Soft Tissue Sarcoma Cell Research Articles

Hypoxia-related proteins VEGF A and CA IX and resistance of soft tissue sarcoma cells to chemotherapy: <i>ex vivo</i> pilot study

Introduction. The identification of predictive factors is a cornerstone task of modern oncology. The development of new targeted drugs determines the need for prediction of chemosensitivity of each patient to the prescribed therapy, in this regard, the search for biomarkers of predictive response to therapy is actively conducted.The study objective to investigate the relationship between tumor cell resistance and the expression levels of CA IX (carbonic anhydrase IX) and VEGF A (vascular endothelial growth factor А) in patient-derived cultures of soft tissue sarcomas.Materials and methods: ex vivo soft tissue sarcoma cell culture, resazurin test, immunoblotting.Results. We obtained 46 ex vivo samples of soft tissue sarcoma cultures for which chemosensitivity to doxorubicin, ifosfamide, docetaxel, gemcitabine, and their combinations was assessed by the resazurin cytotoxicity test. We analyzed the relationship between the expression of hypoxic proteins VEGF A and CA IX and the resistance to drugs. A correlation between the CA IX expression in hypoxia and cell resistance to ifosfamide and its combination with doxorubicin was found. Soft tissue sarcomas with high VEGF A index were resistant to doxorubicin, docetaxel, and its combination with gemcitabine (p <0.05).Conclusion. The data obtained on patient-derived cultures indicate the relationship between hypoxic signaling and resistance of soft tissue sarcomas to chemotherapeutics.

Abstract 3937: PRMT5 inhibition suppress soft tissue sarcomas tumorigenicity through the regulation of glucose metabolism

Abstract Introduction: Soft tissue sarcomas (STS) are a heterogeneous group of rare tumors representing 1% of solid tumors in adults and 15% in children. Despite adequate locoregional treatment, up to 40% of patients will develop metastatic disease. Doxorubicin represents the 1st line standard of care for patients in this setting. However, its activity is limited with a response rate of only 10%, a progression-free survival of less than 6-months. New therapeutic strategies are therefore urgently needed. PRMT5 belongs to a family of enzymes that catalyze the methylation of arginine residues on both histones and non-histone proteins. Recent evidence shows that PRMT5 is upregulated in a wide variety of cancers. Its expression and activity are associated with tumorigenesis and with a decrease in patient survival in several cancers, a rationale for developing highly potent inhibitors. Methods: We analyzed the transcriptome data from 255 translocation-related STS and 389 complex genomics STS with annotated clinical data from the French Sarcoma Group. We then investigated the anti-tumor activity of the first -in class PRMT5 specific inhibitor, GSK3226593 in a panel of 7 cell lines and in two xenograft models established at Institut Bergonié (Bordeaux, France). Results: We found that high PRMT5 gene expression level was significantly associated with worse metastases-free survival in both translocation-related and complex genomics STS (p=0.003 and p=0.009, respectively), support the relevance of PRMT5 as a potential therapeutic target in this indication. We then found that pharmacological inhibition of PRMT5 by using GSK3226593, suppressed the viability and the cell proliferation of 7 STS cell lines in vitro (IC50 range between 0.01 and 1.4 µM) as well as in vivo in two models of xenograft cell lines. This inhibition of cell growth was concomitant with an increase of p53 and p21 expression. However, anti-tumor activity of GSK3226593 was independent of p53 since the compound was still active in shp53 expressing cell lines. Moreover, we did not notice any significant effect of GSK3226593 on cell cycle phases or apoptosis. To decipher the mechanism of action of PRMT5 inhibition in STS, we did a comparative transcriptomic analysis (RNAseq) of STS cells before and after 10 days of treatment with GSK3226593. Gene Ontology and gene set enrichment analysis revealed downregulation of metabolic processes especially glycolysis and mTORC1 pathways. A specific metabolic pathway panel was used to confirm and deepen the underlying molecular mechanism. The downregulation of several genes by GSK3226593 such as PCK2 was confirmed at the protein level by Western blot. Glycolysis inhibition was confirmed by a decrease of glucose uptake. Conclusion: PRMT5 inhibition has anti-tumor activity in vitro and in vivo through the regulation of STS cell metabolism providing a clear rationale for its clinical investigation in this indication. Citation Format: Stephanie Verbeke, Laura Leroy, Aurelien Bourdon, Vanessa Chaire, Elodie Richard, Jean-Philippe Guegan, Alban Bessede, Antoine Italiano. PRMT5 inhibition suppress soft tissue sarcomas tumorigenicity through the regulation of glucose metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3937.

