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Women with ovarian cancer have limited therapy options, with immunotherapy being unsatisfactory for a large group of patients. Tumor cells spread from the ovary or the fallopian tube into the abdominal cavity, which is commonly accompanied with massive ascites production. The ascites represents a unique peritoneal liquid tumor microenvironment with the presence of both tumor and immune cells, including cytotoxic lymphocytes. We characterized lymphocytes in ascites from patients with high-grade serous ovarian cancer. Our data reveal the presence of NK and CD8+ T lymphocytes expressing CD103 and CD49a, which are markers of tissue residency. Moreover, these cells express high levels of the inhibitory NKG2A receptor, with the highest expression level detected on tissue-resident NK cells. Lymphocytes with these features were also present at the primary tumor site. Functional assays showed that tissue-resident NK cells in ascites are highly responsive towards ovarian tumor cells. Similar results were observed in an in vivo mouse model, in which tissue-resident NK and CD8+ T cells were detected in the peritoneal fluid upon tumor growth. Together, our data reveal the presence of highly functional lymphocyte populations that may be targeted to improve immunotherapy for patients with ovarian cancer.

Funder: Biomedical Research Centre, Funder: European Regional Development Fund, Funder: Mayo Foundation for Medical Education and Research, Funder: Pomeranian Medical University, Funder: Pomorski Uniwersytet Medyczny W Szczecinie, Funder: Cancer Council NSW, Funder: Cancer Institute NSW, Funder: Deutsches Krebsforschungszentrum, Funder: The BC Cancer Foundation, Funder: University College London Hospitals Biomedical Research Centre, Funder: Breast Cancer Now, Funder: Cancer Council Tasmania, Funder: Clinical Academic Reserve, Funder: ELAN Funds of the University of Erlangen-Nuremberg, Funder: Fondo Europeo de Desarrollo Regional, Funder: National Institute for Health Research (NIHR), Funder: National Institute for Health and Care Research, Funder: Ovarian Cancer Australia, Funder: Queensland Cancer Fund, Funder: Cancer Council New South Wales, Funder: Fred C. and Katherine B. Andersen Foundation, Funder: German Cancer Research Center, Funder: Institute of Cancer Research, Funder: Mayo Foundation, Funder: Minnesota Ovarian Cancer Alliance, Funder: Peter MacCallum Foundation, Funder: University of Cambridge, Funder: Cancer Foundation of Western Australia, Funder: VGH and UBC Hospital Foundation, Funder: Cancer Council Victoria, Funder: NHS, Funder: UK National Institute for Health Research, Funder: Cancer Council South Australia, Funder: Oak Foundation, Funder: Sydney West Translational Cancer Research Centre, Our objective was to test whether p53 expression status is associated with survival for women diagnosed with the most common ovarian carcinoma histotypes (high-grade serous carcinoma [HGSC], endometrioid carcinoma [EC], and clear cell carcinoma [CCC]) using a large multi-institutional cohort from the Ovarian Tumor Tissue Analysis (OTTA) consortium. p53 expression was assessed on 6,678 cases represented on tissue microarrays from 25 participating OTTA study sites using a previously validated immunohistochemical (IHC) assay as a surrogate for the presence and functional effect of TP53 mutations. Three abnormal expression patterns (overexpression, complete absence, and cytoplasmic) and the normal (wild type) pattern were recorded. Survival analyses were performed by histotype. The frequency of abnormal p53 expression was 93.4% (4,630/4,957) in HGSC compared to 11.9% (116/973) in EC and 11.5% (86/748) in CCC. In HGSC, there were no differences in overall survival across the abnormal p53 expression patterns. However, in EC and CCC, abnormal p53 expression was associated with an increased risk of death for women diagnosed with EC in multivariate analysis compared to normal p53 as the reference (hazard ratio [HR] = 2.18, 95% confidence interval [CI] 1.36-3.47, p = 0.0011) and with CCC (HR = 1.57, 95% CI 1.11-2.22, p = 0.012). Abnormal p53 was also associated with shorter overall survival in The International Federation of Gynecology and Obstetrics stage I/II EC and CCC. Our study provides further evidence that functional groups of TP53 mutations assessed by abnormal surrogate p53 IHC patterns are not associated with survival in HGSC. In contrast, we validate that abnormal p53 IHC is a strong independent prognostic marker for EC and demonstrate for the first time an independent prognostic association of abnormal p53 IHC with overall survival in patients with CCC.

