Imatinib is the first-line treatment for advanced gastrointestinal stromal tumors (GISTs) harboring KIT or PDGFRA mutations. Unfortunately, resistance invariably develops, typically through secondary KIT/PDGFRA mutations. Here, we describe an unprecedented case of acquired imatinib resistance associated with an oncogenic driver switch, from a KIT mutation to an NTRK3 fusion. The index case was a KIT exon 11-mutated gastric GIST that progressed on imatinib. Despite retaining the original KIT mutation and DOG1 expression, the relapsed tumor lost KIT expression and exhibited a dedifferentiated phenotype. Transcriptomic profiling revealed a de novo EML4::NTRK3 gene fusion. In vitro modeling demonstrated that EML4::NTRK3 confers imatinib resistance, while sensitizing GIST cells to NTRK inhibitors. This first reported instance of an NTRK fusion as a secondary event in GIST progression underscores the importance of testing for NTRK alterations in tumors that have developed resistance to tyrosine kinase inhibitors to ensure patients are offered all available therapeutic options.

Background. Epithelioid sarcoma (EpS) is an ultra-rare, aggressive soft tissue sarcoma (STS) driven by loss of the tumor suppressor INI1, encoded by SMARCB1 , which triggers hyperactivation of the chromatin-modifying enzyme EZH2. However, objective responses to the EZH2 inhibitor (EZH2i) tazemetostat (TZM) remain limited to approximately 15% of patients. Combination strategies incorporating EZH2i with doxorubicin (DOX), the first-line standard for advanced STSs, may enhance the activity of either agent alone, warranting investigation of determinants of response and resistance. Methods. Two INI1-deficient in-house generated proximal-type EpS (P-EpS) patient-derived xenograft (PDX) models, EpS-1 and EpS-2, and their matched cell lines were established. EpS-1 and EpS-2 models harbored a homozygous SMARCB1 deletion and underwent comprehensive genomic, epigenomic, transcriptomic, protein-level, and histological characterization. Both models were used to evaluate TZM and DOX, alone and in combination, and to investigate determinants of response and resistance. Results. TZM + DOX greatly inhibited tumor growth in EpS-1 in vivo, though regrowth occurred approximately three weeks after treatment cessation. EpS-2 displayed intrinsic resistance to all treatments. RNA sequencing of post-treatment PDXs revealed divergent transcriptional responses: in EpS-1 but not in EpS-2 TZM + DOX downregulated gene sets linked to chromatin organization and histone deacetylases (HDACs). Consistently, H3K27 acetylation (H3K27ac) accumulated exclusively in EpS-1, suggesting HDAC activity limits EZH2i-induced chromatin remodeling in resistant EpS. In EpS-2, co-treatment with TZM, DOX, and the pan-HDAC inhibitor vorinostat (SAHA) restored H3K27ac, induced apoptosis, and showed tendency toward synergism. Conclusions. This study identifies distinct resistance profiles to EZH2i-based therapy in P-EpS PDXs. HDAC activity limits chromatin remodeling induced by EZH2i (± DOX), and its inhibition with SAHA restores sensitivity, supporting HDAC inhibition as a rational strategy to overcome EZH2i resistance.

