Australian Ovarian Cancer Study Research Articles

Mathematical modeling of the evolution of resistance and aggressiveness of high-grade serous ovarian cancer from patient CA-125 time series.

A time-series analysis of serum Cancer Antigen 125 (CA-125) levels was performed in 791 patients with high-grade serous ovarian cancer (HGSOC) from the Australian Ovarian Cancer Study to evaluate the development of chemoresistance and response to therapy. To investigate chemoresistance and better predict the treatment effectiveness, we examined two traits: resistance (defined as the rate of CA-125 change when patients were treated with therapy) and aggressiveness (defined as the rate of CA-125 change when patients were not treated). We found that as the number of treatment lines increases, the data-based resistance increases (a decreased rate of CA-125 decay). We use mathematical models of two distinct cancer cell types, treatment-sensitive cells and treatment-resistant cells, to estimate the values and evolution of the two traits in individual patients. By fitting to individual patient HGSOC data, our models successfully capture the dynamics of the CA-125 level. The parameters estimated from the mathematical models show that patients with inferred low growth rates of treatment-sensitive cells and treatment-resistant cells (low model-estimated aggressiveness) and a high death rate of treatment-resistant cells (low model-estimated resistance) have longer survival time after completing their second-line of therapy. These findings show that mathematical models can characterize the degree of resistance and aggressiveness in individual patients, which improves our understanding of chemoresistance development and could predict treatment effectiveness in HGSOC patients.

Inferring Homologous Recombination Deficiency of Ovarian Cancer From the Landscape of Copy Number Variation at Subchromosomal and Genetic Resolutions.

BackgroundHomologous recombination deficiency (HRD) is characterized by overall genomic instability and has emerged as an indispensable therapeutic target across various tumor types, particularly in ovarian cancer (OV). Unfortunately, current detection assays are far from perfect for identifying every HRD patient. The purpose of this study was to infer HRD from the landscape of copy number variation (CNV).MethodsGenome-wide CNV landscape was measured in OV patients from the Australian Ovarian Cancer Study (AOCS) clinical cohort and >10,000 patients across 33 tumor types from The Cancer Genome Atlas (TCGA). HRD-predictive CNVs at subchromosomal resolution were identified through exploratory analysis depicting the CNV landscape of HRD versus non-HRD OV patients and independently validated using TCGA and AOCS cohorts. Gene-level CNVs were further analyzed to explore their potential predictive significance for HRD across tumor types at genetic resolution.ResultsAt subchromosomal resolution, 8q24.2 amplification and 5q13.2 deletion were predominantly witnessed in HRD patients (both p < 0.0001), whereas 19q12 amplification occurred mainly in non-HRD patients (p < 0.0001), compared with their corresponding counterparts within TCGA-OV. The predictive significance of 8q24.2 amplification (p < 0.0001), 5q13.2 deletion (p = 0.0056), and 19q12 amplification (p = 0.0034) was externally validated within AOCS. Remarkably, pan-cancer analysis confirmed a cross-tumor predictive role of 8q24.2 amplification for HRD (p < 0.0001). Further analysis of CNV in 8q24.2 at genetic resolution revealed that amplifications of the oncogenes, MYC (p = 0.0001) and NDRG1 (p = 0.0004), located on this fragment were also associated with HRD in a pan-cancer manner.ConclusionsThe CNV landscape serves as a generalized predictor of HRD in cancer patients not limited to OV. The detection of CNV at subchromosomal or genetic resolution could aid in the personalized treatment of HRD patients.

1466Vitamin D status before, during and after treatment and ovarian cancer survival

Abstract Background Previous work found higher serum 25-hydroxyvitamin D (25(OH)D) [circulating form of vitamin D] concentrations at diagnosis were associated with longer survival in patients with ovarian cancer (OvCa). There was no evidence for an association with 25(OH)D after primary treatment, but power was limited. Our aim was to reassess this association in a larger sample, including measures collected during treatment and using techniques to deseasonalise 25(OH)D. Methods Participants were diagnosed between 2002-2006 and 2012-2015 from the Australian Ovarian Cancer Study (AOCS) and the Ovarian Cancer, Prognosis and Lifestyle (OPAL) study, respectively. 25(OH)D concentrations were available for 676 at diagnosis (AOCS), 805 during treatment (AOCS:208; OPAL:597) and 861 after completion of primary treatment and before recurrence (AOCS:342; OPAL:519); 1006 AOCS samples were included in the previous analysis. Sociodemographic, diet and lifestyle data came from questionnaires self-completed at recruitment, and clinical/survival data from medical records, supplemented with National Death Index linkage. We will use Cox regression and non-parametric models to examine associations with survival. Results Median 25(OH)D concentrations were lowest during treatment, intermediate at diagnosis and highest after treatment (AOCS 51, 64, and 71 nmol/L, respectively). 5-year survival was 50% in AOCS and 59% in OPAL. Updated survival results will be presented. Conclusions If the association is confirmed in this updated analysis, then increasing vitamin D concentrations may provide a way to improve survival following OvCa. Key messages Higher circulating vitamin D concentrations may improve survival in OvCa.

