Functional Homologous Recombination Assay on FFPE Specimens of Advanced High-Grade Serous Ovarian Cancer Predicts Clinical Outcomes.

PURPOSE: Deficiency in homologous recombination (HR) repair of DNA damage is characteristic of many high-grade serous ovarian cancers (HGSC). It is imperative to identify patients with homologous recombination-deficient (HRD) tumors as they are most likely to benefit from platinum-based chemotherapy and PARP inhibitors (PARPi). Existing methods measure historical, not necessarily current HRD and/or require high tumor cell content, which is not achievable for many patients. We set out to develop a clinically feasible assay for identifying functionally HRD tumors that can predict clinical outcomes. EXPERIMENTAL DESIGN: We quantified RAD51, a key HR protein, in immunostained formalin-fixed, paraffin-embedded (FFPE) tumor samples obtained from chemotherapy-naïve and neoadjuvant chemotherapy (NACT)-treated HGSC patients. We defined cutoffs for functional HRD separately for these sample types, classified the patients accordingly as HRD or HR-proficient, and analyzed correlations with clinical outcomes. From the same specimens, genomics-based HRD estimates (HR gene mutations, genomic signatures, and genomic scars) were also determined, and compared with functional HR (fHR) status. RESULTS: fHR status significantly predicted several clinical outcomes, including progression-free survival (PFS) and overall survival (OS), when determined from chemo-naïve (PFS, P < 0.0001; OS, P < 0.0001) as well as NACT-treated (PFS, P < 0.0001; OS, P = 0.0033) tumor specimens. The fHR test also identified as HRD those PARPi-at-recurrence-treated patients with longer OS (P = 0.0188). CONCLUSIONS: We developed an fHR assay performed on routine FFPE specimens, obtained from either chemo-naïve or NACT-treated HGSC patients, that can significantly predict real-world platinum-based chemotherapy and PARPi response. See related commentary by Garg and Oza, p. 2957.

Drug response profiles in patient-derived cancer cells across histological subtypes of ovarian cancer: real-time therapy tailoring for a patient with low-grade serous carcinoma.

Many efforts are underway to develop novel therapies against the aggressive high-grade serous ovarian cancers (HGSOCs), while our understanding of treatment options for low-grade (LGSOC) or mucinous (MUCOC) of ovarian malignancies is not developing as well. We describe here a functional precision oncology (fPO) strategy in epithelial ovarian cancers (EOC), which involves high-throughput drug testing of patient-derived ovarian cancer cells (PDCs) with a library of 526 oncology drugs, combined with genomic and transcriptomic profiling. HGSOC, LGSOC and MUCOC PDCs had statistically different overall drug response profiles, with LGSOCs responding better to targeted inhibitors than HGSOCs. We identified several subtype-specific drug responses, such as LGSOC PDCs showing high sensitivity to MDM2, ERBB2/EGFR inhibitors, MUCOC PDCs to MEK inhibitors, whereas HGSOCs showed strongest effects with CHK1 inhibitors and SMAC mimetics. We also explored several drug combinations and found that the dual inhibition of MEK and SHP2 was synergistic in MAPK-driven EOCs. We describe a clinical case study, where real-time fPO analysis of samples from a patient with metastatic, chemorefractory LGSOC with a CLU-NRG1 fusion guided clinical therapy selection. fPO-tailored therapy with afatinib, followed by trastuzumab and pertuzumab, successfully reduced tumour burden and blocked disease progression over a five-year period. In summary, fPO is a powerful approach for the identification of systematic drug response differences across EOC subtypes, as well as to highlight patient-specific drug regimens that could help to optimise therapies to individual patients in the future.

Effective Combination Immunotherapy with Oncolytic Adenovirus and Anti-PD-1 for Treatment of Human and Murine Ovarian Cancers.

