Undifferentiated Round Cell Sarcomas Research Articles

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.

Para-Testicular Mesenchymal Chondrosarcomas: an Updated Review of Clinical Insights and Therapeutic Considerations

Para-testicular mesenchymal chondrosarcoma, an uncommon malignancy originating in the para-testicular region, poses distinctive diagnostic and therapeutic challenges. This comprehensive review offers insights into its incidence, epidemiology, clinical presentation, histopathological features, diagnostic methods, treatment strategies, and prognosis. The recent WHO classification for Soft Tissue Tumors and Bone is a pivotal tool in refining pathologic diagnoses and therapeutic decisions. Notably, the inclusion of skilled clinicians’ insights underscores the pivotal role of accurate pathologic diagnoses in guiding treatment choices. Advancements include risk assessment for solitary fibrous tumors, the recognition of NTRK-rearranged soft tissue tumors as an “emerging entity,” and the classification of undifferentiated round cell sarcomas. The tumor’s rarity, intrinsic and technological complexities, and limited educational impact contribute to the intricate landscape of diagnosis and treatment for mesenchymal tumors.

Primary CIC-rearranged Gastric sarcoma: A rare diagnosis

Gastric sarcoma is rare, accounting for 1-3% of all gastric neoplasms. WHO has published a new classification of soft tissue tumors and a group of undifferentiated round cell sarcomas, including CIC-rearranged sarcoma has been added. CIC-rearranged sarcoma has not been reported till date. We report a case of CIC-rearranged gastric sarcoma which was managed with chemotherapy and multivisceral resection.

Undifferentiated Small Round Cell Sarcoma of the Hypopharynx in a 7-Month-Old Patient.

Undifferentiated small round cell sarcoma is an extremely rare and highly aggressive tumor touching the soft tissues and bones. Here, we report the case of a 7-month-old girl who presented to our department with respiratory distress due to a hypopharyngeal mass causing airway obstruction. The patient underwent a tracheotomy and had a direct laryngoscopy. Histological examination confirmed the diagnosis of an undifferentiated small round cell sarcoma. The patient received chemotherapy and radiotherapy and underwent neck dissection. One year later, the patient was diagnosed with local recurrence as well as metastatic cervical nodes and liver and pulmonary metastases. The case is original by the tumor's location and the age of onset. To the best of our knowledge, this could be the first case in English and French literature reporting an undifferentiated small round cell sarcoma affecting the hypopharynx among infants. The purpose of this article is to report our case and discuss its clinical and anatomopathological features as well as its prognosis and treatment options.

PATH-63. NEXT GENERATION SEQUENCING FOR CNS TUMORS IN CHILDREN; DOES IT ADD VALUE IN A MIDDLE-INCOME COUNTRY SETUP?

Abstract INTRODUCTION Advances in molecular diagnostics led to improved targeted interventions in the treatment of pediatric CNS tumors. However, the capacity to do these diagnostics is limited in LMICs, and consequently their value is questionable. METHODS We reviewed our experience of Next Generation Sequencing (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 Sickkids laboratory based on local multidisciplinary team recommendations. We reviewed patients’ clinical characteristics, tumor types and the impact on treatment. RESULTS There were 16 boys and 16 girls; median age at time of testing was 9.5 years (range, 0.9- 21.9 years). Twenty samples were sent at diagnosis and twelve upon tumor progression. Main diagnoses were Low-grade glioma (15), High-grade glioma (10), and other tumors (7). Reasons for NGS testing were: to find a molecular target (20), and to characterize more tumor/behavior (12). The median time from shipping the samples to receive the NGS result was 22 days (range, 15-49 days) at a cost of US$1000. Twenty (63%) tumors had targetable alterations (MAPK/PI3K inhibitors (16), NTRK inhibitors (2), check point inhibitors (2)). Two rare BRAF mutations were identified (BRAFp.G469A, BRAFp.K601E). One tumor diagnosed initially as undifferentiated round-cell sarcoma found to have NAB2::STAT6 fusion and re-diagnosed as aggressive metastatic solitary fibrous tumor. Eight patients received targeted therapy; Dabrafenib/Trametinib (5), Pembrolizumab (2), Entrectinib (1), while radio-chemotherapy was used in the others. Three patients died from disease progression (one used Entrectinib). CONCLUSIONS Sent abroad NGS testing was feasible, however local capacity building is necessary. In this highly selected tumor cohort, high percentage of targetable alterations were identified. NGS was helpful to characterize tumors more and to offer alternative therapies. Nevertheless, interpretation of the significance of NGS results and access to those “new” therapies continues to be a challenge in LMICs