737P Phase II study of IV vinorelbine in relapsed platinum resistant or refractory C5 high grade serous primary peritoneal, fallopian tube or ovarian cancer (VIP)

The Australian Ovarian Cancer Study identified 4 distinct molecular subtypes of high-grade serous ovarian cancer (HGSOC). C5 subtype is linked to poor prognosis and stemness behavior. We have previously described dependency of C5 HGSOC on genes related to microtubule dynamics. In vitro and vivo studies have shown sensitivity of C5 subtype HGSOC cell lines and patient-derived xenografts to tubulin-depolymerizing vinca alkaloids such as vinorelbine.

Abstract IA04: Drug resistance in end-stage high-grade serous ovarian cancer

Abstract Background: Acquired drug resistance is the major obstacle in controlling high-grade serous ovarian cancer (HGSC) and leads to poor overall survival. Comparatively little massively parallel sequencing data exist from HGSC patients with recurrent or end-stage disease who have been extensively treated with chemotherapy or newer targeted agents, such as antiangiogenics or PARP inhibitors (PARPi). We previously identified four mechanisms of acquired resistance from whole-genome sequencing (WGS) of 23 HGSC patients with recurrent disease; a resistance mechanism of any kind was only identified in half of the patients. Aim: To characterize genomic features and identify mechanisms of acquired drug resistance in end-stage HGSC patients. Methods: Since 2012, we have collected tumor samples from 19 HGSC patients through our rapid-autopsy program. On average, we collect 17 tumor sites per patient during the autopsy, which is performed a median 6 hours after death. SNP array, WGS, and RNA sequencing were performed to detect germline and somatic mutations, copy number alterations and gene expression patterns. Results: A total of 89 end-stage tumor samples from 15 patients with germline or somatic mutations in components of the homologous recombination (HR) DNA repair pathway have been analyzed by SNP array, and a subset of those have undergone WGS (n=44) and RNAseq (n=47). As expected, all samples had a somatic pathogenic TP53 mutation and have highly aberrant somatic copy number profiles. Prominent mutational signatures (single- and double-base substitutions) are associated with defective HR and prior treatment with platinum drugs. The total single-base substitution mutation burden is similar across tumor samples within patients; however, many mutations are unique to individual tumor samples. Currently, reversion mutations, which are secondary mutations in HR genes, are the most common resistance mechanism identified in our cohort of end-stage patients. Conclusion: Our data are allowing us to understand HGSC at end stage, by examining the catalogue of resistance mechanisms within an individual patient and reconstructing the natural history of HGSC. Citation Format: Elizabeth L. Christie, Kathryn Alsop, George Au-Yeung, Ahwan Pandey, Australian Ovarian Cancer Study, David D.L. Bowtell. Drug resistance in end-stage high-grade serous ovarian cancer [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 IA04.

Abstract A48: Antiapoptotic gene expression and sensitivity to BH3-mimetics in chemoresistant, high-grade serous ovarian cancer cell lines

Abstract Introduction: The development of chemotherapy resistance is common in relapsed high-grade serous ovarian cancer (HGSOC). An understanding of mechanisms that are associated with the development of chemotherapy resistance in the clinical setting is needed to improve treatment outcomes. In this study, cell lines newly derived from patients with relapsed HGSOC were characterized at a molecular level to determine associations with response to commonly used chemotherapy drugs. RNA sequencing combined with high-throughput drug library screening was used to determine potential resistance mechanisms and identify drugs and combinations that may be effective in chemoresistant HGSOC. Methods: Six ovarian cancer cell lines derived from ascites of relapsed HGSOC patients who had developed chemoresistant disease were characterized using multigene panel mutation testing, whole-genome DNA copy number analysis, and RNA sequencing. Drug responses to carboplatin and paclitaxel were determined using the CellTitre 96® AQueous One Solution Cell Proliferation Assay (Promega). For two of the resistant cell lines, high-throughput drug screens using three drug libraries—NCI-Approved Oncology Drug set (101 compounds), Kinase Inhibitor Library (210 compounds), and Targeted Agent Library (73 compounds)—were carried out to identify agents with activity in these resistant models. Apoptosis activation was measured using CellPlayer Caspase 3/7 assay (Essen Bioscience). Sensitivity to BH3-mimetics, ABT-737 and navitoclax (ABT-263), was tested in three of the cell lines and in one line, the effects of combining these agents with paclitaxel were assessed using CompuSyn. Results: TP53 mutations were identified in all cell lines, consistent with high-grade serous histology, and all lines were relatively resistant to carboplatin. AOCS5 and AOCS15 were the most resistant to carboplatin and AOCS5 was also highly resistant to paclitaxel. Drug library screens carried out on both AOCS5 and AOCS15 showed differential sensitivity to a number of drug classes. While AOCS5 was found to be generally resistant to most drugs, it was sensitive to ABT-737. RNA-seq identified relative overexpression of antiapoptotic genes in AOCS5, in particular BCL2L1, BCL2, and BCL2L2, in comparison to the other five cell lines. AOCS5 was more sensitive to navitoclax and ABT-737 compared with cell lines that did not overexpress antiapoptotic genes. Combining BH3-mimetics and paclitaxel showed synergism in the highly paclitaxel-resistant cell line AOCS5. Conclusion: Inhibition of apoptosis was identified as a potential mechanism of paclitaxel resistance, and overexpression of antiapoptotic genes may constitute a biomarker profile to predict sensitivity to BH3-mimetics in relapsed HGSOC. Citation Format: Cristina Mapagu, Sivatharsny Srirangan, George Au-Yeung, Australian Ovarian Cancer Study, Paul R. Harnett, David D. Bowtell, Anna deFazio. Antiapoptotic gene expression and sensitivity to BH3-mimetics in chemoresistant, high-grade serous ovarian cancer cell lines [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 A48.

