Alternative lengthening of telomeres (ALT) is a telomere elongation mechanism activated during oncogenesis and primarily acting in tumors of mesenchymal origin. Although the proteins involved in the machinery enabling ALT to elongate telomeres are becoming better understood, the underlying biology of this mechanism remains unclear. In the present study, we took advantage of a fully characterized cohort of 98 leiomyosarcomas (LMS) from the French Sarcoma Group to further our understanding of the ALT mechanism. We first compared the transcriptomic profiles of ALT+ and TERT+ LMS and demonstrated a strong enrichment of the CINSARC signature in ALT+ tumors. The establishment of an ALT+-related signature in these LMS confirmed the close association between CINSARC and ALT in two additional cohorts of non-translocation-related sarcomas. In vitro mesenchymal models of spontaneous ALT induction showed increased CINSARC expression following acquisition of the ALT mechanism. Conversely, ALT inactivation, through BLM inhibition, led to decreased CINSARC expression. These results establish CINSARC as a new hallmark of the ALT mechanism in non-translocation-related sarcomas and demonstrate the association of a cellular biological process with the CINSARC prognostic signature, namely the ALT mechanism.

Abstract Background Gastrointestinal stromal tumors (GIST) are mainly caused by gain-of-function mutations in KIT or PDGFRA genes and constitute the most common malignant neoplasm of mesenchymal origin. Dysregulation of the Hippo pathway and its downstream effectors YAP1 and TAZ has been implicated in GIST progression, yet their individual contributions remain unclear. Given emerging evidence of non‑redundant YAP1/TAZ functions in cancer, we sought to dissect their respective roles in GIST tumorigenic properties. Methods We developed sequence‑specific siRNAs targeting YAP1 or TAZ, individually or in combination, and delivered them into GIST-T1, GIST‑430, and GIST‑882 cells using WRAP5 peptide‑based nanoparticles. Silencing efficiency and specificity were confirmed by Western blot, RT-qPCR, and immunofluorescence. Functional assays, including wound healing, Transwell migration, MTT metabolic activity, and cell counting, were used to evaluate proliferation and migration. Downstream transcriptional targets (CYR61, CTGF) and signaling proteins (KIT/AKT/ERK pathways) were quantified. Clinical relevance was assessed using progression‑free survival and metastasis data from the ATGsarc GIST cohort. Results WRAP5 nanoparticles enabled specific and efficient knockdown of YAP1, TAZ, or both, with no cytotoxicity. Across GIST cell lines, TAZ silencing consistently reduced migration and proliferation, whereas YAP1 knockdown had minimal or cell-line-dependent effects. TAZ depletion markedly downregulated CYR61 and CTGF expression, while YAP1 silencing showed limited impact. CYR61 knockdown impaired proliferation and migration in all models, identifying it as a key mediator of TAZ-driven oncogenicity. Clinical analysis confirmed that high TAZ and high CYR61 expression were associated with shorter disease-free survival and metastasis. Conclusion TAZ, more than YAP1, acts as a major regulator of GIST proliferation and migration through transcriptional control of CYR61. These findings highlight TAZ as a promising therapeutic target and demonstrate the utility of WRAP5-based nanoparticles for selective gene silencing in GIST.

This study was performed to assess whether the Complexity INdex in SARComas (CINSARC) signature is applicable in dogs with soft tissue sarcomas (STSs) and how it compares to conventional histological grading in this regard. Twenty-four dogs with well-established low- or high-grade STSs were retrospectively evaluated between 2018 and 2023 at two referral centers. Formalin-fixed paraffin-embedded (FFPE) blocks were tested for the CINSARC signature, with the histological grade used as the endpoint. The CINSARC analysis classified the samples into two groups: 14 dogs classified as C1 (favorable prognosis) and 10 dogs classified as C2 (poor prognosis). All dogs with Grade I tumors and one dog with Grade III disease were classified as C1, indicating an almost perfect agreement (κ = 0.92; 95% CI: 0.75-1.00). This proof-of-concept study confirmed that CINSARC analysis can be performed on canine FFPE tissue blocks, even with highly degraded mRNA. CINSARC effectively classified dogs carrying STSs, highlighting that this signature appears to be consistent with conventional histopathology. However, it remains to be determined whether this classification has prognostic value in a larger cohort.

