Interfaces of Malignant and Immunologic Clonal Dynamics in Ovarian Cancer.

High-grade serous ovarian cancer (HGSC) exhibits extensive malignant clonal diversity with widespread but non-random patterns of disease dissemination. We investigated whether local immune microenvironment factors shape tumor progression properties at the interface of tumor-infiltrating lymphocytes (TILs) and cancer cells. Through multi-region study of 212 samples from 38 patients with whole-genome sequencing, immunohistochemistry, histologic image analysis, gene expression profiling, and T and B cell receptor sequencing, we identified three immunologic subtypes across samples and extensive within-patient diversity. Epithelial CD8+ TILs negatively associated with malignant diversity, reflecting immunological pruning of tumor clones inferred by neoantigen depletion, HLA I loss of heterozygosity, and spatial tracking between T cell and tumor clones. In addition, combinatorial prognostic effects of mutational processes and immune properties were observed, illuminating how specific genomic aberration types associate with immune response and impact survival. We conclude that within-patient spatial immune microenvironment variation shapes intraperitoneal malignant spread, provoking new evolutionary perspectives on HGSC clonal dispersion.

Ovarian cancer mutational processes drive site-specific immune evasion.

High-grade serous ovarian cancer (HGSOC) is an archetypal cancer of genomic instability1-4 patterned by distinct mutational processes5,6, tumour heterogeneity7-9 and intraperitoneal spread7,8,10. Immunotherapies have had limited efficacy in HGSOC11-13, highlighting an unmet need to assess how mutational processes and the anatomical sites of tumour foci determine the immunological states of the tumour microenvironment. Here we carried out an integrative analysis of whole-genome sequencing, single-cell RNA sequencing, digital histopathology and multiplexed immunofluorescence of 160 tumour sites from 42 treatment-naive patients with HGSOC. Homologous recombination-deficient HRD-Dup (BRCA1 mutant-like) and HRD-Del (BRCA2 mutant-like) tumours harboured inflammatory signalling and ongoing immunoediting, reflected in loss of HLA diversity and tumour infiltration with highly differentiated dysfunctional CD8+ T cells. By contrast, foldback-inversion-bearing tumours exhibited elevated immunosuppressive TGFß signalling and immune exclusion, with predominantly naive/stem-like and memory T cells. Phenotypic state associations were specific to anatomical sites, highlighting compositional, topological and functional differences between adnexal tumours and distal peritoneal foci. Our findings implicate anatomical sites and mutational processes as determinants of evolutionary phenotypic divergence and immune resistance mechanisms in HGSOC. Our study provides a multi-omic cellular phenotype data substrate from which to develop and interpret future personalized immunotherapeutic approaches and early detection research.

Single-cell genomic variation induced by mutational processes in cancer.

How cell-to-cell copy number alterations that underpin genomic instability1 in human cancers drive genomic and phenotypic variation, and consequently the evolution of cancer2, remains understudied. Here, by applying scaled single-cell whole-genome sequencing3 to wild-type, TP53-deficient and TP53-deficient;BRCA1-deficient or TP53-deficient;BRCA2-deficient mammary epithelial cells (13,818 genomes), and to primary triple-negative breast cancer (TNBC) and high-grade serous ovarian cancer (HGSC) cells (22,057 genomes), we identify three distinct 'foreground' mutational patterns that are defined by cell-to-cell structural variation. Cell- and clone-specific high-level amplifications, parallel haplotype-specific copy number alterations and copy number segment length variation (serrate structural variations) had measurable phenotypic and evolutionary consequences. In TNBC and HGSC, clone-specific high-level amplifications in known oncogenes were highly prevalent in tumours bearing fold-back inversions, relative to tumours with homologous recombination deficiency, and were associated with increased clone-to-clone phenotypic variation. Parallel haplotype-specific alterations were also commonly observed, leading to phylogenetic evolutionary diversity and clone-specific mono-allelic expression. Serrate variants were increased in tumours with fold-back inversions and were highly correlated with increased genomic diversity of cellular populations. Together, our findings show that cell-to-cell structural variation contributes to the origins of phenotypic and evolutionary diversity in TNBC and HGSC, and provide insight into the genomic and mutational states of individual cancer cells.