Abstract 467: Deep single-cell RNA-seq of the putative cell of origin revealed a novel molecular subtype of high-grade serous ovarian cancer with poor prognosis

Abstract Accurate molecular classification in cancer is essential for rationalized therapy. However, achieving stable subtyping is highly challenging due to the underlying genomic complexity of tumors. An example of the most successful classification is in breast cancer where it is possible to link individual tumor types, basal vs luminal, to individual cell types of origin. However, the generalization of such an approach has not been easy because of the lack of knowledge about subtypes of the putative cells of origin. For instance, it has been difficult to establish a stable molecular classification of high-grade serous ovarian cancer (HGSOC). More recent evidence strongly supports the notion that HGSOC originates from the secretory cells in fallopian tube epithelium (FTE). However, whether or not there are multiple subtypes of secretory cells and how such subtypes are linked to tumor molecular subtypes have remained elusive. To address this issue, we applied single-cell RNA sequencing (Smart-Seq2) on around 4000 FTE cells from patients with ovarian or endometrial cancers. We developed and employed an in-house differential-expression-based clustering method, termed ClinCluster, for clinical samples that takes into account inter-patient variability. Apart from the established secretory and ciliated cell types, we discovered four novel subtypes of secretory cells and a rare intermediate cell type that co-expresses secretory and ciliated markers. We termed these four novel secretory subtypes as cell cycle, progenitor, epithelial-mesenchymal transition (EMT) and immunoreactive subtypes. Based on fifty-three biomarkers of these subtypes, we designed a panel of transcriptomic signatures and exploited them to deconvolute two bulk expression datasets of ovarian cancer: the RNA-seq dataset of TCGA (The Cancer Genome Atlas) and the microarray dataset of AOCS (Australian Ovarian Cancer Study). The deconvolution analysis revealed a poor-prognostic EMThigh subtype of HGSOC, which was not driven by copy number aberrations. Cox regression analysis indicated that the EMThigh tumors were strongly associated with poor overall survival in the two independent datasets tested (hazard ratio = 2.7, 95% confidence interval = 1.4 - 5.1, P = 0.002, n = 307 for TCGA; hazard ratio = 3.2, 95% confidence interval = 1.7 - 6.1, P = 0.0002, n = 253 for AOCS). Importantly, we found that the PI3K-Akt signaling pathway is upregulated in the EMThigh tumors (FDR &amp;lt; 0.002 for the two independent datasets) giving a strong potential for effective rationalized therapies. In summary, our work portrays a new model for the landscape of fallopian tube epithelium, the putative origin of HGSOC, and illustrates that the cellular subtypes in FTE are related to the HGSOC tumor types with important prognostic and therapeutic implications. Citation Format: Zhiyuan Hu, Abdulkhaliq Alsaadi, Nina Wietek, Laura Santana González, Christopher Yau, Ahmed Ashour Ahmed. Deep single-cell RNA-seq of the putative cell of origin revealed a novel molecular subtype of high-grade serous ovarian cancer with poor prognosis [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 467.