Our objective was to test whether p53 expression status is associated with survival for women diagnosed with the most common ovarian carcinoma histotypes (high-grade serous [HGSC], endometrioid [EC] and clear cell carcinomas [CCC]) using a large multi-institutional cohort from the Ovarian Tumor Tissue Analysis (OTTA) consortium. p53 expression was assessed on 6678 cases represented on tissue microarrays from 25 participating OTTA study sites using a previously validated immunohistochemical (IHC) assay as a surrogate for the presence and functional effect of TP53 mutations. Three abnormal expression patterns (overexpression, complete absence, cytoplasmic) and the normal (wild type) pattern were recorded. Survival analyses were performed by histotype. The frequency of abnormal p53 expression was 93% (4630/4957) in HGSC compared to 11.9% (116/973) in EC and 11.5% (86/748) in CCC. In HGSC, there were no differences in overall survival across the abnormal p53 expression patterns. However, in EC and CCC abnormal p53 expression was associated with an increased risk of death for women diagnosed with EC in multivariate analysis compared to normal p53 as the reference (HR=2.18, 95% CI 1.36-3.47, p=0.0011) as well as with CCC (HR=1.57, 95% CI 1.11-2.22, p=0.012). Abnormal p53 was also associated with shorter overall survival in FIGO stage I/II EC and CCC. Our study provides further evidence that functional groups of TP53 mutations assessed by abnormal surrogate p53 IHC patterns are not associated with survival in HGSC. In contrast, we validate that abnormal p53 IHC is a strong independent prognostic marker for EC and demonstrate for the first time an independent prognostic association of abnormal p53 IHC with overall survival in patients with CCC., CRUK

Tubo-ovarian high-grade serous carcinomas (HGSC) are highly proliferative neoplasms that generally respond well to platinum/taxane chemotherapy. We recently identified minichromosome maintenance complex component 3 (MCM3), which is involved in the initiation of DNA replication and proliferation, as a favorable prognostic marker in HGSC. Our objective was to further validate whether MCM3 mRNA expression and possibly MCM3 protein levels are associated with survival in patients with HGSC. MCM3 mRNA expression was measured using NanoString expression profiling on formalin-fixed and paraffin-embedded tissue (N = 2355 HGSC) and MCM3 protein expression was assessed by immunohistochemistry (N = 522 HGSC) and compared with Ki-67. Kaplan-Meier curves and the Cox proportional hazards model were used to estimate associations with survival. Among chemotherapy-naïve HGSC, higher MCM3 mRNA expression (one standard deviation increase in the score) was associated with longer overall survival (HR = 0.87, 95% CI 0.81-0.92, p

BACKGROUND: Ovarian cancer is the eighth most common cancer among women and has a 5-year survival of only 30-50%. The survival is close to 90% for patients in stage I but only 20% for patients in stage IV. The presently available biomarkers have insufficient sensitivity and specificity for early detection and there is an urgent need to identify novel biomarkers. METHODS: We employed the Explore PEA technology for high-precision analysis of 1463 plasma proteins and conducted a discovery and replication study using two clinical cohorts of previously untreated patients with benign or malignant ovarian tumours (N = 111 and N = 37). RESULTS: The discovery analysis identified 32 proteins that had significantly higher levels in malignant cases as compared to benign diagnoses, and for 28 of these, the association was replicated in the second cohort. Multivariate modelling identified three highly accurate models based on 4 to 7 proteins each for separating benign tumours from early-stage and/or late-stage ovarian cancers, all with AUCs above 0.96 in the replication cohort. We also developed a model for separating the early-stage from the late-stage achieving an AUC of 0.81 in the replication cohort. These models were based on eleven proteins in total (ALPP, CXCL8, DPY30, IL6, IL12, KRT19, PAEP, TSPAN1, SIGLEC5, VTCN1, and WFDC2), notably without MUCIN-16. The majority of the associated proteins have been connected to ovarian cancer but not identified as potential biomarkers. CONCLUSIONS: The results show the ability of using high-precision proteomics for the identification of novel plasma protein biomarker candidates for the early detection of ovarian cancer.