11546 Background: The prognosis of patients (pts) with retroperitoneal dedifferentiated liposarcoma (DDLPS) is mainly driven by tumor size, grade, patient age, and completeness of resection. Furthermore, the expression of myogenic markers has been associated with poorer outcomes. Prognostic nomograms, such as Sarculator, and transcriptomic signatures—most notably the Complexity INdex in SARComas (CINSARC)—have improved risk stratification. However, given the rarity of DDLPS and the limited evidence guiding treatment, identifying robust prognostic biomarkers remains a major challenge. This study evaluated the prognostic impact of clinical, morphological, and molecular features and assessed the performance of Sarculator and CINSARC in retroperitoneal DDLPS. Methods: A retrospective observational study was conducted on pts with retroperitoneal DDLPS treated at the Veneto Institute of Oncology (IOV)-IRCCS between 2004 and 2024. Tumor samples with adequate material underwent morphological, immunohistochemical, and molecular analyses, including CINSARC assessment. Disease-free survival (DFS) and overall survival (OS) were evaluated in surgically treated pts (Cohort 1), while progression-free survival (PFS) and OS were assessed in pts receiving first-line chemotherapy (Cohort 2). Sarculator was applied to Cohort 1, and its discriminative ability was evaluated using ROC curves. Concordance between CINSARC and Sarculator was also examined. Results: A total of 119 pts were included. In the overall population, tumor grade (3 vs 2: HR, 2.23; 95% CI 1.06 - 4.70; p = 0.03) and age (<65 vs ≥65: HR, 0.43; 95% CI 0.22 - 0.85; p = 0.02) were the most significant predictors of OS. In Cohort 1, these variables remained determinants of survival. In Cohort 2, sex, tumor size and differentiation significantly affected survival, with rhabdomyoblastic differentiation conferring the worst prognosis (p = 0.04). When Sarculator was applied to Cohort 1, a moderate discriminative ability was observed for OS (AUC = 0.75), with a lower performance for DFS (AUC = 0.64). Molecular analyses were performed on 32 samples; MYOG transcripts, indicative of rhabdomyoblastic differentiation, were detected in 8 cases. Based on CINSARC, 11 pts (34.4%) were low risk and 21 (65.6%) high risk. Agreement between Sarculator and CINSARC was moderate for both OS (Cohen’s Kappa, κ = 0.40) and DFS (κ = 0.55). Conclusions: To our knowledge, our study is among the largest conducted in retroperitoneal DDLPS specifically evaluating clinico-pathological features and the performance of Sarculator and CINSARC. Our findings support the negative prognostic impact of rhabdomyoblastic differentiation. Further investigation of differentiation patterns, combined with prognostic nomograms and transcriptomic signatures, may improve the identification of high-risk pts and support more personalized therapeutic strategies.

Tumors that retain wild-type TP53 and rely on MDM2 overexpression to dampen the p53 response were considered prime candidates for therapies based on MDM2 inhibitors (MDM2i). However, clinical trials to date have been disappointing, with limited improvements in progression-free survival, pointing to the existence of intrinsic resistance mechanisms. Building on our previous work demonstrating that TWIST1 attenuates the p53 response in sarcomas, we hypothesized that TWIST1 plays a role in modulating MDM2i efficacy. RNA-sequencing data were integrated with cytotoxicity measurements across a large panel of TP53 wild-type sarcoma cell models. Perturbation experiments were performed using shRNA or CRISPR/Cas9-mediated inhibition, as well as ectopic TWIST1 expression. Cell viability, pathway activation, and transcriptional profile following MDM2i treatment were evaluated in TWIST1-proficient and -deficient models. Co-precipitations, TurboID, molecular docking experiments, and in vitro and in vivo functional assays were employed to characterize the interplay between TWIST1, p53, and MDM2. TWIST1 expression correlated with reduced sensitivity to multiple MDM2i. TWIST1 inhibition enhanced p53 pathway activation and cell death in response to MDM2i, while TWIST1 overexpression conferred resistance. Mechanistically, we found that TWIST1 directly binds both p53 and MDM2, forming a trimeric complex that facilitates p53:MDM2 interaction, thereby promoting p53 degradation and limiting MDM2i efficacy. Notably, harmine, a compound that promotes TWIST1 degradation, phenocopied the effects of TWIST1 genetic inhibition in augmenting MDM2i sensitivity. TWIST1 confers resistance to MDM2i in TP53 wild-type sarcomas. Targeting TWIST1 restores p53 pathway activation and sensitizes sarcoma cells to MDM2 blockade, establishing TWIST1 as a promising predictive biomarker and therapeutic vulnerability for improving MDM2i efficacy.