The efficacy of the TRK inhibitor entrectinib in patients with extracranial &lt;i&gt;NTRK&lt;/i&gt; fusion-positive tumors

Somatic translocations involving the NTRK genes occur in 0.34–2.2% of all malignant neoplasms in children. TRK inhibitors whose efficacy has been demonstrated in prospective clinical studies expand treatment options for patients with solid tumors harboring NTRK gene rearrangements. The aim of our study was to summarize the first Russian experience with the use of the TRK inhibitor entrectinib in patients with extracranial NTRK fusion-positive solid tumors included in the compassionate use program. This study was approved by the Independent Ethics Committee and the Academic Council of the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology. The study included 8 patients who had been treated at the Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology and the N.N. Blokhin National Medical Research Center of Oncology. The main criteria for inclusion in the compassionate use program were a confirmed rearrangement of either NTRK1/2/3 genes in a solid tumor in patients with unresectable disease for whom no effective standard systemic therapy was available, progressive or recurrent disease during therapy prescribed according to the established diagnosis, risk group and risk stratification criteria, and the infeasibility of non-mutilating radical surgery. The median age at diagnosis was 4.3 months (range 1.2–83.6). The male to female ratio was 1:1. The primary site distribution was as follows: head and neck (n = 6; 75%), chest wall (n = 1; 12.5%), pelvis (n = 1; 12.5%). None of the patients had regional lymph node involvement or distant metastases at diagnosis. The distribution by histology (according to histopathology reports) was as follows: infantile fibrosarcoma (n = 4; 50%), undifferentiated round cell sarcoma, low-grade (n = 1; 12.5%), undifferentiated spindle cell sarcoma, high-grade (n = 1; 12.5%), NTRK-rearranged spindle cell sarcoma, low-grade (n = 1; 12.5%), spindle cell tumor associated with an NTRK rearrangement (n = 1; 12.5%). Immunohistochemistry (IHC) with a pan-Trk monoclonal antibody was performed in 7/8 (87.5%) patients. Pan-Trk IHC was positive in 4/7 (57%) patients. Rearrangements in the NTRK1 and NTRK3 genes were confirmed in all the patients. The final methods used for the detection of fusion transcripts were as follows: reverse transcription polymerase chain reaction (n = 4; 50%) and RNA-based next-generation sequencing (n = 4; 50%). NTRK1 and NTRK3 gene translocations were detected in 3/8 (37.5%) and 5/8 (62.5%) patients, respectively. The following fusion transcripts were identified: ETV6::NTRK3 (n = 4), DIP2C::NTRK3 (n = 1), TPR::NTRK1 (n = 1), TPM3::NTRK1 (n = 1), MYH10::NTRK1 (n = 1). One (12.5%) patient received entrectinib as first-line therapy, other patients (7/8, 87.5%) received entrectinib as secondor subsequent-line therapy. Three (37.5%) patients had undergone surgery before treatment with entrectinib: 2 had R2 resection, 1 had R0/R1 resection (resection margins were not evaluated). None of the patients received radiation therapy. The median duration of entrectinib therapy at the time of analysis was 11.8 months (range 2.3–20.1). Delayed surgery was performed in 2/8 patients; according to the histopathology reports, they achieved grade IV pathomorphosis. Three patients experienced adverse events during treatment with entrectinib. The median time to adverse events was 0.23 months (range 0.2–7.96). Three patients required temporary interruption in treatment to relieve symptoms, a subsequent dose reduction by one dose level was necessary when resuming therapy in two patients. The median follow-up since diagnosis was 19.5 months (range 14.9–75.0). All the patients included in our analysis were alive, three of them had no radiologic evidence of disease. Fifty percent of the patients completed targeted therapy, another 50% of the patients continued treatment with entrectinib. Complete and very good partial response was achieved in 3/8 and 2/8 patients, respectively. Partial response, minor partial response and stable disease were observed in one patient each. These results indicate high efficacy and safety of entrectinib in pediatric patients with extracranial NTRK fusion-positive solid tumors. Further studies are needed to determine the therapeutic potential of TRK inhibitors in the treatment of different solid malignant neoplasms in children and to assess long-term treatment results.

