Undifferentiated Round Cell Sarcomas Research Articles

The Capicua C1 Domain is Required for Full Activity of the CIC::DUX4 Fusion Oncoprotein.

Rearrangements between genes can yield neomorphic fusions that drive oncogenesis. Fusion oncogenes are made up of fractional segments of the partner genes that comprise them, with each partner potentially contributing some of its own function to the nascent fusion oncoprotein. Clinically, fusion oncoproteins driving one diagnostic entity are typically clustered into a single molecular subset and are often treated a similar fashion. However, knowledge of where specific fusion breakpoints occur in partner genes, and the resulting retention of functional domains in the fusion, is an important determinant of fusion oncoprotein activity and may differ between patients. This study investigates this phenomena through the example of CIC::DUX4, a fusion between the transcriptional repressor capicua (CIC) and the double homeobox 4 gene (DUX4), which drives an aggressive subset of undifferentiated round cell sarcoma. Using a harmonized dataset of over 100 patient fusion breakpoints from the literature, we show that most bona fide CIC::DUX4 fusions retain the C1 domain, which is known to contribute to DNA binding by wild type CIC. Mechanistically, deletion or mutation of the C1 domain reduces, but does not eliminate, activation of CIC target genes by CIC::DUX4. We also find that expression of C1-deleted CIC::DUX4 is capable of exerting intermediate transformation-related phenotypes compared with those imparted by full-length CIC::DUX4, but was not sufficient for tumorigenesis in a subcutaneous mouse model. In summary, our results suggest a supercharging role for the C1 domain in the activity of CIC::DUX4.

High-Throughput Hybridization Assay as First-Line Diagnostic Test for Sarcomas: Clinical Assessment in a Tertiary Referral Center.

Sarcomas are rare and highly heterogeneous mesenchymal tumors with deceptive morphologic features that pose a challenge for precise diagnostics. Chromosomal rearrangements generating pathognomonic gene fusions are useful diagnostic markers, traditionally tested using single-plex standard of care assays with limited diagnostic yield. NanoString nCounter technology has emerged as a robust solution with multiplexing capabilities. To optimize NanoString effective coverage of specific entities and conduct a validation study to support its clinical implementation. We reconfigured a NanoString's codeset by including a set of probes for detecting gene fusion variants of solitary fibrous tumors, low-grade fibromyxoid sarcomas/sclerosing epithelioid fibrosarcomas, and undifferentiated small round cell sarcomas, totaling 188 probes. A technical validation study was conducted with 96 retrospective samples. Additionally, 76 prospective samples were evaluated to assess the assay's clinical performance. Both technical and clinical validation studies showed that NanoString's codeset reached >88% sensitivity and 100% specificity, compared with standard of care methods, and superior diagnostic yield as a first-line test. Our design enabled the detection of almost all fusion variants of NGFI-A binding protein 2 (NAB2) with signal transducer and activator of transcription 6 (STAT6) in solitary fibrous tumors, as well as cAMP responsive element binding protein 3 like 1/2 (CREB3L1/2) rearrangements in all low-grade fibromyxoid sarcoma/sclerosing epithelioid fibrosarcoma cases. Identification of specific gene fusions of undifferentiated small round cell sarcoma was also improved, but additional strategies are necessary to attain full coverage. The NanoString platform demonstrated good sensitivity, specificity, and superior diagnostic yield. It is a cost-effective assay with rapid turnaround time, low sample consumption, streamlined analysis, and easy customization. Therefore, it is a promising alternative first-line diagnostic tool for routine sarcoma testing.

Nuclear DUX4 immunohistochemistry is a highly sensitive and specific marker for the presence of CIC::DUX4 fusion in CIC-rearranged sarcomas: a study of 48 molecularly confirmed cases.

