Publications by authors named "Stephen X Skapek"

78 Publications

Genomic Classification and Clinical Outcome in Rhabdomyosarcoma: A Report From an International Consortium.

J Clin Oncol 2021 09 24;39(26):2859-2871. Epub 2021 Jun 24.

Department of Pediatrics, Seattle Children's Hospital, Fred Hutchinson Cancer Research Center, University of Washington, Seattle, WA.

Purpose: Rhabdomyosarcoma is the most common soft tissue sarcoma of childhood. Despite aggressive therapy, the 5-year survival rate for patients with metastatic or recurrent disease remains poor, and beyond fusion status, no genomic markers are available for risk stratification. We present an international consortium study designed to determine the incidence of driver mutations and their association with clinical outcome.

Patients And Methods: Tumor samples collected from patients enrolled on Children's Oncology Group trials (1998-2017) and UK patients enrolled on malignant mesenchymal tumor and RMS2005 (1995-2016) trials were subjected to custom-capture sequencing. Mutations, indels, gene deletions, and amplifications were identified, and survival analysis was performed.

Results: DNA from 641 patients was suitable for analyses. A median of one mutation was found per tumor. In fusion-negative cases, mutation of any RAS pathway member was found in > 50% of cases, and 21% had no putative driver mutation identified. (15%), (15%), and (13%) mutations were found at a higher incidence than previously reported and mutations were associated with worse outcomes in both fusion-negative and fusion-positive cases. Interestingly, mutations in isoforms predominated in infants < 1 year (64% of cases). Mutation of was associated with histologic patterns beyond those previously described, older age, head and neck primary site, and a dismal survival. Finally, we provide a searchable companion database (ClinOmics), containing all genomic variants, and clinical annotation including survival data.

Conclusion: This is the largest genomic characterization of clinically annotated rhabdomyosarcoma tumors to date and provides prognostic genetic features that refine risk stratification and will be incorporated into prospective trials.
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http://dx.doi.org/10.1200/JCO.20.03060DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8425837PMC
September 2021

Pathogenic Germline Variants in Cancer Susceptibility Genes in Children and Young Adults With Rhabdomyosarcoma.

JCO Precis Oncol 2021 11;5. Epub 2021 Jan 11.

Clinical Genetics Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, Rockville, MD.

Rhabdomyosarcoma (RMS) is the most common pediatric soft-tissue sarcoma and accounts for 3% of all pediatric cancer. In this study, we investigated germline sequence and structural variation in a broad set of genes in two large, independent RMS cohorts.

Materials And Methods: Genome sequencing of the discovery cohort (n = 273) and exome sequencing of the secondary cohort (n = 121) were conducted on germline DNA. Analyses were performed on 130 cancer susceptibility genes (CSG). Pathogenic or likely pathogenic (P/LP) variants were predicted using the American College of Medical Genetics and Genomics (ACMG) criteria. Structural variation and survival analyses were performed on the discovery cohort.

Results: We found that 6.6%-7.7% of patients with RMS harbored P/LP variants in dominant-acting CSG. An additional approximately 1% have structural variants (, ) in CSGs. CSG variants did not influence survival, although there was a significant correlation with an earlier age of tumor onset. There was a nonsignificant excess of P/LP variants in dominant inheritance genes in the patients with fusion-negative RMS patients versus the patients with fusion-positive RMS. We identified pathogenic germline variants in CSGs previously (, , , mismatch repair genes), rarely (, , , ), or never () reported in RMS. Numerous genes (, , mismatch repair) were on the ACMG Secondary Findings 2.0 list.

Conclusion: In two cohorts of patients with RMS, we identified pathogenic germline variants for which gene-specific therapies and surveillance guidelines may be beneficial. In families with a proband with an RMS-risk P/LP variant, genetic counseling and cascade testing should be considered, especially for ACMG Secondary Findings genes and/or with gene-specific surveillance guidelines.
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http://dx.doi.org/10.1200/PO.20.00218DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8169077PMC
January 2021

Prioritization of Novel Agents for Patients with Rhabdomyosarcoma: A Report from the Children's Oncology Group (COG) New Agents for Rhabdomyosarcoma Task Force.

J Clin Med 2021 Apr 1;10(7). Epub 2021 Apr 1.

Department of Pediatrics and Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

Rhabdomyosarcoma is the most common soft tissue sarcoma diagnosed in children and adolescents. Patients that are diagnosed with advanced or relapsed disease have exceptionally poor outcomes. The Children's Oncology Group (COG) convened a rhabdomyosarcoma new agent task force in 2020 to systematically evaluate novel agents for inclusion in phase 2 or phase 3 clinical trials for patients diagnosed with rhabdomyosarcoma, following a similar effort for Ewing sarcoma. The task force was comprised of clinicians and basic scientists who collectively identified new agents for evaluation and prioritization in clinical trial testing. Here, we report the work of the task force including the framework upon which the decisions were rendered and review the top classes of agents that were discussed. Representative agents include poly-ADP-ribose polymerase (PARP) inhibitors in combination with cytotoxic agents, mitogen-activated protein kinase (MEK) inhibitors in combination with type 1 insulin-like growth factor receptor (IGFR1) inhibitors, histone deacetylase (HDAC) inhibitors, and novel cytotoxic agents.
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http://dx.doi.org/10.3390/jcm10071416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8037615PMC
April 2021

Germline Cancer Predisposition Variants in Pediatric Rhabdomyosarcoma: A Report From the Children's Oncology Group.

J Natl Cancer Inst 2021 Jul;113(7):875-883

Department of Pediatrics, Section of Hematology-Oncology, Baylor College of Medicine, Houston, TX, USA.

Background: Several cancer-susceptibility syndromes are reported to underlie pediatric rhabdomyosarcoma (RMS); however, to our knowledge there have been no systematic efforts to characterize the heterogeneous genetic etiologies of this often-fatal malignancy.

Methods: We performed exome-sequencing on germline DNA from 615 patients with newly diagnosed RMS consented through the Children's Oncology Group. We compared the prevalence of cancer predisposition variants in 63 autosomal-dominant cancer predisposition genes in these patients with population controls (n = 9963). All statistical tests were 2-sided.

Results: We identified germline cancer predisposition variants in 45 RMS patients (7.3%; all FOXO1 fusion negative) across 15 autosomal dominant genes, which was statistically significantly enriched compared with controls (1.4%, P = 1.3 × 10-22). Specifically, 73.3% of the predisposition variants were found in predisposition syndrome genes previously associated with pediatric RMS risk, such as Li-Fraumeni syndrome (TP53) and neurofibromatosis type I (NF1). Notably, 5 patients had well-described oncogenic missense variants in HRAS (p.G12V and p.G12S) associated with Costello syndrome. Also, genetic etiology differed with histology, as germline variants were more frequent in embryonal vs alveolar RMS patients (10.0% vs 3.0%, P = .02). Although patients with a cancer predisposition variant tended to be younger at diagnosis (P = 9.9 × 10-4), 40.0% of germline variants were identified in those older than 3 years of age, which is in contrast to current genetic testing recommendations based on early age at diagnosis.