Ovarian cancer (OvCa) is one of the most common gynecological cancers and has the highest mortality in this category. Tumors are often detected late, and unfortunately over 70% of OvCa patients experience relapse after first-line treatments. OvCa has shown low response rates to immune checkpoint inhibitor (ICI) treatments, thus leaving room for improvement. We have shown that oncolytic adenoviral therapy with Ad5/3-E2F-d24-hTNFa-IRES-hIL2 (aka. TILT-123) is promising for single-agent treatment of cancer, but also for sensitizing tumors for T-cell dependent immunotherapy approaches, such as ICI treatments. Therefore, this study set out to determine the effect of inhibition of the immune checkpoint inhibitors (ICI), in the context of TILT-123 therapy of OvCa. We show that simultaneous treatment of patient derived samples with TILT-123 and ICIs anti-PD-1 or anti-PD-L1 efficiently reduced overall viability. The combinations induced T cell activation, T cells expressed activation markers more often, and the treatment caused positive microenvironment changes, measured by flow cytometric assays. Furthermore, in an immunocompetent in vivo C57BL/6NHsda mouse model, tumor growth was hindered, when treated with TILT-123, ICI or both. Taken together, this study provides a rationale for using TILT-123 virotherapy in combination with TILT-123 and immune checkpoint inhibitors together in an ovarian cancer OvCa clinical trial.

Local delivery of interleukin 7 with an oncolytic adenovirus activates tumor-infiltrating lymphocytes and causes tumor regression.

Cytokines have proven to be effective for cancer therapy, however whilst low-dose monotherapy with cytokines provides limited therapeutic benefit, high-dose treatment can lead to a number of adverse events. Interleukin 7 has shown promising results in clinical trials, but anti-cancer effect was limited, in part due to a low concentration of the cytokine within the tumor. We hypothesized that arming an oncolytic adenovirus with Interleukin 7, enabling high expression localized to the tumor microenvironment, would overcome systemic delivery issues and improve therapeutic efficacy. We evaluated the effects of Ad5/3-E2F-d24-hIL7 (TILT-517) on tumor growth, immune cell activation and cytokine profiles in the tumor microenvironment using three clinically relevant animal models and ex vivo tumor cultures. Our data showed that local treatment of tumor bearing animals with Ad5/3- E2F-d24-hIL7 significantly decreased cancer growth and increased frequency of tumor-infiltrating cells. Ad5/3-E2F-d24-hIL7 promoted notable upregulation of pro-inflammatory cytokines, and concomitant activation and migration of CD4+ and CD8 + T cells. Interleukin 7 expression within the tumor was positively correlated with increased number of cytotoxic CD4+ cells and IFNg-producing CD4+ and CD8+ cells. These findings offer an approach to overcome the current limitations of conventional IL7 therapy and could therefore be translated to the clinic.

Parity, menopausal hormone therapy, and risk of ovarian granulosa cell tumor - A population-based case-control study.

OBJECTIVE: Adult-type ovarian granulosa cell tumors (AGCTs) are hormonally active neoplasms with limited epidemiological data available. We evaluated the effect of parity and postmenopausal hormone therapy (HT) use on the risk of AGCT in a population-based case-control setting. METHODS: We identified all women diagnosed with AGCT during 1994-2015 (n = 505) from the Finnish Cancer Registry. For each case, five controls matched for age were selected from the National Population Registry, which also provided data on parity and ages at deliveries. Information on postmenopausal HT by different regimens (estradiol-only, sequential estrogen-progestin and continuous estrogen-progestin) was obtained from nationwide Prescription Register. The association between parity, ages at deliveries, HT use, and AGCT incidence was evaluated by odds ratios (ORs) using a conditional logistic regression model and stratified by age at index date (<55 years or ≥ 55 years). RESULTS: Parity and age at first or last delivery had no significant effect on AGCT risk. Systemic postmenopausal HT had been used by 20.4% of women who were later diagnosed with AGCT. The risk for subsequent AGCT was significantly decreased among users of estradiol-only therapy for at least five years (OR 0.28; 95% confidence interval 0.08-0.94) and continuous estradiol-progestin therapy for 6 months to 5 years (0.23; 0.08-0.71). CONCLUSIONS: Unlike in epithelial ovarian cancer, AGCT development is not clearly associated with parity, and users of postmenopausal HT do not seem to carry an excess risk for AGCT formation.

Glucocorticoids induce differentiation and chemoresistance in ovarian cancer by promoting ROR1-mediated stemness.