CIC-rearranged sarcomas (CRS) are clinically aggressive undifferentiated round cell sarcomas (URCS), commonly driven by CIC::DUX4. Due to the repetitive nature of DUX4 and the variability of the fusion breakpoints, CIC::DUX4 fusion may be missed by molecular testing. Immunohistochemical (IHC) stains have been studied as surrogates for the CIC::DUX4 fusion. We aim to assess the performance of DUX4 IHC in the work-up of CRS and its expression in non-CRS round cell or epithelioid neoplasms. Cases of molecularly confirmed CRS (n = 48) and non-CRS (n = 105) were included. CRS cases consisted of 35 females and 13 males, with ages ranging from less than 1 year to 67 years (median = 41 years). Among the molecularly confirmed non-CRS cases, C-terminal DUX4 expression was investigated in Ewing sarcomas (38 cases), alveolar rhabdomyosarcomas (18 cases), desmoplastic smallround cell tumours (12 cases) and synovial sarcomas (n = five), as well as in non-mesenchymal neoplasms such as SMARCA4/SMARCB1-deficient tumours (n = five), carcinomas of unknown primary (n = three) and haematolymphoid neoplasms (four cases). DUX4 IHC was considered positive when strong nuclear expression was detected in more than 50% of neoplastic cells. When used as a surrogate for the diagnosis of CRS, the sensitivity and specificity of DUX4 IHC was 98 and 100%, respectively. Only one CRS case was negative for DUX4 IHC and harboured a CIC::FOXO4 fusion. DUX4 IHC is a highly sensitive and specific surrogate marker for the presence of CIC::DUX4 fusion, demonstrating its utility in establishing a diagnosis of CRS.

Undifferentiated Round Cell Sarcoma With CRTC1::SS18 Fusion: Expanding Clinicopathologic Features of a Rare Translocation Sarcoma With Prominent Desmoplastic Stroma

Undifferentiated round cell sarcomas (URCS) represent a diverse group of tumors, including conventional Ewing sarcoma, round cell sarcoma with EWSR1/FUS–non-ETS fusions, CIC-rearranged sarcoma, and sarcoma with BCOR alterations. Since 2018, 3 cases of URCS with a novel CRTC1::SS18 gene fusion have been reported in the literature. Herein, we report 3 additional cases of CRTC1::SS18 sarcoma, thereby doubling the number of described cases and expanding the clinicopathologic features of this rare translocation sarcoma. Together with the previously reported cases, we show that the male-to-female ratio is 1:2 with a median age of 34 years (range, 12-42 years). Tumors occurred primarily in intramuscular locations involving the lower extremity. Histologically, all tumors contained uniform round-to-epithelioid cells with a moderate amount of eosinophilic cytoplasm growing in sheets and nests with prominent desmoplastic stroma reminiscent of desmoplastic small round cell tumor. Immunohistochemical results were nonspecific, demonstrating variable expression of CD99 (patchy), ALK, GATA3, and cyclin D1. RNA sequencing revealed CRTC1::SS18 gene fusions in all cases, involving exons 1 to 2 of CRTC1 (the 5′ partner gene) on chromosome 19 and either exon 2 or exon 4 of SS18 (the 3′ partner gene) on chromosome 18. The clinical course was variable. Although 1 previously reported case demonstrated aggressive behavior with a fatal outcome, 2 others had a relatively indolent course with gradual growth for 6 to 7 years prior to resection. Two cases developed metastatic disease, including 1 case with bilateral lung metastasis and 1 with locoregional spread to a lymph node. By analyzing the clinicopathologic features, we aimed to improve recognition of this rare translocation sarcoma to better understand its biologic potential, optimize patient management, and expand the current classification of URCS.

Expanding the Spectrum of NUTM1 -Rearranged Sarcoma : A Clinicopathologic and Molecular Genetic Study of 8 Cases.

