Integrative multi-omics analyses to identify the genetic and functional mechanisms underlying ovarian cancer risk regions.

To identify credible causal risk variants (CCVs) associated with different histotypes of epithelial ovarian cancer (EOC), we performed genome-wide association analysis for 470,825 genotyped and 10,163,797 imputed SNPs in 25,981 EOC cases and 105,724 controls of European origin. We identified five histotype-specific EOC risk regions (p value <5 × 10-8) and confirmed previously reported associations for 27 risk regions. Conditional analyses identified an additional 11 signals independent of the primary signal at six risk regions (p value <10-5). Fine mapping identified 4,008 CCVs in these regions, of which 1,452 CCVs were located in ovarian cancer-related chromatin marks with significant enrichment in active enhancers, active promoters, and active regions for CCVs from each EOC histotype. Transcriptome-wide association and colocalization analyses across histotypes using tissue-specific and cross-tissue datasets identified 86 candidate susceptibility genes in known EOC risk regions and 32 genes in 23 additional genomic regions that may represent novel EOC risk loci (false discovery rate <0.05). Finally, by integrating genome-wide HiChIP interactome analysis with transcriptome-wide association study (TWAS), variant effect predictor, transcription factor ChIP-seq, and motifbreakR data, we identified candidate gene-CCV interactions at each locus. This included risk loci where TWAS identified one or more candidate susceptibility genes (e.g., HOXD-AS2, HOXD8, and HOXD3 at 2q31) and other loci where no candidate gene was identified (e.g., MYC and PVT1 at 8q24) by TWAS. In summary, this study describes a functional framework and provides a greater understanding of the biological significance of risk alleles and candidate gene targets at EOC susceptibility loci identified by a genome-wide association study.

Mathematical modeling of the evolution of resistance and aggressiveness of high-grade serous ovarian cancer from patient CA-125 time series.

A time-series analysis of serum Cancer Antigen 125 (CA-125) levels was performed in 791 patients with high-grade serous ovarian cancer (HGSOC) from the Australian Ovarian Cancer Study to evaluate the development of chemoresistance and response to therapy. To investigate chemoresistance and better predict the treatment effectiveness, we examined two traits: resistance (defined as the rate of CA-125 change when patients were treated with therapy) and aggressiveness (defined as the rate of CA-125 change when patients were not treated). We found that as the number of treatment lines increases, the data-based resistance increases (a decreased rate of CA-125 decay). We use mathematical models of two distinct cancer cell types, treatment-sensitive cells and treatment-resistant cells, to estimate the values and evolution of the two traits in individual patients. By fitting to individual patient HGSOC data, our models successfully capture the dynamics of the CA-125 level. The parameters estimated from the mathematical models show that patients with inferred low growth rates of treatment-sensitive cells and treatment-resistant cells (low model-estimated aggressiveness) and a high death rate of treatment-resistant cells (low model-estimated resistance) have longer survival time after completing their second-line of therapy. These findings show that mathematical models can characterize the degree of resistance and aggressiveness in individual patients, which improves our understanding of chemoresistance development and could predict treatment effectiveness in HGSOC patients.

A comparison of DNA sequencing and gene expression profiling to assist tissue of origin diagnosis in cancer of unknown primary.