Conclusions: These findings demonstrate that genetic risk of RMS results from germline predisposition variants associated with a wide spectrum of cancer susceptibility syndromes. Germline genetic testing for children with RMS should be informed by RMS subtypes and not be limited to only young patients.
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http://dx.doi.org/10.1093/jnci/djaa204DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8246828PMC
July 2021

HDAC6 promotes growth, migration/invasion, and self-renewal of rhabdomyosarcoma.

Oncogene 2021 01 16;40(3):578-591. Epub 2020 Nov 16.

Department of Laboratory Medicine and Pathology, University of Washington, Seattle, WA, USA.

Rhabdomyosarcoma (RMS) is a devastating pediatric sarcoma. The survival outcomes remain poor for patients with relapsed or metastatic disease. Effective targeted therapy is lacking due to our limited knowledge of the underlying cellular and molecular mechanisms leading to disease progression. In this study, we used functional assays in vitro and in vivo (zebrafish and xenograft mouse models) to demonstrate the crucial role of HDAC6, a cytoplasmic histone deacetylase, in driving RMS tumor growth, self-renewal, and migration/invasion. Treatment with HDAC6-selective inhibitors recapitulates the HDAC6 loss-of-function phenotypes. HDAC6 regulates cytoskeletal dynamics to promote tumor cell migration and invasion. RAC1, a Rho family GTPase, is an essential mediator of HDAC6 function, and is necessary and sufficient for RMS cell migration and invasion. High expression of RAC1 correlates with poor clinical prognosis in RMS patients. Targeting the HDAC6-RAC1 axis represents a promising therapeutic option for improving survival outcomes of RMS patients.
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http://dx.doi.org/10.1038/s41388-020-01550-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7855743PMC
January 2021

Functional imaging of RAS pathway targeting in malignant peripheral nerve sheath tumor cells and xenografts.

Pediatr Blood Cancer 2020 12 25;67(12):e28639. Epub 2020 Sep 25.

Department of Pediatrics Division of Hematology/Oncology, University of Texas Southwestern Medical Center, Dallas, Texas.

Background: Malignant peripheral nerve sheath tumor (MPNST) is an aggressive form of soft-tissue sarcoma (STS) in children. Despite intensive therapy, relatively few children with metastatic and unresectable disease survive beyond three years. RAS pathway activation is common in MPNST, suggesting MEK pathway inhibition as a targeted therapy, but the impact on clinical outcome has been small to date.

Procedure: We conducted preclinical pharmacokinetic (PK) and pharmacodynamic studies of two MEK inhibitors, trametinib and selumetinib, in two MPNST models and analyzed tumors for intratumor drug levels. We then investigated 3'-deoxy-3'-[ F]fluorothymidine ( F-FLT) PET imaging followed by F-FDG PET/CT imaging of MPNST xenografts coupled to short-term or longer-term treatment with selumetinib focusing on PET-based imaging as a biomarker of MEK inhibition.

Results: Trametinib decreased pERK expression in MPNST xenografts but did not prolong survival or decrease Ki67 expression. In contrast, selumetinib prolonged survival of animals bearing MPNST xenografts, and this correlated with decreased pERK and Ki67 staining. PK studies revealed a significantly higher fraction of unbound selumetinib within a responsive MPNST xenograft model. Thymidine uptake, assessed by F-FLT PET/CT, positively correlated with Ki67 expression in different xenograft models and in response to selumetinib.

Conclusion: The ability of MEK inhibitors to control MPNST growth cannot simply be predicted by serum drug levels or drug-induced changes in pERK expression. Tumor cell proliferation assessed by F-FLT PET imaging might be useful as an early response marker to targeted therapies, including MEK inhibition, where a primary effect is cell-cycle arrest.
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http://dx.doi.org/10.1002/pbc.28639DOI Listing
December 2020

Transcriptome analysis of desmoplastic small round cell tumors identifies actionable therapeutic targets: a report from the Children's Oncology Group.

Sci Rep 2020 07 23;10(1):12318. Epub 2020 Jul 23.

Department of Translational Genomics, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA.

To further understand the molecular pathogenesis of desmoplastic small round cell tumor (DSRCT), a fatal malignancy occurring primarily in adolescent/young adult males, we used next-generation RNA sequencing to investigate the gene expression profiles intrinsic to this disease. RNA from DSRCT specimens obtained from the Children's Oncology Group was sequenced using the Illumina HiSeq 2000 system and subjected to bioinformatic analyses. Validation and functional studies included WT1 ChIP-seq, EWS-WT1 knockdown using JN-DSRCT-1 cells and immunohistochemistry. A panel of immune signature genes was also evaluated to identify possible immune therapeutic targets. Twelve of 14 tumor samples demonstrated presence of the diagnostic EWSR1-WT1 translocation and these 12 samples were used for the remainder of the analysis. RNA sequencing confirmed the lack of full-length WT1 in all fusion positive samples as well as the JN-DSRCT-1 cell line. ChIP-seq for WT1 showed significant overlap with genes found to be highly expressed, including IGF2 and FGFR4, which were both highly expressed and targets of the EWS-WT1 fusion protein. In addition, we identified CD200 and CD276 as potentially targetable immune checkpoints whose expression is independent of the EWS-WT1 fusion gene in cultured DSCRT cells. In conclusion, we identified IGF2, FGFR4, CD200, and CD276 as potential therapeutic targets with clinical relevance for patients with DSRCT.
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http://dx.doi.org/10.1038/s41598-020-69015-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7378211PMC
July 2020

Development of a Data Model and Data Commons for Germ Cell Tumors.

JCO Clin Cancer Inform 2020 06;4:555-566

Quantitative Biomedical Research Center, Department of Population and Data Sciences, University of Texas Southwestern Medical Center, Dallas, TX.

