Soft Tissue Sarcoma Cell Research Articles

The BCL-2 selective inhibitor ABT-199 sensitizes soft tissue sarcomas to proteasome inhibition by a concerted mechanism requiring BAX and NOXA

Soft tissue sarcomas (STS) are a heterogeneous group of malignancies predominantly affecting children and young adults. Despite improvements in multimodal therapies, 5-year survival rates are only 50% and new treatment options in STS are urgently needed. To develop a rational combination therapy for the treatment of STS we focused on ABT-199 (Venetoclax), a BCL-2 specific BH3-mimetic, in combination with the proteasome inhibitor bortezomib (BZB). Simultaneous inhibition of BCL-2 and the proteasome resulted in strongly synergistic apoptosis induction. Mechanistically, ABT-199 mainly affected the multidomain effector BAX by liberating it from BCL-2 inhibition. The combination with BZB additionally resulted in the accumulation of BOK, a BAX/BAK homologue, and of the BH3-only protein NOXA, which inhibits the anti-apoptotic protein MCL-1. Thus, the combination of ABT-199 and BZB sensitizes STS cells to apoptosis by simultaneously releasing several defined apoptotic restraints. This synergistic mechanism of action was verified by CRISPR/Cas9 knock-out, showing that both BAX and NOXA are crucial for ABT-199/BZB-induced apoptosis. Noteworthy, efficient induction of apoptosis by ABT-199/BZB was not affected by the p53 status and invariably detected in cell lines and patient-derived tumor cells of several sarcoma types, including rhabdomyo-, leiomyo-, lipo-, chondro-, osteo-, or synovial sarcomas. Hence, we propose the combination of ABT-199 and BZB as a promising strategy for the treatment of STS, which should warrant further clinical investigation.

TGF-βR inhibitor SB431542 restores immune suppression induced by regulatory B-T cell axis and decreases tumour burden in murine fibrosarcoma.

The contribution of immune cells in soft tissue sarcomas (STS) is not completely known and understanding their role is very essential for employing immunotherapy strategies. Here, we show that murine fibrosarcoma-conditioned medium promoted total spleen cell proliferation but inhibited T cell responses to mitogenic and allo-antigen-mediated stimulation. This increased proliferation was found to be in B cells resulting in generation of Breg further leading to Treg population. This was found to be the same in vitro and in vivo. The phenotype of these B cells was CD19+CD81+CD27+CD25+PD-L1hi and they secreted both IL-10 and TGF-β. These tumor evoked Bregs (tBreg), when co-cultured with B depleted T cells, suppressed their proliferation in response to anti-CD3/CD28 stimulation. tBreg-induced suppression of T cell responses wasnot abrogated by the inhibition or neutralization of IL-10 but by the small molecule inhibitor of TGFβ Receptor type I, SB431542. While SB531542 per se was not cytotoxic to tumor cells, administration of SB431542 in tumor-bearing mice (TBM) significantly reduced the tumor burden. In addition, the treatment significantly reduced Treg cells and rescued proliferation of T cells in response to mitogen and allo-antigen. Collectively, our results identify that tumor evoked Breg cells mediate T cell immune suppression through TGFβ-mediated pathway and that targeting the Breg-Treg axis can be potentially used as an immunotherapy agent.

COMPARATIVE ANALYSIS OF MIGRATION ACTIVITY AND INVASIVE POTENTIAL OF CULTURED SOLID TUMOR CELLS

Understanding of the sequence of events that ensure invasiveness of malignant cells is important for prognostic purposes. The study of the cellular and molecular pathways in the metastatic process lays the foundation for further progress in the treatment of cancer patients.Purpose: a comparative analysis of in vitro migration and invasion of human solid tumor cells isolated from primary and metastatic lesions.Material and Methods. Cell cultures of skin melanoma (SM, n=29), renal cell cancer (RCC, n=2), colorectal cancer (CRC, n=1), soft tissue and bone sarcomas (STBS, n=39) isolated from solid human tumors were studied. Cell migration and invasion were assessed using xCelligence (ACEA Bioscience Inc., USA).Results. All solid tumor cell cultures demonstrated in vitro invasive potential (IP), which was 73.79 % for RCC; 53.16 % for SM; 43.96 % for STBS and 5.16 % for CRC. The rates of migration and invasion (SlopeInv) in STBS cells were higher than those in SM cells (39.33 and 25.3 μm/h (p<0.05), 95.32 and 59.82е-3, respectively (p<0.05). The differences in IP values depending on the origin of STBC cells (primary tumor, relapse, and metastasis) were revealed: 18.11 ± 3.05 %, 25.75 ± 5.57 %, 52.97 ± 5.64 %, respectively (p<0.05). We found a correlation between migration and invasion parameters of solid tumor cells and the expression of factors ensuring their mobility and affecting other cellular components of the tumor microenvironment, including cells of the immune system.Conclusion. The biologically «aggressive» phenotype of SM and STBS cells is associated with the expression of the cancer-testis genes, such as PRAME, PASD1, SSX1 and with the production of HB-EGF, IGFBP, PLGF, PECAM-1, FST, SCF, IL-8. These products can be considered as new targets for therapeutic technologies aimed at influencing metastatic disease.

