Osteoclast-like stromal giant cells in breast cancer likely belong to the spectrum of immunosuppressive tumor-associated macrophages.

Background: Breast cancer with osteoclast-like stromal giant cells (OSGC) is an exceedingly rare morphological pattern of invasive breast carcinoma. The tumor immune microenvironment (TIME) of these tumors is populated by OSGC, which resemble osteoclasts and show a histiocytic-like immunophenotype. Their role in breast cancer is unknown. The osteoclast maturation in the bone is regulated by the expression of cytokines that are also present in the TIME of tumors and in breast cancer tumor-associated macrophages (TAMs). TAMs-mediated anti-tumor immune pathways are regulated by miRNAs akin to osteoclast homeostasis. Here, we sought to characterize the different cellular compartments of breast cancers with OSGC and investigate the similarities of OSGC with tumor and TIME in terms of morphology, protein, and miRNA expression, specifically emphasizing on monocytic signatures. Methods and Results: Six breast cancers with OSGC were included. Tumor-infiltrating lymphocytes (TILs) and TAMs were separately quantified. The different cellular populations (i.e., normal epithelium, cancer cells, and OSGC) were isolated from tissue sections by laser-assisted microdissection. After RNA purification, 752 miRNAs were analyzed using a TaqMan Advanced miRNA Low-Density Array for all samples. Differentially expressed miRNAs were identified by computing the fold change (log2Ratio) using the Kolmogorov-Smirnov test and p values were corrected for multiple comparisons using the false discovery rate (FDR) approach. As a similarity analysis among samples, we used the Pearson test. The association between pairs of variables was investigated using Fisher exact test. Classical and non-classical monocyte miRNA signatures were finally applied. All OSGC displayed CD68 expression, TILs (range, 45-85%) and high TAMs (range, 35-75%). Regarding the global miRNAs profile, OSGC was more similar to cancer cells than to non-neoplastic ones. Shared deregulation of miR-143-3p, miR-195-5p, miR-181a-5p, and miR-181b-5p was observed between OSGC and cancer cells. The monocyte-associated miR-29a-3p and miR-21-3p were dysregulated in OSGCs compared with non-neoplastic or breast cancer tissues. Conclusion: Breast cancers with OSGC have an activated TIME. Shared epigenetic events occur during the ontogenesis of breast cancer cells and OSGC but the innumophenotype and miRNA profiles of the different cellular compartmens suggest that OSGC likely belong to the spectrum of M2 TAMs.

HDAC2 Facilitates Pancreatic Cancer Metastasis.

The mortality of patients with pancreatic ductal adenocarcinoma (PDAC) is strongly associated with metastasis, a multistep process that is incompletely understood in this disease. Although genetic drivers of PDAC metastasis have not been defined, transcriptional and epigenetic rewiring can contribute to the metastatic process. The epigenetic eraser histone deacetylase 2 (HDAC2) has been connected to less differentiated PDAC, but the function of HDAC2 in PDAC has not been comprehensively evaluated. Using genetically defined models, we show that HDAC2 is a cellular fitness factor that controls cell cycle in vitro and metastasis in vivo, particularly in undifferentiated, mesenchymal PDAC cells. Unbiased expression profiling detected a core set of HDAC2-regulated genes. HDAC2 controlled expression of several prosurvival receptor tyrosine kinases connected to mesenchymal PDAC, including PDGFRα, PDGFRß, and EGFR. The HDAC2-maintained program disabled the tumor-suppressive arm of the TGFß pathway, explaining impaired metastasis formation of HDAC2-deficient PDAC. These data identify HDAC2 as a tractable player in the PDAC metastatic cascade. The complexity of the function of epigenetic regulators like HDAC2 implicates that an increased understanding of these proteins is needed for implementation of effective epigenetic therapies. SIGNIFICANCE: HDAC2 has a context-specific role in undifferentiated PDAC and the capacity to disseminate systemically, implicating HDAC2 as targetable protein to prevent metastasis.

An EBC/Plasma miRNA Signature Discriminates Lung Adenocarcinomas From Pleural Mesothelioma and Healthy Controls.

