Single cell transcriptomes of normal endometrial derived organoids uncover novel cell type markers and cryptic differentiation of primary tumours.

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.

Differential expression and prognostic relevance of autophagy-related markers ATG4B, GABARAP, and LC3B in breast cancer.

PURPOSE: Previous studies indicate that breast cancer molecular subtypes differ with respect to their dependency on autophagy, but our knowledge of the differential expression and prognostic significance of autophagy-related biomarkers in breast cancer is limited. METHODS: Immunohistochemistry (IHC) was performed on tissue microarrays from a large population of 3992 breast cancer patients divided into training and validation cohorts. Consensus staining scores were used to evaluate the expression levels of autophagy proteins LC3B, ATG4B, and GABARAP and determine the associations with clinicopathological variables and molecular biomarkers. Survival analyses were performed using the Kaplan-Meier function and Cox proportional hazards regression models. RESULTS: We found subtype-specific expression differences for ATG4B, with its expression lowest in basal-like breast cancer and highest in Luminal A, but there were no significant associations with patient prognosis. LC3B and GABARAP levels were highest in basal-like breast cancers, and high levels were associated with worse outcomes across all subtypes (DSS; GABARAP: HR 1.43, LC3B puncta: HR 1.43). High ATG4B levels were associated with ER, PR, and BCL2 positivity, while high LC3B and GABARAP levels were associated with ER, PR, and BCL2 negativity, as well as EGFR, HER2, HER3, CA-IX, PD-L1 positivity, and high Ki67 index (p < 0.05 for all associations). Exploratory multi-marker analysis indicated that the combination of ATG4B and GABARAP with LC3B could be useful for further stratifying patient outcomes. CONCLUSIONS: ATG4B levels varied across breast cancer subtypes but did not show prognostic significance. High LC3B expression and high GABARAP expression were both associated with poor prognosis and with clinicopathological characteristics of aggressive disease phenotypes in all breast cancer subtypes.

STING pathway expression in low-grade serous carcinoma of the ovary: an unexpected therapeutic opportunity?

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.

Proteomic analysis of transitional cell carcinoma-like variant of tubo-ovarian high-grade serous carcinoma.

The current World Health Organization classification does not distinguish transitional cell carcinoma of the ovary (TCC) from conventional tubo-ovarian high-grade serous carcinoma (HGSC), despite evidence suggesting improved prognosis for patients with TCC; instead, it is considered a morphologic variant of HGSC. The immunohistochemical (IHC) markers applied to date do not distinguish between TCC and HGSC. Therefore, we sought to compare the proteomic profiles of TCC and conventional HGSC to identify proteins enriched in TCC. Prognostic biomarkers in HGSC have proven to be elusive, and our aim was to identify biomarkers of TCC as a way of reliably and reproducibly identifying patients with a favorable prognosis and better response to chemotherapy compared with those with conventional HGSC. Quantitative global proteome analysis was performed on archival material of 12 cases of TCC and 16 cases of HGSC using SP3 (single-pot, solid phase-enhanced, sample preparation)-Clinical Tissue Proteomics, a recently described protocol for full-proteome analysis from formalin-fixed paraffin-embedded tissues. We identified 430 proteins that were significantly enriched in TCC over HGSC. Unsupervised co-clustering perfectly distinguished TCC from HGSC based on protein expression. Pathway analysis showed that proteins associated with cell death, necrosis, and apoptosis were highly expressed in TCCs, whereas proteins associated with DNA homologous recombination, cell mitosis, proliferation and survival, and cell cycle progression pathways had reduced expression. From the proteomic analysis, three potential biomarkers for TCC were identified, claudin-4 (CLDN4), ubiquitin carboxyl-terminal esterase L1 (UCHL1), and minichromosome maintenance protein 7 (MCM7), and tested by IHC analysis on tissue microarrays. In agreement with the proteomic analysis, IHC expression of those proteins was stronger in TCC than in HGSC (p < 0.0001). Using global proteomic analysis, we are able to distinguish TCC from conventional HGSC. Follow-up studies will be necessary to confirm that these molecular and morphologic differences are clinically significant.