Small bowel gastrointestinal stromal tumors (GISTs) are more aggressive than gastric GISTs, yet the biologic basis for this difference remains poorly understood. We hypothesized that differential expression of immune checkpoints contributes to this site-specific behavior. Bulk RNA sequencing of 42 primary GISTs (36 gastric, 6 small bowel) revealed marked upregulation of VTCN1, which encodes the inhibitory checkpoint B7-H4, in small bowel tumors (log2FC = 7.95, adjusted P < 0.001). In contrast, expression of the therapeutically targeted checkpoints PD-L1, PD-1, and CTLA-4 was comparable between sites. Concordantly, B7-H4 enrichment was accompanied by an immunosuppressive tumor microenvironment, characterized by reduced antigen-presenting cells, fewer effector-memory CD8+ T cells, lower granzyme B expression, and suppression of interferon and inflammatory signaling pathways. Notably, the differences in B7-H4 expression were independent of imatinib-treatment status. These findings were corroborated in an external cohort of 77 untreated GISTs, in which VTCN1 was similarly enriched in small bowel tumors. Independent immunohistochemical analysis of a tissue microarray comprising 68 untreated primary GISTs confirmed the pattern, showing median B7-H4 positivity of 78.6% in duodenal, 20.5% in jejunal/ileal, and 0% in gastric tumors, with staining localized to tumor cells rather than stroma. Collectively, these data identify B7-H4 as a site-specific feature of small bowel GISTs and a potential therapeutic target for tumors that have not responded to conventional checkpoint blockade.

Cancer resistance is one of the major challenges in oncology, often resulting in disease relapse and poor patient outcomes. Within the RNA family, microRNAs (miRNAs) regulate core biological processes and have been recognized also as critical contributors of tumor resistance and therapy failure. Being pivotal, they are increasingly exploited as biomarkers in various settings. Although in silico analyses facilitate miRNAs identification, PCR-based approaches remain essential to validate their expression. Currently, a plethora of well-established, single-target methods exist but multiplex detection from the same input have been only rarely explored. We present miRquad, the first-in-class digital PCR (dPCR) TaqMan™ multiplex clinical research assay for miRNA detection in head and neck (HNC) cancers. Based on a patented prognostic signature including miR-21-5p, miR-96-5p, miR-21-3p and miR-429, the assay would enable simultaneous miRNA analysis via qPCR and dPCR on multiple clinically relevant sample types. We designed and optimized miRquad using both synthetic controls and retrospective patient-derived tissues, sera and saliva. A multicentre ring study was conducted to evaluate assay reliability across different platforms, demonstrating strong correlation with commercial singleplexes, broad applicability, reduced turnaround time (TAT) and cost-effectiveness. Finally, we provide evidence for its potential clinical application to predict disease outcome in HNC, testing miRquad on tumoral and peritumoral tissues, sera and saliva samples collected throughout patient follow up. The assay overcomes common challenges associated with multiple miRNAs detection, particularly in liquid biopsy samples (e.g., multiple pipetting issues, increased consumption of sample for multiple assessment, extended TAT for complete profiling) and provides robust and accurate detection, demonstrating potential for real-time patient monitoring and prognostication in HNC.

Extended molecular profiling using massive parallel sequencing (MPS) technologies, commonly referred to as next-generation sequencing (NGS), has revolutionized cancer diagnosis and treatment, including in bone and soft tissue sarcomas (BSTS). This heterogeneous group of mesenchymal tumors presents a complex spectrum of genetic alterations, such as chromosomal rearrangements, point mutations, and copy number variations. Unlike carcinomas, where driver mutations are often well defined, the role of specific genomic signatures in dictating BSTS prognosis and therapy response remains to be fully elucidated. Despite its promise, the adoption of MPS/NGS in BSTS is limited by variability in testing access, turnaround times, specimen quality, costs, and data interpretation. Although identified alterations are often not yet directly targetable, they provide critical insights that can refine diagnosis, enable better patient stratification, and guide treatment strategies. To optimize the use of MPS/NGS in BSTS, harmonization and multidisciplinary collaboration within molecular tumor boards (MTBs) are essential. With this aim, the Italian Sarcoma Group ETS (ISG) convened a consensus meeting to establish best practices for integrating MPS/NGS into everyday clinical care. ISG experts developed ten consensus statements: the first five address the role of extended molecular profiling in BSTS diagnostics, while the others offer guidance on MPS/NGS use and interpretation when searching for potentially actionable targets in the treatment of advanced disease. Furthermore, collaboration with the National Rare Cancer Network to offer expert consultation and systematically correlate MPS/NGS findings with clinical outcomes for BSTS cases undergoing extended molecular profiling will be critical to advancing precision medicine in this field.

