Resistance to platinum-based chemotherapy remains a major impediment in the treatment of serous epithelial ovarian cancer. The objective of this study was to use gene expression and copy number profiling to delineate major deregulated pathways and biomarker networks associated with the development of intrinsic chemotherapy resistance with exposure to standard first-line therapy for ovarian cancer. The study cohort comprised 28 high grade serous ovarian cancer patients divided into two groups based on their varying sensitivity to first-line chemotherapy using progression free survival (PFS) as a surrogate of response. Twelve patient tumors demonstrating relative resistance to platinum based chemotherapy corresponding to shorter PFS (less than 6 months) were compared to 16 tumors from platinum-sensitive patients (PFS more than 18months). Molecular profiling was performed using Affymetrix high-resolution microarray platforms to permit global comparisons of gene expression levels and copy number profiles between tumors from the resistant group with the sensitive group. Microarray data analysis using statistical methods revealed a set of 204 discriminating genes of which expression levels may be influencing differential chemotherapy response between the two groups. Pathway analysis of the differentiating genes showed IGF1 network to be significantly altered between the two groups in addition to PI3K, NFkB, distinguishing the chemotherapy resistant with the sensitive group. Copy number analysis showed differences in the chromosomal regions, 4q31.22, 5q13.2, 9p24.3, 2p23.2, 16q21, 6q14.1, 7p22.3, 12p13 and Xq. Integrative copy number and gene expression profiling will delineate the drivers of chemotherapy resistance in patients undergoing standard platinum-based treatment of ovarian cancer. Future studies to validate these markers are necessary to apply this knowledge to biomarker-based clinical trials. Citation Format: Madhuri Koti, Robert J. Gooding, Paulo Nuin, Alexandria Haslehurst, Colleen Crane, Johanne Weberpals, Timothy Chids, Peter Bryson, Moyez Dharsee, Kenneth R. Evans, Harriet E. Feilotter, Paul C. Park, Jeremy A. Squire. Biomarkers of chemotherapy resistance in serous epithelial ovarian cancer identified by integrative genomic and transcriptomic analysis. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A53.

Background Resistance to platinum-based chemotherapy remains a major impediment in the treatment of serous epithelial ovarian cancer. The objective of this study was to use gene expression profiling to delineate major deregulated pathways and biomarkers associated with the development of intrinsic chemotherapy resistance upon exposure to standard first-line therapy for ovarian cancer. Methods The study cohort comprised 28 patients divided into two groups based on their varying sensitivity to first-line chemotherapy using progression free survival (PFS) as a surrogate of response. All 28 patients had advanced stage, high-grade serous ovarian cancer, and were treated with standard platinum-based chemotherapy. Twelve patient tumours demonstrating relative resistance to platinum chemotherapy corresponding to shorter PFS (< eight months) were compared to sixteen tumours from platinum-sensitive patients (PFS > eighteen months). Whole transcriptome profiling was performed using an Affymetrix high-resolution microarray platform to permit global comparisons of gene expression profiles between tumours from the resistant group and the sensitive group. Results Microarray data analysis revealed a set of 204 discriminating genes possessing expression levels which could influence differential chemotherapy response between the two groups. Robust statistical testing was then performed which eliminated a dependence on the normalization algorithm employed, producing a restricted list of differentially regulated genes, and which found IGF1 to be the most strongly differentially expressed gene. Pathway analysis, based on the list of 204 genes, revealed enrichment in genes primarily involved in the IGF1/PI3K/NF κ B/ERK gene signalling networks. Conclusions This study has identified pathway specific prognostic biomarkers possibly underlying a differential chemotherapy response in patients undergoing standard platinum-based treatment of serous epithelial ovarian cancer. In addition, our results provide a pathway context for further experimental validations, and the findings are a significant step towards future therapeutic interventions.

