Compartment-specific multiomic profiling identifies SRC and GNAS as candidate drivers of epithelial-to-mesenchymal transition in ovarian carcinosarcoma.

BACKGROUND: Ovarian carcinosarcoma (OCS) is an exceptionally aggressive and understudied ovarian cancer type harbouring distinct carcinomatous and sarcomatous compartments. Here, we seek to identify shared and compartment-specific events that may represent potential therapeutic targets and candidate drivers of sarcomatous compartment formation through epithelial-to-mesenchymal transition (EMT). METHODS: We performed multiomic profiling (exome sequencing, RNA-sequencing, microRNA profiling) of paired carcinomatous and sarcomatous components in 12 OCS cases. RESULTS: While paired sarcomatous and carcinomatous compartments demonstrate substantial genomic similarities, multiple loci are recurrently copy number-altered between components; regions containing GNAS and SRC are recurrently gained within the sarcomatous compartment. CCNE1 gain is a common event in OCS, occurring more frequently than in high grade serous ovarian carcinoma (HGSOC). Transcriptomic analysis suggests increased MAPK activity and subtype switching toward poor prognosis HGSOC-derived transcriptomic subtypes within the sarcomatous component. The two compartments show global differences in microRNA profiles, with differentially expressed microRNAs targeting EMT-related genes (SIRT1, ZEB2) and regulators of pro-tumourigenic pathways (TGFß, NOTCH); chrX is a highly enriched target of these microRNAs and is also frequently deleted across samples. The sarcomatous component harbours significantly fewer CD8-positive cells, suggesting poorer immune engagement. CONCLUSION: CCNE1 gain and chrX loss are frequent in OCS. SRC gain, increased GNAS expression and microRNA dysregulation represent potential mechanisms driving sarcomatous compartment formation.

High throughput screening identifies dasatinib as synergistic with trametinib in low grade serous ovarian carcinoma.

BACKGROUND: Low grade serous ovarian carcinoma (LGSOC) is a distinct histotype of ovarian cancer characterised high levels of intrinsic chemoresistance, highlighting the urgent need for new treatments. High throughput screening in clinically-informative cell-based models represents an attractive strategy for identifying candidate treatment options for prioritisation in clinical studies. METHODS: We performed a high throughput drug screen of 1610 agents across a panel of 6 LGSOC cell lines (3 RAS/RAF-mutant, 3 RAS/RAF-wildtype) to identify novel candidate therapeutic approaches. Validation comprised dose-response analysis across 9 LGSOC models and 5 high grade serous comparator lines. RESULTS: 16 hits of 1610 screened compounds were prioritised for validation based on >50% reduction in nuclei counts in over half of screened cell lines at 1000 nM concentration. 11 compounds passed validation, and the four agents of greatest interest (dasatinib, tyrosine kinase inhibitor; disulfiram, aldehyde dehydrogenase inhibitor; carfilzomib, proteasome inhibitor; romidepsin, histone deacetylase inhibitor) underwent synergy profiling with the recently approved MEK inhibitor trametinib. Disulfiram demonstrated excellent selectivity for LGSOC versus high grade serous ovarian carcinoma comparator lines (P = 0.003 for IC50 comparison), while the tyrosine kinase inhibitor dasatinib demonstrated favourable synergy with trametinib across multiple LGSOC models (maximum zero interaction potency synergy score 46.9). The novel, highly selective Src family kinase (SFK) inhibitor NXP900 demonstrated a similar trametinib synergy profile to dasatinib, suggesting that SFK inhibition is the likely driver of synergy. CONCLUSION: Dasatinib and other SFK inhibitors represent novel candidate treatments for LGSOC and demonstrate synergy with trametinib. Disulfiram represents an additional treatment strategy worthy of investigation.

FANCD2 expression affects platinum response and further characteristics of high grade serous ovarian cancer in cells with different genetic backgrounds.

High-grade serous ovarian cancer (HGSOC) is the most prevalent subtype of ovarian cancer and demonstrates 5-year survival of just 40%. One of the major causes of mortality is the development of tumour resistance to platinum-based chemotherapy, which can be modulated by dysregulation of DNA damage repair pathways. We therefore investigated the contribution of the DNA interstrand crosslink repair protein FANCD2 to chemosensitivity in HGSOC. Increased FANCD2 protein expression was observed in some cell line models of platinum resistant HGSOC compared with paired platinum sensitive models. Knockdown of FANCD2 in some cell lines, including the platinum resistant PEO4, led to increased carboplatin sensitivity. Investigation into mechanisms of FANCD2 regulation showed that increased FANCD2 expression in platinum resistant cells coincides with increased expression of mTOR. Treatment with mTOR inhibitors resulted in FANCD2 depletion, suggesting that mTOR can mediate platinum sensitivity via regulation of FANCD2. Tumours from a cohort of HGSOC patients showed varied nuclear and cytoplasmic FANCD2 expression, however this was not significantly associated with clinical characteristics. Knockout of FANCD2 was associated with increased cell migration, which may represent a non-canonical function of cytoplasmic FANCD2. We conclude that upregulation of FANCD2, possibly mediated by mTOR, is a potential mechanism of chemoresistance in HGSOC and modulation of FANCD2 expression can influence platinum sensitivity and other tumour cell characteristics.