PURPOSE:Genomic instability (GIN) plays a critical role in cancer progression and treatment responses. Soft tissue sarcomas (STSs) are characterized by extensive chromosomal rearrangements and transcription-associated stress, both of which contribute to poor clinical outcomes. Current standard grading systems including fédération nationale des centres de lutte contre le cancer (FNCLCC) have limited prognostic accuracy for STS, necessitating improved risk stratification. To address this gap, we developed the Mixed Transcription- and Replication-Associated GIN Classifier (MAGIC) and assessed its translatability from whole-genome sequencing to RNA sequencing (RNA-seq). METHODS:This study analyzed RNA-seq from 226 localized STS tumors to analyze the fusion transcript breakpoint distribution and assess GIN. We computed MAGIC indices transcription association chromosomal instability index (iTRAC) and Replication-Associated Chromosomal INstability index (iRACIN), which are based on GIN linked to transcription and replication processes, respectively. The iTRAC biomarker was evaluated using FNCLCC and Complexity INdex in SARComas (CINSARC) for metastatic risk stratification. Kaplan-Meier and iterative multi-thresholds partitioning analyses assessed prognostic relevance. RESULTS:iTRAC significantly stratified patients with distinct metastatic outcomes, outperforming the FNCLCC and CINSARC grading systems. STS patients with medium iTRAC levels showed the poorest metastasis-free survival. Patients classified into iTRAC-high and iTRAC-low groups achieved a better prognosis. Furthermore, iTRAC stratified patients' metastatic risk in treated and nontreated patients, indicating poorer prognosis with chemotherapy in patients with low iTRAC and better prognosis for those with medium iTRAC. By contrast, iRACIN was not measurable in the RNA-seq-based analysis. CONCLUSION:iTRAC demonstrates superior prognostic utility in STS over the current grading systems, effectively stratifying metastatic risk for patients who might benefit from alternative therapeutic strategies. iTRAC holds the potential for personalizing chemotherapeutic approaches, paving the way for a new precision oncology approach in STS.

This study was performed to assess whether the Complexity INdex in SARComas (CINSARC) signature is applicable in dogs with soft tissue sarcomas (STSs) and how it compares to conventional histological grading in this regard. Twenty-four dogs with well-established low- or high-grade STSs were retrospectively evaluated between 2018 and 2023 at two referral centers. Formalin-fixed paraffin-embedded (FFPE) blocks were tested for the CINSARC signature, with the histological grade used as the endpoint. The CINSARC analysis classified the samples into two groups: 14 dogs classified as C1 (favorable prognosis) and 10 dogs classified as C2 (poor prognosis). All dogs with Grade I tumors and one dog with Grade III disease were classified as C1. This proof-of-concept study confirmed that CINSARC analysis can be performed on canine FFPE tissue blocks, even with highly degraded mRNA. CINSARC effectively classified dogs carrying STS, highlighting that this signature appears to be consistent with conventional histopathology. However, it remains to be determined whether this classification has prognostic value in a larger cohort.

PURPOSE:The Complexity INdex in SARComas (CINSARC) predicts the metastatic risk in patients with soft-tissue sarcoma. The aims of this study were to provide the first independent validation of CINSARC in patients with retroperitoneal sarcoma (RPS) and evaluate whether CINSARC could enhance the performance of Sarculator. EXPERIMENTAL DESIGN:A retrospective cohort included patients with primary localized RPS resected with curative intent (2011-2015) at a single institution. The STRASS cohort comprised patients from the surgery-only arm of the EORTC-STBSG-62092 (STRASS) trial who had undergone CINSARC categorization. Patients were classified as CINSARC low-risk (C1) versus high-risk (C2). Primary study endpoints were overall survival (OS) and disease-free survival (DFS). Sarculator performance was assessed in terms of discrimination (the Harrell C-index) and calibration (calibration plots and the Brier score) before and after adding CINSARC. RESULTS:The study cohorts included 104 and 69 patients, respectively, with similar OS. In a pooled cohort, in multivariable analysis for OS considering Sarculator and CINSARC, only Sarculator was significantly associated with OS [HR, 1.93; 95% confidence interval (CI), 1.35-2.74; P < 0.001]. In multivariable analysis for DFS, both Sarculator (HR, 1.51; 95% CI, 1.09-2.09; P = 0.013) and CINSARC (HR, 2.01; 95% CI, 1.26-3.23; P = 0.004) were significantly associated with DFS. However, the addition of CINSARC did not improve Sarculator's discrimination or calibration for either OS or DFS. CONCLUSIONS:This study validates CINSARC as a prognostic predictor for OS and DFS in patients with primary RPS. CINSARC did not improve the performance of Sarculator, suggesting that its addition to the Sarculator may not provide added clinical benefit.