In precision medicine, DNA-based assays are currently necessary but not always sufficient for predicting therapeutic efficacy of cancer drugs based on the mutational findings in a patient's tumor specimen. Most drugs target proteins, but it is challenging and not yet cost-effective to perform high-throughput proteomics profiling, including mutational analysis, on cancer specimens. RNA may be an effective mediator for bridging the "DNA to protein divide" and provide more clarity and therapeutic predictability for precision oncology. While RNA sequencing (RNA-seq) has been increasingly used alongside DNA cancer mutation screening panels to assess the impact of variants on gene transcript expression and splicing, comprehensive evaluations of RNA panels and the integration of expressed mutation data analytics to supplement DNA panels are still limited. In this study, we conducted targeted RNA-seq on a reference sample set for expressed variant detection to explore its potential capability to complement DNA variant results or detect variants independently. The results indicated that, with a carefully controlled false positive rate ensuring high accuracy, RNA-seq uniquely identified variants with significant pathological relevance that were missed by DNA-seq, demonstrating its potential to uncover clinically actionable mutations. On the other hand, while some variants were detected by both approaches, others were missed by one or the other, reflecting either the nature of these variants or limitations of the bioinformatics tools used. Variants missed by RNA-seq are often not expressed or expressed at very low levels, suggesting they may be of lower clinical relevance. Incorporating RNA-seq into clinical biomarker panels will ultimately advance precision medicine and improve patient outcomes by improving the strength and reliability of somatic mutation findings for clinical diagnosis, prognosis and prediction of therapeutic efficacy.

Background: Despite remarkable therapeutic progress, cancer resistance remains one of the major challenges in oncology, often resulting in disease relapse and poor patient outcomes. Resistance arises from multiple genetic and non-genetic mechanisms, ultimately limiting the effectiveness of chemo- and targeted therapies. Within the RNA family, microRNAs (miRNAs) regulate core biological processes and have been recognized also as critical contributors of tumor resistance and therapy failure. Being pivotal, they have been increasingly exploited as biomarkers in various settings. Although in silico analyses facilitate miRNAs identification, PCR-based approaches remain essential to validate their expression. Currently, a plethora of well-established methods exist but multiplex detection from the same input have been only rarely explored. Methods: We present miRquad, the first-in-class digital PCR (dPCR) TaqMan™ multiplex RUO assay for miRNA detection in head and neck (HNC) cancers. Based on a patented prognostic signature including miR-21-5p, miR-96-5p, miR-21-3p and miR-429, the assay enables simultaneous miRNA analysis via qPCR and dPCR across multiple clinically relevant sample types. Results: We designed and optimized miRquad using both synthetic controls and retrospective patient-derived tissues, sera and saliva. A multicentre ring study was conducted to evaluate assay reliability across different platforms, demonstrating strong correlation with commercial singleplexes, broad applicability, and cost-effectiveness. Finally, we provide evidence for its potential clinical application in different HNC settings, testing miRquad on tumoral and peritumoral tissues, sera and saliva samples collected throughout patient follow up. Conclusions : The assay overcomes common challenges associated with multiple miRNAs detection, particularly in liquid biopsy samples, and provides robust and accurate detection, demonstrating potential for real-time patient monitoring and prognostication in HNC.