Abstract 1509: Genome-wide CRISPR/Cas9 library screening identified THRAP3 as a critical driver for resistance to ATR inhibitor in sarcomas revealing synthetic lethality with ATM inhibition

Abstract Background: ATR plays a crucial role in maintaining genomic integrity by responding to a large spectrum of DNA damage, including double strand breaks (DSBs) that interfere with replication. We have previously demo started that ATR inhibition is associated with anti-tumor activity in pre-clinical models of soft-tissue sarcomas (STS) (Laroche et al., 2020). We decided to investigate the mechanisms of resistance to ATR inhibition in sarcomas. Methods: We used a CRISPRa was to identify genes that mediate resistance to AZD6738 in STS cells. The anti-tumor activity of AZD6738 (ATR inhibitor) and AZD0156 (ATM inhibitor) was investigated in a panel for 10 STS cell lines and two patient-derived xenografts (PDX) established at Institut Bergonié (Bordeaux, France). Results: We used a CRISPR approach in a STS cell line resistant to AZD6738 and we made a negative-selection screen, to identify genes whose silencing conferred sensitivity to low doses of ATR inhibition. Among these genes, we identified THRAP3 which encodes a protein phosphorylated in response to DNA damage and primarily by ATR. THRAP3 is involved in the maturation and further export of transcripts encoding the ATM and its depletion results in deficient processing of transcripts encoding the ATM kinase. We validate the result of our CRISPR analysis by demonstrating that selective THRAP3 inhibition reduce significantly ATRi IC50. Given the role of role of THRAP3 on the regulation of ATM activity, we decided to investigate the potential synergy between ATR and ATM inhibition in vivo and in vitro. AZD6738 and AZD0156 displayed a synergistic or additive effect in above all the STS cell lines tested. We noted that ATRi treatment reduced phosphorylation of the ATR kinase target CHK1 but increased ATM phosphorylation, suggesting that ATM pathway takes over for DNA Damage Response and cells survival. In contrast, both ATR and ATM pathway were inhibited when AZD6738 and AZD0156 were used in combination, as shown by reduction of CHK1 and ATM phosphorylation. We validated our in vitro results in an in vivo model of liposarcoma xenograft in which AZD6738 and AZD0156 had synergistic anti-tumor activity. Conclusion: Our study demonstrates synthetically lethal interactions between ATM and ATR in vitro and in vivo. This combination deserves further investigaitons in sarcomas. Citation Format: Mariella Spalato, Audrey Laroche-Clary, Vanessa Chaire, Antoine Italiano. Genome-wide CRISPR/Cas9 library screening identified THRAP3 as a critical driver for resistance to ATR inhibitor in sarcomas revealing synthetic lethality with ATM inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1509.

Abstract CT206: EVT801, a novel selective VEGFR-3 inhibitor targeting tumor angiogenesis, started enrollment for its phase I first-in-human study

Abstract Introduction: EVT801 is a highly selective, orally available VEGFR3 inhibitor that strongly inhibits angiogenesis without inducing hypoxia, considered one of the main causes of cancer-associated immunosuppression. EVT801 has shown compelling single agent efficacy in multiple in vivo models. In addition, combination of EVT801 and Immune Checkpoint Therapy (ICT) agents shows additive effects, and thus holds promise for combination treatment without induction of hypoxia-induced-immunosuppression. A phase I clinical trial is underway. Methods: The phase I trial will consist of two sequential stages. During the first stage, EVT801 will be administered to patients with advanced solid tumors in a multiple ascending dose study using an accelerated 3+3 design (1 patient per dose until grade 2 toxicities are observed) in up to 48 patients in 8 dose levels. The primary objective is to determine the maximum tolerated dose (MTD) and recommended phase 2 dose (RP2D). Stage 2 will focus on validation of this RP2D in two 6-patient cohorts diagnosed with soft tissue sarcoma or renal cell carcinoma. These patients will participate in intensive analyses involving several biomarkers to better understand the pharmacological activity of the drug. A potential third stage, consisting of a multiple ascending dose evaluation of the combination of EVT801 with immuno-oncology drugs, may be added to the ongoing trial, pending further technical discussions with physicians and regulators. In addition to conventional measures of safety, tolerability, efficacy, and pharmacokinetics, the phase I study will employ a rich suite of histological, immunological, and radiological biomarkers to provide early insights into the activity of EVT801. Citation Format: Carlos Gomez roca, Philippe Cassier, Marie Mandron, Myriam Estrabaut, Nathalie Delesque-Touchard, Adam C. Smith, Amy Klawitter, Leesa Gentry, Pierre Fons, Michael R. Paillasse, Lise Davenne, Michael Fitzgerald, James Garner, Jean-Pierre Delord. EVT801, a novel selective VEGFR-3 inhibitor targeting tumor angiogenesis, started enrollment for its phase I first-in-human study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr CT206.

