Mucinous ovarian carcinoma (MOC) is an epithelial ovarian cancer subtype that is frequently misclassified as extraovarian mucinous metastasis (EOM) because of overlapping features. To address this diagnostic challenge, we perform genome-wide DNA methylation profiling of 58 MOCs, 38 EOMs, and 18 mucinous borderline ovarian tumors (mBOTs) collected from six institutions. Methylation analysis defines two mBOT groups, one epigenetically similar to normal ovary and one resembling MOC. Unsupervised clustering reveals two distinct MOC methylation subtypes with potential prognostic relevance in the internal cohort. Using these data together with 389 external profiles, we develop and validate a three-step machine-learning classifier that distinguishes MOC from EOM with 95.5% accuracy. External validation of this classifier on 21 MOCs and 24 EOMs yields an accuracy of 91.11% for differentiating MOC from EOM. These findings establish an epigenetic framework for mucinous ovarian tumors and provide a robust clinical classification tool.

PURPOSE OF REVIEW:Mesothelin (MSLN) is a CA125-binding surface glycoprotein that mediates cell adhesion and peritoneal metastasis development in mesothelioma, high-grade serous ovarian cancer (HGSOC), pancreatic ductal adenocarcinoma, and cholangiocarcinoma. RECENT FINDINGS:Because of its tumor-restricted expression and functional role in dissemination, MSLN is represents an attractive molecule to target in solid tumors. Several antibody-based therapeutic agents, vaccine and chimeric antigen receptor therapy directed against MSLN are object of clinical evaluation. MSLN-targeted therapies are limited by antigen shedding and on-target/off-target effects. SUMMARY:MSLN is expressed in solid tumor patients, with no differences in expression among histologies. MSLN expression is associated with Fédération Internationale de Gynécologie et d'Obstétrique (FIGO) stage and platinum sensitivity. Higher MSLN expression is detected among primary ovarian cancer patients and correlates with better survival data in HGSOC patients only. According to our data, treatment strategies targeting MSLN should be offered in first line setting rather than in relapse.

Supplementary Figure 5 | Clustering (n=106) and survival analysis (n=70) on kinome mutations and CNA. (A) Disease-specific survival of patients (n=70) grouped on ARID1A status, (B) PIK3CA status and (C) ARID1A + PIK3CA status. (D) Consensus clustering, with maximum group count set to 10 and clustering optimization with 1000 repetitions maximum, shows adding of tumors (n=106) as consensus index (horizontally) and empirical cumulative distribution (vertically) for 1-10 clusters. (E) Area under the curve plot of decrease in friction from 1-10 clusters. (F) Heatmap distribution of tumors in 8 clusters. (G) Disease-specific survival of patients (n=70) in 8 clusters, (H) cluster 3 vs. other clusters. (I) Disease-specific survival in advanced stage OCCC patients (FIGO 2C-4), Log rank (Mantel-Cox) was used for statistical analysis. (J) Nonsynonymous mutation distribution in genes involved in the frequently mutated PI3K/AKT/mTOR (blue) and MAPK pathway (yellow), ERBB family of receptor tyrosine kinases (green) and DNA repair pathway (red) as well as ARID1A and PALB2 are shown with OncoPrint. CNA in each mutated gene are added. The 106 OCCC tumors that were both kinome sequenced and SNP arrayed are shown on the horizontal axis grouped on tumor clusters and ordered on total event frequency in the subsequently altered pathways. (K) Oncoprint from advanced stage patients in cluster 3 vs. other clusters, shown on the horizontal axis grouped on tumor clusters and ordered on total event frequency in the subsequent altered pathways, ordered as aforementioned.

Supplementary Figure 4 | Whole genome CNA heatmap. (A) Genome-wide CNA heatmap profiles of all 108 OCCC tumors (above), 63 ARID1A wildtype tumors (middle) and 45 ARID1A mutant tumors (below). (B) Significant CNA plot of only ARID1A wildtype tumors (n=63) and (C) only ARID1A mutant tumors (n=45) as determined by GISTIC analysis. All kinases and cancer-related genes from the kinome sequencing gene panel that were focally significantly amplified (red) or deleted (blue) are indicated along the chromosomes vertically. Chromosomal location and total amount of tumors harboring the event are annotated with each gene name. False-discovery rate (FDR) 0.05 threshold, indicated by the green line, and G-score are shown along the horizontal axis.

Supplementary Table 3 | Binomial testing of kinome sequencing variants.

Supplementary Table 1 | Gene set for kinome sequencing.

