INTRODUCTION:Phyllodes tumors (PT) are rare breast lesions arising from fibroepithelial stroma and may be hard to clinically distinguish from fibroadenomas. They are defined as benign, borderline or malignant. The purpose of this study was to describe diagnostic workup and surgical management, and to investigate incidence of local recurrence (LR) and overall survival (OS) in relation to tumor subtype, size, age, surgical margins, surgical method, and year of diagnosis. METHODS:Retrospective cohort study of all patients surgically treated for a PT in Stockholm, Sweden from 1999 to 2018. Descriptive analyses were performed, and regression models were used to analyze associations between selected covariates and LR and OS. RESULTS:Among 191 patients, 132 were treated for a benign PT, 40 for a borderline PT and 19 for a malignant PT. Preoperatively, results from diagnostic workup were often ambiguous, and only 45.6 % of cases had a preoperative diagnosis of PT. Initial surgery was breast-conserving in 93.2 % of patients. Recurrences occurred in 10.5 % of the total cohort. 5-year and 10-year OS was 96.1 % and 93.5 %, respectively, for the entire cohort. In a multivariable analysis, neither covariate was associated with risk of LR. Distant recurrences were only detected among patients with malignant PT. CONCLUSIONS:In the workup of PT, common diagnostic methods such as FNAC, CNB, and mammography may be unreliable, and clinical suspicion plays a critical role in guiding pre-operative decision-making. We found no association between surgical margins and rate of LR or OS. We found no evidence of metastatic potential in benign or borderline PT.

Synovial Sarcomas (SS) are characterized by the presence of the SS18::SSX fusion gene, which protein product induce chromatin changes through remodeling of the BAF complex. To elucidate the genomic events that drive phenotypic diversity in SS, we performed RNA and targeted DNA sequencing on 91 tumors from 55 patients. Our results were verified by proteomic analysis, public gene expression cohorts and single-cell RNA sequencing. Transcriptome profiling identified three distinct SS subtypes resembling the known histological subtypes: SS subtype I and was characterized by hyperproliferation, evasion of immune detection and a poor prognosis. SS subtype II and was dominated by a vascular-stromal component and had a significantly better outcome. SS Subtype III was characterized by biphasic differentiation, increased genomic complexity and immune suppression mediated by checkpoint inhibition, and poor prognosis despite good responses to neoadjuvant therapy. Chromosomal abnormalities were an independent significant risk factor for metastasis. KRT8 was identified as a key component for epithelial differentiation in biphasic tumors, potentially controlled by OVOL1 regulation. Our findings explain the histological grounds for SS classification and indicate that a significantly larger proportion of patients have high risk tumors (corresponding to SS subtype I) than previously believed.

Abstract Introduction Colorectal cancer management is evolving towards personalized medicine, with genetic testing playing a crucial role in treatment decisions. The Reflex Testing for Colorectal Cancer initiative aimed to integrate comprehensive genetic testing into routine care. Specifically, the aim was to promptly determine treatment predictive genetic factors and hereditary cancer risk syndromes that would lead to potential prophylactic surgery as well as identify DPYD deficiency that affects oncological treatment. Method The study, conducted from October 2021 to December 2023, included 370 patients diagnosed with colorectal cancer. Genetic analysis was performed on DNA extracted from blood and from tumor tissue taken from biopsy during endoscopy, using a gene panel of 385 genes (GMCK panel) designed to identify variants relevant to treatment prediction, prognosis, and heredity for all solid tumors. Result In the first 100 cases (48 females, 52 males), 7% were diagnosed before 50 years and 18% between 50 and 60 years of age. The tumor localization varied, with 23% in the right colon, 9% in the transverse colon, 27% in the left colon and 41% in the rectum.15% showed high microsatellite instability. Additionally, four patients (4%) were identified with Lynch syndrome (disease-associated variants in MLH1(x2) or MSH6(x2)). Regarding treatment-predictive biomarkers, pathogenic variants were found in KRAS in 44% and in BRAF in 20%. Discussion The analysis of paired tumor and blood data can identify both somatic and germline aberrations that are of clinical significance and thus enable personalized therapeutic decisions at diagnosis. Grants: Regional Cancer Center Stockholm-Gotland funded this project.

