High expression of the p53 isoform γ is associated with reduced progression-free survival in uterine serous carcinoma.

BACKGROUND: Uterine serous carcinoma (USC) is a rare but aggressive subtype of endometrial carcinoma. Large-scale comprehensive efforts have resulted in an improved molecular understanding of its pathogenesis, and the p53 pathway has been proposed as a key player and is potentially targetable. Here we attempt to further portray the p53 pathway in USC by assessing p53 isoform expression. METHODS: We applied quantitative Real-Time PCRs (RT-qPCR) for expression analyses of total p53 mRNA as well as quantitative distinction of p53ß, p53γ, and the total mRNA of amino-terminal truncated Δ40p53 and Δ133p53 in a retrospective cohort of 37 patients with USC. TP53 mutation status was assessed by targeted massive parallel sequencing. Findings were correlated with clinical data. RESULTS: The p53 isoform expression landscape in USCs was heterogeneous and dominated by total Δ133p53, while the distinct p53ß and p53γ variants were found at much lower levels. The isoform expression profiles varied between samples, while their expression was independent of TP53 mutation status. We found high relative p53γ expression to be associated with reduced progression-free survival (PFS). CONCLUSIONS: This is the first indication that elevated p53γ expression is associated with reduced PFS in USC. This single-center study may offer some insight in the landscape of p53 isoform expression in USC, but further validation studies are crucial to understand the context-dependent and tissue-specific role of the p53 isoform network in gynecological cancer.

Occludin is overexpressed in tubo-ovarian high-grade serous carcinoma compared to mesothelioma and is a marker of tumor progression and chemoresistance.

The objective of this study was to analyze the expression and prognostic role of the tight junction protein occludin in high-grade serous carcinoma (HGSC). Occludin protein expression by immunohistochemistry was analyzed in 602 HGSC (417 effusions, 185 surgical specimens). Expression in mesothelioma (n = 87; 45 effusions, 42 surgical specimens) was studied for comparative purposes. Occludin protein expression was found in 587/602 (98%) HGSC vs. 40/87 (46%) mesotheliomas and was predominantly limited to < 5% of cells in the latter (p < 0.001). Occludin was additionally overexpressed in HGSC effusions compared to surgical specimens (p < 0.001) and was overexpressed in post-chemotherapy effusions compared to chemo-naive effusions tapped at diagnosis (p = 0.015). Occludin expression in HGSC surgical specimens was associated with poor chemoresponse (p < 0.001) and primary resistance (p = 0.001). Expression in effusions and surgical specimens was unrelated to survival (p > 0.05). In conclusion, occludin expression is higher in HGSC compared to mesothelioma, and this protein is overexpressed in HGSC effusions, possibly reflecting changes in adhesion related to anchorage-independent growth in this microenvironment. Overexpression in post-chemotherapy compared to chemo-naïve effusions suggest a role in disease progression. Occludin expression in surgical specimens may be related to chemoresistance.

Patient-derived acellular ascites fluid affects drug responses in ovarian cancer cell lines through the activation of key signalling pathways.

Malignant ascites is commonly produced in advanced epithelial ovarian cancer (EOC) and serves as unique microenvironment for tumour cells. Acellular ascites fluid (AAF) is rich in signalling molecules and has been proposed to play a role in the induction of chemoresistance. Through in vitro testing of drug sensitivity and by assessing intracellular phosphorylation status in response to mono- and combination treatment of five EOC cell lines after incubation with AAFs derived from 20 different patients, we investigated the chemoresistance-inducing potential of ascites. We show that the addition of AAFs to the culture media of EOC cell lines has the potential to induce resistance to standard-of-care drugs (SCDs). We also show that AAFs induce time- and concentration-dependent activation of downstream signalling to signal transducer and activator of transcription 3 (STAT3), and concomitantly altered phosphorylation of mitogen-activated protein kinase kinase (MEK), phosphoinositide 3-kinase (PI3K)-protein kinase B (AKT) and nuclear factor NF-kappa-B (NFκB). Antibodies targeting the interleukin-6 receptor (IL6R) effectively blocked phosphorylation of STAT3 and STAT1. Treatments with SCDs were effective in reducing cell viability in only a third of 30 clinically relevant conditions examined, defined as combinations of drugs, different cell lines and AAFs. Combinations of SCDs and novel therapeutics such as trametinib, fludarabine or rapamycin were superior in another third. Notably, we could nominate effective treatment combinations in almost all conditions except in 4 out of 30 conditions, in which trametinib or fludarabine showed higher efficacy alone. Taken together, our study underscores the importance of the molecular characterisation of individual patients' AAFs and the impact on treatment resistance as providing clinically meaningful information for future precision treatment approaches in EOC.

