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SummaryER and PR regulate growth and differentiation in normal ovaries and fallopian tubes and in HGSC transformation and progression. Higher PR expression was associated with improved survival outcomes, while high ER expression was associated with worse survival in patients with HGSC. Here, we show that patients with ER+PR+ tumors have longer overall survival and confirm the role of PR as a prognostic marker of survival and response to chemotherapy. Gene expression analysis demonstrated up-regulation of the ATM signaling pathway in the ER+PR+ subgroup when compared to ER+PR− tumors. Up-regulation of interferon alpha, beta and gamma signaling, and antigen presentation pathways were identified in ER+PR− compared to ER−PR+. In summary, this study elucidated that the genomic and transcriptomic signatures related to ER/PR status in HGSC have clinical prognostic value.

Heatmap of protein functions

The human microbiome has been strongly correlated with disease pathology and outcomes, yet remains relatively underexplored in patients with malignant endometrial disease. In this study, vaginal microbiome samples were prospectively collected at the time of hysterectomy from 61 racially and ethnically diverse patients from three disease conditions: (i) benign gynecologic disease (controls, n = 11), (ii) low-grade endometrial carcinoma (n = 30), and (iii) high-grade endometrial carcinoma (n = 20). Extracted DNA underwent shotgun metagenomics sequencing, and microbial α and β diversities were calculated. Hierarchical clustering was used to describe community state types (CST), which were then compared by microbial diversity and grade. Differential abundance was calculated, and machine learning utilized to assess the predictive value of bacterial abundance to distinguish grade and histology. Both α- and β-diversity were associated with patient tumor grade. Four vaginal CST were identified that associated with grade of disease. Different histologies also demonstrated variation in CST within tumor grades. Using supervised clustering algorithms, critical microbiome markers at the species level were used to build models that predicted benign versus carcinoma, high-grade carcinoma versus benign, and high-grade versus low-grade carcinoma with high accuracy. These results confirm that the vaginal microbiome segregates not just benign disease from endometrial cancer, but is predictive of histology and grade. Further characterization of these findings in large, prospective studies is needed to elucidate their potential clinical applications.Significance:The vaginal microbiome reliably segregates not just benign gynecologic condition from endometrial cancer, but also predicts cancer grade and histology. Patterns of microbial abundance and gene expression should be increasingly considered as a factor in the evolution of precision medicine approaches, especially as they relate to cancer screening, disease pathogenesis, and patient-centered outcomes.

Heatmap of abundance values

Additional statistical methods

Biomarker discovery by histology

Individual Specimen Reads

Analysis of Confounders

Functional pathway analysis

Microbial alpha diversities

Microbial abundance by histotype

The human microbiome has been strongly correlated with disease pathology and outcomes, yet remains relatively underexplored in patients with malignant endometrial disease. In this study, vaginal microbiome samples were prospectively collected at the time of hysterectomy from 61 racially and ethnically diverse patients from three disease conditions: (i) benign gynecologic disease (controls, n = 11), (ii) low-grade endometrial carcinoma (n = 30), and (iii) high-grade endometrial carcinoma (n = 20). Extracted DNA underwent shotgun metagenomics sequencing, and microbial α and β diversities were calculated. Hierarchical clustering was used to describe community state types (CST), which were then compared by microbial diversity and grade. Differential abundance was calculated, and machine learning utilized to assess the predictive value of bacterial abundance to distinguish grade and histology. Both α- and β-diversity were associated with patient tumor grade. Four vaginal CST were identified that associated with grade of disease. Different histologies also demonstrated variation in CST within tumor grades. Using supervised clustering algorithms, critical microbiome markers at the species level were used to build models that predicted benign versus carcinoma, high-grade carcinoma versus benign, and high-grade versus low-grade carcinoma with high accuracy. These results confirm that the vaginal microbiome segregates not just benign disease from endometrial cancer, but is predictive of histology and grade. Further characterization of these findings in large, prospective studies is needed to elucidate their potential clinical applications. Significance: The vaginal microbiome reliably segregates not just benign gynecologic condition from endometrial cancer, but also predicts cancer grade and histology. Patterns of microbial abundance and gene expression should be increasingly considered as a factor in the evolution of precision medicine approaches, especially as they relate to cancer screening, disease pathogenesis, and patient-centered outcomes.

