures may alter the risk of developing therapeutic resistance. Thus, our results suggest that HGSOC is a continuum of genomes. We derived signatures using inexpensive sWGS of DNA from core biopsies. These approaches are rapid and cost effective, thus providing a clear path to clinical implementation. By dissecting the mutational forces shaping HGSOC genomes, our study paves the way to understanding extreme genomic complexity, as well as revealing the evolution of tumors as they relapse and acquire resistance to therapy. Citation Format: Geoff Macintyre, Teodora E. Goranova, Dilrini De Silva, Darren Ennis, Anna M. Piskorz, Matthew Eldridge, Daoud Sie, Liz-Anne Lewsley, Aishah Hanif, Cheryl Wilson, Suzanne Dowson, Rosalind M. Glasspool, Michelle Lockley, Elly Brockbank, Ana Montes, Axel Walther, Sudha Sundar, Richard Edmondson, Geoff D. Hall, Andrew Clamp, Charlie Gourley, Marcia Hall, Christina Fotopoulou, Hani Gabra, James Paul, Anna Supernat, David Millan, Aoisha Hoyle, Gareth Bryson, Craig Nourse, Laura Mincarelli, Luis Navarro Sanchez, Bauke Ylstra, Mercedes Jimenez-Linan, Luiza Moore, Oliver Hofmann, Florian Markowetz, Iain A. McNeish, James D. Brenton. COPY-NUMBER SIGNATURES AND MUTATIONAL PROCESSES IN HIGH GRADE SEROUS OVARIAN CARCINOMA [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr AP09.

Abstract GMM-053: ROLE OF LATS AND DYRK1A KINASES IN THE PATHOGENESIS AND TREATMENT OF HGSOC

Abstract Epithelial ovarian cancer (EOC) is the most lethal gynecological malignancy, with 75% of women presenting with metastatic disease at the time of diagnosis. High Grade Serous Ovarian Carcinoma (HGSOC) is the most common and aggressive type of epithelial ovarian cancer, with 5-year survival rate of 30% for most patients. Developing of accurate experimental models of HGSOC is necessary to elucidate the disease pathogenesis and to evaluate new treatments. Our study is aimed at determining the roles of the Large Tumor Suppressor 1 and 2 kinases (LATS1/2), and their downstream effector Dual-specificity tyrosine(Y)-Regulated Protein Kinase 1A (DYRK1A), in HGSOC pathogenesis and treatment sensitivity. The Cancer Genome Atlas study revealed copy number losses of the LATS1, LATS2 and DYRK1A genes in 65%, 59% and 38% of HGSOC cases, respectively. Previous studies found that LATS1/2 and DYRK1A mediate a crosstalk between the Hippo tumor suppressor pathway and the G0/G1 cell cycle checkpoint. However, the role of these kinases in HGSOC pathogenesis is not known. We found that loss of both LATS1 and LATS2, or DYRK1A results in increased proliferation of SKOV3 ovarian cancer cells in 2D or 3D cultures, mediated in part by disruption of the DREAM complex that represses cell cycle-regulated genes. Interestingly, we observed upregulation of cyclin D1 and increased phosphorylation of retinoblastoma (RB) family proteins in SKOV3 cells depleted of LATS1/2 or DYRK1A kinases, indicative of aberrant activation of CDK4/6. Similar changes were observed upon depletion of LATS1/2 or DYRK1A using non-transformed human fallopian epithelial cells, suggesting that genetic losses of these kinases could contribute to the HGSOC pathogenesis. Furthermore, we tested whether an aberrant activation of CDK4/6 in SKOV3 cells with low levels of LATS1/2 or DYRK1A could increase their sensitivity to pharmacological CDK4/6 inhibitors such as palbociclib. Using metabolic cell proliferation assays, we found that loss of LATS1/2 or DYRK1A resulted in a significant decrease of palbociclib IC50 in SKOV3 cells. Interestingly, palbociclib treatment increased the DREAM assembly in these cells, resulting in downregulation of DREAM-regulated cell cycle factors such as cyclin A or B-Myb. Our results show that loss of LATS1/2 or DYRK1A could contribute to HGSOC pathogenesis by CDK4/6-mediated disruption of RB family function. Further studies are underway to validate our findings in vivo using orthotopic ovarian tumor xenografts in mice and a bioluminescence imaging approach. In addition, these models will be used for validation of novel therapeutic approaches for HGSOC, such as palbociclib, as well as for the mechanistic studies of the EOC pathogenesis. Citation Format: Fatmata Sesay, Siddharth Saini, Larisa Litovchick. ROLE OF LATS AND DYRK1A KINASES IN THE PATHOGENESIS AND TREATMENT OF HGSOC [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr GMM-053.

