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BACKGROUND: Cyclin E1 (CCNE1) is a potential predictive marker and therapeutic target in tubo-ovarian high-grade serous carcinoma (HGSC). Smaller studies have revealed unfavorable associations for CCNE1 amplification and CCNE1 overexpression with survival, but to date no large-scale, histotype-specific validation has been performed. The hypothesis was that high-level amplification of CCNE1 and CCNE1 overexpression, as well as a combination of the two, are linked to shorter overall survival in HGSC. METHODS: Within the Ovarian Tumor Tissue Analysis consortium, amplification status and protein level in 3029 HGSC cases and mRNA expression in 2419 samples were investigated. RESULTS: High-level amplification (>8 copies by chromogenic in situ hybridization) was found in 8.6% of HGSC and overexpression (>60% with at least 5% demonstrating strong intensity by immunohistochemistry) was found in 22.4%. CCNE1 high-level amplification and overexpression both were linked to shorter overall survival in multivariate survival analysis adjusted for age and stage, with hazard stratification by study (hazard ratio [HR], 1.26; 95% CI, 1.08-1.47, p = .034, and HR, 1.18; 95% CI, 1.05-1.32, p = .015, respectively). This was also true for cases with combined high-level amplification/overexpression (HR, 1.26; 95% CI, 1.09-1.47, p = .033). CCNE1 mRNA expression was not associated with overall survival (HR, 1.00 per 1-SD increase; 95% CI, 0.94-1.06; p = .58). CCNE1 high-level amplification is mutually exclusive with the presence of germline BRCA1/2 pathogenic variants and shows an inverse association to RB1 loss. CONCLUSION: This study provides large-scale validation that CCNE1 high-level amplification is associated with shorter survival, supporting its utility as a prognostic biomarker in HGSC. ispartof: CANCER vol:129 issue:5 pages:697-713 ispartof: location:United States status: published

Background Recently, we showed a >60% difference in 5-year survival for patients with tubo-ovarian high-grade serous carcinoma (HGSC) when stratified by a 101-gene mRNA expression prognostic signature. Given the varied patient outcomes, this study aimed to translate prognostic mRNA markers into protein expression assays by immunohistochemistry and validate their survival association in HGSC. Methods Two prognostic genes, FOXJ1 and GMNN, were selected based on high-quality antibodies, correlation with protein expression and variation in immunohistochemical scores in a preliminary cohort (n = 134 and n = 80, respectively). Six thousand four hundred and thirty-four (FOXJ1) and 5470 (GMNN) formalin-fixed, paraffin-embedded ovarian neoplasms (4634 and 4185 HGSC, respectively) represented on tissue microarrays from the Ovarian Tumor Tissue Analysis consortium underwent immunohistochemical staining and scoring, then univariate and multivariate survival analysis. Results Consistent with mRNA, FOXJ1 protein expression exhibited a linear, increasing association with improved overall survival in HGSC patients. Women with >50% expression had the most favourable outcomes (HR = 0.78, 95% CI 0.67–0.91, p 35% GMNN expression showed a trend for better outcomes, though this was not significant. Conclusion We provide foundational evidence for the prognostic value of FOXJ1 in HGSC, validating the prior mRNA-based prognostic association by immunohistochemistry.

Purpose: Advanced-stage mucinous ovarian carcinoma (MOC) has poor chemotherapy response and prognosis and lacks biomarkers to aid stage I adjuvant treatment. Differentiating primary MOC from gastrointestinal (GI) metastases to the ovary is also challenging due to phenotypic similarities. Clinicopathologic and gene-expression data were analyzed to identify prognostic and diagnostic features. Experimental Design: Discovery analyses selected 19 genes with prognostic/diagnostic potential. Validation was performed through the Ovarian Tumor Tissue Analysis consortium and GI cancer biobanks comprising 604 patients with MOC (n = 333), mucinous borderline ovarian tumors (MBOT, n = 151), and upper GI (n = 65) and lower GI tumors (n = 55). Results: Infiltrative pattern of invasion was associated with decreased overall survival (OS) within 2 years from diagnosis, compared with expansile pattern in stage I MOC [hazard ratio (HR), 2.77; 95% confidence interval (CI), 1.04–7.41, P = 0.042]. Increased expression of THBS2 and TAGLN was associated with shorter OS in MOC patients (HR, 1.25; 95% CI, 1.04–1.51, P = 0.016) and (HR, 1.21; 95% CI, 1.01–1.45, P = 0.043), respectively. ERBB2 (HER2) amplification or high mRNA expression was evident in 64 of 243 (26%) of MOCs, but only 8 of 243 (3%) were also infiltrative (4/39, 10%) or stage III/IV (4/31, 13%). Conclusions: An infiltrative growth pattern infers poor prognosis within 2 years from diagnosis and may help select stage I patients for adjuvant therapy. High expression of THBS2 and TAGLN in MOC confers an adverse prognosis and is upregulated in the infiltrative subtype, which warrants further investigation. Anti-HER2 therapy should be investigated in a subset of patients. MOC samples clustered with upper GI, yet markers to differentiate these entities remain elusive, suggesting similar underlying biology and shared treatment strategies.

