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Abstract Background Vascular anomalies caused by somatic (postzygotic) variants are clinically and genetically heterogeneous diseases with overlapping or distinct entities. The genetic knowledge in this field is rapidly growing, and genetic testing is now part of the diagnostic workup alongside the clinical, radiological and histopathological data. Nonetheless, access to genetic testing is still limited, and there is significant heterogeneity across the approaches used by the diagnostic laboratories, with direct consequences on test sensitivity and accuracy. The clinical utility of genetic testing is expected to increase progressively with improved theragnostics, which will be based on information about the efficacy and safety of the emerging drugs and future molecules. The aim of this study was to make recommendations for optimising and guiding the diagnostic genetic testing for somatic variants in patients with vascular malformations. Results Physicians and lab specialists from 11 multidisciplinary European centres for vascular anomalies reviewed the genes identified to date as being involved in non-hereditary vascular malformations, evaluated gene–disease associations, and made recommendations about the technical aspects for identification of low-level mosaicism and variant interpretation. A core list of 24 genes were selected based on the current practices in the participating laboratories, the ISSVA classification and the literature. In total 45 gene–phenotype associations were evaluated: 16 were considered definitive, 16 strong, 3 moderate, 7 limited and 3 with no evidence. Conclusions This work provides a detailed evidence-based view of the gene–disease associations in the field of vascular malformations caused by somatic variants. Knowing both the gene–phenotype relationships and the strength of the associations greatly help laboratories in data interpretation and eventually in the clinical diagnosis. This study reflects the state of knowledge as of mid-2023 and will be regularly updated on the VASCERN-VASCA website (VASCERN-VASCA, https://vascern.eu/groupe/vascular-anomalies/ ).

Abstract Background Uterine clear cell carcinoma (CCC) consists of either pure clear cell histology but can also display other histological components (mixed uterine CCCs). In this study, the molecular and immunohistochemical background of pure and mixed uterine CCC was compared. Secondly, it was evaluated whether histological classification and molecular background affected clinical outcome. Methods A retrospective multicenter study was performed comparing pure uterine CCCs (n = 22) and mixed uterine CCCs (n = 21). Targeted next‐generation sequencing using a 12‐gene targeted panel classified cases as polymerase‐ε (POLE) mutated, microsatellite instable (MSI), TP53 wildtype or TP53 mutated. Immunohistochemistry was performed for estrogen receptor, progesterone receptor, L1 cell adhesion molecule, MSH6, and PMS2. Results The following molecular subgroups were identified for pure and mixed uterine CCCs, respectively: POLE mutated 0% (0/18) and 6% (1/18); MSI in 6% (1/18) and 50% (9/18); TP53 wildtype in 56% (10/18) and 22% (4/18); TP53 mutated in 39% (7/18) and 22% (4/18) (p = 0.013). Patients with mixed CCCs had improved outcome compared to patients with pure CCCs. Frequent TP53 mutations were found in pure CCCs and frequent MSI in mixed CCCs, associated with clinical outcome. Conclusion Pure and mixed uterine CCCs are two entities with different clinical outcomes, which could be explained by different molecular backgrounds. These results underline the relevance of both morphological and molecular evaluation, and may assist in tailoring treatment.

Abstract Background Sensitive and reliable molecular diagnostics is needed to guide therapeutic decisions for cancer patients. Although less material becomes available for testing, genetic markers are rapidly expanding. Simultaneous detection of predictive markers, including mutations, gene amplifications and MSI, will save valuable material, time and costs. Methods Using a single-molecule molecular inversion probe (smMIP)-based targeted next-generation sequencing (NGS) approach, we developed an NGS panel allowing detection of predictive mutations in 33 genes, gene amplifications of 13 genes and microsatellite instability (MSI) by the evaluation of 55 microsatellite markers. The panel was designed to target all clinically relevant single and multiple nucleotide mutations in routinely available lung cancer, colorectal cancer, melanoma, and gastro-intestinal stromal tumor samples, but is useful for a broader set of tumor types. Results The smMIP-based NGS panel was successfully validated and cut-off values were established for reliable gene amplification analysis (i.e. relative coverage ≥3) and MSI detection (≥30% unstable loci). After validation, 728 routine diagnostic tumor samples including a broad range of tumor types were sequenced with sufficient sensitivity (2.4% drop-out), including samples with low DNA input (

