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.
Mesonephric carcinoma is a rare malignancy, thought to derive from Wolffian remnants. To date, no targeted molecular therapeutic options have been identified. On the basis of limited case reports, c-KIT immunohistochemical expression has been reported in female adnexal tumors of Wolffian origin, and targeted therapy with Imatinib has been attempted with mixed success. Currently, it is unclear whether c-KIT immunohistochemical expression is seen in mesonephric carcinoma, a tumor that is thought to be related to female adnexal tumors of Wolffian origin, and how this correlates with KIT mutational status. In this study, we assessed the immunohistochemical expression of c-KIT and KIT mutational status, in a series of 13 mesonephric neoplasms (5 cervical [including 2 cervical carcinosarcomas], 3 uterine corpora, 4 ovarian, and 1 vaginal/pelvic). The intensity of staining and proportion of cells showing cytoplasmic/membranous staining for c-KIT were recorded. KIT was sequenced using a next-generation sequencing panel that targeted 120 hotspots and 17 exons in 33 known actionable cancer genes. This panel included KIT exons 9, 11, and 13, and 6 hotspots (T670, D816, D820, N822, Y823, A829). Although c-KIT immunohistochemical expression was observed in the majority of mesonephric carcinomas (10/12 cases; 83%), no KIT mutations were detected. This cautions pathologists against the use of c-KIT immunohistochemistry as a surrogate marker for KIT-activating mutations in this setting. Consistent with previous studies, the majority of mesonephric neoplasms (10/13; 77%) harbored KRAS mutations. Additional mutations were found in CTNNB1 (2/13, 15%), TP53 (2/13, 15%), and PIK3CA (1/13, 8%).
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.
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