Epithelioid sarcoma (ES) is a rare tumor hallmarked by the loss of INI1/SMARCB1 expression. Apart from this alteration, little is known about the biology of ES. Despite recent advances in treatment, the prognosis of ES remains unsatisfactory. To elucidate the molecular underpinnings of ES, and to identify diagnostic biomarkers and potential therapeutic vulnerabilities, we performed an integrated omics profiling (RNA sequencing and methylation array) of 24 primary, untreated ESs. Transcriptome and methylome analysis identified 2 distinct molecular clusters that essentially corresponded to the morphologic variants of ES, classic ES (C-ES) and the more aggressive proximal ES (P-ES). The P-ES group was characterized by hyperactivation of GATA3 and MYC pathways, with extensive epigenetic rewiring associated with EZH2 overexpression. Both DNA methylation and gene expression analysis indicated a striking similarity with the "MYC subgroup" of atypical teratoid/ rhabdoid tumor, another SMARCB1-deficient tumor, implying a shared molecular background and potential therapeutic vulnerabilities. Conversely, the C-ES group exhibited an endothelial-like molecular profile, with expression of vascular genes and elevated proangiogenic SOX17 signaling. Immunohistochemistry validated the overexpression of the chromatin regulators GATA3 (9/12 vs 0/ 16) and EZH2 (7/7 vs 2/6) in P-ESs, and of the vascular factors SOX17 (8/8 vs 1/10) and N-cadherin (5/9 vs 0/10) in C-ESs. Therefore, these molecules emerge as potential diagnostic tools to fill the gap represented by the lack of ES subtype-specific biomarkers. In summary, our study shows that P-ES and C-ES represent distinct molecular entities defined by MYC/GATA3 and SOX17/endothelial molecular traits, respectively. Besides providing insights into the biology of ES, our study pinpoints subtype-specific biomarkers and potential therapeutic vulnerabilities. (c) 2024 THE AUTHORS. Published by Elsevier Inc. on behalf of the United States & Canadian Academy of Pathology. This is an open access article under the CC BY license (http://creativecommons.org/ licenses/by/4.0/).

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 epithelial‑to‑mesenchymal 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 Beclin‑1, also exert autophagy‑independent functions in a variety of biological processes. The present review aimed to summarize autophagy‑independent functions of molecules of the autophagy machinery and how the activity of these molecules can influence signaling pathways that are deregulated in cancer progression.

One of the fundamental aspects of genomic research is the identification of differentially expressed (DE) genes between two conditions. In the past decade, numerous DE analysis tools have been developed, employing various normalization methods and statistical modelling approaches. In this article, we introduce DElite, an R package that leverages the capabilities of four state-of-the-art DE tools: edgeR, limma, DESeq2, and dearseq. DElite returns the outputs of the four tools with a single command line, thus providing a simplified way for non-expert users to perform DE analysis. Furthermore, DElite provides a statistically combined output of the four tools, and in vitro validations support the improved performance of these combination approaches for the detection of DE genes in small datasets. Finally, DElite offers comprehensive and well-documented plots and tables at each stage of the analysis, thus facilitating result interpretation. Although DElite has been designed with the intention of being accessible to users without extensive expertise in bioinformatics or statistics, the underlying code is open source and structured in such a way that it can be customized by advanced users to meet their specific requirements. DElite is freely available for download from https://gitlab.com/soc-fogg-cro-aviano/DElite.

Clinical/pathologic characteristics of the patients from whom the tissue samples molecularly profiled were derived; bold/underlined type indicates the tumor tissue from which the sample for the molecular analysis was derived.

Supplementary Figure 1. Evidence of tumor regression and inflammatory infiltration in IM-treated metastatic FS-DFSP. IHC staining of a metastatic FS-DFSP lesions derived from patient #4 in Table 1 and 2. Panel A and B show haematoxylin and eosin stains of (a, b) pre-IM and (c, d, e, f) post-IM lesions. Micrograph picture c shows the presence of a cellular depleted area replaced by hyalinized stroma. Panel B displays the intra-tumor presence of inflammatory cells (indicated by the arrows). Original magnification: a and c 25x; d and e 50x; b and f 100x.

AimsEpithelioid 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 resultsMultifocal 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. ConclusionsThese 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.

Supplemental methodological information

ABSTRACT Molecular characterization of a biological sample, e.g., with omics approaches, is fundamental for the development and implementation of personalized and precision medicine approaches. In this context, quality assessment is one of the most critical aspects. Accurate performance and interpretation of omics techniques is based on consensus, harmonization, and standardization of protocols, procedures, data analysis and reference values and materials. EATRIS, the European Infrastructure for Translational Medicine ( www.EATRIS.eu ), brings together resources and services to support researchers in developing their biomedical discoveries into novel translational tools and interventions for better health outcomes. Here we describe activities of member facilities of EATRIS towards quality assessment of pre-clinical sample processing, clinical omics data generation, multi-omics data integration, and dissemination of the resources in a Multi-Omics Toolbox, the principal deliverable of the EATRIS Plus project for the consolidation of EATRIS towards translational Medicine.

Additional gene mutations detected in the GISTs series