Supplementary Methods

Supplementary Figures S1-S13

Purpose:Advanced-stage mucinous ovarian carcinoma (MOC) has poor chemotherapy response and prognosis and lacks biomarkers to aid stage I adjuvant treatment. Differentiating primary MOC from gastrointestinal (GI) metastases to the ovary is also challenging due to phenotypic similarities. Clinicopathologic and gene-expression data were analyzed to identify prognostic and diagnostic features.Experimental Design:Discovery analyses selected 19 genes with prognostic/diagnostic potential. Validation was performed through the Ovarian Tumor Tissue Analysis consortium and GI cancer biobanks comprising 604 patients with MOC (n = 333), mucinous borderline ovarian tumors (MBOT, n = 151), and upper GI (n = 65) and lower GI tumors (n = 55).Results:Infiltrative pattern of invasion was associated with decreased overall survival (OS) within 2 years from diagnosis, compared with expansile pattern in stage I MOC [hazard ratio (HR), 2.77; 95% confidence interval (CI), 1.04–7.41, P = 0.042]. Increased expression of THBS2 and TAGLN was associated with shorter OS in MOC patients (HR, 1.25; 95% CI, 1.04–1.51, P = 0.016) and (HR, 1.21; 95% CI, 1.01–1.45, P = 0.043), respectively. ERBB2 (HER2) amplification or high mRNA expression was evident in 64 of 243 (26%) of MOCs, but only 8 of 243 (3%) were also infiltrative (4/39, 10%) or stage III/IV (4/31, 13%).Conclusions:An infiltrative growth pattern infers poor prognosis within 2 years from diagnosis and may help select stage I patients for adjuvant therapy. High expression of THBS2 and TAGLN in MOC confers an adverse prognosis and is upregulated in the infiltrative subtype, which warrants further investigation. Anti-HER2 therapy should be investigated in a subset of patients. MOC samples clustered with upper GI, yet markers to differentiate these entities remain elusive, suggesting similar underlying biology and shared treatment strategies.

Supplementary Tables S1-S11

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 1,623 endometriosis-associated ovarian carcinomas, including 1,078 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+ TIL), and DNA mismatch repair deficiency (MMRd). ARID1A loss was observed in 42% of CCOC and 25% of ENOC. 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 associated with MMRd (p

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 < 0.0001, N = 1840) in multivariable analysis. MCM3 mRNA expression was highest in the HGSC C5.PRO molecular subtype, although no interaction was observed between MCM3, survival and molecular subtypes. MCM3 and Ki-67 protein levels were significantly lower after exposure to neoadjuvant chemotherapy compared to chemotherapy-naïve tumors: 37.0% versus 46.4% and 22.9% versus 34.2%, respectively. Among chemotherapy-naïve HGSC, high MCM3 protein levels were also associated with significantly longer disease-specific survival (HR = 0.52, 95% CI 0.36–0.74, p = 0.0003, N = 392) compared to cases with low MCM3 protein levels in multivariable analysis. MCM3 immunohistochemistry is a promising surrogate marker of proliferation in HGSC.

