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High-grade serous ovarian cancer (HGSOC) is an archetypal cancer of genomic instability patterned by distinct mutational processes, intratumoral heterogeneity and intraperitoneal spread. We investigated determinants of immune recognition and evasion in HGSOC to elucidate co- evolutionary processes underlying malignant progression and tumor immunity. Mutational processes and anatomic sites of tumor foci were key determinants of tumor microenvironment cellular phenotypes, inferred from whole genome sequencing, single-cell RNA sequencing, digital histopathology and multiplexed immunofluorescence of 160 tumor sites from 42 treatment-naive HGSOC patients. Homologous recombination-deficient (HRD)-Dup (BRCA1 mutant-like) and HRD- Del (BRCA2 mutant-like) tumors harbored increased neoantigen burden, inflammatory signaling and ongoing immunoediting, reflected in loss of HLA diversity and tumor infiltration with highly- differentiated dysfunctional CD8+ T cells. Foldback inversion (FBI, non-HRD) tumors exhibited elevated TGFβ signaling and immune exclusion, with predominantly naive/stem-like and memory T cells. Our findings implicate distinct immune resistance mechanisms across HGSOC subtypes which can inform future immunotherapeutic strategies.HIGHLIGHTSMulti-region, multi-modal profiling of malignant and immune cell phenotypes in ovarian cancerAnatomic site specificity is a determinant of cancer cell and intratumoral immune phenotypesTumor mutational processes impact mechanisms of immune control and immune evasionSpatial topology of HR-deficient tumors is defined by immune interactions absent from immune inert HR-proficient subtypes

Single-cell RNA sequencing (scRNA-seq) has transformed biomedical research, enabling decomposition of complex tissues into disaggregated, functionally distinct cell types. For many applications, investigators wish to identify cell types with known marker genes. Typically, such cell type assignments are performed through unsupervised clustering followed by manual annotation based on these marker genes, or via "mapping" procedures to existing data. However, the manual interpretation required in the former case scales poorly to large datasets, which are also often prone to batch effects, while existing data for purified cell types must be available for the latter. Furthermore, unsupervised clustering can be error-prone, leading to under- and over- clustering of the cell types of interest. To overcome these issues we present CellAssign, a probabilistic model that leverages prior knowledge of cell type marker genes to annotate scRNA-seq data into pre-defined and de novo cell types. CellAssign automates the process of assigning cells in a highly scalable manner across large datasets while simultaneously controlling for batch and patient effects. We demonstrate the analytical advantages of CellAssign through extensive simulations and exemplify real-world utility to profile the spatial dynamics of high-grade serous ovarian cancer and the temporal dynamics of follicular lymphoma. Our analysis reveals subclonal malignant phenotypes and points towards an evolutionary interplay between immune and cancer cell populations with cancer cells escaping immune recognition.

The telomerase reverse transcriptase (TERT) gene is highly expressed in stem cells and silenced upon differentiation. Cancer cells can attain immortality by activating TERT to maintain telomere length and telomerase activity, which is a crucial step of tumorigenesis. Two somatic mutations in the TERT promoter (C228T; C250T) have been identified as gain-of-function mutations that promote transcriptional activation of TERT in multiple cancers, such as melanoma and glioblastoma. A recent study investigating TERT promoter mutations in ovarian carcinomas found C228T and C250T mutations in 15.9% of clear cell carcinomas. However, it is unknown whether these mutations are frequent in other ovarian cancer subtypes, in particular, sex cordstromal tumors including adult granulosa cell tumors (AGCTs). We performed whole genome sequencing on ten AGCTs with matched normal blood and identified a TERT C228T promoter mutation in 50% of tumors. We found that AGCT with mutated TERT promoter have increased expression of TERT mRNA compared to those with wild-type TERT promoter. AGCT with TERT C228T mutation exhibited significantly longer telomeres compared to AGCT with TERT wild-type promoter. Extension cohort analysis using allelic discrimination revealed the TERT C228T mutation in 51 of 229 primary AGCTs (22%), 24 of 58 recurrent AGCTs (41%), and 1 of 22 other sex cord-stromal tumors (5%). There was a significant difference in overall survival between patients with TERT C228T promoter mutation in the primary tumors and those without it (p = 0.00253, log rank test). In seven AGCTs, we found the TERT C228T mutation present in recurrent tumors and absent in the corresponding primary tumor. Our data suggests that TERT C228T mutations may have an important role in progression of AGCT. Telomeres are conserved, repetitive (TTAGGG) DNA-protein complexes that are added to the ends of chromosomes by the enzyme telomerase to prevent DNA damage and maintain replicative potential. Telomere attrition during DNA replication induces genomic instability that can result in tumorigenesis. Telomerase consists of a catalytic protein subunit known as telomerase reverse transcriptase (TERT) and a functional RNA called telomerase RNA component (TERC). TERT is highly expressed in stem cells and is silenced upon differentiation in somatic cells. Most cancer cells attain proliferative immortality by upregulating the TERT gene to maintain telomere length and telomerase activity. The known mechanisms of telomerase activation include mutations in the TERT promoter, TERT gene amplification, CpG methylation at the TERT promoter, changes in alternative splicing of TERT pre-mRNA and upregulation of transcriptional activators. Approximately 90% of cancers express TERT, while the remaining 10-15% of cancers maintain their telomere length through a telomerase-independent method called alternative lengthening of telomeres. TERT promoter mutations were first reported in familial melanoma and subsequently in sporadic melanoma. There are two hot-spot TERT promoter mutations, C228T and C250T, each generates an identical 11 base pair sequence containing a consensus binding motif for ETS transcription factors, and functions as either a transcriptional activator or repressor to regulate telomerase expression. These two mutations are implicated in the activation of telomerase in other malignances such as central nervous system tumors, hepatocellular carcinomas, bladder cancers and thyroid cancers. A recent study on TERT promoter mutations in gynecological malignancies, including ovarian and uterine carcinomas, reported TERT hot-spot mutations in 15.9% of ovarian clear cell carcinomas. However, it is unknown whether TERT promoter mutations are frequent in sex cord-stromal tumors, including adult granulosa cell tumors (AGCTs). In this study, we evaluated the biological and clinical significance of TERT promoter mutations, specifically C228T, in total of 251 primary ovarian sex cord-stromal tumors.