Germ cell tumors (GCTs) are considered a rare disease but are the most common solid tumors in adolescents and young adults, accounting for 15% of all malignancies in this age group. The rarity of GCTs in some groups, particularly children, has impeded progress in treatment and biologic understanding. The most effective GCT research will result from the interrogation of data sets from historical and prospective trials across institutions. However, inconsistent use of terminology among groups, different sample-labeling rules, and lack of data standards have hampered researchers' efforts in data sharing and across-study validation. To overcome the low interoperability of data and facilitate future clinical trials, we worked with the Malignant Germ Cell International Consortium (MaGIC) and developed a GCT clinical data model as a uniform standard to curate and harmonize GCT data sets. This data model will also be the standard for prospective data collection in future trials. Using the GCT data model, we developed a GCT data commons with data sets from both MaGIC and public domains as an integrated research platform. The commons supports functions, such as data query, management, sharing, visualization, and analysis of the harmonized data, as well as patient cohort discovery. This GCT data commons will facilitate future collaborative research to advance the biologic understanding and treatment of GCTs. Moreover, the framework of the GCT data model and data commons will provide insights for other rare disease research communities into developing similar collaborative research platforms.
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http://dx.doi.org/10.1200/CCI.20.00025DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7328105PMC
June 2020

A risk-based treatment strategy for non-rhabdomyosarcoma soft-tissue sarcomas in patients younger than 30 years (ARST0332): a Children's Oncology Group prospective study.

Lancet Oncol 2020 01 27;21(1):145-161. Epub 2019 Nov 27.

Seattle Children's Hospital, Seattle, WA, USA.

Background: Tumour grade, tumour size, resection potential, and extent of disease affect outcome in paediatric non-rhabdomyosarcoma soft-tissue sarcoma (NRSTS), but no risk stratification systems exist and the standard of care is poorly defined. We developed a risk stratification system from known prognostic factors and assessed it in the context of risk-adapted therapy for young patients with NRSTS.

Methods: In this prospective study, eligible patients enrolled in 159 hospitals in three countries were younger than 30 years, had a Lansky (patients ≤16 years) or Karnofsky (patients >16 years) performance status score of at least 50, and a new diagnosis of a WHO (2002 criteria) intermediate (rarely metastasising) or malignant soft-tissue tumour (apart from tumour types eligible for other Children's Oncology Group studies and tumours for which the therapy in this trial was deemed inappropriate), malignant peripheral nerve sheath tumour, non-metastatic and grossly resected dermatofibrosarcoma protuberans, undifferentiated embryonal sarcoma of the liver, or unclassified malignant soft-tissue sarcoma. Each patient was assigned to one of three risk groups and one of four treatment groups. Risk groups were: low (non-metastatic R0 or R1 low-grade, or ≤5 cm R1 high-grade tumour); intermediate (non-metastatic R0 or R1 >5 cm high-grade, or unresected tumour of any size or grade); or high (metastatic tumour). The treatment groups were surgery alone, radiotherapy (55·8 Gy), chemoradiotherapy (chemotherapy and 55·8 Gy radiotherapy), and neoadjuvant chemoradiotherapy (chemotherapy and 45 Gy radiotherapy, then surgery and radiotherapy boost based on margins with continued chemotherapy). Chemotherapy included six cycles of ifosfamide 3 g/m per dose intravenously on days 1-3 and five cycles of doxorubicin 37·5 mg/m per dose intravenously on days 1-2 every 3 weeks with sequence adjusted on the basis of timing of surgery or radiotherapy. The primary outcomes were event-free survival, overall survival, and the pattern of treatment failure. Analysis was done per protocol. This study has been completed and is registered with ClinicalTrials.gov, NCT00346164.

Findings: Between Feb 5, 2007, and Feb 10, 2012, 550 eligible patients were enrolled, of whom 21 were treated in the incorrect group and excluded from this analysis. 529 evaluable patients were included in the analysis: low-risk (n=222), intermediate-risk (n=227), high-risk (n=80); surgery alone (n=205), radiotherapy (n=17), chemoradiotherapy (n=111), and neoadjuvant chemoradiotherapy (n=196). At a median follow-up of 6·5 years (IQR 4·9-7·9), 5-year event-free survival and overall survival were: 88·9% (95% CI 84·0-93·8) and 96·2% (93·2-99·2) in the low-risk group; 65·0% (58·2-71·8) and 79·2% (73·4-85·0) in the intermediate-risk group; and 21·2% (11·4-31·1) and 35·5% (23·6-47·4) in the high-risk group, respectively. Risk group predicted event-free survival and overall survival (p<0·0001). No deaths from toxic events during treatment were reported. Nine patients had unexpected grade 4 adverse events (chemoradiotherapy group, n=2; neoadjuvant chemoradiotherapy group, n=7), including three wound complications that required surgery (all in the neoadjuvant chemoradiotherapy group).

Interpretation: Pre-treatment clinical features can be used to effectively define treatment failure risk and to stratify young patients with NRSTS for risk-adapted therapy. Most low-risk patients can be cured without adjuvant therapy, thereby avoiding known long-term treatment complications. Survival remains suboptimal for intermediate-risk and high-risk patients and novel therapies are needed.

Funding: National Institutes of Health, St Baldrick's Foundation, Seattle Children's Foundation, American Lebanese Syrian Associated Charities.
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http://dx.doi.org/10.1016/S1470-2045(19)30672-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6946838PMC
January 2020

Bayesian Modeling Identifies PLAG1 as a Key Regulator of Proliferation and Survival in Rhabdomyosarcoma Cells.

Mol Cancer Res 2020 03 22;18(3):364-374. Epub 2019 Nov 22.

Department of Pediatrics, Division of Hematology/Oncology, University of Texas Southwestern Medical Center, Dallas, Texas.

We recently developed a novel computational algorithm that incorporates Bayesian methodology to identify rhabdomyosarcoma disease genes whose expression level correlates with copy-number variations, and we identified as a candidate oncogenic driver. Although PLAG1 has been shown to contribute to other type of cancers, its role in rhabdomyosarcoma has not been elucidated. We observed that PLAG1 mRNA is highly expressed in rhabdomyosarcoma and is associated with gene copy-number gain. Knockdown of PLAG1 dramatically decreased cell accumulation and induced apoptosis in rhabdomyosarcoma cells, whereas its ectopic expression increased cell accumulation and as a xenograft and promoted G to S-phase cell-cycle progression. We found that PLAG1 regulates IGF2 expression and influences AKT and MAPK pathways in rhabdomyosarcoma, and IGF2 partially rescues cell death triggered by PLAG1 knockdown. The expression level of PLAG1 correlated with the IC of rhabdomyosarcoma cells to BMS754807, an IGF receptor inhibitor. IMPLICATIONS: Our data demonstrate that PLAG1 contributes to proliferation and survival of rhabdomyosarcoma cells at least partially by inducing IGF2, and this new understanding may have the potential for clinical translation.
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http://dx.doi.org/10.1158/1541-7786.MCR-19-0764DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7056573PMC
March 2020

Novel rearrangement in multifocal infantile myofibromatosis is tumorigenic and sensitive to imatinib.