Abstract 2564: CDK9 inhibition via KB-0742 is a potential strategy to treat transcriptionally addicted cancers

Abstract Lineage-specific transcriptional networks drive cellular differentiation and development. Disruption of these specific cell programs can result in cancer and create a subset of tumors that are “transcriptionally addicted.” Sarcomas, for example, are characterized by an oncogenic fusion protein consisting of a FET family RNA-binding protein fused to a transcription factor (TF). The oncogenic fusions result in a restructuring of the transcriptome promoting cancer. In chordoma, TBXT (brachyury)—normally involved in notochord differentiation—is aberrantly expressed, resulting in promotion of cancer. As these oncogenic TFs are difficult to target directly, we and others have proposed targeting associated transcriptional regulators to inhibit their activity. Cyclin-dependent kinase 9 (CDK9) interacts with TFs to promote activation of target genes and promotes transcription elongation through phosphorylation of RNA polymerase II. We have developed a potent, selective, and orally bioavailable CDK9 inhibitor, KB-0742. Here we present the preclinical activity of KB-0742 in models of sarcoma and chordoma. We first evaluated KB-0742 activity in sarcoma in a 300-immortalized cell line screen containing 18 soft tissue sarcoma cell lines. The median IC50 of the cell lines in the study was 0.8705 µM. Sarcoma cell lines were enriched for sensitivity to KB-0742 with 61% (11/18) of the lines with an IC50 below the median. The 11 cell lines had a median IC50 of 0.679 µM. We then evaluated the activity of KB-0742 in 5 patient-derived cell line (PDC) models, with all 5 showing a cytotoxic response to treatment as measured by negative GRmax values. Last, a single patient-derived organoid (PDO) model of adult rhabdomyosarcoma was evaluated. KB-0742 treatment resulted in an IC50 of 2.75 µM and a max inhibition rate of 98.61%. For chordoma we examined the activity of KB-0742 in vivo using 2 patient-derived xenograft (PDX) models. In model CF466, we observed a dose-dependent response with increased TGI and greater reductions in RNA polymerase II phosphorylation in tumors treated with KB-0742 at 60 mg/kg as compared to 30 mg/kg. We then evaluated KB-0742 as a single agent and in combination with afatinib in the model CF539. KB-0742 as a single agent showed similar TGI activity as afatinib, whereas the combination showed increased response as compared to the two single-agent arms. These data show that transcriptionally addicted tumors are sensitive to CDK9 inhibition via KB-0742 treatment, and support the continued development of our compound to potentially treat sarcoma and chordoma. KB-0742 is currently being evaluated in a phase 1/2 clinical trial (NCT04718675) for relapsed or refractory solid tumors or non-Hodgkin lymphoma. Once a recommended phase 2 dose is established, expansion cohorts for patients with sarcoma, chordoma, and other transcriptionally addicted tumors may be opened. Citation Format: Melinda A. Day, Douglas C. Saffran, Tressa Hood, Nikolaus Obholzer, Akanksha Pandey, Charles Y. Lin, Pavan Kumar, Daniel M. Freed, Jorge DiMartino. CDK9 inhibition via KB-0742 is a potential strategy to treat transcriptionally addicted cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2564.

Ribavirin inhibits cell proliferation and metastasis and prolongs survival in soft tissue sarcomas by downregulating both protein arginine methyltransferases 1 and 5.

Protein arginine methyltransferases 1 and 5 (PRMT1 and PRMT5) are frequently overexpressed in diverse types of cancers and correlate with poor prognosis, thus making these enzymes potential therapeutic targets. The aim of this study was to assess and elucidate the anti-tumour effect and epigenetic regulatory mechanism of ribavirin in soft tissue sarcomas (STS). We showed that ribavirin inhibited growth and metastasis and prolonged survival in animals bearing STS cells by downregulating the mRNA and protein levels of PRMT1/PRMT5 and attenuating the accumulation of asymmetric and symmetric di-methylation of arginine (ADMA and SDMA). Furthermore, ribavirin lowered the permeability of the peritoneum in KM mice bearing S180 ascites via decreasing the level of vascular endothelial growth factor (VEGF). Ribavirin was a potent inhibitor of cell proliferation and metastasis in STS cells through downregulation of both type I PRMT1 and type II PRMT5. Ribavirin could be used to enhance the efficacy of doxorubicin in STS allograft tumour models.

Identifying the uncommon solitary fibrous tumour in a rare location - A case report.

Introduction and importanceThe authors describe a case work up of an unusual natal cleft soft tissue tumour which eventually was concluded as solitary fibrous tumour. Solitary fibrous tumour is an uncommon fibroblastic tumour which commonly presents in pleural region and very rarely in extrapleural location such as natal cleft. Accurate identification of this tumour, considering the indeterminate nature of the tumour irrespective of the location, avoided misdiagnosis and played a vital role in follow up management of the patient.Case presentation43 year old female patient presented with a painless lump in the natal cleft region for a short duration of 7 months with no other significant medical or surgical or family or psychosocial history. She was not on any regular medications.Clinical discussionDue to unusual location of the tumour broad range of differential diagnosis including benign and malignant entities were discussed based on histological findings and immunophenotyping. Mammary type myofibroblastoma, glomus tumour, clear cell renal cell carcinoma and clear cell sarcoma of soft tissue were few to note. STAT6 immunohistochemical stain played a crucial role in unravelling this case. Due to gradual increase in size of the lump complete surgical excision was performed with no local recurrence of the tumour.ConclusionIt is important to recognise this rare mesenchymal tumour with intermediate malignant potential occurring at uncommon natal cleft region for which complete surgical enucleation is curative.