Apart from the lethal midline carcinoma (NUT carcinoma), NUTM1 translocation has also been reported in mesenchymal tumors, but is exceedingly rare. Here, we describe a series of 8 NUTM1 -rearranged sarcomas to further characterize the clinicopathologic features of this emerging entity. This cohort included 2 males and 6 females with age ranging from 24 to 64 years (mean: 51y; median: 56y). Tumors occurred in the colon (2), abdomen (2), jejunum (1), esophagus (1), lung (1) and infraorbital region (1). At diagnosis, 6 patients presented with metastatic disease. Tumor size ranged from 1 to 10.5cm (mean: 6cm; median: 5.5 cm). Histologically, 4 tumors were composed of primitive small round cells to epithelioid cells intermixed with variable spindle cells, while 3 tumors consisted exclusively of small round cells to epithelioid cells and 1 tumor consisted predominantly of high-grade spindle cells. The neoplastic cells were arranged in solid sheets, nests, or intersecting fascicles. Mitotic activity ranged from 1 to 15/10 HPF (median: 5/10 HPF). Other features included rhabdoid phenotype (4/8), pronounced nuclear convolutions (2/8), prominent stromal hyalinization (2/8), focally myxoid stroma (1/8), foci of osteoclasts (1/8), and necrosis (1/8). By immunohistochemistry, all tumors showed diffuse and strong nuclear staining of NUT protein, with variable expression of pancytokeratin (AE1/AE3) (2/8), CK18 (1/8), CD99 (3/8), NKX2.2 (2/8), cyclin D1 (2/8), desmin (2/8), BCOR (2/8), S100 (1/8), TLE1 (1/8), and synaptophysin (1/8). Seven of 8 tumors demonstrated NUTM1 rearrangement by fluorescence in situ hybridization analysis. RNA-sequencing analysis identified MXD4::NUTM1 (3/7), MXI1::NUTM1 (3/7), and MGA::NUTM1 (1/7) fusions, respectively. DNA-based methylation profiling performed in 2 cases revealed distinct methylation cluster differing from those of NUT carcinoma and undifferentiated small round cell and spindle cell sarcomas. At follow-up (range: 4 to 24mo), 1 patient experienced recurrence at 8.5 months, 4 patients were alive with metastatic disease (5, 10, 11, and 24mo after diagnosis), 3 patients remained well with no signs of recurrence or metastasis (4, 6, and 12mo after diagnosis). Our study further demonstrated that NUTM1 -rearranged sarcoma had a broad range of clinicopathologic spectrum. NUT immunohistochemistry should be included in the diagnostic approach of monotonous undifferentiated small round, epithelioid to high-grade spindle cell malignancies that difficult to classify by conventional means. DNA-based methylation profiling might provide a promising tool in the epigenetic classification of undifferentiated sarcomas.

Epigenetic determinants of fusion-driven sarcomas: paradigms and challenges.

We describe exciting recent advances in fusion-driven sarcoma etiology, from an epigenetics perspective. By exploring the current state of the field, we identify and describe the central mechanisms that determine sarcomagenesis. Further, we discuss seminal studies in translational genomics, which enabled epigenetic characterization of fusion-driven sarcomas. Important context for epigenetic mechanisms include, but are not limited to, cell cycle and metabolism, core regulatory circuitry, 3-dimensional chromatin architectural dysregulation, integration with ATP-dependent chromatin remodeling, and translational animal modeling. Paradoxically, while the genetic requirements for oncogenic transformation are highly specific for the fusion partners, the epigenetic mechanisms we as a community have uncovered are categorically very broad. This dichotomy prompts the question of whether the investigation of rare disease epigenomics should prioritize studying individual cell populations, thereby examining whether the mechanisms of chromatin dysregulation are specific to a particular tumor. We review recent advances focusing on rhabdomyosarcoma, synovial sarcoma, alveolar soft part sarcoma, clear cell sarcoma, undifferentiated round cell sarcoma, Ewing sarcoma, myxoid/round liposarcoma, epithelioid hemangioendothelioma and desmoplastic round cell tumor. The growing number of groundbreaking discoveries in the field, motivated us to anticipate further exciting advances in the area of mechanistic epigenomics and direct targeting of fusion transcription factors in the years ahead.

Efficacy of Pazopanib on Primary Patient-derived Undifferentiated Malignant Round Cell Sarcoma Line

Abstract Introduction: Undifferentiated round cell tumours (URCTs) are rare and less researched Ewing-like mesenchymal tumours that primarily affect bones and soft tissues. Therapeutic options for advanced URCT are limited. Pazopanib, a multi-tyrosine kinase and angiogenesis inhibitor, is currently used for managing advanced soft-tissue sarcoma. However, its efficacy in the treatment of URCT remains uncertain and has not been established. In this study, we have cultured and characterised a patient-derived URCT cell line to understand the in vitro growth properties and anti-cancer agent sensitivity. Materials and Methods: Primary cell culture was performed by mechanical and enzymatic dissociation of URCT tissue specimens to derive a cell line. Morphology, growth properties and immunological features characterised the cells. Further, the in vitro sensitivity for clinically used anti-cancer drugs was evaluated. Results: The URCT cell line was established from a high-grade round-cell tumour patient. The cells had mesenchymal morphology and showed cyclin B3 (CCNB3) positivity, which was confirmed in the tissue from the patient. The cells exhibited an anchorage-independent growth property and aggregated to form spheroids in a non-adherent in vitro system. Anti-cancer agents vincristine, doxorubicin, etoposide and pazopanib inhibited URCT cell proliferation. Pazopanib exhibited cytotoxic action in URCT cells, leading to cell death. Conclusions: This is an early report of cultured URCT cells expressing CCNB3, studied in vitro. The patient-derived model suggests the efficacy of pazopanib in URCT cells. The characterised line will be helpful to advance sarcoma studies.

