Endometrial carcinoma, the most common gynaecological cancer, develops from endometrial epithelium which is composed of secretory and ciliated cells. Pathologic classification is unreliable and there is a need for prognostic tools. We used single cell sequencing to study organoid model systems derived from normal endometrial endometrium to discover novel markers specific for endometrial ciliated or secretory cells. A marker of secretory cells (MPST) and several markers of ciliated cells (FAM92B, WDR16, and DYDC2) were validated by immunohistochemistry on organoids and tissue sections. We performed single cell sequencing on endometrial and ovarian tumours and found both secretory‐like and ciliated‐like tumour cells. We found that ciliated cell markers (DYDC2, CTH, FOXJ1, and p73) and the secretory cell marker MPST were expressed in endometrial tumours and positively correlated with disease‐specific and overall survival of endometrial cancer patients. These findings suggest that expression of differentiation markers in tumours correlates with less aggressive disease, as would be expected for tumours that retain differentiation capacity, albeit cryptic in the case of ciliated cells. These markers could be used to improve the risk stratification of endometrial cancer patients, thereby improving their management. We further assessed whether consideration of MPST expression could refine the ProMiSE molecular classification system for endometrial tumours. We found that higher expression levels of MPST could be used to refine stratification of three of the four ProMiSE molecular subgroups, and that any level of MPST expression was able to significantly refine risk stratification of the copy number high subgroup which has the worst prognosis. Taken together, this shows that single cell sequencing of putative cells of origin has the potential to uncover novel biomarkers that could be used to guide management of cancers. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
Ovarian carcinoma histotypes are distinct diseases with variable clinical outcomes and response to treatment. There is a need for new subtype-specific treatment modalities, especially for women with widespread and chemo-resistant disease. Stimulator of interferon genes (STING) is a part of the cGAS-STING pathway that mediates innate immune defence against infectious DNA-containing pathogens and also detects tumour-derived DNA and generates intrinsic antitumour immunity. The STING signalling pathway is suppressed by several mechanisms in a variety of malignant diseases and, in some cancers that may be a requirement for cellular transformation. The aim of this study was to use immunohistochemistry to evaluate STING protein expression across normal tissue, paratubal and ovarian cysts, and ovarian tumour histotypes including ovarian carcinomas. Herein, we show that the fallopian tube ciliated cells express STING protein, whereas the secretory cells are negative. STING expression differs among ovarian cancer histotypes; low-grade serous ovarian carcinomas and serous borderline tumours have uniform high STING expression, while high-grade serous and endometrioid carcinomas have heterogeneous expression, and clear cell and mucinous carcinomas show low expression. As low-grade serous carcinomas are known to be genomically stable and typically lack a prominent host immune response, the consistently high STING expression is unexpected. High STING expression may reflect pathway activation or histogenesis and the mechanisms may be different in different ovarian carcinoma histotypes. Further studies are needed to determine whether the STING signalling pathway is active and whether these tumours would be candidates for therapeutic interventions that trigger innate immunity activation.
BACKGROUND: High grade serous ovarian cancer (HGSOC) accounts for >70% of ovarian cancer related deaths and is the most common ovarian cancer histotype, most originating from pre-cancerous p53 lesions in the fallopian tube (FT) fimbria. Use of oral contraceptive pills (OCPs) for 5 years or more is associated with >40% reduction in risk of HGSOC, but the mechanism is unknown. We hypothesize that OCP use reduces the incidence of p53 lesions. Our preliminary data show higher incidence of p53 lesions in post- compared to pre-menopausal women, therefore we aim quantify p53 lesions in post-menopausal women who previously did or did not use OCPs. This will provide insight into the protective effects of OCPs against HGSOC. PRELIMINARY RESULTS: We determined the presence of p53 lesions by immunohistochemistry (IHC) in FT of women up to 40 years old (n=27) and >60 years old (n=24) who underwent salpingectomies for non-cancer reasons. p53 lesions were identified in 3/27 cases of the younger cohort (11%) and in 10/24 of the older cohort (42%). Thus, we conclude an increased incidence of p53 lesions in older compared to younger women. PROPOSED DESIGN: IHC for p53 will be performed on FT fimbria of women >55 years old who received salpingectomy/hysterectomy for non-cancer reasons. Based on an assumed reduction in p53 lesions of 35% in women who used OCPs for 5 years or more compared to non-users (25 vs. 42%), analysis of 190 cases from each group will provide >80% power (p<0.05). Cases will be identified through Population Data BC and blind analysis by tp53 IHC will be performed at the Vancouver General Hospital. Post-menopausal status will be confirmed by endometrium histology and data flowed back to Pop Data BC to compare to OCP data. CONCLUSION: Our preliminary study found that 42% of post menopausal women had p53 lesions, informing this study design. The study registered through this abstract will be the first to examine the impact of OCPs on the earliest known precursors of HGSOC. Citation Format: Kendall Greening, Anthony Karnezis, Dawn Cochrane, Gillian Hanley, David Huntsman. THE EFFECT OF AGE AND OCP USE ON THE INCIDENCE OF PRE-CANCEROUS P53 LESIONS AND THE DEVELOPMENT OF HIGH GRADE SEROUS OVARIAN CARCINOMA [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-006.