BACKGROUND:The biology of small cell ovarian carcinoma of the hypercalcemic type (SCCOHT), which is a rare and aggressive form of ovarian cancer, is poorly understood. Tumourigenicity, in vitro growth characteristics, genetic and genomic anomalies, and sensitivity to standard and novel chemotherapeutic treatments were investigated in the unique SCCOHT cell line, BIN-67, to provide further insight in the biology of this rare type of ovarian cancer.METHOD:The tumourigenic potential of BIN-67 cells was determined and the tumours formed in a xenograft model was compared to human SCCOHT. DNA sequencing, spectral karyotyping and high density SNP array analysis was performed. The sensitivity of the BIN-67 cells to standard chemotherapeutic agents and to vesicular stomatitis virus (VSV) and the JX-594 vaccinia virus was tested.RESULTS:BIN-67 cells were capable of forming spheroids in hanging drop cultures. When xenografted into immunodeficient mice, BIN-67 cells developed into tumours that reflected the hypercalcemia and histology of human SCCOHT, notably intense expression of WT-1 and vimentin, and lack of expression of inhibin. Somatic mutations in TP53 and the most common activating mutations in KRAS and BRAF were not found in BIN-67 cells by DNA sequencing. Spectral karyotyping revealed a largely normal diploid karyotype (in greater than 95% of cells) with a visibly shorter chromosome 20 contig. High density SNP array analysis also revealed few genomic anomalies in BIN-67 cells, which included loss of heterozygosity of an estimated 16.7 Mb interval on chromosome 20. SNP array analyses of four SCCOHT samples also indicated a low frequency of genomic anomalies in the majority of cases. Although resistant to platinum chemotherapeutic drugs, BIN-67 cell viability in vitro was reduced by > 75% after infection with oncolytic viruses.CONCLUSIONS:These results show that SCCOHT differs from high-grade serous carcinomas by exhibiting few chromosomal anomalies and lacking TP53 mutations. Although BIN-67 cells are resistant to standard chemotherapeutic agents, their sensitivity to oncolytic viruses suggests that their therapeutic use in SCCOHT should be considered.

Human biological specimens are important for translational research programs such as the Canadian Ovarian Experimental Unified Resource (COEUR) funded by the Terry Fox Research Institute. Sample quality is an important consideration, as it directly impacts the quality of ensuing research. The aim of the present study was to determine the quality of tissues collected from different sites contributing to the COEUR cohort. Samples from high-grade serous ovarian tumors (fresh frozen and corresponding paraffin-embedded tissues) were provided by nine participating Canadian biobanks. All samples were shipped to a central site using a Standard Operating Protocol (SOP). DNA and RNA extraction was conducted by the quality control division of the Canadian Tumor Repository Network (CTRNet). DNA quality was determined by ß-globin gene PCR amplification, and RNA quality by the RNA integrity number (RIN), as measured by the Agilent BioAnalyzer. DNA of acceptable quality had at least three bands of ß-globin amplified from DNA (n=115/135), and a RIN number ≥7 was considered very good for RNA (n=80/135). Sample preparation and storage time had little effect on RNA or DNA quality. Protein expression was assessed on tissue microarray by immunohistochemistry with antibodies against p53, WT1, E-cadherin, CK-7, and Ki67 from formalin fixed-paraffin embedded (FFPE) tissues. As seen with a nonhierarchical clustering statistical method, there was no significant difference in immunostaining of paraffin tissues among specimens from different biobanks. Interestingly, patients with worse outcome were highly positive for p53 and weak for WT1. In conclusion, while there was no common SOP for retrospectively collected material across Canadian biobanks, these results indicate that specimens collected at these multiple sites are of comparable quality, and can serve as an adequate resource to create a national cohort for the validation of molecular biomarkers in ovarian cancer.