Cold Spring Harb Mol Case Stud 2019 10 23;5(5). Epub 2019 Oct 23.

Division of Hematology/Oncology, Departments of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

Infantile myofibromatosis (IM) is an aggressive neoplasm composed of myofibroblast-like cells in children. Although typically localized, it can also present as multifocal disease, which represents a challenge for effective treatment. IM has previously been linked to activating somatic and germline point mutations in the PDGFRβ tyrosine kinase encoded by the gene. Clinical panel-based targeted tumor sequencing of a tumor from a newborn with multifocal IM revealed a novel rearrangement, which was reported as being of unclear significance. Additional sequencing of cDNA from tumor and germline DNA confirmed a complex somatic/mosaic rearrangement with an apparent partial tandem duplication disrupting the juxtamembrane domain. Ectopic expression of cDNA encoding the mutant form of PDGFRB markedly enhanced cell proliferation of mouse embryo fibroblasts (MEFs) compared to wild-type PDGFRB and conferred tumor-forming capacity on nontumorigenic 10T1/2 fibroblasts. The mutated protein enhanced MAPK activation and retained sensitivity to the PDGFRβ inhibitor imatinib. Our findings reveal a new mechanism by which PDGFRB can be activated in IM, suggest that therapy with tyrosine kinase inhibitors including imatinib may be beneficial, and raise the possibility that this receptor tyrosine kinase might be altered in a similar fashion in additional cases that would similarly present annotation challenges in clinical DNA sequencing analysis pipelines.
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http://dx.doi.org/10.1101/mcs.a004440DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6824247PMC
October 2019

Testis-specific Arf promoter expression in a transposase-aided BAC transgenic mouse model.

Mol Biol Rep 2019 Dec 3;46(6):6243-6252. Epub 2019 Oct 3.

Division of Hematology/Oncology, Department of Pediatrics, UT Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX, 75390, USA.

CDKN2A is an evolutionarily conserved gene encoding proteins implicated in tumor suppression, ocular development, aging, and metabolic diseases. Like the human form, mouse Cdkn2a encodes two distinct proteins-p16, which blocks cyclin-dependent kinase activity, and p19, which is best known as a positive regulator of the p53 tumor suppressor-and their functions have been well-studied in genetically engineered mouse models. Relatively little is known about how expression of the two transcripts is controlled in normal development and in certain disease states. To better understand their coordinate and transcript-specific expression in situ, we used a transposase-aided approach to generate a new BAC transgenic mouse model in which the first exons encoding Arf and Ink4a are replaced by fluorescent reporters. We show that mouse embryo fibroblasts generated from the transgenic lines faithfully display induction of each transgenic reporter in cell culture models, and we demonstrate the expected expression of the Arf reporter in the normal testis, one of the few places where that promoter is normally expressed. Interestingly, the TGFβ-2-dependent induction of the Arf reporter in the eye-a process essential for normal eye development-does not occur. Our findings illustrate the value of BAC transgenesis in mapping key regulatory elements in the mouse by revealing the genomic DNA required for Cdkn2a induction in cultured cells and the developing testis, and the apparent lack of elements driving expression in the developing eye.
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http://dx.doi.org/10.1007/s11033-019-05063-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6883165PMC
December 2019

Refinement of risk stratification for childhood rhabdomyosarcoma using FOXO1 fusion status in addition to established clinical outcome predictors: A report from the Children's Oncology Group.

Cancer Med 2019 10 27;8(14):6437-6448. Epub 2019 Aug 27.

Department of Pediatric and Adolescent Medicine, Mayo Clinic, Rochester, Minnesota.

Background: Previous studies of the prognostic importance of FOXO1 fusion status in patients with rhabdomyosarcoma (RMS) have had conflicting results. We re-examined risk stratification by adding FOXO1 status to traditional clinical prognostic factors in children with localized or metastatic RMS.

Methods: Data from six COG clinical trials (D9602, D9802, D9803, ARST0331, ARTS0431, ARST0531; two studies each for low-, intermediate- and high-risk patients) accruing previously untreated patients with RMS from 1997 to 2013 yielded 1727 evaluable patients. Survival tree regression for event-free survival (EFS) was conducted to recursively select prognostic factors for branching and split. Factors included were age, FOXO1, clinical group, histology, nodal status, number of metastatic sites, primary site, sex, tumor size, and presence of metastases in bone/bone marrow, soft tissue, effusions, lung, distant lymph nodes, and other sites. Definition and outcome of the proposed risk groups were compared to existing systems and cross-validated results.

Results: The 5-year EFS and overall survival (OS) for evaluable patients were 69% and 79%, respectively. Extent of disease (localized versus metastatic) was the first split (EFS 73% vs 30%; OS 84% vs. 42%). FOXO1 status (positive vs negative) was significant in the second split both for localized (EFS 52% vs 78%; OS 65% vs 88%) and metastatic disease (EFS 6% vs 46%; OS 19% vs 58%).

Conclusions: After metastatic status, FOXO1 status is the most important prognostic factor in patients with RMS and improves risk stratification of patients with localized RMS. Our findings support incorporation of FOXO1 status in risk stratified clinical trials.
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http://dx.doi.org/10.1002/cam4.2504DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6797586PMC
October 2019

Identification of Enhancers Activated by TGFβ to Drive Expression of and in HeLa Cells.

Mol Cancer Res 2019 09 12;17(9):1854-1866. Epub 2019 Jun 12.

Division of Hematology/Oncology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, Texas.

Disruption of the () and () genes, which encode three function-independent tumor suppressors, is one of the most common events in human cancer. Because their relative importance in tumor prevention appears to be species- and context-specific, studying their regulation can shed light on mechanisms by which they are bypassed in malignant transformation. We previously unveiled a new pathway in which TGFβ selectively induces at mouse in eye development and cultured fibroblasts. As TGFβ signaling is often derailed in cancer development or progression, we investigated its control of in human cancer. Computational analyses of sequencing and array data from nearly 11,000 patients with cancer in TCGA showed discordant expression of and in most cancer subtypes, with gene copy-number loss and promoter methylation involved in only a subset. Using HeLa cells as a model, we found that exogenous TGFβ induced mRNA and protein, and knockdown limited TGFβ-mediated growth suppression. TGFβ-mediated mRNA induction required SMAD2/3, p38MAPK, and SP1, and mRNA was induced without added RNAPII recruitment. Chromatin immunoprecipitation unveiled a remote enhancer element engaged by TGFβ by a mechanism that partially depended on p38MAPK. CRISPR-based editing of this enhancer limited induction of and by TGFβ, but not by oncogenic RAS. IMPLICATIONS: Our findings reveal new molecular mechanisms by which regulation is coupled to external cues, and those findings represent entry points to further explore pharmacologic strategies to restore their expression in cancer.
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http://dx.doi.org/10.1158/1541-7786.MCR-19-0289DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726575PMC
September 2019

Twist2 amplification in rhabdomyosarcoma represses myogenesis and promotes oncogenesis by redirecting MyoD DNA binding.

