Perivascular epithelioid cell neoplasms (PEComas) are ultra-rare mesenchymal tumors lacking a molecular classification to guide therapy. Here we perform comprehensive multi-omic profiling of an unselected PEComa cohort. We identify frequent MITF rearrangements involving actin gene partners (ACTA2, ACTG1 and ACTB). Anatomical stratification reveals cyclin-dependent kinase module mutations in gynecologic tumors, whereas soft tissue, gastrointestinal, and pelvic tumors lacked mTOR pathway alterations but are enriched for TFE3/MITF rearrangements. Transcriptomic analysis defines four subtypes with distinct lineage programs-melanocytic, mesenchymal, or adipogenic-as well as unique mutational patterns and clinical behaviors. Notably, an aggressive stem-like subtype enriched for TP53/RB1 mutations exhibits high proliferation, activation of embryonic and Hedgehog signaling, immune infiltration, and resistance to mTOR inhibitors, but potential responsiveness to immunotherapy. Single-nucleus RNA sequencing reveals intra-tumoral heterogeneity within this subtype, including divergent inflammatory states. Together, these findings establish a molecular classification framework and identify actionable vulnerabilities in PEComa.

AIMS:Myxomas are benign soft tissue neoplasms typically driven by activating GNAS mutations. However, a subset lacks this alteration and remains molecularly undefined. Recent reports have implicated MAP3K family gene fusions in rare myxoid neoplasms sharing features with myxoma. METHODS AND RESULTS:In this study, we characterise 39 MAP3K-rearranged myxoid tumours by integrating clinical, histopathological, immunohistochemical and molecular data. The cohort comprised predominantly middle-aged adults, with tumours distributed across a wide anatomic range (limbs, trunk, face), often in superficial locations. Histologically, the tumours were composed of bland spindle or histiocytoid cells embedded in an abundant myxoid stroma with features mimicking cellular myxoma, such as intermediate-to-high cellularity and broad fibrous septa. High-grade pleomorphism and necrosis were consistently absent. Immunohistochemistry revealed frequent CD34 and HMGA2 positivity, a low Ki-67 proliferation index, and preserved PRKAR1A expression. RNA sequencing identified diverse MAP3K gene fusions, most notably MSI2::MAP3K3 and MAP3K8::MAP3K3, alongside several unique fusion partners. Transcriptomic clustering demonstrated a relationship between MAP3K-rearranged tumours, GNAS-mutant myxomas and a proximity with myxofibrosarcomas, while clearly distinguishing them from other myxoid sarcomas. Clinical follow-up indicated an indolent course with no evidence of metastasis and rare local recurrences; notably, one patient with positive surgical margins remained disease-free several years postoperatively. CONCLUSION:MAP3K-rearranged myxoid tumours represent a molecularly distinct subset of benign neoplasms that overlap histologically with cellular myxoma and must be distinguished from low-grade myxofibrosarcoma.

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.

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.

(1) Background: Genomic Instability (GIN) plays a critical role in cancer progression and treatment response. Soft tissue sarcomas (STS), are characterized by high levels of chromosomal rearrangements and transcription-associated stress, both of which contribute to poor clinical outcomes. Current standard grading systems, such as FNCLCC, are limited in prognostic accuracy for STS, necessitating novel approaches for risk stratification and treatment guidance. To address this gap, we developed a holistic classifier, the MAGIC (Mixed transcription- and replication-associated GIN classifier), combining transcription- and replication-related GIN indices (iTRAC and iRACIN) to predict metastatic risk. (2) Patients and Methods: This study utilized RNA sequencing (RNAseq) on 226 STS tumor samples to analyze fusions transcripts break points (BP) distribution and assess GIN. We computed MAGIC indices iTRAC and iRACIN, which are based on chromosomal instability linked to transcription and replication processes, respectively. iTRAC biomarker was evaluated against FNCLCC and CINSARC for metastatic risk stratification. Kaplan-Meier and iPART analyses were used to determine the prognostic relevance of iTRAC levels. (3) Results: iTRAC significantly stratified patients with distinct metastatic outcomes, outperforming FNCLCC and CINSARC grading systems. STS patients with medium level of iTRAC showed the poorest metastasis-free survival. Patients classified as iTRAC high- and low-risk groups, achieved better prognosis. Furthermore, iTRAC stratified patients' metastatic risk in treated and not treated patients, indicating poorer prognosis with chemotherapy in patients with low iTRAC and better for those with with medium iTRAC. (4) Conclusion(s): iTRAC demonstrates a superior prognostic utility in STS over current grading systems, effectively stratifying metastatic risk for patients who might benefit from alternative therapeutic strategies. iTRAC holds potential for personalizing chemotherapeutic approaches, paving the way for a new precision oncology approach in STS. ### Competing Interest Statement Ataaillah Benhaddou is a founder of Magic Genomix, which co-owns a patent application (PCT/EP2021/085491) related to the MAGIC method. Frederic Chibon is a consultant for Magic Genomix ### Funding Statement This work was supported by Region Occitanie, INCa (Institut National du Cancer), Inserm, Inserm Transfert, IUCT/ICR (Institut universitaire de cancerologie de Toulouse/Institut Claudius Regaud). We thank these institutions for their financial and strategic support of this research. ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The samples used in this study are part of the Biological Resources Center of Bergonie Cancer Institute (CRB-IB). Accordance with the French Public Health Code (articles L. 1243-4 and R. 1243-61), the CRB-IB has received the agreement from the French authorities to delivered samples for scientific research (number AC-2008-812). The samples come from care and are re-qualified for research as part of the ICGC program (International Cancer Genome Consortium), with patient consent. The project was approved by the Bergonie Institute ethic committee (scientific advisory board). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All data produced in the present work are contained in the manuscript