Cancer of unknown primary (CUP) is a syndrome defined by clinical absence of a primary cancer after standardised investigations. Gene expression profiling (GEP) and DNA sequencing have been used to predict primary tissue of origin (TOO) in CUP and find molecularly guided treatments; however, a detailed comparison of the diagnostic yield from these two tests has not been described. Here, we compared the diagnostic utility of RNA and DNA tests in 215 CUP patients (82% received both tests) in a prospective Australian study. Based on retrospective assessment of clinicopathological data, 77% (166/215) of CUPs had insufficient evidence to support TOO diagnosis (clinicopathology unresolved). The remainder had either a latent primary diagnosis (10%) or clinicopathological evidence to support a likely TOO diagnosis (13%) (clinicopathology resolved). We applied a microarray (CUPGuide) or custom NanoString 18-class GEP test to 191 CUPs with an accuracy of 91.5% in known metastatic cancers for high-medium confidence predictions. Classification performance was similar in clinicopathology-resolved CUPs - 80% had high-medium predictions and 94% were concordant with pathology. Notably, only 56% of the clinicopathology-unresolved CUPs had high-medium confidence GEP predictions. Diagnostic DNA features were interrogated in 201 CUP tumours guided by the cancer type specificity of mutations observed across 22 cancer types from the AACR Project GENIE database (77,058 tumours) as well as mutational signatures (e.g. smoking). Among the clinicopathology-unresolved CUPs, mutations and mutational signatures provided additional diagnostic evidence in 31% of cases. GEP classification was useful in only 13% of cases and oncoviral detection in 4%. Among CUPs where genomics informed TOO, lung and biliary cancers were the most frequently identified types, while kidney tumours were another identifiable subset. In conclusion, DNA and RNA profiling supported an unconfirmed TOO diagnosis in one-third of CUPs otherwise unresolved by clinicopathology assessment alone. DNA mutation profiling was the more diagnostically informative assay. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Mucinous ovarian carcinoma: A survey of practice in Australia and New Zealand.

BACKGROUND: Mucinous ovarian carcinoma (MOC) is a rare ovarian cancer with limited evidence to support clinical care. AIMS: We undertook a clinician survey to better understand current practice in treating MOC in Australia and New Zealand, and to determine any features associated with variation in care. In addition, we aimed to understand future research priorities. METHODS: A RedCap survey was distributed to clinician members of the Australia New Zealand Gynaecological Oncology Group (ANZGOG). Questions included respondent demographics, three case studies and future research priorities. Clinicians were asked questions specific to their speciality. RESULTS: Respondents (n = 47) were commonly experienced gynae-oncology specialists, most often surgical (38%) or medical (30%) oncologists. There was good consensus for surgical approaches for stage I disease; however, variation in practice was noted for advanced or recurrent MOC. Variation was also observed for medical oncologists; in early-stage disease there was no clear consensus on whether to offer chemotherapy, or which regimen to recommend. For advanced and recurrent disease a wide range of chemotherapy options was considered, with a trend away from an ovarian-type toward gastrointestinal (GI)-type regimens in advanced MOC. This practice was reflected in future research priorities, with 'Is a GI chemotherapy regimen better than an ovarian regimen?' the most highly ranked option, followed by 'Should stage 1C patients receive chemotherapy?' CONCLUSIONS: Although the number of respondents limited the analyses, it was clear that chemotherapy selection was a key point of divergence for medical oncologists. Future research is needed to establish well-evidenced guidelines for clinical care of MOC.

Pathways to diagnosis of endometrial and ovarian cancer in the 45 and Up Study cohort.

PURPOSE: To determine pathways to endometrial or ovarian cancer diagnosis by comparing health service utilization between cancer cases and matched cancer-free controls, using linked health records. METHODS: From cancer registry records, we identified 238 incident endometrial and 167 ovarian cancer cases diagnosed during 2006-2013 in the Australian 45 and Up Study cohort (142,973 female participants). Each case was matched to four cancer-free controls on birthdate, sex, place of residence, smoking status, and body mass index. The use of relevant health services during the 13-18-, 7-12-, 0-6-, and 0-1-months pre-diagnosis for cases and the corresponding dates for their matched controls was determined through linkage with subsidized medical services and hospital records. RESULTS: Healthcare utilization diverged between women with cancer and controls in the 0-6-months, particularly 0-1 months, pre-diagnosis. In the 0-1 months, 74.8% of endometrial and 50.3% of ovarian cases visited a gynecologist/gynecological oncologist, 11.3% and 59.3% had a CA125 test, 5.5% and 48.5% an abdominal pelvic CT scan, and 34.5% and 30.5% a transvaginal pelvic ultrasound, respectively (versus ≤ 1% of matched controls). Moreover, 25.1% of ovarian cancer cases visited an emergency department in the 0-1-months pre-diagnosis (versus 1.3% of matched controls), and GP visits were significantly more common for cases than controls in this period. CONCLUSION: Most women with endometrial or ovarian cancer accessed recommended specialists and tests in the 0-1-months pre-diagnosis, but a high proportion of women with ovarian cancer visited an emergency department. This reinforces the importance of timely specialist referral.