Supplementary Figure 7 | PDX alteration status and p-S6 staining. (A) Nonsynonymous mutation distribution in PDX.155, PDX.180 and PDX.247 in genes involved in the frequently mutated PI3K/AKT/mTOR (blue) and MAPK pathway (yellow), ERBB family of receptor tyrosine kinases (green) and DNA repair pathway (red) as well as ARID1A and PALB2 are shown with OncoPrint. CNA in each mutated gene are added. (B) PDX.155, (C) PDX.180 and (D) PDX.247 representative p-S6 expression after 21 days of vehicle or AZD8055 treatment.

Supplementary Figure 2 | Significantly mutated genes AKT1, PIK3R1, ERBB3, FBXW7, ATM, CHEK2 and MYO3A. Schematics of the identified novel significantly mutated genes (A) AKT1, (B) PIK3R1, (C) ERBB3, (D) FBXW7, (E) ATM, (F) CHEK2 and (G) MYO3A in OCCC. Mutation marks are shown in black (truncating), red (SIFT and PolyPhen damaging prediction), yellow (SIFT or PolyPhen damaging prediction) or white (SIFT and PolyPhen benign prediction). Mutation effects are indicated with a black spot when paired control was available and written in black (previously described mutation) or red (novel mutations).

Supplementary Figure 3 | Mutation distribution in OCCC. (A) Nonsynonymous mutation distribution in genes involved in the frequently mutated PI3K/AKT/mTOR (blue) and MAPK pathway (yellow), ERBB family of receptor tyrosine kinases (green) and DNA repair (red) pathway is shown. In this OncoPrint, kinome sequenced OCCC tumors are depicted on the horizontal axis and ordered on mutation frequency in the subsequent altered pathways, represented vertically on the right. ARID1A mutant tumors are displayed at the top. (B) BioVenn diagrams demonstrating overlap in ARID1A mutant tumors and PI3K/AKT/mTOR, MAPK and DNA repair pathway and ERBB family of receptor tyrosine kinases mutant tumors (n=103). On the right, PI3K/AKT/mTOR and MAPK pathway, the ERBB family of receptor tyrosine kinases and DNA repair pathway mutations within only ARID1A wildtype tumors (n=49) and only ARID1A mutant tumors (n=44). All data in A and B is derived from 122 kinome-sequenced OCCC tumors, overlap in BioVenn diagram circles is proportional to group overlap.

Legends of supplementary figures and tables

Supplementary Table 4 | Clinicopathological characteristics (n=70 patients).

Supplementary Figure 6 | mTORC1, mTORC1/2 and PI3K-mTORC1/2 inhibitor sensitivities. (A) IC50 of the mTORC1 inhibitor temsirolimus from COSMICs drug screening database in all cancer cell lines vs. ovarian cancer cell lines, horizontal lines indicate geometric mean. (B) MTT assay curves from AZD8055 (left) or everolimus (right) treatment on the aforementioned cell line panel. (C) Expression of p-AKT308, p-AKT473 and p-S6 after 24 (up) and 72h (down) exposure to 100 nM everolimus, AZD8055 or MLN0128 in the OCCC cell lines KOC7C or JHOC5 determined by Western blot. β-Actin was used as loading control. Results are representative from n=2 experiments. (D) Expression of p-AKT308, p-AKT473, p-S6 and (cleaved) PARP after 48h exposure to increasing concentrations of everolimus, AZD8055 and dactolisib in the OCCC cell lines KOC7C (up) and JHOC5 (down) determined by Western blot. β-Actin was used as loading control. Results are representative from n=2 experiments. (E) Everolimus, AZD8055, GDC0941 and selumetinib IC50 determined for 14 OCCC cell lines (ES2, KOC7C, SMOV2, JHOC5, RMG1, OVMANA, HAC2, OV207, OVTOKO, TOV21G, OVAS, OVCA429, TUOC1 and RMG2) and dactolisib IC50 determined for 7 OCCC cell lines (ES2, KOC7C, SMOV2, JHOC5, RMG1, OVMANA and HAC2) by MTT assay. Selumetinib IC50 of KOC7C was not reached at maximum used concentration of 25 uM. Horizontal lines indicate geometric mean. Data is derived from n{greater than or equal to}2 experiments. (F) Long-term proliferation assay after exposure to increasing concentrations of AZD8055 and dactolisib. Results are representative from n=3 experiments.