11534 Background: Soft Tissue Sarcomas (STS), known for their extensive Genomic instability (GIN), often result in poor clinical outcomes. Grading systems, such as FNCLCC, have limited prognostic accuracy in stratifying metastatic risk for STS patients. We introduce transcription-associated GIN indice (iTRAC) to improve prognostic precision and guide treatment decisions. Methods: This study analyzed 226 STS tumor samples using RNA sequencing (RNAseq) to assess breakpoint distribution from fusion transcripts as a surrogate for GIN. We calculated iTRAC to quantify transcription-associated GIN. Kaplan-Meier survival analysis were used to evaluate prognostic relevance in patients receiving or not chemotherapy. Multivariate analysis was performed to evaluate the iTRAC compared to FNCLCC and CINSARC for metastatic risk stratification. Results: STS patients with medium iTRAC level had the poorest metastasis-free survival (MFS) compared to low and high iTRAC levels. Importantly, patients with low iTRAC have a poorer outcome when treated with chemotherapy than those untreated, but at the contrary patients with medium iTRAC and treated by chemotherapy have a better outcome, raising the question of the potential predictive value of iTRAC for adjuvant chemotherapy in STS patients. FNCLCC and CINSARC groups did not show significant difference in MFS between treated and not treated patients. Conclusions: iTRAC is a novel biomarker for stratifying metastatic risk and guiding personalized treatment in STS. It outperforms molecular and histological prognosis systems like CINSARC and FNCLCC grade by revealing distinct MFS between patients receiving or not chemotherapy. This could enable better identification of patients who may benefit from chemotherapy and alternative options for those with poor responses. Prospective clinical trials are needed for validation and integration for patients care.

Supplementary Table 2: univariate and multivariable analyses for OS in specific subgroups

Dedifferentiated and Well-differentiated liposarcoma are characterized by a systematic amplification of the Murine Double Minute 2 (MDM2) oncogene. We demonstrate that p53-independent metabolic functions of chromatin-bound MDM2 are exacerbated in liposarcoma and mediate an addiction to serine metabolism to sustain tumor growth. However, the origin of exogenous serine remains unclear. Here, we show that elevated serine levels in mice harboring liposarcoma-patient derived xenograft, released by distant muscle is essential for liposarcoma cell survival. Repressing interleukine-6 expression, or treating liposarcoma cells with Food and Drugs Administration (FDA) approved anti-interleukine-6 monoclonal antibody, decreases de novo serine synthesis in muscle, impairs proliferation, and increases cell death in vitro and in vivo. This work reveals a metabolic crosstalk between muscle and liposarcoma tumor and identifies anti-interleukine-6 as a plausible treatment for liposarcoma patients.

Adult-type gynecological soft tissue and visceral sarcomas are rare tumors, with an estimated incidence of 13% of all sarcomas and 4% of all gynecological malignancies. They most often develop in the uterus (83%), followed by the ovaries (8%), vulva and vagina (5%), and other gynecological organs (2%). The objective of this review is to provide an overview of the current management of gynecological sarcomas, according to international guidelines. The management of gynecological sarcomas should follow the recommendations for the management of soft tissue and visceral sarcomas. Centralizing cases in expert centers improves patient survival, both for the diagnostic phase and for multidisciplinary therapeutic management. In the case of pelvic soft tissue sarcomas, a radiological biopsy is essential before any surgical decision is taken. In the case of a myometrial tumour which may correspond to a sarcoma, if conservative surgery such as myomectomy or morcellation is planned, an ultrasound-guided biopsy with pathological analysis including comparative genomic hybridization analysis must be carried out. In all cases, en bloc surgery, without rupture, is mandatory. Many rare histological subtypes require specific surgical management.