The Capicua C1 Domain is Required for Full Activity of the CIC::DUX4 Fusion Oncoprotein.

Rearrangements between genes can yield neomorphic fusions that drive oncogenesis. Fusion oncogenes are made up of fractional segments of the partner genes that comprise them, with each partner potentially contributing some of its own function to the nascent fusion oncoprotein. Clinically, fusion oncoproteins driving one diagnostic entity are typically clustered into a single molecular subset and are often treated a similar fashion. However, knowledge of where specific fusion breakpoints occur in partner genes, and the resulting retention of functional domains in the fusion, is an important determinant of fusion oncoprotein activity and may differ between patients. This study investigates this phenomena through the example of CIC::DUX4, a fusion between the transcriptional repressor capicua (CIC) and the double homeobox 4 gene (DUX4), which drives an aggressive subset of undifferentiated round cell sarcoma. Using a harmonized dataset of over 100 patient fusion breakpoints from the literature, we show that most bona fide CIC::DUX4 fusions retain the C1 domain, which is known to contribute to DNA binding by wild type CIC. Mechanistically, deletion or mutation of the C1 domain reduces, but does not eliminate, activation of CIC target genes by CIC::DUX4. We also find that expression of C1-deleted CIC::DUX4 is capable of exerting intermediate transformation-related phenotypes compared with those imparted by full-length CIC::DUX4, but was not sufficient for tumorigenesis in a subcutaneous mouse model. In summary, our results suggest a supercharging role for the C1 domain in the activity of CIC::DUX4.

CIC::NUTM1 sarcomas occurred in soft tissues of upper limbs : a rare case report and literature review

BackgroundCIC-rearranged sarcomas (CRS) represent a new entity of undifferentiated small round cell sarcoma belonging to the Ewing-like sarcomas family. CRS are the most common type. Fusion partners for the CIC gene include DUX4, FOXO4, and the recently recognizedNUTM1. Rare cases of CIC::NUTM1 sarcoma in pediatric patients have recently been reported in brain, kidney, bone, and soft tissues. However, such cases have not been identified in the soft tissues of the limbs.Case presentationWe reported a case of CIC::NUTM1 sarcoma located in the right upper limb of an 18-year-old man. The tumor displayed morphologic features typical of CIC::DUX4 sarcomas, with small- to medium-sized round cells, a lobular pattern, focal spindling, myxoid stroma, and patchy necrosis. The tumor diffusely expressed NUTM1, was positive for WT1cter at weak to moderate intensity, and was focally positive for CD99, while it was negative for keratins, EMA, P40, MyoD1, myogenin, NKX2.2, BCOR, and pan-TRK. Fluorescence in situ hybridization analyses revealed cleavage of the CIC and NUTM1 genes.ConclusionCIC::NUTM1 sarcomas represent a novel molecular variant of CRS with a preference for the central nervous system and younger pediatric persons. Its morphology and phenotype may be mistaken for NUT carcinomas, and the behavior is more progressive than other forms of CRS. For this rare and newly discovered gene fusion variant, it is necessary to integrate molecular and immunohistochemical findings with morphologic features in the diagnosis of undifferentiated neoplasms.

Biological and therapeutic insights from animal modeling of fusion-driven pediatric soft tissue sarcomas.

Survival for children with cancer has primarily improved over the past decades due to refinements in surgery, radiation and chemotherapy. Although these general therapies are sometimes curative, the cancer often recurs, resulting in poor outcomes for patients. Fusion-driven pediatric soft tissue sarcomas are genetically defined by chromosomal translocations that create a chimeric oncogene. This distinctive, almost 'monogenic', genetic feature supports the generation of animal models to study the respective diseases in vivo. This Review focuses on a subset of fusion-driven pediatric soft tissue sarcomas that have transgenic animal tumor models, which includes fusion-positive and infantile rhabdomyosarcoma, synovial sarcoma, undifferentiated small round cell sarcoma, alveolar soft part sarcoma and clear cell sarcoma. Studies using the animal models of these sarcomas have highlighted that pediatric cancers require a specific cellular state or developmental stage to drive tumorigenesis, as the fusion oncogenes cause different outcomes depending on their lineage and timing of expression. Therefore, understanding these context-specific activities could identify targetable activities and mechanisms critical for tumorigenesis. Broadly, these cancers show dependencies on chromatin regulators to support oncogenic gene expression and co-opting of developmental pathways. Comparative analyses across lineages and tumor models will further provide biological and therapeutic insights to improve outcomes for these children.

