Chromosomal instability and a deregulated cell cycle are intrinsic features of high-risk gastrointestinal stromal tumours with a metastatic potential.

Patients with localised, high-risk gastrointestinal stromal tumours (GIST) benefit from adjuvant imatinib treatment. Still, approximately 40% of patients relapse within 3 years after adjuvant therapy and the clinical and histopathological features currently used for risk classification cannot precisely predict poor outcomes after standard treatment. This study aimed to identify genomic and transcriptomic profiles that could be associated with disease relapse and thus a more aggressive phenotype. Using a multi-omics approach, we analysed a cohort of primary tumours from patients with untreated, resectable high-risk GISTs. We compared patients who developed metastatic disease within 3 years after finishing adjuvant imatinib treatment and patients without disease relapse after more than 5 years of follow-up. Combining genomics and transcriptomics data, we identified somatic mutations and deregulated mRNA and miRNA genes intrinsic to each group. Our study shows that increased chromosomal instability (CIN), including chromothripsis and deregulated kinetochore and cell cycle signalling, separates high-risk samples according to metastatic potential. The increased CIN seems to be an intrinsic feature for tumours that metastasise and should be further validated as a novel prognostic biomarker for high-risk GIST.

Clinical and molecular implications of NAB2-STAT6 fusion variants in solitary fibrous tumour.

Solitary fibrous tumour (SFT) is a mesenchymal neoplasm characterised by pathognomonic NAB2-STAT6 gene fusions. The clinical implications and prognostic value of different fusion variants has not been clarified. In the current study, we explore the clinicopathological, prognostic and molecular differences between tumours with different fusions. Thirty-nine patients with localised, extrameningeal SFT were included, of whom 20 developed distant recurrence and 19 were without recurrence after long term follow-up. Capture-based RNA sequencing identified 12 breakpoint variants, which were categorised into two groups based on the STAT6 domain composition in the predicted chimeric proteins. Twenty-one of 34 (62%) sequenced tumours had fusions with most of the STAT6 domains intact and were classified as STAT6-Full. Thirteen tumours (38%) contained only the transactivation domain of STAT6 and were classified as STAT6-TAD. Tumours with STAT6-TAD fusions had a higher mitotic count (p=0.016) and were associated with inferior recurrence-free interval (p=0.004) and overall survival (p=0.012). Estimated 10-year recurrence-free survival was 25% for patients with STAT6-TAD tumours compared to 78% for the STAT6-Full group. Distinct transcriptional signatures between the fusion groups were identified, including higher expression of FGF2 in the STAT6-TAD group and IGF2, EGR2, PDGFRB, STAT6 and several extracellular matrix genes in STAT6-Full tumours. In summary, we demonstrate that NAB2-STAT6 fusion variants are associated with distinct clinicopathological and molecular characteristics and have prognostic significance in extrameningeal SFT.

Cell-free DNA in blood as a noninvasive insight into the sarcoma genome.

Sarcomas are malignant tumors of mesenchymal origin that arise mainly from connective and supportive tissue. Sarcomas include a wide range of histological subtypes, showing a large diversity at the molecular level, from simple to highly complex karyotypes but with few recurrent somatic changes. Therapeutic decisions increasingly rely on the molecular characteristics of the individual tumor. Circulating cell-free DNA (ctDNA) is released into peripheral blood and can be used for the genomic analysis of sarcomas. However, the diversity and heterogeneity of somatic changes observed in sarcomas pose a challenge when choosing an adequate assay for the detection of ctDNA in body fluids. In this review, we provide an overview of different studies on ctDNA from blood in bone and soft tissue sarcomas, including gastrointestinal stromal tumors. We will specifically address the technological challenges that must be considered to achieve the sensitive detection of ctDNA and discuss the clinical applications of ctDNA in the management and treatment of sarcomas.

ctDNA detected by ddPCR reveals changes in tumour load in metastatic malignant melanoma treated with bevacizumab.

