Recurrent High-grade Serous Ovarian Carcinoma Research Articles

The CHK1 inhibitor prexasertib in BRCA wild-type platinum-resistant recurrent high-grade serous ovarian carcinoma: a phase 2 trial

The multi-cohort phase 2 trial NCT02203513 was designed to evaluate the clinical activity of the CHK1 inhibitor (CHK1i) prexasertib in patients with breast or ovarian cancer. Here we report the activity of CHK1i in platinum-resistant high-grade serous ovarian carcinoma (HGSOC) with measurable and biopsiable disease (cohort 5), or without biopsiable disease (cohort 6). The primary endpoint was objective response rate (ORR). Secondary outcomes were safety and progression-free survival (PFS). 49 heavily pretreated patients were enrolled (24 in cohort 5, 25 in cohort 6). Among the 39 RECISTv1.1-evaluable patients, ORR was 33.3% in cohort 5 and 28.6% in cohort 6. Primary endpoint was not evaluable due to early stop of the trial. The median PFS was 4 months in cohort 5 and 6 months in cohort 6. Toxicity was manageable. Translational research was an exploratory endpoint. Potential biomarkers were investigated using pre-treatment fresh biopsies and serial blood samples. Transcriptomic analysis revealed high levels of DNA replication-related genes (POLA1, POLE, GINS3) associated with lack of clinical benefit [defined post-hoc as PFS < 6 months]. Subsequent preclinical experiments demonstrated significant cytotoxicity of POLA1 silencing in combination with CHK1i in platinum-resistant HGSOC cell line models. Therefore, POLA1 expression may be predictive for CHK1i resistance, and the concurrent POLA1 inhibition may improve the efficacy of CHK1i monotherapy in this hard-to-treat population, deserving further investigation.

AHRR and SFRP2 in primary versus recurrent high-grade serous ovarian carcinoma and their prognostic implication

BackgroundThe aim of this study was to analyse transcriptomic differences between primary and recurrent high-grade serous ovarian carcinoma (HGSOC) to identify prognostic biomarkers.MethodsWe analysed 19 paired primary and recurrent HGSOC samples using targeted RNA sequencing. We selected the best candidates using in silico survival and pathway analysis and validated the biomarkers using immunohistochemistry on a cohort of 44 paired samples, an additional cohort of 504 primary HGSOCs and explored their function.ResultsWe identified 233 differential expressed genes. Twenty-three showed a significant prognostic value for PFS and OS in silico. Seven markers (AHRR, COL5A2, FABP4, HMGCS2, ITGA5, SFRP2 and WNT9B) were chosen for validation at the protein level. AHRR expression was higher in primary tumours (p < 0.0001) and correlated with better patient survival (p < 0.05). Stromal SFRP2 expression was higher in recurrent samples (p = 0.009) and protein expression in primary tumours was associated with worse patient survival (p = 0.022). In multivariate analysis, tumour AHRR and SFRP2 remained independent prognostic markers. In vitro studies supported the anti-tumorigenic role of AHRR and the oncogenic function of SFRP2.ConclusionsOur results underline the relevance of AHRR and SFRP2 proteins in aryl-hydrocarbon receptor and Wnt-signalling, respectively, and might lead to establishing them as biomarkers in HGSOC.

A B7-H4-Targeting Antibody-Drug Conjugate Shows Antitumor Activity in PARPi and Platinum-Resistant Cancers with B7-H4 Expression.

Platinum and PARP inhibitors (PARPi) demonstrate activity in breast and ovarian cancers, but drug resistance ultimately emerges. Here, we examine B7-H4 expression in primary and recurrent high-grade serous ovarian carcinoma (HGSOC) and the activity of a B7-H4-directed antibody-drug conjugate (B7-H4-ADC), using a pyrrolobenzodiazepine-dimer payload, in PARPi- and platinum-resistant HGSOC patient-derived xenograft (PDX) models. B7-H4 expression was quantified by flow cytometry and IHC. B7-H4-ADC efficacy was tested against multiple cell lines in vitro and PDX in vivo. The effect of B7-H4-ADC on cell cycle, DNA damage, and apoptosis was measured using flow cytometry. B7-H4 is overexpressed in 92% of HGSOC tumors at diagnosis (n = 12), persisted in recurrent matched samples after platinum treatment, and was expressed at similar levels across metastatic sites after acquired multi-drug resistance (n = 4). Treatment with B7-H4-ADC resulted in target-specific growth inhibition of multiple ovarian and breast cancer cell lines. In platinum- or PARPi-resistant ovarian cancer cells, B7-H4-ADC significantly decreased viability and colony formation while increasing cell-cycle arrest and DNA damage, ultimately leading to apoptosis. Single-dose B7-H4-ADC led to tumor regression in 65.5% of breast and ovarian PDX models (n = 29), with reduced activity in B7-H4 low or negative models. In PARPi and platinum-resistant HGSOC PDX models, scheduled B7-H4-ADC dosing led to sustained tumor regression and increased survival. These data support B7-H4 as an attractive ADC target for treatment of drug-resistant HGSOC and provide evidence for activity of an ADC with a DNA-damaging payload in this population. See related commentary by Veneziani et al., p. 1434.

