Anca Milea

Low malignant potential tumors with micropapillary features are molecularly similar to low-grade serous carcinoma of the ovary

OBJECTIVE Low-grade serous carcinoma (LGSC) is a chemoresistant ovarian neoplasm thought to potentially arise in a background of low malignant potential tumors (LMP), which are typically non-aggressive. However, LMP with micropapillary features (LMP-MP) have more aggressive clinical behavior and may represent an intermediate in progression to LGSC. The objective of this study was to obtain and compare gene expression profiles of LMP, LMP-MP and LGSC to determine if LMP-MP more closely resembles LGSC, and to identify genes involved in LGSC carcinogenesis. METHODS Epithelial cells from LMP (n=17), LMP-MP (n=9) and LGSC (n=11) were isolated by laser capture microdissection. RNA was extracted, reverse transcribed to cDNA, amplified and hybridized to Affymetrix U133 Plus2 genechip arrays. Gene expression data were checked for quality, filtered and significantly altered genes between subgroups were identified. Differential expression of selected genes was verified by RT-qPCR and immunohistochemistry. RESULTS Gene expression analysis identified differential expression between LMP and LMP-MP, LMP and LGSC but not LMP-MP and LGSC. Integration of differentially expressed genes into the protein interaction database CytoScape highlighted gene products in the MAPK pathway as differentially regulated between LMP and LGSC. Four genes were selected and validated by RT-qPCR performed on microarray samples (n=15) and immunohistochemistry on a representative microarray (n=57). CONCLUSION The gene expression profile of LMP-MP is similar to LGSC and distinct from LMP, reflecting their more aggressive clinical behavior. Candidate genes in the MAPK pathway were highlighted which may play a role in LGSC carcinogenesis and indicate potential therapeutic targets.

Proliferation in the normal FTE is a hallmark of the follicular phase not BRCA mutation status

Purpose: Women who have inherited germline mutations of BRCA1/BRCA2 are at increased risk of developing high-grade serous carcinoma, and many of these cancers arise in the distal fimbriated end of the fallopian tube. We have previously shown that the fallopian tube epithelia of BRCA1 mutation carriers (FTE-BRCA) have altered signaling pathways compared to nonmutation carriers. In this study, we sought to determine whether these differences result in a proliferative advantage to the epithelia in this high-risk patient population and to investigate whether the postovulation environment of the FTE-BRCA compared to FTE from nonmutation carriers experiences a differential abundance of immune cells. Method: Immunohistochemistry for Ki67, CD3, CD8, CD20, and CD68 was performed on histologically normal tubal epithelium (ampulla, n = 83), fimbria (n = 18) with known ovarian cycle status and germline mutation status and for Ki67 on fimbrial epithelium from women (n = 144) with and without BRCA1 or BRCA2 mutations who underwent risk-reducing salpingo-oophorectomy (RRSO). Serous tubal intraepithelial carcinomas (STIC) with concomitant cancer (n = 15) were also analyzed for presence of immune infiltrates. All slides were digitized and analyzed using automated image analysis software. Results: There was no significant difference in the proliferative index in histologically normal FTE between BRCA1/BRCA2 and non-BRCA, in 144 fimbriae and 83 ampullae. The FTE-BRCA1 epithelia did not exhibit a differential presence of lymphocytes or macrophages, however more macrophages were present in the luteal phase compared to the follicular phase epithelia. In STICs macrophages were more abundant than lymphocytes with an incremental increase noted with disease progression. Conclusions: BRCA1/2 mutation carriers exhibited no significant increase in proliferation in the fallopian tube epithelial cells either in the ampulla or fimbriated ends of the tube. Rather, a significant proliferative increase was defined in the cases determined to be in the follicular, or proliferative, preovulatory phase of the ovarian cycle. Finally, we also show an incremental increase in leukocytes invading the STICs and HGSC, implicating a possible role of the leukocytes early in the progression or inhibition of tumor formation, which is independent of ovarian cycle status. Clin Cancer Res; 18(22); 6199–207. ©2012 AACR.