An investigation of kV mini-GRID spatially fractionated radiation therapy: dosimetry and preclinical trial

Objective. To develop and characterize novel methods of extreme spatially fractionated kV radiation therapy (including mini-GRID therapy) and to evaluate efficacy in the context of a pre-clinical mouse study. Approach. Spatially fractionated GRIDs were precision-milled from 3 mm thick lead sheets compatible with mounting on a 225 kVp small animal irradiator (X-Rad). Three pencil-beam GRIDs created arrays of 1 mm diameter beams, and three ‘bar’ GRIDs created 1 × 20 mm rectangular fields. GRIDs projected 20 × 20 mm2 fields at isocenter, and beamlets were spaced at 1, 1.25, and 1.5 mm, respectively. Peak-to-valley ratios and dose distributions were evaluated with Gafchromic film. Syngeneic transplant tumors were induced by intramuscular injection of a soft tissue sarcoma cell line into the gastrocnemius muscle of C57BL/6 mice. Tumor-bearing mice were randomized to four groups: unirradiated control, conventional irradiation of entire tumor, GRID therapy, and hemi-irradiation (half-beam block, 50% tumor volume treated). All irradiated mice received a single fraction of 15 Gy. Results. High peak-to-valley ratios were achieved (bar GRIDs: 11.9 ± 0.9, 13.6 ± 0.4, 13.8 ± 0.5; pencil-beam GRIDs: 18.7 ± 0.6, 26.3 ± 1.5, 31.0 ± 3.3). Pencil-beam GRIDs could theoretically spare more intra-tumor immune cells than bar GRIDs, but they treat less tumor tissue (3%–4% versus 19%–23% area receiving 90% prescription, respectively). Bar GRID and hemi-irradiation treatments significantly delayed tumor growth (P < 0.05), but not as much as a conventional treatment (P < 0.001). No significant difference was found in tumor growth delay between GRID and hemi-irradiation. Significance. High peak-to-valley ratios were achieved with kV grids: two-to-five times higher than values reported in literature for MV grids. GRID irradiation and hemi-irradiation delayed tumor growth, but neither was as effective as conventional whole tumor uniform dose treatment. Single fraction GRID therapy could not initiate an anti-cancer immune response strong enough to match conventional RT outcomes, but follow-up studies will evaluate the combination of mini-GRID with immune checkpoint blockade.

HDAC Inhibition for Optimized Cellular Immunotherapy of NY-ESO-1-Positive Soft Tissue Sarcoma.

Adoptive cell therapy with NY-ESO-1-specific T cells is a promising option for the treatment of soft tissue sarcoma (STS) but achieves only transient tumor control in the majority of cases. A strategy to optimize this cell therapeutic approach might be the modulation of the expression of the cancer-testis antigen NY-ESO-1 using histone deacetylase inhibitors (HDACis). In this study, the ex vivo effect of combining NY-ESO-1-specific T cells with the clinically approved pan HDACis panobinostat or vorionstat was investigated. Our data demonstrated that STS cells were sensitive to HDACis. Administration of HDACi prior to NY-ESO-1-specific T cells exerted enhanced lysis against the NY-ESO-1+ STS cell line SW982. This correlated with an increase in the NY-ESO-1 and HLA-ABC expression of SW982 cells, as well as increased CD25 expression on NY-ESO-1-specific T cells. Furthermore, the immune reactivity of NY-ESO-1-specific CD8+ T cells in terms of cytokine release was enhanced by HDACis. In summary, pretreatment with HDACis represents a potential means of enhancing the cytotoxic efficacy of NY-ESO-1-specific T cells against NY-ESO-1-positive STS.

HIWI2 induces G2/M cell cycle arrest and apoptosis in human fibrosarcoma via the ROS/DNA damage/p53 axis

AimsPiwi-like RNA-mediated gene silencing 4 (PIWIL4) or HIWI2, are seen deregulated in human cancers and possibly play critical roles in tumorigenesis. It is unknown what role HIWI2 plays in the regulation of fibrosarcoma, an early metastatic lethal type of soft tissue sarcoma (STS). The present study aimed to investigate the role of HIWI2 in the tumorigenesis of fibrosarcoma. Main methodsThe expression of HIWI2 in HT1080 fibrosarcoma cells was determined by qRT-PCR and western blotting. The MTT assay, colony formation assay, cell cycle, and PE-AnnexinV/7AAD apoptosis assay using flow cytometry, DNA laddering assay, comet assay, and γH2AX accumulation assay were performed to study the effect of HIWI2 overexpression in HT1080 cells. Further, the effect of silencing of HIWI2 was determined by cell viability assay, transwell migration, and invasion assay. Key findingsHIWI2 is under-expressed in STS cell lines and tissues, which is associated with poor disease-free survival, disease-specific survival, and progression-free survival of the patients. Overexpression of HIWI2 in HT1080 cells causes DNA damage by increasing intracellular ROS by inhibiting the expression of antioxidant genes (SOD1, SOD2, GPX1, GPX4, and CAT). Furthermore, an increase in H2AX phosphorylation was observed, which activates p53 that promotes p21 expression and caspase-3 activation, leading to G2/M phase cell cycle arrest and apoptosis. HIWI2 silencing, on the contrary, promotes cell growth, migration, and invasion by activating MMP2 and MMP9. SignificanceThese results are the first to show that HIWI2 acts as a tumor suppressor in fibrosarcoma by modulating the ROS/DNA damage/p53 pathway.