BackgroundThe vaginal microbiome is an underexplored environment which may yield insights about endometrial cancer (EC) pathophysiology and serve as an early marker of disease. Our objective was to evaluate the preoperative vaginal microbiome in women undergoing hysterectomy for benign conditions and EC to identify patterns which could segregate benign disease from EC, and to assess if the microbiome distinguishes low-grade (LG) from high-grade (HG) histologies.MethodsVaginal microbiome samples were prospectively collected at the time of hysterectomy, and clinicopathologic data collected. Extracted DNA underwent shotgun metagenomics sequencing. Microbial diversity was calculated using the Shannon Index (α-diversity) and PERMANOVA (β-diversity). Hierarchical clustering was used to describe community state types (CST), which were then compared by microbial diversity and grade. Machine learning was utilized to assess the predictive value of grade and histology based on relative bacterial abundance.Results61 patients participated: 1) benign gynecologic disease (n=11), 2) LG EC (n=30), and 3) HG EC (n=20). 40 (62.5%) were White, 22 (34.3%) were Black, and 37 (57.8%) were Hispanic. Both α- and β-diversity were associated with tumor grade (p□=□0.026 and p□=□0.035, respectively). Four CST were identified that associated with grade of disease (p=0.036). Different histologies demonstrated variation in CST even within tumor grades (p=0.017). Markers at the species level informed models that predicted benign vs cancer (AUC 0.878), HG versus benign (AUC=0.803), and HG versus LG (AUC=0.771) with high accuracy.ConclusionsThe vaginal microbiome segregates benign disease from EC, and is predictive of histology and grade, suggesting it may be an effective tool for screening.

Introduction Variability in endometrial cancer (EC) risk and outcomes exist, with Black women disproportionately negatively affected related to White women. The biologic etiologies of these observations are poorly understood. Our objective was to describe the somatic mutations of the tumor genome and compare them to RNA expression in self-identified Black and White women diagnosed with EC. Methods Women with newly diagnosed EC and undergoing hysterectomy were recruited prospectively. Fresh tumors obtained at the time of surgery were flash-frozen, underwent DNA and RNA extraction and were submitted for whole exome sequencing (WES) analysis (DNA) or bulk RNA sequencing analysis. Differentially expressed genes between racial groups and tumor grade were calculated using Deseq2. Genomic ancestry was determined using ADMIXTURE. Chi-square and Mann Whitney U tests were used for analyses, with a threshold for significance of p14% African ancestry. The remaining 13 (26.5%) patients had admixed European and Native American ancestry, with Citation Format: Alex P. Sanchez-Covarrubias, Ramlogan Sowamber, Leah Dodds, Andre Pinto, Sophia George, Matthew Schlumbrecht. Endometrial cancer genomics varies by race [abstract]. In: Proceedings of the 15th AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2022 Sep 16-19; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2022;31(1 Suppl):Abstract nr C052.

The purpose of this protocol is to grow primary fallopian tube epithelial cells as 2D culture. This protocol explains the method of culturing and maintaining fallopian tube epithelial cells derived from processing human fallopian tube tissue.

Epithelial ovarian cancer represents a group of heterogeneous diseases with high grade serous cancer (HGSC) representing the most common histotype. Molecular profiles of precancerous lesions found in the fallopian tube have implicated this tissue as the presumptive site of origin of HGSC. Precancerous lesions are primarily found in the distal fallopian tube (fimbria), near the ovary relative to the proximal tissue (ampulla), nearer to the uterus. The proximity of the fimbria to the ovary and the link between ovulation, through follicular fluid release, and ovarian cancer risk led us to examine transcriptional responses of fallopian tube epithelia (FTE) at the different anatomical sites of the human fallopian tube. Gene expression profiles of matched FTE from the fimbria and from premenopausal women resulted in differentially expressed genes (DEGs): CYYR1, SALL1, FOXP2, TAAR1, AKR1C2/C3/C4, NMBR, ME1 and GSTA2. These genes are part of the antioxidant, stem and inflammation pathways. Comparisons between the luteal phase (post-ovulation) to the follicular phase (pre-ovulation) demonstrated greater differences in DEGs than a comparison between fimbria and fallopian tube anatomical differences alone. This data suggests that cyclical transcriptional changes experienced in pre-menopause are inherent physiological triggers that expose the FTE in the fimbria to cytotoxic stressors. These cyclical exposures induce transcriptional changes reflective of genotoxic and cytotoxic damage to the FTE in the fimbria which are closely related to transcriptional and genomic alterations observed in ovarian cancer.

