Beyond self­eating: Emerging autophagy­independent functions for the autophagy molecules in cancer (Review).

Autophagy is a conserved catabolic process that controls organelle quality, removes misfolded or abnormally aggregated proteins and is part of the defense mechanisms against intracellular pathogens. Autophagy contributes to the suppression of tumor initiation by promoting genome stability, cellular integrity, redox balance and proteostasis. On the other hand, once a tumor is established, autophagy can support cancer cell survival and promote epithelial­to­mesenchymal transition. A growing number of molecules involved in autophagy have been identified. In addition to their key canonical activity, several of these molecules, such as ATG5, ATG12 and Beclin­1, also exert autophagy­independent functions in a variety of biological processes. The present review aimed to summarize autophagy­independent functions of molecules of the autophagy machinery and how the activity of these molecules can influence signaling pathways that are deregulated in cancer progression.

Fusion transcriptome profiling defines the monoclonal origin of multifocal epithelioid haemangioma of bone.

AIMS: Epithelioid haemangioma (EH) of bone remains a highly controversial entity. Indeed, the WHO classifies EHs of soft tissues as benign tumours, whereas bone EHs are considered intermediate-locally aggressive tumours due to common multifocal presentation and local destructive growth. To gain insights into the clinical behaviour and biology of EH of bone we retrospectively analysed 42 patients treated in a single institution from 1978 to 2021. METHODS AND RESULTS: Multifocal presentation was detected in 17 of 42 patients (40%) primarily as synchronous lesions. Patients were treated with curettage (57%), resection (29%) or biopsy, followed by radiotherapy or embolisation (14%). Follow-up (minimum 24 months) was available for 38 patients, with only five local recurrences (13%) and no death of disease. To clarify whether the synchronous bone lesions in multifocal EH represent multicentric disease or clonal dissemination, four cases were profiled by RNA-sequencing. Separate lesions from the same patient, which showed a similar transcriptional profile, expressed the same fusion transcript (involving FOS or FOSB) with identical gene breakpoints. CONCLUSIONS: These results indicate that, in EH of bone, multifocal lesions are clonally related and therefore represent the spread of a same neoplastic clone rather than simultaneous independent tumours. This finding is in apparent contradiction with the benign clinical course of the disease, and suggests that tumour dissemination in bone EH probably reflects a phenomenon of passive spreading, with tumour cells colonising distal sites while maintaining their benign biological nature.

In Silico Identification of a BRCA1:miR-29:DNMT3 Axis Involved in the Control of Hormone Receptors in BRCA1-Associated Breast Cancers.

Germline inactivating mutations in the BRCA1 gene lead to an increased lifetime risk of ovarian and breast cancer (BC). Most BRCA1-associated BC are triple-negative tumors (TNBC), aggressive forms of BC characterized by a lack of expression of estrogen and progesterone hormone receptors (HR) and HER2. How BRCA1 inactivation may favor the development of such a specific BC phenotype remains to be elucidated. To address this question, we focused on the role of miRNAs and their networks in mediating BRCA1 functions. miRNA, mRNA, and methylation data were retrieved from the BRCA cohort of the TCGA project. The cohort was divided into a discovery set (Hi-TCGA) and a validation set (GA-TCGA) based on the platform used for miRNA analyses. The METABRIC, GSE81002, and GSE59248 studies were used as additional validation data sets. BCs were differentiated into BRCA1-like and non-BRCA1-like based on an established signature of BRCA1 pathway inactivation. Differential expression of miRNAs, gene enrichment analysis, functional annotation, and methylation correlation analyses were performed. The miRNAs downregulated in BRCA1-associated BC were identified by comparing the miRNome of BRCA1-like with non-BRCA1-like tumors from the Hi-TCGA discovery cohort. miRNAs:gene-target anticorrelation analyses were then performed. The target genes of miRNAs downregulated in the Hi-TCGA series were enriched in the BRCA1-like tumors from the GA-TCGA and METABRIC validation data sets. Functional annotation of these genes revealed an over-representation of several biological processes ascribable to BRCA1 activity. The enrichment of genes related to DNA methylation was particularly intriguing, as this is an aspect of BRCA1 functions that has been poorly explored. We then focused on the miR-29:DNA methyltransferase network and showed that the miR-29 family, which was downregulated in BRCA1-like tumors, was associated with poor prognosis in these BCs and inversely correlated with the expression of the DNA methyltransferases DNMT3A and DNMT3B. This, in turn, correlated with the methylation extent of the promoter of HR genes. These results suggest that BRCA1 may control the expression of HR via a miR-29:DNMT3:HR axis and that disruption of this network may contribute to the receptor negative phenotype of tumors with dysfunctional BRCA1.