Standardization of evaluation method and prognostic significance of histological response to preoperative chemotherapy in high-grade non-round cell soft tissue sarcomas

BackgroundPreoperative chemotherapy is widely applied to high-grade localized soft tissue sarcomas (STSs); however, the prognostic significance of histological response to chemotherapy remains controversial. This study aimed to standardize evaluation method of histological response to chemotherapy with high agreement score among pathologists, and to establish a cut-off value closely related to prognosis.MethodsUsing data and specimens from the patients who had registered in the Japan Clinical Oncology Group study, JCOG0304, a phase II trial evaluating the efficacy of perioperative chemotherapy with doxorubicin (DOX) and ifosfamide (IFO), we evaluated histological response to preoperative chemotherapy at the central review board.ResultsA total of 64 patients were eligible for this study. The percentage of viable tumor area ranged from 0.1% to 97.0%, with median value of 35.7%. Regarding concordance proportion between pathologists, the weighted kappa coefficient (κ) score in all patients was 0.71, indicating that the established evaluation method achieved substantial agreement score. When the cut-off value of the percentage of the residual tumor area was set as 25%, the p-value for the difference in overall survival showed the minimum value. Hazard ratio of the non-responder with percentage of the residual tumor < 25%, to the responder was 4.029 (95% confidence interval 0.893–18.188, p = 0.070).ConclusionThe standardized evaluation method of pathological response to preoperative chemotherapy showed a substantial agreement in the weighted κ score. The evaluation method established here was useful for estimating of the prognosis in STS patients who were administered perioperative chemotherapy with DOX and IFO.Trial registrationUMIN Clinical Trials Registry C000000096. Registered 30 August, 2005 (retrospectively registered).

Early Complications after Limb Salvage Procedures: Prospective Cross Sectional Study

ABSTRACT Introduction: Sarcomas are tumors that are mesenchymal in origin involving bone and soft tissues occurring in all age groups. Giant cell tumor, although benign, is locally aggressive and can also destroy the bone. Amputation was once considered primary treatment but now with advancements in diagnostic imaging modalities, surgical procedures and adjuvant therapies, limb salvage surgeries have become the preferred treatment modality. The primary objective of our study was to determine the incidence of early complications occurring within the first thirty days in patients who underwent limb salvage surgery at our hospital. The secondary objective was to assess the association of these complications with patient-related factors, surgical modality, tumor characteristics and neoadjuvant therapy. Methodology: All the patients with bone or soft tissue sarcoma and aggressive giant cell tumors of the extremities who underwent wide excision and limb salvage surgery from February 2020 to December 2021 were included in this study. Data were collected prospectively on follow-up visits in the clinic for one month. Data were compiled and analyzed using SPSS version 22. A p-value of &lt;0.05 was considered significant throughout the study. Results: A total of 60 patients were included in this study. The mean age of the patients was 39.90 years. Bone and soft tissue tumors were found in 35 and 25 patients respectively. A total of 13 patients experienced postoperative complications within 30 days of surgery. The association of these postoperative complications with gender distribution, prior comorbid conditions, type of tumor, anatomical location of the tumor and neoadjuvant therapy was found to be statistically insignificant. Conclusion: The most frequently occurring postoperative complications are wound-related and the incidence of these complications is independent of the proposed risk factors. Research registry number: Researchregistry4695. Highlights

Ribonucleotide reductase subunit M2 is a potential prognostic marker and therapeutic target for soft tissue sarcoma.

Soft tissue sarcomas (STSs) are highly aggressive malignant tumors that exhibit poor therapeutic outcomes. Hence, we aimed to track down a potential gene that can be used as a prognostic marker and therapeutic target for this malignancy. We integrated omics analysis of clinical data and in vitro studies and identified Ribonucleotide reductase subunit M2 (RRM2) as a potential oncogene associated with STS prognosis. We found RRM2 is highly expressed in STS cell lines and tissues. STS patients with increased RRM2 levels showed worse overall survival, disease-free survival, progression-free survival, and disease-specific survival. Further, overexpression of RRM2 in HT1080 cells induces proliferation, migration, invasion, and colony formation, whereas its silencing arrest the cell cycle at G0/G1 phase and induces apoptosis. Taken together, we established RRM2 to be positively associated with oncogenesis and prognosis of STS and therefore could be a promising prognostic marker and therapeutic target.

Trends of lymph node sampling and metastasis in pediatric and young adult patients with clear cell, epithelioid, and synovial sarcomas.