A close relationship has been demonstrated between genomic complexity and clinical outcome in uterine smooth muscle tumors. We studied the genomic profiles by array-CGH of 28 fumarate hydratase deficient leiomyomas and 37 leiomyomas with bizarre nuclei (LMBN) from 64 patients. Follow-up was available for 46 patients (from three to 249 months, mean 87.3 months). All patients were alive without evidence of disease. For 51 array-CGH interpretable tumors the mean Genomic Index (GI) was 16.4 (median: 9.8; from 1 to 57.8), significantly lower than the mean GI in LMS (mean GI 51.8, p < 0.001). We described three groups: (1) a group with FH deletion (24/58) with low GI (mean GI: 11 vs. 22,4, p = 0.02), (2) a group with TP53 deletion (17/58) with higher GI (22.4 vs. 11 p = 0.02), and (3) a group without genomic events on FH or TP53 genes (17/58) (mean GI:18.3; from 1 to 57.8). Because none of these tumors recurred and none showed morphological features of LMS we concluded that GI at the cut-off of 10 was not applicable in these subtypes of LM. By integration of all those findings, a GI <10 in LMBN remains a valuable argument for benignity. Conversely, in LMBN a GI >10 or alteration in tumor suppressor genes, should not alone warrant a diagnosis of malignancy. Nine tumors were tested with Nanocind CINSARC® signature and all were classified in low risk of recurrence. We propose, based on our observations, a diagnostic approach of these challenging lesions.

Supplementary Table 1: RT-PCR primers used for fusion transcripts detection. Supplementary Table 2: Primers used for fusion transcript quantification. Supplementary Table 3: shRNA sequences.Supplementary Table 4: Sarcomas cohort analysed in aCGH (n=24).Supplementary Table 5: 69 differentially overexpressed genes in six fused TRIO samples versus 111 non-fused TRIO samples. Supplementary Table 6: 354 differentially underexpressed genes in six fused TRIO samples versus 111 non-fused TRIO samples.

Abstract Purpose: Despite various differences, nontranslocation-related sarcomas (e.g., comprising undifferentiated pleomorphic sarcoma, leiomyosarcoma, myxofibrosarcoma) are unified by their complex genetics. Extensive analysis of the tumor genome using molecular cytogenetic approaches showed many chromosomal gains, losses, and translocations per cell. Genomic quantitative alterations and expression variations have been extensively studied by adapted high-throughput approaches, yet translocations still remained unscreened. We therefore analyzed 117 nontranslocation-related sarcomas by RNA sequencing to identify fusion genes. Experimental design: We performed RNA sequencing and applied a bioinformatics pipeline dedicated to the detection of fusion transcripts. RT-PCR and Sanger sequencing were then applied to validate predictions and to search for recurrence and specificity. Results: Among the 6,772 predicted fusion genes, 420 were in-frame. One recurrent rearrangement, consistently involving TRIO with various partners, was identified in 5.1% of cases. TRIO translocations are either intrachromosomal with TERT or interchromosomal with LINC01504 or ZNF558. Our results suggest that all translocations led to a truncated TRIO protein either directly or indirectly by alternative splicing. TRIO rearrangement is associated with a modified transcriptomic program to immunity/inflammation, proliferation and migration, and an increase in proliferation. Conclusions: TRIO fusions have been identified in four different sarcoma histotypes, likely meaning that they are not related to a primary oncogenic event but rather to a secondary one implicated in tumor progression. Moreover, they appear to be specific to nontranslocation-related sarcomas, as no such rearrangement was identified in sarcomas with simple genetics. More cases could lead to a significant association of these fusions to a specific clinical behavior. Clin Cancer Res; 23(3); 857–67. ©2016 AACR.

Supplementary Figure 1: Identification of FARSA-SYCE2 and GSE1-RP11-680G10.1 read-throughs in sarcoma CL1 and normal tissue. Supplementary Figure 2: aCGH (SNP-array) profile of CL1 which present a genomic rearrangement of TRIO and TERT genes. Supplementary Figure 3: Expression percentage of the two TRIO-TERT isoforms in three cases. Supplementary Figure 4: Predicted acceptor splicing regions in intron 2 and 3 of LINC01504. Supplementary Figure 5: Transcriptomic profile clustering of the 117 non-translocation-related sarcomas. Supplementary Figure 6: Volcano plot of the 117 sarcomas with six fused TRIO cases versus 111 non-fused TRIO cases. Supplementary Figure 7: Exonic expression of fused TRIO fused and non-fused TRIO cases.Supplementary Figure 8: Enrichment plot for significant Hallmarks in six fused TRIO samples included in the MSig5.0 database according to GSEA. Supplementary Figure 9: Enrichment plot for significant Hallmarks in 111 non-fused TRIO samples included in the MSig5.0 database according to GSEA. Supplementary Figure 10: ALT mechanism status determined by PML/TERF2 immunofluorescence analysis.