Evolutionary mode and timing of dissemination of high-grade serous carcinomas.

Dissemination within the peritoneal cavity is a main determinant of poor patient outcomes from high-grade serous carcinomas (HGSCs). The dissemination process is poorly understood from a cancer evolutionary perspective. We reconstructed the evolutionary trajectories across a median of 5 tumor sites and regions from each of 23 patients based on deep whole-exome sequencing. Polyclonal cancer origin was detected in 1 patient. Ovarian tumors had more complex subclonal architectures than other intraperitoneal tumors in each patient, which indicated that tumors developed earlier in the ovaries. Three common modes of dissemination were identified, including monoclonal or polyclonal dissemination of monophyletic (linear) or polyphyletic (branched) subclones. Mutation profiles of initial or disseminated clones varied greatly among cancers, but recurrent mutations were found in 7 cancer-critical genes, including TP53, BRCA1, BRCA2, and DNMT3A, and in the PI3K/AKT1 pathway. Disseminated clones developed late in the evolutionary trajectory models of most cancers, in particular in cancers with DNA damage repair deficiency. Polyclonal dissemination was predicted to occur predominantly as a single and rapid wave, but chemotherapy exposure was associated with higher genomic diversity of disseminated clones. In conclusion, we described three common evolutionary dissemination modes across HGSCs and proposed factors associated with dissemination diversity.

Cross-Cancer Genome-Wide Association Study of Endometrial Cancer and Epithelial Ovarian Cancer Identifies Genetic Risk Regions Associated with Risk of Both Cancers.

BACKGROUND: Accumulating evidence suggests a relationship between endometrial cancer and ovarian cancer. Independent genome-wide association studies (GWAS) for endometrial cancer and ovarian cancer have identified 16 and 27 risk regions, respectively, four of which overlap between the two cancers. We aimed to identify joint endometrial and ovarian cancer risk loci by performing a meta-analysis of GWAS summary statistics from these two cancers. METHODS: Using LDScore regression, we explored the genetic correlation between endometrial cancer and ovarian cancer. To identify loci associated with the risk of both cancers, we implemented a pipeline of statistical genetic analyses (i.e., inverse-variance meta-analysis, colocalization, and M-values) and performed analyses stratified by subtype. Candidate target genes were then prioritized using functional genomic data. RESULTS: Genetic correlation analysis revealed significant genetic correlation between the two cancers (rG = 0.43, P = 2.66 × 10-5). We found seven loci associated with risk for both cancers (P Bonferroni < 2.4 × 10-9). In addition, four novel subgenome-wide regions at 7p22.2, 7q22.1, 9p12, and 11q13.3 were identified (P < 5 × 10-7). Promoter-associated HiChIP chromatin loops from immortalized endometrium and ovarian cell lines and expression quantitative trait loci data highlighted candidate target genes for further investigation. CONCLUSIONS: Using cross-cancer GWAS meta-analysis, we have identified several joint endometrial and ovarian cancer risk loci and candidate target genes for future functional analysis. IMPACT: Our research highlights the shared genetic relationship between endometrial cancer and ovarian cancer. Further studies in larger sample sets are required to confirm our findings.

CD24-targeted fluorescence imaging in patient-derived xenograft models of high-grade serous ovarian carcinoma.