Lifestyle and personal factors associated with having macroscopic residual disease after ovarian cancer primary cytoreductive surgery.

OBJECTIVE: The presence of macroscopic residual disease after primary cytoreductive surgery (PCS) is an important factor influencing survival for patients with high-grade serous ovarian cancer (HGSC). More research is needed to identify factors associated with having macroscopic residual disease. We analyzed 12 lifestyle and personal exposures known to be related to ovarian cancer risk or inflammation to identify those associated with having residual disease after surgery. METHODS: This analysis used data on 2054 patients with advanced stage HGSC from the Ovarian Cancer Association Consortium. The exposures were body mass index, breastfeeding, oral contraceptive use, depot-medroxyprogesterone acetate use, endometriosis, first-degree family history of ovarian cancer, incomplete pregnancy, menopausal hormone therapy use, menopausal status, parity, smoking, and tubal ligation. Logistic regression models were fit to assess the association between these exposures and having residual disease following PCS. RESULTS: Menopausal estrogen-only therapy (ET) use was associated with 33% lower odds of having macroscopic residual disease compared to never use (OR = 0.67, 95%CI 0.46-0.97, p = 0.033). Compared to nulliparous women, parous women who did not breastfeed had 36% lower odds of having residual disease (OR = 0.64, 95%CI 0.43-0.94, p = 0.022), while there was no association among parous women who breastfed (OR = 0.90, 95%CI 0.65-1.25, p = 0.53). CONCLUSIONS: The association between ET and having no macroscopic residual disease is plausible given a strong underlying biologic hypothesis between this exposure and diagnosis with HGSC. If this or the parity finding is replicated, these factors could be included in risk stratification models to determine whether HGSC patients should receive PCS or neoadjuvant chemotherapy.

Profiling the immune landscape in mucinous ovarian carcinoma.

OBJECTIVE: Mucinous ovarian carcinoma (MOC) is a rare histotype of ovarian cancer, with low response rates to standard chemotherapy, and very poor survival for patients diagnosed at advanced stage. There is a limited understanding of the MOC immune landscape, and consequently whether immune checkpoint inhibitors could be considered for a subset of patients. METHODS: We performed multicolor immunohistochemistry (IHC) and immunofluorescence (IF) on tissue microarrays in a cohort of 126 MOC patients. Cell densities were calculated in the epithelial and stromal components for tumor-associated macrophages (CD68+/PD-L1+, CD68+/PD-L1-), T cells (CD3+/CD8-, CD3+/CD8+), putative T-regulatory cells (Tregs, FOXP3+), B cells (CD20+/CD79A+), plasma cells (CD20-/CD79a+), and PD-L1+ and PD-1+ cells, and compared these values with clinical factors. Univariate and multivariable Cox Proportional Hazards assessed overall survival. Unsupervised k-means clustering identified patient subsets with common patterns of immune cell infiltration. RESULTS: Mean densities of PD1+ cells, PD-L1- macrophages, CD4+ and CD8+ T cells, and FOXP3+ Tregs were higher in the stroma compared to the epithelium. Tumors from advanced (Stage III/IV) MOC had greater epithelial infiltration of PD-L1- macrophages, and fewer PD-L1+ macrophages compared with Stage I/II cancers (p = 0.004 and p = 0.014 respectively). Patients with high epithelial density of FOXP3+ cells, CD8+/FOXP3+ cells, or PD-L1- macrophages, had poorer survival, and high epithelial CD79a + plasma cells conferred better survival, all upon univariate analysis only. Clustering showed that most MOC (86%) had an immune depleted (cold) phenotype, with only a small proportion (11/76,14%) considered immune inflamed (hot) based on T cell and PD-L1 infiltrates. CONCLUSION: In summary, MOCs are mostly immunogenically 'cold', suggesting they may have limited response to current immunotherapies.

Validated biomarker assays confirm that ARID1A loss is confounded with MMR deficiency, CD8+ TIL infiltration, and provides no independent prognostic value in endometriosis-associated ovarian carcinomas.