Ayurveda Maintenance Therapy in Recurrent Ovarian Cancer

AbstractDespite optimal surgery and first-line platinum-based doublet chemotherapy, approximately 70 to 80% of patients with epithelial ovarian cancers relapse. Two cases of recurrent ovarian cancer (ROC) were treated with non–platinum-based Ayurveda maintenance therapy (AMT) consisting of drugs having a herbal and herbomineral origin. This regimen was followed over a period of 3 years and progression-free survival (PFS) was noted along with platinum-free interval (PFI). Two patients were diagnosed with BRCA1 mutated recurrent high-grade serous ovarian carcinoma and treated with the per-oral AMT regimen labeled as ZINCA-30 in our hospital after completion of standard of care treatment and followed up until progression. The ZINCA-30 regimen comprising Jasada (traditional Zinc preparation), Indukanth kwatham and Curcuma amada powder in combination was prescribed based on Rasayana chikitsa postulated in Ayurveda. The patients were followed up every 3 months. The progression-free survival observed in these patients was 28 months and 45 months, respectively. These two pilot cases suggested an increased platinum-free interval (PFI), improved progression-free survival (PFS) in recurrent ovarian cancer (ROC), with the AMT labeled as ZINCA-30 after chemotherapy.

Genome-wide DNA methylome analysis identifies methylation signatures associated with survival and drug resistance of ovarian cancers

BackgroundIn contrast to stable genetic events, epigenetic changes are highly plastic and play crucial roles in tumor evolution and development. Epithelial ovarian cancer (EOC) is a highly heterogeneous disease that is generally associated with poor prognosis and treatment failure. Profiling epigenome-wide DNA methylation status is therefore essential to better characterize the impact of epigenetic alterations on the heterogeneity of EOC.MethodsAn epigenome-wide association study was conducted to evaluate global DNA methylation in a retrospective cohort of 80 mixed subtypes of primary ovarian cancers and 30 patients with high-grade serous ovarian carcinoma (HGSOC). Three demethylating agents, azacytidine, decitabine, and thioguanine, were tested their anti-cancer and anti-chemoresistant effects on HGSOC cells.ResultsGlobal DNA hypermethylation was significantly associated with high-grade tumors, platinum resistance, and poor prognosis. We determined that 9313 differentially methylated probes (DMPs) were enriched in their relative gene regions of 4938 genes involved in small GTPases and were significantly correlated with the PI3K-AKT, MAPK, RAS, and WNT oncogenic pathways. On the other hand, global DNA hypermethylation was preferentially associated with recurrent HGSOC. A total of 2969 DMPs corresponding to 1471 genes were involved in olfactory transduction, and calcium and cAMP signaling. Co-treatment with demethylating agents showed significant growth retardation in ovarian cancer cells through differential inductions, such as cell apoptosis by azacytidine or G2/M cell cycle arrest by decitabine and thioguanine. Notably, azacytidine and decitabine, though not thioguanine, synergistically enhanced cisplatin-mediated cytotoxicity in HGSOC cells.ConclusionsThis study demonstrates the significant association of global hypermethylation with poor prognosis and drug resistance in high-grade EOC and highlights the potential of demethylating agents in cancer treatment.Graphic abstract

Co-expression patterns of chimeric antigen receptor (CAR)-T cell target antigens in primary and recurrent ovarian cancer