Cytomorphological spectrum, including immunohistochemical results of 16 cases of clear cell sarcoma of soft tissue, along with positive EWSR1 gene rearrangement result in two cases.

To describe the cytopathological features of clear cell sarcomas (CCSs), including immunohistochemical and molecular results, the latter in selected cases. Sixteen consecutively diagnosed cases of CCS of soft tissue, over 6-year duration were included. Fine needle aspiration cytology was performed for primary diagnosis in three and for recurrent/metastatic lesions in 12 cases. Cytopathological features in 16 cases (conventional Papanicolaou- and May-Grünwald Giemsa-stained smears) were critically analysed. Corresponding histopathological and immunostained sections were available in 15 cases. Two cases were tested for EWSR1 gene rearrangement by fluorescence in-situ hybridisation. Sixteen tumours occurred in patients with age ranging from 18 to 56years (median=33.5); M: F ratio=1:1; in deep soft tissues, mostly in extremities. Primary cytopathological diagnosis (3 cases) was CCS with a differential diagnosis of melanoma (1 case) and poorly differentiated malignant tumour (2 cases). On review, smears were predominantly hypercellular (n=14), invariably composed of monomorphic appearing epithelioid/polygonal cells (n=16), including spindle cells (n=6); mostly singly scattered (n=16), in loose clusters (n=12); with prominent nucleolisation (n=16); granular to vacuolated, well-defined cytoplasm (n=12), binucleation/multinucleation (n=9); mitoses (n=6); sudden anisonucleosis; racquet-shaped cells (n=3), against a tigroid background (n=2), along with focal intracytoplasmic pigment deposition (n=2). Immunohistochemically, tumour cells were positive for S-100P (15/15), HMB-45 (15/15) and melan-A(6/12). Two cases tested for EWSR1 rearrangement displayed red-green split signals. This constitutes one of the largest series describing the cytomorphological spectrum of CCS of soft tissue. Certain features, such as singly scattered monomorphic, epithelioid cells with prominent nucleolisation are useful diagnostic clues. Immunohistochemical stains are necessary and molecular testing is further helpful in reinforcing a diagnosis in certain cases. A correct diagnosis has crucial treatment implications.

Cancer Stem Cells in Soft-Tissue Sarcomas.

Soft tissue sarcomas (STS) are a rare group of mesenchymal solid tumors with heterogeneous genetic profiles and clinical features. Systemic chemotherapy is the backbone treatment for advanced STS; however, STS frequently acquire resistance to standard therapies, which highlights the need to improve treatments and identify novel therapeutic targets. Increases in the knowledge of the molecular pathways that drive sarcomas have brought to light different molecular alterations that cause tumor initiation and progression. These findings have triggered a breakthrough of targeted therapies that are being assessed in clinical trials. Cancer stem cells (CSCs) exhibit mesenchymal stem cell (MSC) features and represent a subpopulation of tumor cells that play an important role in tumor progression, chemotherapy resistance, recurrence and metastasis. In fact, CSCs phenotypes have been identified in sarcomas, allied to drug resistance and tumorigenesis. Herein, we will review the published evidence of CSCs in STS, discussing the molecular characteristic of CSCs, the commonly used isolation techniques and the new possibilities of targeting CSCs as a way to improve STS treatment and consequently patient outcome.

Cabozantinib as an emerging treatment for sarcoma.

Sarcomas are a diverse group of rare solid tumors with limited treatment options for patients with advanced, inoperable disease. Cabozantinib is a tyrosine kinase inhibitor currently approved for advanced renal cell, hepatocellular, and medullary thyroid carcinoma. Cabozantinib has potent activity against a variety of kinases, including MET, vascular endothelial growth factor receptor, and AXL, that are associated with sarcoma growth and development. Here we review the preclinical findings and clinical development of cabozantinib in the treatment of soft tissue sarcoma, gastrointestinal stromal tumors (GIST), osteosarcoma, and Ewing sarcoma. In vitro, cabozantinib has shown relevant activity in inhibiting the growth and viability of soft tissue sarcoma, GIST, osteosarcoma, and Ewing sarcoma tumor cell lines. Cabozantinib also promoted the regression of GIST in various murine xenografts, including imatinib-resistant models. More than 10 prospective trials with cabozantinib that included patients with sarcomas have been completed or are currently ongoing. Clinical activity with cabozantinib has been recently reported in phase 2 clinical trials for patients with GIST and for patients with osteosarcoma or Ewing sarcoma. Cabozantinib has shown promising activity for the treatment of various sarcomas, supporting further evaluation in this setting.