Clinical and molecular characterization of ovarian carcinoma displaying isolated lymph node relapse

BackgroundDisease relapse is the primary cause of death from ovarian carcinoma. Isolated lymph node relapse is a rare pattern of ovarian carcinoma recurrence, with a reported median postrelapse survival of 2.5 to 4 years. To date, investigations have not compared isolated lymph node relapse ovarian carcinoma directly to a matched extranodal relapse cohort or performed molecular characterization of cases that subsequently experience isolated lymph node relapse.ObjectiveHere we seek to compare the clinical outcome, tumor-infiltrating lymphocyte burden, and frequency of known prognostic genomic events in isolated lymph node relapse ovarian carcinoma vs extranodal relapse ovarian carcinoma.Study DesignForty-nine isolated lymph node relapse ovarian carcinoma patients were identified and matched to 49 extranodal relapse cases using the Edinburgh Ovarian Cancer Database, from which the clinical data for identified patients were retrieved. Matching criteria were disease stage, histologic subtype and grade, extent of residual disease following surgical debulking, and age at diagnosis. Clinicopathologic factors and survival data were compared between the isolated lymph node relapse and extranodal relapse cohorts. Genomic characterization of tumor material from diagnosis was performed using panel-based high-throughput sequencing and tumor-infiltrating T cell burden was assessed using immunohistochemistry for CD3+ and CD8+ cells.ResultsIsolated lymph node relapse cases demonstrated significantly prolonged postrelapse survival and overall survival vs extranodal relapse upon multivariable analysis (HRmulti = 0.52 [0.33–0.84] and 0.51 [0.31–0.84]). Diagnostic specimens from high-grade serous ovarian carcinomas that subsequently displayed isolated lymph node relapse harbored significantly greater CD3+ and CD8+ cell infiltration compared to extranodal relapse cases (P = .001 and P = .009, Bonferroni-adjusted P = .003 and P = .019). Isolated lymph node relapse high-grade serous ovarian carcinoma cases did not show marked enrichment or depletion of cases with BRCA1/2 mutation or CCNE1 copy number gain when compared to their extranodal relapse counterparts (24.4% vs 19.4% and 18.2% vs 22.6%, P = .865 and P = .900).ConclusionIsolated lymph node relapse ovarian carcinoma represents a distinct clinical entity with favorable outcome compared to extranodal relapse. There was no clear enrichment or depletion of BRCA1/2 mutation or CCNE1 gain in the isolated lymph node relapse ovarian carcinoma cohort compared with extranodal relapse cases, suggesting that these known prognostic genomically defined subtypes of disease do not display markedly altered propensity for isolated lymph node relapse. Diagnostic tumor material from isolated lymph node relapse patients demonstrated greater CD3+ and CD8+ cell infiltration, indicating stronger tumor engagement by T cell populations, which may contribute to the more indolent disease course of isolated lymph node relapse.