Retinoblastoma pathway deregulatory mechanisms determine clinical outcome in high-grade serous ovarian carcinoma

Alterations in the retinoblastoma pathway are frequent in ovarian/tubal high-grade serous cancers, but the mechanism of deregulation and the impact on patient outcome are poorly understood. A cohort of 334 high-grade serous carcinomas was studied by immunohistochemical analysis of RB1, p16, cyclin D1, cyclin E1, and Ki67. Additional detailed analyses including RB1 allelic deletion (n=42), mutation (n=75), methylation (n=31), and SNP array analyses (n=75) were performed on cases with clinical parameters, including age, debulking status, treatment, and clinical outcome. p16/RB1 expression results yielded three distinct clinically relevant subgroups upon multivariable analysis controlling for stage, debulking status, and treatment types: p16 homogeneous/RB1+ with the shortest progression-free survival (median 15 months (95% CI: 13–18); P=0.016) compared with the p16 heterogeneous/RB1+ subgroup (median 22 months (95% CI: 16–32)) and the p16 homogeneous/RB1− subgroup (median 20 months (95% CI: 15–24)). Patients in the p16 homo/RB1− subgroup showed a significant increase in overall survival (>60 months; P=0.013), which suggests an increase in sensitivity to cytotoxic agents. Analyses of Rb pathway mechanistic differences among these groups revealed frequent RB1 genomic alterations such as RB1 allelic loss and/or large spanning deletions (83%) in the p16 homo/RB1− subgroups, also indicating that RB1 deletions are frequent in high-grade serous carcinoma. CCNE1 gene gains/amplifications were frequent in the p16 homogeneous/RB1+ subgroup (68%) and cyclin D1 protein overexpression was predominantly characteristic of the p16 heterogeneous/RB1+ subgroup. These subcategories occur early in tumor progression and are seen with similar frequency in the cancer precursor lesion, serous tubal intra-epithelial carcinoma. Overall, this study uniquely identifies multiple non-synonymous mechanisms of retinoblastoma pathway deregulation that correlate with significantly different clinical outcomes. Furthermore, deregulations identified in precursor lesions suggest a key role of this pathway in serous tumor development. Recognition of these categories may identify patients with increased sensitivity to chemotherapy and new opportunities for novel therapeutics.

Abstract A27: Transcriptome analyses of human ampulla and fimbriae highlight similarities and differences.

Background: Recently described precursors of high-grade serous carcinoma (HGSC), the p53 signature, a latent precursor, and Serous Tubal Intraepithelial Carcinoma (STIC), a pre-malignant precursor, occur most frequently at the distal and fimbriated end of the fallopian tube (FTE). In 3 previous reports, we have demonstrated that the FTE of BRCA1 mutation carriers, at genetic risk of HGSC, have altered signaling pathways compared to controls. Ovarian production of ROS is released after the LH surge to induce ovulation. Reactive oxygen species (ROS) have been implicated in serous carcinogenesis. The objective of this study is to compare the transcriptome profiles of normal fimbria (high-risk epithelia prone to transformation) FTE and normal ampulla (low-risk epithelia) FTE which may lead to understanding the distal end of the fallopian tube as the preferential anatomic location of the fallopian to tube for cellular transformation. Method: Snap-frozen matched fimbria and ampulla tissues were controlled for age and ovarian cycle status. Cases included 12 luteal phase and 12 follicular phase women at no known risk for ovarian cancer. Laser capture microscopy was used to microdissect FTE cells, using 7-10 sections per case. Total RNA was isolated, RNA extracted and cDNA amplified. The expression profiles were generated using Affymetrix Human Genome HTA-2.0 Array. Bayes moderated t-statistics implemented in the R/Bioconductor package LIMMA to compare mRNA expression values for probes of samples based on their ‘phase’ and ‘anatomy’, respectively. t-statistics was generated from the aforementioned respective analyses, and ranked based Wilcoxon tests on gene sets from the Reactome, GO and Cytoband databases. Results: The cases clustered predominantly by ovarian cycle status rather than by their differences in anatomical origin or their matched pair. There were 75 unique probe IDs that were significantly different (p Conclusions: The epithelia of the anatomically high-risk fallopian tube – the fimbria, show few differences in gene expression profiles compared to the lower risk portion – the ampulla. Expression differences predominantly are in response to the hormonal milieu, post-ovulation with an antioxidant program. The increased anatomic risk of the fimbria is likely due to effects of the microenvironment, such as repeated exposure to follicular fluid at ovulation (higher ROS) and antioxidant genes and their interaction with DNA repair genes (like TP53), rather than intrinsic differences of the FTE in the two sites Citation Format: Sophia George, Anca Milea, Sowamber Ramlogan, Michael Considine, Leslie Cope, Noor Salman, Patricia Shaw. Transcriptome analyses of human ampulla and fimbriae highlight similarities and differences. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr A27.

