Microsatellite instability (MSI) refers to the hypermutability of short repetitive sequences in the genome caused by impaired DNA mismatch repair. Although MSI has been studied for decades, large amounts of sequencing data now available allows us to examine the molecular fingerprints of MSI in greater detail. Here, we analyse ∼8,000 exomes and ∼1,000 whole genomes of cancer patients across 23 cancer types. Our analysis reveals that the frequency of MSI events is highly variable within and across tumour types. We also identify genes in DNA repair and oncogenic pathways recurrently subject to MSI and uncover non-coding loci that frequently display MSI. Finally, we propose a highly accurate exome-based predictive model for the MSI phenotype. These results advance our understanding of the genomic drivers and consequences of MSI, and our comprehensive catalogue of tumour-type-specific MSI loci will enable panel-based MSI testing to identify patients who are likely to benefit from immunotherapy.

Purpose: Histologic features of diffuse-type gastric cancer indicate that the tumor microenvironment (TME) may substantially impact tumor invasiveness. However, cellular components and molecular features associated with cancer invasiveness in the TME of diffuse-type gastric cancers are poorly understood.Experimental Design: We performed single-cell RNA-sequencing (scRNA-seq) using tissue samples from superficial and deep invasive layers of cancerous and paired normal tissues freshly harvested from five patients with diffuse-type gastric cancer. The scRNA-seq results were validated by immunohistochemistry (IHC) and duplex in situ hybridization (ISH) in formalin-fixed paraffin-embedded tissues.Results: Seven major cell types were identified. Fibroblasts, endothelial cells, and myeloid cells were categorized as being enriched in the deep layers. Cell type–specific clustering further revealed that the superficial-to-deep layer transition is associated with enrichment in inflammatory endothelial cells and fibroblasts with upregulated CCL2 transcripts. IHC and duplex ISH revealed the distribution of the major cell types and CCL2-expressing endothelial cells and fibroblasts, indicating tumor invasion. Elevation of CCL2 levels along the superficial-to-deep layer axis revealed the immunosuppressive immune cell subtypes that may contribute to tumor cell aggressiveness in the deep invasive layers of diffuse-type gastric cancer. The analyses of public datasets revealed the high-level coexpression of stromal cell–specific genes and that CCL2 correlated with poor survival outcomes in patients with gastric cancer.Conclusions: This study reveals the spatial reprogramming of the TME that may underlie invasive tumor potential in diffuse-type gastric cancer. This TME profiling across tumor layers suggests new targets, such as CCL2, that can modify the TME to inhibit tumor progression in diffuse-type gastric cancer.See related commentary by Huang and Brekken, p. 6284

Background: A spatially resolved, niche-level analysis of tumour microenvironments (TME) can provide insights into cellular interactions and their functional impacts in gastric cancers (GC). Objective: Our goal was to translate the spatial organisation of GC ecosystems into a functional landscape of cellular interactions involving malignant, stromal and immune cells. Design: We performed spatial transcriptomics on nine primary GC samples using the Visium platform to delineate the transcriptional landscape and dynamics of malignant, stromal and immune cells within the GC tissue architecture, highlighting cellular crosstalks and their functional consequences in the TME. Results: GC spatial transcriptomes with substantial cellular heterogeneity were delineated into six regional compartments. Specifically, the fibroblast-enriched TME upregulates epithelial-to-mesenchymal transformation and immunosuppressive response in malignant and TME cells, respectively. Cell type-specific transcriptional dynamics revealed that malignant and endothelial cells promote the cellular proliferations of TME cells, whereas the fibroblasts and immune cells are associated with procancer and anticancer immunity, respectively. Ligand-receptor analysis revealed that CCL2 -expressing fibroblasts promote the tumour progression via JAK-STAT3 signalling and inflammatory response in tumour-infiltrated macrophages. CCL2+ fibroblasts and STAT3 -activated macrophages are co-localised and their co-abundance was associated with unfavourable prognosis. We experimentally validated that CCL2+ fibroblasts recruit myeloid cells and stimulate STAT3 activation in recruited macrophages. The development of immunosuppressive TME by CCL2+ fibroblasts were also validated in syngeneic mouse models. Conclusion: GC spatial transcriptomes revealed functional cellular crosstalk involving multiple cell types among which the interaction between CCL2+ fibroblasts and STAT3 -activated macrophages plays roles in establishing immune-suppressive GC TME with potential clinical relevance.

