Identifying specific TLS-associated genes as potential biomarkers for predicting prognosis and evaluating the efficacy of immunotherapy in soft tissue sarcoma.

Background: The tertiary lymphatic structure (TLS) is an important component of the tumor immune microenvironment and has important significance in patient prognosis and response to immune therapy. However, the underlying mechanism of TLS in soft tissue sarcoma remains unclear. Methods: A total of 256 RNAseq and 7 single-cell sequencing samples were collected from TCGA-SARC and GSE212527 cohorts. Based on published TLS-related gene sets, four TLS scores were established by GSVA algorithm. The immune cell infiltration was calculated via TIMER2.0 and "MCPcounter" algorithms. In addition, the univariate, LASSO, and multivariate-Cox analyses were used to select TLS-related and prognosis-significant hub genes. Single-cell sequencing dataset, clinical immunohistochemical, and cell experiments were utilized to validate the hub genes. Results: In this study, four TLS-related scores were identified, and the total-gene TLS score more accurately reflected the infiltration level of TLS in STS. We further established two hub genes (DUSP9 and TNFSF14) prognosis markers and risk scores associated with soft tissue sarcoma prognosis and immune therapy response. Flow cytometry analysis showed that the amount of CD3, CD8, CD19, and CD11c positive immune cell infiltration in the tumor tissue dedifferentiated liposarcoma patients was significantly higher than that of liposarcoma patients. Cytological experiments showed that soft tissue sarcoma cell lines overexpressing TNFSF14 could inhibit the proliferation and migration of sarcoma cells. Conclusion: This study systematically explored the TLS and related genes from the perspectives of bioinformatics, clinical features and cytology experiments. The total-gene TLS score, risk score and TNFSF14 hub gene may be useful biomarkers for predicting the prognosis and immunotherapy efficacy of soft tissue sarcoma.

Acquired NF2 mutation confers resistance to TRK inhibition in an ex vivo LMNA::NTRK1-rearranged soft-tissue sarcoma cell model.

Genomic rearrangements of the neurotrophic receptor tyrosine kinase genes (NTRK1, NTRK2, and NTRK3) are the most common mechanism of oncogenic activation for this family of receptors, resulting in sustained cancer cell proliferation. Several targeted therapies have been approved for tumours harbouring NTRK fusions and a new generation of TRK inhibitors has already been developed due to acquired resistance. We established a patient-derived LMNA::NTRK1-rearranged soft-tissue sarcoma cell model ex vivo with an acquired resistance to targeted TRK inhibition. Molecular profiling of the resistant clones revealed an acquired NF2 loss of function mutation that was absent in the parental cell model. Parental cells showed continuous sensitivity to TRK-targeted treatment, whereas the resistant clones were insensitive. Furthermore, resistant clones showed upregulation of the MAPK and mTOR/AKT pathways in the gene expression based on RNA sequencing data and increased sensitivity to MEK and mTOR inhibitor therapy. Drug synergy was seen using trametinib and rapamycin in combination with entrectinib. Medium-throughput drug screening further identified small compounds as potential drug candidates to overcome resistance as monotherapy or in combination with entrectinib. In summary, we developed a comprehensive model of drug resistance in an LMNA::NTRK1-rearranged soft-tissue sarcoma and have broadened the understanding of acquired drug resistance to targeted TRK therapy. Furthermore, we identified drug combinations and small compounds to overcome acquired drug resistance and potentially guide patient care in a functional precision oncology setting. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Imaging Features of Primary Intracranial Sarcoma with DICER1 Mutation: A Multicenter Case Series.

Primary intracranial sarcoma, DICER1-mutant, is a rare, recently described entity in the fifth edition of the WHO Classification of CNS Tumors. Given the entity's rarity and recent description, imaging data on primary intracranial sarcoma, DICER1-mutant, remains scarce. In this multicenter case series, we present detailed multimodality imaging features of primary intracranial sarcoma, DICER1-mutant, with emphasis on the appearance of the entity on MR imaging. In total, 8 patients were included. In all 8 patients, the lesion demonstrated blood products on T1WI. In 7 patients, susceptibility-weighted imaging was obtained and demonstrated blood products. Primary intracranial sarcoma, DICER1-mutant, is a CNS neoplasm that primarily affects pediatric and young adult patients. In the present case series, we explore potential imaging findings that are helpful in suggesting this diagnosis. In younger patients, the presence of a cortical lesion with intralesional blood products on SWI and T1-weighted MR imaging, with or without extra-axial blood products, should prompt the inclusion of this entity in the differential diagnosis.