Introduction: Here, we report the first case of a soft tissue tumor with a PAK2::RAF1 fusion. Case Presentation: The patient was an 11-year-old female presenting with a 5 cm intramuscular mass in the lower leg. Microscopic examination revealed a mitotically active spindle cell lesion with monomorphic and moderately atypical cells growing in a patternless pattern with the presence of stromal and perivascular keloidal collagen with focal immunoreactivity for smooth muscle actin and S100; negative stains included cytokeratins, CD34, and caldesmon. Whole genome and RNA sequencing detected a chromosome 3 inversion and a resultant PAK2::RAF1 fusion as well as a CDKN2A homozygous deletion. The patient was treated with neoadjuvant chemoradiotherapy with only minimal response and the tumor was excised surgically. There was no evidence of disease progression at 4 months. Conclusion: This is the first case of a soft tissue tumor harboring a PAK2::RAF1 fusion with histological features in keeping with previous cases of RAF1 and other kinase fusion soft tissue tumors.

Synovial sarcoma (SS) is driven by a unique t(18;X) chromosomal translocation resulting in expression of the SS18-SSX fusion oncoprotein, a transcriptional regulator with both activating and repressing functions. However, the manner in which SS18-SSX contributes to the development of SS is not entirely known. Here, we show that SS18-SSX drives the expression of Preferentially Expressed Antigen in Melanoma (PRAME), which is highly expressed in SS but whose function remains poorly understood. The fusion protein directly binds and activates the PRAME promoter and we found that expression of SS18-SSX and PRAME are positively correlated. We provide evidence that PRAME modulates retinoic acid (RA) signaling, forming a ternary complex with the RA receptor α (RARα) and the Enhancer of Zeste Homolog 2 (EZH2). Knockdown of PRAME suppressed the response to all-trans retinoic acid (ATRA) supporting PRAME's role in modulating RA-signaling. Notably, we demonstrate that combined pharmacological inhibition of EZH2 and treatment with ATRA reconstituted RA signaling followed by reduced proliferation and induction of cellular senescence. In conclusion, our data provides new insights on the role of the SS18-SSX fusion protein in regulation of PRAME expression and RA signaling, highlighting the therapeutic potential of disrupting the RARα-PRAME-EZH2 complex in SS. Schematic presentation of the proposed model. A The RARα-PRAME-EZH2 ternary complex in SS. The fusion SS18-SSX oncoprotein binds to the PRAME promoter and activates its expression. PRAME in turn interacts with RARα-RXR heterodimers as well as with EZH2, and the complex binds to retinoic acid response elements (RAREs) in the DNA. This results in transcriptional repression of retinoic acid (RA) responsive genes and thus inhibition of RA-signaling, allowing tumor cell proliferation. B Therapeutic strategy. Treatment with an EZH2 inhibitor, such as GSK343, or activation of RAR receptors via all-trans retinoic acid (ATRA), disrupts the RARα-PRAME-EZH2 ternary complex and restores RA-signaling. Exposure to GSK343 or ATRA results in inhibition of cell proliferation and induction of cellular senescence, where GSK343 shows a dominant effect. The Figure was created with Biorender.com.

Background: The etiology of most mesenchymal tumors is unknown, and knowledge about syndromes with an increased risk of tumors in bone or soft tissue is sparse. Methods: We present a prospective germline analysis of 312 patients with tumors suspected of being sarcomas at a tertiary sarcoma center. Germline and tumor whole genome sequencing, tumor transcriptome, and methylome analyses were performed. Results: Germline pathogenic or likely pathogenic variants associated with an increased risk of tumors were detected in 24 patients (8%), of which 11 (4%) harbored a detectable second hit in the tumor. Second hits were confirmed in genes with (NF1, RB1, TP53, EXT2, and SDHC) and without (ATM, CDC73, MLH1, MSH6, POLG, and KCNQ1) known association with mesenchymal tumor predisposition. Sarcomas from two Lynch syndrome patients showed mismatch repair deficiency, predicting a treatment response to immune checkpoint inhibitors (Level 1 biomarker according to the FDA (Federal Drug Administration) and ESMO (European Society for Medical Oncology)). None of the three CHEK2 carriers had a second hit in the tumor, suggesting a weak link to sarcoma. Conclusions: We conclude that second-hit analyses can be used in standard of care to identify syndrome-related tumors. This approach can help distinguish true manifestations of tumor syndromes from unrelated germline findings and enhance the understanding of germline predisposition in soft tissue tumors. Prospective screening using germline whole genome sequencing should be considered when comprehensive somatic sequencing is introduced into clinical practice.