Closing the loop - the role of pathologists in digital and computational pathology research.

An increasing number of manuscripts related to digital and computational pathology are being submitted to The Journal of Pathology: Clinical Research as part of the continuous evolution from digital imaging and algorithm-based digital pathology to computational pathology and artificial intelligence. However, despite these technological advances, tissue analysis still relies heavily on pathologists' annotations. There are three crucial elements to the pathologist's role during annotation tasks: granularity, time constraints, and responsibility for the interpretation of computational results. Granularity involves detailed annotations, including case level, regional, and cellular features; and integration of attributions from different sources. Time constraints due to pathologist shortages have led to the development of techniques to expedite annotation tasks from cell-level attributions up to so-called unsupervised learning. The impact of pathologists may seem diminished, but their role is crucial in providing ground truth and connecting pathological knowledge generation with computational advancements. Measures to display results back to pathologists and reflections about correctly applied diagnostic criteria are mandatory to maintain fidelity during human-machine interactions. Collaboration and iterative processes, such as human-in-the-loop machine learning are key for continuous improvement, ensuring the pathologist's involvement in evaluating computational results and closing the loop for clinical applicability. The journal is interested particularly in the clinical diagnostic application of computational pathology and invites submissions that address the issues raised in this editorial.

RFWD3 modulates response to platinum chemotherapy and promotes cancer associated phenotypes in high grade serous ovarian cancer.

Background: DNA damage repair is frequently dysregulated in high grade serous ovarian cancer (HGSOC), which can lead to changes in chemosensitivity and other phenotypic differences in tumours. RFWD3, a key component of multiple DNA repair and maintenance pathways, was investigated to characterise its impact in HGSOC. Methods: RFWD3 expression and association with clinical features was assessed using in silico analysis in the TCGA HGSOC dataset, and in a further cohort of HGSOC tumours stained for RFWD3 using immunohistochemistry. RFWD3 expression was modulated in cell lines using siRNA and CRISPR/cas9 gene editing, and cells were characterised using cytotoxicity and proliferation assays, flow cytometry, and live cell microscopy. Results: Expression of RFWD3 RNA and protein varied in HGSOCs. In cell lines, reduction of RFWD3 expression led to increased sensitivity to interstrand crosslinking (ICL) inducing agents mitomycin C and carboplatin. RFWD3 also demonstrated further functionality outside its role in DNA damage repair, with RFWD3 deficient cells displaying cell cycle dysregulation, reduced cellular proliferation and reduced migration. In tumours, low RFWD3 expression was associated with increased tumour mutational burden, and complete response to platinum chemotherapy. Conclusion: RFWD3 expression varies in HGSOCs, which can lead to functional effects at both the cellular and tumour levels.

The survival benefit associated with complete macroscopic resection in epithelial ovarian cancer is histotype specific.

BACKGROUND: Complete macroscopic resection is a key factor associated with prolonged survival in ovarian cancer. However, most evidence derives from high-grade serous ovarian carcinoma, and the benefit of complete macroscopic resection in other histotypes is poorly characterized. We sought to determine which histotypes derive the greatest benefit from complete macroscopic resection to better inform future decisions on radical cytoreductive efforts. METHODS: We performed multivariable analysis of disease-specific survival across 2 independent patient cohorts to determine the magnitude of benefit associated with complete macroscopic resection within each histotype. RESULTS: Across both cohorts (Scottish: n = 1622; Surveillance, Epidemiology, and End Results [SEER]: n = 18 947), complete macroscopic resection was associated with prolonged disease-specific survival; this was more marked in the Scottish cohort (multivariable hazard ratio [HR] = 0.44, 95% confidence interval [CI] = 0.37 to 0.52 vs HR = 0.59, 95% CI = 0.57 to 0.62 in SEER). In both cohorts, clear cell ovarian carcinoma was among the histotypes to benefit most from complete macroscopic resection (multivariable HR = 0.23 and HR = 0.50 in Scottish and SEER cohorts, respectively); high-grade serous ovarian carcinoma patients demonstrated highly statistically significant and clinically meaningful survival benefit, but this was of lower magnitude than in clear cell ovarian carcinoma and endometrioid ovarian carcinoma across both cohorts. The benefit derived in low-grade serous ovarian carcinoma is also high (multivariable HR = 0.27 in Scottish cohort). Complete macroscopic resection was associated with prolonged survival in mucinous ovarian carcinoma patients in the SEER cohort (multivariable HR = 0.65), but the association failed to reach statistical significance in the Scottish cohort. CONCLUSIONS: The overall ovarian cancer patient population demonstrates clinically significant survival benefit associated with complete macroscopic resection; however, the magnitude of benefit differs between histotypes.