Detection and surveillance of circulating tumor cells in sarcoma for predicting therapy response and prognosis.

e23573 Background: Generally sarcomas are categorized as soft tissue and bone sarcomas and represent heterogenous group of malignancies with more than 70 distinct histologic subtypes. Due to their rarity and heterogeneity, sarcomas present particular challenges for accurate diagnosis, prognosis, and treatment. The presence and persistence of circulating tumor cells (CTCs) in the peripheral blood are believed to be associated with poor prognosis and distant metastases. A blood-based CTCs test is thus greatly needed for monitoring disease progression and predicting clinical outcomes. Methods: In this study, we developed a CTCs test for the detection and surveillance of sarcoma patients’ CTCs, and subsequently explored its clinical value. 123 patients with sarcomas were enrolled as the cohort for serial CTCs tests. Dynamic CTCs counting, in combination with therapy evaluation and post-treatment follow-up, was used to explore predicting pre- and post-chemotherapy evaluation and prognosis. Results: Quantification of CTCs demonstrated a significant increase in sarcoma patients compared to healthy individuals. Furthermore, patients in stage III-IV had a higher CTC count than those in stage I-II (p<0.01), suggesting a correlation between CTCs and disease development. Remarkably, there was variation in the quantity of CTCs among different types of sarcomas. The highest number of CTCs was observed in undifferentiated small round cell sarcoma, followed by soft tissue tumor, while bone tumors had the lowest number. In addition, the number of CTCs in patients with positive PD-L1 expression was considerably greater than in those with negative expression (p<0.01). The count of CTCs shown a substantial decrease following neoadjuvant chemotherapy treatment. There was a decline in the number of CTCs both before and after postoperative adjuvant chemotherapy treatment, however, there was no significant statistical discrepancy. The number of CTCs at the baseline was shown to be considerably larger in the group of patients with progressive disease or stable disease (PD+SD) compared to the group with partial response or complete response (PR+CR) (p<0.05). This difference was particularly pronounced for interstitial-type CTCs (p<0.01). Conclusions: The rate of presence of CTCs in the peripheral blood of sarcoma patients is high, and patients with an increased percentage of CTCs after treatment have a poor treatment effect. The dynamic monitoring of changes in CTCs counts after treatment has clinical significance for the timely detection of recurrence or metastasis. CTCs also showed a clinical value in prediction of therapy efficiency or prognosis, and may be clinically valuable for the whole course management of patients.

Proteomic Characterization of Undifferentiated Small Round Cell Sarcomas With EWSR1 and CIC::DUX4 Translocations Reveals Diverging Tumor Biology and Distinct Diagnostic Markers

Undifferentiated small round cell sarcomas (USRS) of bone and soft tissue are a group of tumors with heterogenic genomic alterations sharing similar morphology. In the present study, we performed a comparative large-scale proteomic analysis of USRS (n = 42) with diverse genomic translocations including classic Ewing sarcomas with EWSR1::FLI1 fusions (n = 24) or EWSR1::ERG fusions (n = 4), sarcomas with an EWSR1 rearrangement (n = 2), CIC::DUX4 fusion (n = 8), as well as tumors classified as USRS with no genetic data available (n = 4). Proteins extracted from formalin-fixed, paraffin-embedded pretherapeutic biopsies were analyzed qualitatively and quantitatively using shotgun mass spectrometry (MS). More than 8000 protein groups could be quantified using data-independent acquisition. Unsupervised hierarchical cluster analysis based on proteomic data allowed stratification of the 42 cases into distinct groups reflecting the different molecular genotypes. Protein signatures that significantly correlated with the respective genomic translocations were identified and used to generate a heatmap of all 42 sarcomas with assignment of cases with unknown molecular genetic data to either the EWSR1- or CIC-rearranged groups. MS-based prediction of sarcoma subtypes was molecularly confirmed in 2 cases where next-generation sequencing was technically feasible. MS also detected proteins routinely used in the immunohistochemical approach for the differential diagnosis of USRS. BCL11B highly expressed in Ewing sarcomas, and BACH2 as well as ETS-1 highly expressed in CIC::DUX4-associated sarcomas, were among proteins identified by the present proteomic study, and were chosen for immunohistochemical confirmation of MS data in our study cohort. Differential expressions of these 3 markers in the 2 genetic groups were further validated in an independent cohort of n = 34 USRS. Finally, our proteomic results point toward diverging signaling pathways in the different USRS subgroups.