Introduction: The luteal phase of BRCA mutation carrier tissues has distinct profiles of differentially expressed genes compared to wild-type fimbria. High Grade Serous Ovarian Carcinoma (HGSOC) is one of the most lethal gynecological diseases and presents at late tumor stage. Studies focusing on the early events of the disease have implicated the fimbriated end of the fallopian tube as the high-risk zone for cancer development. In this study, we aim to identify BRCA transcriptomic signatures in the fimbria of patients with a BRCA mutation and compare the luteal and follicular phases to capture the effect of the extrinsic milieu in response to stressors. Experimental Procedures: Laser capture microdissection (LCM) was performed on 68 formalin fixed paraffin embedded (FFPE) fallopian tube samples based on BRCA mutation status and ovulatory cycle: Normal-Follicular, Normal-Luteal, BRCA-Follicular, BRCA-Luteal. Nine HGSC samples were derived from patients with a mutation in BRCA1. Six-Twelve, 10um FFPE sections were cut and stained with hematoxylin prior to LCM. RNA was extracted using the Roche High Pure FFPE Micro Kit and samples and were processed using Illumina Tru-Seq Stranded Total RNA Kit with RiboGold ready and sequenced on the Illumina Hi-seq 2000 V3. Bioinformatic analysis performed on multiple comparisons highlighted differentially expressed genes. Gene Set Enrichment Analysis (GSEA) performed on subgroups highlighted pathways of interest for each comparison. Genes highly expressed or strongly suppressed were shortlisted for further analysis. Western blot analysis performed on wildtype patients (WT), BRCA1 mutation and BRCA2 mutation showed protein expression of selected genes. Results: The fimbriae compared to ampullae have more stem-like features, have up-regulated antioxidant/xenobiotic genes, and have more inflammatory cells intercalated within the epithelia. The fimbria of FTE-BRCA have distinctly up-regulated genes involved in DDR (DNA damage response) which are exacerbated in the luteal phase of the ovulatory cycle. BRCA1/2 luteal versus follicular phases are involved in endoplasmic reticulum stress, migration, MAPKK. Whereas decreased differentially expressed genes are involved with G2/M checkpoint, DNA replication and ER-associated degradation pathway. Post-ovulatory phase of the ovarian cycle drives inflammation, differentiation and DNA damage-repair in FTE cells. Furthermore, GSEA analysis of the luteal and follicular phases showed positive enrichment of the inflammatory response pathway and negative enrichment of oxidative phosphorylation in the luteal phase. Conclusions: Our results highlight distinct pathways related to repair and inflammation for the BRCA1 mutation carriers and luteal phase of the fimbria. These results are consistent with previous examination of the fallopian tube and provide insight into targetable genes associated with the development of HGSC. Citation Format: Ramlogan Sowamber, Iru Paudel, Alex Sanchez, Alexandra Diaz, Patricia Shaw, Matthew Schlumbrecht, Sophia George. BRCAness in HGSC cancer initiation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2155.

