Rhabdomyosarcoma Samples Research Articles

Schwann Cell-Mediated M2-Like Macrophage Polarization in Rhabdomyosarcoma.

To investigate the cellular components and immunological characteristics of the head and neck rhabdomyosarcoma (RMS) microenvironment. We conducted single-cell transcriptomics to analyze the cellular components of the RMS microenvironment. CellChat was utilized for analyzing intercellular interactions. The cancer genome atlas database was used for validation. CIBERSORT was applied for immune infiltration profiling. Functional enrichment analyses were performed using the Kyoto Encyclopedia of Genes and Genomes. Gene set scores were calculated using single-sample gene set enrichment analysis. Subcutaneous allograft models and bulk RNA sequencing were used for validation. Flow cytometry and immunohistochemistry were used to identify M2-like macrophages. Our findings revealed an extremely low presence of neutrophils in RMS samples compared with normal sample. RMS sample with high Schwann cell infiltration exhibited an increase in M2-like macrophage infiltration. Receptor-ligand pairs, specifically MIF-CD74 and PTN-SDC3, were identified between Schwann cells and M2-like macrophages. In the RMS sample characterized by significant Schwann cell infiltration, M2-like macrophages demonstrate robust expression of axon guidance factors and are enriched in the axon guidance pathway. Our study provides valuable insights into the microenvironment and immunological characteristics of RMS, offering crucial information for further research and potential therapeutic strategies.

Abstract 73: Spatial profiling of pediatric rhabdomyosarcoma to elucidate their immunosuppressive tumor microenvironment

Abstract Background: Pediatric rhabdomyosarcoma (RMS) is a mesenchymal soft tissue malignancy. Despite intensive chemotherapy regimens, there is currently a gap in improving treatment outcomes for RMS. Chemotherapy can modulate the tumor microenvironment (TME) by altering the immune cell composition or their functions. However, the impact of chemotherapy in pediatric RMS TME is unclear. RMS tumors are also considered as “cold” tumors due to low immune infiltration, which could be a determinant of why immunotherapy is unlikely to work. Additional investigations are required to determine the reasons of why RMS is “cold” and whether a combined immunotherapy and chemotherapy approach should be administered for this hard-to-cure cancer. Methods We conducted for the first time spatial GeoMx whole transcriptome atlas and 51-plex Akoya CODEX profiling for a tissue microarray (TMA) from a cohort of pediatric RMS patients (n = 12) with pre-chemotherapy (n = 5) and post-chemotherapy (n = 5) samples. Regions of interest (ROIs) were selected based on tumor areas with immune cell infiltration, with the lymphocytes, tumor cells, and nuclei visualized using fluorescent morphological markers CD45, Desmin, and DAPI. ROIs were segmented into tumor stroma compartments and immune cell compartments. Computational analyses were conducted on the transcriptomics data using R package standR including quality control, normalization, differential expression analysis, gene set enrichment analysis, and cell type deconvolution. For CODEX data, we preformed cell segmentation, followed by further quality control, cell phenotyping, cellular neighborhood and cellular interaction analysis. Results: From differential expression analysis of the immune compartment, we have identified type I interferon-related genes (DTX3L, ADAP2, RNF135) and reactive oxygen species (ROS)-induced gene (NCF1) are upregulated, and an antioxidant enzyme gene (PRDX2) is downregulated in post-chemotherapy samples compared to pre-chemotherapy samples. Gene set enrichment analysis of the immune compartment revealed an enriched antigen presentation pathway in post-chemotherapy samples. In the tumor stroma compartment, we identified increased TGF-β binding pathway in post-chemotherapy samples. Cellular deconvolution revealed a higher proportion of endothelial cells in post-chemotherapy samples, which is validated via CODEX imaging analysis. Cellular neighborhoods and cell-cell interaction analysis for CODEX data also reveal how chemotherapy affect functional units and cellular interactions in RMS TME with spatial context preserved. Conclusion: By applying spatial transcriptomic and multiplexed imaging to RMS samples, we have revealed key genes, pathways and cell types involved in chemotherapy-induced alterations in the TME. This will provide a new perspective for developing novel chemoimmunotherapy for RMS patients. Citation Format: Cui Tu, Chin Wee Tan, Ning Liu, Arutha Kulasinghe, Fernando Souza-Fonseca-Guimaraes. Spatial profiling of pediatric rhabdomyosarcoma to elucidate their immunosuppressive tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 73.