[Primary pulmonary myxoid sarcoma with EWSR1-CREB1 fusion]. / Pervichnaya legochnaya miksoidnaya sarkoma so sliyaniem genov EWSR1-CREB1.

Primary pulmonary myxoid sarcoma with EWSR1-CREB1 fusion is an extremely rare tumor. Its clinical manifestation is unspecific and only molecular genetic method can proof this diagnosis. This paper describes an unusual clinical presentation of primary pulmonary myxoid sarcoma in a 68-year-old patient with involvement of both lungs.

Reclassification of a spindle cell sarcoma after identification of a TFG-ROS1 fusion: A case demonstrating the clinical benefit of next-generation sequencing in sarcoma.

BACKGROUND: Inflammatory myofibroblastic tumors (IMTs) are rare mesenchymal soft tissue sarcomas that often present diagnostic challenges due to their wide and varied morphology. A subset of IMTs have fusions involving ALK or ROS1. The role of next-generation sequencing (NGS) for classification of unselected sarcomas remains controversial. METHODS AND RESULTS: We report a case of a metastatic sarcoma in a 34-year-old female originally diagnosed as an unclassified spindle cell sarcoma with myofibroblastic differentiation and later reclassified as IMT after NGS revealed a TFG-ROS1 rearrangement. Histologically, the neoplasm had spindle cell morphology with a lobulated to focally infiltrative growth pattern with scant inflammatory cell infiltrate. Immunohistochemistry demonstrated focal desmin and variable smooth muscle actin staining but was negative for SOX10, S100, and CD34. Fluorescence in situ hybridization was negative for USP6 or ALK gene rearrangements. NGS revealed a TFG-ROS1 rearrangement and the patient was treated with crizotinib with clinical benefit. CONCLUSIONS: We discuss the role of NGS as well as its potential benefit in patients with unresectable, ALK-negative metastatic disease. Considering this case and previous literature, we support the use of NGS for patients requiring systemic treatment.

Targeting RCC1 to block the human soft-tissue sarcoma by disrupting nucleo-cytoplasmic trafficking of Skp2.

Soft-tissue sarcomas (STS) emerges as formidable challenges in clinics due to the complex genetic heterogeneity, high rates of local recurrence and metastasis. Exploring specific targets and biomarkers would benefit the prognosis and treatment of STS. Here, we identified RCC1, a guanine-nucleotide exchange factor for Ran, as an oncogene and a potential intervention target in STS. Bioinformatics analysis indicated that RCC1 is highly expressed and correlated with poor prognosis in STS. Functional studies showed that RCC1 knockdown significantly inhibited the cell cycle transition, proliferation and migration of STS cells in vitro, and the growth of STS xenografts in mice. Mechanistically, we identified Skp2 as a downstream target of RCC1 in STS. Loss of RCC1 substantially diminished Skp2 abundance by compromising its protein stability, resulting in the upregulation of p27Kip1 and G1/S transition arrest. Specifically, RCC1 might facilitate the nucleo-cytoplasmic trafficking of Skp2 via direct interaction. As a result, the cytoplasmic retention of Skp2 would further protect it from ubiquitination and degradation. Notably, recovery of Skp2 expression largely reversed the phenotypes induced by RCC1 knockdown in STS cells. Collectively, this study unveils a novel RCC1-Skp2-p27Kip1 axis in STS oncogenesis, which holds promise for improving prognosis and treatment of this formidable malignancy.

Low-grade undifferentiated sarcoma with MEIS1::NCOA2-rearrangement primary to the lung: a case report.