Genomic characterizations of KIT and PDGFRA in Chinese pan-cancer patients.

e13654 Background: KIT and PDGFRA gene mutations have been identified as biomarkers for targeted therapy in gastrointestinal stromal tumor. Mutations and amplification of KIT and PDGFRA are also considered as potential targets in solid tumors in clinical trials. We assessed the characteristics of KIT and PDGFRA alterations in a variety of cancers and tried to provide genomic landscape of these genes. Methods: Formalin-fixed, paraffin-embedded (FFPE) tumors and their matched blood samples were collected from 10,010 patients for 450 cancer genes targeted next-generation sequencing (NGS) panel.The testing was carried out in a College of American Pathologists (CAP) accredited and Clinical Laboratory Improvement Amendments (CLIA) certified laboratory. Genomic alterations including single nucleotide variations, short and long insertions/deletions, copy number variations, and gene rearrangements were assessed. Results: Alterations of KIT and PDGFRA were detected 2.1% and 1.6% respectively in Chinese pan-cancer patients. KIT alterations was detected most frequently in gastrointestinal stromal tumor (68.8%), soft tissue sarcoma (10.8%), melanoma (10.3 %), thymic tumors (7.7%) and small cell lung cancer (5.1%), and PDGFRA alterations was detected in gastrointestinal stromal tumor (18.7%), melanoma (5.2 %), primary hepatic carcinoma (4.3%), bone sarcoma (4.2%) and soft tissue sarcoma (3.4%). Particularly, KIT and PDGFRA amplifications accounted for 0.68% and 0.58% respectively in pan-cancer patients and were highly co-existed. Conversely, KIT and PDGFRA mutations were mutually exclusive which indicated an independent role of driver genes. Furthermore, tumor with microsatellite instability high (MSI-H) status had a significantly higher rate of KIT or PDGFRA mutations compared to microsatellite stable (MSS) tumors (p < 0.001). Conclusions: KIT and PDGFRA alterations occurred in 2.1% and 1.6% respectively in Chinese pan-cancer patients and mutually exclusive with known driver genes. KIT and PDGFRA alterations may act as driving oncogenic factors across cancers and warrant drug development strategy.

Therapeutic drug monitoring of pazopanib: Using cost-neutral PK-guided interventions to optimize exposure.

3598 Background: Pazopanib is an approved treatment for renal cell carcinoma (RCC) and soft tissue sarcoma (STS). At the currently registered fixed dose of 800 mg QD, 20% of patients (pts) do not attain the efficacy threshold of Cmin ≥ 20.5 mg/L (Suttle et al, 2014), providing a strong rationale for therapeutic drug monitoring (i.e. individualizing the dose based on measured plasma drug concentrations). Previous studies provided cost-neutral alternatives to absolute dose increments to optimize exposure (i.e. splitting intake moments or concomitant intake with food (Groenland et al, 2020; Lubberman et al, 2019)). This study aimed to investigate the feasibility, tolerability and efficacy of TDM of pazopanib, using cost-neutral interventions. Methods: Patients starting treatment with pazopanib at the standard dose of 800 mg QD in modified fasting state were included in the prospective DPOG TDM study (www.trialregister.nl, NL6695). PK sampling occurred 4, 8 and 12 weeks after start of treatment, and every 12 weeks thereafter. Pazopanib concentrations were measured with LC-MS/MS and Cmin was calculated. In case of Cmin < 20.5 mg/L and acceptable toxicity, a dose intervention was recommended. As a first step, intake moments were split (i.e. 400 mg BID). Secondly, concomitant intake with food was recommended. Results: In total, 34 pts were included (19 STS, 15 RCC), of whom 158 PK samples were collected. Eleven pts (32%) were underdosed and had at least 1 PK sample below the target. In 24% of the pts a PK-guided intervention could be performed, which was successful in 6 pts (75%). Median Cmin increased from 15 mg/L to 32 mg/L (p = 0.027). Eventually, 3 pts went back to 800 mg QD due to toxicity, after which Cmin remained ≥ 20.5 mg/L in 2 pts. In pts with adequate exposure throughout the study, median Cmin was 32 mg/L (range 23 – 65 mg/L). In 3 pts, a PK-guided intervention could not be performed, due to progression (n = 1) or logistical issues (n = 2). Twelve pts (35%) received a dose reduction due to toxicity (lowest dose was 200 mg QAD), exposure remained adequate at this reduced dose in all pts. For STS pts, median PFS was 19.8 months in pts with Cmin < 20.5 mg/L who did need an intervention vs. 6.4 months in pts with all Cmin ≥ 20.5 mg/L (not significant). For RCC pts, this was 15.5 months vs. 7.4 months, respectively (not significant). Conclusions: This prospective study shows that PK-guided dose optimization of pazopanib using cost-neutral interventions is feasible in daily practice. A PK-guided intervention was performed in 24% of the patients, which was successful in 75% of these patients. Clinical trial information: NL6695 .