Analysis of anaplastic lymphoma kinase status in rhabdomyosarcomas: correlation with clinicopathological findings

Introduction. Rhabdomyosarcoma (RMS) is a highly malignant tumor occurring mostly in children. Multimodal therapy is currently promising, however, in patients with high risk (per the stratification scale) low survival rates are observed. There remains a necessity for new possibilities in treatment of this category of patients. Currently, anaplastic lymphoma kinase (ALK) is of considerable interest.Aim. To study expression of ALK as a potential target marker in RMS tissues.Materials and methods. In total, 202 histological RMS blocks were studied. ALK expression was qualitatively measured using immunohistochemistry, and its correlation with histological tumors subtype and patient’s clinical data was evaluated.Results. ALK expression in samples of alveolar RMS was observed in 68 % (41/60) of cases, samples of embryonal RMS in 30 % (26/87) cases, samples of spindle cell/sclerosing RMS in 7 % (1/14) of cases, samples of epithelioid RMS (EpiRMS) in 100 % (2/2) of cases. The study showed correlation between ALK expression and alveolar RMS subtype (р <0.05). In all cases of alveolar RMS with focal ALK D5F3 expression, primarily low or moderate activity levels against anti-myogenin and MyoD1 were observed. ALK-positive samples in the embryonal and spindle cell/sclerosing RMS samples were rarer.Conclusion. The results show that ALK is a promising target for treatment of patients with RMS. Further study of additional genetic aberrations of ALK is planned which could serve as a springboard for development of targeted therapy.

Machine learning for rhabdomyosarcoma histopathology

AbstractCorrectly diagnosing a rare childhood cancer such as sarcoma can be critical to assigning the correct treatment regimen. With a finite number of pathologists worldwide specializing in pediatric/young adult sarcoma histopathology, access to expert differential diagnosis early in case assessment is limited for many global regions. The lack of highly-trained sarcoma pathologists is especially pronounced in low to middle-income countries, where pathology expertise may be limited despite a similar rate of sarcoma incidence. To address this issue in part, we developed a deep learning convolutional neural network (CNN)-based differential diagnosis system to act as a pre-pathologist screening tool that quantifies diagnosis likelihood amongst trained soft-tissue sarcoma subtypes based on whole histopathology tissue slides. The CNN model is trained on a cohort of 424 centrally-reviewed histopathology tissue slides of alveolar rhabdomyosarcoma, embryonal rhabdomyosarcoma and clear-cell sarcoma tumors, all initially diagnosed at the originating institution and subsequently validated by central review. This CNN model was able to accurately classify the withheld testing cohort with resulting receiver operating characteristic (ROC) area under curve (AUC) values above 0.889 for all tested sarcoma subtypes. We subsequently used the CNN model to classify an externally-sourced cohort of human alveolar and embryonal rhabdomyosarcoma samples and a cohort of 318 histopathology tissue sections from genetically engineered mouse models of rhabdomyosarcoma. Finally, we investigated the overall robustness of the trained CNN model with respect to histopathological variations such as anaplasia, and classification outcomes on histopathology slides from untrained disease models. Overall positive results from our validation studies coupled with the limited worldwide availability of sarcoma pathology expertise suggests the potential of machine learning to assist local pathologists in quickly narrowing the differential diagnosis of sarcoma subtype in children, adolescents, and young adults.

Tumor suppressive functions of WNT5A in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is a highly aggressive soft tissue malignancy that predominantly affects children. The main subtypes are alveolar RMS (ARMS) and embryonal RMS (ERMS) and the two show an impaired muscle differentiation phenotype. One pathway involved in muscle differentiation is WNT signaling. However, the role of this pathway in RMS is far from clear. Our recent data showed that the canonical WNT/β-Catenin pathway serves a subordinate role in RMS, whereas non-canonical WNT signaling probably is more important for this tumor entity. The present study investigated the role of WNT5A, which is the major ligand of non-canonical WNT signaling, in ERMS and ARMS. Gene expression analysis showed that WNT5A was expressed in human RMS samples and that its expression is more pronounced in ERMS. When stably overexpressed in RMS cell lines, WNT5A decreased proliferation and migration of the cells as demonstrated by BrdU incorporation and Transwell migration or scratch assay, respectively. WNT5A also decreased the self-renewal capacity and the expression of stem cell markers and modulates the levels of muscle differentiation markers as shown by sphere assay and western blot analysis, respectively. Finally, overexpression of WNT5A can destabilize active β-Catenin of RMS cells. A WNT5A knockdown has opposite effects. Together, the results suggest that WNT5A has tumor suppressive functions in RMS, which accompanies downregulation of β-Catenin.

Clinical significance of circulating tumor cells and cell-free DNA in pediatric rhabdomyosarcoma.

Liquid biopsy analysis represents a powerful and noninvasive tool to uncover biomarkers for disseminated disease assessment and longitudinal monitoring of patients. Herein, we explored the value of circulating and disseminated tumor cells (CTC and DTC, respectively) and cell‐free DNA (cfDNA) in pediatric rhabdomyosarcoma (RMS). Peripheral blood and bone marrow samples were analyzed to detect and enumerate CTC and DTC, respectively. We used the epithelial cellular adhesion molecule (EpCAM)‐based CellSearch platform coupled with an automatic device to collect both EpCAM‐positive and EpCAM‐low/negative CTCs. The standard assay was implemented, including the mesenchymal marker desmin. For selected cases, we molecularly profiled primary tumors and liquid biopsy biomarkers using whole‐exome sequencing and droplet digital PCR, respectively. RMS patients with metastatic disease had a significantly higher number of CTCs compared to those with localized disease, whereas DTCs were detected independently of disease presentation. The use of the desmin marker remarkably increased the identification of CTCs and DTCs in RMS samples. Of note, CTC clusters were detected in RMS patients with disseminated disease. Further, cfDNA and CTC molecular features closely reflected the molecular makeup of primary tumors and informed of disease course.