Clear cell sarcoma of soft tissue (CCS), epithelioid sarcoma, and synovial sarcoma are rare tumors historically identified as high risk for lymph node metastasis. This study investigates incident nodal metastasis and associated survival in children and young adults with these subtypes. Using the National Cancer Database (2004-2015), we created a retrospective cohort of 1303 patients (aged ≤25years) who underwent local control therapy for CCS, epithelioid sarcoma, and synovial sarcoma. Kaplan-Meier curves estimated overall survival (OS) by subtype. Stratifying on subtype, Cox regressions assessed OS by lymph node sampling status and nodal metastasis. There were 103 (7.9%) patients with CCS, 221 (17.0%) with epithelioid sarcoma, and 979 (75.1%) with synovial sarcoma. Lymph node sampling was more frequent in patients with CCS (56.3%) and epithelioid sarcoma (52.5%) versus synovial sarcoma (20.5%, p<.001). Synovial sarcoma metastasized to lymph nodes less frequently than CCS or epithelioid sarcoma (2.1% vs. 14.6% and 14.9%, p<.001). Across all subtypes, lymph node metastasis was associated with inferior OS (HR 2.02, CI 1.38-2.95, p<.001). Lymph node sampling was associated with improved OS in CCS (HR 0.35, CI: 0.15-0.78, p=.010), inferior OS in synovial sarcoma (HR 1.60, CI: 1.13-2.25, p=.007), and no statistical association with OS in epithelioid sarcoma. Lymph node metastasis is rare in children and young adults with synovial sarcoma. Lymph node sampling procedures were not consistently performed for patients with CCS or epithelioid sarcoma, but improved OS supports routine lymph node sampling in children and young adults with CCS.

Whole‐exome sequencing in clear cell sarcoma of soft tissue uncovers novel prognostic categorization and drug targets

Whole‐exome sequencing in clear cell sarcoma of soft tissue uncovers novel prognostic categorization and drug targets

Abstract P082: Distinct and significant anti-cancer efficacy of plinabulin in patient derived small cell lung cancer (SCLC) 3D soft agar clonogenic assays

Abstract Plinabulin (Plin) is a new chemical entity and a selective immunomodulating microtubule-targeting agent (SIMBA), which exerts immune-enhancing effects through increasing dendritic cell (DC) maturation and DC-dependent antigen presentation to CD4+ and CD8+ T-Cells. Here we evaluated whether Plin also exerts direct anti-cancer effects against approximately 80 patient derived (PDX) models. Plin was characterized for its ability to inhibit anchorage independent growth and ex vivo colony formation of PDX tumor cells in semi-solid medium. The compound was investigated in PDX models of various cancer types, using a 3D clonogenic assay in a 96-well format. Briefly, tumor cell suspensions were prepared from subcutaneous xenografts in NMRI nu/nu mice and seeded into 96 well ultra-low attachment plates within a layer of semi-solid medium. Plin was added to the assay plate at either day 2 or day 7 and tested at 9 concentrations in half-log increments up to a concentration of 3 µM. Supernatant was exchanged 24 h after dosing and cells were further incubated at 37°C and 7.5 % CO2 up to a total assay time of 8 or 13 days. Colony counts based on image analysis were utilized to evaluate efficacy. Plin inhibited tumor colony formation in a concentration-dependent manner. With Plin treatment beginning 2 days after PDX cell seeding, the most sensitive cancer types were small cell lung cancer (mean absolute IC70 = 0.035 μM; n = 7), bladder cancer (mean absolute IC70 = 0.038 μM; n = 9), and soft tissue sarcoma (mean absolute IC70 = 0.057 μM; n = 10). Melanoma models were observed to be the least sensitive to Plin monotherapy, with a mean absolute IC70 of 1.105 μM (n = 19). With Plin treatment beginning 7 days after PDX cell seeding, based on absolute IC70 values, 9 out of 68 tumor models were sensitive towards plinabulin. The most sensitive histotypes were gastric cancer (Asian, mean abs. IC70 = 0.319 µM, n = 3), small cell lung cancer (mean abs. IC70 = 0.385 µM, n = 7; 3 below 50 nM), osteosarcoma (mean abs. IC70 = 0.624 µM, n = 3), and central nervous system cancer (mean abs. IC70 = 1.521 µM, n = 6). Overall, the most responsive models based on IC70 were the small cell lung cancer models 2156 (abs. IC70 = 0.015 µM), 1129 (abs. IC70 = 0.032 µM) and 650 (abs. IC70 = 0.032 µM), and the osteosarcoma model 1186 (abs. IC70 = 0.027 µM). Soft tissue sarcoma models (n = 8), Her2-enriched breast cancer models (n = 6), melanoma models (n = 9), bladder cancer models (n = 6), and gastric cancer models (Caucasian, n = 3) were observed to be the least sensitive cancer types with a mean IC70 value &amp;gt; 3 µM. In conclusion, using patient-derived tumor models in a 3D clonogenic assay, SCLC tumor types were the most sensitive to Plin. These results provide mechanistic support for the preliminary positive results recently reported with Plin in combination with Nivolumab and Ipilimumab in SCLC patients (Malhotra ASCO 2021). Citation Format: George K. Lloyd, James Tonra, Claudia Goettlich, Tobias Deigner, Ramon Mohanlal, Lan Huang. Distinct and significant anti-cancer efficacy of plinabulin in patient derived small cell lung cancer (SCLC) 3D soft agar clonogenic assays [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P082.