ATR Inhibition Broadly Sensitizes Soft-Tissue Sarcoma Cells to Chemotherapy Independent of Alternative Lengthening Telomere (ALT) Status

Only few drugs have shown activity in patients with advanced soft-tissue and the median overall survival is only 18 months. Alterations of genes involved in the DNA damage repair pathway have been associated with sarcoma risk and prognosis. 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. The objective of this study is to evaluate the pre-clinical activity of ATR inhibition in soft tissue sarcomas (STS). We explored the ability of the ATR inhibitor, VE-822, to prevent chemotherapy-induced intra-S-phase checkpoint activation and evaluated the antitumor potential of this combination in vitro and in vivo in STS cell lines and in a patient-derived xenograft model. The combination of VE-822 and gemcitabine in vitro was synergistic, inhibited cell proliferation, induced apoptosis, and accumulated in the S phase of the cell cycle with higher efficacy than either single agent alone. The combination also resulted in enhanced γH2AX intranuclear accumulation as a result of DNA damage induction. These effects were unrelated to the alternative lengthening of telomeres pathway. In vivo, the combination of VE-822 and gemcitabine significantly enhanced tumor growth inhibition and progression-free survival in an aggressive model of undifferentiated pleomorphic sarcoma. The combination of ATR inhibitor and chemotherapy is beneficial in pre-clinical models of soft-tissue sarcoma and deserves further exploration in the clinical setting.

Possibility for Dose Optimization of Pazopanib from Its Plasma Concentration in Japanese Patients with Cancer.

The currently approved dose of pazopanib (800 mg) is being re-examined owing to its adverse effects. The aim of this study was to evaluate the relationships among starting or maintenance doses of pazopanib, estimated pazopanib Cmin, and other clinical factors, including albumin and α-1 acid glycoprotein levels, in soft-tissue sarcoma and renal cell carcinoma. We also determined whether therapeutic drug monitoring of pazopanib concentrations may be used to improve its therapeutic efficacy and prevent adverse effects. Forty patients who received pazopanib for renal cancer or soft-tissue sarcoma at the Hokkaido Cancer Center were evaluated prospectively. Cmin for pazopanib was calculated based on the measured values from the plasma samples. The efficacy and time to treatment failure were then assessed. The pazopanib maintenance doses were 200 (n = 4), 400 (n = 34), 600 (n = 4), and 800 mg (n = 1). Most patients (65%) who received a 400 mg dose had an effective pazopanib concentration (≧20 µg/mL), whereas 35% of patients who received the 400 mg dose had ineffective concentrations (<20 µg/mL). Logistic regression analysis revealed that only the albumin level was significantly associated with effective pazopanib concentrations (odds ratio: 1.37, p = 0.0234). In conclusion, a dose of 400 mg had been effective and well tolerated in more than half of patients in this study. However, therapeutic drug monitoring is necessary during pazopanib therapy.

Abstract A23: Identification of prevalent targets for the development of tailored sarcoma immunotherapies using a rapid clinic-to-bench immunoprofiling pipeline