Background: Epithelial ovarian cancer (EOC) is the most lethal gynecologic cancer. The 5-year survival is about 30%, a consequence of failure to establish early diagnosis due to unspecific symptoms. The biomarker serum CA125 and transvaginal ultrasound are used as the “golden standards” for assessing ovarian cysts and pelvic tumors of unknown origin, but early diagnosis is still not achieved and specificity is low. Increased knowledge about EOC specific mutations has revised our understanding of ovarian cancer etiology and heterogeneity. Rare tumor mutations can be detected in liquid biopsies from different compartments. Aims: To investigate established EOC biomarkers and algorithms in an unselected population of women with ovarian cysts/pelvic tumors. To compare and combine established and new biomarkers and algorithms to improve differential diagnosis of EOC (paper I-III). To explore new ways for early detection of gynecologic cancer, from circulating tumor DNA (ctDNA) and somatic mutations, by mutation specific analysis in liquid biopsies from the genital tract and plasma (paper IV). Methods: A prospective multicenter trial (paper I-III) was conducted and we also participated in an international multicenter trial (paper IV) with the aim to improve diagnostic accuracy of EOC and to find new screening methods. Serum was collected for analysis of the biomarkers CA125 and HE4. Risk of Malignancy Index (RMI), Risk of Ovarian Malignancy Algorithm (ROMA) and new algorithms were explored. Patient and tumor characteristics were recorded at the time of inclusion and evaluated by multivariate regression analysis (paper I-III). Liquid biopsies were collected from genital tract thin-prep liquids and plasma. Corresponding formalin fixed and paraffin imbedded tissue biopsies were retrieved from the pathology repository. They were analyzed with a multiplex PCR based barcoding of DNA for mutation detection using next generation sequencing (PapSEEK) for rare mutations (paper IV). Results: Paper I: At the recommended cut-off level off >35, CA125 achieved highest sensitivity (SN) for both pre- and postmenopausal women (SN 95.7%; 92.0%), but low specificity (SP) in both pre- and postmenopausal women (Pre-M 59.6%; Post-M 79.5%). HE4 was inferior compared to CA125 in SN but increased in diagnostic performance with the highest SP (Pre-M 90.9%; Post-M 92.1%). RMI and ROMA were identical in their predictive ability. Paper II: Three new algorithms were tested and found to perform better than RMI, ROMA or CA125 alone (GOT-1, GOT-2, GOT-3). The addition of HE4 to CA125 or RMI increased SP without hampering SN. Paper III: Smoking, heart- or kidney failure and endometriosis should be considered when evaluating CA125 and HE4 levels in women assessed for an ovarian cyst/pelvic tumor of unknown origin. Paper IV: The PapSEEK technique showed impressively high SP (98.6% cervical; 100% endometrial), and when cervical sampling was combined with plasma ctDNA analysis, SN for ovarian cancer was 63%, with retained SP of 100%. Conclusion: CA125 was superior to HE4 to identify women with ovarian cancer and HE4 was superior to CA125 to identify benign lesions, in this unselected population of women with an ovarian cyst/pelvic mass from the Western region of Sweden. Addition of HE4 to CA125 increased diagnostic accuracy and decreased false positives. Combining established and novel biomarkers and algorithms improved diagnostic accuracy of ovarian tumors. We suggest that HE4 should be incorporated for the differential diagnosis of women with ovarian cysts/pelvic tumors of unknown origin to decrease unnecessary oophorectomies. It is possible to detect somatic mutations and ctDNA from ovarian cancer in plasma, cervical- and endometrial liquid biopsies with high SP. This thesis demonstrate the potential of a protein and gene mutation-based diagnostic test to detect EOC. With improved technique, this might be a potential test for ovarian cancer screening, in the future.

Additional file 1: Table 1. P-values of individual proteins in the discovery step.

Additional file 2: Table 2. CA125 values in the clinical analyses and the PEA NPX values for some of the benign and ovarian cancer cohorts used in the discovery analysis.