Differential cyclin-E1 expression in CIC-rearranged sarcoma

CIC-rearranged sarcoma (CRS) is a group of high-grade undifferentiated small round cell sarcomas examined as a separate entity in the current WHO classification; since it shows more aggressive clinical behavior and distinct morphological and molecular features compared to Ewing sarcoma (ES). As CCNE1 expression is associated with tumor growth in CIC::DUX4 sarcomas, we aimed to demonstrate the value of cyclin E1 expression in CRS. Cyclin E1 immunohistochemistry and break-apart FISH for EWSR1 and CIC gene rearrangements were performed on 3-mm tissue microarrays composed of 40 small round cell tumors. Five cases were classified as CRS, whereas 22 were ES and 13 were unclassified (EWSR1-/CIC-). Among all three diagnostic groups, we found cyclin E1 expression level to be higher in CRS (80 %) and unclassified groups (61.5 %) compared to ES (4.5 %, p < 0.001). In addition, high cyclin E1 expression levels were associated with higher mean age at diagnosis, presence of atypical histology and myxoid stroma, low CD99 expression, and presence of metastasis at diagnosis. The sensitivity and specificity of high cyclin E1 expression in detecting non-ES cases were 95.5 % and 66.7 %, respectively. However, the correlation between cyclin E1 expression level and survival was not statistically significant. This is the first study that shows cyclin E1 immunohistochemical expression in EWSR1-negative undifferentiated small cell sarcomas, particularly CRS.

A Rare Case of a Malignant Epithelioid Neoplasm With an Underlying Novel EWSR1::ZBT44 Fusion, Identified on Next-Generation Sequencing (NGS).

The clinicopathological spectrum of undifferentiated round cell sarcomas of bone and soft tissues is expanding after the 5th edition of the WHO classification. A 23-year-old male patient presented with a lump in his left thigh of 3 months' duration. Radiological examination revealed a well-defined, solid-cystic lobulated, soft tissue lesion in the proximal medial region of his left thigh, measuring 7.7 cm in the largest dimension. The referring diagnosis was an epithelioid sarcoma. Histopathological review of the tumor sections revealed a cellular tumor composed of malignant epithelioid to focally "rhabdoid-like" cells in a variable hyalinized and myxoid stroma with geographic areas of necrosis. In addition, there were areas reminiscent of hemangiopericytomatous vasculature. By immunohistochemistry, the tumor cells were diffusely positive for CD34, focally and distinctly for pan keratin (AE1/AE3). INI1/SMARCB1 and SMARCA4 (BRG1) were diffusely positive (normal). Next-generation sequencing with a wide sarcoma panel revealed EWSR1exon8::ZBT44exon4 fusion. The present example constitutes the first malignant epithelioid tumor with a hemangiopericytomatous growth pattern, exhibiting this rare fusion. The differential diagnoses of this tumor and their corresponding immunohistochemical profile are discussed. This example highlights the value of NGS in unraveling rare fusions and in differentiating these tumors from their several mimics.

Spontaneous expression of the CIC::DUX4 fusion oncoprotein from a conditional allele potently drives sarcoma formation in genetically engineered mice.

CIC::DUX4 sarcoma (CDS) is a rare but highly aggressive undifferentiated small round cell sarcoma driven by a fusion between the tumor suppressor Capicua (CIC) and DUX4. Currently, there are no effective treatments and efforts to identify and translate better therapies are limited by the scarcity of patient tumor samples and cell lines. To address this limitation, we generated three genetically engineered mouse models of CDS (Ch7CDS, Ai9CDS, and TOPCDS). Remarkably, chimeric mice from all three conditional models developed spontaneous soft tissue tumors and disseminated disease in the absence of Cre-recombinase. The penetrance of spontaneous (Cre-independent) tumor formation was complete irrespective of bi-allelic Cic function and the distance between adjacent loxP sites. Characterization of soft tissue and presumed metastatic tumors showed that they consistently expressed the CIC::DUX4 fusion protein and many downstream markers of the disease credentialing the models as CDS. In addition, tumor-derived cell lines were generated and ChIP-seq was preformed to map fusion-gene specific binding using an N-terminal HA epitope tag. These datasets, along with paired H3K27ac ChIP-sequencing maps, validate CIC::DUX4 as a neomorphic transcriptional activator. Moreover, they are consistent with a model where ETS family transcription factors are cooperative and redundant drivers of the core regulatory circuitry in CDS.