Abstract A53: Hypermethylated RASSF1A as circulating tumor marker in pediatric and adolescent solid tumors

Abstract Background: The tumor suppressor gene RASSF1A is hypermethylated in many adult and pediatric cancers. With the absence of recurrent mutations or translocations in various pediatric and young adult (malignant) tumors, detection of hypermethylated RASSF1A (RASSF1A-M) is an attractive biomarker for diagnosis as well as follow-up. Previously, we showed that RASSF1A-M can be used to detect neuroblastoma cells in bone marrow. Here we provide proof of concept of the detection of RASSF1A-M in plasma and serum of patients with neuroblastoma, renal tumors, rhabdomyosarcoma and germ cell tumors. Method: Plasma was collected at diagnosis from patients with neuroblastoma (n=47), renal tumors (n=13), and rhabdomyosarcoma (n=19) and during treatment and follow-up for neuroblastoma (n=121). Serum was collected from patients with germ cell tumors at diagnosis (n=94). Initially, cell-free DNA (cfDNA) was isolated, bisulfite treated, and tested by qPCR for actin beta (ACTB), unmethylated and hypermethylated RASSF1A (35 diagnostic and 121 follow-up neuroblastoma samples, 19 rhabdomyosarcoma samples). Subsequently, a droplet digital PCR (ddPCR) method for RASSF1A-M and ACTB, including a methylation-specific restriction enzymatic digestion, was applied on the remaining samples to ensure accurate quantification and reducing bisulfite induced cfDNA loss. A threshold for detection of RASSF1A-M was established by testing plasma (73 by qPCR, 25 by ddPCR) and serum samples (21 by ddPCR) from healthy donors. Results: In plasma samples, the total cfDNA level (ng/mL; median) was significantly higher in patients with renal tumors and neuroblastoma (localized and metastatic disease), but not in rhabdomyosarcoma patients, when compared to healthy donors (66.4; 34.4; 80.9; 3.0 and 2.4, respectively). For diagnostic neuroblastoma samples, RASSF1A-M was detected in all 26 patients with metastatic disease and in 10/21 patients with localized disease (mean 71.4% and 8.4% RASSF1A-M, respectively). RASSF1A-M levels decreased during therapy and re-elevated at relapse. RASSF1A-M was detected in 10/13 renal tumor and 8/21 rhabdomyosarcoma diagnostic samples (18.6% and 19.7% RASSF1A-M, respectively). Total cfDNA levels in healthy donor samples were higher in serum when compared to plasma (39.9 versus 2.4 ng/mL). cfDNA levels were significantly higher in patients with germ cell tumors (230.20 ng/mL) compared to healthy donor serum. RASSF1A-M was detected across all subtypes, including 15/16 embryonal carcinomas, 16/18 seminomas, 2/2 yolk sac tumors, 10/11 teratomas, 1/1 choriocarcinoma, and 42/46 mixed tumors. Conclusion: Our findings demonstrate the value of RASSF1A-M as a molecular circulating tumor marker in various solid tumors. We developed a ddPCR-based sensitive and quantitative assay for RASSF1A-M detection. Based on the data presented, RASSF1A hypermethylation is an interesting biomarker for detection of minimal residual disease across various solid tumor types, including those lacking recurrent mutations/translocations. Citation Format: Lieke M. J. van Zogchel, João Lobo, Nathalie Lak, Esther M. van Wezel, Jalenka van Wijk, Janine Stutterheim, Lily Zappeij-Kannegieter, Leendert H.J. Looijenga, Ellen van der Schoot, Godelieve A. M. Tytgat. Hypermethylated RASSF1A as circulating tumor marker in pediatric and adolescent solid tumors [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A53.

VCF2CNA: A tool for efficiently detecting copy-number alterations in VCF genotype data and tumor purity

VCF2CNA is a tool (Linux commandline or web-interface) for copy-number alteration (CNA) analysis and tumor purity estimation of paired tumor-normal VCF variant file formats. It operates on whole genome and whole exome datasets. To benchmark its performance, we applied it to 46 adult glioblastoma and 146 pediatric neuroblastoma samples sequenced by Illumina and Complete Genomics (CGI) platforms respectively. VCF2CNA was highly consistent with a state-of-the-art algorithm using raw sequencing data (mean F1-score = 0.994) in high-quality whole genome glioblastoma samples and was robust to uneven coverage introduced by library artifacts. In the whole genome neuroblastoma set, VCF2CNA identified MYCN high-level amplifications in 31 of 32 clinically validated samples compared to 15 found by CGI’s HMM-based CNA model. Moreover, VCF2CNA achieved highly consistent CNA profiles between WGS and WXS platforms (mean F1 score 0.97 on a set of 15 rhabdomyosarcoma samples). In addition, VCF2CNA provides accurate tumor purity estimates for samples with sufficient CNAs. These results suggest that VCF2CNA is an accurate, efficient and platform-independent tool for CNA and tumor purity analyses without accessing raw sequence data.