Genes Dev 2019 06 11;33(11-12):626-640. Epub 2019 Apr 11.

Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA.

Rhabdomyosarcoma (RMS) is an aggressive pediatric cancer composed of myoblast-like cells. Recently, we discovered a unique muscle progenitor marked by the expression of the Twist2 transcription factor. Genomic analyses of 258 RMS patient tumors uncovered prevalent copy number amplification events and increased expression of in fusion-negative RMS. Knockdown of in RMS cells results in up-regulation of and a decrease in proliferation, implicating TWIST2 as an oncogene in RMS. Through an inducible Twist2 expression system, we identified Twist2 as a reversible inhibitor of myogenic differentiation with the remarkable ability to promote myotube dedifferentiation in vitro. Integrated analysis of genome-wide ChIP-seq and RNA-seq data revealed the first dynamic chromatin and transcriptional landscape of Twist2 binding during myogenic differentiation. During differentiation, Twist2 competes with MyoD at shared DNA motifs to direct global gene transcription and repression of the myogenic program. Additionally, Twist2 shapes the epigenetic landscape to drive chromatin opening at oncogenic loci and chromatin closing at myogenic loci. These epigenetic changes redirect MyoD binding from myogenic genes toward oncogenic, metabolic, and growth genes. Our study reveals the dynamic interplay between two opposing transcriptional regulators that control the fate of RMS and provides insight into the molecular etiology of this aggressive form of cancer.
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http://dx.doi.org/10.1101/gad.324467.119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546057PMC
June 2019

Rhabdomyosarcoma.

Nat Rev Dis Primers 2019 01 7;5(1). Epub 2019 Jan 7.

Seattle Children's Hospital, University of Washington, and Fred Hutchinson Cancer Research Center, Seattle, WA, USA.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and represents a high-grade neoplasm of skeletal myoblast-like cells. Decades of clinical and basic research have gradually improved our understanding of the pathophysiology of RMS and helped to optimize clinical care. The two major subtypes of RMS, originally characterized on the basis of light microscopic features, are driven by fundamentally different molecular mechanisms and pose distinct clinical challenges. Curative therapy depends on control of the primary tumour, which can arise at many distinct anatomical sites, as well as controlling disseminated disease that is known or assumed to be present in every case. Sophisticated risk stratification for children with RMS incorporates various clinical, pathological and molecular features, and that information is used to guide the application of multifaceted therapy. Such therapy has historically included cytotoxic chemotherapy as well as surgery, ionizing radiation or both. This Primer describes our current understanding of RMS epidemiology, disease susceptibility factors, disease mechanisms and elements of clinical care, including diagnostics, risk-based care of newly diagnosed and relapsed disease and the prevention and management of late effects in survivors. We also outline potential opportunities to further translate new biological insights into improved clinical outcomes.
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http://dx.doi.org/10.1038/s41572-018-0051-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7456566PMC
January 2019

Addition of Vincristine and Irinotecan to Vincristine, Dactinomycin, and Cyclophosphamide Does Not Improve Outcome for Intermediate-Risk Rhabdomyosarcoma: A Report From the Children's Oncology Group.

J Clin Oncol 2018 09 9;36(27):2770-2777. Epub 2018 Aug 9.

Douglas S. Hawkins, Erin R. Rudzinski, and Margarett Shnorhavorian, Seattle Children's Hospital, Seattle, WA; Yueh-Yun Chi and Jing Tian, University of Florida, Gainesville, FL; James R. Anderson, Merck Research Laboratories, North Wales, PA; Carola A.S. Arndt, Mayo Clinic, Rochester, MN; Lisa Bomgaars, Texas Children's Hospital; Andrea Hayes-Jordan, MD Anderson Cancer Center, Houston; Stephen X. Skapek, University of Texas Southwestern, Dallas, TX; Sarah S. Donaldson, Lynn Million, and Sheri L. Spunt, Stanford University School of Medicine, Stanford; Leo Mascarenhas and David M. Parham, Children's Hospital of Los Angeles, Los Angeles; Jeannine S. McCune, City of Hope, Duarte, CA; Mary Beth McCarville, St Jude Children's Research Hospital, Memphis, TN; Geoff McCowage, Children's Hospital at Westmead, Westmead, New South Wales, Australia; Carol D. Morris, Johns Hopkins University, Baltimore, MD, David A. Rodeberg, East Carolina University, Greenville, NC; Lisa A. Teot, Boston Children's Hospital; Torunn I. Yock, Massachusetts General Hospital, Boston, MA; Suzanne Wolden, Memorial Sloan Kettering Cancer Center, New York, NY; and William H. Meyer, University of Oklahoma Health Sciences Center, Oklahoma City, OK.

Purpose Intermediate-risk rhabdomyosarcoma (RMS) includes patients with either nonmetastatic, unresected embryonal RMS (ERMS) with an unfavorable primary site or nonmetastatic alveolar RMS (ARMS). The primary aim of this study was to improve the outcome of patients with intermediate-risk RMS by substituting vincristine and irinotecan (VI) for half of vincristine, dactinomycin, and cyclophosphamide (VAC) courses. All patients received a lower dose of cyclophosphamide and earlier radiation therapy than in previous trials. Patients and Methods Patients were randomly assigned at study entry to either VAC (cumulative cyclophosphamide dose, 16.8 g/m) or VAC/VI (cumulative cyclophosphamide dose, 8.4 g/m) for 42 weeks of therapy. Radiation therapy started at week 4, with individualized local control plans permitted for patients younger than 24 months. The primary study end point was event-free survival (EFS). The study design had an 80% power (5% one-sided α-level) to detect an improved long-term EFS from 65% (with VAC) to 76% (with VAC/VI). Results A total of 448 eligible patients were enrolled in the study. At a median follow-up of 4.8 years, the 4-year EFS was 63% with VAC and 59% with VAC/VI ( P = .51), and 4-year overall survival was 73% for VAC and 72% for VAC/VI ( P = .80). Within the ARMS and ERMS subgroups, no difference in outcome by treatment arm was found. Severe hematologic toxicity was less common with VAC/VI therapy. Conclusion The addition of VI to VAC did not improve EFS or OS for patients with intermediate-risk RMS. VAC/VI had less hematologic toxicity and a lower cumulative cyclophosphamide dose, making VAC/VI an alternative standard therapy for intermediate-risk RMS.
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http://dx.doi.org/10.1200/JCO.2018.77.9694DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6145831PMC
September 2018

Integrative Bayesian Analysis Identifies Rhabdomyosarcoma Disease Genes.