BACKGROUND: MEIS1::NCOA2 is a rare fusion gene that has been recently described in a subset of spindle cell rhabdomyosarcomas and multiple low-grade undifferentiated spindle cell sarcomas predominantly arising in the genitourinary and gynecologic tracts with no specific line of differentiation. We present the first documented case of this neoplasm arising as a lung primary tumor. CASE PRESENTATION: A 74-year-old woman with a 40-year smoking history presented with a 2.1 × 1.7 cm lung nodule discovered on computed tomography (CT) scan. A biopsy and subsequent lobe resection were performed, as well as an extensive metastatic work up, which revealed no additional masses. No specific line of differentiation was found by immunohistochemical staining, and an RNA-based fusion panel revealed a MEIS1::NCOA2 fusion, at which point a diagnosis of Low-Grade Undifferentiated Sarcoma with MEIS1::NCOA2-Rearrangement was rendered. CONCLUSIONS: This report represents the first diagnosis of this tumor primary to the lung, and provides additional insight into the origin and localization of these rare tumors.

Elucidating the Diagnostic Complexity of Round Cell Sarcoma with EWSR1-CREM Fusion: A Comprehensive Case Study.

Sarcomas, particularly undifferentiated small round cell sarcomas of bone and soft tissue, pose significant diagnostic challenges due to their nonspecific morphology and the necessity for comprehensive molecular analyses. This paper discusses a rare case of round cell sarcoma exhibiting the EWSR1-CREM fusion, offering insights into the complexities of its diagnosis and management. The patient, a 15-year-old female with a history of Type 1 diabetes, presented with persistent right thigh tenderness and swelling. MRI revealed a large necrotic mass in the retroperitoneal region. Histological analysis showed a well-demarcated tumor with diverse cellular morphologies and distinct necrotic areas. Immunohistochemical (IHC) tests identified dot-like staining for Desmin and Vimentin but negative results for several markers, including Cytokeratin and CD45. Strong ALK positivity was noted. Next-generation sequencing with the Illumina TruSight™ Oncology 500 assay revealed the fusion gene EWSR1-CREM, along with benign and uncertain mutations in other genes. The tumor's morphology and immunoprofile, along with molecular findings, led to a diagnosis of round cell sarcoma with EWSR1-CREM fusion. This case adds to the spectrum of tumors associated with this fusion, often presenting diverse morphologies. The rarity of EWSR1-CREM fusion sarcomas poses a challenge in treatment, highlighted by the development of pulmonary metastases and disease progression after surgical excision in this patient despite the lack of an effective targeted therapy. In conclusion, this case emphasizes the need for a multidisciplinary diagnostic approach in complex sarcomas and highlights the importance of continued research on rare sarcomas, their genetic underpinnings, and potential therapeutic targets.

A Case Study of a Rare Undifferentiated Spindle Cell Sarcoma of the Penis: Establishment and Characterization of Patient-Derived Models.

Rare sarcomas present significant treatment challenges compared to more prevalent soft tissue sarcomas due to limited treatment options and a poor understanding of their biology. This study investigates a unique case of penile sarcoma, providing a comprehensive morphological and molecular analysis. Through the creation of experimental patient-derived models-including patient-derived xenograft (PDX), 3D, and monolayer primary cultures-we successfully replicated crucial molecular traits observed in the patient's tumor, such as smooth muscle actin and CD99 expression, along with specific mutations in genes like TSC2 and FGFR4. These models are helpful in assessing the potential for an in-depth exploration of this tumor's biology. This comprehensive approach holds promise in identifying potential therapeutic avenues for managing this exceedingly rare soft tissue sarcoma.

Habitat escalated adaptive therapy (HEAT): a phase 2 trial utilizing radiomic habitat-directed and genomic-adjusted radiation dose (GARD) optimization for high-grade soft tissue sarcoma.