Abstract Resistance to platinum-based chemotherapy remains a major impediment in the treatment of serous epithelial ovarian cancer. The objective of this study was to use gene expression and copy number profiling to delineate major deregulated pathways and biomarker networks associated with the development of intrinsic chemotherapy resistance with exposure to standard first-line therapy for ovarian cancer. The study cohort comprised 28 high grade serous ovarian cancer patients divided into two groups based on their varying sensitivity to first-line chemotherapy using progression free survival (PFS) as a surrogate of response. Twelve patient tumors demonstrating relative resistance to platinum based chemotherapy corresponding to shorter PFS (less than 6 months) were compared to 16 tumors from platinum-sensitive patients (PFS more than 18months). Molecular profiling was performed using Affymetrix high-resolution microarray platforms to permit global comparisons of gene expression levels and copy number profiles between tumors from the resistant group with the sensitive group. Microarray data analysis revealed a set of 227 discriminating genes of which expression levels may be influencing differential chemotherapy response between the two groups. Pathway analysis of these genes showed the,PI3K,NFkB and IGF1 networks as some of the significant networks distinguishing the chemotherapy resistant with the sensitive group. Copy number analysis performed using Nexus copy number version 6.1 revealed differences in the chromosomal regions, 4q31.22, 5q13.2, 9p24.3, 2p23.2, 16q21, 6q14.1, 7p22.3, 12p13 and Xq. Integrative copy number and gene expression profiling will delineate the drivers of chemotherapy resistance in patients undergoing standard platinum-based treatment of ovarian cancer. Future studies to validate these markers are necessary to apply this knowledge to biomarker-based clinical trials. Citation Format: Madhuri Koti, Robert J. Gooding, Paulo Nuin, Alexandria Haslehurst, Colleen Crane, Johanne Weberpals, Timothy Childs, Peter Bryson, Moyez Dharsee, Kenneth Evans, Harriet E. Feilotter, Paul C. Park, Jeremy A. Squire. Integrative genomic and transcriptomic analysis in idenfitication of biomarkers of chemoresistance in serous epithelial ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 810. doi:10.1158/1538-7445.AM2013-810

BACKGROUND:Granulosa-cell tumors (GCTs) are the most common type of malignant ovarian sex cord-stromal tumor (SCST). The pathogenesis of these tumors is unknown. Moreover, their histopathological diagnosis can be challenging, and there is no curative treatment beyond surgery.METHODS:We analyzed four adult-type GCTs using whole-transcriptome paired-end RNA sequencing. We identified putative GCT-specific mutations that were present in at least three of these samples but were absent from the transcriptomes of 11 epithelial ovarian tumors, published human genomes, and databases of single-nucleotide polymorphisms. We confirmed these variants by direct sequencing of complementary DNA and genomic DNA. We then analyzed additional tumors and matched normal genomic DNA, using a combination of direct sequencing, analyses of restriction-fragment-length polymorphisms, and TaqMan assays.RESULTS:All four index GCTs had a missense point mutation, 402C-->G (C134W), in FOXL2, a gene encoding a transcription factor known to be critical for granulosa-cell development. The FOXL2 mutation was present in 86 of 89 additional adult-type GCTs (97%), in 3 of 14 thecomas (21%), and in 1 of 10 juvenile-type GCTs (10%). The mutation was absent in 49 SCSTs of other types and in 329 unrelated ovarian or breast tumors.CONCLUSIONS:Whole-transcriptome sequencing of four GCTs identified a single, recurrent somatic mutation (402C-->G) in FOXL2 that was present in almost all morphologically identified adult-type GCTs. Mutant FOXL2 is a potential driver in the pathogenesis of adult-type GCTs.

Improvement of ovarian cancer patient outcome requires well-characterized animal models in which to evaluate novel therapeutics. Xenograft models are frequently used, but with little discussion of disease histology. The objectives of this study were to inject 11 ovarian cancer cell lines intraperitoneally (ip), and a subset intrabursally (ib; orthotopic), into nude mice and to analyze the resulting pathologies. Eight of 11 lines injected ip formed tumors within 3 months at variable rates with the following histological subtype distribution: one endometrioid, one serous, one clear cell, and five undifferentiated. Only mice injected with A2780-cp cells presented with ovarian-specific metastases (11 of 88), and the survival time of these animals was significantly shorter, which may be attributed to the higher proliferation rate as determined by Ki67 positivity. Additional analysis of the influence of the ovarian microenvironment on cell characteristics was conducted with ib injection of two cell lines (OVCA 429 and ES-2). The site of injection did not affect the tumor histology, the effect on proliferation was cell-type dependent, and the tumor take rate (cell survival) was negatively affected for OVCA 429 cells. The animal models described herein represent histologically distinct models of both early and late stage ovarian cancer useful for evaluation of therapeutics.