The EWSR1::CREM rearranged intra-abdominal malignant epithelioid neoplasm is an emerging tumor, with only a few publications describing it to date. Here, we report two new cases of this highly aggressive tumor, primarily involving the peritoneal surface. The tumors presented as a widespread diffuse peritoneal lesion associated with a 4-cm pelvic mass in a 28-year-old woman (Case 1) and as a 10-cm intra-abdominal mass infiltrating the stomach with multiple hepatic metastases in a 53-year-old woman (Case 2). The tumors shared predominant epithelioid morphology with minimal nuclear polymorphism. One of them additionally harbored spindle and rhabdoid cell populations. Both tumors displayed immunoreactivity for pan-cytokeratins, EMA, and CD99, and variable positivity for MUC4, progesterone and estrogen receptors, pan-NTRK, and synaptophysin. This misleading histology and immunophenotype give rise to a wide spectrum of differential diagnoses and highlight the crucial role of RNA sequencing in diagnostic accuracy and thus in appropriate therapeutic approaches.

La stadification des sarcomes des tissus mous est une pierre angulaire de la décision thérapeutique. Alors que la TEP-TDM au 18F-FDG est une modalité clé dans le staging de nombreux cancers (incluant notamment sarcomes d'Ewing et ostéosarcomes), sa place dans la stadification initiale ou la récidive des sarcomes des tissus mous n'est pas clairement définie. Nous avons réalisé une évaluation rétrospective de l'impact clinique de la TEP-TDM au 18F-FDG dans l'évaluation des sarcomes des tissus mous, en situation initiale ou de récidive, chez 122 patients à l'Institut universitaire du cancer de Toulouse, un centre accrédité NetSarc+. Les paramètres semi-quantitatifs de captation du FDG (SUVs) et les volumes tumoraux métaboliques ont été mesurés. Une analyse descriptive des modifications d'intention de traitement consécutives à la TEP-TDM a été réalisée. Un test de Wilcoxon-Mann Whitney a été réalisé afin d'étudier l'association entre les paramètres TEP et les types et grades histologiques. Enfin, des analyses de survie ont été réalisées à l'aide de la méthode Kaplan-Meier et du test Log-Rank. La TEP-TDM au 18F-FDG a conduit à un upstaging (mise en évidence du statut M1) chez 20 % des patients, influençant considérablement l'évaluation pronostique et la proposition thérapeutique formulée par la RCP régionale. Dans les autres cas, la TEP-TDM a soit confirmé le statut localisé de la maladie, soit mieux cartographié la maladie métastatique. Par ailleurs, le SUVmax tumoral était corrélé aux sous-types et grades histologiques et prédictif de la survie globale. Ce travail démontre l'apport de la TEP-TDM au 18F-FDG dans le staging des sarcomes des tissus mous et son impact sur la stratégie thérapeutique. Cette modalité permet, par ailleurs, une évaluation non invasive de l'agressivité des sarcomes des tissus mous et participe à l'évaluation du pronostic des patients.