Dynamic of molecular subtypes of high‐grade serous ovarian cancer in paired primary and relapsed biopsies.

e17091 Background: Previously four molecular subtypes of high grade serous ovarian cancer (HGSOC) with distinct biological features and clinical outcome have been described: C1 (mesenchymal), C2 (immunoreactive), C4 (differentiated), and C5 (proliferative). Using Nanostring technique and a minimal signature of 39 classifier genes could reproduce the subtypes identified by microarray gene expression profiling (Leong HS et al. Australian Ovarian Cancer Study. J Pathol. 2015). Methods: We characterized paraffin embedded tissue samples from 279 HGSOC patients for molecular subtypes, utilizing the 39 classifier signature and 9 control genes by Nanostring nCounter Analysis System. From 16 patients paired primary and relapsed samples were available. Only chemonaive primary HGSOC patients were included in the study. FFPEs and clinical data were provided by TOC ( www.toc-network.de ). For each sample, probability scores for the four molecular subtypes (C1, C2, C4, and C5) were calculated. The highest calculated score determined the most likely subtype of the tumor. Results: Of all analyzed primary tumor samples, 88 (31.5%) were classified as C1, 83 (29.8%), 53 (19.0%) and 55 (19.7%) as subtypes C2, C4 and C5, respectively. Our results confirmed data by the AOCS study, which described the distribution of HGSOC with 40.2% (C1), 22.5% (C2), 20.1% (C4) and 17.2% (C5), respectively. Within the paired samples, for 12 of the 16 patients dynamic changes in the molecular subtypes between primary and relapse occurred, while in the remaining 4 patients the phenotype was stable. Conclusions: Molecular subtypes of HGSOC using Nanostring technology with a small panel of classifier genes can be confirmed. Furthermore, the data showed that a change of the established molecular subtype might occur during the evolution of the disease, and therefore translate in a different clinical outcome.

Abstract IA15: Early detection of ovarian cancer: An update

Abstract IA15: Early detection of ovarian cancer: An update

Abstract IA12: Acquired chemotherapy resistance in high-grade serous ovarian cancer patients

Abstract High-grade serous ovarian cancer (HGSC) was one of the first cancer types subjected to comprehensive genomic analysis, and over the last 5-7 years gene expression and DNA sequence data have been generated from hundreds of samples. A large majority of this data has been obtained from surgical samples collected following primary debulking surgery or following a few cycles of neoadjuvant chemotherapy. By contrast, comparatively little data exist from patients who have been extensively treated with chemotherapy or newer targeted agents, such as antiangiogenics or PARP inhibitors (PARPi). Of particular interest are samples collected from patients whose cancer was initially responsive to treatment but has become resistant to therapy (acquired resistance) at the time of collection. The presentation will describe data obtained from whole-genome (N=73 samples, 36 patients) and targeted (N=65 samples, 48 patients) sequence analysis of recurrent or end-stage HGSC samples, focusing in particular on two mechanisms of acquired resistance–fusions involving the ABCB1 gene and reversion of germline BRCA1/2 mutations. ABCB1 encodes the multidrug resistance transporter MDR1, also known as P-glycoprotein. Data will be presented on the frequency and mechanisms of ABCB1 deregulation in recurrent HGSC, and approaches to clinical intervention in fusion-positive patients. Reversions in BRCA1/2 appear to render tumors that were defective in homologous recombination (HR) repair, HR proficient and therefore may have important implications for likely treatment response. The presentation will also discuss approaches to evaluating reversion status in patients with mutation in BRCA1/2. Citation Format: Elizabeth L. Christie, Jessica Beach, Dariush Etemadmoghadam, Dale Garsed, Ann-Marie Patch, Sian Fereday, Swetansu Pattnaik, Australian Ovarian Cancer Study, Samuel Brady, Andrea Bild, David D.L. Bowtell. Acquired chemotherapy resistance in high-grade serous ovarian cancer patients. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr IA12.

Abstract B50: YAP induces development of mesenchymal subtype of high-grade serous ovarian cancer from granulosa cells