Abstract Sarcomas are rare malignancies of mesenchymal origin for which current treatment options are very limited. Due to the vast heterogeneity of the various subtypes, the development of new targeted therapies requires a customized approach. Checkpoint blockade therapies are a promising alternative that still have not been systematically investigated for sarcoma patients. Thus, utilizing the infrastructure of the Cell Therapy Core Facility (CTC) at Nova Southeastern University (NSU), we developed a clinic-to-bench pipeline that enables the processing of fresh tumor material after surgical excision to generate primary sarcoma cell lines for biobanking and research purposes. Using this pipeline, we have extensively characterized mRNA, miRNA and cell surface expression profiles from more than 40 sarcoma cell lines to date, as well as validated their tumorigenicity by assessing expression of sarcoma/cancer associated genes such as Birc5, Birc2, Bcl2, Ewsr1-fli1, Syt/Ssx and Pcna. Moreover, we assessed the expression of immunomodulatory molecules that may suppress antitumor responses, such as PD-L1, PD-L2, OX40L and CD40L, using multicolor flow cytometry. Detailed characterization of the immune profile of 7 bone-related and 7 soft tissue sarcoma cell lines, assessed after 12 weeks of in vitro serial passaging, revealed a common signature in the cell surface expression of proliferative marker PCNA, and lymphocyte ligands CD112 and CD115. Moreover, immunoprofiling of the PCNA/CD112/CD155+ sarcoma cell lines (n=14) demonstrated persistent expression of PD-L2 in all cell lines and the potential applications of currently clinically approved anti-PD-1 checkpoint inhibitors for sarcoma treatment. Importantly, the conserved expression of these proteins enabled the distinction of sarcoma tumor cells from other cells in the fresh primary tumor mix directly after isolation. Thus, we next performed a comparative analysis of the immune profile of 6 freshly isolated sarcoma tumors and their respective cell lines. Comparison of the surface expression profiles of the generated cell lines and the corresponding primary tumor cells showed that the initial phenotype was preserved after more than 12 weeks of expansion and after cryopreservation. This proves that the generated cell lines are an accurate tool that can be used for the development of novel personalized immunotherapies. Most importantly, our results enabled the design of a translational research pipeline that allows the systematic characterization of the immune profile of understudied sarcomas for the first time in order to gain insight into which patients may directly benefit from available immunotherapies and which may be eligible to enroll in clinical trials assessing the efficacy of checkpoint inhibitors. In summary, the generated sarcoma biobank serves as a unique and highly representative primary resource that can be utilized to identify potential candidates for novel targeted and personalized immunotherapies for the treatment of sarcomas. Citation Format: Anna-Maria Georgoudaki, Robin Krueger, Michelle Hartman, Tamara Chinn, Dustin Tran, Reneé Potens, Wendy Weston, H. Thomas Temple, Adil D. Duru. Identification of prevalent targets for the development of tailored sarcoma immunotherapies using a rapid clinic-to-bench immunoprofiling pipeline [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr A23.

Induction of Myogenic Differentiation Improves Chemosensitivity of Chemoresistant Cells in Soft-Tissue Sarcoma Cell Lines.

Rhabdomyosarcoma (RMS) and rhabdoid tumors (RT) are rare soft-tissue malignancies with the highest incidence in infants, children, and adolescents. Advanced, recurrent, and/or metastatic RMS and RT exhibit poor response to treatment. One of the main mechanisms behind resistance to treatment is believed to be intratumoral heterogeneity. In this study, we investigated the myogenic determination factor 1 (MYOD1) and Noggin (NOG) markers in an embryonal RMS (ERMS) cell line and an RT cell line and the differential response of the MYOD1 and NOG expressing subpopulations to chemotherapy. Importantly, we found that these markers together identify a subpopulation of cells (MYOD1+ NOG+ cells) with primary resistance to Vincristine and Doxorubicin, two commonly used chemotherapies for ERMS and RT. The chemoresistant MYOD1+ NOG+ cells express markers of undifferentiated cells such as myogenin and ID1. Combination of Vincristine with TPA/GSK126, a drug combination shown to induce differentiation of RMS cell lines, is able to partially overcome MYOD1/NOG cells chemoresistance.

Bone and Soft-Tissue Sarcoma: A New Target for Telomerase-Specific Oncolytic Virotherapy.

Adenovirus serotype 5 (Ad5) is widely and frequently used as a virus vector in cancer gene therapy and oncolytic virotherapy. Oncolytic virotherapy is a novel antitumor treatment for inducing lytic cell death in tumor cells without affecting normal cells. Based on the Ad5 genome, we have generated three types of telomerase-specific replication-competent oncolytic adenoviruses: OBP-301 (Telomelysin), green fluorescent protein (GFP)-expressing OBP-401 (TelomeScan), and tumor suppressor p53-armed OBP-702. These viruses drive the expression of the adenoviral E1A and E1B genes under the control of the hTERT (human telomerase reverse transcriptase-encoding gene) promoter, providing tumor-specific virus replication. This review focuses on the therapeutic potential of three hTERT promoter-driven oncolytic adenoviruses against bone and soft-tissue sarcoma cells with telomerase activity. OBP-301 induces the antitumor effect in monotherapy or combination therapy with chemotherapeutic drugs via induction of autophagy and apoptosis. OBP-401 enables visualization of sarcoma cells within normal tissues by serving as a tumor-specific labeling reagent for fluorescence-guided surgery via induction of GFP expression. OBP-702 exhibits a profound antitumor effect in OBP-301-resistant sarcoma cells via activation of the p53 signaling pathway. Taken together, telomerase-specific oncolytic adenoviruses are promising antitumor reagents that are expected to provide novel therapeutic options for the treatment of bone and soft-tissue sarcomas.