Abstract Background Intramuscular myxoma (IM) is a hypocellular benign soft tissue neoplasm characterized by abundant myxoid stroma and occasional hypercellular areas. These tumors can, especially on biopsy material, be difficult to distinguish from low-grade fibromyxoid sarcoma or low-grade myxofibrosarcoma. GNAS mutations are frequently involved in IM, in contrast to these other malignant tumors. Therefore, sensitive molecular techniques for detection of GNAS aberrations in IM, which frequently yield low amounts of DNA due to poor cellularity, will be beneficial for differential diagnosis. Methods In our study, a total of 34 IM samples from 33 patients were analyzed for the presence of GNAS mutations, of which 29 samples were analyzed using a gene-specific TaqMan genotyping assay for the detection of GNAS hotspot mutations c.601C > T and c602G > A in IM, and 32 samples using a novel next generation sequencing (NGS)-based approach employing single-molecule tagged molecular inversion probes (smMIP) to identify mutations in exon 8 and 9 of GNAS. Results between the two assays were compared for their ability to detect GNAS mutations with high confidence. Results In total, 23 of 34 samples were successfully analyzed with both techniques showing GNAS mutations in 12 out of 23 (52%) samples. The remaining 11 samples were analyzed with either TaqMan assay or smMIP assay only. The TaqMan assay revealed GNAS mutations in 16 out of 29 samples (55%), with six samples c.601C > T (p.R201C; 38%) and ten samples c.602G > A (p.R201H; 62%) missense mutations. The smMIP assay identified mutations in 16 out of 28 samples (57%), with five samples c.601C > T (p.R201C; 31%) and seven samples c.602G > A (p.R201H; 44%) missense mutations. In addition, four samples (25%) revealed novel IM-associated mutations, including c.601C > A (p.R201S), c.602G > T (p.R201L), c.602G > C (p.R201P) and c.680A > G (p.Q227R). Combining the results of both tests, 23 out of 34 sporadic IM samples (68%) showed a GNAS mutation. Conclusions Both the TaqMan and the smMIP assay a show a high degree of concordance in detecting GNAS hotspot mutations in IM with comparable sensitivity. However, since the NGS-based smMIP assay permits mutation detection in whole exons of GNAS, a broader range of GNAS mutations can be identified by the smMIP approach.

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Forkhead box O (FOXO) transcription factors regulate diverse cellular processes, affecting tumorigenesis, metabolism, stem cell maintenance, and lifespan. We show that FOXO3 transcription regulation mainly proceeds through the most active subset of enhancers. In addition to the general distinction between “open” and “closed” chromatin, we show that the level of activity marks (H3K27ac, RNAPII, enhancer RNAs) of these open chromatin regions prior to FOXO3 activation largely determines FOXO3 DNA binding. Consequently, FOXO3 amplifies the levels of these activity marks and their absolute rather than relative changes associate best with FOXO3 target gene regulation. The importance of preexisting chromatin state in directing FOXO3 gene regulation, as shown here, provides a mechanism whereby FOXO3 can regulate cell-specific homeostasis. Genetic variation is reported to affect these chromatin signatures in a quantitative manner, and, in agreement, we observe a correlation between cancer-associated genetic variations and the amplitude of FOXO3 enhancer binding.

Abstract Forkhead box O (FOXO) transcription factors are key players in diverse cellular processes affecting tumorigenesis, stem cell maintenance and lifespan. To gain insight into the mechanisms of FOXO‐regulated target gene expression, we studied genome‐wide effects of FOXO3 activation. Profiling RNA polymerase II changes shows that FOXO3 regulates gene expression through transcription initiation. Correlative analysis of FOXO3 and RNA polymerase II ChIP‐seq profiles demonstrates FOXO3 to act as a transcriptional activator. Furthermore, this analysis reveals a significant part of FOXO3 gene regulation proceeds through enhancer regions. FOXO3 binds to pre‐existing enhancers and further activates these enhancers as shown by changes in histone acetylation and RNA polymerase II recruitment. In addition, FOXO3‐mediated enhancer activation correlates with regulation of adjacent genes and pre‐existence of chromatin loops between FOXO3 bound enhancers and target genes. Combined, our data elucidate how FOXOs regulate gene transcription and provide insight into mechanisms by which FOXOs can induce different gene expression programs depending on chromatin architecture.