Abstract The Hippo pathway plays critical roles in both organ development and tumorigenesis. Our previous studies demonstrated that Yes-Associated Protein 1 (YAP), a transcription coactivator and the major effector of the Hippo pathway, plays a critical role in ovarian cancer development. The present study investigates the role of the Hippo/YAP signaling pathway in the tumorigenesis of ovarian granulosa cells. Fluorescent and chromogenic immunohistochemistry were used to examine YAP expression in human and mouse ovaries. Western blot and qRT-PCR were used to determine protein and mRNA expression. Retrovirus-based YAP expressing constructs and YAP siRNAs were utilized to manipulate YAP levels in primary or immortalized human granulosa cells. A new three-dimensional hanging-drop culture system was used to mimic the growth of granulosa cells in vivo. The soft agar assay and xenograft mouse models were employed to investigate the role of YAP in transformation and tumorigenesis of granulosa cells. Immunohistochemical analyses showed that the active form of YAP (nuclear YAP) was expressed in proliferative granulosa cells in human and mouse ovaries, whereas the inactive form of YAP (cytoplasmic YAP) was detected predominantly in terminally differentiated luteal cells, indicating that YAP activity may be involved in the regulation of granulosa cell proliferation and differentiation. Overexpression of wild-type or constitutively active YAP compromised the expression of genes required for steroidogenesis and induced de-differentiation of differentiated granulosa cells. Ectopic expression of the constitutively active form of YAP in less-differentiated human granulosa cells derived from growing follicles induced tumors in a xenograft mouse model. Intriguingly, we found that these tumors resembled human high-grade serous ovarian cancer (HGSOC) morphologically, histologically, and genetically. The tumors were accompanied by accumulation of large amount of ascites in the peritoneal cavity. Highly proliferative and aggressive tumor cells invaded into omentum, visceral fat tissue, diaphragm, pancreas, liver, and gastrointestinal system. Deletion of the BRCA1 gene in these tumor cells promoted tumor progression and significantly reduced survival rate. Interestingly, these tumors have high expression of N-cadherin but very low expression of CA125 and E-cadherin, which are molecular characteristics of a recently identified mesenchymal subtype of HGSOC. Studies from The Cancer Genome Atlas and the Australian Ovarian Cancer Study Group indicate that the mesenchymal subtype of HGSOC has the poorest prognosis among all subtypes of ovarian cancers. In conclusion, results from this study indicate that YAP plays a critical role in regulating granulosa cell proliferation and differentiation. Overactivation of YAP can result in de-differentiation and reprogramming of ovarian granulosa cells, leading to development of mesenchymal subtype of HGSOC. Our results identify early-stage granulosa cells as a cell of origin of newly identified mesenchymal type of HGSOC. Citation Format: Xiangmin Lv, Chunbo He, Cong Huang, Guohua Hua, Bowen Ma, Xingcheng Chen, Jin Zhou, Zhengfeng Wang, Jixin Dong, Bo R. Rueda, John S. Davis, Cheng Wang. YAP induces development of mesenchymal subtype of high-grade serous ovarian cancer from granulosa cells. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B50.

The association between the inflammatory potential of diet and risk of developing, and survival following, a diagnosis of ovarian cancer.

Inflammation has been implicated in ovarian carcinogenesis. This study evaluated two dietary indices: the Dietary Inflammatory Index (DII®) and the Empirical Dietary Inflammatory Pattern (EDIP), in relation to risk of developing, and survival following, a diagnosis of ovarian cancer. Data came from the Australian Ovarian Cancer Study (1375 cases, 1415 population controls). DII and EDIP scores were computed from dietary information obtained using a semiquantitative food-frequency questionnaire. Logistic regression was used to assess the association between DII and EDIP scores and risk of ovarian cancer and proportional hazards models were used for survival analysis. A high DII score, reflecting a more pro-inflammatory diet, was associated with a modest increased risk of ovarian cancer [odds ratio (OR) DII scoreQ4 vs.Q1 = 1.31, 95% CI 1.06-1.63, ptrend = 0.014]. Likewise a high EDIP score was associated with an increase in risk of ovarian cancer [OR EDIP scoreQ4 vs.Q1 = 1.39, 95% confidence interval (CI) 1.12-1.73, ptrend = 0.002]. We found no association between DII or EDIP score and overall or ovarian cancer-specific survival. In conclusion, our results suggest that a pro-inflammatory diet modestly increases the risk of developing ovarian cancer.

Abstract 2584: Mutations in low-grade serous ovarian cancer and response to BRAF and MEK inhibitors