Genetically Engineered Mouse Models of CIC-DUX4 Sarcoma Reveal an Unexpected Sensitivity to Histone Deacetylase (HDAC) Inhibition

Undifferentiated small round cell sarcomas constitute a highly aggressive subtype of soft tissue sarcoma that affects adolescents and young adults. Frequently, these cancers are caused by a gene fusion between the DNA binding domain of capicua (CIC), a canonical tumor suppressor that normally acts as a transcriptional repressor, with the transcriptional activation domain of double homeobox 4 (DUX4). Given the rare nature of CIC-DUX4 sarcoma (CDS), obtaining patient tissues to study the mechanisms of disease is very challenging. Moreover, conducting clinical trials to identify radiosensitizers or other novel treatments is not possible underscoring the need for a primary animal model. Here, we report the development and characterization of the first genetically engineered mouse model of CDS and the use of this model to investigate novel therapies. Using Cre-loxP technology, three genetically engineered mouse models of CDS were developed. The resulting tumors were characterized by hematoxylin and eosin (H&E) staining, immunohistochemistry (IHC), and RNA-sequencing. Cell lines from multiple primary and metastatic tumors were derived and used for Chromatin Immunoprecipitation (ChiP) sequencing, Rapid Immunoprecipitation mass spectrometry of Endogenous proteins (RIME), and targeted drug screening. In all three models, spontaneous (Cre-independent) recombination occurred in chimeric animals resulting in the formation of soft tissue tumors, widespread metastasis to the lungs, liver, and brain, and 100% lethality within 4-8 weeks of life. The tumors histologically resembled human CDS and stained positive for an HA (hemagglutinin) epitope tag confirming transgene expression. Of 108 chimeric pups (generation 0, G0), only one male bred before succumbing to metastatic disease. G2 progeny now exhibit resistance to spontaneous recombination and tumor development enabling reliable propagation of one of the models for experimentation. Using tumor derived cell lines for integrative analyses of multi-omics datasets, we find evidence for a model where CIC-DUX4 functions as a neomorphic and oncogenic transcriptional activator, which cooperates with ETS family transcription factors to drive the CDS transcriptional program. Lastly, small molecule epigenetic drug screens in mouse and human CDS cell lines point to an unexpected sensitivity to HDAC inhibitors, which we hypothesize may be attributable to ETS factor hyperactivity. Genetically engineered mouse models of CIC-DUX4 sarcoma form tumors that recapitulate the human disease. Using mouse tumors and tumor-derived cell lines, we identify a mechanistic role and potential therapeutic vulnerability that converges on the ETS family transcription factors.

Giant middle mediastinal lesions: when tumor size correlates with mesenchymal origin-a retrospective single-center analysis.

The International Thymic Malignancy Interest Group (ITMIG) proposed an internationally accepted division of the mediastinum into three compartments based on computed tomography (CT): anterior (prevascular), middle (visceral) and posterior (paravertebral) compartment. There is no generally accepted definition for the term "giant" when applied to middle mediastinal lesions. We defined the term "giant" and described our surgical experience in treating patients with giant lesions of the middle mediastinum. CT imaging of patients operated in our center from January 2016 to August 2021 for mediastinal lesions was reviewed. Lesions were categorized to one of the ITMIG-defined compartments. Lesion size at diagnosis was measured at its largest diameter on axial CT imaging. Giant middle mediastinal lesions were defined as lesions having a size ≥90th percentile of our middle mediastinal lesion cohort. Patients with giant middle mediastinal lesions were further analyzed. Thirty-six patients (23%) had lesions located in the middle mediastinal compartment. Most common diagnoses were mediastinal cysts (n=10, 28%), metastatic lesions (n=6, 17%), lymphomas (n=5, 14%), and sarcomas (n=3, 8%). Ninetieth percentile lesion size was 73 mm. As per definition, four patients had giant middle mediastinal lesions. All these four lesions were of mesenchymal origin including oesophageal leiomyoma, synovial sarcoma, leiomyosarcoma and undifferentiated round cell sarcoma. Resection was performed through posterolateral thoracotomy or sternotomy, with or without cardiopulmonary bypass. The term "giant" could be defined as a mass larger or equal to 73 mm. This definition selected specifically lesions with mesenchymal origin and may therefore guide diagnostic algorithm and patient management.