Abstract 2017: Phenotypic plasticity and class switching in ovarian cancer

Abstract The multifaceted evolution of cancer involves differential responses of tumor cell populations to intrinsic and extrinsic stimuli. Numerous reports have examined the role of epithelial-mesenchymal transition (EMT) in the progression of high-grade serous ovarian carcinoma (HGSC). An earlier study from our group successfully resolved molecular subclasses in HGSC wherein metastasis was mediated by cooperative cell migration in the epithelial subclass and EMT in the mesenchymal subclass. We now explore the transcriptional circuitry in expression datasets and cell lines regulating these states. This led to the identification that Tcf21 and Slug expression correlated with the epithelial and mesenchymal phenotypes, respectively, and inversely correlated with each other. Probing the differential altered localization and expression of TCF21 and Slug further resolved a spectrum of intermediary phenotypic states along the epithelial to mesenchymal spectrum, each of which is associated with the expression of specific markers. Further, functional assays reveal the inherent plasticity of each phenotype with migratory mechanisms emerging as a differential parameter. Rigidity and high degree of differentiation of the epithelial and mesenchymal state is evident from a lack of responsiveness to specific microenvironmental conditions. Importantly, such cellular plasticity induced shifts from a steady-state phenotype following modulation of the microenvironment only in the intermediate phenotypes to exhibit flexibility to transit towards either the two ends of the spectrum. Specifically, a tendency to acquire an epithelial phenotype on growth factor deprivation was noted. The effects of BMP7 and TGFβ to induce epithelial or mesenchymal phenotypes, respectively, were also noted exclusively within the intermediary states. The stress generated by chemotherapeutic agents like paclitaxel led to enrichment of and dominant effects of the epithelial state. We further queried if this plasticity is also a feature of HGSC tumors. Marker expression (Tcf21, E-cadherin, Parp1, Slug, Anxa2 and Hyaluronan) was scored based on their frequency, intensity and location in tumors to derive a definitive scoring system comprising biomarker and tumor class indices. Application of such scoring could effectively stratifiy 95 human HGSC cases comprising primary and metastatic tumors into epithelial and mesenchymal classes, and further resolve a third class likely to represent the intermediate states. A salient feature noted was that metastases and chemotherapy could lead to class switching. Our study thus provides a comprehensive view of the intrinsic and extrinsic molecular events governing cellular plasticity in HGSC. Citation Format: Sharmila A. Bapat, Sagar Varankar, Swapnil Kamble. Phenotypic plasticity and class switching in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2017.

Abstract 5435: Role of the WT1 transcription factor in high-grade serous ovarian cancer

Abstract The Wilms Tumor protein 1 (WT1) transcription factor (TF) is a ubiquitous biomarker of high-grade serous ovarian carcinoma (HGSOC), which is the most common and aggressive subtype of epithelial ovarian cancer (EOC). This TF interacts with a host of cellular factors including the tumor suppressor P53, which is mutated in virtually all HGSOC cases. Studies have suggested a role for WT1 in HGSOC development, but little is known about the underlying mechanism. We have generated unique catalogues of epigenomic landscapes (specifically H3K27ac) in primary HGSOC using chromatin immunoprecipitation sequencing (ChIP-seq). Using Bayesian analyses of H3K27ac profiling in HGSOCs, we identified significant tissue specific enrichment of active chromatin at the WT1 locus in HGSOC when compared to all other EOC tumor histotypes. Furthermore, this locus contains a superenhancer in HGSOCs and in normal fallopian tube secretory epithelial cells (FTSECs). This locus features a bidirectional promoter that is shared by WT1-AS lncRNA and that contains a WT1 binding site. LncRNAs are essential in many cellular processes, and have been shown to play pivotal roles in tumorigenicity. The WT1-AS lncRNA is implicated in other diseases but little is known of its function in EOC. We have investigated the mechanisms of interaction between WT1 and WT1-AS. We observed that both protein and transcript expression for WT1 and WT1-AS are positively correlated in HGSOC cell lines. Analysis of data for &amp;gt;400 primary HGSOCs from TCGA further supported this correlation. We hypothesized that WT1 is a critical TF in the development of HGSOC, and that WT1-AS plays a key role in its regulatory pathway. We evaluated the regulatory role of WT1 first by analyzing cellular phenotypes and target gene expression changes in HGSOC cell lines after shRNA mediated knockdown (KD) of WT1. HGSOC cells with stable WT1 knockdown had impaired proliferation and concomitant reduction of WT1-AS expression. We also identified a decrease in expression of SHMT1 in WT1 KD cells, which is a known WT1 target gene. Integrated genome-wide analysis of the WT1 transcriptome by RNA-seq and the WT1 cistrome by ChIP-seq, in parallel with ChIP-MS, is being used to establish the architecture of the WT1 regulome, specifically to establish the regulatory mechanisms and relationships between WT1 and WT1-AS. Ultimately, these studies aim to establish the mechanisms of action for the WT1 TF in neoplastic transformation of HGSOC. Citation Format: Norma I. Rodriguez-Malave, Justyna E. Kanska, Kevin C. Vavra, Dennis Hazelett, Rosario I. Corona, Ji-Heui Seo, Matthew Freedman, Simon R. Knott, Sandra Orsulic, Beth Y. Karlan, Kate Lawrenson, Simon A. Gayther. Role of the WT1 transcription factor in high-grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5435.