Abstract 3013: Acquired resistance to immune checkpoint inhibition by melanoma phenotypic transformation

Abstract Immune checkpoint blockade (ICB) has revolutionized the treatment of metastatic melanoma, however, cases of resistance have now begun to emerge. We report here a case of acquired resistance by melanoma phenotypic transformation in a patient treated on a Phase 1b/2 clinical trial (NCT02437136) with anti-PD1 (Pembrolizumab) and a histone deacetylase (HDAC) inhibitor (Entinostat). The patient presented to MGH with metastatic melanoma confirmed by core-needle biopsy of a liver lesion. A PET/CT scan one month after starting the trial revealed interval regression of liver and bone lesions, however, four months later the patient presented with disease progression. A tumor debulked from the L2 vertebral body at that time revealed the presence of a pleomorphic rhabdomyosarcoma. The patient was started on anti-CTLA4 (Ipilumimab) with further disease progression, and surgical specimens thereafter revealed either melanoma or rhabdomyosarcoma. Tumor tissue was evaluated for markers by immunohistochemistry. All tumors (5 specimens in total) were subjected to whole exosome sequencing (WES), RNA-sequencing (RNAseq) and epigenomic sequencing (ATACseq). WES revealed 844 mutations were shared between all tumors, suggesting a common ancestor including driver mutations in NRAS (G13D), NF1 (E1121* and W1314*) and the TERT2 promoter, among others. The pre-treatment melanoma biopsy had positive staining for S100 and MART-1, nuclear staining patterns for SOX10 and MITF, and were BRAF V600E negative. Importantly, 80% of the tumor cells had strong staining for PD-L1 expression and CD8 CTL infiltration was associated with 25% of the tumor. The post-treatment rhabdomyosarcoma biopsy was notable for diffusely positive staining for Desmin and MyoD1, and multifocal positivity for myogenin. The tumor stained negative for S100 and SOX10, and there was no PD-L1 staining and less than 5% tumor CD8 CTL infiltration. Gene expression analysis revealed distinct expression patterns between melanoma and rhabdomyosarcoma samples, with 150 differentially expressed genes implicated in processes such as antigen presentation, T cell activation, cell death and protein metabolism. In addition, each tumor was found to have a unique immune cell composition and T-cell receptor repertoire, suggesting an interplay between phenotypic transformation and immune response. Results from epigenomic sequencing are pending, but may shed light on the mechanisms of transformation leading to resistance. These data therefore elucidate a previously undescribed mechanism of acquired resistance to ICB through melanoma phenotypic transformation to a rhabdomyosarcoma. Whereas these tumors were genetically similar, they expressed distinct gene expression patterns suggestive of underlying epigenomic differences in the setting of an HDAC inhibitor. Citation Format: Arnav Mehta, David Liu, Alvin Shi, Dennie T. Frederick, Li-Lun Ho, Ana B. Larque, Benchun Miao, Rumya S. Raghavan, Tatyana Sharova, John H. Shin, Nicola Rinaldi, Ivan A. Chebib, Manolis Kellis, Eliezer Van Allen, Nir Hacohen, Keith T. Flaherty, Genevieve M. Boland, Ryan J. Sullivan. Acquired resistance to immune checkpoint inhibition by melanoma phenotypic transformation [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 3013.

Cell-Free DNA in Pediatric Rhabdomyosarcoma: Potential and Challenges.

Rhabdomyosarcoma is an aggressive solid tumor that may disseminate hematogenously giving metastasis which represents the most important prognostic factor. Chances of an effective cure in childhood cancer rely on the capacity to make an early and accurate diagnosis, detect metastatic disease or relapse, and predict the response to treatment.Liquid biopsy is a very promising blood test for cancer detection and noninvasive disease monitoring. This method has a great advantage to use blood and plasma, a more accessible biological material, quick and easy to obtain with minimal pain and risk for patients. In particular, circulating free DNA (cfDNA) represents a tumor biomarker detected in plasma that gives information on biology and genetic background of tumor.Moreover, cfDNA mutation detection could be a reliable method to monitor the efficacy of treatment and to discover novel targets for a personalized treatment in pediatric solid tumor. Here, we describe an optimized protocol to cfDNA isolation from small amount of plasma, as well as a method to assess the quantity and quality of cfDNA. Finally, we propose ddPCR as a reliable method to detect mutations at low frequency in cfDNA obtained from pediatric rhabdomyosarcoma samples.