Introduction/Background* Mesothelin (MSLN) is a CA125 binding protein that mediates cell adhesion. This interaction was suggested to play a role in the peritoneal metastasis development. In preclinical models, MSLN overexpression activates the PI3K/AkT, NFκB, and MAPK/ERK pathways, to promote cell proliferation, migration and metastasis. For these reasons, MSLN represents an attractive molecule for targeted ovarian cancer (OC) therapies. Methodology Paraffin-embedded tumor tissue samples from 113 primary OC patients were selected from TOC biobank (www.toc-network.de) and assessed for the immunohistochemical expression of MSLN on Tissue Microarray. For 51 included HGSOC patients, also paired recurrent samples were available and selected for MSLN evaluation. All patients were treated at Charité Medical University Berlin, Germany, through primary cytoreduction followed by platinum-based chemotherapy. MSLN expression profiles were correlated with patients' clinic-pathological and survival data. MSLN expression was also compared between paired primary and recurrent HGSOC samples. Result(s)* 164 samples were assessed for MSLN expression (113 primary OC and 51 recurrent OC). Among the primary OC cohort, results showed that MSLN(+) samples were 85% of cases (96/113), whereas MSLN was negative in the remaining 15% of cases (17/113). MSLN expression did not differ among different OC histological subtypes (serous, clear cells and endometrioid), but MSLN(+) samples were diagnosed more frequent in the group of advanced FIGO stage (65/96 vs 31/96, p=0.022) and in platinum sensitive patients (85/96 vs 11/96, p=0.001). Survival analysis showed that MSLN(+) was associated with a significant survival advantage at 5yOS (p=0.022) in HGSOC patients. No survival impact (5yPFS and/or 5yOS) of MSLN expression could be detected for other OC histologies. Pairwise analysis on paired primary and recurrent HGSOC, also revealed that MSLN(+) tumors were more frequent among primary rather than recurrent HGSOC (46/51 vs 38/51,p=0.012); Furthermore, Spearman test showed a significant correlation among primary and recurrent samples in terms of MSLN expression decrease at recurrence (p=0.003). Conclusion* Overexpression of MSLN was observed in FIGO advanced stage and in platinum sensitive primary OC patients. MSLN expression was equally distributed among different OC histologies, but in HGSOC conferred survival advantage. Moreover, its expression significantly decreased from primary to recurrent OC.

Introduction/Background* Recently 3 Phase III first-line studies, PAOLA-1/ENGOT-ov25 (Ray-Coquard et al. NEJM 2019). PRIMA/ENGOT-ov26/GOG-3012 (Gonzales Martin et al. NEJM 2019) and VELIA/GOG-3005 (Coleman et al. NEJM 2019) have demonstrated that the addition of a PARP inhibitor (PARPi) to platinum-based therapy+/-bevacizumab improved progression-free survival (PFS) in advanced ovarian cancer (AOC) patients. The benefit was greater when the tumor was homologous recombination deficient (HRD) according to Myriad myChoice test, independently of BRCA status. The PAOLA-1 olaparib+bevacizumab maintenance regimen was approved in USA/Europe/Japan for HRD positive patients. The European initiative aims at evaluating various HRD tests on PAOLA-1 tumor samples to identify new reliable and feasible HRD tests Methodology The HRD initiative has 2 components; one based on artificial intelligence with various partners and the other, the European HRD ENGOT initiative (EHEI), is led by academic research laboratories (RL) from ENGOT groups. The HRD test evaluation protocol for the EHEI RL includes 3 phases. The phase 1 (2019/12) brought together European RL. Because non-BRCA Homologous Recombination Repair (HRR) mutations have not been found predictive of PARPi activity in PAOLA-1 (Pujade-Lauraine et al, SGO 2021)) RL tests based on these mutations were not selected for the next phases. Phase 2 evaluated the correlation between RL tests and the Myriad myChoice test on tumor samples from 85 PAOLA-1 BRCA wild type patients using the KAPPA statistics. Phase 3 is the final PFS evaluation on more than 350 additional patient samples. Result(s)* A total of 20 RL from 21 ENGOT groups participated to the EHEI phase 1. Half of them had a test mainly based on an HRR gene panel. Three RL did not pursue for various reasons (capacity, financial or regulatory). The remaining 7 RL from 6 countries (table 1) completed the phase 2 in May 2021 and may proceed to phase 3. Conclusion* The EHEI is a unique collaboration of European academic laboratories involved in gynecologic oncology translational research with the aim of providing a reliable biomarker (HRD) for selecting AOC patients who could benefit most from PARPi+/-bevacizumab in first-line therapy. HRD tests performance will be described after their phase 3 is completed.