ARID1A (BAF250a) is a component of the SWI/SNF chromatin modifying complex, plays an important tumour suppressor role, and is considered prognostic in several malignancies. However, in ovarian carcinomas there are contradictory reports on its relationship to outcome, immune response, and correlation with clinicopathological features. We assembled a series of 1623 endometriosis-associated ovarian carcinomas, including 1078 endometrioid (ENOC) and 545 clear cell (CCOC) ovarian carcinomas, through combining resources of the Ovarian Tumor Tissue Analysis (OTTA) Consortium, the Canadian Ovarian Unified Experimental Resource (COEUR), local, and collaborative networks. Validated immunohistochemical surrogate assays for ARID1A mutations were applied to all samples. We investigated associations between ARID1A loss/mutation, clinical features, outcome, CD8+ tumour-infiltrating lymphocytes (CD8+ TILs), and DNA mismatch repair deficiency (MMRd). ARID1A loss was observed in 42% of CCOCs and 25% of ENOCs. We found no associations between ARID1A loss and outcomes, stage, age, or CD8+ TIL status in CCOC. Similarly, we found no association with outcome or stage in endometrioid cases. In ENOC, ARID1A loss was more prevalent in younger patients (p = 0.012) and was associated with MMRd (p < 0.001) and the presence of CD8+ TILs (p = 0.008). Consistent with MMRd being causative of ARID1A mutations, in a subset of ENOCs we also observed an association with ARID1A loss-of-function mutation as a result of small indels (p = 0.035, versus single nucleotide variants). In ENOC, the association with ARID1A loss, CD8+ TILs, and age appears confounded by MMRd status. Although this observation does not explicitly rule out a role for ARID1A influence on CD8+ TIL infiltration in ENOC, given current knowledge regarding MMRd, it seems more likely that effects are dominated by the hypermutation phenotype. This large dataset with consistently applied biomarker assessment now provides a benchmark for the prevalence of ARID1A loss-of-function mutations in endometriosis-associated ovarian cancers and brings clarity to the prognostic significance. © 2021 The Pathological Society of Great Britain and Ireland.

Follow-up assessment of sleep-related symptoms in patients after treatment for cancer: responses to continuous positive airway pressure treatment for co-morbid obstructive sleep apnoea.

PURPOSE: To assess changes in sleep-related symptoms in patients with breast cancer, endometrial cancer and melanoma previously examined for sleep-related symptoms and the presence of PSG (polysomnography)-determined OSA, ≥ 3 years post-treatment; and to evaluate how CPAP treatment affects sleep-related symptoms in patients previously diagnosed with OSA. METHODS: Patients initially recruited from breast cancer, endometrial cancer, and melanoma follow-up clinics at Westmead Hospital (Sydney, Australia) participated in this questionnaire-based study. Demographic and change in cancer status data were collected at follow-up. Patients completed the Pittsburgh Sleep Quality Index [poor sleep quality, PSQITOTAL ≥ 5au], Insomnia Severity Index, Epworth Sleepiness Scale and Functional Outcomes of Sleep Questionnaire; with ΔPSQITOTAL ≥ 3au indicating a clinically meaningful change in sleep quality over follow-up. PSG-determined OSA was confirmed using the apnoea-hypopnoea index. CPAP compliance was determined via self-report (CPAP compliant, CPAP; not compliant, non-CPAP). Logistic regression models determined if changes in cancer status, AHI, cancer subgroup or CPAP treatment was predictive of ΔPSQITOTAL ≥ 3 au and p < 0.05 indicated statistical significance. RESULTS: The 60 patients recruited had breast cancer (n = 22), endometrial cancer (n = 15), and melanoma (n = 23). Cancer subgroups were similarly aged, and all had median follow-up PSQITOTAL scores ≥ 5au; breast cancer patients scoring the highest (p < 0.05). The CPAP group had significantly reduced PSQITOTAL scores (p = 0.02) at follow-up, unlike the non-CPAP group. Cancer subgroups had similar median ISITOTAL, ESSTOTAL and FOSQ-10TOTAL scores at follow-up, regardless of CPAP treatment. There were no significant predictors of ΔPSQITOTAL ≥ 3 au at follow-up. CONCLUSION: Sleep-related symptoms persist in patients with cancer ≥ 3 years after treatment, although these symptoms improve with CPAP. Future studies should evaluate how CPAP affects survival outcomes in cancer patients with comorbid OSA.