BACKGROUND: Despite significant improvement in screening programs for cancers of the respiratory district, especially in at-risk subjects, early disease detection is still a major issue. In this scenario, new molecular and non-invasive biomarkers are needed to improve early disease diagnosis. METHODS: We profiled the miRNome in exhaled breath condensate (EBC) and plasma samples from fourteen patients affected by lung AdCa, nine healthy subjects. miRNA signatures were then analyzed in another neoplasia of the respiratory district, i.e. pleural mesothelioma (n = 23) and subjects previously exposed to asbestos were used as controls for this cohort (n = 19). Selected miRNAs were analyzed in purified pulmonary neoplastic or normal epithelial and stromal cell subpopulation from AdCa patients. Finally, the plasmatic miRNA signature was analyzed in a publicly available cohort of NSCLC patients for data validation and in silico analysis was performed with predicted miRNA targets using the multiMiR tool and STRING database. RESULTS: miR-597-5p and miR-1260a are significantly over-expressed in EBC from lung AdCa and are associated with AdCa. Similarly, miR-1260a is also up-regulated in the plasma of AdCa patients together with miR-518f-3p and correlates with presence of lung cancer, whereas let-7f-5p is under-expressed. Analysis of these circulating miRNAs in pleural mesothelioma cases confirmed that up-regulation of miR-518f-3p, -597-5p and -1260a, is specific for lung AdCa. Lastly, quantification of the miRNAs in laser-assisted microdissected lung tissues revealed that miR-518f-3p, 597-5p and miR-1260a are predominantly expressed in tumor epithelial cells. Validation analysis confirmed miR-518f-3p as a possible circulating biomarker of NSCLC. In silico analysis of the potentially modulated biological processes by these three miRNAs, shows that tumor bioenergetics are the most affected pathways. CONCLUSIONS: Overall, our data suggest a 3-miRNAs signature as a non-invasive and accurate biomarker of lung AdCa. This approach could supplement the current screening approaches for early lung cancer diagnosis.

The Oncosuppressors MEN1 and CDC73 Are Involved in lncRNA Deregulation in Human Parathyroid Tumors.

A role for long non-coding RNAs (lncRNAs) in endocrine cancer pathogenesis is emerging. However, knowledge regarding their expression pattern, correlation with known genetic defects, and clinical implications in parathyroid tumors is still unclear. Here, we profiled 90 known lncRNAs in a first series of normal (PaN = 2), adenomatous (PAd = 12), and carcinomatous (PCa = 4) parathyroid glands and we confirmed deregulation of 11 lncRNAs using an independent cohort of patients (PaN = 4; PAd = 26; PCa = 9). Expression of lncRNAs was correlated with cytogenetic aberrations, status of genes multiple endocrine neoplasia 1 (MEN1) and cell division cycle 73 (CDC73), or clinical features. Globally, lncRNAs discriminate according to tissue histology. BC200 consistently identifies parathyroid cancers from adenomas and atypical adenomas. Loss-of-heterozygosity (LOH) at chromosomes 1, 11, 15, 21, and 22 significantly impacts expression of lncRNAs in PAds. Silencing of the key parathyroid gene MEN1 modulates the expression of six lncRNAs in primary PAds-derived cultures. Analogous levels of lncRNAs are measured in PAds with the mutation in the MEN1 gene compared with PAds with wild-type MEN1. Similarly, carcinomas with mutated CDC73 differ from PCas with wild-type protein in terms of expression of lncRNAs. PCas harboring CDC73 mutations overexpress BC200 compared to wild-type carcinomas. Overall, these findings shed light on deregulation of lncRNAs in human parathyroid tumors and propose that circuits between lncRNAs and the oncosuppressors MEN1 or CDC73 may have a role in parathyroid tumorigenesis as epigenetic modulators. © 2020 American Society for Bone and Mineral Research (ASBMR).

Interplay Between V-ATPase G1 and Small EV-miRNAs Modulates ERK1/2 Activation in GBM Stem Cells and Nonneoplastic Milieu.