Introduction/Background* Malignant ovarian germ cell tumours (OGCT) often affect women of younger age at an early stage of disease who may desire fertility conservation. The Arbeitsgemeinschaft fuer Gynaekologische Onkologie (AGO) has established a clinicopathological (Current Ovarian geRm cell and SEx cord stromal Tumour Treatment strategies, CORSETT) database to describe treatment strategies and outcomes for these women. Methodology 20 German centres entered mixed retro- and prospective data of 56 FIGO stage I OGCT patients treated between 2000 to 2014 into the CORSETT database. An independent CORSETT pathology reference panel re-evaluated the primary histological diagnosis. A descriptive analysis of the treatment strategies, pregnancy rates and disease recurrence was conducted. Result(s)* Median age at diagnosis of patients with malignant dysgerminoma, mixed OGCT and teratoma was 28 (IQR 26 – 33), 33 (30 – 40) and 38 (29 – 44) years. FIGO IA/IB/IC stage distribution was 13/2/11 for dysgerminoma, 3/0/9 for mixed OGCT and 8/0/8 for teratoma patients. Laparoscopy was performed for 23 (69.7%) dysgerminoma, six (35.3%) mixed OGCT and eight (40%) teratoma patients and fertility-sparing surgery was provided for > 80% of all FIGO I OGCT patients. Intra-operative cyst rupture occurred in six (19.4%) dysgerminoma, five (41.6%) mixed OGCT and four (25.5%) teratoma patients and adjuvant chemotherapy was consequently given in one (3.5%) dysgerminoma, nine (81.2%) mixed OGCT and eight (47%) teratoma patients. Four (14.3%) dysgerminoma, three (27. 3%) mixed OGCT and four (23.5%) teratoma patients conceived after first line treatment. The disease reoccurred in two (7.1%) dysgerminoma, five (45.5%) mixed OGCT and two (11.8%) teratoma patients, predominantly intraperitoneally. No FIGO I OGCT patient died due to disease recurrence. Conclusion* Women with mixed OGCTs had a high risk of intra-operative cyst rupture and high recurrence rates despite FIFO stage I disease. These events had no impact though on overall survival rates.

Introduction/Background* Malignant sex cord-stromal cell tumours (SCST) account for about 7% of all ovarian malignancies and higher-level evidence on these tumours is scarce. The Arbeitsgemeinschaft fuer Gynaekologische Onkologie (AGO) has established a clinico-pathological database (Current Ovarian geRm cell and SEx cord stromal Tumour Treatment strategies, CORSETT) for this rare disease. Methodology 20 German centres entered mixed retro- and prospective data of SCST patients treated between 2000 to 2014 into the CORSETT database. Descriptive analyses of the treatment strategies, Kaplan-Meier curves and cox regression analyses for the survival analysis were conducted. Tumour reference pathology was provided. Result(s)* 235 patients with granulosa cell tumour (GCT, median age 52 years) and 24 patients with sertoli-leydig cell tumour (SLCT, median age 33.5 years) were included. Main surgical treatment strategy was laparoscopy (50.9%) and fertility-sparing surgery was performed in 105 GCT (54.9%) and in 14 (58.3%) SLCT patients. In 38 GCT (23.6%) and two (9.25%) SLCT patients, the tumour ruptured intra-operatively. 44 GCT (18.7%) and two (8.3%) SLCT patients received adjuvant systemic treatment (most common regimen: paclitaxel/carboplatinum, 52.3%). Median PFS was 80.4 months (95% CI 67.8 – 99.6) in GCT and was not reached in SLCT patients. 105 GCT patients (44.5%) experienced disease recurrence. Advanced FIGO stage (p = 0.04) and intra-operative cyst rupture (p = 0.02) was associated with disease recurrence. Conclusion* Fertility-sparing surgery was offered in most SCST patients in Germany over the past years, and intra-operative tumour rupture occurred in almost every fourth GCT patient. The most commonly applied adjuvant chemotherapy regimen was taxane and carboplatinum, which had however no effect on disease recurrence. Despite excellent overall survival rates in GCT and SLCT patients, recurrence rate in GCT patients was high.