Introduction: The histologically normal BRCA1 mutation carrier fallopian tube epithelia (FTE), compared to controls, showed that CEBPD was upregulated in the luteal phase of the ovulatory cycle. CEBPD is involved with the maintenance of genomic stability, promoting cellular differentiation, and regulating the cell cycle in response to cytotoxic stressors. In breast epithelial cells, CEBPD protein expression correlated with estrogen receptor (ER) and progesterone receptor (PR) and was found to be associated with increased progression-free survival in breast cancer patients. In fallopian tube epithelia (FTE), CEBPD was also found to modulate the epithelial-to-mesenchymal (EMT)/mesenchymal-to-epithelial transition (MET) by modulating target genes of this pathway. Given the hormonal response of this gene and its function in modulating an EMT/MET, the objective of this study was to determine whether sex hormones influence CEBPD regulation of EMT/MET in the fallopian tube and thus ovarian cancer. Methods: Fresh fallopian tube (FTE) tissues were obtained from patients approved for collection by IRB. Immunohistochemical profiling on normal fallopian tube tissue and HGSC was performed using CEBPD, ER, and PR protein markers. FTE cell lines with a p53 mutation (R175H) were subjected to estradiol (50nM and 100nM) and 4-hydroxytamoxifen (10nM) and assayed using qRT-PCR and PCR. ANOVA and t-tests were conducted in GraphPad Prism software with significance set at p Results: E-cadherin in normal fallopian tube tissue was highly expressed in both the luteal and follicular phase whereas vimentin was highly expressed in the follicular phase but showed a range of expression (low expression to high expression) in the luteal phase. CEBPD overexpression increased SNAIL expression (p Conclusion: Together these results demonstrate a role for CEBPD in modulating EMT/MET in normal FTE in a hormonally regulated manner, which during cancer formation and spread is critical for anoikis and metastasis. Furthermore, these data will facilitate an understanding of the early events of carcinogenesis in fallopian tube epithelia. Citation Format: Ramlogan Sowamber, Leah V. Dodds, Patricia Shaw, Sophia H.L. George. Ovarian hormones regulate C/EBPD induced EMT/MET transition in the human fallopian tube epithelia [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 B29.

Introduction: High-grade serous ovarian cancer (HGSC) is one of the deadliest gynecologic malignancies due to presentation at advanced stages and the development of chemotherapy resistance. The recent discovery that HGSC arises from fallopian tube epithelia (FTE) and the analysis results obtained from TCGA study are helping in understanding the genes involved in the progression of the disease. HGSC is genetically unstable due to the loss of function mutations in BRCA1, BRCA2, PALB2, ATM, and RAD51 genes involved in DNA damage response. These genes also regulate cellular metabolism, antioxidant, and detoxification responses. Preliminary data show that phase II antioxidant enzymes are significantly reduced in HGSC and there is an increase in reactive oxygen species (ROS) in cells harboring p53 and BRCA1 mutations in precursor lesions of FTE and HGSC. We also observed differential mRNA expression of PCK1 and PCK2, GLUT1 genes involved in gluconeogenesis and glycolysis. Mitochondria are one of the major organelles for the production of ROS, and it is a well-known fact that metabolism is drastically changed in cancer cells compared to normal cells. Our aim was to determine the metabolic and fuel dependency in preneoplastic transformed cells, characterize the mitochondrial phenotype, and link these dependencies to transformation in FTE. Methods: Fallopian tube specimens were obtained from the Biospecimen Shared Resources Core at SCCC. Patients undergoing risk reduction surgery were consent and FTE cells were obtained from BRCA and non-BRCA. Preneoplastic cell lines were then generated by hTERT, p53 mutation (R175H), and HPVE7. To mimic the genetic changes of in vivo HGSC, oncogenes CCNE1 and PIK3CA-H1047R were then overexpressed (OE) in these immortalized cell lines. Xenografts were generated from the transformed FTE cells lines. Agilent Seahorse assays were used to study the fuel dependency, oxidative phosphorylation, and glycolysis. Results: FTE-BRCA1 and FTE-BRCA2 cells showed differences in both glycolytic and oxidative metabolism depending on the oncogenic type. The transformed cells had lower oxidative metabolism and there was also discrepancy in the dependency of metabolites utilized by these cell lines for energy. As seen by mRNA, FTE-BRCA cells had more PCK2 protein and were characterized by higher metabolic rates; these observations were augmented in the presence of PIK3CA-H1047R. However, these FTE preneoplastic and oncogene transformed cells showed no difference in their mitochondrial mass or membrane potential. Conclusion: FTE-BRCA mutant cells utilize glycolysis or gluconeogenesis pathways to overcome metabolic stress depending on which oncogene is driving carcinogenesis. Further work is required to elucidate the mechanism of PCK2 and BRCA link in ovarian cancer carcinogenesis. Citation Format: Iru Paudel, Ramlogan Sowamber, Leah Dodds, Alex Sanchez, Marilyn Huang, Matthew Schlumbrecht, Brian Slomovitz, Sophia George. BRCA haploinsufficiency promotes gluconeogenesis in fallopian tube epithelial cells [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 B15.