Routine Molecular Profiling in Both Resectable and Unresectable Pancreatic Adenocarcinoma: Relevance of Cytologic Samples.

BACKGROUND & AIMS: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive disease, for which it is crucial to promptly detect actionable and prognostic alterations to drive specific therapeutic decisions, regardless of tumor resectability status. Endoscopic ultrasonography-guided fine-needle aspiration (EUS-FNA) is of key importance for PDAC diagnosis and can contribute significantly to tumor molecular profiling. METHODS: Comprehensive genomic profile by targeted next-generation sequencing (NGS) was performed on 2 independent PDAC patient cohorts. Cohort 1 consisted of 77 patients with resectable PDAC for whom the histologic sample at the time of resection was available; for 56 patients cytologic specimens at the time of diagnosis also were obtained by EUS-FNA. Cohort 2 consisted of 20 patients with unresectable PDAC, for whom only the EUS-FNA cytologic sample was available. RESULTS: In cohort 1, a complete concordant mutational profile between the cytologic sample at diagnosis and the corresponding histologic specimen after surgery was observed in 88% of the cases, proving the ability to detect potential clinically relevant alterations in cytologic samples by NGS analysis. Notably, clinically actionable mutations were identified in 20% of patients. In cohort 2, comprehensive mutational profiling was obtained successfully for all samples. Consistent with the findings of cohort 1, KRAS, TP53, CDKN2A, and SMAD4 were the most altered genes. Most importantly, 15% of the patients harbored actionable mutations. CONCLUSIONS: Our findings show the feasibility of an NGS approach using both surgical specimens and cytologic samples. The model proposed in this study can be included successfully in the clinical setting for comprehensive molecular profiling of all PDAC patients irrespective of their surgical eligibility.

CINSARC in high-risk soft tissue sarcoma patients treated with neoadjuvant chemotherapy: Results from the ISG-STS 1001 study.

BACKGROUND: The Complexity INdex in SARComas (CINSARC) is a transcriptional signature derived from the expression of 67 genes involved in mitosis control and chromosome integrity. This study aims to assess CINSARC value of in an independent series of high-risk patients with localized soft tissue sarcoma (STS) treated with preoperative chemotherapy within a prospective, randomized, phase III study (ISG-STS 1001). PATIENTS AND METHODS: Patients with available pre-treatment samples, treated with 3 cycles of either standard (ST) preoperative or histotype-tailored (HT) chemotherapy, were scored according to CINSARC (low-risk, C1; high-risk, C2). The 10-year overall survival probability (pr-OS) according to SARCULATOR was calculated, and patients were classified accordingly (low-risk, Sarc-LR, 10-year pr-OS>60%; high-risk, Sarc-HR, 10-year pr-OS<60%). Survival functions were estimated using the Kaplan-Meier method and compared using log-rank test. RESULTS: Eighty-six patients were included, 30 C1 and 56 C2, 49 Sarc-LR and 37 Sarc-HR. A low level of agreement between CINSARC and SARCULATOR was observed (Cohen's Kappa = 0.174). The 5-year relapse-free survival in C1 and C2 were 0.57 and 0.55 (p = 0.481); 5-year metastases-free survival 0.63 and 0.64 (p = 0.740); 5-year OS 0.80 and 0.72 (p = 0.460). The 5-year OS in C1 treated with ST and HT chemotherapy was 0.84 and 0.76 (p = 0.251) respectively; in C2 treated it was 0.72 and 0.70 (p = 0.349). The 5-year OS in Sarc-LR treated with S and HT chemotherapy was 0.80 and 0.82 (p = 0.502) respectively; in Sarc-HR it was 0.70 and 0.61 (p = 0.233). CONCLUSIONS: Our results, although constrained by the small size of the series, suggest that CINSARC has weak prognostic power in high-risk, localized STS treated with neoadjuvant chemotherapy.