INTRODUCTION: Ovarian cancer is the 5th deadliest cancer found in women and is the deadliest involving the gynecological tract. Most epithelial ovarian cancers have extra-ovarian origins and can be stratified into various histotypes: high and low-grade serous (HGS and LGS), endometrioid (ENOC), clear cell (CCOC), and mucinous – each of which are proposed to have distinct precursor lesions. We present organoids as a useful model to study precursor lesions and the process of tumorigenesis in epithelial ovarian carcinomas. Organoids recapitulate the in vivo growth microenvironment and are useful to study the mechanisms of tumorigenesis from healthy cells. We have previously proposed that ENOC arise from the secretory cell lineage, while CCOC originate from the ciliated cell lineage, and organoids are an ideal model to examine in greater depth the impact of mutation on specific cell populations, such as ciliated cells. METHODS: Surgical fallopian tube and endometrial tissues, removed for non-cancer reasons, were cultured in 2D followed by plating into Matrigel. Matrigel cultures were supplemented with media containing stem/progenitor differentiation factors promoting organoid growth. To study the effect of mutations often found in ovarian cancers on organoid growth and development, gene knockouts were produced using CRISPR lentiviruses on cells prior to Matrigel culture. Lentiviral transductions were optimized for organoid formation and for minimizing invasiveness accrued on cells. CRISPR gRNA constructs were validated by Western Blot and qPCR. Organoids containing knockouts of p53, BRCA1 and BRCA2 were used to model precursor lesions of HGS, whereas ARID1A knockouts and an inducible PIK3CA activating mutations were used to model CCOC. To gain further insight into ciliated cells of the endometrium, organoids were treated with the notch inhibitor-DBZ to drive differentiation of cells towards a ciliated cell lineage. We analyzed organoids by single-cell RNA sequencing (scRNA-seq), immunohistochemistry (IHC), and immunofluorescence staining (IF). Single cells were derived by purifying the organoids from Matrigel followed by a chemical and physical digestion. scRNA-seq was performed utilizing the 10X Genomics Platform and analyzed by in-house bioinformaticians. Bioinformatic analyses included stringent QC to remove low-quality and dead cells, before applying unsupervised learning algorithms like PCA and Gaussian mixture modeling as well as differential expression analysis to understand both how samples relate to each other and cell types discovered within each sample. RESULTS: We successfully recapitulated the histology observed in tissues by growing endometrial and fallopian tube organoids. The notch inhibitor, DBZ forced ciliated cell differentiation, as observed by IHC, IF and scRNA-seq. scRNA-seq clustering of DBZ-treated organoid cultures revealed a possible intermediary state between progenitor and ciliated cells. Initial IHC and IF analyses of CRISPR-mediated organoids reveal successful gene manipulation. CONCLUSIONS: Organoid cultures present as a powerful method for modelling precursor lesions; they can be readily manipulated genetically and with rapid turnaround compared to conventional mouse models. Organoids are also amenable to sequencing at single-cell resolution. The ability to model ovarian cancers with permanent knockouts in human tissue serves as a necessary link between animal models and human therapy. Citation Format: Germain C. Ho, Dawn R. Cochrane, Evan W. Gibbard, Kieran Campbell, Basile Tessier-Cloutier, Kendall Greening, Forouh Kalantari, Genny Trigo-Gonzalez, Yemin Wang, Jessica N. McAlpine, Sohrab P. Shah, David G. Huntsman. MODELS AND ANALYTIC TECHNIQUES OF MULLERIAN TISSUE-DERIVED ORGANOIDS [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 GMM-030.