The ATP6V1G1 subunit (V1G1) of the vacuolar proton ATPase (V-ATPase) pump is crucial for glioma stem cells (GSC) maintenance and in vivo tumorigenicity. Moreover, V-ATPase reprograms the tumor microenvironment through acidification and release of extracellular vesicles (EV). Therefore, we investigated the role of V1G1 in GSC small EVs and their effects on primary brain cultures. To this end, small EVs were isolated from patients-derived GSCs grown as neurospheres (NS) with high (V1G1HIGH-NS) or low (V1G1LOW-NS) V1G1 expression and analyzed for V-ATPase subunits presence, miRNA contents, and cellular responses in recipient cultures. Our results show that NS-derived small EVs stimulate proliferation and motility of recipient cells, with small EV derived from V1G1HIGH-NS showing the most pronounced activity. This involved activation of ERK1/2 signaling, in a response reversed by V-ATPase inhibition in NS-producing small EV. The miRNA profile of V1G1HIGH-NS-derived small EVs differed significantly from that of V1G1LOW-NS, which included miRNAs predicted to target MAPK/ERK signaling. Mechanistically, forced expression of a MAPK-targeting pool of miRNAs in recipient cells suppressed MAPK/ERK pathway activation and blunted the prooncogenic effects of V1G1HIGH small EV. These findings propose that the GSC influences the brain milieu through a V1G1-coordinated EVs release of MAPK/ERK-targeting miRNAs. Interfering with V-ATPase activity could prevent ERK-dependent oncogenic reprogramming of the microenvironment, potentially hampering local GBM infiltration. IMPLICATIONS: Our data identify a novel molecular mechanism of gliomagenesis specific of the GBM stem cell niche, which coordinates a V-ATPase-dependent reprogramming of the brain microenvironment through the release of specialized EVs.

Bronchoalveolar Lavage-microRNAs Are Potential Novel Biomarkers of Outcome After Lung Transplantation.

Primary graft dysfunction, infections, and acute rejection (AR) worsen lung transplantation (LTx) outcome and patient survival. Despite significant efforts, reliable biomarkers of acute lung allograft dysfunction are lacking. To address this issue, we profiled the bronchoalveolar lavage (BAL) miRNome in LTx patients. METHODS: BAL-microRNAs (miRNAs) from 16 patients were collected 7 days (T0), 15 days (T1), and 3 months (T2) after bilateral LTx and profiled on low-density array. Unsupervised and supervised analyses were used to identify miRNAs associated with clinical features, pneumonia, or AR. Prognostic markers were identified using the Cox model. Targeted signaling pathways were predicted in silico. A second series of 11 patients were used to validate AR-associated miRNAs. RESULTS: Variation in BAL-miRNAs was associated with acute lung allograft dysfunction. Increased levels of miR-23b-3p at T2 were detected in patients with pneumonia, whereas let-7f-5p, miR-146b-3p, miR-22-5p, miR-29c-5p, miR-362-5p, and miR-452-5p were upregulated at T2 in patients with AR. miR-148b-5p and miR-744-3p distinguished LTx patients with AR in both cohorts. Low miR-148b-5p and high miR-744-3p expression levels were significantly associated with a shorter time to AR either within the first year after LTx or during follow-up. Combination of the 2 miRNAs identified LTx patients with higher AR risk independently of clinical variables. CONCLUSIONS: Our data provide new insights into the roles of BAL-miRNAs in regulating the pulmonary environment after transplantation and suggest that these miRNAs could serve as biomarkers of early- or mid-stage events. If validated, these findings could pave the way to a personalized clinical approach in LTx patients.

Deregulation of miRNAs-cMYC circuits is a key event in refractory celiac disease type-2 lymphomagenesis.

A percentage of celiac disease (CD) patients develop refractory type-2 disease (RCD2), a condition associated with increased risk of enteropathy-associated T-cell-lymphoma (EATL) and without therapeutic option. Therefore, we profiled the miRNome in series of peripheral T-cell lymphomas (PTCLs), CD, RCD1 or 2 and in the murine interleukin-15 (IL15)-transgenic (TG) model of RCD. The transcriptome was analyzed in 18 intestinal T-cell lymphomas (ITLs). Bioinformatics pipelines provided significant microRNA (miRNA) lists and predicted targets that were confirmed in a second set of patients. Our data show that ITLs have a unique miRNA profile with respect to other PTCLs. The c-MYC regulated miR-17/92 cluster distinguishes monomorphic epitheliotropic ITL (MEITL) from EATL and prognosticates EATL outcome. These miRNAs are decreased in IL15-TG mice upon Janus kinase (JAK) inhibition. The random forest algorithm identified a signature of 38 classifier miRNAs, among which, the miR-200 and miR-192/215 families were progressively lost in RCD2 and ITL-CD, whereas miR-17/92 and C19MC miRNAs were up-regulated. Accordingly, SMAD3, MDM2, c-Myc and activated-STAT3 were increased in RCD2 and EATL tissues while JAK inhibition in IL15-TG mice restored their levels to baseline. Our data suggest that miRNAs circuit supports activation of STAT3 and c-Myc oncogenic signaling in RCD2, thus contributing to lymphomagenesis. This novel understanding might pave the way to personalized medicine approaches for RCD and EATL.