458 Primary Renal Mesenchymal Neoplasms: A Retrospective Clinicopathological Study

Abstract Aim Mesenchymal tumors are rare primary neoplasms of the kidney with limited demographic data in literature. We sought to study the prevalence of this rare subset of tumors in adults, at our tertiary care hospital focusing on the pathological and radiological characteristics. Method Clinico-radiological and intra-operative details were retrospectively analyzed for adult primary renal mesenchymal neoplasms received between 2006 and 2021 and correlated with the histopathology data. Results Of 2164 nephrectomies performed for neoplastic conditions, 97 (4.5%) were diagnosed as renal mesenchymal tumors. There were 59 (60.8%) benign, 10 (10.3%) with intermediate biologic behavior and 28 (28.9%) malignant tumors. The mean age was similar across these categories. Hematuria was seen in 11 of 38 (29%) intermediate grade and malignant tumors and in only 6 of 59 (10%) benign tumors a difference that was statistically significant (p = 0.017). Tumors ≥ 7cm were more likely to be malignant/intermediate grade than benign (p = 0.027). Classical angiomyolipomas constituted 90% of the benign tumors. Epithelioid angiomyolipomas and solitary fibrous tumors were the common intermediate grade tumors. Undifferentiated small round cell sarcoma was the most common malignant neoplasm. Immunohistochemical and molecular studies were useful for confirmation of diagnosis, particularly in cases of intermediate/malignant tumors. Conclusions Mesenchymal tumors constitute 4.5% of adult primary renal tumors, are predominantly benign tumors but are malignant in 1/4 th of cases. Malignant tumors are typically larger and more often present with hematuria. Immunohistochemistry (IHC) and molecular studies play a role in the diagnosis of intermediate and malignant mesenchymal tumors.

Identification of novel actionable SNVs using targeted comprehensive genome profiling in patients with sarcoma.

e23520 Background: The utility of comprehensive genome profiling is not well validated in early-stage sarcoma. The 5-year survival rate for metastatic sarcoma is less than 20%. The application of comprehensive Next Generation Sequencing (NGS) on tumor DNA has led to the identification of a multitude of actionable alterations in oncogenes &amp; tumor suppressor genes alongside the detection of immunotherapy markers like microsatellite instability, tumor mutational burden, and homologous recombinant deficiency. Methods: We retrospectively analyzed ccfDNA and gDNA in 13 sarcoma patients by targeted Next Generation Sequencing (NGS) using OncoIndx proprietary gene panel that targets exonic regions of 600 cancer-related genes. Illumina-compatible libraries were prepared by the target hybridization method and sequenced on Nextseq 2000 platform in a pair-end fashion. Variant calling was performed using an indigenously developed bioinformatics pipeline. Results: Here we report for the first time the identification of several, novel mutations in rare sarcomas such as IDH2R140W (in epithelioid hemangioendothelioma), CTNNB1D32V (in undifferentiated small round cell sarcoma) and DDR2M754I (in osteosarcoma). Furthermore, we observed, JAK1 amplification and BRAF-GUSBP1 fusion in osteosarcoma and a sarcomatoid carcinoma case, respectively. JAK1 amplification and DDR2M754I were found in the same patient. The IDH2 mutation and JAK1 amplification could find potential applications for JAK1 and IDH2 inhibitors respectively. Similarly, cancers having DDR2 mutations are known to respond to multikinase inhibitors such as Imatinib and Dasatinib, thus making a strong case for the potential repurposing of these drugs in sarcomas. ~93% of patients demonstrated genome-wide instabilities as evidenced by LOH, LST, TAI, and high TMB. Homologous Recombinant deficiency was found in 66.7% of patients. Although this is a small patient population, the high HRD+ percentages point to the underappreciated importance of HRD testing in sarcoma and herald the use of PARPi therapy for sarcoma. Conclusions: We identified several actionable mutations in rare sarcoma cases. These findings highlight the power of comprehensive genome profiling like the OncoIndx panel and warrant further studies on the role of IDH2, JAK1, and BRAF fusion in sarcomas. Moreover, they also present a strong potential case for repurposing of TKI such as Imatinib and Dasatinib in sarcomas.

The clinical value of tumor-informed minimal residual disease detection in sarcoma.

