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

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. 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. 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. 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

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

Abstract 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 Rb's 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<0.001). Protein expression determined by IHC analysis of HGSC revealed a similar pattern of expression when compared to normal fallopian tube, the site of origin of this carcinoma. There were important differences in the expression of these proteins between HGSC subgroups, where up-regulation was observed for p16, CCNE, CCND1 and BIRC5 in 58.3%, 57%, 33.3%, and 58.3% respectively. Rb however, showed no statistically significant differences at the RNA level, but 40% of all HGSC profiled had a significant decrease in protein expression. Interestingly, LOH analysis revealed 76% of HGSC had Rb inactivation at the gene level. Furthermore, we observed statistically significant correlations (p=0.029) between p16 over-expression and Rb protein loss, using Fisher's Exact test. 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.AM2011-33

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

Abstract 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<0.05) and Fisher's Exact Test p<0.05). 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.AM2011-283

Integrative Transcriptome Analyses of the Human Fallopian Tube: Fimbria and Ampulla-Site of Origin of Serous Carcinoma of the Ovary

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

A distinct innate lymphoid cell population regulates tumor-associated T cells.

Antitumor T cells are subject to multiple mechanisms of negative regulation. Recent findings that innate lymphoid cells (ILCs) regulate adaptive T cell responses led us to examine the regulatory potential of ILCs in the context of cancer. We identified a unique ILC population that inhibits tumor-infiltrating lymphocytes (TILs) from high-grade serous tumors, defined their suppressive capacity in vitro, and performed a comprehensive analysis of their phenotype. Notably, the presence of this CD56+CD3- population in TIL cultures was associated with reduced T cell numbers, and further functional studies demonstrated that this population suppressed TIL expansion and altered TIL cytokine production. Transcriptome analysis and phenotypic characterization determined that regulatory CD56+CD3- cells exhibit low cytotoxic activity, produce IL-22, and have an expression profile that overlaps with those of natural killer (NK) cells and other ILCs. NKp46 was highly expressed by these cells, and addition of anti-NKp46 antibodies to TIL cultures abrogated the ability of these regulatory ILCs to suppress T cell expansion. Notably, the presence of these regulatory ILCs in TIL cultures corresponded with a striking reduction in the time to disease recurrence. These studies demonstrate that a previously uncharacterized ILC population regulates the activity and expansion of tumor-associated T cells

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

A distinct innate lymphoid cell population regulates tumor-associated T cells.

Antitumor T cells are subject to multiple mechanisms of negative regulation. Recent findings that innate lymphoid cells (ILCs) regulate adaptive T cell responses led us to examine the regulatory potential of ILCs in the context of cancer. We identified a unique ILC population that inhibits tumor-infiltrating lymphocytes (TILs) from high-grade serous tumors, defined their suppressive capacity in vitro, and performed a comprehensive analysis of their phenotype. Notably, the presence of this CD56+CD3- population in TIL cultures was associated with reduced T cell numbers, and further functional studies demonstrated that this population suppressed TIL expansion and altered TIL cytokine production. Transcriptome analysis and phenotypic characterization determined that regulatory CD56+CD3- cells exhibit low cytotoxic activity, produce IL-22, and have an expression profile that overlaps with those of natural killer (NK) cells and other ILCs. NKp46 was highly expressed by these cells, and addition of anti-NKp46 antibodies to TIL cultures abrogated the ability of these regulatory ILCs to suppress T cell expansion. Notably, the presence of these regulatory ILCs in TIL cultures corresponded with a striking reduction in the time to disease recurrence. These studies demonstrate that a previously uncharacterized ILC population regulates the activity and expansion of tumor-associated T cells