BACKGROUND: Soft tissue sarcomas (STS), have significant inter- and intra-tumoral heterogeneity, with poor response to standard neoadjuvant radiotherapy (RT). Achieving a favorable pathologic response (FPR ≥ 95%) from RT is associated with improved patient outcome. Genomic adjusted radiation dose (GARD), a radiation-specific metric that quantifies the expected RT treatment effect as a function of tumor dose and genomics, proposed that STS is significantly underdosed. STS have significant radiomic heterogeneity, where radiomic habitats can delineate regions of intra-tumoral hypoxia and radioresistance. We designed a novel clinical trial, Habitat Escalated Adaptive Therapy (HEAT), utilizing radiomic habitats to identify areas of radioresistance within the tumor and targeting them with GARD-optimized doses, to improve FPR in high-grade STS. METHODS: Phase 2 non-randomized single-arm clinical trial includes non-metastatic, resectable high-grade STS patients. Pre-treatment multiparametric MRIs (mpMRI) delineate three distinct intra-tumoral habitats based on apparent diffusion coefficient (ADC) and dynamic contrast enhanced (DCE) sequences. GARD estimates that simultaneous integrated boost (SIB) doses of 70 and 60 Gy in 25 fractions to the highest and intermediate radioresistant habitats, while the remaining volume receives standard 50 Gy, would lead to a > 3 fold FPR increase to 24%. Pre-treatment CT guided biopsies of each habitat along with clip placement will be performed for pathologic evaluation, future genomic studies, and response assessment. An mpMRI taken between weeks two and three of treatment will be used for biological plan adaptation to account for tumor response, in addition to an mpMRI after the completion of radiotherapy in addition to pathologic response, toxicity, radiomic response, disease control, and survival will be evaluated as secondary endpoints. Furthermore, liquid biopsy will be performed with mpMRI for future ancillary studies. DISCUSSION: This is the first clinical trial to test a novel genomic-based RT dose optimization (GARD) and to utilize radiomic habitats to identify and target radioresistance regions, as a strategy to improve the outcome of RT-treated STS patients. Its success could usher in a new phase in radiation oncology, integrating genomic and radiomic insights into clinical practice and trial designs, and may reveal new radiomic and genomic biomarkers, refining personalized treatment strategies for STS. TRIAL REGISTRATION: NCT05301283. TRIAL STATUS: The trial started recruitment on March 17, 2022.

Cytomorphology of metastatic colonic MXD4::NUTM1-rearranged sarcoma.

This report describes the cytologic features of a recently described MXD4::NUTM1-rearranged colonic sarcoma metastatic to the midclavicular soft tissue. Thirteen years ago, a 65-year-old woman presented with a cecal mass and subsequent liver mass. The cecal mass was diagnosed as malignant undifferentiated spindled and epithelioid neoplasm based on morphology and lack of tumor immunoreactivity with a panel of epithelial, smooth muscle, skeletal, melanoma, hematologic, and GIST markers. The liver mass showed morphologic and immunophenotypic similarity to the epithelioid component of the patient's cecal mass, albeit devoid of the spindled component. Fine needle aspiration of the midclavicular soft tissue mass showed singly scattered to clustered epithelioid to rhabdoid tumor cells with centrally to eccentrically located nuclei, prominent nucleoli, and moderate eosinophilic cytoplasm. Immunohistochemical stains performed on the concurrent biopsy showed the tumor cells were positive for NUT and negative for all other additional markers with retained normal expression of SMARCA2 and SMARCA4. Next-generation sequencing showed the presence of MXD4::NUTM1 gene fusion. Due to the identical cytomorphologic findings with the epithelioid component of the patient's prior cecal and liver masses, the tumor was deemed as consistent with a NUTM1-rearranged sarcoma. To our knowledge, this case represents the first reported cytologic features of a NUTM1-rearranged sarcoma on fine needle aspiration. Familiarity with the cytologic features, inclusion of this entity in the differential diagnosis of tumors with epithelioid and/or rhabdoid morphology, and performance of ancillary tests (immunohistochemistry and molecular) will be helpful in arriving at the right diagnosis.

NTRK-rearranged spindle cell neoplasm: Initial observation of imaging appearance and clinicopathologic correlation.

OBJECTIVE: To explore pre-treatment imaging findings of neurotrophic tyrosine receptor kinase (NTRK)-rearranged spindle cell neoplasm, an emerging group of molecularly defined soft tissue tumors and summarize the clinical course, including TRK inhibitor therapy response. MATERIALS AND METHODS: This retrospective study included 8 women and 4 men with NTRK-rearranged spindle cell neoplasm (median age, 35.5 years, range, 0-66). Available pre-treatment MRI, CT, PET, and US imaging were reviewed. Tumor histology and the patients' clinical course were reviewed. RESULTS: Primary tumors were located within the soft tissue, lungs, kidney, and breast with soft tissue being the most prevalent site (n = 6). Pre-treatment MRI (n = 4) revealed linear hypointense signal foci and contrast enhancement in all patients with hemorrhage in half of the tumors. A tail sign (n = 1) and fluid levels (n = 1) were less frequent. Ultrasound showed well-marginated hypoechoic masses with internal flow. Primary tumors were all non-calcified on CT (4/4). Metastases were FDG-avid (4/4). Among the 8 patients who developed metastasis, 7 developed pulmonary metastases. All four patients who received NTRK inhibitor therapy showed an initial decrease in tumor size or FDG uptake. CONCLUSION: NTRK-rearranged neoplasms may occur as enhancing masses with linear hypointense signal foci on MRI and FDG avid metastases on PET. Pulmonary metastases were frequent in our study. Initial treatment response is observed in most patients.