Ovarian carcinosarcoma is highly aggressive compared to other ovarian cancer histotypes.

Background: Ovarian carcinosarcoma (OCS) is an unusual ovarian cancer type characterized by distinct carcinomatous and sarcomatous components. OCS has been excluded from many of the pan-histotype studies of ovarian carcinoma, limiting our understanding of its behavior. Methods: We performed a multi-cohort cross-sectional study of characteristics and outcomes in ovarian cancer patients from Scotland (n=2082) and the Surveillance, Epidemiology and End Results Program (SEER, n=44946) diagnosed with OCS or one of the other major histotypes: high grade serous (HGSOC), endometrioid (EnOC), clear cell (CCOC), mucinous (MOC) or low grade serous ovarian carcinoma (LGSOC). Differences in overall survival were quantified using Cox regression models to calculate hazard ratios (HR). Results: Across both cohorts, OCS patients were significantly older at diagnosis compared to all other histotypes (median age at diagnosis 69 and 67 in Scottish and SEER cohorts) and demonstrated the shortest survival time upon univariable analysis. Within the Scottish cohort, 59.3% and 16.9% of OCS patients presented with FIGO stage III and IV disease, respectively; this was significantly higher than in EnOC, CCOC or MOC (P<0.0001 for all), but lower than in HGSOC (P=0.004). Multivariable analysis accounting for other prognostic factors identified OCS as independently associated with significantly shorter survival time compared to HGSOC, EnOC, LGSOC and MOC in both the Scottish (multivariable HR vs OCS: HGSOC 0.45, EnOC 0.39, LGSOC 0.26, MOC 0.43) and SEER cohorts (multivariable HR vs OCS: HGSOC 0.59, EnOC 0.34, LGSOC 0.30, MOC 0.81). Within the SEER cohort, OCS also demonstrated shorter survival compared to CCOC (multivariable HR 0.63, 95% CI 0.58-0.68), but this was not replicated within the Scottish cohort (multivariable HR for CCOC: 1.05, 95% CI 0.74-1.51). Within early-stage disease specifically (FIGO I-II or SEER localized stage), OCS was associated with the poorest survival of all histotypes across both cohorts. In the context of late-stage disease (FIGO III-IV or SEER distant stage), OCS, MOC and CCOC represented the histotypes with poorest survival. Conclusion: OCS is a unique ovarian cancer type that affects older women and is associated with exceptionally poor outcome, even when diagnosed at earlier stage. New therapeutic options are urgently required to improve outcomes.

Whole exome sequencing reveals diverse genomic relatedness between paired concurrent endometrial and ovarian carcinomas.

INTRODUCTION: Concurrent non-serous endometrial and ovarian tumours are often managed clinically as two separate primary tumours, but almost all exhibit evidence of a genomic relationship. METHODOLOGY: This study investigates the extent of relatedness using whole exome sequencing, which was performed on paired non-serous endometrial and ovarian carcinomas from 27 patients with concurrent tumours in a cohort with detailed clinicopathological annotation. Four whole exome sequencing-derived parameters (mutation, mutational burden, mutational signatures and mutant allele tumour heterogeneity scores) were used to develop a novel unsupervised model for the assessment of genomic similarity to infer genomic relatedness of paired tumours. RESULTS: This novel model demonstrated genomic relatedness across all four parameters in all tumour pairs. Mutations in PTEN, ARID1A, CTNNB1, KMT2D and PIK3CA occurred most frequently and 24 of 27 (89 %) tumour pairs shared identical mutations in at least one of these genes, with all pairs sharing mutations in a number of other genes. Ovarian endometriosis, CTNNB1 exon 3 mutation, and progression and death from disease were present across the similarity ranking. Mismatch repair deficiency was associated with less genomically similar pairs. DISCUSSION: Although there was diversity across the cohort, the presence of genomic similarity in all paired tumours supports the hypothesis that concurrent non-serous endometrial and ovarian carcinomas are genomically related and may have arisen from a common origin.