Next-generation sequencing for pediatric CNS tumors: does it add value in a middle-income country setup?

Advances in molecular diagnostics led to improved targeted interventions in the treatment of pediatric CNS tumors. However, the capacity to test for these is limited in LMICs, and thus their value needs exploration. We reviewed our experience with NGS testing (TruSight RNA Pan-Cancer-seq panel) for pediatric CNS tumors at KHCC/Jordan (March/2022-April/2023). Paraffin blocks' scrolls were shipped to the SickKids laboratory based on the multidisciplinary clinic (MDC) recommendations. We reviewed the patients' characteristics, the tumor types, and the NGS results' impact on treatment. Of 237 patients discussed during the MDC meetings, 32 patients (14%) were included. They were 16 boys and 16 girls; the median age at time of testing was 9.5 years (range, 0.9-21.9 years). There were 21 samples sent at diagnosis and 11 upon tumor progression. The main diagnoses were low-grade-glioma (15), high-grade-glioma (10), and other histologies (7). Reasons to request NGS included searching for a targetable alteration (20) and to better characterize the tumor behavior (12). The median turnaround time from samples' shipment to receiving the results was 23.5 days (range, 15-49 days) with a median laboratory processing time of 16 days (range, 8-39 days) at a cost of US$1,000/sample. There were 19 (59%) tumors that had targetable alterations (FGFR/MAPK pathway inhibitors (14), checkpoint inhibitors (2), NTRK inhibitors (2), and one with PI3K inhibitor or IDH1 inhibitor). Two rare BRAF mutations were identified (BRAFp.G469A, BRAFp.K601E). One tumor diagnosed initially as undifferentiated round cell sarcoma harbored NAB2::STAT6 fusion and was reclassified as an aggressive metastatic solitary fibrous tumor. Another tumor initially diagnosed as grade 2 astroblastoma grade 2 was reclassified as low-grade-glioma in the absence of MN1 alteration. NGS failed to help characterize a tumor that was diagnosed histologically as small round blue cell tumor. Nine patients received targeted therapy; dabrafenib/trametinib (6), pembrolizumab (2), and entrectinib (1), mostly upon tumor progression (7). In this highly selective cohort, a high percentage of targetable mutations was identified facilitating targeted therapies. Outsourcing of NGS testing was feasible; however, criteria for case selection are needed. In addition, local capacity-building in conducting the test, interpretation of the results, and access to "new drugs" continue to be a challenge in LMICs.

Clinicopathological Features of Three Rare EWSR1::NFATC2 Sarcomas of Bone and Soft Tissues.

Certain undifferentiated round cell sarcomas displaying EWSR1::NFATC2 fusion have recently been reported, mostly in the bones. This report presents clinicopathological features of 3 additional EWSR1::NFATC2 fusion sarcomas of bone and soft tissues. We present 2 soft tissue and 1 bone tumors: A 62-year-old man with pain and a slowly growing, 8-cm-sized soft tissue mass in the anterolateral compartment of his right calf, along with multiple pulmonary metastatic lesions; a 63-year-old man with a 5-cm sized axillary mass of 4 months duration and a cystic renal mass; and a 53-year-old man with a complaint of leg pain was found to have a 2-cm diameter, intramedullary, lytic mass in the diaphysis of his left femur. Microscopic examination of the tumors in all patients revealed round to epithelioid cells arranged in cords and trabeculae in a myxohyaline stroma. Immunohistochemically, the tumor cells were positive for MIC2/CD99 (3/3), EMA (3/3), NKX3.1 (3/3), NKX2.2 (2/2), CD10 (2/2), and aggrecan (1/1), while negative for S100P and GFAP. Various keratins were also negative except focal AE1/AE3 positivity in the third tumor. By fluorescence in-situ hybridization, 2 tumors (#1 and #3) revealed EWSR1 gene rearrangement and amplification. Furthermore, 2 tumors (#1 and #2) displayed EWSR1ex8::NFATC2ex3 fusion with next-generation sequencing (NGS). The first patient was offered chemotherapy. However, he died of pulmonary metastasis. This report highlights the value of combining histopathological features and immunostains such as NXK3.1, NKX2.2, CD10, and aggrecan, along with EWSR1 testing for triaging these tumors for rare gene fusions by NGS that has prognostic implications.