Clinical parameters affecting survival outcomes in patients with low-grade serous ovarian carcinoma: an international multicentre analysis.

BACKGROUND: Women with low-grade ovarian serous carcinoma (LGSC) benefit from surgical treatment; however, the role of chemotherapy is controversial. We examined an international database through the Ovarian Cancer Association Consortium to identify factors that affect survival in LGSC. METHODS: We performed a retrospective cohort analysis of patients with LGSC who had had primary surgery and had overall survival data available. We performed univariate and multivariate analyses of progression-free survival and overall survival, and generated Kaplan-Meier survival curves. RESULTS: Of the 707 patients with LGSC, 680 (96.2%) had available overall survival data. The patients' median age overall was 54 years. Of the 659 patients with International Federation of Obstetrics and Gynecology stage data, 156 (23.7%) had stage I disease, 64 (9.7%) had stage II, 395 (59.9%) had stage III, and 44 (6.7%) had stage IV. Of the 377 patients with surgical data, 200 (53.0%) had no visible residual disease. Of the 361 patients with chemotherapy data, 330 (91.4%) received first-line platinum-based chemotherapy. The median follow-up duration was 5.0 years. The median progression-free survival and overall survival were 43.2 months and 110.4 months, respectively. Multivariate analysis indicated a statistically significant impact of stage and residual disease on progression-free survival and overall survival. Platinum-based chemotherapy was not associated with a survival advantage. CONCLUSION: This multicentre analysis indicates that complete surgical cytoreduction to no visible residual disease has the most impact on improved survival in LGSC. This finding could immediately inform and change practice.

Germline BRCA variants, lifestyle and ovarian cancer survival.

OBJECTIVE: Women with ovarian cancer who have a pathogenic germline variant in BRCA1 or BRCA2 (BRCA) have been shown to have better 5-year survival after diagnosis than women who are BRCA-wildtype (non-carriers). Modifiable lifestyle factors, including smoking, physical activity and body mass index (BMI) have previously been associated with ovarian cancer survival; however, it is unknown whether these associations differ by germline BRCA status. METHODS: We investigated measures of lifestyle prior to diagnosis in two cohorts of Australian women with invasive epithelial ovarian cancer, using Cox proportional hazards regression to calculate adjusted hazard ratios (HRs) and 95% confidence intervals (CIs). RESULTS: In the combined studies (n = 1923), there was little association between physical activity, BMI or alcohol intake and survival, and no difference by BRCA status. However, the association between current smoking status before diagnosis and poorer survival was stronger for BRCA variant carriers (HR 1.98; 95% CI 1.20-3.27) than non-carriers (HR 1.18; 95% CI 0.96-1.46; p-interaction 0.02). We saw a similar differential association with smoking when we pooled results from two additional cohorts from the USA and UK (n = 2120). Combining the results from all four studies gave a pooled-HR of 1.94 (95% CI 1.28-2.94) for current smoking among BRCA variant carriers compared to 1.08 (0.90-1.29) for non-carriers. CONCLUSIONS: Our results suggest that the adverse effect of smoking on survival may be stronger for women with a BRCA variant than those without. Thus, while smoking cessation may improve outcomes for all women with ovarian cancer, it might provide a greater benefit for BRCA variant carriers.

Evaluating patient-reported symptoms and late adverse effects following completion of first-line chemotherapy for ovarian cancer using the MOST (Measure of Ovarian Symptoms and Treatment concerns).