The diagnostic utility of cytology specimen in a case of EWSR1::NFATC2 sarcoma.

EWSR1::NFATC2 sarcoma, a rare round cell sarcoma constituting the majority of EWSR1::non-ETS sarcomas, has recently been defined in the latest WHO classification. To date, the cytological findings of EWSR1::NFATC2 sarcoma remain undocumented. We present the case of a 25-year-old man with a history of polyostotic fibrous dysplasia in the right leg, referred to our hospital with left thigh pain. Cytological findings included metachromasia, minimally pleomorphic round cells, and eosinophilic infiltration. There was no precursor fibrous dysplasia and the initial diagnosis was undifferentiated pleomorphic sarcoma. Following histologic review, we successfully performed immunocytochemistry and fluorescence in situ hybridization (FISH) on archival cytology specimens. The tumor cells were positive for NKX2-2, NKX3-1, and PAX7 and showed amplified 5' single signals of EWSR1 gene. Reverse transcriptase-polymerase chain reaction revealed an in-frame fusion of EWSR1 and NFATC2. This report describes the cytological features of EWSR1::NFATC2 sarcoma and highlights the diagnostic utility of archival cytology specimens.

A recurrent NTRK1 tyrosine kinase domain mutation pair is characteristic in a subset of dedifferentiated liposarcomas.

INTRODUCTION: Dedifferentiated liposarcoma (DDLPS) is a common form of liposarcoma with challenging treatment modalities. Pan-TRK immunopositivity can be often observed without NTRK gene fusion in soft tissue sarcomas with myogenic differentiation. Expression and the role of NTRK in DDLPS are under-studied. We sought to identify activating mutations of the NTRK genes. MATERIALS AND METHODS: 131 DDLPS patients were selected for pan-TRK immunohistochemistry and positive cases were analyzed by Sanger sequencing for NTRK1, NTRK2 and NTRK3 genes. Functional assays were performed using a lentiviral transduction system to study the effect of NTRK variants in fibroblast, immortalized fibroblast, and dedifferentiated liposarcoma cell lines. RESULTS: Out of the 131 DDLPS cases, 75 immunohistochemical staining positive cases, 46 were successfully Sanger sequenced. A recurrent somatic mutation pair in cis position (NGS) of the NTRK1 c.1810C>T (p.H604Y) and c.1838G>T (p.G613V) was identified in six cases (13%) that have never been reported in DDLPS. NTRK fusions were excluded in all six cases by FISH and NGS. The phospho-AKT immunopositivity among the six mutated cases suggested downstream activation of the NTRK signaling pathway. Functional assays showed no transforming effects, but resistance to first- and second-line TRK inhibitors of the p.G613V and p.H604Y variant. CONCLUSIONS: We detected (de novo/somatic) missense mutation variants in cis position of the NTRK1 gene in a subset of DDLPS indicating modifying mutations that may contribute to tumorigenesis in a subset of DDLPS. These variants beget resistance to TRK inhibitors indicating an interesting biomarker for other studies with TRK inhibitors.

Opportunity in Complexity: Harnessing Molecular Biomarkers and Liquid Biopsies for Personalized Sarcoma Care.