ObjectiveChimeric antigen receptor (CAR)-T cell strategies ideally target a surface antigen that is exclusively and uniformly expressed by tumors; however, no such antigen is known for high-grade serous ovarian carcinoma (HGSC). A potential solution involves combinatorial antigen targeting with AND or OR logic-gating. Therefore, we investigated co-expression of CA125, Mesothelin (MSLN) and Folate Receptor alpha (FOLRA) on individual tumor cells in HGSC. MethodsRNA expression of CA125, MSLN, and FOLR1 was assessed using TCGA (HGSC) and GTEx (healthy tissues) databases. Antigen expression profiles and CD3+, CD8+ and CD20+ tumor-infiltrating lymphocyte (TIL) patterns were assessed in primary and recurrent HGSC by multiplex immunofluorescence and immunohistochemistry. ResultsAt the transcriptional level, each antigen was overexpressed in >90% of cases; however, MSLN and FOLR1 showed substantial expression in healthy tissues. At the protein level, CA125 was expressed by the highest proportion of cases and tumor cells per case, followed by MSLN and FOLRA. The most promising pairwise combination was CA125 and/or MSLN (OR gate), with 51.9% of cases containing ≥90% of tumor cells expressing one or both antigens. In contrast, only 5.8% of cases contained ≥90% of tumor cells co-expressing CA125 and MSLN (AND gate). Antigen expression patterns showed modest correlations with TIL. Recurrent tumors retained expression of all three antigens and showed increased TIL densities. ConclusionsAn OR-gated CAR-T cell strategy against CA125 and MSLN would target the majority of tumor cells in most cases. Antigen expression and T-cell infiltration patterns are favorable for this strategy in primary and recurrent disease.

Multiomic Analysis of Subtype Evolution and Heterogeneity in High-Grade Serous Ovarian Carcinoma.

Multiple studies have identified transcriptome subtypes of high-grade serous ovarian carcinoma (HGSOC), but their interpretation and translation are complicated by tumor evolution and polyclonality accompanied by extensive accumulation of somatic aberrations, varying cell type admixtures, and different tissues of origin. In this study, we examined the chronology of HGSOC subtype evolution in the context of these factors using a novel integrative analysis of absolute copy-number analysis and gene expression in The Cancer Genome Atlas complemented by single-cell analysis of six independent tumors. Tumor purity, ploidy, and subclonality were reliably inferred from different genomic platforms, and these characteristics displayed marked differences between subtypes. Genomic lesions associated with HGSOC subtypes tended to be subclonal, implying subtype divergence at later stages of tumor evolution. Subclonality of recurrent HGSOC alterations was evident for proliferative tumors, characterized by extreme genomic instability, absence of immune infiltration, and greater patient age. In contrast, differentiated tumors were characterized by largely intact genome integrity, high immune infiltration, and younger patient age. Single-cell sequencing of 42,000 tumor cells revealed widespread heterogeneity in tumor cell type composition that drove bulk subtypes but demonstrated a lack of intrinsic subtypes among tumor epithelial cells. Our findings prompt the dismissal of discrete transcriptome subtypes for HGSOC and replacement by a more realistic model of continuous tumor development that includes mixtures of subclones, accumulation of somatic aberrations, infiltration of immune and stromal cells in proportions correlated with tumor stage and tissue of origin, and evolution between properties previously associated with discrete subtypes. SIGNIFICANCE: This study infers whether transcriptome-based groupings of tumors differentiate early in carcinogenesis and are, therefore, appropriate targets for therapy and demonstrates that this is not the case for HGSOC.

Predictive Relevance of Circulating miR-622 in Patients with Newly Diagnosed and Recurrent High-Grade Serous Ovarian Carcinoma.

Identifying patients with high-grade serous ovarian cancer (HGSOC) who will respond to treatment remains a clinical challenge. We focused on miR-622, a miRNA involved in the homologous recombination repair (HRR) pathway, and we assessed its predictive value in serum prior to first-line chemotherapy and at relapse. Serum miR-622 expression was assessed in serum prior to first-line platinum-based chemotherapy in a prospective multicenter study (miRNA Serum Analysis, miRSA, NCT01391351) and a retrospective cohort (Biological Resource Center, BRC), and was also studied at relapse. Progression-free survival (PFS) and overall survival (OS) were used as primary and secondary endpoints prior to first-line chemotherapy and OS as a primary endpoint at relapse. The group with high serum miR-622 expression was associated with a significantly lower PFS (15.4 versus 24.4 months; adjusted HR 2.11, 95% CI 1.2 3.8, P = 0.015) and OS (29.7 versus 40.6 months; adjusted HR 7.68, 95% CI 2.2-26.2, P = 0.0011) in the miRSA cohort. In the BRC cohort, a high expression of miR-622 was also associated with a significantly lower OS (22.8 versus 35.9 months; adjusted HR 1.98, 95% CI 1.1-3.6, P = 0.026). At relapse, high serum miR-622 was associated with a significantly lower OS (7.9 versus 20.6 months; adjusted HR 3.15, 95% CI 1.4-7.2, P = 0.0062). Serum miR-622 expression is a predictive independent biomarker of response to platinum-based chemotherapy for newly diagnosed and recurrent HGSOC. These results may open new perspectives for HGSOC patient stratification and monitoring of resistance to platinum-based and poly(ADP-ribose)-polymerase-inhibitor-maintenance therapies, facilitating better and personalized treatment decisions.