<p>Supplementary figures 1-3 and tables 1-5. Supplementary figure 1. Survival analysis of the 77 cases using the FNCLCC grading system (grade 1 and 2 versus grade 3). Supplementary figure 2. WGD refers to whole genome doubling; CNA refers to copy-number alteration; ITH refers to intra-tumour heterogeneity. Supplementary figure 3. WGD refers to whole genome doubling; CNA refers to copy-number alteration; Meth refers to methylation; ITH refers to intra-tumour heterogeneity. Supplementary table 1. Clinical information for the 77 cases. Supplementary table 2. Genomic coordinates of the 13 differentially (adjusted P < 0.05 and absolute M-value difference > 1) methylated probes with distances from nearest CpG islands and transcription start site. Supplementary table 3. Enrichment for CINSARC genes in top anti-correlated genes to each miRNA of interest. Supplementary table 4. Top 20 Gene Ontologies (GO) enriched in genes having validated interactions with miRNA. Supplementary table 5. Cancer Gene Census information for genes significantly anti-correlated with miRNAs of interest.</p>

In leiomyosarcoma (LMS), a very aggressive disease, a relatively transcriptionally uniform subgroup of well-differentiated tumors has been described and is associated with poor survival. The question raised how differentiation and tumor progression, two apparently antagonist processes, coexist and allow tumor malignancy. We first identified the most transcriptionally homogeneous LMS subgroup in three independent cohorts, which we named 'hLMS'. The integration of multi-omics data and functional analysis suggests that hLMS originate from vascular smooth muscle cells and show that hLMS transcriptional program reflects both modulations of smooth muscle contraction activity controlled by MYOCD/SRF regulatory network and activation of the cell cycle activity controlled by E2F/RB1 pathway. We propose that the phenotypic plasticity of vascular smooth muscle cells coupled with MYOCD/SRF pathway amplification, essential for hLMS survival, concomitant with PTEN absence and RB1 alteration, could explain how hLMS balance this uncommon interplay between differentiation and aggressiveness.

BACKGROUND:The management of soft-tissue sarcoma (STS) relies on a multidisciplinary approach involving specialized oncological surgery combined with other adjuvant therapies to achieve optimal local disease control. Purpose and Results: Genomic and transcriptomic pseudocapsules of 20 prospective sarcomas were analyzed and revealed to be correlated with a higher risk of recurrence after surgery.CONCLUSIONS:A peritumoral environment that has been remodeled and infiltrated by M2 macrophages, and is less expressive of healthy tissue, would pose a significant risk of relapse and require more aggressive treatment strategies.

The exonuclease domain of DNA polymerases epsilon's catalytic subunit (POLE) removes misincorporated nucleotides, called proofreading. POLE-exonuclease mutations cause colorectal- and endometrial cancers with an extreme burden of single nucleotide substitutions. We recently reported that particularly the hereditary POLE exonuclease mutation N363K predisposes in addition to aggressive giant cell glioblastomas. We knocked-in this mutation homozygously into human cell lines and compared its properties to knock-ins of the likewise hereditary POLE L424V mutation and to a complete proofreading-inactivating mutation (exo-null). We found that N363K cells have higher mutation rates as both L424V- or exo-null mutant cells. In contrast to L424V cells, N363K cells expose a growth defect, replication stress and DNA damage. In non-transformed cells, these burdens lead to aneuploidy but macroscopically normal nuclei. In contrast, transformed N363K cells phenocopy the enlarged and disorganized nuclei of giant cell glioblastomas. Taken together, our data characterize a POLE exonuclease domain mutant that not only causes single nucleotide hypermutation, but in addition DNA damage and chromosome instability, leading to an extended tumor spectrum. Our results expand the understanding of the polymerase exonuclease domain and suggest that an assessment of both the mutational potential and the genetic instability might refine classification and treatment of POLE-mutated tumors.