Abstract 2080: Differential transcription profile of epithelia in fimbria versus the ampulla of the fallopian tube

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Recently described precursors of high-grade serous carcinoma (HGSC), the p53 signature, a latent precursor, and Serous Tubal Intraepithelial Carcinoma (STIC), a pre-malignant precursor, occur most frequently at the distal and fimbriated end of the fallopian tube (FTE). In 3 previous reports, we have demonstrated that the FTE of BRCA1 mutation carriers, at genetic risk of HGSC, have altered signaling pathways compared to controls. Ovarian production of ROS is released after the LH surge to induce ovulation. Reactive oxygen species (ROS) have been implicated in serous carcinogenesis. The objective of this study is to compare the transcriptome profiles of normal fimbria (high-risk epithelia prone to transformation) FTE and normal ampulla (low-risk epithelia) FTE which may lead to understanding the distal end of the fallopian tube as the preferential anatomic location of the fallopian to tube for cellular transformation. Method: Snap-frozen matched fimbria and ampulla tissues were controlled for age and ovarian cycle status. Cases included 12 luteal phase and 12 follicular phase women at no known risk for ovarian cancer. Laser capture microscopy was used to microdissect FTE cells, using 7-10 sections per case. Total RNA was isolated, RNA extracted and cDNA amplified. The expression profiles were generated using Affymetrix Human Genome HTA-2.0 Array. Results: Using gene level differential expression analysis with the Affymetrix Expression Console software, we performed unsupervised hierarchical clustering analysis with all 24 samples. We used a fold change of 2 and ANOVA p-value < 0.05 as a cut-off criteria for selecting genes. The cases clustered predominantly by ovarian cycle status rather than by their differences in anatomical origin or their matched pair. There were 427 genes differentially expressed amongst the 4 groups– Fim-Luteal, Fim-Follicular, FT-Luteal and FT-Follicular. Independent of ovarian cycle status, very few differences (35 genes– SALL1, ME1– higher in the ampulla; GSTA1, GSTA2, SERPINA3-higher in the fimbria– genes involved in metabolic pathways) were observed between the ampulla and fimbria FTE. Conclusions: The epithelia of the anatomically high-risk fallopian tube– the fimbria, show few differences in gene expression profiles compared to the lower risk portion– the ampulla. Expression differences predominantly are in response to the hormonal milieu, post-ovulation with an antioxidant program The increased anatomic risk of the fimbria is likely due to effects of the microenvironment, such as repeated exposure to follicular fluid at ovulation (higher ROS) and antioxidant genes and their interaction with DNA repair genes (like TP53), rather than intrinsic differences of the FTE in the two sites. Citation Format: Sophia H. George, Anca Milea, Noor Hera Salman, Patricia Ann Shaw. Differential transcription profile of epithelia in fimbria versus the ampulla of the fallopian tube. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2080. doi:10.1158/1538-7445.AM2015-2080

Abstract 4765: The role of estrogen receptor signalling in serous ovarian cancer.