Abstract NT-094: TARGETED INHIBITION OF HSP90 IMPAIRS DNA-DAMAGE RESPONSE PROTEINS AND INCREASES THE SENSITIVITY OF OVARIAN CARCINOMA CELLS TO PARP INHIBITORS

Abstract Poly (ADP-ribose) Polymerase inhibitors (PARPi) are a promising class of inhibitors for the treatment of high grade serous ovarian carcinoma (HGSOC). The greatest activity of these agents is seen in patients with defects in DNA damage repair (DDR) mechanisms, including mutation or epigenetic inactivation of BRCA1 and BRCA2 genes and alterations in expression and/or function of DNA repair genes/proteins. PARPi are approved as second-line and maintenance therapies in recurrent HGSOCs. Notably, clinical trials have shown that single agent PARPi show activity in a significant number of HGSOC patients in the absence of alterations in BRCA genes, particularly in patients with platinum sensitive disease, possibly those with tumors exhibiting defects in homologous recombination (HR), or ‘BRCAness'. To extend the benefit of these agents beyond patients with inherent defects in HR, we wished to test the idea that combination of PARPi with agents that functionally abrogate HR could extend the benefit of PARPi's. An attractive molecular target for this purpose is heat shock protein 90 (HSP90) based on its essential role in mediating the maturation and stability of several key proteins required for the DDR. The goal of this study was to test the hypothesis that targeted inhibition of HSP90 with a small-molecule inhibitor ganetespib (STA-9090) would sensitize non-BRCA mutant OC cells to the PARPi talazoparib (BMN-673). To test this hypothesis, we used established OC cell lines (OVCAR3, UWB1.289), and novel OC cells lines (OC-38, OC-1) derived in our laboratory from de-identified tumors isolated from patients with HGSOC. Cells were treated with talazoparib and ganetespib alone and in combination, and the effects of drug treatment on expression of DDR proteins, ionizing radiation (IR)-induced RAD51+ and γH2AX+ foci, and cell viability was assessed. Ganetespib treatment led to dose- and time-dependent depletion of HSP90 client proteins participating in DDR including BRCA1 and 2, MRE11, CDK1, CHK1, RAD51. Treatment with ganetespib also led to a significant reduction in the percentage of RAD51+ nuclei following IR. The quantity of DNA double-strand break marker γH2Ax foci/nucleus decreased over time in vehicle treated, but not in ganetespib-treated cells. We next conducted a comprehensive analysis of cytotoxicity in cells treated with ganetespib and talazoparib alone, in combination and at differing molar ratios. Combination indexes (CI) were calculated to assess additive, synergistic and antagonistic effects using the methods of Greco plus the bootstrap to determine statistical significance. Ganetespib sensitized BRCA1-null UWB1.289 cells to the effects of talazoparib (CI=0.73, p&amp;lt;0.0005). Among the non-BRCA mutant cell lines analyzed, the combination of ganetespib and talazoparib were synergistic in some patient-derived cell lines, but antagonistic in others. Together, our data suggest that ganetespib effectively disrupts critical DDR pathway proteins in HGSOC cells and may sensitize non-BRCA-mutant OC cells to PARPi. From clinical perspective, this implicates the potential of sensitization of some HGSOC patients without HR pathway alterations to PARPi, and potentially other DNA-damage inducing agents. Citation Format: Rashid Gabbasov, I. Daniel Benrubi, Shane W. O'Brien, John J. Krais, Neil Johnson, Samuel Litwin, Denise C. Connolly. TARGETED INHIBITION OF HSP90 IMPAIRS DNA-DAMAGE RESPONSE PROTEINS AND INCREASES THE SENSITIVITY OF OVARIAN CARCINOMA CELLS TO PARP INHIBITORS [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 NT-094.