Apart from their killer identity, natural killer (NK) cells have integral roles in shaping the tumor microenvironment. Through immune gene deconvolution, the present study revealed an interplay between NK cells and myeloid-derived suppressor cells (MDSCs) in nonresponders of immune checkpoint therapy. Given that the mechanisms governing the outcome of NK cell–to–myeloid cell interactions remain largely unknown, we sought to investigate the cross-talk between NK cells and suppressive myeloid cells. Upon contact with tumor-experienced NK cells, monocytes and neutrophils displayed increased expression of MDSC-related suppressive factors along with increased capacities to suppress T cells. These changes were accompanied by impaired antigen presentation by monocytes and increased ER stress response by neutrophils. In a cohort of patients with sarcoma and breast cancer, the production of interleukin-6 (IL-6) by tumor-infiltrating NK cells correlated with S100A8/9 and arginase-1 expression by MDSCs. At the same time, NK cell–derived IL-6 was associated with tumors with higher major histocompatibility complex class I expression, which we further validated with b2m -knockout (KO) tumor mice models. Similarly in syngeneic wild-type and IL-6 KO mouse models, we then demonstrated that the accumulation of MDSCs was influenced by the presence of such regulatory NK cells. Inhibition of the IL-6/signal transducer and activator of transcription 3 (STAT3) axis alleviated suppression of T cell responses, resulting in reduced tumor growth and metastatic dissemination. Together, these results characterize a critical NK cell–mediated mechanism that drives the development of MDSCs during tumor immune escape.

Amplification of the MDM2 and CDK4 genes on chromosome 12 is commonly associated with low-grade osteosarcomas. In this study, we conducted high-resolution genomic and transcriptomic analyses on 33 samples from 25 osteosarcomas, encompassing both high- and low-grade cases with MDM2 and/or CDK4 amplification. We discerned four major subgroups, ranging from nearly intact genomes to heavily rearranged ones, each harbouring CDK4 and MDM2 amplification or CDK4 amplification with TP53 structural alterations. While amplicons involving MDM2 exhibited signs of an initial chromothripsis event, no evidence of chromothripsis was found in TP53-rearranged cases. Instead, the initial disruption of the TP53 locus led to co-amplification of the CDK4 locus. Additionally, we observed recurring promoter swapping events involving the regulatory regions of the FRS2, PLEKHA5, and TP53 genes. These events resulted in ectopic expression of partner genes, with the ELF1 gene being upregulated by the FRS2 and TP53 promoter regions in two distinct cases.

Sarcomas are a diverse group of malignant tumors arising in the bone and soft tissues and can affect patients of any age. For an increasing number of subtypes, there are already pathognomonic genetic alterations described. However, such alterations remain to be identified for other sarcomas, primarily those with a massive amount of genomic copy number and structural alterations. We have developed SarcDBase, a database-building method that matches genetic variants detected in high-resolution genomic and transcriptomic data from tumour biopsies with information on established biomarkers, eliminating the need for prior knowledge and manual screening of data. The performance of the database was tested on genomic and transcriptomic data from a diverse cohort of sarcomas (n=50). SarcDBase was able to detect mutations that confirmed the diagnosis of some patients, such as NAB2::STAT6, typical of solitary fibrous tumor, and H3F3A p.G35W, typical of giant cell tumor of bone. It was also able to identify new genetic alterations of likely biological significance, such as new combinations of partner genes in gene fusions. SarcDBase can be used on any samples with deep sequencing data.

AbstractPurpose: Tumor classification is a key component in personalized cancer care. For soft-tissue and bone tumors, this classification is currently based primarily on morphology assessment and IHC staining. However, these standard-of-care methods can pose challenges for pathologists. We therefore assessed how whole-genome and whole-transcriptome sequencing (WGTS) impacted tumor classification and clinical management when interpreted together with histomorphology. Experimental Design: We prospectively evaluated WGTS in routine diagnostics of 200 soft-tissue and bone tumors suspicious for malignancy, including DNA and RNA isolation from the tumor, and DNA isolation from a peripheral blood sample or any non-tumor tissue. Results: On the basis of specific genomic alterations or absence of presumed findings, WGTS resulted in reclassification of 7% (13/197) of the histopathologic diagnoses. Four cases were downgraded from low-grade sarcomas to benign lesions, and two cases were reclassified as metastatic malignant melanomas. Fusion genes associated with specific tumor entities were found in 30 samples. For malignant soft-tissue and bone tumors, we identified treatment relevant variants in 15% of cases. Germline pathogenic variants associated with a hereditary cancer syndrome were found in 22 participants (11%). Conclusions: WGTS provides an important dimension of data that aids in the classification of soft-tissue and bone tumors, correcting a significant fraction of clinical diagnoses, and identifies molecular targets relevant for precision medicine. However, genetic findings need to be evaluated in their morphopathologic context, just as germline findings need to be evaluated in the context of patient phenotype and family history.