Liver kinase B1 (LKB1) is a tumor suppressor ubiquitously expressed serine/threonine protein kinase involved in energy metabolism and cellular polarity. In microarray experiments that compared normal tubal epithelium with high-grade serous carcinoma (HGSC), we observed a decrease in LKB1 mRNA expression in HGSC. In this study, we demonstrate that loss of cytoplasmic and nuclear LKB1 protein expression is frequently observed in tubal cancer precursor lesions as well as in both sporadic and hereditary HGSCs compared with other ovarian cancer histotypes. Bi-allelic genomic loss of LKB1 in HGSC did not account for the majority of cases with a decrease in protein expression. In vitro, shLKB1-fallopian tube epithelial (FTE) cells underwent premature cellular arrest and in ex vivo FTE culture, LKB1 loss and p53 mutant synergized to disrupt apical to basal polarity and decrease the number of ciliated cells. Overexpression of cyclin E1 allowed for bypass of LKB1-induced cellular arrest, and increased both proliferation and anchorage-independent growth of transformed FTE cells. These data suggest that LKB1 loss early in ovarian serous tumorigenesis has an integral role in tumor promotion by disrupting apical to basal polarity in the presence of mutated p53 in fallopian tube cells.

Inheritance of a deleterious mutation in the BRCA1/2 genes increases the risk of the disease by up to 40%. The origin of the disease is still debated; however considerable evidence suggests the fallopian tube as the primary site of disease origin. The fallopian tube epithelium changes morphologically and genomically with the ovulatory cycle. To further understand the transcriptomic profile of the fallopian tube epithelium amongst patients with and without a BRCA mutation who have undergone prophylactic surgery, samples with clinically annotated ovulatory cycle status were analyzed by next-generation sequencing (RNA-Seq). The results from this study will provide an understanding of the origins of the disease and how new therapeutic and preventative interventions can be applied early in the development of the disease. METHODS: In total a cohort of 68 archival formalin fixed paraffin embedded (FFPE) fallopian tube -fimbria was amassed: FTE-BRCA1 (n=32), FTE-BRCA2 (n=4), chemonaive HGSC-BRCA1 (n=9), FTE-nonBRCA (n=26). The pre-menopausal fallopian tube specimens were sub-divided by ovulatory cycle: normal-Follicular, normal-Luteal, BRCA-Follicular, BRCA-Luteal. The majority of the FTE specimen were obtained from pre-menopausal women (post-menopausal n=2). Laser capture microdissection was performed on the distal end of the fallopian tubes – the fimbria. Six-twelve, 10um FFPE sections were cut and stained with hematoxylin prior to LCM. RNA was extracted using the Roche High Pure FFPE Micro Kit and samples were processed using Illumina Tru-Seq Stranded Total RNA Kit with RiboGold ready and sequenced on the Illumina Hi-seq 2000 V3. Raw results, in FASTQ format, were then processed through the RNA sequencing pipeline to generate results including gene expression data. RESULTS: Gene expression differences between carriers and non-carriers (FTE-BRCA1/2 and FTE-nonBRCA) revealed genes involved in metabolic pathways namely: oxidative phosphorylation, mitochondrial functions, glycolysis/gluconeogenesis and glycan biosynthesis and metabolism: UGT2A1, ST6GALNNAC6, Complex 1 (ND1, ND2, ND4, NDL4, ND5), ATP6/ATP8, and COX1-3. A comparison between the tumor and fimbria cases showed increased activity in the HMGBI Signaling (increase in HAT1, KAT2B, LIF, JAK3 and PIK3CA and decrease in RAP1A/B, ATM and HMGB1). CONCLUSION: These results highlight BRCA1/2 distinct unique preneoplastic processes not previously identified. The fallopian tube epithelial in the fimbria in BRCA1 and BRCA2 mutation carriers have increased metabolic activity, indicated by their gene expression profiles. HGSC developed BRCA1 germline mutation carriers have an increased histone acetyltransferase activity shown to be involved in BRCA mediated DNA damage repair. Further analysis is required to understand how the ovulatory cycle influences the fallopian tube epithelium transcriptome, but preliminary results suggest that pathways altered include: metabolic pathways, apoptosis pathways, p53 pathways and mismatch repair pathways. These results support previous findings in addition to providing new insight into the early development of the disease. Citation Format: Ramlogan Sowamber, Leah Dodds, Anca Milea, Iru Paudel, Michael Considine, Leslie Cope, Patricia A Shaw and Sophia HL George. RNA-SEQUENCING OF FALLOPIAN TUBE – FIMBRIA AND HGSC FROM BRCA MUTATION CARRIERS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr DP-005.