Abstract Background: EVT801 is a highly selective, orally available VEGFR3 inhibitor that modulates tumor angiogenesis without inducing hypoxia, one of the main causes of cancer-associated immunosuppression. EVT801 has shown compelling efficacy as single agent in multiple in vivo models. In addition, combination of EVT801 and Immune Checkpoint Therapy (ICT) agents shows additive effects and thus holds promises for combination treatment without induction of hypoxia-induced immunosuppression. Clinical trial design: A phase I clinical study (NCT05114668) is underway to evaluate safety, tolerability, pharmacokinetics of EVT801 and explore patient characterization which is key for further development. The phase I trial consists in two sequential stages. During the first stage, currently ongoing, EVT801 is orally administered to patients with advanced solid tumors in a multiple ascending dose study using an accelerated 3+3 design (1 patient per dose until grade 2 toxicities are observed) in up to 48 patients in 8 dose levels. The primary objective is to determine the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D). Stage 2 will focus on validation of this RP2D in two 6-patient cohorts diagnosed with 2 indications. In addition to conventional measures of safety, tolerability, efficacy, and pharmacokinetics, the phase I study employs a rich suite of biomarkers analysis using histology, transcriptomics and immunomonitoring to provide early insights into the pharmacological activity of EVT801. At end of September 2023, 20 patients have been treated in five different cohorts for EVT801 doses ranging from 50mg QD to 400mg BID. At this date, only 1 dose limiting toxicity was declared at the higher dose. Conclusion: Collection of safety data on these first enrolled patients showed no safety alert which allows further dose escalation and reinforces the good safety profile of EVT801 observed during the preclinical toxicology studies. Citation Format: Carlos Gomez-Roca, Philippe Cassier, Michael Fitzgerald, Lise Davenne, Cristina Costantin, Philippe Rochaix, Jean-Pierre Delord, John Friend, Andrea Nizzardo, Alessia Tagliavini, Marco Pergher, Pierre Fons, Marie Mandron. EVT801, a novel selective VEGFR-3 inhibitor targeting tumor angiogenesis, is pursuing dose escalation stage of phase I first-in-human study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT088.

Abstract Introduction: EVT801 is a highly selective small molecule inhibitor of VEGFR3 that acts by inducing tumor (lymph)-angiogenesis normalization in and around the tumor. EVT801 has shown compelling activity in a wide range of cancer models showing a decrease of tumor hypoxia and an increase of CD8pos T-cells infiltration into the tumor micro-environment. EVT801 was well-tolerated in preclinical toxicology studies. A phase I study is underway focusing primarily on understanding the safety, tolerability, and pharmacokinetics of EVT801 across a range of doses (NCT05114668). Methods: The first stage of the phase I trial aims to determine the maximum tolerated dose (MTD) and recommended Phase II dose (RP2D) for EVT801. Clinical samples from enrolled patients have been collected to explore preliminary signals of the biological activity of the drug and characterize tumor phenotypes. Patient characterization includes histology and mRNA signature in archival biopsies. Target engagement and pharmacodynamics effects are investigated by immunomonitoring as well as assessment of a defined set of proteins as markers of inflammation and angiogenesis as identified in preclinical in vivo models. Conclusion: Our first biomarkers analyses on a subpopulation of patients with High Grade Serous Ovarian Cancer (HGS-OC) enrolled in the first stage of the study showed that levels of VEGFR3 expression had a negative correlation with CD8 positive T-cells infiltration in the tumor microenvironment and a tendency to positive correlation with a PD1 mAb resistance signature. VEGFR3 expression also tends to be correlated with higher levels of hypoxia (CAIX labelling). Our working hypothesis is that patients with hypoxic HGS-OC tumors with high VEGFR3 expression may benefit from EVT801 treatment; this will need to be reinforced by inclusion of additional patients in dedicated PD biomarkers cohorts during stage 2 of the clinical trial. Citation Format: Lise Davenne, Michael Fitzgerald, Pierre-Benoit Ancey, Oona Delpuech, Céline Poussereau-Pomié, Michael Esquerre, Michael R. Paillasse, Marie Mandron, Philippe Rochaix, Maha Ayyoub, Clara Maria Scarlata, Christine Caux, Christophe Caux, Philippe Cassier, Carlos Gomez-Roca, Jean-Pierre Delord, John Friend, Pierre Fons. Biomarkers analysis on samples from patients in EVT801 clinical trial: Patient characterization and immunomonitoring [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1059.

Supplementary Figure S1. TNF does not modulate in vitro B16 proliferation.