Bevacizumab is included in an increasing number of clinical trials. To find biomarkers to predict and monitor treatment response, cancer and angiogenesis relevant mutations in tumour and circulating tumour DNA (ctDNA) were investigated in 26 metastatic melanoma patients treated with bevacizumab. Patients with >1% BRAF/NRAS ctDNA at treatment start had significantly decreased progression free survival (PFS) and overall survival (OS) (PFS: p = 0.019, median 54 vs 774 days, OS: p = 0.026, median 209 vs 1064 days). Patients with >1% BRAF/NRAS ctDNA during treatment showed similar results (PFS: p = 0.002, OS: p = 0.003). ≤1% BRAF/NRAS ctDNA and normal lactate dehydrogenase (LDH) levels both significantly predicted increased response to treatment, but BRAF/NRAS ctDNA was better at predicting response compared to LDH at treatment start (OR 16.94, p = 0.032 vs OR 4.57, p = 0.190), and at predicting PFS (HR 6.76, p = 0.002) and OS (HR 6.78, p = 0.002) during therapy. ctDNA BRAF p.V600D/E/K and NRAS p.G12V/p.Q61K/L/R were better biomarkers for response prediction than TERT promoter mutations (OR 1.50, p = 0.657). Next generation sequencing showed that all patients with ≥2 mutations in angiogenesis-relevant genes had progressive disease, but did not reveal other biomarkers identifying responders. To conclude, ctDNA and LDH are useful biomarkers for both monitoring and predicting response to bevacizumab.

Diagnostic and prognostic gene expression signatures in 177 soft tissue sarcomas: hypoxia-induced transcription profile signifies metastatic potential.

BACKGROUND: Soft tissue sarcoma (STS) diagnosis is challenging because of a multitude of histopathological subtypes, different genetic characteristics, and frequent intratumoral pleomorphism. One-third of STS metastasize and current risk-stratification is suboptimal, therefore, novel diagnostic and prognostic markers would be clinically valuable. We assessed the diagnostic and prognostic value of array-based gene expression profiles using 27 k cDNA microarrays in 177, mainly high-grade, STS of 13 histopathological subtypes. RESULTS: Unsupervised analysis resulted in two major clusters--one mainly containing STS characterized by type-specific genetic alterations and the other with a predominance of genetically complex and pleomorphic STS. Synovial sarcomas, myxoid/round-cell liposarcomas, and gastrointestinal stromal tumors clustered tightly within the former cluster and discriminatory signatures for these were characterized by developmental genes from the EGFR, FGFR, Wnt, Notch, Hedgehog, RAR and KIT signaling pathways. The more pleomorphic STS subtypes, e.g. leiomyosarcoma, malignant fibrous histiocytoma/undifferentiated pleomorphic sarcoma and dedifferentiated/pleomorphic liposarcoma, were part of the latter cluster and were characterized by relatively heterogeneous profiles, although subclusters herein were identified. A prognostic signature partly characterized by hypoxia-related genes was identified among 89 genetically complex pleomorphic primary STS and could, in a multivariate analysis including established prognostic markers, independently predict the risk of metastasis with a hazard ratio of 2.2 (P = 0.04). CONCLUSION: Diagnostic gene expression profiles linking signaling pathways to the different STS subtypes were demonstrated and a hypoxia-induced metastatic profile was identified in the pleomorphic, high-grade STS. These findings verify diagnostic utility and application of expression data for improved selection of high-risk STS patients.

Generation and characterization of an immortalized human mesenchymal stromal cell line.

Human mesenchymal stromal cells (hMSCs) show great potential for clinical and experimental use due to their capacity to self-renew and differentiate into multiple mesenchymal lineages. However, disadvantages of primary cultures of hMSCs are the limited in vitro lifespan, and the variable properties of cells from different donors and over time in culture. In this article, we describe the generation of a telomerase-immortalized nontumorigenic human bone marrow-derived stromal mesenchymal cell line, and its detailed characterization after long-term culturing (up to 155 population doublings). The resulting cell line, iMSC#3, maintained a fibroblast-like phenotype comparable to early passages of primary hMSCs, and showed no major differences from hMSCs regarding surface marker expression. Furthermore, iMSC#3 had a normal karyotype, and high-resolution array comparative genomic hybridization confirmed normal copy numbers. The gene expression profiles of immortalized and primary hMSCs were also similar, whereas the corresponding DNA methylation profiles were more diverse. The cells also had proliferation characteristics comparable to primary hMSCs and maintained the capacity to differentiate into osteoblasts and adipocytes. A detailed characterization of the mRNA and microRNA transcriptomes during adipocyte differentiation also showed that the iMSC#3 recapitulates this process at the molecular level. In summary, the immortalized mesenchymal cells represent a valuable model system that can be used for studies of candidate genes and their role in differentiation or oncogenic transformation, and basic studies of mesenchymal biology.