Background: New targets for ovarian cancer treatment are critically needed. The Wnt receptors ROR1 and ROR2 are overexpressed in all subtypes of ovarian cancer and appear to play a role in both the tumor and surrounding microenvironment (1). In vitro studies support the approach of targeting these receptors together to inhibit ovarian cancer migration and invasion (2, 3). Aim: To investigate the role of ROR1 and ROR2 in ovarian cancer progression, survival and chemoresistance, to determine their feasibility as therapeutic targets. Methods: Analysis of RNA-Seq data from publicly available ovarian cancer datasets was performed to determine associations with overall survival and platinum resistance. ROR1 IHC using a recently released monoclonal antibody (4A5, BD Bioscience) was performed on a cohort of ovarian cancer patients. ROR1 and/or ROR2 were silenced in a unique preclinical organotypic model of platinum-resistant ovarian cancer metastasis. Results: There is a significant increase (p=0.01) in ROR1 expression in both primary resistant and acquired resistant (p=0.03) high-grade serous ovarian cancer (HGSOC). ROR2 expression is not significantly associated with chemoresistance. High ROR1 (P=0.002, HR 1.93) or high ROR2 (P=0.004, HR 1.9) expression is associated with a significantly shorter overall survival (OS) in the Tothill cohort (4). Patients expressing both high ROR1 and high ROR2 have the shortest OS (P=0.0003, HR 2.38). ROR1 protein expression was detected in 97% of HGSOC patients via IHC, with 59% of HGSOC expressing “high ROR1” (IHC score 2 or 3). Overall, patients with higher-grade tumors expressed significantly higher ROR1 levels (P=0.001) than patients with low-grade tumors. Silencing ROR1 and ROR2 in combination inhibits adhesion and invasion of platinum resistant ovarian cancer in a 3D organotypic coculture model. Conclusion: Our data support targeting both ROR1 and ROR2 as a new approach to treating ovarian cancer. The development of a number of monoclonal antibodies targeting ROR1 and ROR2 that are currently in phase 1 trials for other tumor types makes this clinically feasible in the future. These data also suggest that this may be most effective in the context of HGSOC with platinum resistance. References: 1. Henry CE et al. Distinct patterns of stromal and tumor expression of ROR1 and ROR2 in histological subtypes of epithelial ovarian cancer. Transl Oncol 2017;10(3):346-56. 2. Henry C, Hacker NF, Ford CE. Silencing ROR1 and ROR2 inhibits invasion and adhesion in an organotypic model of ovarian cancer metastasis. Oncotarget 2017;8(68):112727-38. 3. Henry C et al. ROR1 and ROR2 are upregulated in chemoresistant ovarian cancer and promote migration and invasion. Oncogenesis 2016;5(5):doi: 10.1038/oncsis.2016.32. 4. Tothill RW et al. Novel molecular subtypes of serous and endometrioid ovarian cancer linked to clinical outcome. Clin Cancer Res 2008;14(16):5198-208. Citation Format: Dongli Liu, Miya John, Claire E. Henry, Elizabeth L. Christie, David D.L. Bowtell, Kristina Tang, Viola Heinzelmann-Schwarz, Catherine Kennedy, Jessica Boros, Anna DeFazio, Caroline E. Ford. ROR1 is associated with ovarian cancer progression and chemoresistance [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A41.

An eclectic range of ocular growth factors with differing actions are present within the aqueous and vitreous humors that bathe the lens. Growth factors that exert their actions via receptor tyrosine kinases (RTKs), such as FGF, play a normal regulatory role in lens; whereas other factors, such as TGFβ, can lead to an epithelial to mesenchymal transition (EMT) that underlies several forms of cataract. The respective downstream intracellular signaling pathways of these factors are in turn tightly regulated. One level of negative regulation is thought to be through RTK-antagonists, namely, Sprouty (Spry), Sef and Spred that are all expressed in the lens. In this study, we tested these different negative regulators and compared their ability to block TGFβ-induced EMT in rat lens epithelial cells. Spred expression within the rodent eye was confirmed using RT-PCR, western blotting and immunofluorescence. Rat lens epithelial explants were used to examine the morphological changes associated with TGFβ-induced EMT over 3 days of culture, as well as α-smooth muscle actin (α-sma) immunolabeling. Cells in lens epithelial explants were transfected with either a reporter (EGFP) vector (pLXSG), or with plasmids also coding for different RTK-antagonists (i.e. pLSXG-Spry1, pLSXG-Spry2, pLXSG-Sef, pLSXG-Spred1, pLSXG-Spred2, pLSXG-Spred3), before treating with TGFβ for up to 3 days. The percentages of transfected cells that underwent TGFβ-induced morphological changes consistent with an EMT were determined using cell counts and validated with a paired two-tailed t-test. Explants transfected with pLXSG demonstrated a distinct transition in cell morphology after TGFβ treatment, with ∼60% of the cells undergoing fibrotic-like cell elongation. This percentage was significantly reduced in cells overexpressing the different antagonists, indicative of a block in lens EMT. Of the antagonists tested under these in vitro conditions, Spred1 was the most potent demonstrating the greatest block in TGFβ-induced fibrotic cell elongation/EMT. Through the overexpression of RTK-antagonists in lens epithelial cells we have established a novel role for Spry, Spred and Sef as negative regulators of TGFβ-induced EMT. Further investigations may help us develop a better understanding of the molecular mechanisms involved in maintaining the integrity of the normal lens epithelium, with these antagonists serving as putative therapeutic agents for prevention of EMT, and hence cataractogenesis.