Abstract A27: Ligand-dependent Hedgehog pathway activation in rhabdomyosarcoma

Abstract The Hedgehog (HH) pathway is known to develop an oncogenic role in some cancers, including rhabdomyosarcoma (RMS). However, the molecular mechanism that drives activation of the pathway in RMS is not well understood. Currently, a consistent activation of the pathway is well established and generally accepted in RMS. However, despite controversy regarding the importance of mutation-driven constitutive activation of the pathway in RMS patients, the activator mutations reported to date can only account---in the best scenario---for the activation of the pathway in small subsets of patients; therefore, the mechanism of activation remains elusive for the majority of cases. Purposes of the Study: The main purpose of this study is to demonstrate the importance of the ligand-dependent activation of Hedgehog pathway in this neoplasia. A secondary purpose is the description and characterization of a deleterious effect of the SMO inhibitor vismodegib, which promotes cell invasiveness throughout activation of Unfolded Protein Response (UPR). Experimental Procedures: The expression of Hedgehog ligands was studied in RMS samples by quantitative PCR, Western blot, and immunohistochemistry. Functional assays (cell cycle, cell proliferation, cell invasion, and apoptosis) and animal model studies were carried out with cells transduced with shRNAs (against the 3 Hedgehog ligands and Gli1) or treated with Hedgehog-specific inhibitors (vismodegib and MEDI-5304). Finally, the molecular characterization of an off-target effect of vismodegib was also made by cDNA microarrays, Transwell assay, Western blot, and shRNA-mediated TRIB3 inhibition. Summary of New Data: Results showed a prominent expression of HH ligands (especially Indian and Desert Hedgehog) in RMS cells and tumors, thereby supporting the existence of autocrine ligand-dependent activation of the pathway in this neoplasia. Moreover, although the shRNA-mediated downregulation of the ligands was not complete and performed separately for each ligand, the effects observed on tumor growth were remarkable. Thus, the downregulation of each single HH ligand clearly impaired tumor growth, suggesting that the participation of all ligands is crucial for RMS pathogenesis. Interestingly, a first description of pernicious off-target effect of vismodegib is also reported. Unexpectedly, vismodegib enhanced the invasive properties of all 3 cell lines by 2-fold. The mechanistic characterization of this biologic response suggested an off-target effect of vismodegib on cell invasiveness and chemoresistance via UPR activation. Given that the cell lines studied covered all the main subtypes of RMS, the results strongly suggest that UPR activation may be a general mechanism of cell invasiveness in RMS under vismodegib treatment and not a negligible peculiarity of a single cell line. Conclusions: The clarification of the HH pathway activation mechanism in RMS opens a door for targeted therapies against HH ligands as a possible alternative in the future development of better treatment protocols for RMS and other ligand-dependent cancers. Moreover, the description of a pernicious off-target effect of vismodegib may mechanistically explain its previously reported inefficiency in several ligand-dependent cancers or even the premature halting of some of them owing to survival worsening. Therefore, the findings herein described may have significant translational implication not only for RMS but also for other HH ligand-dependent cancers. Citation Format: Josep Roma, Ana Almazán-Moga, Patricia Zarzosa, Pablo Velasco, Carla Molist, Irina Giralt, Isaac Vidal, Natalia Navarro, Miguel Segura, Aroa Soriano, Josep Sánchez de Toledo, Soledad Gallego. Ligand-dependent Hedgehog pathway activation in rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr A27.

C-Myc promotes tumor proliferation and anti‑apoptosis by repressing p21 in rhabdomyosarcomas.

v-myc avian myelocytomatosis viral oncogene homolog (c-Myc) is an important member protein of the Myc family that is important in cell cycle progression, apoptosis and tumorigenesis. In the present study, the role of c‑Myc in rhabdomyosarcoma (RMS) was assessed. Firstly, expression of endogenous c‑Myc and cyclin dependent kinase inhibitor 1A (p21) was examined in normal skeletal muscle, RMS specimens and TE671 RMS cells by immunohistochemistry, reverse transcription‑quantitative polymerase chain reaction and western blotting. Furthermore, cell cycle progression and apoptosis were assessed in TE671 RMS cells following treatment with a c‑Myc inhibitor, 10058‑F4. The results demonstrated that c‑Myc was overexpressed in clinical RMS tissues and TE671 cells, with the highest expression observed in the most RMS samples. Expression of p21 protein and apoptosis function were increased following treatment with 10058‑F4, but no difference was observed in cell cycle progression. In conclusion, the present study indicated that c‑Myc promotes RMS development by inhibiting apoptosis through repression of p21 transcription. Further studies will be required to evaluate c‑Myc as a target for RMS clinical treatment.

Abstract 5522: Novel myogenic differentiation transcription factor CASZ1 suppresses rhabdomyosarcoma tumor growth