Cell Rep 2018 07;24(1):238-251

Department of Pediatrics, Division of Hematology/Oncology, University of Texas Southwestern Medical Center, Dallas, TX; Harold C. Simmons Comprehensive Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX; Gill Center for Cancer and Blood Disorders, Children's Health, Children's Medical Center, Dallas, TX. Electronic address:

Identifying oncogenic drivers and tumor suppressors remains a challenge in many forms of cancer, including rhabdomyosarcoma. Anticipating gene expression alterations resulting from DNA copy-number variants to be particularly important, we developed a computational and experimental strategy incorporating a Bayesian algorithm and CRISPR/Cas9 "mini-pool" screen that enables both genome-scale assessment of disease genes and functional validation. The algorithm, called iExCN, identified 29 rhabdomyosarcoma drivers and suppressors enriched for cell-cycle and nucleic-acid-binding activities. Functional studies showed that many iExCN genes represent rhabdomyosarcoma line-specific or shared vulnerabilities. Complementary experiments addressed modes of action and demonstrated coordinated repression of multiple iExCN genes during skeletal muscle differentiation. Analysis of two separate cohorts revealed that the number of iExCN genes harboring copy-number alterations correlates with survival. Our findings highlight rhabdomyosarcoma as a cancer in which multiple drivers influence disease biology and demonstrate a generalizable capacity for iExCN to unmask disease genes in cancer.
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http://dx.doi.org/10.1016/j.celrep.2018.06.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7293769PMC
July 2018

transgenic zebrafish models identify as a mediator of rhabdomyosarcoma tumorigenesis.

Elife 2018 06 5;7. Epub 2018 Jun 5.

Department of Pediatrics, UT Southwestern Medical Center, Dallas, United States.

Alveolar rhabdomyosarcoma is a pediatric soft-tissue sarcoma caused by fusion oncogenes and is characterized by impaired skeletal muscle development. We developed human -driven zebrafish models of tumorigenesis and found that exhibits discrete cell lineage susceptibility and transformation. Tumors developed by 1.6-19 months and were primitive neuroectodermal tumors or rhabdomyosarcoma. We applied this transgenic zebrafish model to study how leverages early developmental pathways for oncogenesis and found that is a unique target. Ectopic expression of the human ortholog, , inhibits myogenesis in zebrafish and mammalian cells, recapitulating the arrested muscle development characteristic of rhabdomyosarcoma. In patients, is overexpressed in fusion-positive versus fusion-negative tumors. Finally, overexpression is associated with reduced survival in patients in the context of the fusion. Our novel zebrafish rhabdomyosarcoma model identifies a new target, /, that contributes to impaired myogenic differentiation and has prognostic significance in human disease.
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http://dx.doi.org/10.7554/eLife.33800DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5988421PMC
June 2018

Undifferentiated Sarcomas in Children Harbor Clinically Relevant Oncogenic Fusions and Gene Copy-Number Alterations: A Report from the Children's Oncology Group.

Clin Cancer Res 2018 08 24;24(16):3888-3897. Epub 2018 Apr 24.

University of Texas Southwestern Medical Center, Dallas, Texas.

A comprehensive analysis of the genomics of undifferentiated sarcomas (UDS) is lacking. We analyzed copy-number alterations and fusion status in patients with UDS prospectively treated on Children's Oncology Group protocol ARST0332. Copy-number alterations were assessed by OncoScan FFPE Express on 32 UDS. Whole-exome and transcriptome libraries from eight tumors with sufficient archived material were sequenced on HiSeq (2 × 100 bp). Targeted RNA-sequencing using Archer chemistry was performed on two additional cases. Five-year overall survival for patients with UDS was 83% (95% CI, 69%-97%) with risk-adapted therapy (surgery, chemotherapy, and radiotherapy). Both focal and arm-level copy-number alterations were common including gain of 1q (8/32, 25%) and loss of 1p (7/32, 22%), both of which occurred more often in clinically defined high-risk tumors. Tumors with both loss of 1p and gain of 1q carried an especially poor prognosis with a 5-year event-free survival of 20%. GISTIC analysis identified recurrent amplification of FGF1 on 5q31.3 ( = 0.03) and loss of and on 9p21.3 ( = 0.07). Known oncogenic fusions were identified in eight of 10 cases analyzed by next-generation sequencing. Pediatric UDS generally has a good outcome with risk-adapted therapy. A high-risk subset of patients whose tumors have copy-number loss of 1p and gain of 1q was identified with only 20% survival. Oncogenic fusions are common in UDS, and next-generation sequencing should be considered for children with UDS to refine the diagnosis and identify potentially targetable drivers. .
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http://dx.doi.org/10.1158/1078-0432.CCR-18-0672DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6335968PMC
August 2018

Clinical and mutational spectrum of highly differentiated, paired box 3:forkhead box protein o1 fusion-negative rhabdomyosarcoma: A report from the Children's Oncology Group.

Cancer 2018 05 20;124(9):1973-1981. Epub 2018 Feb 20.

Division of Pediatric Hematology and Oncology, Department of Pediatric and Adolescent Medicine, Faculty of Medicine, University of Freiburg, Germany.

Background: Pediatric paired box 3:forkhead box protein O1 fusion-negative (PF-) rhabdomyosarcoma (RMS) represents a diverse spectrum of tumors with marked differences in histology, myogenic differentiation, and clinical behavior.

Methods: This study sought to evaluate the clinical and mutational spectrum of 24 pediatric PF- human RMS tumors with high levels of myogenic differentiation. Tumors were sequenced with OncoPanel v.2, a panel consisting of the coding regions of 504 genes previously linked to human cancer.

Results: Most of the tumors (19 of 24) arose at head/neck or genitourinary sites, and the overall survival rate was 100% with a median follow-up time of 4.6 years (range, 1.4-8.6 years). RAS pathway gene mutations were the most common mutations in PF-, highly differentiated RMS tumors. In addition, Hedgehog (Hh) and mechanistic target of rapamycin (mTOR) gene mutations with evidence for functional relevance (high-impact) were identified in subsets of tumors. The presence of Hh and mTOR pathway gene mutations was mutually exclusive and was associated with high-impact RAS pathway gene mutations in 3 of 4 Hh-mutated tumors and in 1 of 6 mTOR-mutated tumors.