Beyond SMARCB1 Loss: Recent Insights into the Pathobiology of Epithelioid Sarcoma.

Epithelioid sarcoma (ES) is a very rare and aggressive mesenchymal tumor of unclear origin and uncertain lineage characterized by a prevalent epithelioid morphology. The only recurrent genetic alteration reported in ES as yet is the functional inactivation of SMARCB1 (SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily B member 1), a key component of the SWI/SNF (SWItch/Sucrose Non-Fermentable) chromatin remodeling complexes. How SMARCB1 deficiency dictates the clinicopathological characteristics of ES and what other molecular defects concur to its malignant progression is still poorly understood. This review summarizes the recent findings about ES pathobiology, including defects in chromatin remodeling and other signaling pathways and their role as therapeutic vulnerabilities.

Retrospective observational studies in ultra-rare sarcomas: A consensus paper from the Connective Tissue Oncology Society (CTOS) community of experts on the minimum requirements for the evaluation of activity of systemic treatments.

BACKGROUND: In ultra-rare sarcomas (URS) the conduction of prospective, randomized trials is challenging. Data from retrospective observational studies (ROS) may represent the best evidence available. ROS implicit limitations led to poor acceptance by the scientific community and regulatory authorities. In this context, an expert panel from the Connective Tissue Oncology Society (CTOS), agreed on the need to establish a set of minimum requirements for conducting high-quality ROS on the activity of systemic therapies in URS. METHODS: Representatives from > 25 worldwide sarcoma reference centres met in November 2020 and identified a list of topics summarizing the main issues encountered in ROS on URS. An online survey on these topics was distributed to the panel; results were summarized by descriptive statistics and discussed during a second meeting (November 2021). RESULTS: Topics identified by the panel included the use of ROS results as external control data, the criteria for contributing centers selection, modalities for ensuring a correct pathological diagnosis and radiologic assessment, consistency of surveillance policies across centers, study end-points, risk of data duplication, results publication. Based on the answers to the survey (55 of 62 invited experts) and discussion the panel agreed on 18 statements summarizing principles of recommended practice. CONCLUSIONS: These recommendations will be disseminated by CTOS across the sarcoma community and incorporated in future ROS on URS, to maximize their quality and favor their use as control data when results from prospective studies are unavailable. These recommendations could help the optimal conduction of ROS also in other rare tumors.

ISG15 as a prognostic biomarker in solitary fibrous tumour.

BACKGROUND: Solitary fibrous tumour (SFT) is a rare mesenchymal malignancy that lacks robust prognostic and predictive biomarkers. Interferon-stimulated gene 15 (ISG15) is a ubiquitin-like modifier, associated with tumour progression, and with poor survival of SFT patients, as previous published by our group. Here, we describe the role of ISG15 in the biology of this rare tumour. METHODS: ISG15 expression was assessed by immunohistochemistry in tissue microarrays from SFT patients and tested for correlation with progression-free survival and overall survival (OS). The effects of ISG15 knockdown or induction were investigated for cancer stem cell (CSC) characteristics and for drug sensitivity in unique in vitro models of SFT. RESULTS: The prognostic value of ISG15 for OS was validated at protein level in malignant SFT patients, prospectively treated with pazopanib and enrolled in GEIS-32 trial. In SFT in vitro models, ISG15 knockdown lead to a decrease in the expression of CSC-related genes, including SOX2, NANOG, ALDH1A1, ABCB1 and ABCC1. Likewise, ISG15 downregulation decreased the clonogenic/ tumoursphere-forming ability of SFT cells, while enhancing apoptotic cell death after doxorubicin, pazopanib or trabectedin treatment in 3D cell cultures. The regulation of CSC-related genes by ISG15 was confirmed after inducing its expression with interferon-ß1; ISG15 induction upregulated 1.28- to 451-fold the expression of CSC-associated genes. CONCLUSIONS: ISG15 is a prognostic factor in malignant SFT, regulating the expression of CSC-related genes and CSCs maintenance. Our results suggest that ISG15 could be a novel therapeutic target in SFT, which could improve the efficacy of the currently available treatments.