Abstract Disruption of muscle regulatory factors (MRFs) in muscle progenitor cells results in a failure to withdraw from cell cycle and terminal differentiation and is proposed to contribute to rhabdomyosarcoma (RMS) tumorigenesis. Zinc finger transcription factor CASZ1 is a tumor suppressor gene and regulates normal nervous system and heart development. CASZ1 is known to regulate a subset of genes that are involved in skeletal muscle development although the function of CASZ1 in normal myogenesis and RMS tumorigenesis is unknown. CASZ1 levels increase over 20-fold when C2C12 myoblasts differentiate into myotubes. To probe its role in normal myogenesis we performed genetic knockdown or overexpression experiments in C2C12 myoblasts. When CASZ1 expression is silenced using RNAi, there is a significant reduction in myotubes upon induction of differentiation. Realtime PCR shows that knockdown of CASZ1 increases Myf5, a MRF that determines early commitment of muscle precursor cells, but decreases Myogenin (2-fold, p<0.01), a MRF required for terminal differentiation and myotube formation. Knockdown of CASZ1 also decreases late skeletal differentiation genes Acta1, Ckm, Tnnt1 and Tnni2 (> 2-fold, p<0.01). Overexpression of CASZ1 induces expression of Myogenin, Acta1, Ckm and Tnnt1, represses Myf5 and accelerates myotube formation. This indicates that CASZ1 regulates genes important in the transition from early to late myogenic differentiation. To investigate the role of CASZ1 in RMS, we first evaluated the expression of CASZ1 in primary RMS tumors using public available microarray database. CASZ1 mRNA levels are ~1.5-fold lower in embryonal RMS (ERMS) samples compared to normal muscle (p<0.001), while levels of CASZ1 in alveolar (ARMS) are similar to levels in normal muscle. CASZ1 levels increase over 3-fold when RD cells (ERMS) were cultured in differentiation medium (100 nM 12-O-tetradecanoylphorbol-13-acetate, TPA). Knockdown of CASZ1 in the RD cells suppressed TPA induced expression of myogenic markers TNNT1 and TNNI2 while CASZ1 overexpression stimulated expression of TNNT1 and TNNI2 (all p<0.05). An in vivo spontaneous xenograft model showed that overexpression of CASZ1 significantly suppressed RD tumor growth (p<0.005). In RMS patients, NexGen sequencing of 85 tumors identified 4 samples with nonsynonymous single nucleotide variants (SNVs) in CASZ1 that were absent in the 1000 Genomes databases. We engineered these SNVs into CASZ1 construct and transfected them into C2C12 cells. We found that the R25C SNV had impaired nuclear localization and failed to activate skeletal muscle gene transcription (p<0.03). Taken together, our results suggest that the regulation of muscle differentiation program by CASZ1 in C2C12 is integral to proper myogenic differentiation, and genetic variants of CASZ1 disrupt early myogenesis and may contribute to ERMS tumorigenesis and progression. Citation Format: Zhihui Liu, Norris Lam, Arnulfo Mendoza, Jun S. Wei, John F. Shern, Marielle Yohe, Javed Khan, Carol J. Thiele. Novel myogenic differentiation transcription factor CASZ1 suppresses rhabdomyosarcoma tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5522. doi:10.1158/1538-7445.AM2017-5522

NELL1, whose high expression correlates with negative outcomes, has different methylation patterns in alveolar and embryonal rhabdomyosarcoma.

Rhabdomyosarcoma (RMS), which represents the most frequent soft tissue sarcoma in pediatric populations, is classified into two major subtypes: embryonal RMS (ERMS) and alveolar RMS (ARMS). ARMS subtype, which shows greater aggressiveness and proneness to metastasis with respect to ERMS, are characterized, in about 75% of cases, by specific chromosomal translocations that involve PAX and FOXO1 genes. Many findings have demonstrated that PAX/FOXO1-positive ARMS have a worse prognosis than PAX/FOXO1-negative ones and that distinct molecular features characterize RMS with different gene fusion statuses. DNA methylation, which presently represents a challenging research area, is involved in the modulation of gene expression.We performed a genome-wide DNA methylation analysis using reduced-representation bisulfite sequencing (RRBS) in RMS samples and we found that fusion-positive alveolar and embryonal subgroups have different DNA methylation signatures and that ARMS fusion-positive subtypes are characterized by overall hypomethylation levels. While NELL1 was found to be hypomethylated and transcriptionally enhanced in RMS alveolar subtypes, high NELL1 expression levels, which proved to be correlated with negative RMS prognostic factors such as fusion status and histology (P < 0.0001), were found to discriminate between RMS patients with different outcomes (P < 0.05).In conclusion, our results demonstrated that different DNA methylation patterns distinguish between different RMS subgroups and they suggest that epigenetic signatures could be useful for risk stratification of patients.

MYOD1 (L122R) mutations are associated with spindle cell and sclerosing rhabdomyosarcomas with aggressive clinical outcomes