EVT801 is a highly selective small molecule inhibitor of VEGFR-3 and acts by inhibiting tumor lymphangiogenesis and angiogenesis. A phase I clinical study (NCT05114668) has started to characterize safety, tolerability, pharmacokinetics of EVT801. Clinical samples from these patients will be used to explore preliminary signals of clinical efficacy and investigate the biological activity of the drug using several biomarkers. Among them, we have investigated VEGFR3 expression profiling by histology. We validated a highly specific protocol for VEGFR-3 immunohistochemistry labelling and a VEGFR-3 mRNA signature, readily transferable to clinical centers. Initially, VEGFR-3 expression was assessed in 29 primary kidney cancer samples and 23 metastatic kidney cancer samples with CD34 to stain vessels and D2-40 to track lymphatics. We detected VEGFR-3 expression in the endothelial cells within the kidney tumor but not in the tumor cells. Remarkably, we observed a very distinct delineation between the kidney tumor vasculature, which showed high VEGFR-3 expression, and the normal adjacent tissue, which was devoid of VEGFR-3 expression. In addition, we found that VEGFR-3 expression in metastases from kidney tumors matched the expression pattern of the primary tumor. Then, We have demonstrated that it exists a strong correlation between VEGFR-3 expression by histology and VEGFR-3 mRNA signature. We applied both VEGFR-3 signatures in different indications of interest like soft tissue sarcomas and lung cancers. We were able to quantify VEGFR-3 expression by histology and mRNA signature in the kidney and soft tissue sarcoma cohorts plus several others, such as hepatocarcinoma and non-small cell lung cancer. This enables us to select indications that might benefit from EVT801 as a monotherapy (e.g., clear cell renal cell carcinoma and soft tissue sarcomas) or in combination with standard of care. Accordingly, VEGFR-3 expression is retrospectively quantified during the EVT801 phase 1 clinical trial and may be used to stratify patients in the future.

BACKGROUND:Using adaptive radiotherapy (ART), to determine objective clinical criteria that identify extremity soft tissue sarcoma (ESTS) patients requiring adaptation of their preoperative radiotherapy (RT) plan. PATIENTS AND METHODS:We included 17 patients with a lower extremity ESTS treated between 2019 and 2021 with preoperative RT, using helicoidal intensity-modulated RT (IMRT) tomotherapy, before surgical resection. We collected clinical, tumor parameters and treatment data. Repositioning was ascertained by daily Megavoltage computed tomography (MVCT) imaging. Using the PreciseART technology we retrospectively manually delineated at least one MVCT for each patient per week and recorded volume and dosimetric parameters. A greater than 5% change between target volume and planned target volume (PTV) dosimetric coverage from the initial planning CT scan to at least one MVCT was defined as clinically significant. RESULTS:All 17 patients experienced significant tumor volume changes during treatment; 7 tumors grew (41%) and 10 shrank (59%). Three patients (18%), all undifferentiated pleomorphic sarcomas (UPS) with increased volume changes, experienced significant reductions in tumor dose coverage. Seven patients required a plan adaptation, as determined by practical criteria applied in our departmental practice. Among these patients, only one ultimately experienced a significant change in PTV coverage. Three patients had a PTV decrease of coverage. Among them, 2 did not receive plan adaptation according our criteria. None of the patients with decreased tumor volumes had reduced target volume coverage. Monitoring volume variations by estimating gross tumor volume (GTV) on MVCT, in addition to axial and sagittal linear tumor dimensions, appeared to be most effective for detecting reductions in PTV coverage throughout treatment. CONCLUSIONS:Variations in ESTS volume are evident during preoperative RT, but significant dosimetric variations are rare. Specific attention should be paid to grade 2-3 UPSs during the first 2 weeks of treatment. In the absence of dedicated software in routine clinical practice, monitoring of tumor volume changes by estimating GTV may represent a useful strategy for identifying patients whose treatment needs to be replanned.

Supplementary Figure S3. Analysis of the immune response in spleen from WT and TNF-deficient mice.

PDF file - 364K, B16K1, LL2 and 4T1 characterization

Supplementary Figure S3 shows the expression of VEGFR3 altogether with vascular and lymphatic markers in primary kidney tumors

Supplementary Figure S7. Effect of Etanercept injection in mice.

Supplementary Figure S5. Analysis of Treg and MDSC content in the tumors from WT and TNF-deficient mice.

Supplementary Table S3 shows pharmacokinetic parameters of EVT801 in mice and rats

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.