MET Inhibition Sensitizes Rhabdomyosarcoma Cells to NOTCH Signaling Suppression.

Rhabdomyosarcoma (RMS) is a pediatric myogenic soft tissue sarcoma. The Fusion-Positive (FP) subtype expresses the chimeric protein PAX3-FOXO1 (P3F) while the Fusion-Negative (FN) is devoid of any gene translocation. FP-RMS and metastatic FN-RMS are often unresponsive to conventional therapy. Therefore, novel therapeutic approaches are needed to halt tumor progression. NOTCH signaling has oncogenic functions in RMS and its pharmacologic inhibition through γ-secretase inhibitors blocks tumor growth in vitro and in vivo. Here, we show that NOTCH signaling blockade resulted in the up-regulation and phosphorylation of the MET oncogene in both RH30 (FP-RMS) and RD (FN-RMS) cell lines. Pharmacologic inhibition of either NOTCH or MET signaling slowed proliferation and restrained cell survival compared to control cells partly by increasing Annexin V and CASP3/7 activation. Co-treatment with NOTCH and MET inhibitors significantly amplified these effects and enhanced PARP1 cleavage in both cell lines. Moreover, it severely hampered cell migration, colony formation, and anchorage-independent growth compared to single-agent treatments in both cell lines and significantly prevented the growth of FN-RMS cells grown as spheroids. Collectively, our results unveil the overexpression of the MET oncogene by NOTCH signaling targeting in RMS cells and show that MET pathway blockade sensitizes them to NOTCH inhibition.

Interference of p53:Twist1 interaction through competing nanobodies.

Twist1 promote the bypass of p53 response by interacting with p53 and facilitating its MDM2-mediated degradation. We reasoned that reagents able to interfere with the p53:Twist1 complex might alleviate Twist1 inhibitory effect over p53, thus representing potential therapeutic tools in p53 wild type tumors. From a pre-immune library of llama nanobodies (VHH), we isolated binders targeting the p53 C-terminal region (p53-CTD) involved in the interaction with Twist1 by using recombinant Twist1 as an epitope-specific competitor during elution. Positive hits were validated by proving their capacity to immunoprecipitate p53 and to inhibit Twist1:p53 binding in vitro. Molecular modeling confirmed a preferential docking of positive hits with p53-CTD. D11 VHH activity was validated in human cell models, succeeded in immunoprecipitating endogenous p53 and, similarly to Twist1 knock-down, interfered with p53 turnover, p53 phosphorylation at Serine 392 and affected cell viability. Despite the limited functional effect determined by D11 expression in target cells, our results provide the proof of principle that nanobodies ectopically expressed within a cell, have the capacity to target the assembly of the pro-tumorigenic Twist1:p53 complex. These results disclose novel tools for dissecting p53 biology and lay down the grounds for the development of innovative targeted therapeutic approaches.

The Autophagic Route of E-Cadherin and Cell Adhesion Molecules in Cancer Progression.