Probabilistic cell-type assignment of single-cell RNA-seq for tumor microenvironment profiling.

Single-cell RNA sequencing has enabled the decomposition of complex tissues into functionally distinct cell types. Often, investigators wish to assign cells to cell types through unsupervised clustering followed by manual annotation or via 'mapping' to existing data. However, manual interpretation scales poorly to large datasets, mapping approaches require purified or pre-annotated data and both are prone to batch effects. To overcome these issues, we present CellAssign, a probabilistic model that leverages prior knowledge of cell-type marker genes to annotate single-cell RNA sequencing data into predefined or de novo cell types. CellAssign automates the process of assigning cells in a highly scalable manner across large datasets while controlling for batch and sample effects. We demonstrate the advantages of CellAssign through extensive simulations and analysis of tumor microenvironment composition in high-grade serous ovarian cancer and follicular lymphoma.

Correction: TERT promoter mutation in adult granulosa cell tumor of the ovary.

The original version of this Article omitted the author Hannah van Meurs from the Department of Gynecology, Center for Gynecologic Oncology Amsterdam, Academic Medical Center, 1100 DD Amsterdam, The Netherlands. This has been corrected in both the PDF and HTML versions of the article.

TERT promoter mutation in adult granulosa cell tumor of the ovary.

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 cord-stromal tumors including adult granulosa cell tumors. We performed whole-genome sequencing on ten adult granulosa cell tumors with matched normal blood and identified a TERT C228T promoter mutation in 50% of tumors. We found that adult granulosa cell tumors with mutated TERT promoter have increased expression of TERT mRNA and exhibited significantly longer telomeres compared to those with wild-type TERT promoter. Extension cohort analysis using allelic discrimination revealed the TERT C228T mutation in 51 of 229 primary adult granulosa cell tumors (22%), 24 of 58 recurrent adult granulosa cell tumors (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 adult granulosa cell tumors, we found the TERT C228T mutation present in recurrent tumors and absent in the corresponding primary tumor. Our data suggest that TERT C228T promoter mutations may have an important role in progression of adult granulosa cell tumors.

Dissociation of solid tumor tissues with cold active protease for single-cell RNA-seq minimizes conserved collagenase-associated stress responses.

BACKGROUND: Single-cell RNA sequencing (scRNA-seq) is a powerful tool for studying complex biological systems, such as tumor heterogeneity and tissue microenvironments. However, the sources of technical and biological variation in primary solid tumor tissues and patient-derived mouse xenografts for scRNA-seq are not well understood. RESULTS: We use low temperature (6 °C) protease and collagenase (37 °C) to identify the transcriptional signatures associated with tissue dissociation across a diverse scRNA-seq dataset comprising 155,165 cells from patient cancer tissues, patient-derived breast cancer xenografts, and cancer cell lines. We observe substantial variation in standard quality control metrics of cell viability across conditions and tissues. From the contrast between tissue protease dissociation at 37 °C or 6 °C, we observe that collagenase digestion results in a stress response. We derive a core gene set of 512 heat shock and stress response genes, including FOS and JUN, induced by collagenase (37 °C), which are minimized by dissociation with a cold active protease (6 °C). While induction of these genes was highly conserved across all cell types, cell type-specific responses to collagenase digestion were observed in patient tissues. CONCLUSIONS: The method and conditions of tumor dissociation influence cell yield and transcriptome state and are both tissue- and cell-type dependent. Interpretation of stress pathway expression differences in cancer single-cell studies, including components of surface immune recognition such as MHC class I, may be especially confounded. We define a core set of 512 genes that can assist with the identification of such effects in dissociated scRNA-seq experiments.