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 Fisher9s 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

Abstract 333: The role of the retinoblastoma pathway (Rb) in high grade serous ovarian varcinoma (HGSC)

The Rb pathway functions as a cell cycle checkpoint and deregulation of its components, commonly found in malignancies, causes progression from G1 to S phase, promoting cellular proliferation. Because of Rb9s central role in checkpoint regulation, abrogation of the pathway can occur through multiple non-redundant mechanisms including Rb loss, hypermethylation or mutation, and CCND1 or CCNE amplification. Emerging evidence shows that tumour types can often be distinguished by particular alterations in one member of the pathway suggesting that different mechanisms of Rb abrogation may regulate tumour behaviour. We hypothesize that Rb pathway deregulation is frequent in HGSC, the most common and most aggressive histotype of ovarian cancer, and that the mechanism of Rb pathway deregulation identifies clinically distinct subgroups of HGSC. Micro-dissected epithelium from HGSC and normal FTE samples were analyzed for differential gene expression using the Affymetrix U133 Plus 2.0 gene-chips, and expression values for p53, p21, p27, p16, CCND1, CCNE and Rb genes determined. Protein expression was assessed by immunohistochemistry (IHC) on tissue microarrays composed of ovarian/tubal carcinomas inclusive of the major histotypes. Digitized stained slides were quantified using automated image analysis and correlated with clinico-pathologic variables including outcome. Rb loss of heterozygosity (LOH) was tested by an Rb diagnostics protocol involving D13S153 and RB1.2 polymorphic marker analyses using PCR amplification, followed by comparisons of the tumour and its corresponding normal sample by MicroGene Clipper sequencers. Gene expression analysis showed statistically significant over-expression of p53, CCNE E2F1/3 and p16 and down-regulation of p21 and CCND1 in HGSC compared to normal fallopian tube epithelium (p HGSC is characterized by both genetic and protein abrogation in the Rb pathway. Additionally, we observed differences in the mechanism of this G1/S checkpoint inactivation amongst HGSC patient samples which may represent important biological/clinical differences amongst sub-groups of serous cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 333. doi:10.1158/1538-7445.AM2011-333

Abstract 2835: BRCA1 signature in high-risk fallopian tube epithelium

Background: High-grade serous ovarian cancer is rarely diagnosed at an early and potentially curable stage, effective early detection and preventative strategies are few. Recently the discovery of occult invasive and intraepithelial tubal carcinomas in BRCA1 mutation carriers, who are at high risk of serous cancer, undergoing prophylactic surgery has focused attention on the fallopian tube epithelium as the cell of origin and has led to the reporting of putative serous cancer precursor lesions. However, little is known of the early molecular events of serous oncogenesis, or why cancers in BRCA 1 mutation carriers are found preferentially in tissues which are responsive to reproductive hormones. We hypothesize that molecular alterations are present in morphologically normal tubal epithelium from BRCA1 heterozygotes, and that these changes may reflect the earliest alterations in serous carcinogenesis and may be markers of increased cancer risk as well as targets for risk reduction. To identify cancer predisposition candidate genes of high-risk fallopian tube epithelium, we compared gene expression profiles of microdissected tubal epithelium from BRCA1 mutation carriers, control women and patients with HGSC. Methodology: Snap-frozen tissues were selected from the UHN Biobank; control and BRCA cases controlled for age, ovarian cycle status at surgery, and hormone therapy. Cases included 12 BRCA1 mutation carriers and 12 control women, undergoing salpingo-oophorectomy for reasons other than adnexal malignancy or family history, 15 HGSC and 8 contralateral normal tubes. Epithelium was microdissected in all cases using laser capture, RNA extracted and cDNA amplified. The gene expression profiles were generated using Affymetrix Human Genome U133 Plus 2.0 Array. Candidate genes were validated by qPCR and IHC on 2 fallopian tube TMAs. IHC was scored using Spectrum. Statistical analysis was performed using ANOVA (p Results: We focused the microarray analyses on identifying differential expression between BRCA1 heterozygotes and controls. Comparisons between FTE-nonBRCA and FTE-BRCA, resulted in 440 probe sets with more than 2-FC in gene expression. 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 histological normal fallopian tube epithelium from BRCA1 heterozygotes have altered gene expression and these differences are most pronounced in the post-ovulatory phase of the ovarian cycle in pre-menopausal women, suggesting that factors associated with the luteal phase are implicated in the increased risk of HGSC in BRCA1 mutation carriers. We also show that genes involved in inflammation pathways are up-regulated in mutation carriers and that these genetic signatures are maintained in HGSC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2835. doi:10.1158/1538-7445.AM2011-2835