Abstract GMM-048: CTDNA RESPONSE TO THE PARP INHIBITOR RUCAPARIB PREDICTS PROGRESSION-FREE SURVIVAL AND BEST OVERALL RESPONSE ON THE ARIEL2 TRIAL

Abstract BACKGROUND: High grade serous ovarian carcinoma (HGSOC) is characterized by ubiquitous TP53 mutation and has the highest genomic complexity of all epithelial neoplasms. Sensitivity to PARP inhibitor therapy is strongly associated with homologous recombination deficiency (HRD). Genomic biomarkers of HRD such as genome-wide loss of heterozygosity (LOH) scores predict HRD and response to rucaparib. We hypothesized that functional testing of response during PARP inhibitor treatment using changes in circulating tumour DNA (ctDNA) could improve prediction of patient outcome. We tested whether the change in ctDNA TP53 mutant allele fraction (MAF) after one cycle of rucaparib treatment was predictive of progression free survival (PFS) and response in patients from the phase 2 ARIEL2 trial in women with platinum-sensitive recurrent high grade ovarian cancer (NCT01891344). METHODS: We analyzed serial plasma samples (n = 636) from 142 HGSOC patients during screening, on day 1 of each treatment cycle, and at the end of rucaparib treatment. Targeted amplicon deep sequencing (TADS) of TP53 was performed on DNA extracted from plasma (median depth 6916×). Somatic TP53 mutation and loss of heterozygosity score (LOH) were available from archival and biopsy specimens. Statistical analyses were pre-specified and ctDNA analysis was carried out blinded to visit and response data. TP53 MAF changes after one cycle of treatment were compared with PFS and best overall response assessed by RECIST v1.1 and GCIG CA-125 criteria. Optimal cut points for ctDNA response were determined using a cross-validation analysis. In cases with &amp;gt;1 TP53 mutation, response assessment was performed using the mutation with highest MAF. RESULTS: We detected TP53 mutations in plasma from 134 patients; all cases were concordant between tumour and plasma except for one patient (present in plasma but not tumour). In 18 patients (13%), 2 or more TP53 mutations were detected in ctDNA. The median TP53 MAF prior to cycle 1 was 2.6% (IQR 0.3–8.6). Reduction of &amp;gt;70% of TP53 MAF in ctDNA between cycle 1 and 2 was significantly predictive of improved PFS (n = 97; HR = 0.53, 95% CI 0.34-0.85, p = 0.008, median 273 vs 158 days, sensitivity 76%, specificity 62%) and best overall response (n = 97; OR = 7.04, 95% CI 2.69–21.06, p &amp;lt; 0.001). Combining ctDNA and LOH scores did not improve prediction of response. CONCLUSIONS Response measured by &amp;gt;70% fall in TP53 ctDNA between pre-cycle 1 and pre-cycle 2 of rucaparib therapy was significantly associated with best overall response and improved PFS. Similar findings were observed in a retrospective study of recurrent HGSOC treated with standard of care chemotherapy. The pathological or genomic factors causing multiple TP53 mutations in ctDNA are unknown. The association between early fall in ctDNA and validated RECIST and CA-125 response markers provides strong evidence that ctDNA may have utility for detecting early response to targeted therapy. Further analyses in randomized studies should be performed to confirm that ctDNA response has strong predictive value. Citation Format: Anna Piskorz, David Robertson, Kevin K. Lin, James Morris, Elaina Mann, Amit Oza, Robert L. Coleman, David M. O'Malley, Michael Friedlander, Janiel M. Cragun, Ling Ma, Heidi Giordano, Iain A. McNeish, Elizabeth Swisher, James Wason, James D. Brenton. CTDNA RESPONSE TO THE PARP INHIBITOR RUCAPARIB PREDICTS PROGRESSION-FREE SURVIVAL AND BEST OVERALL RESPONSE ON THE ARIEL2 TRIAL [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-048.

Activation of Wnt signaling promotes olaparib resistant ovarian cancer.