The Impact of Obesity on Tumor Glucose Uptake in Breast and Lung Cancer.

Obesity confers an increased incidence and poorer clinical prognosis in more than 10 cancer types. Paradoxically, obesity may provide protection from poor outcomes in lung cancer. Mechanisms for the obesity-cancer links are not fully elucidated, with altered glucose metabolism being a promising candidate. Using 18F-fluorodeoxyglucose positron-emission-tomography/computed tomography images from The Cancer Imaging Archive, we explored the relationship between body mass index (BMI) and glucose metabolism in several cancers. In 188 patients (BMI mean [SD] = 27.7 [5.1], range = 17.4–49.3 kg/m2), higher BMI was associated with greater tumor glucose uptake in breast cancer (r = 0.36; P = .02) and with lower tumor glucose uptake in non-small cell lung cancer (r = -0.26; P = .048) using two-sided Pearson correlations. No relationship was observed in soft tissue sarcoma or squamous cell carcinoma. Harnessing the National Cancer Institute’s open-access database, we demonstrate altered tumor glucose metabolism as a potential mechanism for the detrimental and protective effects of obesity on breast and lung cancer, respectively.

Cancer/testis antigens expression during cultivation of melanoma and soft tissue sarcoma cells

BackgroundAutologous dendritic cells (DC) loaded with tumor-associated antigens (TAAs) are a promising approach for anticancer immunotherapy. Polyantigen lysates appear to be an excellent source of TAAs for loading onto the patient’s dendritic cells. Cancer/testis antigens (CTA) are expressed by a wide range of tumors, but are minimally expressed on normal tissues, and could serve as a universal target for immunotherapy. However, CTA expression levels can vary significantly in patients with the same tumor type. We proposed that patients who do not respond to DC-based therapy may have distinct features of the CTA expression profile on tumor cells.Patients and methodsWe compared the gene expression of the principal families CTA in 22 melanoma and 27 soft tissue and bone sarcomas cell lines (STBS), received from patients and used for DC vaccine preparation.ResultsThe majority (47 of 49, 95.9%) cell lines showed CTA gene activity. The incidence of gene expression of GAGE, NYESO1, MAGEA1, PRAME’s was significantly different (adj. p < 0.05) between melanoma and sarcoma cell lines. The expression of the SCP1 gene was detected neither in melanoma cells nor in the STBS cells. Clustering by the gene expression profile revealed four different expression patterns. We found three main patterns types: hyperexpression of multiple CTA, hyperexpression of one CTA with almost no expression of others, and no expression of CTA. All clusters types exist in melanoma and sarcoma cell lines. We observed dependence of killing efficacy from the PRAME (rho = 0.940, adj. p < 0.01) expression during real-time monitoring with the xCELLigence system of the interaction between melanoma or sarcoma cells with the T-lymphocytes activated by the lysate of selected allogenous melanoma cell lines with high expression of CTA.ConclusionOur results demonstrate that one can use lysates from allogeneic melanoma cell lines as a source of CTA for DC load during the production of anticancer vaccines for the STBS treatment. Patterns of CTA expression should be evaluated as biomarkers of response in prospective clinical trials.

The epidemiology of musculoskeletal tumors in Aseer Central Hospital, south of Saudi Arabia

Background: Musculoskeletal neoplasms lead to significantly high morbidity and mortality rates among cases. Musculoskeletal tumors (MSTs) are relatively uncommon. The current study aimed to assess the magnitude, pattern, and predictors of MSTs among cases. Methodology: A record-based retrospective approach was applied for the current study. The study was conducted at Aseer Central Hospital. Medical records for patients with complete files and no errors and confirmed cases of any type of musculoskeletal tumor were included during the period from 2018 to 2020. Data on both patient and tumor, including patient’s demographic data, such as name, gender, and age, were collected. Results: The study included 69 cases with MSTs. The cases’ age ranged from 8 to 72 years, with a mean age of 31.5 ± 16.3 years. There were 35 female (50.7%) and 34 male (49.3%) participants. The femur was the most reported site for MSTs in the study (30.4%; 21), followed by foot and tibia (11.6%; 8 for each), hand (7.2%; 5), and elbow (5.8%; 4). The most recorded diagnosis was osteochondroma (12.7%), followed by ganglion cyst (9.1%), Ewing’s sarcoma (7.3%), soft tissue sarcoma (5.5%), osteosarcoma (5.5%), lipoma (5.5%), chondroblastoma (5.5%), non-ossifying fibroma (3.6%), benign lesion (3.6%), and Giant cell tumours (3.6%). Conclusions: This study revealed that most of the diagnosed MSTs among the studied cases were benign (osteochondroma and ganglion cyst) with uniform distribution of incidence by age and gender. Also, lower extremities were the most reported sites for MSTs, followed by upper extremities.