Abstract Low-grade serous ovarian cancer (LGSC) responds poorly to platinum based chemotherapy and is characterized by activating RAS-MAPK pathway mutations, including oncogenic BRAF. Drugs that target this pathway are effective in BRAF-mutant melanoma but other cancer types, such as colorectal cancers, are less sensitive. Early phase trials report a 15% response rate to MEK inhibitors in LGSC patients, however it is not known which features may predict response. We aimed to determine clinical characteristics and treatment response in women with LGSC and to determine whether response to targeted pathway inhibition is associated with specific mutation profiles in LGSC cell lines. Tumor samples from a cohort of grade 1 and 2 serous ovarian cancer patients were analyzed using targeted, exome or whole genome sequencing. Patient characteristics, treatment and clinical outcome were assessed. Cell lines derived from patients with LGSC with known mutation profiles, AOCS2 (KRAS/BRAF/NRAS wild-type), MPSC1 (BRAFV600L, NRASQ16R), VOA1056 (NRASQ16R) and HCC5075 (KRASG12V), were treated with BRAF (dabrafenib) or MEK inhibitors (trametinib, pimasertib and binimetinib) and response was compared to cell lines derived from high-grade serous cancer (HGSC) and BRAF-mutant melanoma. Women diagnosed with grade 1 or 2 serous carcinoma between 1994-2015 were identified from 1654 invasive ovarian serous cancer cases in the Australian Ovarian Cancer Study and the GynBiobank. HGSC cases were excluded following histopathology review and TP53 mutation screening. Amongst 65 confirmed LGSC patients, 18 (27.7%) had a KRAS mutation, 9 (13.8%) had a BRAF mutation and 7 (10.8%) had a NRAS mutation. Women with advanced stage LGSC and residual disease following debulking surgery had a similarly poor progression-free and overall-survival compared with HGSC patients. We saw a dramatic response to BRAF inhibition in a patient with BRAFV600E-positive LGSC, however, the LGSC cell lines did not respond to dabrafenib. This is not surprising as the cell lines did not harbour the hot-spot BRAFV600E mutation. MPSC1 has a BRAFV600L and a NRASQ16R mutation, but dabrafenib did not inhibit growth. HCC5075 (KRASG12V) was sensitive to all three MEK inhibitors, but response to MEK inhibition in the other LGSC cell lines was modest. In conclusion, LGSC are generally chemotherapy resistant and molecular analyses can identify targetable mutations. However, LGSC are heterogenous with respect to underlying mutations and response to pathway inhibitors is likely to depend on which mutations and pathways are activated. BRAF mutations are not uncommon in patients with LGSC and should be routinely tested as BRAF inhibitors can be an effective treatment for these patients. MEK inhibitors may also be effective in a subset of cases. The results highlight the need for novel clinical trial design, as traditional clinical trials are unlikely to be effective in rare ovarian cancer sub-types. Citation Format: Tania Moujaber, Dariush Etemadmoghadam, Cristina Mapagu, Catherine Kennedy, Yoke-Eng Chiew, Casina Kan, Nikilyn Nevins, Sivatharsny Srirangan, Sian Fereday, Nadia Traficante, Australian Ovarian Cancer Study group, David Bowtell, Rosemary Balleine, Paul Harnett, Anna deFazio. Mutations in low-grade serous ovarian cancer and response to BRAF and MEK inhibitors [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 2584.

Abstract 3393: Characterizing recurrent high-grade serous ovarian cancer through whole-genome sequencing

Abstract Acquired drug resistance is the major obstacle in controlling high-grade serous ovarian cancer (HGSC) and leads to poor overall survival. Comparatively little massively parallel sequencing data exist from HGSC patients with recurrent disease who have been extensively treated with chemotherapy or newer targeted agents, such as anti-angiogenics or PARP inhibitors (PARPi). We previously identified four mechanisms of acquired resistance from whole-genome sequencing (WGS) of 23 HGSC patients with recurrent disease; a resistance mechanism of any kind was only identified in half of the patients. Since 2012, we have collected tumor samples from 16 HGSC patients through our rapid-autopsy program. On average, we collect 17 tumor sites per patient during the autopsy, and to date we have performed WGS on 28 autopsy samples from 8 patients. These data are allowing us to understand HGSC at end-stage, by examining the complete catalog of resistance mechanisms within an individual patient and reconstructing the natural history of HGSC. One acquired resistance mechanism we identified in HGSC involves transcriptional fusion of the drug efflux pump ABCB1 to an upstream gene that causes its overexpression. ABCB1 encodes the multidrug resistance transporter MDR1, also known as P-glycoprotein. Through WGS and targeted RNA sequencing on end-stage tumor samples as well as recurrent ascites samples, we identified multiple fusion partners to ABCB1, with more than 15% of recurrent HGSC patients harboring ABCB1 transcriptional fusions. A number of HGSC patients carry multiple ABCB1 fusions, demonstrating convergent evolution within patients and the strong selective advantage of ABCB1 overexpression by gene fusion. Extending our analysis to recurrent and autopsy samples from breast and prostate cancer patients also identified ABCB1 transcriptional fusions as a resistance mechanism in these cancers. Our findings suggest that ABCB1 transcriptional fusions are a common mechanism of acquired chemotherapy resistance in ovarian, breast and prostate cancer patients, and that characterizing end-stage disease is particularly informative for understanding resistance mechanisms. Citation Format: Elizabeth L. Christie, Swetansu Pattnaik, Sian Fereday, Australian Ovarian Cancer Study, Heather Thorne, Andrea Bild, David D. Bowtell. Characterizing recurrent high-grade serous ovarian cancer through whole-genome sequencing [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 3393.

Transducin-Like Enhancer of Split 3 (TLE3) Expression Is Associated with Taxane Sensitivity in Nonserous Ovarian Carcinoma in a Three-Cohort Study.