Objective. Circulating tumor cells (CTCs) may represent a chronic stimulus for the immune system. In the present study we investigated the potential association of CTCs, the immune activation marker neopterin, and the ratio of kynurenine to tryptophan (Kyn/Trp) as a measure for tryptophan breakdown. Methods. Neopterin, tryptophan and kynurenine levels were measured in plasma samples from patients with benign gynecological diseases (n = 65) and with primary advanced epithelial ovarian cancer (EOC) at diagnosis (n = 216) and six months after adjuvant platinum-based chemotherapy (n = 45) by an enzyme-linked immunosorbent assay and high performance liquid chromatography. The presence of CTCs had been assessed in a previous study by qPCR-based analysis of CTC-related transcripts in the blood. The respective plasma levels in EOC and benign samples were compared using a two-tailed Chi(2) or Fisher's exact test. The associations of the analytes and Kyn/Trp with clinicopathological parameters, platinum-sensitivity, and the presence of CTC-related transcripts were assessed using a two-sided t-test. Associations with patient outcome were evaluated using Cox regression analysis. Results. In EOC, elevated Kyn/Trp and neopterin levels were associated with advanced disease, peritoneal carcinomatosis, ascites, sub-optimal debulking, poor response to therapy and worse outcome. Likewise, neopterin and Kyn/Trp were elevated in CTC-positive patients, both at diagnosis and at follow-up in platinum-sensitive disease. Conclusions. We observed concomitant alterations of CTCs and immune system related biomarkers suggesting that immune responses along with increase of neopterin and Kyn/Trp concentrations are not necessarily only located at the site of the tumor, but may also go on in the circulation. (C) 2018 Elsevier Inc. All rights reserved.

Abstract Purpose: Advanced-stage ovarian clear cell carcinoma (OCCC) is unresponsive to conventional platinum-based chemotherapy. Frequent alterations in OCCC include deleterious mutations in the tumor suppressor ARID1A and activating mutations in the PI3K subunit PIK3CA. In this study, we aimed to identify currently unknown mutated kinases in patients with OCCC and test druggability of downstream affected pathways in OCCC models. Experimental Design: In a large set of patients with OCCC (n = 124), the human kinome (518 kinases) and additional cancer-related genes were sequenced, and copy-number alterations were determined. Genetically characterized OCCC cell lines (n = 17) and OCCC patient–derived xenografts (n = 3) were used for drug testing of ERBB tyrosine kinase inhibitors erlotinib and lapatinib, the PARP inhibitor olaparib, and the mTORC1/2 inhibitor AZD8055. Results: We identified several putative driver mutations in kinases at low frequency that were not previously annotated in OCCC. Combining mutations and copy-number alterations, 91% of all tumors are affected in the PI3K/AKT/mTOR pathway, the MAPK pathway, or the ERBB family of receptor tyrosine kinases, and 82% in the DNA repair pathway. Strong p-S6 staining in patients with OCCC suggests high mTORC1/2 activity. We consistently found that the majority of OCCC cell lines are especially sensitive to mTORC1/2 inhibition by AZD8055 and not toward drugs targeting ERBB family of receptor tyrosine kinases or DNA repair signaling. We subsequently demonstrated the efficacy of mTORC1/2 inhibition in all our unique OCCC patient–derived xenograft models. Conclusions: These results propose mTORC1/2 inhibition as an effective treatment strategy in OCCC. Clin Cancer Res; 24(16); 3928–40. ©2018 AACR.

Tumor recurrence, following initial response to adjuvant chemotherapy, is a major problem in women with high-grade serous ovarian cancer (HGSOC). Microarray analysis of primary tumors has identified genes that may be useful in risk stratification/overall survival, but are of limited value in predicting the >70% rate for tumor recurrence. In this study, we performed RNA-Seq analysis of primary and recurrent HGSOC to first identify unique differentially expressed genes. From this dataset, we selected 21 archetypical coding genes and one noncoding RNA, based on statistically significant differences in their expression profile between tumors, for validation by qPCR in a larger cohort of 110 ovarian tumors (71 primary and 39 recurrent) and for testing association of specific genes with time-to-recurrence (TTR). Kaplan-Meier tests revealed that high expression of collagen type II, alpha 1 (COL2A1) was associated with delayed TTR (HR = 0.47, 95% CI: 0.27-0.82, p = 0.008), whereas low expression of the pseudogene, solute carrier family 6 member 10 (SLC6A10P), was associated with longer TTR (HR = 0.53, 95% CI: 0.30-0.93, p = 0.027). Notably, TTR was significantly delayed for tumors that simultaneously highly expressed COL2A1 and lowly expressed SLC6A10P (HR = 0.21, 95% CI: 0.082-0.54, p = 0.0011), an estimated median of 95 months as compared to an estimated median of 16 months for subjects expressing other levels of COL2A1 and SLC6A10P. Thus, evaluating expression levels of COL2A1 and SLC6A10P at primary surgery could be beneficial for clinically managing recurrence of HGSOC.