Cell-to-cell adhesion is a key element in epithelial tissue integrity and homeostasis during embryogenesis, response to damage, and differentiation. Loss of cell adhesion and gain of mesenchymal features, a phenomenon known as epithelial to mesenchymal transition (EMT), are essential steps in cancer progression. Interestingly, downregulation or degradation by endocytosis of epithelial adhesion molecules (e.g., E-cadherin) associates with EMT and promotes cell migration. Autophagy is a physiological intracellular degradation and recycling process. In cancer, it is thought to exert a tumor suppressive role in the early phases of cell transformation but, once cells have gained a fully transformed phenotype, autophagy may fuel malignant progression by promoting EMT and conferring drug resistance. In this review, we discuss the crosstalk between autophagy, EMT, and turnover of epithelial cell adhesion molecules, with particular attention to E-cadherin.

Whole genome and exome sequencing reference datasets from a multi-center and cross-platform benchmark study.

With the rapid advancement of sequencing technologies, next generation sequencing (NGS) analysis has been widely applied in cancer genomics research. More recently, NGS has been adopted in clinical oncology to advance personalized medicine. Clinical applications of precision oncology require accurate tests that can distinguish tumor-specific mutations from artifacts introduced during NGS processes or data analysis. Therefore, there is an urgent need to develop best practices in cancer mutation detection using NGS and the need for standard reference data sets for systematically measuring accuracy and reproducibility across platforms and methods. Within the SEQC2 consortium context, we established paired tumor-normal reference samples and generated whole-genome (WGS) and whole-exome sequencing (WES) data using sixteen library protocols, seven sequencing platforms at six different centers. We systematically interrogated somatic mutations in the reference samples to identify factors affecting detection reproducibility and accuracy in cancer genomes. These large cross-platform/site WGS and WES datasets using well-characterized reference samples will represent a powerful resource for benchmarking NGS technologies, bioinformatics pipelines, and for the cancer genomics studies.

mTORC1 promotes malignant large cell/anaplastic histology and is a targetable vulnerability in SHH-TP53 mutant medulloblastoma.

Medulloblastoma (MB), one of the most malignant brain tumors of childhood, comprises distinct molecular subgroups, with p53 mutant sonic hedgehog-activated (SHH-activated) MB patients having a very severe outcome that is associated with unfavorable histological large cell/anaplastic (LC/A) features. To identify the molecular underpinnings of this phenotype, we analyzed a large cohort of MB developing in p53-deficient Ptch+/- SHH mice that, unexpectedly, showed LC/A traits that correlated with mTORC1 hyperactivation. Mechanistically, mTORC1 hyperactivation was mediated by a decrease in the p53-dependent expression of mTORC1 negative regulator Tsc2. Ectopic mTORC1 activation in mouse MB cancer stem cells (CSCs) promoted the in vivo acquisition of LC/A features and increased malignancy; accordingly, mTORC1 inhibition in p53-mutant Ptch+/- SHH MB and CSC-derived MB resulted in reduced tumor burden and aggressiveness. Most remarkably, mTORC1 hyperactivation was detected only in p53-mutant SHH MB patient samples, and treatment with rapamycin of a human preclinical model phenocopying this subgroup decreased tumor growth and malignancy. Thus, mTORC1 may act as a specific druggable target for this subset of SHH MB, resulting in the implementation of a stringent risk stratification and in the potentially rapid translation of this precision medicine approach into the clinical setting.

Filling the gap: A thorough investigation for the genetic diagnosis of unsolved polyposis patients with monoallelic MUTYH pathogenic variants.