Background: Chemoresistance is a major challenge in ovarian cancer treatment, resulting in poor survival rates. Identifying markers of treatment response is imperative for improving outcome while minimizing unnecessary side effects. We have previously demonstrated that expression of transducin-like enhancer of split 3 (TLE3) is associated with favorable progression-free survival in taxane-treated ovarian cancer patients with nonserous histology. The purpose of this study was to perform an independent evaluation of the association of TLE3 expression with response to taxane-based chemotherapy in nonserous ovarian cancer, to validate its role as a potential therapeutic response marker for taxane-based chemotherapy.Methods: We performed immunohistochemical staining of TLE3 on ovarian cancer specimens from the Australian Ovarian Cancer Study, the Westmead Gynaecological Oncology Biobank, and Memorial Sloan Kettering Cancer Center. Progression-free survival and overall survival were assessed to validate an association between TLE3 expression and response to taxane therapy that we previously observed in a smaller study.Results: Expression of TLE3 was associated with favorable outcome only in patients who had received paclitaxel as part of their treatment regimen for both 3-year progression-free survival (n = 160; HR, 0.56; P = 0.03) and 5-year overall survival (HR, 0.53; P = 0.04). Further analysis revealed that the predictive association between TLE3 expression and outcome was strongest in tumors with clear cell histology.Conclusions: The association between high TLE3 expression and a favorable response to taxane-containing chemotherapy regimens was validated in patients with nonserous ovarian cancer.Impact: TLE3 expression may serve as a marker of chemosensitivity in taxane-treated patients with nonserous histologies. Cancer Epidemiol Biomarkers Prev; 27(6); 680-8. ©2018 AACR.

Response rates to second-line platinum-based therapy in ovarian cancer patients challenge the clinical definition of platinum resistance

ObjectiveThe aim of this study was to compare response rates and survival in women with “platinum resistant” epithelial ovarian cancer (EOC) who received further platinum-based or non‑platinum chemotherapy for treatment at first relapse. MethodsPatients with high-grade EOC (including fallopian tube and peritoneal cancer) of all histologies recruited to the Australian Ovarian Cancer Study (AOCS) and treated with platinum-based primary chemotherapy were included. Response to second-line chemotherapy, overall survival (OS) and survival after treatment for first progression (OS2) were determined in all histologies and separately in women with high-grade serous tumors. ResultsOf the 341 patients classified as platinum-resistant by the 6-month threshold, 243 (71%) were treated with chemotherapy at relapse. CA-125 response rates to platinum-based chemotherapy were significantly higher compared to non‑platinum chemotherapy (51% vs 21%, P < 0.001). Among patients with a platinum-free interval (PFI) of 3–6 months, OS2 in patients treated with platinum was significantly longer compared to individuals receiving non‑platinum-based treatment (median 17.67 months, 95% CI: 14.79–20.75 vs. 10.62 months, 95% CI: 8.02–12.72, P = 0.022). The patterns were similar when restricted to patients with high-grade serous histology. In patients with PFI <3 months, there was no significant difference in response or survival according to type of second-line treatment. ConclusionsOur findings further question the use of a 6-month PFI as an arbitrary threshold for subsequent treatment decision-making. Some patients considered “platinum resistant” still derive clinical benefit from platinum-based chemotherapy. Biomarkers of platinum sensitivity are needed in clinical practice to identify potential responders who should be offered re-treatment with platinum.

Chemotherapy weakly contributes to predicted neoantigen expression in ovarian cancer

BackgroundPatients with highly mutated tumors, such as melanoma or smoking-related lung cancer, have higher rates of response to immune checkpoint blockade therapy, perhaps due to increased neoantigen expression. Many chemotherapies including platinum compounds are known to be mutagenic, but the impact of standard treatment protocols on mutational burden and resulting neoantigen expression in most human cancers is unknown.MethodsWe sought to quantify the effect of chemotherapy treatment on computationally predicted neoantigen expression for high grade serous ovarian carcinoma patients enrolled in the Australian Ovarian Cancer Study. In this series, 35 of 114 samples were collected after exposure to chemotherapy; 14 are matched with an untreated sample from the same patient. Our approach integrates whole genome and RNA sequencing of bulk tumor samples with class I MHC binding prediction and mutational signatures extracted from studies of chemotherapy-exposed Caenorhabditis elegans and Gallus gallus cells. We additionally investigated the relationship between neoantigens, tumor infiltrating immune cells estimated from RNA-seq with CIBERSORT, and patient survival.ResultsGreater neoantigen burden and CD8+ T cell infiltration in primary, pre-treatment samples were independently associated with improved survival. Relapse samples collected after chemotherapy harbored a median of 78% more expressed neoantigens than untreated primary samples, a figure that combines the effects of chemotherapy and other processes operative during relapse. The contribution from chemotherapy-associated signatures was small, accounting for a mean of 5% (range 0–16) of the expressed neoantigen burden in relapse samples. In both treated and untreated samples, most neoantigens were attributed to COSMIC Signature (3), associated with BRCA disruption, Signature (1), associated with a slow mutagenic process active in healthy tissue, and Signature (8), of unknown etiology.ConclusionRelapsed ovarian cancers harbor more predicted neoantigens than primary tumors, but the increase is due to pre-existing mutational processes, not mutagenesis from chemotherapy.