Endometrial epithelium gives rise to both endometrial and ovarian cancers (of clear-cell and endometrioid subtypes), the latter arising from ectopic endometrium (endometriosis). Endometrial epithelium comprises mainly secretory cells, with a minor ciliated cell population. Due to their scarcity, little is known about the biology or function of endometrial ciliated cells. To understand the biology of endometrial epithelium, and by extension the cancers that arise from it, organoids derived from normal endometrial tissue were cultured. Notch signaling inhibitors were used to induce ciliated cell differentiation. Through single-cell RNA sequencing, distinct secretory and ciliated cell populations were observed, with the ciliated cell population increasing with Notch signaling inhibition. Many novel markers of ciliated cells were observed, but no highly specific markers of secretory cell differentiation. A marker of secretory cells (MST) and several markers of ciliated cells (FAM92B, WDR16 and DYDC2) were validated by immunohistochemistry on organoids and tissue sections. In endometrial tumors, both MST and FAM92B exhibited diffuse staining and were markers of better prognosis. This suggests that tumors expressing differentiation markers have better prognosis, whether it is a marker of secretory or ciliated cells. Interestingly, a small number of endometrial tumors stained positive for DYDC2; however, these tumors exhibited a variable staining pattern with 25-50% tumor cells staining intensely, and the remaining tumor cells not staining at all. A similar variable staining pattern had been observed previously with CTH, another ciliated cell marker. Endometrial and ovarian tumor tissue microarrays were stained with DYDC2, CTH and two ciliated cell markers, FOXJ1 and p73. For all these markers, a subset of tumors displayed a variable staining pattern and for endometrial cancers, the variable staining was a good prognostic indicator. Single-cell sequencing of endometrial tumors has been able to capture these two populations of tumor cells. In ovarian tumors, only variable CTH staining was a significant prognostic indicator. Normal endometrial secretory cells are able to differentiate into ciliated cells, and the variable staining pattern suggests that a subset of tumors retains this ability, and these are clinically less aggressive. Using single-cell sequencing technology on normal tissues to guide development of prognostic markers and provide insight into the biology of the tumors arising from these tissues may be useful for many other tumor types. Citation Format: Dawn R. Cochrane, Kieran R. Campbell, Kendall Greening, Germain C. Ho, James Hopkins, Minh Bui, Vassilena Sharlandjieva, Daniel Lai, Maya DeGrood, Evan W. Gibbard, Samuel Leung, Angela S. Cheng, Jamie L.P. Lim, Samantha Neilson, David Farnell, Friedrich Kommoss, Jessica N. McAlpine, Sohrab P. Shah, David G. Huntsman. Single-cell RNA sequencing of normal endometrial organoids uncovers novel cell-type markers for prognostication of primary tumor samples [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 B09.
Background: High-grade serous ovarian carcinoma (HGSOC) accounts for >70% of ovarian cancer-related deaths and is the most common ovarian cancer histotype, most originating from precancerous p53 lesions in the fallopian tube (FT) fimbria. Use of oral contraceptive pills (OCPs) for 5 years or more is associated with a >40% reduction in risk of HGSOC, but the mechanism is unknown. We hypothesize that OCP use reduces the incidence of p53 lesions. Our preliminary data show higher incidence of p53 lesions in post- compared to premenopausal women; therefore we aim to quantify p53 lesions in postmenopausal women who previously did or did not use OCPs. This will provide insight into the protective effects of OCPs against HGSOC. Preliminary Results: We determined the presence of p53 lesions by immunohistochemistry (IHC) in FT of women up to 40 years old (n=27) and >60 years old (n=24) who underwent salpingectomies for noncancer reasons. p53 lesions were identified in 3/27 cases of the younger cohort (11%) and in 10/24 of the older cohort (42%). Thus, we conclude an increased incidence of p53 lesions in older compared to younger women. Proposed Design: IHC for p53 will be performed on FT fimbria of women >55 years old who received salpingectomy for noncancer reasons. Based on an assumed reduction in p53 lesions of 35% in women who used OCPs for 5 years or more compared to nonusers (25 vs. 42%), analysis of 190 cases from each group will provide >80% power (p<0.05). Cases will be identified through Population Data BC and blind analysis by p53 IHC will be performed at the Vancouver General Hospital. Data will be flowed back to Pop Data BC to compare to OCP data. Conclusion: Our preliminary study found that 42% of postmenopausal women had p53 lesions, informing this study design. The study registered through this abstract will be the first to examine the impact of OCPs on the earliest known precursors of HGSOC. Citation Format: Kendall Greening, Anthony Karnezis, Dawn Cochrane, David Farnell, Lien Hoang, Gillian Hanley, David Huntsman. The effect of OCP use on the incidence of precancerous p53 lesions in fallopian tube fimbria [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 B30.
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