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Ovarian carcinosarcoma (OCS) is an aggressive and rare tumor type with limited treatment options. OCS is hypothesized to develop via the combination theory, with a single progenitor resulting in carcinomatous and sarcomatous components, or alternatively via the conversion theory, with the sarcomatous component developing from the carcinomatous component through epithelial-to-mesenchymal transition (EMT). In this study, we analyzed DNA variants from isolated carcinoma and sarcoma components to show that OCS from 18 women is monoclonal. RNA sequencing indicated that the carcinoma components were more mesenchymal when compared with pure epithelial ovarian carcinomas, supporting the conversion theory and suggesting that EMT is important in the formation of these tumors. Preclinical OCS models were used to test the efficacy of microtubule-targeting drugs, including eribulin, which has previously been shown to reverse EMT characteristics in breast cancers and induce differentiation in sarcomas. Vinorelbine and eribulin more effectively inhibited OCS growth than standard-of-care platinum-based chemotherapy, and treatment with eribulin reduced mesenchymal characteristics and N-MYC expression in OCS patient-derived xenografts. Eribulin treatment resulted in an accumulation of intracellular cholesterol in OCS cells, which triggered a downregulation of the mevalonate pathway and prevented further cholesterol biosynthesis. Finally, eribulin increased expression of genes related to immune activation and increased the intratumoral accumulation of CD8+ T cells, supporting exploration of immunotherapy combinations in the clinic. Together, these data indicate that EMT plays a key role in OCS tumorigenesis and support the conversion theory for OCS histogenesis. Targeting EMT using eribulin could help improve OCS patient outcomes. Significance: Genomic analyses and preclinical models of ovarian carcinosarcoma support the conversion theory for disease development and indicate that microtubule inhibitors could be used to suppress EMT and stimulate antitumor immunity.

Background Uterine leiomyosarcoma (uLMS) is a rare and aggressive gynaecological malignancy, with individuals with advanced uLMS having a five-year survival of Methods A cohort of 58 individuals with uLMS were screened for HR Deficiency (HRD) using whole genome sequencing (WGS), whole exome sequencing (WES) or NGS panel testing. Individuals identified to have HRD uLMS were offered PARPi therapy and clinical outcome details collected. Patient-derived xenografts (PDX) were generated for therapeutic targeting. Results All 13 uLMS samples analysed by WGS had a dominant COSMIC mutational signature 3; 11 of these had high genome-wide loss of heterozygosity (LOH) (> 0.2) but only two samples had a CHORD score > 50%, one of which had a homozygous pathogenic alteration in an HR gene (deletion in BRCA2). A further three samples harboured homozygous HRD alterations (all deletions in BRCA2), detected by WES or panel sequencing, with 5/58 (9%) individuals having HRD uLMS. All five individuals gained access to PARPi therapy. Two of three individuals with mature clinical follow up achieved a complete response or durable partial response (PR) with the subsequent addition of platinum to PARPi upon minor progression during initial PR on PARPi. Corresponding PDX responses were most rapid, complete and sustained with the PARP1-specific PARPi, AZD5305, compared with either olaparib alone or olaparib plus cisplatin, even in a paired sample of a BRCA2-deleted PDX, derived following PARPi therapy in the patient, which had developed PARPi-resistance mutations in PRKDC, encoding DNA-PKcs. Conclusions Our work demonstrates the value of identifying HRD for therapeutic targeting by PARPi and platinum in individuals with the aggressive rare malignancy, uLMS and suggests that individuals with HRD uLMS should be included in trials of PARP1-specific PARPi.

A list of copy number alterations, rearrangements and short variants detected by Foundation Medicine NGS

Supplementary Tables, Figures and Video legends, Tables 2,3,5 and all Supplementary Figures. Supplementary Table 2. Confirmation of cis configuration of BRCA1 primary and secondary mutations in case 4 by colony PCR. Supplementary Table 3. IC50 (mircoM) values of the PARPi and platinum drugs in parental OVCAR8 cell line and OVCAR8 RAD51C KO clone, and the fold change in IC50 values. Supplementary Table 5. Sequences of primers used for site-directed mutagenesis. Supplementary Figure 1. Foundation Medicine NGS analysis of the 12 cases with archival tissue and/or pre-treatment and post-progression biopsies. Supplementary Figure 2. Sanger sequencing trace of the primary and secondary BRCA1 mutations in cis configuration in case 4 post-progression biopsy sample. Supplementary Figure 3. In vitro response to PARP inhibitor therapy and platinum agents in RAD51C deficient cell lines, with primary or secondary mutations in RAD51C. Supplementary Figure 4. RAD51 foci formation in geminin positive cells deficient for RAD51C, complemented with primary or secondary mutations in RAD51C. Supplementary Figure 5. Diagram of HR reporter assay. Supplementary Figure 6. RAD51C expression in MCF10A cells and in yeast. Supplementary Figure 7. Analysis of serial sections by direct PCR sequencing approach of a post-progression biopsy containing multiple secondary mutations in RAD51C. Supplementary Figure 8. Molecular Dynamics Modeling of WT RAD51D protein and RAD51D protein with secondary mutation c.770_776delinsA, p.S257_R259delinsK. Supplementary Figure 9. In vitro response to PARP inhibitor therapy and cisplatin in RAD51D deficient CHO cell line, with primary or secondary mutation in RAD51D. Supplementary Figure 10. Examination of the parental PEO4 cell line, PEO4 cells with the homozygous frameshift RAD51D mutation (c.762_763del, D254E*fs72) in the same exon as the primary mutation and PEO4 cells with the homozygous secondary RAD51D mutation (c.770_776delinsA, S257_R259delinsK). Supplementary Figure 11. Modeling of tumor clonal fractions in the post-progression biopsy sample with germline RAD51C mutation and multiple secondary mutations.