BACKGROUND: The survival rate of patients with advanced high-grade serous ovarian carcinoma (HGSOC) remains disappointing. Clinically translatable orthotopic cell line xenograft models and patient-derived xenografts (PDXs) may aid the implementation of more personalised treatment approaches. Although orthotopic PDX reflecting heterogeneous molecular subtypes are considered the most relevant preclinical models, their use in therapeutic development is limited by lack of appropriate imaging modalities. METHODS: We developed novel orthotopic xenograft and PDX models for HGSOC, and applied a near-infrared fluorescently labelled monoclonal antibody targeting the cell surface antigen CD24 for non-invasive molecular imaging of epithelial ovarian cancer. CD24-Alexa Fluor 680 fluorescence imaging was compared to bioluminescence imaging in three orthotopic cell line xenograft models of ovarian cancer (OV-90luc+, Skov-3luc+ and Caov-3luc+, n = 3 per model). The application of fluorescence imaging to assess treatment efficacy was performed in carboplatin-paclitaxel treated orthotopic OV-90 xenografts (n = 10), before the probe was evaluated to detect disease progression in heterogenous PDX models (n = 7). FINDINGS: Application of the near-infrared probe, CD24-AF680, enabled both spatio-temporal visualisation of tumour development, and longitudinal therapy monitoring of orthotopic xenografts. Notably, CD24-AF680 facilitated imaging of multiple PDX models representing different histological subtypes of the disease. INTERPRETATION: The combined implementation of CD24-AF680 and orthotopic PDX models creates a state-of-the-art preclinical platform which will impact the identification and validation of new targeted therapies, fluorescence image-guided surgery, and ultimately the outcome for HGSOC patients. FUNDING: This study was supported by the H2020 program MSCA-ITN [675743], Helse Vest RHF, and Helse Bergen HF [911809, 911852, 912171, 240222, HV1269], as well as by The Norwegian Cancer Society [182735], and The Research Council of Norway through its Centers of excellence funding scheme [223250, 262652].

Influence of p53 Isoform Expression on Survival in High-Grade Serous Ovarian Cancers.

High-grade serous ovarian carcinoma (HGSOC) is characterised by alterations in the p53 pathway. The expression levels of p53 isoforms have been shown to be associated with patient survival in several cancers. This study examined the predictive and prognostic effects of the expression levels of TP53 pre-mRNA splicing isoforms and TP53 mutations in tumour tissues in 40 chemotherapy responders and 29 non-responders with HGSOC. The mRNA expression levels from total p53, and total Δ133p53, p53ß, p53γ isoforms were determined by RT-qPCR, and TP53 mutation status by targeted massive parallel sequencing. The results from these analyses were correlated with the clinical outcome parameters. No differential expression of p53 isoforms could be detected between the chemosensitive and chemoresistant subgroups. In a multivariate Cox regression model, high levels of total Δ133p53 were found to be an independent prognosticator for improved overall survival (HR = 0.422, p = 0.018, 95% CI: 0.207-0.861) and reached borderline significance for progression-free survival (HR = 0.569, p = 0.061, 95% CI: 0.315-1.027). TP53 mutations resulting in loss of function or located at known hotspots were predictive of tumour characteristics and disease progression. These findings suggest that total Δ133p53 mRNA can be a biomarker for survival in HGSOC.

The HDACi Panobinostat Shows Growth Inhibition Both In Vitro and in a Bioluminescent Orthotopic Surgical Xenograft Model of Ovarian Cancer.

BACKGROUND: In most epithelial ovarian carcinomas (EOC), epigenetic changes are evident, and overexpression of histone deacetylases (HDACs) represents an important manifestation. In this study, we wanted to evaluate the effects of the novel HDAC inhibitor (HDACi) panobinostat, both alone and in combination with carboplatin, on ovarian cancer cell lines and in a murine bioluminescent orthotopic surgical xenograft model for EOC. METHODS: The effects of panobinostat, both alone and in combination with carboplatin, on proliferation and apoptosis in ovarian cancer cell lines, were evaluated using colony and WST-1 assays, Hoechst staining and flow cytometry analysis. In addition, mechanisms were characterised by western blotting and phosphoflow analysis. Immuno-deficient mice were engrafted orthotopically with SKOV-3luc+ cells and serial bioluminescence imaging monitored the effects of treatment with panobinostat and/or carboplatin and/or surgery. Survival parameters were also measured. RESULTS: Panobinostat treatment reduced cell growth and diminished cell viability, as shown by the induced cell cycle arrest and apoptosis in vitro. We observed increased levels of cleaved PARP and caspase-3, downregulation of cdc2 protein kinase, acetylation of H2B and higher pH2AX expression. The combined administration of carboplatin and panobinostat synergistically increased the anti-tumour effects compared to panobinostat or carboplatin treatment alone. In our novel ovarian cancer model, the mice showed significantly higher rates of survival when treated with panobinostat, carboplatin or a combination of both, compared to the controls. Panobinostat was as efficient as carboplatin regarding prolongation of survival. No significant additional effect on survival was observed when surgery was combined with carboplatin/panobinostat treatment. CONCLUSIONS: Panobinostat demonstrates effective in vitro growth inhibition in ovarian cancer cells. The efficacy of panobinostat and carboplatin was equal in the orthotopic EOC model used. We conclude that panobinostat is a promising therapeutic alternative that needs to be further assessed for the treatment of EOC.