Semi-supervised identification of cancer subgroups using survival outcomes and overlapping grouping information.

Identification of cancer patient subgroups using high throughput genomic data is of critical importance to clinicians and scientists because it can offer opportunities for more personalized treatment and overlapping treatments of cancers. In spite of tremendous efforts, this problem still remains challenging because of low reproducibility and instability of identified cancer subgroups and molecular features. In order to address this challenge, we developed Integrative Genomics Robust iDentification of cancer subgroups (InGRiD), a statistical approach that integrates information from biological pathway databases with high-throughput genomic data to improve the robustness for identification and interpretation of molecularly-defined subgroups of cancer patients. We applied InGRiD to the gene expression data of high-grade serous ovarian cancer from The Cancer Genome Atlas and the Australian Ovarian Cancer Study. The results indicate clear benefits of the pathway-level approaches over the gene-level approaches. In addition, using the proposed InGRiD framework, we also investigate and address the issue of gene sharing among pathways, which often occurs in practice, to further facilitate biological interpretation of key molecular features associated with cancer progression. The R package "InGRiD" implementing the proposed approach is currently available in our research group GitHub webpage ( https://dongjunchung.github.io/INGRID/ ).

The impact on high-grade serous ovarian cancer of obesity and lipid metabolism-related gene expression patterns: the underestimated driving force affecting prognosis.

To investigate whether specific obesity/metabolism‐related gene expression patterns affect the survival of patients with ovarian cancer. Clinical and genomic data of 590 samples from the high‐grade ovarian serous carcinoma (HGOSC) study of The Cancer Genome Atlas (TCGA) and 91 samples from the Australian Ovarian Cancer Study were downloaded from the International Cancer Genome Consortium (ICGC) portal. Clustering of mRNA microarray and reverse‐phase protein array (RPPA) data was performed with 83 consensus driver genes and 144 obesity and lipid metabolism‐related genes. Association between different clusters and survival was analyzed with the Kaplan–Meier method and a Cox regression. Mutually exclusive, co‐occurrence and network analyses were also carried out. Using RNA and RPPA data, it was possible to identify two subsets of HGOSCs with similar clinical characteristics and cancer driver mutation profiles (e.g. TP53), but with different outcome. These differences depend more on up‐regulation of specific obesity and lipid metabolism‐related genes than on the number of gene mutations or copy number alterations. It was also found that CD36 and TGF‐ß are highly up‐regulated at the protein levels in the cluster with the poorer outcome. In contrast, BSCL2 is highly up‐regulated in the cluster with better progression‐free and overall survival. Different obesity/metabolism‐related gene expression patterns constitute a risk factor for prognosis independent of the therapy results in the Cox regression. Prognoses were conditioned by the differential expression of obesity and lipid metabolism‐related genes in HGOSCs with similar cancer driver mutation profiles, independent of the initial therapeutic response.

Elevation of TP53 Autoantibody Before CA125 in Preclinical Invasive Epithelial Ovarian Cancer.

Purpose: The TP53 tumor-suppressor gene is mutated in >95% of high-grade serous ovarian cancers. Detecting an autologous antibody response to TP53 that might improve early detection.Experimental Design: An immunoassay was developed to measure TP53 autoantibody in sera from 378 cases of invasive epithelial ovarian cancer and 944 age-matched healthy controls from the United States, Australia, and the United Kingdom. Serial preclinical samples from cases and controls were also assayed from the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS).Results: Using a cutoff value of 78 U/mL to achieve a specificity of 97.4%, TP53 autoantibody was elevated in 30% of 50 cases from MD Anderson, 21.3% of 108 cases from the Australian Ovarian Cancer Study, and 21% of 220 cases from the UKCTOCS. Among 164 cases with rising CA125 detected with the UKCTOCS risk of ovarian cancer algorithm (ROCA), 20.7% had elevated TP53 autoantibody. In cases missed by the ROCA, 16% of cases had elevated TP53 autoantibody. Of the 34 ovarian cancer cases detected with the ROCA, TP53 autoantibody titers were elevated 11.0 months before CA125. In the 9 cases missed by the ROCA, TP53 autoantibody was elevated 22.9 months before cancer diagnosis. Similar sensitivity was obtained using assays with specific mutant and wild-type TP53.Conclusions: TP53 autoantibody levels provide a biomarker with clinically significant lead time over elevation of CA125 or an elevated ROCA value. Quantitative assessment of autoantibodies in combination with CA125 holds promise for earlier detection of invasive epithelial ovarian cancer. Clin Cancer Res; 23(19); 5912-22. ©2017 AACR.