Molecular Dynamics Modeling of WT RAD51D monofilament interaction with dsDNA.

Copy number estimation by SNP array in the archival tumor tissue of the patient identified to have a germline RAD51C mutation (c.577C

In high-grade serous ovarian carcinoma (HGSC), deleterious mutations in DNA repair gene RAD51C are established drivers of defective homologous recombination and are emerging biomarkers of PARP inhibitor (PARPi) sensitivity. RAD51C promoter methylation (meRAD51C) is detected at similar frequencies to mutations, yet its effects on PARPi responses remain unresolved.In this study, three HGSC patient-derived xenograft (PDX) models with methylation at most or all examined CpG sites in the RAD51C promoter show responses to PARPi. Both complete and heterogeneous methylation patterns were associated with RAD51C gene silencing and homologous recombination deficiency (HRD). PDX models lost meRAD51C following treatment with PARPi rucaparib or niraparib, where a single unmethylated copy of RAD51C was sufficient to drive PARPi resistance. Genomic copy number profiling of one of the PDX models using SNP arrays revealed that this resistance was acquired independently in two genetically distinct lineages.In a cohort of 12 patients with RAD51C-methylated HGSC, various patterns of meRAD51C were associated with genomic “scarring,” indicative of HRD history, but exhibited no clear correlations with clinical outcome. Differences in methylation stability under treatment pressure were also observed between patients, where one HGSC was found to maintain meRAD51C after six lines of therapy (four platinum-based), whereas another HGSC sample was found to have heterozygous meRAD51C and elevated RAD51C gene expression (relative to homozygous meRAD51C controls) after only neoadjuvant chemotherapy.As meRAD51C loss in a single gene copy was sufficient to cause PARPi resistance in PDX, methylation zygosity should be carefully assessed in previously treated patients when considering PARPi therapy.Significance:Homozygous RAD51C methylation is a positive predictive biomarker for sensitivity to PARP inhibitors, whereas a single unmethylated gene copy is sufficient to confer resistance.

All Supplementary Methods for the manuscript

Supplementary methods and figures

All supplementary tables for the manuscript

Ovarian carcinosarcoma (OCS) is an aggressive and rare tumor type with limited treatment options. OCS is hypothesized to develop via the combination theory, with a single progenitor resulting in carcinomatous and sarcomatous components, or alternatively via the conversion theory, with the sarcomatous component developing from the carcinomatous component through epithelial-to-mesenchymal transition (EMT). In this study, we analyzed DNA variants from isolated carcinoma and sarcoma components to show that OCS from 18 women is monoclonal. RNA sequencing indicated that the carcinoma components were more mesenchymal when compared with pure epithelial ovarian carcinomas, supporting the conversion theory and suggesting that EMT is important in the formation of these tumors. Preclinical OCS models were used to test the efficacy of microtubule-targeting drugs, including eribulin, which has previously been shown to reverse EMT characteristics in breast cancers and induce differentiation in sarcomas. Vinorelbine and eribulin more effectively inhibited OCS growth than standard-of-care platinum-based chemotherapy, and treatment with eribulin reduced mesenchymal characteristics and N-MYC expression in OCS patient-derived xenografts. Eribulin treatment resulted in an accumulation of intracellular cholesterol in OCS cells, which triggered a downregulation of the mevalonate pathway and prevented further cholesterol biosynthesis. Finally, eribulin increased expression of genes related to immune activation and increased the intratumoral accumulation of CD8+ T cells, supporting exploration of immunotherapy combinations in the clinic. Together, these data indicate that EMT plays a key role in OCS tumorigenesis and support the conversion theory for OCS histogenesis. Targeting EMT using eribulin could help improve OCS patient outcomes.Significance:Genomic analyses and preclinical models of ovarian carcinosarcoma support the conversion theory for disease development and indicate that microtubule inhibitors could be used to suppress EMT and stimulate antitumor immunity.