Molecular signatures of BRCAness analysis identifies PARP inhibitor Niraparib as a novel targeted therapeutic strategy for soft tissue Sarcomas.

Background: Patients with advanced soft tissue sarcomas (STS) have a dismal prognosis with few effective therapeutic options. A defect in the homologous recombination repair (HRR) pathway can accumulate DNA repair errors and gene mutations, which can lead to tumorigenesis. BRCAness describes tumors with an HRR deficiency (HRD) in the absence of a germline BRCA1/2 mutation. However, the characteristics of BRCAness in STS remain largely unknown. Thus, this study aimed to explore the genomic and molecular landscape of BRCAness using whole exome sequencing (WES) in STS, aiming to find a potential target for STS treatment.Methods: WES was performed in 22 STS samples from the First Affiliated Hospital of Sun Yat-sen University to reveal the possible genomic and molecular characteristics. The characteristics were then validated using data of 224 STS samples from The Cancer Genome Atlas (TCGA) database and in vitro data. The analysis of the potential biomarker for BRCAness was performed. Targeted drug susceptibility and combination therapy screening of chemotherapeutics for STS were evaluated in STS cell lines, cell-line-derived xenografts (CDX), and patient-derived xenografts (PDX).Results: Compared with 30 somatic mutation signatures of cancers, high cosine-similarity (0.75) was identified for HRD signatures in the 22 STS samples using nonnegative matrix factorization. Single nucleotide polymorphism indicated a low mutation rate of BRCA1/2 in the 22 STS samples (11.76% and 5.88%, respectively). However, copy number variation analyses demonstrated widespread chromosomal instability; furthermore, 54.55% of STS samples (12/22) carried BRCAness traits. Subsequently, similar genomic and molecular characteristics were also detected in the 224 STS samples from TCGA and in vitro. Poly (ADP-ribose) polymerases (PARP)-1 could be a promising reflection of HRD and therapeutic response. Furthermore, the level of PAR formation was found to be correlated with PARP-1. Subsequently, STS cell lines were determined to be sensitive to PARP inhibitor (PARPi), niraparib. Moreover, based on the screening test of the five common PARPis and combination test among doxorubicin, ifosfamide, dacarbazine, and temozolomide (TMZ), niraparib and TMZ were the most synergistic in STS cell lines. The synergistic effect and safety of niraparib and TMZ combination were also shown in CDX and PDX.Conclusions: BRCAness might be the common genomic and molecular characteristics of majority of STS cases. PARP-1 and PAR could be potential proper and feasible theranostic biomarkers for assessing HRD in patients. STSs were sensitive to PARPi. Moreover, the combination of niraparib and TMZ showed synergistic effect. Niraparib and TMZ could be a promising targeted therapeutic strategy for patients with STS.

Expanding the Phenotypic Spectrum of Mesenchymal Tumors Harboring the EWSR1-CREM Fusion.

ATF1, CREB1, and CREM constitute the CREB family of transcription factors. The genes encoding these factors are involved in gene fusion events in human tumors. EWSR1-ATF1 and EWSR1-CREB1 are the 2 most characterized fusions, whereas EWSR1-CREM has been less studied. To better understand the phenotypic spectrum of mesenchymal tumors associated with the EWSR1-CREM fusion, we investigated archival cases using fluorescence in situ hybridization and/or RNA sequencing. Among 33 clear cell sarcomas of soft tissue tested, we found 1 specimen, a hand tumor bearing the rearrangements of EWSR1 and CREM, with classic histology and immunophenotype. None of 6 clear cell sarcoma-like tumors of the gastrointestinal tract tested harbored the EWSR1-CREM fusion. Among 11 angiomatoid fibrous histiocytomas, we found that 3 tumors of myxoid variant harbored the rearrangements of EWSR1 and CREM. All 3 tumors occurred in middle-aged men and involved the distal extremities (N=2) and the lung (N=1). Prominent lymphoid cuff, fibrous pseudocapsule, and amianthoid fiber were present in 3, 2, and 2 tumors, respectively, whereas none showed pseudoangiomatoid spaces. All 3 tumors were immunohistochemically positive for epithelial membrane antigen and desmin. These cases suggested a closer relationship between angiomatoid fibrous histiocytoma and a recently proposed novel group of myxoid tumors with CREB family fusions. Our cohort also included 2 unclassifiable sarcomas positive for EWSR1-CREM. One of these was an aggressive pediatric tumor of the abdominal cavity characterized by proliferation of swirling spindle cells immunopositive for cytokeratin and CD34. The other tumor derived from the chest wall of an adult and exhibited a MUC4-positive sclerosing epithelioid fibrosarcoma-like histology. Our study demonstrates that a wider phenotypic spectrum is associated with the EWSR1-CREM fusion than previously reported.