BACKGROUNDS: MUTYH-associated polyposis (MAP) is an autosomal recessive disease caused by biallelic pathogenic variants (PV) of the MUTYH gene. The aim of this study was to investigate the genetic causes of unexplained polyposis patients with monoallelic MUTYH PV. The analysis focused on 26 patients with suspected MAP, belonging to 23 families. Ten probands carried also one or more additional MUTYH variants of unknown significance. METHODS: Based on variant type and on the collected clinical and molecular data, these variants were reinterpreted by applying the ACMG/AMP rules. Moreover, supplementary analyses were carried out to investigate the presence of other variants and copy number variations in the coding and promoter regions of MUTYH, as well as other polyposis genes (APC, NTHL1, POLE, POLD1, MSH3, RNF43, and MCM9). RESULTS: We reclassified 4 out of 10 MUTYH variants as pathogenic or likely pathogenic, thus supporting the diagnosis of MAP in only four cases. Two other patients belonging to the same family showed a previously undetected deletion of the APC gene promoter. No PVs were found in the other investigated genes. However, 6 out of the 18 remaining families are still interesting MAP candidates, due to the co-presence of a class 3 MUTYH variant that could be reinterpreted in the next future. CONCLUSION: Several efforts are necessary to fully elucidate the genetic etiology of suspected MAP patients, especially those with the most severe polyposis/tumor phenotype. Clinical data, tumor molecular profile, family history, and polyposis inheritance mode may guide variant interpretation and address supplementary studies.

Toward best practice in cancer mutation detection with whole-genome and whole-exome sequencing.

Clinical applications of precision oncology require accurate tests that can distinguish true cancer-specific mutations from errors introduced at each step of next-generation sequencing (NGS). To date, no bulk sequencing study has addressed the effects of cross-site reproducibility, nor the biological, technical and computational factors that influence variant identification. Here we report a systematic interrogation of somatic mutations in paired tumor-normal cell lines to identify factors affecting detection reproducibility and accuracy at six different centers. Using whole-genome sequencing (WGS) and whole-exome sequencing (WES), we evaluated the reproducibility of different sample types with varying input amount and tumor purity, and multiple library construction protocols, followed by processing with nine bioinformatics pipelines. We found that read coverage and callers affected both WGS and WES reproducibility, but WES performance was influenced by insert fragment size, genomic copy content and the global imbalance score (GIV; G > T/C > A). Finally, taking into account library preparation protocol, tumor content, read coverage and bioinformatics processes concomitantly, we recommend actionable practices to improve the reproducibility and accuracy of NGS experiments for cancer mutation detection.

Establishing community reference samples, data and call sets for benchmarking cancer mutation detection using whole-genome sequencing.

The lack of samples for generating standardized DNA datasets for setting up a sequencing pipeline or benchmarking the performance of different algorithms limits the implementation and uptake of cancer genomics. Here, we describe reference call sets obtained from paired tumor-normal genomic DNA (gDNA) samples derived from a breast cancer cell line-which is highly heterogeneous, with an aneuploid genome, and enriched in somatic alterations-and a matched lymphoblastoid cell line. We partially validated both somatic mutations and germline variants in these call sets via whole-exome sequencing (WES) with different sequencing platforms and targeted sequencing with >2,000-fold coverage, spanning 82% of genomic regions with high confidence. Although the gDNA reference samples are not representative of primary cancer cells from a clinical sample, when setting up a sequencing pipeline, they not only minimize potential biases from technologies, assays and informatics but also provide a unique resource for benchmarking 'tumor-only' or 'matched tumor-normal' analyses.

Crosstalk between Macrophages and Myxoid Liposarcoma Cells Increases Spreading and Invasiveness of Tumor Cells.

Myxoid liposarcoma (MLPS) is the second most common subtype of liposarcoma and has tendency to metastasize to soft tissues. To date, the mechanisms of invasion and metastasis of MLPS remain unclear, and new therapeutic strategies that improve patients' outcomes are expected. In this study, we analyzed by immunohistochemistry the immune cellular components and microvessel density in tumor tissues from patients affected by MLPS. In order to evaluate the effects of primary human MLPS cells on macrophage polarization and, in turn, the ability of macrophages to influence invasiveness of MLPS cells, non-contact and 3D organotypic co-cultures were set up. High grade MLPS tissues were found heavily vascularized, exhibited a CD3, CD4, and CD8 positive T lymphocyte-poor phenotype and were massively infiltrated by CD163 positive M2-like macrophages. Conversely, low grade MLPS tissues were infiltrated by a discrete amount of CD3, CD4, and CD8 positive T lymphocytes and a scarce amount of CD163 positive macrophages. Kaplan-Meier analysis revealed a shorter Progression Free Survival in MLPS patients whose tumor tissues were highly vascularized and heavily infiltrated by CD163 positive macrophages, indicating a clear-cut link between M2-like macrophage abundance and poor prognosis in patients. Moreover, we documented that, in co-culture, soluble factors produced by primary human MLPS cells induce macrophage polarization toward an M2-like phenotype which, in turn, increases MLPS cell capability to spread into extracellular matrix and to cross endothelial monolayers. The identification of M2-like polarization factors secreted by MLPS cells may allow to develop novel targeted therapies counteracting MLPS progression.