Surgical archives of tumor specimens are often impure. The presence of RNA transcripts from nontumor cells, such as immune and stromal cells, can impede analyses of cancer expression profiles. To systematically analyze the impact of tumor purity, the gene expression profiles and tumor purities were obtained for 7,794 tumor specimens across 21 tumor types (available in The Cancer Genome Atlas consortium). First, we observed that genes with roles in immunity and oxidative phosphorylation were significantly inversely correlated and correlated with the tumor purity, respectively. The expression of genes implicated in immunotherapy and specific immune cell genes, along with the abundance of immune cell infiltrates, was substantially inversely correlated with tumor purity. This relationship may explain the correlation between immune gene expression and mutation burden, highlighting the need to account for tumor purity in the evaluation of expression markers obtained from bulk tumor transcriptome data. Second, examination of cluster membership of gene pairs, with or without controlling for tumor purity, revealed that tumor purity may have a substantial impact on gene clustering across tumor types. Third, feature genes for molecular taxonomy were analyzed for correlation with tumor purity, and for some tumor types, feature genes representing the mesenchymal and classical subtypes were inversely correlated and correlated with tumor purity, respectively. Our findings indicate that tumor purity is an important confounder in evaluating the correlation between gene expression and clinicopathologic features such as mutation burden, as well as gene clustering and molecular taxonomy. Cancer Immunol Res; 6(1); 87–97. ©2017 AACR.

Human mesenchymal stromal cells (MSCs) have served as a major cellular resource for cell-based immunomodulatory and regenerative therapies. However, genomic instability may accumulate during ex vivo expansion of MSCs, thereby increasing the potential of malignant transformation. Here, we performed whole genome sequencing of two peripheral blood-derived MSC lines (MSC1 and MSC2) at various passages (passage 1 [P1] to P9). The majority of single-nucleotide variations (SNVs) occurred in later passages; specifically, 90% and 70% of all SNVs in MSC1 and MSC2 were observed in P9 and P7/P9, respectively. These late-occurring SNVs were enriched with C > A transversions and were overrepresented in intronic regions compared to intergenic regions, suggesting that the mutational forces are not constant across the passages. Clonality analyses also distinguished early-occurring, subclonal SNVs from late-occurring, clonally fixed SNVs. In addition, MSCs were largely devoid of copy number alterations (CNAs) (i.e., 0–2 CNAs per passage), with one exception (MSC2-P3) harboring 29 passage-specific CNAs. Our findings suggest that the SNVs found to be abundant at later passages likely resulted from the accumulation of replication stress, which can be associated with proliferation activity. Thus, the genomic instability associated with proliferation records should be considered for clinical applications of MSCs.

Papillary thyroid carcinoma (PTC) represents a heterogeneous disease with diverse clinical outcomes highlighting a need to identify robust biomarkers with clinical relevance. We applied non-negative matrix factorization-based deconvolution to publicly available gene expression profiles of thyroid cancers in the Cancer Genome Atlas (TCGA) consortium. Among three metagene signatures identified, two signatures were enriched in canonical BRAF-like and RAS-like thyroid cancers with up-regulation of genes involved in oxidative phosphorylation and cell adhesions, respectively. The third metagene signature representing up-regulation of immune-related genes further segregated BRAF-like and RAS-like PTCs into their respective subgroups of immunoreactive (IR) and immunodeficient (ID), respectively. BRAF-IR PTCs showed enrichment of tumor infiltrating immune cells, tall cell variant PTC, and shorter recurrence-free survival compared to BRAF-ID PTCs. RAS-IR and RAS-ID PTC subtypes included majority of normal thyroid tissues and follicular variant PTC, respectively. Immunopathological features of PTC subtypes such as immune cell fraction, repertoire of T cell receptors, cytolytic activity, and expression level of immune checkpoints such as and PD-L1 and CTLA-4 were consistently observed in two different cohorts. Taken together, an immune-related metagene signature can classify PTCs into four molecular subtypes, featuring the distinct histologic type, genetic and transcriptional alterations, and potential clinical significance.