Abstract LB-323: Atrx deletion delays tumor formation and increases radiosensitivity of a primary mouse model of soft tissue sarcoma

Abstract Soft tissue sarcomas are tumors of the connective tissue that account for an estimated 12,000 new cancer cases annually and carry a poor prognosis with a five year survival rate of 50% despite treatment. An important research objective in improving therapy for soft tissue sarcoma patients is to understand how genetic mutations affect soft tissue sarcoma development and radiation response. Intriguingly, next-generation sequencing data from The Cancer Genome Atlas and other massive cancer sequencing efforts have identified Alpha Thalassemia and Mental Retardation X-linked, or ATRX, as the second most frequently mutated gene in soft tissue sarcoma. The vast majority of these ATRX mutations are frameshift mutations. To dissect the role of ATRX in soft tissue sarcoma development and therapeutic response, we generated a primary mouse model of soft tissue sarcoma. This model utilizes Cre-LoxP technology to conditionally activate oncogenic KrasG12D, delete p53, and delete both alleles of Atrx (KPA) in mouse hindlimb Pax7+ muscle satellite cells. When compared to mice with activation of oncogenic KrasG12D, deletion of p53 and at least one wild-type Atrx allele (KP), KPA mice showed a significant delay in time to tumor formation (35 vs. 58 days median), but no significant difference in tumor growth rate as measured by time to tumor quintupling. Next, we generated primary soft tissue sarcoma cell lines from our KP mouse model, and performed CRISPR-Cas9 mediated knockout of Atrx to generate isogenic cell lines. Testing of these isogenic cell lines revealed that Atrx deletion in soft tissue sarcoma cells increased sensitivity to double strand break inducing chemotherapeutics and ionizing radiation, but not to single strand break inducing chemotherapeutics in vitro. In clonogenic assays, Atrx deletion increased radiosensitivity of soft tissue sarcoma cell lines more than fourfold across all radiation doses tested. We next used our KP and KPA models to test the effect of Atrx deletion on radiosensitivity in vivo. Results showed that, relative to the KP model, Atrx deletion improved the response of soft tissue sarcomas to radiation therapy in vivo, increasing the time to tumor quintupling after radiation by 20%. Further analysis demonstrated that Atrx deletion significantly increases mitotic segregation defects after ionizing radiation, suggesting a potential mechanism for the radiosensitivity observed in our studies. Our in vivo study provides the first insight into how ATRX alters the development and therapeutic response of soft tissue sarcoma. Citation Format: Robert W. Floyd, Lixia Luo, David Kirsch. Atrx deletion delays tumor formation and increases radiosensitivity of a primary mouse model of soft tissue sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-323.

Recurrent DMD Deletions Highlight Specific Role of Dp71 Isoform in Soft-Tissue Sarcomas.

Soft-tissue sarcomas (STS) are rare tumors whose oncogenesis remains unknown and for which no common therapeutic target has yet been identified. Analysis of 318 STS by CGH array evidenced a frequent deletion affecting the DMD gene (encoding dystrophin isoforms) in 16.5% of STS, including sarcomas with complex genomics, gastrointestinal tumors (GIST), and synovial sarcomas (SS). These deletions are significantly associated with metastatic progression, thus suggesting the role of DMD downregulation in the acquisition of aggressive phenotypes. We observed that targeted deletions of DMD were restricted to the 5’ region of the gene, which is responsible for the transcription of Dp427. Analysis of STS tumors and cell lines by RNA sequencing revealed that only the Dp71 isoform was widely expressed. Dp427 depletion had no effect on cell growth or migration. However, Dp71 inhibition by shRNA dramatically reduced the cell proliferation and clonogenicity of three STS cell lines, likely by altering the cell cycle progression through the G2/M-phase. Our work demonstrates that DMD deletions are not restricted to myogenic tumors and could be used as a biomarker for metastatic evolution in STS. Dp71 seems to play an essential role in tumor growth, thus providing a potential target for future STS treatments.