Conclusions: Interestingly, Hh and mTOR gene mutations were previously associated with rhabdomyomas, which are also known to preferentially arise at head/neck and genitourinary sites. Findings from this study further support the idea that PF-, highly differentiated RMS tumors and rhabdomyomas may represent a continuous spectrum of tumors. Cancer 2018;124:1973-81. © 2018 American Cancer Society.
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http://dx.doi.org/10.1002/cncr.31286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5910184PMC
May 2018

Histology, fusion status, and outcome in metastatic rhabdomyosarcoma: A report from the Children's Oncology Group.

Pediatr Blood Cancer 2017 Dec 18;64(12). Epub 2017 May 18.

Department of Pathology and Laboratory Medicine, Children's Hospital Los Angeles and Keck School of Medicine, University of Southern California, Los Angeles, California.

Background: Distinguishing alveolar rhabdomyosarcoma (ARMS) from embryonal rhabdomyosarcoma (ERMS) has historically been of prognostic and therapeutic importance. However, classification has been complicated by shifting histologic criteria required for an ARMS diagnosis. Children's Oncology Group (COG) studies after IRS-IV, which included the height of this diagnostic shift, showed both an increased number of ARMS and an increase in the proportion of fusion-negative ARMS. Following diagnostic standardization and histologic re-review of ARMS cases enrolled during this era, analysis of low-risk (D9602) and intermediate-risk (D9803) rhabdomyosarcoma (RMS) studies showed that fusion status rather than histology best predicts prognosis for patients with RMS. This analysis remains to be completed for patients with high-risk RMS.

Procedure: We re-reviewed cases on high-risk COG studies D9802 and ARST0431 with an enrollment diagnosis of ARMS. We compared the event-free survival (EFS) and overall survival by histology, PAX-FOXO1 fusion, and clinical risk factors (Oberlin score) for patients with metastatic RMS using the log-rank test.

Results: Histology re-review resulted in reclassification as ERMS for 12% of D9802 cases and 5% of ARST0431 cases. Fusion-negative RMS had a superior EFS to fusion-positive RMS; however, poorer outcome for metastatic RMS was most related to clinical risk factors including age, primary site, and number of metastatic sites.

Conclusions: In contrast to low- or intermediate-risk RMS, in metastatic RMS, clinical risk factors have the most impact on patient outcome. PAX-FOXO1 fusion is more common in patients with a high Oberlin score, but fusion status is not an independent biomarker of prognosis.
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http://dx.doi.org/10.1002/pbc.26645DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5647228PMC
December 2017

Potential pitfalls of mass spectrometry to uncover mutations in childhood soft tissue sarcoma: A report from the Children's Oncology Group.

Sci Rep 2016 09 19;6:33429. Epub 2016 Sep 19.

Department of Pediatrics Division of Hematology/Oncology, and the Harold C. Simmons Cancer Center, University of Texas Southwestern Medical Center, and the Pauline Allen Gill Center for Cancer and Blood Disorders, Children's Medical Center, Dallas, TX, USA.

Mass spectrometry-based methods have been widely applied - often as the sole method - to detect mutations in human cancer specimens. We applied this approach to 52 childhood soft tissue sarcoma specimens in an attempt to identify potentially actionable mutations. This analysis revealed that 25% of the specimens harbored high-confidence calls for mutated alleles, including a mutation encoding FLT3(I836M) that was called in four cases. Given the surprisingly high frequency and unusual nature of some of the mutant alleles, we carried out ultra-deep next generation sequencing to confirm them. We confirmed only three mutations, which encoded NRAS(A18T), JAK3(V722I) and MET(R970C) in three specimens. Beyond highlighting those mutations, our findings demonstrate potential pitfalls of primarily utilizing a mass spectrometry-based approach to broadly screen for DNA sequence variants in archived, clinical-grade tumor specimens. Duplicate mass spectrometric analyses and confirmatory next generation sequencing can help diminish false positive calls, but this does not ameliorate potential false negatives due in part to evaluating a limited panel of sequence variants.
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http://dx.doi.org/10.1038/srep33429DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5027578PMC
September 2016

The Role of Childhood Infections and Immunizations on Childhood Rhabdomyosarcoma: A Report From the Children's Oncology Group.

Pediatr Blood Cancer 2016 09 19;63(9):1557-62. Epub 2016 May 19.

Department of Pediatrics, Texas Children's Cancer Center, Baylor College of Medicine, Houston, Texas.

Background: Rhabdomyosarcoma (RMS) is a rare, highly malignant tumor arising from primitive mesenchymal cells that differentiate into skeletal muscle. Relatively little is known about RMS susceptibility. Based on growing evidence regarding the role of early immunologic challenges on RMS development, we evaluated the role of infections and immunizations on this clinically significant pediatric malignancy.

Procedure: RMS cases (n = 322) were enrolled from the third trial coordinated by the Intergroup Rhabdomyosarcoma Study Group. Population-based controls (n = 322) were pair matched to cases on race, sex, and age. The following immunizations were assessed: diphtheria, pertussis, and tetanus (DPT); measles, mumps, and rubella; and oral polio vaccine. We also evaluated if immunizations were complete versus incomplete. We examined selected infections including chickenpox, mumps, pneumonia, scarlet fever, rubella, rubeola, pertussis, mononucleosis, and lung infections. Conditional logistic regression models were used to calculate an odds ratio (OR) and 95% confidence interval (CI) for each exposure, adjusted for maternal education and total annual income.

Results: Incomplete immunization schedules (OR = 5.30, 95% CI: 2.47-11.33) and incomplete DPT immunization (OR = 1.56, 95% CI: 1.06-2.29) were positively associated with childhood RMS. However, infections did not appear to be associated with childhood RMS.

Conclusions: This is the largest study of RMS to date demonstrating a possible protective effect of immunizations against the development of childhood RMS. Further studies are needed to validate our findings. Our findings add to the growing body of literature, suggesting a protective role of routine vaccinations in childhood cancer and specifically in childhood RMS.
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http://dx.doi.org/10.1002/pbc.26065DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4955701PMC
September 2016

Current state of pediatric sarcoma biology and opportunities for future discovery: A report from the sarcoma translational research workshop.

Cancer Genet 2016 05 5;209(5):182-94. Epub 2016 Apr 5.

Moffitt Cancer Center, Sarcoma Department, Adolescent and Young Adult Program, Tampa, FL, USA.