OBJECTIVES: Knowledge on the course of symptoms patients with ovarian cancer experience is limited. We documented the prevalence and trajectories of symptoms after first-line chemotherapy using the Measure of Ovarian Symptoms and Treatment concerns (MOST). METHODS: A total of 726 patients who received platinum-based chemotherapy for ovarian cancer were asked to complete the MOST every 3 months, beginning 6 months post-diagnosis and continuing for up to 4 years. We used descriptive statistics to examine temporal changes in MOST-S26 index scores for disease or treatment-related (MOST-DorT), neurotoxicity (MOST-NTx), abdominal (MOST-Abdo), and psychological (MOST-Psych) symptoms, and wellbeing (MOST-Wellbeing) and selected individual symptoms. We used group-based trajectory models to identify groups with persistently poor symptoms. RESULTS: The median MOST-Abdo, MOST-DorT and MOST-Wellbeing score were worst at chemotherapy-end but improved and stabilised by 1, 3 and 12 months after treatment, respectively. The median MOST-NTx score peaked at 1 month after treatment before improving, while the median MOST-Psych score did not change substantially over time. Long-term moderate-to-severe fatigue (32%), trouble sleeping (31%), sore hands and feet (21%), pins and needles (20%) and anxiety (18%) were common. Trajectory models revealed groups of patients with persistent symptoms had MOST-DorT scores above 30 and MOST-NTx scores above 40 at treatment-end. CONCLUSIONS: Although many patients report improvements in symptoms by 3 months after first-line chemotherapy for ovarian cancer, patients who score > 30/100 on MOST-S26-DorT or > 40/100 on MOST-S26-NTx at the end of chemotherapy are likely to have persistent symptoms. The MOST could triage this at-risk subset for early intervention.

Genomic analysis of low-grade serous ovarian carcinoma to identify key drivers and therapeutic vulnerabilities.

Low-grade serous ovarian carcinoma (LGSOC) is associated with a poor response to existing chemotherapy, highlighting the need to perform comprehensive genomic analysis and identify new therapeutic vulnerabilities. The data presented here represent the largest genetic study of LGSOCs to date (n = 71), analysing 127 candidate genes derived from whole exome sequencing cohorts to generate mutation and copy-number variation data. Additionally, immunohistochemistry was performed on our LGSOC cohort assessing oestrogen receptor, progesterone receptor, TP53, and CDKN2A status. Targeted sequencing identified 47% of cases with mutations in key RAS/RAF pathway genes (KRAS, BRAF, and NRAS), as well as mutations in putative novel driver genes including USP9X (27%), MACF1 (11%), ARID1A (9%), NF2 (4%), DOT1L (6%), and ASH1L (4%). Immunohistochemistry evaluation revealed frequent oestrogen/progesterone receptor positivity (85%), along with CDKN2A protein loss (10%) and CDKN2A protein overexpression (6%), which were linked to shorter disease outcomes. Indeed, 90% of LGSOC samples harboured at least one potentially actionable alteration, which in 19/71 (27%) cases were predictive of clinical benefit from a standard treatment, either in another cancer's indication or in LGSOC specifically. In addition, we validated ubiquitin-specific protease 9X (USP9X), which is a chromosome X-linked substrate-specific deubiquitinase and tumour suppressor, as a relevant therapeutic target for LGSOC. Our comprehensive genomic study highlighted that there is an addiction to a limited number of unique 'driver' aberrations that could be translated into improved therapeutic paths. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Uncertainty and the unmet informational needs of patients with cancer of unknown primary (CUP): a cross-sectional multi-site study.

OBJECTIVE: This study aimed to determine the healthcare experiences, quality of life, and psychosocial needs of patients with cancer of unknown primary (CUP) early after diagnosis; comparing their experiences to patients with advanced cancer of a known primary (non-CUP control patients) and published general population reference data where available. METHODS: This study was a cross-sectional, multi-site study comparing CUP patients (n = 139) compared to non-CUP controls (n = 45). Demographic, clinical information and patient-reported outcome questionnaire data were collected at baseline. RESULTS: Differences in healthcare experienced were found between CUP and non-CUP controls with CUP patients reporting higher scores for unmet medical communication/information needs compared with non-CUP control patients (p = 0.013) as well as greater uncertainty in illness (p = 0.042). Whilst no differences were found between CUP and non-CUP controls on the EORTC and PROMIS measures, of those that 'received written information about your cancer…' and asked '…how useful was it?' fewer CUP patients reported finding the information useful 40% vs 61%, and more were likely to not have received written information at all 59% vs 32%; (p = 0.002). Additionally, of those that found information about their cancer online, fewer patients with CUP reported finding it useful 32% vs 48% control patients (p = 0.005). CONCLUSIONS: CUP patients have unmet medical communication/information needs and greater uncertainty in illness but do not differ in health-related quality of life domains compared to patients with advanced cancer of a known primary.