Ultra-rare sarcomas: A consensus paper from the Connective Tissue Oncology Society community of experts on the incidence threshold and the list of entities.

BACKGROUND: Among sarcomas, which are rare cancers, many types are exceedingly rare; however, a definition of ultra-rare cancers has not been established. The problem of ultra-rare sarcomas is particularly relevant because they represent unique diseases, and their rarity poses major challenges for diagnosis, understanding disease biology, generating clinical evidence to support new drug development, and achieving formal authorization for novel therapies. METHODS: The Connective Tissue Oncology Society promoted a consensus effort in November 2019 to establish how to define ultra-rare sarcomas through expert consensus and epidemiologic data and to work out a comprehensive list of these diseases. The list of ultra-rare sarcomas was based on the 2020 World Health Organization classification, The incidence rates were estimated using the Information Network on Rare Cancers (RARECARENet) database and NETSARC (the French Sarcoma Network's clinical-pathologic registry). Incidence rates were further validated in collaboration with the Asian cancer registries of Japan, Korea, and Taiwan. RESULTS: It was agreed that the best criterion for a definition of ultra-rare sarcomas would be incidence. Ultra-rare sarcomas were defined as those with an incidence of approximately ≤1 per 1,000,000, to include those entities whose rarity renders them extremely difficult to conduct well powered, prospective clinical studies. On the basis of this threshold, a list of ultra-rare sarcomas was defined, which comprised 56 soft tissue sarcoma types and 21 bone sarcoma types. CONCLUSIONS: Altogether, the incidence of ultra-rare sarcomas accounts for roughly 20% of all soft tissue and bone sarcomas. This confirms that the challenges inherent in ultra-rare sarcomas affect large numbers of patients.

Lynch syndrome and Muir-Torre phenotype associated with a recurrent variant in the 3'UTR of the MSH6 gene.

A MSH6 3'UTR variant (c.*23_26dup) was found in 13 unrelated families consulted for Lynch/Muir-Torre Syndrome. This variant, which is very rare in the genomic databases, was absent in healthy controls and strongly segregated with the disease in the studied pedigrees. All tumors were defective for MSH2/MSH6/MSH3 proteins expression, but only MSH2 somatic pathogenic mutations were found in 5 of the 12 sequenced tumors. Moreover, we had no evidence of MSH6 transcript decrease in carriers, whereas MSH2 transcript was downregulated. Additional evaluations performed in representative carriers, including karyotype, arrayCGH and Linked-Reads whole genome sequencing, failed to evidence any MSH2 germline pathogenic variant. Posterior probability of pathogenicity for MSH6 c.*23_26dup was obtained from a multifactorial analysis incorporating segregation and phenotypic data and resulted >0.999, allowing to classify the variant as pathogenic (InSiGHT Class 5). Carriers shared a common haplotype involving MSH2/MSH6 loci, then a cryptic disease-associated variant, linked with MSH6 c.*23_26dup, cannot be completely excluded. Even if it is not clear whether the MSH6 variant is pathogenic per se or simply a marker of a disease-associated MSH2/MSH6 haplotype, all data collected on patients and pedigrees prompted us to manage the variant as pathogenic and to offer predictive testing within these families.