11510 Background: The high recurrence and metastasis rates lead to poor survival of patients with sarcoma. At present, circulating tumor DNA-based minimal residual disease (MRD) has been studied in varies solid tumors and proved to be valuable in guiding treatment and predicting recurrence, but its application in sarcomas is rarely reported. Methods: Tumor tissue samples were used for whole exome sequencing (WES) and clonal mutations were selected for the customized MRD panel. Blood samples were collected for MRD detection. Experiments were conducted at OrigiMed, a College of American Pathologists accredited and Clinical Laboratory Improvement Amendments certified laboratory. Results: A total of 84 sarcoma patients including 47 bone tumor (BT), 32 soft-tissue sarcoma (STS), and 5 undifferentiated small round cell sarcoma (USRCS) patients were enrolled. Patients consisted of 49 (58.3%) males and 35 (41.7%) females, with a median age of 23 years old (1-85 years old). Based on WES, we found that 64% (4936/7715) of the mutated genes were unique to each patient, and 70.4% (859/1220) of selected tumor-informed single nucleotide variants (SNV) were variants with unknown significance, suggesting that tumor-informed MRD is superior to panel-based MRD in sarcomas. In this cohort, 78.6% (66/84) of patients successfully constructed MRD panels, including 85.1% (40/47) of BT patients, 75% (24/32) of STS patients, and 40% (2/5) of USRCS patients. The main reason for failure was the scarcity of SNVs detected. Preoperative MRD detection was performed on 6 cases and revealed a positive rate of 100%. MRD detection was performed twice on 2 patients during preoperative neoadjuvant therapy. For the patient with constant allele frequencies detected throughout the treatment course, imaging showed tumor enlargement; While for another patient with a trend of reduction in allelic frequency, imaging showed tumor shrinkage and necrotic tumor tissue was observed in surgery one month later. These two patients had not recurred after surgery at the time of analysis. Post-surgery MRD detection were performed on 27 patients, of which 1 STS and 7 BTs were positive (30%, 8/27). Three of them confirmed recurrence by imaging at 12, 30, and 101 days after detection of positive MRD, respectively. However, there were still no imaging recurrence in the other patients so far, including the 5 remaining patients with positive MRD and 19 patients with negative MRD. Follow-up is still ongoing. Conclusions: MRD tests had been successfully performed in most of the sarcoma patients. Due to the heterogeneity of sarcomas, about 64% of mutated genes of sarcomas are unique individually, which may be more suitable for monitoring with tumor-informed MRD panel. Tumor-informed MRD could be used to predict the efficacy of preoperative neoadjuvant therapy and discover recurrence earlier than imaging in sarcoma.

Impact of age on survival according to molecular tumor findings in children and adolescents with soft-tissue and bone sarcoma: The BIOSCA project

BackgroundAdolescents (15–19 years) with sarcoma are known to have significantly worse survival than children (0–14 years). One possible reason may be that the adolescent sarcomas exhibit specific biological characteristics resulting in differences in clinical presentation and treatment resistance behaviors. The BIOSCA project aims to further explore these age-related differences in survival accounting for molecular tumor characteristic in children and adolescents with sarcoma. MethodsA retrospective national population-based observational study with documented somatic genetic analyses was conducted between 2011 and 2016 of all patients aged from 0 to 17 years with a diagnosis of sarcoma using the National Registry of Childhood Cancers Database. ResultsA total of 1637 children (0–9years: 40%), preadolescents (10–14years: 35%) and adolescents (15–17 years: 25%) with a diagnosis of bone (N = 845) or soft-tissue (N = 792) sarcoma were included. Adolescents had significantly worse outcome for undifferentiated small round cell sarcoma (USRCS), alveolar rhabdomyosarcoma (ARMS), and epithelioid sarcoma. Five-year overall survivals were worse among CIC-rearranged USRCS cases (47% [95%CI:21–69]) as compared to other USRCS, and PAX3::FOXO1 ARMS patients (44% [95%CI:32–55]) as compared to other ARMS. Adjusting for stage and genomic-profiling status, adolescents with USRCS were 1.6-fold more likely to die than children (P = 0.05), while the difference in survival between age of ARMS patients was weaken. Indeed, the prevalence of PAX3::FOXO1 increased significantly with age. ConclusionAge was an independent prognostic factor of outcome only in patients with USRCS, while the association between age and survival of patients with ARMS could be partly explained by differences in prevalence of PAX3::FOXO1.