Recurrent mutations in the myogenic transcription factor MYOD1 and PIK3CA were initially described in a subset of embryonal rhabdomyosarcomas. Recently, two independent studies demonstrated presence of MYODI (L122R) mutations as the basis to re-classify a spindle cell rhabdomyosarcoma, along with a sclerosing rhabdomyosarcoma, distinct from an embryonal rhabdomyosarcoma. We analyzed a much larger cohort of 49 primary rhabdomyosarcoma tumor samples of various subtypes, collected over a period of 9 years, for the presence of MYOD1 (L122R), PIK3CA (H1047), and PIK3CA (E542/E545) mutations, along with immunohistochemical analysis of desmin, myogenin, and MYOD1. Although activating PIK3CA mutations were absent across the sample set analyzed, we report 20% MYOD1 (L122R) mutation in rhabdomyosarcomas, found exclusively in 10 of 21 spindle cell and sclerosing rhabdomyosarcomas, occurring mostly in the head and neck region along with extremity sites (64%), than the paratesticular and intra-abdominal sites. Furthermore, while all 10 MYOD1 mutant spindle cell and sclerosing rhabdomyosarcoma samples showed diffuse and strong MYOD1 immunoexpression, 7 of 31 samples of rhabdomyosarcoma with wild-type MYOD1 were negative for MYOD1 expression. Clinically, a striking correlation was found between MYOD1 mutation and the clinical outcomes available for 15 of 21 cases: 5 of 7 patients with spindle cell and sclerosing rhabdomyosarcomas, harboring MYOD1 mutation, were alive-with-disease and 2 of 8 patients with spindle cell and sclerosing rhabdomyosarcomas, with mutant MYOD1, were free-of-disease. Taken together, we present the first report of MYOD1 (L122R) mutation in the largest cohort of 49 rhabdomyosarcomas reported so far, that are associated with a relatively aggressive clinical course. Moreover, consistent with the earlier two studies, this study further reinforces a relationship between spindle cell and the sclerosing rhabdomyosarcoma—now recognized as a single subtype, distinct from an embryonal rhabdomyosarcoma.

TBX2 represses PTEN in rhabdomyosarcoma and skeletal muscle.

Rhabdomyosarcoma (RMS) is the most frequent soft tissue sarcoma in children that shares many features of developing skeletal muscle. TBX2, a T-box family member, is highly up regulated in tumor cells of both major RMS subtypes where it functions as an oncogene. TBX2 is a repressor that is often over expressed in cancer cells and functions in bypassing cell growth control, including the repression of the cell cycle regulators p14 and p21. We have found that TBX2 directly represses the tumor suppressor PTEN in both RMS and normal muscle. Exogenous expression of TBX2 in normal muscle cells down regulates PTEN, and depletion or interference with TBX2 in RMS cells up regulates PTEN. Human RMS tumors show high levels of TBX2 and correspondingly low levels of PTEN. The expression of PTEN in clinical RMS samples is relatively uncharacterized and we establish that suppression of PTEN is a frequent event in both subtypes of RMS. TBX2 represses PTEN by directly binding to the promoter and recruiting the histone deacetylase, HDAC1. RMS cells have high levels of activated AKT due to the deregulation of PI3K signaling, and depletion or interference with TBX2, which up regulates PTEN, results in a reduction of phospho-AKT. We have also found that the highly related T-box family member TBX3 does not repress PTEN in the muscle lineage. This work suggests that TBX2 is a central component of the PTEN/PI3K/AKT signaling pathway deregulation in RMS cells and that targeting TBX2 in RMS tumors may offer a novel therapeutic approach for RMS.

Hyper-Activation of Notch3 Amplifies the Proliferative Potential of Rhabdomyosarcoma Cells

Rhabdomyosarcoma (RMS) is a pediatric myogenic-derived soft tissue sarcoma that includes two major histopathological subtypes: embryonal and alveolar. The majority of alveolar RMS expresses PAX3-FOXO1 fusion oncoprotein, associated with the worst prognosis. RMS cells show myogenic markers expression but are unable to terminally differentiate. The Notch signaling pathway is a master player during myogenesis, with Notch1 activation sustaining myoblast expansion and Notch3 activation inhibiting myoblast fusion and differentiation. Accordingly, Notch1 signaling is up-regulated and activated in embryonal RMS samples and supports the proliferation of tumor cells. However, it is unable to control their differentiation properties. We previously reported that Notch3 is activated in RMS cell lines, of both alveolar and embryonal subtype, and acts by inhibiting differentiation. Moreover, Notch3 depletion reduces PAX3-FOXO1 alveolar RMS tumor growth in vivo. However, whether Notch3 activation also sustains the proliferation of RMS cells remained unclear. To address this question, we forced the expression of the activated form of Notch3, Notch3IC, in the RH30 and RH41 PAX3-FOXO1-positive alveolar and in the RD embryonal RMS cell lines and studied the proliferation of these cells. We show that, in all three cell lines tested, Notch3IC over-expression stimulates in vitro cell proliferation and prevents the effects of pharmacological Notch inhibition. Furthermore, Notch3IC further increases RH30 cell growth in vivo. Interestingly, knockdown of Notch canonical ligands JAG1 or DLL1 in RMS cell lines decreases Notch3 activity and reduces cell proliferation. Finally, the expression of Notch3IC and its target gene HES1 correlates with that of the proliferative marker Ki67 in a small cohort of primary PAX-FOXO1 alveolar RMS samples. These results strongly suggest that high levels of Notch3 activation increase the proliferative potential of RMS cells.