A miRNome analysis of drug-free manic psychotic bipolar patients versus healthy controls.

The lifetime presence of psychotic symptoms is associated with more clinical severity, poorer outcome and biological changes in patients affected by bipolar disorder (BD). Epigenetic mechanisms have been evoked to explain the onset of psychotic symptoms in BD as well as the associated biological changes. The main objective of the present study was to evaluate the expression profiles of circulating microRNAs (miRNAs) in drug-free manic psychotic bipolar patients versus healthy controls (HC), to identify possible non-invasive molecular markers of the disorder. 15 drug-free manic psychotic bipolar patients and 9 HC were enrolled and 800 miRNAs expression profile was measured by Nanostring nCounter technology on plasma samples and validated through qPCR. Overall, twelve miRNAs showed a significantly altered expression between the two groups (p < 0.05). Functional annotation of predicted miRNAs targets by MultiMIR R tool showed repression in bipolar patients of genes with a role in neurodevelopment and neurogenesis, and upregulation of genes involved in metabolism regulation. We identified a signature of circulating miRNA characteristic of manic psychotic bipolar patients, suggesting a possible role in neurodevelopment and metabolic processes regulation.

The Genetic Landscape of Human Glioblastoma and Matched Primary Cancer Stem Cells Reveals Intratumour Similarity and Intertumour Heterogeneity.

Glioblastoma (GBM) is the most malignant human brain tumour, characterized by rapid progression, invasion, intense angiogenesis, high genomic instability, and resistance to therapies. Despite countless experimental researches for new therapeutic strategies and promising clinical trials, the prognosis remains extremely poor, with a mean survival of less than 14 months. GBM aggressive behaviour is due to a subpopulation of tumourigenic stem-like cells, GBM stem cells (GSCs), which hierarchically drive onset, proliferation, and tumour recurrence. The morbidity and mortality of this disease strongly encourage exploring genetic characteristics of GSCs. Here, using array-CGH platform, we investigated genetic and genomic aberration profiles of GBM parent tumour (n = 10) and their primarily derived GSCs. Statistical analysis was performed by using R software and complex heatmap and corrplot packages. Pearson correlation and K-means algorithm were exploited to compare genetic alterations and to group similar genetic profiles in matched pairs of GBM and derived GSCs. We identified, in both GBM and matched GSCs, recurrent copy number alterations, as chromosome 7 polysomy, chromosome 10 monosomy, and chromosome 9p21deletions, which are typical features of primary GBM, essential for gliomagenesis. These observations suggest a condition of strong genomic instability both in GBM as GSCs. Our findings showed the robust similarity between GBM mass and GSCs (Pearson corr.≥0.65) but also highlighted a marked variability among different patients. Indeed, the heatmap reporting Gain/Loss State for 21022 coding/noncoding genes demonstrated high interpatient divergence. Furthermore, K-means algorithm identified an impairment of pathways related to the development and progression of cancer, such as angiogenesis, as well as pathways related to the immune system regulation, such as T cell activation. Our data confirmed the preservation of the genomic landscape from tumour tissue to GSCs, supporting the relevance of this cellular model to test in vitro new target therapies for GBM.

A GBM-like V-ATPase signature directs cell-cell tumor signaling and reprogramming via large oncosomes.