Abstract P233: TAS0953/HM06 is effective in preclinical models of diverse tumor types driven by RET alterations

Abstract Fusions involving RET receptor tyrosine kinase are a common driver of tumors across different tissue types, such as lung, thyroid, colorectal, soft tissue and others. TAS0953/HM06 (hereby referred to as HM06) is a novel 2ndgeneration RET-specific inhibitor that is effective against RET solvent front (G810) and gatekeeper (V804) mutations. Here, we evaluated the efficacy of HM06 in lung and thyroid carcinomas, and soft-tissue sarcoma cell lines and PDXs derived from RET inhibitor-naive tumor samples or from tumors with acquired resistance to selpercatinib. HM06 was more effective than the RET multi-kinase inhibitors cabozantinib and vandetanib, and as effective as selpercatinib and pralsetinib in inhibiting growth of patient-derived and isogenic lung, thyroid and sarcoma cell lines (IC50=0.02-0.1 µM) harboring different RET fusions (KIF5B-RET, CCDC6-RET, TRIM33-RET, SPECCL1-RET) or activating mutations (RET C634W). Growth of non-tumor cells was up to 80-fold less sensitive to HM06 (IC50= 1.6 µM). Treatment of RET fusion-positive lung cancer cells with HM06 resulted in a dose-dependent inhibition of RET phosphorylation (Y905 and Y1062) and the downstream effectors AKT, ERK1/2, p70S6K and S6. Caspase 3/7 activity and markers of apoptosis (BIM, cleaved PARP) were induced by HM06 to a similar extent as pralsetinib and selpercatinib (dose range: 0.05-1 µM). HM06 induced changes in the core mediators of cell cycle regulation (upregulation of p27, downregulation of CCND1) and suppressed expression of MYC and ETV5. In vivo, HM06 blocked tumor growth and/or induced regression of up to 65% in seven patient-derived xenograft (PDX) models with RET fusions (five NSCLC PDXs, one sarcoma PDX and one NSCLC cell-line xenograft) to a similar extent as pralsetinib and selpercatinib. However, 6 weeks after cessation of treatment of the SPECCL1-RET-driven sarcoma PDX model, growth of tumors treated with HM06 was suppressed completely, whereas 3/5 pralsetinib-treated tumors and 1/5 selpercatinib-treated tumor regrew. Combination of HM06 and the MET inhibitor capmatinib effectively blocked growth of PDX tumors in a model that was derived from a patient sample that expressed RET fusion and METamplification, and was resistant to selpercatinib. These results suggest that HM06 may be an effective therapy for RET-driven tumors in a tissue-type agnostic manner and can effectively address common on-target and off-target resistance mechanisms such as RET G810X and V804X mutations. HM06 is currently in a phase 1 and 2 clinical trial for patients with advanced solid tumors with RET gene abnormalities (margaRET, NCT 04683250). Citation Format: Igor Odintsov, Renate I. Kurth, Kota Ishizawa, Lukas Delasos, Allan J.W. Lui, Inna Khodos, Connor J. Hagen, Qing Chang, Marissa S. Mattar, Morana Vojnic, Siddharth Kunte, Annalisa Bonifacio, Claudio Giuliano, Elisa De Stanchina, Emily Cheng, Emanuela Lovati, Marc Ladanyi, Romel Somwar. TAS0953/HM06 is effective in preclinical models of diverse tumor types driven by RET alterations [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P233.

Intracranial mesenchymal tumors with FET-CREB fusion are composed of at least two epigenetic subgroups distinct from meningioma and extracranial sarcomas.

‘Intracranial mesenchymal tumor, FET‐CREB fusion‐positive’ occurs primarily in children and young adults and has previously been termed intracranial angiomatoid fibrous histiocytoma (AFH) or intracranial myxoid mesenchymal tumor (IMMT). Here we performed genome‐wide DNA methylation array profiling of 20 primary intracranial mesenchymal tumors with FET‐CREB fusion to further study their ontology. These tumors resolved into two distinct epigenetic subgroups that were both divergent from all other analyzed intracranial neoplasms and soft tissue sarcomas, including meningioma, clear cell sarcoma of soft tissue (CCS), and AFH of extracranial soft tissue. The first subgroup (Group A, 16 tumors) clustered nearest to but independent of solitary fibrous tumor and AFH of extracranial soft tissue, whereas the second epigenetic subgroup (Group B, 4 tumors) clustered nearest to but independent of CCS and also lacked expression of melanocytic markers (HMB45, Melan A, or MITF) characteristic of CCS. Group A tumors most often occurred in adolescence or early adulthood, arose throughout the neuroaxis, and contained mostly EWSR1‐ATF1 and EWSR1‐CREB1 fusions. Group B tumors arose most often in early childhood, were located along the cerebral convexities or spinal cord, and demonstrated an enrichment for tumors with CREM as the fusion partner (either EWSR1‐CREM or FUS‐CREM). Group A tumors more often demonstrated stellate/spindle cell morphology and hemangioma‐like vasculature, whereas Group B tumors more often demonstrated round cell or epithelioid/rhabdoid morphology without hemangioma‐like vasculature, although robust comparison of these clinical and histologic features requires future study. Patients with Group B tumors had inferior progression‐free survival relative to Group A tumors (median 4.5 vs. 49 months, p = 0.001). Together, these findings confirm that intracranial AFH‐like neoplasms and IMMT represent histologic variants of a single tumor type (‘intracranial mesenchymal tumor, FET‐CREB fusion‐positive’) that is distinct from meningioma and extracranial sarcomas. Additionally, epigenomic evaluation may provide important prognostic subtyping for this unique tumor entity.