Growth factor signaling, mediated via receptor tyrosine kinases (RTKs), needs to be tightly regulated in many developmental systems to ensure a physiologically appropriate biological outcome. At one level this regulation may involve spatially and temporally ordered patterns of expression of specific RTK signaling antagonists, such as Sef (similar expression to fgfs). Growth factors, notably FGFs, play important roles in development of the vertebrate ocular lens. FGF induces lens cell proliferation and differentiation at progressively higher concentrations and there is compelling evidence that a gradient of FGF signaling in the eye determines lens polarity and growth patterns. We have recently identified the presence of Sef in the lens, with strongest expression in the epithelial cells. Given the important role for FGFs in lens developmental biology, we employed transgenic mouse strategies to determine if Sef could be involved in regulating lens cell behaviour. Over-expressing Sef specifically in the lens of transgenic mice led to impaired lens and eye development that resulted in microphthalmia. Sef inhibited primary lens fiber cell elongation and differentiation, as well as increased apoptosis, consistent with a block in FGFR-mediated signaling during lens morphogenesis. These results are consistent with growth factor antagonists, such as Sef, being important negative regulators of growth factor signaling. Moreover, the lens provides a useful paradigm as to how opposing gradients of a growth factor and its antagonist could work together to determine and stabilise tissue patterning during development and growth.

In many developmental systems, growth factor signalling must be temporally and spatially regulated, and this is commonly achieved by growth factor antagonists. Here, we describe the expression patterns of newly identified growth factor inhibitors, Sprouty and Sef, in the developing ocular lens. Sprouty and Sef are both expressed in the lens throughout embryogenesis, and become restricted to the lens epithelium, indicating that lens cell proliferation and fibre differentiation may be tightly regulated by such antagonists. Future studies will be aimed at understanding how these negative regulatory molecules modulate growth factor-induced signalling pathways and cellular processes in the lens.

Early in development, the ocular lens establishes its distinctive architecture, and this is maintained throughout life as the lens continues to grow. This growth is tightly regulated through the proliferation of the lens epithelial cells and their subsequent differentiation into specialized elongated fiber cells. Although much work has been carried out to define these patterns of growth, very little has been reported on the detailed fate and kinetics of lens cells during embryogenesis. Using BrdU-incorporation, the present study has attempted to follow the fate of lens cells that have undergone at least one round of DNA synthesis during the early stages of lens morphogenesis. Results from this work have confirmed that the rate of lens cell proliferation and new fiber cell differentiation progressively slows as the lens differentiates and grows. In addition, these studies have shown that early in lens development, not all DNA synthesis is restricted to the lens epithelium, with some elongating fiber cells retaining the ability to undergo DNA synthesis. Adopting this system we have also been able to place the initiation of secondary fiber cell differentiation in the mouse lens by E12.5, concomitant with the loss of the lens vesicle lumen by the elongating primary fiber cells. Overall, this study has allowed us to revisit some of the mechanisms involved in early lens development, has provided us with insights into the fate of cells during this rapid phase of murine lens growth, and has provided a novel method to study the rate of new fiber cell differentiation over a defined period of lens development and growth.