Epithelial ovarian cancer (EOC) has one of the highest death to incidence ratios among all cancers. High grade serous ovarian carcinoma (HGSOC) is the most common and deadliest EOC histotype due to the lack of therapeutic options following debulking surgery and platinum/taxane-based chemotherapies. For recurrent chemosensitive HGSOC, poly(ADP)-ribose polymerase inhibitors (PARPi; olaparib, rucaparib, or niraparib) represent an emerging treatment strategy. While PARPi are most effective in homologous recombination DNA repair-deficient (HRD) HGSOCs, recent studies have observed a significant benefit in non-HRD HGSOCs. However, all HGSOC patients are likely to acquire resistance. Therefore, there is an urgent clinical need to understand PARPi resistance and to introduce novel combinatorial therapies to manage PARPi resistance and extend HGSOC disease-free intervals. In a panel of HGSOC cell lines, we established matched olaparib sensitive and resistant cells. Transcriptome analysis of the matched olaparib-sensitive vs -resistant cells revealed activation of the Wnt signaling pathway and consequently increased TCF transcriptional activity in PARPi-resistant cells. Forced activation of canonical Wnt signaling in several PARPi-sensitive cells via WNT3A reduced olaparib and rucaparib sensitivity. PARPi resistant cells were sensitive to inhibition of Wnt signaling using the FDA-approved compound, pyrvinium pamoate, which has been shown to promote downregulation of β-catenin. In both an HGSOC cell line and a patient-derived xenograft model, we observed that combining pyrvinium pamoate with olaparib resulted in a significant decrease in tumor burden. This study demonstrates that Wnt signaling can mediate PARPi resistance in HGSOC and provides a clinical rationale for combining PARP and Wnt inhibitors.

Methylation of all BRCA1 copies predicts response to the PARP inhibitor rucaparib in ovarian carcinoma

Accurately identifying patients with high-grade serous ovarian carcinoma (HGSOC) who respond to poly(ADP-ribose) polymerase inhibitor (PARPi) therapy is of great clinical importance. Here we show that quantitative BRCA1 methylation analysis provides new insight into PARPi response in preclinical models and ovarian cancer patients. The response of 12 HGSOC patient-derived xenografts (PDX) to the PARPi rucaparib was assessed, with variable dose-dependent responses observed in chemo-naive BRCA1/2-mutated PDX, and no responses in PDX lacking DNA repair pathway defects. Among BRCA1-methylated PDX, silencing of all BRCA1 copies predicts rucaparib response, whilst heterozygous methylation is associated with resistance. Analysis of 21 BRCA1-methylated platinum-sensitive recurrent HGSOC (ARIEL2 Part 1 trial) confirmed that homozygous or hemizygous BRCA1 methylation predicts rucaparib clinical response, and that methylation loss can occur after exposure to chemotherapy. Accordingly, quantitative BRCA1 methylation analysis in a pre-treatment biopsy could allow identification of patients most likely to benefit, and facilitate tailoring of PARPi therapy.

Establishment and Characterization of the Novel High-Grade Serous Ovarian Cancer Cell Line OVPA8.

High-grade serous ovarian carcinoma (HGSOC) is the most frequent histological type of ovarian cancer and the one with worst prognosis. Unfortunately, the majority of established ovarian cancer cell lines which are used in the research have unclear histological origin and probably do not represent HGSOC. Thus, new and reliable models of HGSOC are needed. Ascitic fluid from a patient with recurrent HGSOC was used to establish a stable cancer cell line. Cells were characterized by cytogenetic karyotyping and short tandem repeat (STR) profiling. New generation sequencing was applied to test for hot-spot mutations in 50 cancer-associated genes and fluorescence in situ hybridization (FISH) analysis was used to check for TP53 status. Cells were analyzed for expression of several marker genes/proteins by reverse-transcription polymerase chain reaction (RT-PCR), fluorescence-activated cell sorting (FACS), and immunocytochemistry (ICC). Functional tests were performed to compare OVPA8 cells with five commercially available and frequently used ovarian cancer cell lines: SKOV3, A2780, OVCAR3, ES2, and OAW42. Our newly-established OVPA8 cell line shows morphologic and genetic features consistent with HGSOC, such as epithelial morphology, multiple chromosomal aberrations, TP53 mutation, BRCA1 mutation, and loss of one copy of BRCA2. The OVPA8 line has a stable STR profile. Cells are positive for EpCAM, CK19, and CD44; they have relatively low plating efficiency/ability to form spheroids, a low migration rate, and intermediate invasiveness in matrigel, as compared to other ovarian cancer lines. OVPA8 is sensitive to paclitaxel and resistant to cisplatin. We also tested two FGFR inhibitors; OVPA8 cells were resistant to AZD4547 (AstraZeneca, London, UK), but sensitive to the new inhibitor CPL304-110-01 (Celon Pharma, Łomianki/Kiełpin, Poland). We have established and characterized a novel cell line, OVPA8, which can be a valuable preclinical model for studies on high-grade serous ovarian cancer.