A novel SMARCA2-CREM fusion: expanding the molecular spectrum of intracranial mesenchymal tumors beyond the FET genes

A novel histomolecular tumor of the central nervous system, the “intracranial mesenchymal tumor (IMT), FET-CREB fusion-positive” has recently been identified in the literature and will be added to the 2021 World Health Organization Classification of Tumors of the Central Nervous System. However, our latest study using DNA-methylation analyses has revealed that intracranial FET-CREB fused tumors do not represent a single molecular tumor entity. Among them, the main subgroup presented classical features of angiomatoid fibrous histiocytoma, having ultrastructural features of arachnoidal cells, for. Another tumor type with clear cell component and histopathological signs of aggressivity clustered in close vicinity with clear cell sarcoma of soft tissue. Herein, we report one case of IMT with a novel SMARCA2-CREM fusion which has until now never been described in soft tissue or the central nervous system. We compare its clinical, histopathological, immunophenotypic, genetic and epigenetic features with those previously described in IMT, FET-CREB fusion-positive. Interestingly, the current case did not cluster with IMT, FET-CREB fusion-positive but rather presented histopathological (clear cell morphology with signs of malignancy), clinical (with a dismal course with several recurrences, metastases and finally the patient’s death), genetic (fusion implicating the CREM gene), and epigenetic (DNA-methylation profiling) similarities with our previously reported clear cell sarcoma-like tumor of the central nervous system. Our results added data suggesting that different clinical and histomolecular tumor subtypes or grades seem to be included within the terminology “IMT, FET-CREB fusion-positive”, and that further series of cases are needed to better characterize them.

The C-Circle Biomarker Is Secreted by Alternative-Lengthening-of-Telomeres Positive Cancer Cells inside Exosomes and Provides a Blood-Based Diagnostic for ALT Activity.

Simple SummaryA clinical test for alternative-lengthening-of-telomeres (ALT) could assist with cancer diagnosis and monitoring of disease progression. ALT-targeted anticancer treatments are being developed; however, there is no appropriate companion ALT diagnostic. The C-Circle biomarker is the only known ALT specific molecule and the C-Circle Assay the only quantitative ALT assay that is amenable to clinical use. We show here that C-Circles are secreted by ALT+ cancer cell lines inside the exosomes and are protected from nucleases. We also show that secreted C-Circles, like intracellular C-Circles, are an ALT-specific biomarker, and in high-risk neuroblastoma, the blood-based C-Circle Assay has the potential to be an accurate diagnostic for ALT cancer activity. Therefore, the secretion of C-Circles by ALT+ cancer cells in the exosomes provides a stable blood-based biomarker and, potentially, a clinical diagnostic for ALT activity, which is required for the development of ALT-targeted therapies as well as for the diagnosis and monitoring of ALT+ cancer.C-Circles, self-primed telomeric C-strand templates for rolling circle amplification, are the only known alternative-lengthening-of-telomeres (ALT)-specific molecule. However, little is known about the biology of C-Circles and if they may be clinically useful. Here we show that C-Circles are secreted by ALT+ cancer cells inside exosomes, and that a blood-based C-Circle Assay (CCA) can provide an accurate diagnostic for ALT activity. Extracellular vesicles were isolated by differential centrifugation from the growth media of lung adenocarcinoma, glioblastoma, neuroblastoma, osteosarcoma, and soft tissue sarcoma cell lines, and C-Circles were detected in the exosome fraction from all eleven ALT+ cancer cell lines and not in any extracellular fraction from the eight matching telomerase positive cancer cell lines or the normal fibroblast strain. The existence of C-Circles in ALT+ exosomes was confirmed with exosomes isolated by iodixanol gradient separation and CD81-immunoprecipitation, and C-Circles in the exosomes were protected from nucleases. On average, 0.4% of the total ALT+ intracellular C-Circles were secreted in the exosomes every 24 h. Comparing the serum-based and tumor-based CCAs in 35 high risk neuroblastoma patients divided randomly into ALT+ threshold derivation and validation groups, we found the serum-based CCA to have 100% sensitivity (6/6), 70% specificity (7/10), and 81% concordance (13/16). We conclude that the secretion of C-Circles by ALT+ cancer cells in the exosomes provides a stable blood-based biomarker and a potential clinical diagnostic for ALT activity.