Investigation of IGF2, Hedgehog and fusion gene expression profiles in pediatric sarcomas

The childhood sarcomas are malignant tumors with high mortality rates. They are divided into two genetic categories: a category without distinct pattern karyotypic changes and the other category showing unique translocations that originate gene rearrangements. This category includes rhabdomyosarcoma (RMS), Ewing's sarcoma (ES) and synovial sarcoma (SS). Diverse studies have related development genes, such as; IGF2, IHH, PTCH1 and GLI1 and sarcomatogenesis. ObjectiveTo characterize the RMS, ES and SS rearrangements, we quantify the expression of IGF2 IHH, PTCH1 and GLI1 genes and correlate molecular data with clinical parameters of patients. DesignWe analyzed 29 RMS, 10 SS and 60 ES tumor samples by RT-PCR (polymerase chain reaction-reverse transcription) and qPCR (quantitative PCR). ResultsAmong the samples of ARMS, 50% had rearrangements of PAX3/7–FOXO1, 60% of ES samples were EWS–FLI1 positive and 90% of SS samples were positive for SS18–SSX1/2. In relation to the control reference samples (QPCR Human Reference Total RNA-Stratagene, Human Skeletal Muscle Total RNA-Ambion, Universal RNA Human Normal Tissues-Ambion), RMS samples showed a high IGF2 gene expression (p<0.0001). Moreover, ES samples showed a low IGF2 gene expression (p<0.0001) and high IHH (p<0.0001), PTCH1 (p=0.0173) and GLI1 (p=0.0113) gene expressions. ConclusionsThe molecular characterization of IGF and Hedgehog pathway in these pediatric sarcomas may collaborate to enable a better understanding of the biological behavior of these neoplasms.

Transactivating mutation of theMYOD1gene is a frequent event in adult spindle cell rhabdomyosarcoma

Rhabdomyosarcoma (RMS) is the most common soft-tissue sarcoma in children and adolescents, being characterized by expression of genes and morphological and ultrastructural features of sarcomeric differentiation. The spindle cell variant of rhabdomyosarcoma (spindle cell RMS) in adults has been defined as an entity, separated from embryonal rhabdomyosarcoma (ERMS), with unfavourable clinical outcome. So far, no recurrent genetic alteration has been identified in the adult form of spindle cell RMS. We studied a case of adult spindle cell RMS using next-generation sequencing (NGS) after exome capture. Using this approach, we identified 31 tumour-specific somatic alterations and selected four genes with predicted functional relevance to muscle differentiation and growth. MYOD1, KIF18A, NOTCH1, and EML5 were further tested for mutations using Sanger sequencing on DNA from FFPE samples from 16 additional, adult spindle cell RMS samples. The highly conserved sequence homology of MYOD1 with other myogenic transcription factors prompted us to screen the basic DNA-binding domains of MYF5, MYF6 and MYOG for mutations. From the investigated 17 samples, seven (41%) showed homozygous mutation of MYOD1, indicating a critical role in this rare subtype of adult spindle cell RMS, while no mutations were found in any of the other genes involved in myogenic differentiation. The p.L122R mutation occurs in the conserved DNA binding domain in MYOD1 and leads to transactivation and MYC-like functions. MYOD1 homozygous mutations are frequent, recurrent and pathognomonic events in adult-type spindle cell RMS.

Heparanase-1 and components of the hedgehog signalling pathway are increased in untreated alveolar orbital rhabdomyosarcoma

To assess the activities of heparanase-1 and elements of the hedgehog signalling pathway in alveolar orbital rhabdomyosarcoma. Specimens (n = 23) were divided into two groups, those from patients with preoperative chemoradiotherapy or untreated patients; six samples of normal extraocular muscle were used as a normal muscle group. The presence of heparanase-1, patched, smoothened and glioma-associated oncogene homologue-1 protein expression was determined in 23 cases of archival paraffin-embedded alveolar orbital rhabdomyosarcoma after immunohistochemistry. RNA was extracted from three groups of paraffin-embedded specimens and messenger RNA expressions of heparanase-1, smoothened and glioma-associated oncogene homologue-1 compared using nested reverse transcriptase polymerase chain reaction and a limiting dilution analysis. The heparanase-1, patched, smoothened and glioma-associated oncogene homologue-1 protein was expressed in 91.3%, 87.0%, 91.3% and 78.3%, respectively, of the alveolar orbital rhabdomyosarcoma specimens. Untreated rhabdomyosarcoma samples tended to stain intensely, but staining was relatively weak in tissue obtained from the chemoradiotherapy group. The expression levels of heparanase-1, smoothened and glioma-associated oncogene homologue-1 messenger RNA in untreated and chemoradiotherapy groups paralleled that seen with immunology, and there were no significant differences in heparanase-1, smoothened and glioma-associated oncogene homologue-1 messenger RNA levels between the chemoradiotherapy group and the normal muscle group (P > 0.05). However, the messenger RNA in the untreated group were all significantly higher than those in the chemoradiotherapy and normal muscle groups (P < 0.01). Both heparanase-1 and hedgehog signalling pathway are involved in the pathogenesis of alveolar orbital rhabdomyosarcoma; however, chemotherapy and/or radiotherapy appears to significantly inhibit their upregulation.