Glucocorticoids are routinely used in the clinic as anti-inflammatory and immunosuppressive agents as well as adjuvants during cancer treatment to mitigate the undesirable side effects of chemotherapy. However, recent studies have indicated that glucocorticoids may negatively impact the efficacy of chemotherapy by promoting tumor cell survival, heterogeneity, and metastasis. Here, we show that dexamethasone induces upregulation of ROR1 expression in ovarian cancer (OC), including platinum-resistant OC. Increased ROR1 expression resulted in elevated RhoA, YAP/TAZ, and BMI-1 levels in a panel of OC cell lines as well as primary ovarian cancer patient-derived cells, underlining the translational relevance of our studies. Importantly, dexamethasone induced differentiation of OC patient-derived cells ex vivo according to their molecular subtype and the phenotypic expression of cell differentiation markers. High-throughput drug testing with 528 emerging and clinical oncology compounds of OC cell lines and patient-derived cells revealed that dexamethasone treatment increased the sensitivity to several AKT/PI3K targeted kinase inhibitors, while significantly decreasing the efficacy of chemotherapeutics such as taxanes, as well as anti-apoptotic compounds such as SMAC mimetics. On the other hand, targeting ROR1 expression increased the efficacy of taxane drugs and SMAC mimetics, suggesting new combinatorial targeted treatments for patients with OC.

Functional Profiling of FSH and Estradiol in Ovarian Granulosa Cell Tumors.

Adult-type granulosa cell tumors (AGCTs) are sex-cord derived neoplasms with a propensity for late relapse. Hormonal modulators have been used empirically in the treatment of recurrent AGCT, albeit with limited success. To provide a more rigorous foundation for hormonal therapy in AGCT, we used a multimodal approach to characterize the expressions of key hormone biomarkers in 175 tumor specimens and 51 serum samples using RNA sequencing, immunohistochemistry, RNA in situ hybridization, quantitative PCR, and circulating biomarker analysis, and correlated these results with clinical data. We show that FSH receptor and estrogen receptor beta (ERß) are highly expressed in the majority of AGCTs, whereas the expressions of estrogen receptor alpha (ERα) and G-protein coupled estrogen receptor 1 are less prominent. ERß protein expression is further increased in recurrent tumors. Aromatase expression levels show high variability between tumors. None of the markers examined served as prognostic biomarkers for progression-free or overall survival. In functional experiments, we assessed the effects of FSH, estradiol (E2), and the aromatase inhibitor letrozole on AGCT cell viability using 2 in vitro models: KGN cells and primary cultures of AGCT cells. FSH increased cell viability in a subset of primary AGCT cells, whereas E2 had no effect on cell viability at physiological concentrations. Letrozole suppressed E2 production in AGCTs; however, it did not impact cell viability. We did not find preclinical evidence to support the clinical use of aromatase inhibitors in AGCT treatment, and thus randomized, prospective clinical studies are needed to clarify the role of hormonal treatments in AGCTs.

Pathogenesis and treatment of adult-type granulosa cell tumor of the ovary.

Adult-type granulosa cell tumor is a clinically and molecularly unique subtype of ovarian cancer. These tumors originate from the sex cord stromal cells of the ovary and represent 3-5% of all ovarian cancers. The majority of adult-type granulosa cell tumors are diagnosed at an early stage with an indolent prognosis. Surgery is the cornerstone for the treatment of both primary and relapsed tumor, while chemotherapy is applied only for advanced or non-resectable cases. Tumor stage is the only factor consistently associated with prognosis. However, every third of the patients relapse, typically in 4-7 years from diagnosis, leading to death in 50% of these patients. Anti-Müllerian Hormone and inhibin B are currently the most accurate circulating biomarkers. Adult-type granulosa cell tumors are molecularly characterized by a pathognomonic somatic missense point mutation 402C->G (C134W) in the transcription factor FOXL2. The FOXL2 402C->G mutation leads to increased proliferation and survival of granulosa cells, and promotes hormonal changes. Histological diagnosis of adult-type granulosa cell tumor is challenging, therefore testing for the FOXL2 mutation is crucial for differential diagnosis. Large international collaborations utilizing molecularly defined cohorts are essential to improve and validate new treatment strategies for patients with high-risk or relapsed adult-type granulosa cell tumor. Key Messages: Adult-type granulosa cell tumor is a unique ovarian cancer with an indolent, albeit unpredictable disease course. Adult-type granulosa cell tumors harbor a pathognomonic somatic missense mutation in transcription factor FOXL2. The key challenges in the treatment of patients with adult-type granulosa cell tumor lie in the identification and management of patients with high-risk or relapsed disease.

Roles of human epididymis protein 4, carbohydrate antigen 125, inhibin B and anti-Müllerian hormone in the differential diagnosis and follow-up of ovarian granulosa cell tumors.