Due to their rarity and complexity, sarcomas represent a substantial therapeutic challenge. However, the incredible diversity within and across sarcoma subtypes presents an opportunity for personalized care to maximize efficacy and limit toxicity. A deeper understanding of the molecular alterations that drive sarcoma development and treatment response has paved the way for molecular biomarkers to shape sarcoma treatment. Genetic, transcriptomic, and protein biomarkers have become critical tools for diagnosis, prognostication, and treatment selection in patients with sarcomas. In the future, emerging biomarkers like circulating tumor DNA analysis offer the potential to improve early detection, monitoring response to treatment, and identifying mechanisms of resistance to personalize sarcoma treatment. Here, we review the current state of molecular biomarkers for sarcomas and highlight opportunities and challenges for the implementation of new technologies in the future.

Metabolic targeting of cancer associated fibroblasts overcomes T-cell exclusion and chemoresistance in soft-tissue sarcomas.

T cell-based immunotherapies have exhibited promising outcomes in tumor control; however, their efficacy is limited in immune-excluded tumors. Cancer-associated fibroblasts (CAFs) play a pivotal role in shaping the tumor microenvironment and modulating immune infiltration. Despite the identification of distinct CAF subtypes using single-cell RNA-sequencing (scRNA-seq), their functional impact on hindering T-cell infiltration remains unclear, particularly in soft-tissue sarcomas (STS) characterized by low response rates to T cell-based therapies. In this study, we characterize the STS microenvironment using murine models (in female mice) with distinct immune composition by scRNA-seq, and identify a subset of CAFs we termed glycolytic cancer-associated fibroblasts (glyCAF). GlyCAF rely on GLUT1-dependent expression of CXCL16 to impede cytotoxic T-cell infiltration into the tumor parenchyma. Targeting glycolysis decreases T-cell restrictive glyCAF accumulation at the tumor margin, thereby enhancing T-cell infiltration and augmenting the efficacy of chemotherapy. These findings highlight avenues for combinatorial therapeutic interventions in sarcomas and possibly other solid tumors. Further investigations and clinical trials are needed to validate these potential strategies and translate them into clinical practice.

Primary NTRK -rearranged Spindle Cell Neoplasm of the Gastrointestinal Tract: A Clinicopathological and Molecular Analysis of 8 Cases.

NTRK-rearranged spindle cell neoplasm occurs predominantly in the superficial or deep soft tissues of extremities or trunk. Occurrence in the visceral organs is extremely rare. Herein, we describe 8 cases of NTRK-rearranged spindle cell neoplasm that arose primarily in the gastrointestinal tract. Patients included 5 males and 3 females with age at presentation ranging from 6 to 63 years (median: 29.5 years). Tumors occurred in the colon (n=3), small intestine (n=2), rectum (n=2), and stomach (n=1). Tumor size ranged from 3.5 to 9 cm (median: 5 cm). Morphologically, 4 tumors were low-grade, composed of haphazard or intertwining fascicles of spindle cells, with prominent interstitial collagen fibers and ring-like perivascular hyalinization being present in 2 tumors. The other 4 tumors were histologically high-grade sarcomas, consisting of sweeping fascicles of atypical spindle cells showing increased cellularity and brisk mitotic activity. Immunohistochemically, 6/6 cases (100%) showed diffuse and strong cytoplasmic staining of pan-TRK. Variable expression of TrkA, CD34, and S100 was noted in 5/5 (100%), 5/8 (62.5%), and 4/7 (57.1%) cases, respectively. Fluorescence in situ hybridization analysis showed NTRK1 rearrangement (n=7) and NTRK2 rearrangement (n=1). In cases with available materials, RNA sequencing identified LMNA::NTRK1 (n=3), TPM3::NTRK1 (n=2), and STRN::NTRK2 (n=1) fusions. At follow-up (range: 4 to 30 months; median: 12.5 months), 6 of 7 patients who underwent surgery had no evidence of disease at last follow-up. One patient was succumbed to the disease at 12 months despite adjunctive treatment with TRK inhibitor larotrectinib after surgery. One patient was treated with larotrectinib alone. He showed significant response at 7 months after treatment. NTRK-rearranged spindle cell neoplasm represents an exceptionally rare entity in the gastrointestinal tract. The presence of interstitial collagen fibers and ring-like perivascular hyalinization and co-expression of CD34 and S100 are diagnostic clues to low-grade neoplasms. However, high-grade sarcomas pose a considerable diagnostic challenge to pathologists owing to the lack of specific features. The final diagnosis relies on molecular assays. Patients with advanced disease may benefit from TRK inhibitor treatment.