Refined cut-off for TP53 immunohistochemistry improves prediction of TP53 mutation status in ovarian mucinous tumors: implications for outcome analyses.

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.

Cell-free DNA is abundant in ascites and represents a liquid biopsy of ovarian cancer.

OBJECTIVE: Malignant ascites is a common clinical feature of ovarian cancer and represents a readily accessible sample of tumour cells and tumour DNA. This study aimed to characterise the cell-free DNA (cfDNA) in ascites in terms of its size profile, stability and cell-free tumour DNA (cftDNA) content. METHODS: Cell spheroids, loose cells and cell-free fluid was collected from ascites from 18 patients with ovarian cancer. cfDNA was isolated and assessed for size by electrophoresis, concentration by fluorometry,cftDNA content by methylation specific qPCR of HOXA9 and IFFO1 promoter regions and by targeted sequencing. Stability was assessed after ascites fluid was stored at 4 °C for 24 and 72 h before fractionating. RESULTS: The concentration of cfDNA in ascites ranged from 6.6 to 300 ng/mL. cfDNA size distribution resembled blood plasma-derived cfDNA, with major peaks corresponding to mono- and di-nucleosome DNA fragments. High molecular weight cfDNA was observed in 7 of 18 patients and appeared to be associated with extracellular vesicles. IFFO1 and HOXA9 methylation was proportionately higher in cfDNA than spheroid- and loose-cell fractions and was not observed in healthy primary cells. Variant allele frequency was highest in cfDNA compared to single cells and spheroids from ascites. Though cancer cell numbers in ascites declined to near zero in recurrent ascites from one patient undertaking chemotherapy, cftDNA could still be sampled. cfDNA size, concentration and tumour content was stable over 72 h. CONCLUSION: cfDNA in ovarian cancer ascites demonstrates inter-patient variability, yet is consistently a rich source of cftDNA, which is a stable substrate. This supports the wider clinical use of ascites in the molecular analysis of ovarian cancer.

A healthy lifestyle and survival among women with ovarian cancer.

Ovarian cancer has a poor survival rate and, understandably, women often want to know whether there is anything they can do to improve their prognosis. Our goal was to investigate the association between a healthy lifestyle prediagnosis and postdiagnosis and survival in a cohort of Australian women with invasive epithelial ovarian cancer. We calculated a healthy lifestyle index (HLI) based on women's self-reported smoking status, height, weight, physical activity, diet and alcohol consumption before diagnosis (n = 678) and after completing primary treatment (n = 512). Clinical data and vital status for each woman were ascertained through medical records. Cox proportional hazards regression was conducted to calculate hazard ratios (HR) and 95% confidence interval (CI) for all-cause mortality. There was a suggestive association between a more healthy lifestyle before diagnosis and better survival (HR 0.79, 95% CI: 0.59-1.04), however, the association was stronger for lifestyle after diagnosis, with women in the highest tertile having significantly better survival than women in the lowest tertile (HR 0.61, 95% CI: 0.40-0.93; P-trend = .02). Current smoking, particularly postdiagnosis, was associated with higher mortality (HR 1.68, 95% CI: 1.17-2.42; HR 2.82, 95% CI: 1.29-6.14, for prediagnosis and postdiagnosis smoking, respectively), but women who quit after diagnosis had survival outcomes similar to nonsmokers (HR 0.99, 95% CI: 0.57-1.72). Higher physical activity after diagnosis was associated with better survival (HR 0.60, 95% CI: 0.39-0.92; P-trend = .02). A healthy lifestyle after diagnosis, in particular not smoking and being physically active, may help women with ovarian cancer improve their prognosis.

ABCC4/MRP4 contributes to the aggressiveness of Myc-associated epithelial ovarian cancer.