BACKGROUND: The V-ATPase proton pump controls acidification of intra and extra-cellular milieu in both physiological and pathological conditions. We previously showed that some V-ATPase subunits are enriched in glioma stem cells and in patients with poor survival. In this study, we investigated how expression of a GBM-like V-ATPase pump influences the non-neoplastic brain microenvironment. METHODS: Large oncosome (LO) vesicles were isolated from primary glioblastoma (GBM) neurospheres, or from patient sera, and co-cultured with primary neoplastic or non-neoplastic brain cells. LO transcript and protein contents were analyzed by qPCR, immunoblotting and immunogold staining. Activation of pathways in recipient cells was determined at gene and protein expression levels. V-ATPase activity was impaired by Bafilomycin A1 or gene silencing. FINDINGS: GBM neurospheres influence their non-neoplastic microenvironment by delivering the V-ATPase subunit V1G1 and the homeobox genes HOXA7, HOXA10, and POU3F2 to recipient cells via LO. LOs reprogram recipient cells to proliferate, grow as spheres and to migrate. Moreover, LOs are particularly abundant in the circulation of GBM patients with short survival time. Finally, impairment of V-ATPase reduces LOs activity. INTERPRETATION: We identified a novel mechanism adopted by glioma stem cells to promote disease progression via LO-mediated reprogramming of their microenvironment. Our data provide preliminary evidence for future development of LO-based liquid biopsies and suggest a novel potential strategy to contrast glioma progression. FUND: This work was supported by Fondazione Cariplo (2014-1148 to VV) and by the Italian Minister of Health-Ricerca Corrente program 2017 (to SF).

Specific V-ATPase expression sub-classifies IDHwt lower-grade gliomas and impacts glioma growth in vivo.

BACKGROUND: Cancer cells use specific V-ATPase subunits to activate oncogenic pathways. Therefore, we investigated V-ATPase deregulation in aggressive gliomas and associated signaling. METHODS: V-ATPase genes expression and associated pathways were analyzed in different series of glioma available from public databases, as well as in patients' cohort. Activation of pathways was analyzed at gene and protein expression levels. A genetic model of glioma in Drosophila melanogaster and mice with GBM patients-derived orthotopic xenografts were used as in vivo models of disease. FINDINGS: GBM and recurrent gliomas display a specific V-ATPase signature. Such signature resolves the heterogeneous class of IDH-wild type lower-grade gliomas, identifying the patients with worse prognosis independently from clinical and molecular features (p = 0·03, by Cox proportional-hazards model). In vivo, V-ATPase subunits deregulation significantly impacts tumor growth and proliferation. At the molecular level, GBM-like V-ATPase expression correlates with upregulation of Homeobox genes. INTERPRETATION: Our data identify a V-ATPase signature that accompanies glioma aggressiveness and suggest new entry points for glioma stratification and follow-up. FUND: This work was supported by Fondazione Cariplo (2014-1148 to VV), Fondazione IRCCS Ca' Granda, and Fondazione INGM Grant in Molecular Medicine 2014 (to VV).

Expression of C19MC miRNAs in HCC associates with stem-cell features and the cancer-testis genes signature.

BACKGROUND: Intratumor heterogeneity of hepatocellular carcinoma (HCC) and, among HCC cell subsets, the cancer stem cell population (hCSC), is responsible for therapeutic resistance and disease relapse. AIMS: To characterize hCSC-enriched HCCs at the molecular level. METHODS: Side population (SP) was used to identify the hCSCs in multiple tumor sampling from different patients and primary HCCs cultures. FACS was used to immunoprofile cultures. miRNAs were profiled in samples and correlated to SP. The Cancer Genome Atlas (TCGA) HCC dataset was analyzed to search for signatures associated with C19MC miRNAs expression. Results were confirmed by immunohistochemistry. RESULTS: The miRNA cluster on chromosome 19 (C19MC) was enriched in SP and in HCCs with a high SP fraction. At the molecular level, an elevated C19MC was correlated with expression of precursor transcripts. In TCGA-HCC series, high C19MC expression identified a subset of patients with poorer prognosis, advanced disease and overexpression of the cancer-testis (CT) antigens. These data were confirmed in an independent cohort of HCCs and at the protein level. CONCLUSION: C19MC miRNAs and CT antigens overexpression represents a novel oncogenic pathway in a subset of hCSC-enriched HCCs with dismal prognosis. CT antigens are promising immunotherapy targets. Therefore, these molecular signatures could identify HCCs who could benefit from immunotherapy.

Recurrent NAB2-STAT6 gene fusions and oestrogen receptor-α expression in pulmonary adenofibromas.