Identification of candidate genes associated with tubal origin of high-grade serous ovarian cancer.

Evidence indicates that high-grade serous ovarian carcinoma arises from the fallopian tube, rather than ovarian surface epithelium. This is termed the ‘tubal origin’ theory. The aim of the present study was to compare the immunophenotype and gene expression profiling among high-grade serous ovarian carcinoma (HGSOC), fallopian tube epithelium (FTE) and ovarian surface epithelium (OSE) based on tubal origin theory, and identify the differential genes associated with ovarian carcinogenesis. A total of 61 cases of fresh tissue samples including 21 cases of HGSOC, 20 cases of OSE, and 20 cases of FTE were obtained following surgical resection. Immunostaining was performed to detect the expression of PAX8, which has been considered as a potential immunophenotype marker of Müllerian origin. Illumina BeadChip was applied for gene expression profiling. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to confirm the differential expression of candidate genes between HGSOC and FTE. The results of the present study demonstrated that PAX8 was highly expressed in HGSOC (19/21, 90.4%) and FTE (20/20, 100%), but not in OSE (3/20, 14.3%). A dendrogram generated by cluster analysis indicated a higher similarity of gene expression profile between HGSOC and FTE than OSE. A total of 2,412 differentially expressed genes were identified (absolute fold change >2) between HGSOC and FTE, including 822 upregulated genes in cancer and 1,590 downregulated genes. S100 calcium binding protein P, Ras-interacting protein 1, Wnt family member 5A, tumor-associated calcium signal transducer 2, Dickkopf Wnt signaling pathway inhibitor 3 and tumor suppressor candidate 3 genes were identified as candidate markers, of which the differential gene expression in HGSOC and FTE was confirmed by RT-qPCR (P<0.05). The results indicate the presence of a greater similarity in the immunophenotype and gene expression profile of HGSOC and FTE, when compared with OSE, which was consistent with the tubal origin theory of HGSOC.

Aurora Kinase A expression predicts platinum-resistance and adverse outcome in high-grade serous ovarian carcinoma patients.

High-Grade Serous Ovarian Carcinoma (HGSOC) is the predominant histotype of epithelial ovarian cancer (EOC), characterized by advanced stage at diagnosis, frequent TP53 mutation, rapid progression, and high responsiveness to platinum-based-chemotherapy. To date, standard first-line-chemotherapy in advanced EOC includes platinum salts and paclitaxel with or without bevacizumab. The major prognostic factor is the response duration from the end of the platinum-based treatment (platinum-free interval) and about 10–0 % of EOC patients bear a platinum-refractory disease or develop early resistance (platinum-free interval shorter than 6 months). On these bases, a careful selection of patients who could benefit from chemotherapy is recommended to avoid unnecessary side effects and for a better disease outcome. In this retrospective study, an immunohistochemical evaluation of Aurora Kinase A (AURKA) was performed on 41 cases of HGSOC according to platinum-status. Taking into account the number and intensity of AURKA positive cells we built a predictive score able to discriminate with high accuracy platinum-sensitive patients from platinum-resistant patients (p < 0.001). Furthermore, we observed that AURKA overexpression correlates to worse overall survival (p = 0.001; HR 0.14). We here suggest AURKA as new effective tool to predict the biological behavior of HGSOC. Particularly, our results indicate that AURKA has a role both as predictor of platinum-resistance and as prognostic factor, that deserves further investigation in prospective clinical trials. Indeed, in the era of personalized medicine, AURKA could assist the clinicians in selecting the best treatment and represent, at the same time, a promising new therapeutic target in EOC treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s13048-016-0238-7) contains supplementary material, which is available to authorized users.