All supplementary figures for the manuscript

The RNase III enzyme Drosha has a central role in microRNA (miRNA) biogenesis, where it is required to release the stem-loop intermediate from primary (pri)-miRNA transcripts. However, it can also cleave stem-loops embedded within messenger (m)RNAs. This destabilizes the mRNA causing target gene repression and appears to occur primarily in stem cells. While pri-miRNA stem-loops have been extensively studied, such non-canonical substrates of Drosha have yet to characterized in detail. In this study, we employed high-throughput sequencing to capture all polyA-tailed RNAs that are cleaved by Drosha in mouse embryonic stem cells (ESCs) and compared the features of non-canonical versus miRNA stem-loop substrates. First, mRNA substrates are less efficiently processed than miRNA stem-loops. Sequence and structural analyses revealed that these mRNA substrates are also less stable and more likely to fold into alternative structures than miRNA stem-loops. Moreover, they lack the sequence and structural motifs found in miRNAs stem-loops that are required for precise cleavage. Notably, we discovered a non-canonical Drosha substrate that is cleaved in an inverse manner, which is a process that is normally inhibited by features in miRNA stem-loops. Our study thus provides valuable insights into the recognition of non-canonical targets by Drosha.

TP53 mutations are implicated in the progression of mucinous borderline tumors (MBOT) to mucinous ovarian carcinomas (MOC). Optimized immunohistochemistry (IHC) for TP53 has been established as a proxy for the TP53 mutation status in other ovarian tumor types. We aimed to confirm the ability of TP53 IHC to predict TP53 mutation status in ovarian mucinous tumors and to evaluate the association of TP53 mutation status with survival among patients with MBOT and MOC. Tumor tissue from an initial cohort of 113 women with MBOT/MOC was stained with optimized IHC for TP53 using tissue microarrays (75.2%) or full sections (24.8%) and interpreted using established criteria as normal or abnormal (overexpression, complete absence, or cytoplasmic). Cases were considered concordant if abnormal IHC staining predicted deleterious TP53 mutations. Discordant tissue microarray cases were re-evaluated on full sections and interpretational criteria were refined. The initial cohort was expanded to a total of 165 MBOT and 424 MOC for the examination of the association of survival with TP53 mutation status, assessed either by TP53 IHC and/or sequencing. Initially, 82/113 (72.6%) cases were concordant using the established criteria. Refined criteria for overexpression to account for intratumoral heterogeneity and terminal differentiation improved concordance to 93.8% (106/113). In the expanded cohort, 19.4% (32/165) of MBOT showed evidence for TP53 mutation and this was associated with a higher risk of recurrence, disease-specific death, and all-cause mortality (overall survival: HR = 4.6, 95% CI 1.5-14.3, p = 0.0087). Within MOC, 61.1% (259/424) harbored a TP53 mutation, but this was not associated with survival (overall survival, p = 0.77). TP53 IHC is an accurate proxy for TP53 mutation status with refined interpretation criteria accounting for intratumoral heterogeneity and terminal differentiation in ovarian mucinous tumors. TP53 mutation status is an important biomarker to identify MBOT with a higher risk of mortality.

Poly (ADP-ribose) polymerase inhibitors (PARPis) represent a therapeutic milestone in the management of epithelial ovarian cancer. The concept of ‘synthetic lethality’ is exploited by PARPi in tumors with defects in DNA repair pathways, particularly homologous recombination deficiency. The use of PARPis has been increasing since its approval as maintenance therapy, particularly in the first-line setting. Therefore, resistance to PARPi is an emerging issue in clinical practice. It brings an urgent need to elucidate and identify the mechanisms of PARPi resistance. Ongoing studies address this challenge and investigate potential therapeutic strategies to prevent, overcome, or re-sensitize tumor cells to PARPi. This review aims to summarize the mechanisms of resistance to PARPi, discuss emerging strategies to treat patients post-PARPi progression, and discuss potential biomarkers of resistance.

A central problem in genomics is understanding the effect of individual DNA variants. Multiplexed Assays of Variant Effect (MAVEs) can help address this challenge by measuring all possible single nucleotide variant effects in a gene or regulatory sequence simultaneously. Here we describe MaveDB v2, which has become the database of record for MAVEs. MaveDB now contains a large fraction of published studies, comprising over two hundred datasets and three million variant effect measurements. We created tools and APIs to streamline data submission and access, transforming MaveDB into a hub for the analysis and dissemination of these impactful datasets.

In high-grade serous ovarian carcinoma (HGSC), deleterious mutations in DNA repair gene

High-grade epithelial ovarian carcinomas containing mutated