Epithelial ovarian cancer (EOC) is a complex disease comprising discrete histological and molecular subtypes, for which survival rates remain unacceptably low. Tailored approaches for this deadly heterogeneous disease are urgently needed. Efflux pumps belonging to the ATP-binding cassette (ABC) family of transporters are known for roles in both drug resistance and cancer biology and are also highly targetable. Here we have investigated the association of ABCC4/MRP4 expression to clinical outcome and its biological function in endometrioid and serous tumors, common histological subtypes of EOC. We found high expression of ABCC4/MRP4, previously shown to be directly regulated by c-Myc/N-Myc, was associated with poor prognosis in endometrioid EOC (P = .001) as well as in a subset of serous EOC with a "high-MYCN" profile (C5/proliferative; P = .019). Transient siRNA-mediated suppression of MRP4 in EOC cells led to reduced growth, migration and invasion, with the effects being most pronounced in endometrioid and C5-like serous cells compared to non-C5 serous EOC cells. Sustained knockdown of MRP4 also sensitized endometrioid cells to MRP4 substrate drugs. Furthermore, suppression of MRP4 decreased the growth of patient-derived EOC cells in vivo. Together, our findings provide the first evidence that MRP4 plays an important role in the biology of Myc-associated ovarian tumors and highlight this transporter as a potential therapeutic target for EOC.

FKBPL-based peptide, ALM201, targets angiogenesis and cancer stem cells in ovarian cancer.

BACKGROUND: ALM201 is a therapeutic peptide derived from FKBPL that has previously undergone preclinical and clinical development for oncology indications and has completed a Phase 1a clinical trial in ovarian cancer patients and other advanced solid tumours. METHODS: In vitro, cancer stem cell (CSC) assays in a range of HGSOC cell lines and patient samples, and in vivo tumour initiation, growth delay and limiting dilution assays, were utilised. Mechanisms were determined by using immunohistochemistry, ELISA, qRT-PCR, RNAseq and western blotting. Endogenous FKBPL protein levels were evaluated using tissue microarrays (TMA). RESULTS: ALM201 reduced CSCs in cell lines and primary samples by inducing differentiation. ALM201 treatment of highly vascularised Kuramochi xenografts resulted in tumour growth delay by disruption of angiogenesis and a ten-fold decrease in the CSC population. In contrast, ALM201 failed to elicit a strong antitumour response in non-vascularised OVCAR3 xenografts, due to high levels of IL-6 and vasculogenic mimicry. High endogenous tumour expression of FKBPL was associated with an increased progression-free interval, supporting the protective role of FKBPL in HGSOC. CONCLUSION: FKBPL-based therapy can (i) dually target angiogenesis and CSCs, (ii) target the CD44/STAT3 pathway in tumours and (iii) is effective in highly vascularised HGSOC tumours with low levels of IL-6.

Clinical and pathological associations of PTEN expression in ovarian cancer: a multicentre study from the Ovarian Tumour Tissue Analysis Consortium.

BACKGROUND: PTEN loss is a putative driver in histotypes of ovarian cancer (high-grade serous (HGSOC), endometrioid (ENOC), clear cell (CCOC), mucinous (MOC), low-grade serous (LGSOC)). We aimed to characterise PTEN expression as a biomarker in epithelial ovarian cancer in a large population-based study. METHODS: Tumours from 5400 patients from a multicentre observational, prospective cohort study of the Ovarian Tumour Tissue Analysis Consortium were used to evaluate associations between immunohistochemical PTEN patterns and overall survival time, age, stage, grade, residual tumour, CD8+ tumour-infiltrating lymphocytes (TIL) counts, expression of oestrogen receptor (ER), progesterone receptor (PR) and androgen receptor (AR) by means of Cox proportional hazard models and generalised Cochran-Mantel-Haenszel tests. RESULTS: Downregulation of cytoplasmic PTEN expression was most frequent in ENOC (most frequently in younger patients; p value = 0.0001) and CCOC and was associated with longer overall survival in HGSOC (hazard ratio: 0.78, 95% CI: 0.65-0.94, p value = 0.022). PTEN expression was associated with ER, PR and AR expression (p values: 0.0008, 0.062 and 0.0002, respectively) in HGSOC and with lower CD8 counts in CCOC (p value < 0.0001). Heterogeneous expression of PTEN was more prevalent in advanced HGSOC (p value = 0.019) and associated with higher CD8 counts (p value = 0.0016). CONCLUSIONS: PTEN loss is a frequent driver in ovarian carcinoma associating distinctly with expression of hormonal receptors and CD8+ TIL counts in HGSOC and CCOC histotypes.