SummaryHigh-grade serous ovarian cancer exhibits extensive intratumoral heterogeneity coupled with widespread intraperitoneal disease. Despite this, metastatic spread of tumor clones is non-random, implying the existence of local microenvironmental factors that shape tumor progression. We interrogated the molecular interface between tumor-infiltrating lymphocytes (TIL) and cancer cells in 143 samples from 21 patients using whole-genome sequencing, immunohistochemistry, histologic image analysis, gene expression profiling, and T- and B-cell receptor sequencing. We identify 3 immunologic response categories, which frequently co-exist within individual patients. Furthermore, epithelial CD8+ TIL were inversely associated with malignant cell diversity, evidenced by subclonal neoepitope elimination and spatial tracking between tumor and T-cell clones. Intersecting mutational signatures and immune analysis showed that foldback inversion genomic aberrations lead to worse outcomes even in the presence of cytotoxic TIL (n=433). Thus, regional variation in immune contexture mirrors the pattern of intraperitoneal malignant spread, provoking new perspectives for treatment of this challenging disease.

The telomerase reverse transcriptase (TERT) gene is highly expressed in stem cells and silenced upon differentiation. Cancer cells can attain immortality by activating TERT to maintain telomere length and telomerase activity, a crucial step of tumorigenesis. Two somatic mutations in the TERT promoter (C228T; C250T) have been identified in multiple cancers, such as melanoma and glioblastoma, as gain-of-function mutations that promote transcriptional activation of TERT. A recent study investigating TERT promoter mutations in ovarian carcinomas found mutations in 15% of clear cell carcinomas. However, it is unknown whether these mutations are prevalent in adult-type granulosa cell tumors (AGCTs) of the ovary, Sertoli-Leydig cell tumors (SLCTs), and other malignant sex cord-stromal tumors. We performed whole-genome sequencing on ten AGCT cases with matched normal and identified the TERT C228T promoter mutation in 50% of cases. We found that AGCT cases with mutated TERT promoter have increased expression of TERT mRNA compared to those with wild-type TERT promoter. All five TERT promoter mutated cases had high levels of TERT mRNA expression, whereas three of the five wild-type TERT cases had no measurable TERT mRNA expression. There was a tendency towards longer telomere lengths in AGCT cases with the TERT promoter mutation relative to those without, although it was not statistically significant. These results suggest that telomerase may be activated by a different method in the cases with no TERT promoter mutations but have TERT mRNA expression. The remaining cases with neither TERT promoter mutations nor TERT mRNA expression likely maintain their telomeres using a telomerase-independent method, such as the alternative lengthening of telomeres pathway. TERT C228T allelic discrimination analysis of 331 AGCTs, 5 SLCTs, and 18 other malignant sex cord-stromal tumors detected the mutation in 56/247 (23%) of primary AGCTs, 22/84 (26%) of recurrent AGCTs, 1/5 (20%) of SLCTs and (0/18) 0% of other malignant sex cord-stromal tumors. The single SLCT case with the TERT promoter mutation was poorly differentiated and harbored the pathognomonic FOXL2 mutation of AGCT, suggesting this SLCT case may actually be an AGCT. In 204 AGCT cases with available survival data, there was a trend towards worse disease-specific survival in patients with the TERT promoter mutation compared to those without; however, statistical significance was not reached (p = 0.128, log-ranked test). In 5 AGCT cases with primary and recurrent tissues, we found that the TERT promoter mutation was absent in the primary tumors but present in the recurrent tumors, suggesting that TERT C228T mutation may play an active role in progression of AGCTs. Overall, we found that TERT C228T promoter mutation was most common in AGCTs among the different malignant sex cord-stromal tumors. Our data confirm the activation of telomerase in AGCTs via TERT C228T promoter mutation, although alternative telomerase activation methods in AGCTs may exist. Our results suggest that TERT activation may play a role in AGCT recurrence. As such, telomere biology may be important for the progression of AGCTs. This abstract is also being presented as Poster B54. Citation Format: Jessica A. Pilsworth, Dawn R. Cochrane, Zhouchunyang Xia, Geraldine Aubert, Anniina E. M. Färkkilä, Hugo M. Horlings, Satoshi Yanagida, Winnie Yang, Jamie L. P. Lim, Yikan Wang, Ali Bashashati, Jacqueline Keul, Adele Wong, Esther Oliva, Sohrab P. Shah, Stefan Kommoss, Friedrich Kommoss, Peter M. Lansdorp, Duncan M. Baird, David G. Huntsman. TERT is frequently mutated in adult-type granulosa cell tumors of the ovary compared to other malignant sex cord-stromal tumors. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr PR13.