Introduction: The most common and aggressive histotype of ovarian cancer is high-grade serous carcinoma (HGSC), which accounts for approximately 90% of deaths. Furthermore, the risk of developing HGSC is increased by having a mutation in the BRCA1 and BRCA2 gene. There is considerable evidence detailing the fallopian tube epithelia (FTE) as the site of origin of the disease, yet the ovarian surface epithelium (OSE) is still proposed as the site of origin by some. Currently, no studies have compared in vivo/xenografts models derived from FTE and OSE of BRCA mutation carriers. We suggest that in vivo/xenograft models can prove a useful tool for understanding the disease origins, meanwhile accounting for the genetic characteristics of the disease. Methodology: Immortalized and transformed cell lines, from both FTE and OSE, were derived from both BRCA1 and BRCA2 mutation carriers and non-carriers. In particular, 16 OSE cell lines were created--3 from BRCA1 carriers and noncarriers. Cell lines were transfected with hTERT and SV40, and either vectors overexpressing cMYC and hRASV12. 8 of these lines with cMYC or hRASV12 were injected into the mammary fat pad of 6-week-old female NSG mice (NOD.Cg-Prkdcscid). Similarly, 16 FTE lines were generated from 2-BRCA1 and 1 BRCA2 heterozygous mutation and 3 non-BRCA carriers overexpressing hTERT and SV40 with either cMYC or hRASV12. Results: To date, tumors have developed from mice injected with ovarian surface epithelial cells and fallopian tube epithelial cells at the mammary fat pad and the intraperitoneal cavity. FTE cells injected intraperitoneally produced ascites and displayed tumors lining the abdominal cavity and major organs. The parental immortal lines of 4 cases were karyotyped: FTE-37 and FTE-19 (controls); FTE-13 (BRCA2 mutant) and FTE-98 (BRCA1 mutant) cell lines demonstrated varying types of tetraploidy while the control (BRCA wild type) had fewer tetraploid chromosomes. Western blot analyses demonstrated that tumors express PAX8 and epithelial markers associated with HGSC. STR profiling using GenePrint 10 also revealed that injected FTE cells grown in vitro clustered with xenograft tumors of the same cell line, demonstrating that cell line and tumors retained the same characteristics during tumor development. It is hypothesized that SV40 alone is sufficient to stimulate genomic instability in the FTE cells. Several cell lines grown in culture with expression of hRASV12 demonstrated increased proliferation relative to normal control cells. Conclusions: Our results suggest that tumor development occurs at different rates in vivo and is dependent on multiple factors including genetic mutation, expression of oncogenes, and patient-derived characteristics. We demonstrated that cells grown in culture exhibit unstable chromosomal architecture characteristic of ovarian carcinomas, which is one of many potential factors that promotes neoplastic change in the tissue. Understanding how these factors act in promoting tumorigenesis will enable us to understand the molecular changes that occur in vivo prior to the development of ovarian serous carcinoma. Citation Format: Ramlogan Sowamber, Mauricio Medrano, Noor Salman, Zahra Maamir, Nick Chauvin, Mahmoud Bitar, Patricia Shaw, Sophia HL George. Differences in neoplastic transformation potential between OSE and FTE. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr B12.