OBJECTIVE: Evaluation of circulating tumor markers in ovarian cancer is crucial for optimal patient care. The goal of this study was to verify the most accurate circulating tumor markers for the diagnosis and follow-up of adult-type granulosa cell tumors (AGCTs). METHODS: The levels of circulating human epididymis protein 4 (HE4) and carbohydrate antigen 125 (CA125), together with AGCT markers inhibin B and anti-Müllerian hormone (AMH), were measured in 135 samples from AGCT patients, 37 epithelial ovarian carcinoma (EOC) patients, and 40 endometrioma (ENDO) patients. The levels were plotted with receiver operating characteristic (ROC) graphs, and the area under the curves (AUC) of the different markers were calculated and compared. RESULTS: HE4 levels were significantly lower in AGCTs than in EOCs (p<0.0001). CA125 levels were above 35IU/l in 25% of AGCT patients and 47.5% of ENDO patients, whereas inhibin B and AMH levels were elevated only in patients with AGCTs. In the AUC comparison analyses, inhibin B alone was sufficient to differentiate AGCT from EOC. In differentiating AGCT from ENDO, inhibin B and AMH performed similarly, and the combination of inhibin B and AMH increased the accuracy compared to either marker alone (sensitivity, 100%; specificity, 93%). Among AGCT patients, inhibin B was the best marker for detecting the presence of AGCT. CONCLUSIONS: HE4 and CA125 levels were low in AGCTs, and inhibin B was the most accurate circulating biomarker in distinguishing AGCTs from EOCs and from ENDOs. Inhibin B was also the best single marker for AGCT follow-up.

Characteristics and outcome of recurrence in molecularly defined adult-type ovarian granulosa cell tumors.

OBJECTIVE: Adult-type ovarian granulosa cell tumors (AGCTs) have an unpredictable tendency to relapse. In a carefully validated patient cohort, we evaluated the prognostic factors related to AGCT recurrence. METHODS: We identified all patients diagnosed with AGCT during 1956-2014 in Helsinki University Hospital, with a minimum follow-up of one year (n=240). After a histological review supplemented with FOXL2 (402C-G) mutation status analysis, we analyzed the clinical data for association with relapse. RESULTS: The final cohort included 164 (68%) molecularly defined AGCTs (MD-AGCTs). The majority of the women were postmenopausal (63%), and 92% of tumors were stage I. The median follow-up time was 15.5years. Fifty-two (32%) patients developed tumor recurrence, of whom 55% had successive recurrences. Multiple-site recurrences were common, and nearly half of the recurrences were asymptomatic. The median time to the first relapse was 7.4years, and 75% of relapses occurred within ten years after primary diagnosis. The median disease-free survival was 11.3years. Premenopausal status at initial diagnosis, FIGO stage Ic versus Ia, and tumor rupture associated with relapse. However, tumor rupture was the only independent predictive factor. Of the relapsed patients, 48% died of AGCT in a median time of 15.3years. CONCLUSION: Tumor rupture is the strongest predictive factor for recurrence, and these patients might benefit from a more aggressive initial treatment approach. AGCT requires active follow up for 10 to 15years after primary diagnosis, since recurrences may develop late, asymptomatically and in multiple anatomical locations.

Glycodelin reduces carcinoma-associated gene expression in endometrial adenocarcinoma cells.

OBJECTIVE: Glycodelin is a major secretory glycoprotein of differentiated endometrial epithelium, rarely expressed in proliferative endometrium or endometrial cancer. We aimed to elucidate its role in growth and gene expression of endometrial adenocarcinoma cells, and hypothesized that glycodelin affects cell growth and tumor-associated gene expression. STUDY DESIGN: Endometrial adenocarcinoma HEC-1B cells were transfected with glycodelin cDNA in both antisense and sense orientations. Cellular morphology, cell proliferation, and gene expression were compared between native and transfected cells. RESULTS: Compared with native and antisense-transfected carcinoma cells, sense-transfected, glycodelin-producing carcinoma cells showed reduced proliferation, morphologic changes, and altered expression of cancer-related genes. Notably, anti-apoptotic Bcl-XL and MUC1 genes were down-regulated. CONCLUSION: Reduction by glycodelin transfection of carcinoma cell proliferation and expression of MUC1 and Bcl-XL is significant because these genes are often overexpressed in human cancers--MUC1 is linked to invasive growth and metastases, and both confer resistance to chemotherapy. These results suggest a novel mechanism whereby malignant growth of endometrial adenocarcinoma cells is regulated.