Expression of NKX2.2 in undifferentiated small round cell sarcoma and its diagnostic value

Expression of NKX2.2 in undifferentiated small round cell sarcoma and its diagnostic value

Clinicopathological Features of Primary Renal Mesenchymal Neoplasms in Adults: A Cross-sectional Study

Introduction: Renal mesenchymal tumours are a subset of primary renal tumours arising from the mesenchymal tissue in the kidney. They are a heterogeneous group of mostly benign tumours that exhibit varied behaviours and molecular characteristics. Aim: To analyse the wide spectrum of histological subtypes, their unique clinical presentation, and pathological features of primary renal mesenchymal neoplasms. Materials and Methods: This was a retrospective crosssectional observational clinicopathological study conducted in Department of General Pathology, Christian Medical College, tertiary care hospital, Vellore, Tamil Nadu, India, looking at data of adult Primary Renal Mesenchymal Tumours (PRMT) for a 15- year period between January 2006 and March 2021. Clinical details such as age, presenting symptoms, and tumour size were obtained from the hospital information system. Histopathology and immunohistochemical slides were reviewed for all the cases. Additional Immunohistochemistry (IHC) and molecular studies were performed for the undifferentiated sarcomas. The tumours were categorised into three groups as recommended by the World Health Organisation (WHO) 2020 classification of soft-tissue tumours based on biological behaviour: benign, intermediate, and malignant. Continuous variables are expressed as mean and Standard Deviation (SD). Comparison of categorical variables between groups was performed using the Chi-squared test. Continuous variables between groups were compared using the Student’s t-test for significance. A p-value of less than 0.05 was considered statistically significant. Results: Of the 2164 nephrectomies performed for neoplastic conditions, 97 (4.5%) were diagnosed as renal mesenchymal tumours. There were 59 (60.8%) benign, 10 (10.3%) with intermediate biologic behaviour and 28 (28.9%) malignant tumours. The mean ages at presentation were 40.5 years for benign, 43.4 years for intermediate, and 41 years for malignant tumours, respectively. Haematuria was seen in 11 of 38 (29%) intermediate grade and malignant tumours and in only 6 of 59 (10%) benign tumours, a difference that was statistically significant (p-value=0.017). Malignant tumours 24 of 28 (86%) were more likely to be larger (&gt;7 cm) when compared to benign tumours 28 of 59 (47%) at the time of presentation (p-value=0.027). Classical angiomyolipomas constituted 53/59 (90%) of the benign tumours. Of the 10 intermediate grade tumours, epithelioid angiomyolipomas and solitary fibrous tumours were the most common, accounting for 50% and 30% of the intermediate group, respectively. Undifferentiated small round cell sarcoma was the most common malignant neoplasm, making up 12/28 (43%) of the malignant tumours. Conclusion: The present study found that renal mesenchymal tumours constitute a small but unique group of renal tumours. They are predominantly benign, but up to a quarter are malignant. Malignant tumours tend to be larger and more often present with haematuria. The present study highlights the importance of IHC in the diagnosis of intermediate and malignant mesenchymal tumours and the requirement of exhaustive molecular studies individually tailored to the immuno-profile of malignant tumours.

Molecular and therapeutic advancements in Capicua (CIC)-rearranged sarcoma.

Capicua (CIC)-rearranged sarcomas are an aggressive subset of undifferentiated round cell sarcomas. CIC::DUX4, the proto-typical CIC fusion oncoprotein is associated with rapid clinical progression and chemotherapy resistance leading to poor clinical outcomes. Recent studies have identified additional CIC fusions (CIC::NUTM1, CIC::FOXO4, and CIC::LEUTX) that largely retain CIC-binding specificity but leverage C-terminal binding partners (NUTM1, FOXO4, and LEUTX) to potentially activate transcriptional programs that drive oncogenesis. Moreover, the recent development of preclinical models to study CIC::DUX4 sarcoma have advanced our understanding of the underlying biological mechanisms and uncovered key dependencies that can be translated into rational therapies. In this review, we will highlight these recent advancements in CIC-rearranged sarcoma biology with a vision for clinical translation to improve patient outcomes.