Background: The most common and aggressive type of epithelial ovarian cancers (EOC) is high-grade serous carcinoma (HGSC), which accounts for 90% of ovarian cancer deaths. HGSC is the predominant histotype associated with BRCA1 and BRCA2 mutations. Prophylactic surgery in BRCA mutation carriers has implicated the fallopian tube epithelium (FTE), a hormonal responsive tissue, as the etiologic site of origin for HGSC. Estrogen and its receptors are major regulators of growth and differentiation in normal ovaries and fallopian tubes, and its mutagenic properties have been linked to ovarian carcinogenesis. Estrogen receptors (ER) are rarely mutated, amplified, or deleted in HGSC, yet only 10% of patients respond to antiestrogen treatment. TP53 mutations in the form of the p53 signature have been found in serous tubal intraepithelial carcinomas (STIC) and are ubiquitously present in patients with HGSC. We hypothesized that in the presence of dysfunctional p53, subsequent promiscuous binding of ER will yield aberrant signaling, contributing to cellular transformation. Methods: We used our previously published gene expression profiles to generate a candidate gene list, which was chosen based on the presence of known estrogen-responsive elements. We analyzed expression of 6 ER responsive genes using data collected from laser capture microdissection in normal FTE tissues. Tissue microarray analysis (TMA) was also performed on a subset of HGSC tumor samples from this cohort, staining for PR, ER, and p53, and expression was analyzed alongside their respective outcome and overall survival Finally, to mimic the in vivo environment of early carcinogenesis, FTE-normal and FTE-p53 mutant cell lines were established and treated with 100nM estradiol, an estrogen analog, to observe changes in response. Results: Preliminary data showed that FTE-BRCA and FTE-nonBRCA seemingly look and express ER and PR proteins similarly. Underlying these morphologic similarities is a potential haploinsufficiency predisposing FTE-BRCA to cytotoxic stresses. Microarray gene expression of laser captured FTE-BRCA and FTE-nonBRCA showed varied levels of ER mRNA expression across samples (n=25) while PR transcript levels change dynamically. The data generated have facilitated the development of gene signatures and biomarkers that will predict response to antiestrogen therapy and identify patients who will benefit from hormonal therapies. Citation Format: Leah V. Dodds, Omar Nelson, Patricia Shaw, Anca Milea, Ramlogan Sowamber, Sophia HL George. Estrogen receptor signaling in fallopian tube epithelia of BRCA mutation carriers. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A63.

Genetic and reproductive factors predicate epidemiological risk factors underlying epithelial ovarian cancer. The fallopian tube epithelia (FTE), the presumptive etiological site of high-grade serous ovarian cancer (HGSC), is a hormonal responsive tissue. Estrogen is known to promote cell proliferation and its metabolism produces reactive oxygen species that damage DNA and promote tumorigenesis. Estrogen receptor (ER) is rarely mutated, amplified or deleted in HGSC, yet only 10% of patients respond to anti-estrogen treatment, suggesting that intrinsic variables to the ER pathway contribute to this clinical outcome. TP53 mutations occur in almost 100% of HGSCs, indicating that mutated p53 supports a model as an early event in the pathogenesis of HGSC. We hypothesized that in the presence of dysfunctional p53, subsequent promiscuous binding of ER will yield aberrant signaling, ultimately significantly contributing to cellular transformation. Mutant p53 and ER co-localize in FTE cells, suggesting potential synergy. We established cell lines with p53 mutations and treated them with estradiol, an estrogen analog, to observe any changes in response. The genome binding sites of ER-regulated transcription factors were then identified and mapped by whole genome chromatin immunoprecipitation-deep sequencing (ChIP-Seq). The data generated will facilitate the development of gene signatures that will predict response to anti-estrogen therapy in serous ovarian cancer patients, and contribute to the discovery of biomarkers to more accurately identify patients who will benefit from hormonal therapies. Citation Format: Leah V. Dodds, Omar L. Nelson, Ramlogan Sowamber, Andres Rodrigues, Victoria de Castro, Wendell Henry, Guillermo Morales, Brian Slomovitz, Patricia Shaw, Sophia H. George. Estrogen receptor signaling in FTE of BRCA mutation carriers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1454. doi:10.1158/1538-7445.AM2017-1454