Dynamics of the Intratumoral Immune Response during Progression of High-Grade Serous Ovarian Cancer

PURPOSE: Tumor-infiltrating lymphocytes (TILs) have an established impact on the prognosis of high-grade serous ovarian carcinoma (HGSOC), however, their role in recurrent ovarian cancer is largely unknown. We therefore systematically investigated TIL densities and MHC class I and II (MHC1, 2) expression in the progression of HGSOC. EXPERIMENTAL DESIGN: CD3+, CD4+, CD8+ TILs and MHC1, 2 expression were evaluated by immunohistochemistry on tissue microarrays in 113 paired primary and recurrent HGSOC. TILs were quantified by image analysis. All patients had been included to the EU-funded OCTIPS FP7 project. RESULTS: CD3+, CD4+, CD8+ TILs and MHC1 and MHC2 expression showed significant correlations between primary and recurrent tumor levels (Spearman rho 0.427, 0.533, 0.361, 0.456, 0.526 respectively; P<.0001 each). Paired testing revealed higher CD4+ densities and MHC1 expression in recurrent tumors (Wilcoxon P=.034 and P=.018). There was also a shift towards higher CD3+ TILs levels in recurrent carcinomas when analyzing platinum-sensitive tumors only (Wilcoxon P=.026) and in pairs with recurrent tumor tissue from first relapse only (Wilcoxon P=.031). High MHC2 expression was the only parameter to be significantly linked to prolonged progression-free survival after first relapse (PFS2, log-rank P=.012). CONCLUSIONS: This is the first study that analyzed the development of TILs density and MHC expression in paired primary and recurrent HGSOC. The level of the antitumoral immune response in recurrent tumors was clearly dependent on the one in the primary tumor. Our data contribute to the understanding of temporal heterogeneity of HGSOC immune microenvironment and have implications for selection of samples for biomarker testing in the setting of immune-targeting therapeutics.

Differences of chemoresistance assay between invasive micropapillary/low-grade serous ovarian carcinoma and high-grade serous ovarian carcinoma

The objective of this study was to evaluate the pattern of chemoresistance in invasive micropapillary/low-grade serous ovarian carcinoma (invasive MPSC/LGSC) and high-grade serous ovarian carcinoma (HGSC) according to extreme drug resistance (EDR) assay testing. Surgical specimens of 44 recurrent ovarian cancer patients harvested at the time of cytoreductive surgery between August 1999 and February 2004 were identified retrospectively from the tumor registry database. Thirteen patients (29.5%) had recurrent invasive MPSC/LGSC and 31 (70.5%) patients had recurrent HGSC. Eight drugs were evaluated; EDR assay results were compared between LGSC and HGSC groups using Fisher exact tests and exact logistic regression models. Compared to HGSC, invasive MPSC/LGSC were more likely to manifest EDR to the drugs paclitaxel (69% vs 14%, P < 0.001), carboplatin (50% vs 17%, P= 0.05), cyclophosphamide (40% vs 23%, P= 0.41), gemcitabine (36% vs 19%, P= 0.40), and cisplatin (33% vs 28%, P= 0.72) and less likely to be resistant to etoposide (0% vs 44%, P= 0.007), doxorubicin (8% vs 45%, P= 0.03), and topotecan (8% vs 21%, P= 0.65). Exact logistic regression estimates revealed that invasive MPSC/LGSC patients had significantly increased probabilities of paclitaxel resistance odds ratio (OR) = 12.5 (95% CI: 2.3-100.0), P= 0.001 and carboplatin resistance OR = 4.8 (95% CI: 0.9-25.0), P= 0.07, while the HGSC cases were more likely to be resistant to etoposide OR = 12.1 (95% CI: 1.7-infinity), P=0.009 and doxorubicin OR = 8.6 (95% CI: 1.0-413.7), P= 0.05. In this retrospective analysis, patients with recurrent invasive MPSC/LGSC were more likely to manifest EDR to standard chemotherapy agents (platinum and paclitaxel). These observations may help to guide chemotherapeutic decision making in these patients if confirmed in a large-scale study.