AIMS: Pulmonary adenofibromas are rare benign fibroepithelial tumours of the lung with unknown histogenesis and an indolent clinical behaviour. Their stroma resembles that of solitary fibrous tumours, whereas the glands are composed of respiratory epithelium organized in a phyllodes-like architecture. Differentiation of pulmonary adenofibromas from other more aggressive intrathoracic tumours is clinically relevant. However, their biology is unknown. Here, we sought to characterize pulmonary adenofibromas at a clinicopathological level and to define whether they could be underpinned by a highly recurrent somatic genetic alteration akin to tumours with similar morphology. METHODS AND RESULTS: Seven pulmonary adenofibromas were subjected to immunohistochemical analysis for thyroid transcription factor 1 (TTF1), napsin A, cytokeratin 7, E-cadherin, CD99, CD34, CD31, STAT6, oestrogen receptor (ER), progesterone receptor, androgen receptor, bcl-2, and vimentin, as well as electron microscopy and capillary sequencing on microdissected samples to evaluate the presence of NAB2-STAT6 fusion genes and MED12 exon 2 mutations in their discrete components. A control group comprising pulmonary solitary fibrous tumours, pulmonary hamartomas and breast fibroadenomas was also analysed. We confirmed that the stromal elements of pulmonary adenofibromas pertain to the fibroblastic lineage, and show ER overexpression in 71% of cases, whereas the epithelium consists of TTF1-positive, E-cadherin positive bronchiolar elements. A highly recurrent NAB2-STAT6 fusion variant (exon 4-exon 2) was detected in the stroma but not in the epithelium. No MED12 mutations were identified. CONCLUSIONS: Here, we demonstrate that pulmonary adenofibromas are neoplastic lesions harbouring the molecular hallmark of solitary fibrous tumours.

CD18 promoter methylation is associated with a higher risk of thrombotic complications in primary myelofibrosis.

Morbidity and mortality of BCR-ABL1-negative myeloproliferative neoplasm (MPN) patients are influenced by disease-related hemostatic complications, mostly of thrombotic nature. The pathogenesis of thrombosis is multifactorial: in particular, it has been demonstrated that a deregulated expression of Mac1 (also known as surface receptor integrin CD18/CD11b) by leukocytes has a role in favoring platelets' activation in MPN patients. Based on these data, we investigated the epigenetic status of CD18/CD11b in 78 primary myelofibrosis (PMF) patients to explore any possible association between the epigenetic profiles of these two genes and thrombotic risk. The percentage of CD18 methylation in the PMF samples ranged from hypomethylated to hypermethylated (range: 11-90 %, mean: 64 %), whereas in controls CD18 methylation status clustered in a more restricted interval (range: 24-68 %, mean: 45 %; cases vs. CONTROLS: p = 0.006). Furthermore, the results showed that CD18 hypermethylation (>76 % methylation) was correlated with thrombotic complications. On the contrary, CD11b promoter resulted unmethylated (1-5 %) in both cases and controls. Previous studies showed that older age, JAK2V617F mutation, and thrombophilia might play a role in MPN patients' thrombotic risk. In our cases, the prognostic value of these variables was coherent, being thrombotic events significantly associated with age >65 years (p = 0.001), JAK2 mutation (p = 0.01), and positive thrombophilia tests (p = 0.04). However, multivariate analysis showed that only CD18 methylation and age >65 years were independent prognostic factors of thrombosis (p = 0.02 and p = 0.04, respectively). Taken together, our findings suggest a possible role of CD18 epigenetic regulation in the pathogenesis of the thrombotic complications in PMF patients.

Stromal contribution to the colorectal cancer transcriptome.

Recent studies identified a poor-prognosis stem/serrated/mesenchymal (SSM) transcriptional subtype of colorectal cancer (CRC). We noted that genes upregulated in this subtype are also prominently expressed by stromal cells, suggesting that SSM transcripts could derive from stromal rather than epithelial cancer cells. To test this hypothesis, we analyzed CRC expression data from patient-derived xenografts, where mouse stroma supports human cancer cells. Species-specific expression analysis showed that the mRNA levels of SSM genes were mostly due to stromal expression. Transcriptional signatures built to specifically report the abundance of cancer-associated fibroblasts (CAFs), leukocytes or endothelial cells all had significantly higher expression in human CRC samples of the SSM subtype. High expression of the CAF signature was associated with poor prognosis in untreated CRC, and joint high expression of the stromal signatures predicted resistance to radiotherapy in rectal cancer. These data show that the distinctive transcriptional and clinical features of the SSM subtype can be ascribed to its particularly abundant stromal component.