Introduction: The most common histotype representing 90% of ovarian cancer cases is High Grade Serous Carcinoma. In a previous publication we found C/EBP-δ to be significantly increased in fallopian tube epithelial in BRCA1 mutation carriers and specifically in the luteal phase of the menstrual cycle, by transcription and protein expression. C/EBP-δ represents a master regulator of gene transcription primarily through STAT3 activation in many tumor types. C/EBP-δ is commonly induced by stress and is associated with increased DNA damage. Additionally, studies in breast cancer showed that C/EBP-δ could interact with FANCD2, which is involved in DNA repair along with BRCA1 and BRCA2. C/EBP-δ plays a dichotomous role in tumor suppression and promotion, which is tissue specific. We therefore studied the 1) differential expression of C/EBP-δ in serous carcinoma 2) expression of C/EBP-δ in precursor lesions (STIC) and 3) modeled the effect of over-expression of C/EBP-δ in an in vitro fallopian tube cell culture model. Methods: The study protocol for collection of tissue and clinical information for all patients was approved by the UHN REB. Tissue microarrays containing the different histotypes were used to validate immunohistochemical protein expression. Fallopian tubes with lesions classified as STIC using published diagnostic criteria, were selected (n=15) from women undergoing surgery for HGSC. FTE Cells were propagated on collagen IV coated plates in MCDB105/M199 media supplemented with BPE, EGF, insulin and hydrocortisone. Cells were immortalized via double infection with a lentiviral dominant negative TP53 vector and retroviruses with E7 and hTERT. Statistical analysis was performed using ANOVA (p Results/Conclusion: C/EBP-δ is down-regulated in over 60% of HGSC analyzed, an observation which is also histotype specific as LGSC tumors show high expression levels (p Citation Format: Sophia HL George, Ramlogan Sowamber, Patricia Shaw. The role of BRCA and CEBPD in serous ovarian cancer carcinogenesis [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1312.

Epithelial ovarian cancer is a heterogeneous group of diseases with multiple histotypes which can be broadly divided into a dualistic model based on morphological, molecular genetic, and clinical features. The most common and aggressive of all EOC, accounting for 90% of deaths, is High Grade Serous Carcinoma (HGSC). It is characterized by a genetically unstable, rapidly growing phenotype, is diagnosed at advanced stage with poorly defined cancer precursors compared to the more indolent Low Grade Serous Carcinoma (LGSC). Reproductive hormone receptor status (estrogen/ progesterone receptor) may be an important indicator of response to anti-hormonal therapy in ovarian cancer. There have been few reports detailing the expression of ER in HGSC, LGSC and the normal fallopian tube epithelium, the likely cell of origin of both histotypes. We hypothesize that some ER mediated signaling is maintained despite the loss of progesterone receptor in HGSC compared to the LGSC. Methodology: Snap-frozen tissues (43 HGSC and 18 LGSC) were selected from the UHN Biobank. We used our previously published gene expression profiles to generate a candidate gene list, which was chosen based on the presence of known estrogen responsive elements. They were validated by qPCR and immunohistochemistry on HGSC and LGSC tissue microarrays. Human fallopian tube epithelial cell lines were treated with estradiol at 50nm to determine ‘normal’ ER response. An ER positive cancer cell line (SKOV3) was used to determine the cellular response to candidate genes. IHC was scored using automated image analysis. Statistical analysis was performed using ANOVA and Fisher's Exact Test. Results: 35/43 (81%) of HGSC were ER+/PR- whilst 9/27 of LGSC were ER+/PR- and 18/27 ER+/PR+. Gene expression analysis of ER+ HGSC and ER- HGSC revealed 881 genes with more than a 2-FC in gene expression including 202 genes with known or putative estrogen responsive elements. They are involved in immune response, locomotion, metanephrous development and cellular adhesion. We compared genes that were differentially expressed between normal fallopian tube cells obtained during pre- or post-ovulation (93 genes). 42 ‘FTE-normal’ genes were differentially expressed between ER- versus ER+ HGSC. We selected 5 genes to validate by qPCR and IHC, based on gene ontology and known associations to cancer pathways. Conclusions: These results show that HGSC is predominantly an ER+/ PR- cancer (80%) while LGSC is predominantly an ER positive/ PR positive cancer (66%). Our data also indicate that some ER signalling is maintained in the absence of the progesterone receptor and ER+ HGSC are transcriptionally different from ER- HGSC although, there is no distinct clinical benefit between these two groups. ER is still able to transcriptionally activate a subset of known ER target genes and this information should provide further insight into the use of hormone receptors as indicators of anti-hormone therapy in ovarian cancer. Citation Format: Sophia Hl George, Anca Milea, Ramlogan Sowamber, Danielle Toccalino, Patricia A. Shaw. The role of estrogen receptor signalling in serous ovarian cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4765. doi:10.1158/1538-7445.AM2013-4765