Sarcomas are a rare subgroup of pediatric cancers comprised of a variety of bone and soft-tissue tumors. While significant advances have been made in improving outcomes of patients with localized pediatric sarcomas since the addition of systemic chemotherapy to local control many decades ago, outcomes for patients with metastatic and relapsed sarcoma remain poor with few novel therapeutics identified to date. With the advent of new technologies to study cancer genomes, transcriptomes and epigenomes, our understanding of sarcoma biology has improved tremendously in a relatively short period of time. However, much remains to be accomplished in this arena especially with regard to translating all of this new knowledge to the bedside. To this end, a meeting was convened in Philadelphia, PA, on April 18, 2015 sponsored by the QuadW foundation, Children's Oncology Group and CureSearch for Children's Cancer that brought together sarcoma clinicians and scientists from North America to review the current state of pediatric sarcoma biology and ongoing/planned genomics based clinical trials in an effort to identify and bridge knowledge gaps that continue to exist at present. At the conclusion of the workshop, three key objectives that would significantly further our understanding of sarcoma were identified and a proposal was put forward to develop an all-encompassing pediatric sarcoma biology protocol that would address these specific needs. This review summarizes the proceedings of the workshop.
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http://dx.doi.org/10.1016/j.cancergen.2016.03.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5497490PMC
May 2016

Negative regulation of initial steps in skeletal myogenesis by mTOR and other kinases.

Sci Rep 2016 Feb 5;6:20376. Epub 2016 Feb 5.

Division of Hematology/Oncology, Department of Pediatrics, University of Texas Southwestern Medical Center, Dallas, TX, USA.

The transition from a committed progenitor cell to one that is actively differentiating represents a process that is fundamentally important in skeletal myogenesis. Although the expression and functional activation of myogenic regulatory transcription factors (MRFs) are well known to govern lineage commitment and differentiation, exactly how the first steps in differentiation are suppressed in a proliferating myoblast is much less clear. We used cultured mammalian myoblasts and an RNA interference library targeting 571 kinases to identify those that may repress muscle differentiation in proliferating myoblasts in the presence or absence of a sensitizing agent directed toward CDK4/6, a kinase previously established to impede muscle gene expression. We identified 55 kinases whose knockdown promoted myoblast differentiation, either independently or in conjunction with the sensitizer. A number of the hit kinases could be connected to known MRFs, directly or through one interaction node. Focusing on one hit, Mtor, we validated its role to impede differentiation in proliferating myoblasts and carried out mechanistic studies to show that it acts, in part, by a rapamycin-sensitive complex that involves Raptor. Our findings inform our understanding of kinases that can block the transition from lineage commitment to a differentiating state in myoblasts and offer a useful resource for others studying myogenic differentiation.
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http://dx.doi.org/10.1038/srep20376DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4742887PMC
February 2016

p19(Arf) limits primary vitreous cell proliferation driven by PDGF-B.

Exp Eye Res 2016 04 8;145:224-229. Epub 2016 Jan 8.

Gill Center for Cancer and Blood Disorders, Children's Medical Center, Dallas, TX, USA. Electronic address:

Arf encodes an important tumor suppressor, p19(Arf), which also plays a critical role to control hyperplasia in the primary vitreous during mouse eye development. In the absence of Arf, mice are born blind and display a phenotype closely mimicking severe forms of the human eye disease, persistent hyperplastic primary vitreous (PHPV). In this report, we characterize p19(Arf) expression in perivascular cells that normally populate the primary vitreous and express the Arf promoter. Using a new ex vivo model, we show that these cells respond to exogenous Tgfβ, despite being isolated at a time when Tgfβ has already turned on the Arf promoter. Treatment of the cells with PDGF-B ligand doubles the population of cells in S-phase and ectopic expression of Arf blunts that effect. We show this effect is mediated through Pdgfrβ as expression of Arf represses expression of Pdgfrβ mRNA and protein to approximately 60%. p53 is not required for Arf-dependent blockade of PDGF-B driven proliferation and repression of Pdgfrβ protein as ectopic expression of Arf is still able to inhibit the 2-fold increase in the S-phase fraction of cells upon treatment with PDGF-B. Finally, induction of mature miR-34a, a microRNA previously identified to be regulated by p19(Arf) does not depend on p53 while the expression of the primary transcript does require p53. These data corroborate that, as in vivo, p19(Arf) functions to inhibit PDGF-B driven proliferation ex vivo.
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http://dx.doi.org/10.1016/j.exer.2016.01.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4842104PMC
April 2016

Histology, Fusion Status, and Outcome in Alveolar Rhabdomyosarcoma With Low-Risk Clinical Features: A Report From the Children's Oncology Group.

Pediatr Blood Cancer 2016 Apr 12;63(4):634-9. Epub 2016 Jan 12.

Division of Hematology, Oncology, and Stem Cell Transplantation, Ann & Robert H. Lurie Children's Hospital of Chicago, Northwestern University Feinberg School of Medicine, Chicago, Illinois.

Background: Distinguishing alveolar rhabdomyosarcoma (ARMS) from embryonal rhabdomyosarcoma (ERMS) is of prognostic and therapeutic importance. Criteria for classifying these entities evolved significantly from 1995 to 2013. ARMS is associated with inferior outcome; therefore, patients with alveolar histology have generally been excluded from low-risk therapy. However, patients with ARMS and low-risk stage and group (Stage 1, Group I/II/orbit III; or Stage 2/3, Group I/II) were eligible for the Children's Oncology Group (COG) low-risk rhabdomyosarcoma (RMS) study D9602 from 1997 to 1999. The characteristics and outcomes of these patients have not been previously reported, and the histology of these cases has not been reviewed using current criteria.

Procedure: We re-reviewed cases that were classified as ARMS on D9602 using current histologic criteria, determined PAX3/PAX7-FOXO1 fusion status, and compared these data with outcome for this unique group of patients.

Results: Thirty-eight patients with ARMS were enrolled onto D9602. Only one-third of cases with slides available for re-review (11/33) remained classified as ARMS by current histologic criteria. Most cases were reclassified as ERMS (17/33, 51.5%). Cases that remained classified as ARMS were typically fusion-positive (8/11, 73%), therefore current classification results in a similar rate of fusion-positive ARMS for all clinical risk groups. In conjunction with data from COG intermediate-risk treatment protocol D9803, our data demonstrate excellent outcomes for fusion-negative ARMS with otherwise low-risk clinical features.

Conclusions: Patients with fusion-positive RMS with low-risk clinical features should be classified and treated as intermediate risk, while patients with fusion-negative ARMS could be appropriately treated with reduced intensity therapy.
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http://dx.doi.org/10.1002/pbc.25862DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4755849PMC
April 2016

Recurrent internal tandem duplications of BCOR in clear cell sarcoma of the kidney.

Nat Commun 2015 Nov 17;6:8891. Epub 2015 Nov 17.

Texas Children's Cancer Center, Houston, Texas 77030, USA.

The X-linked BCL-6 co-repressor (BCOR) gene encodes a key constituent of a variant polycomb repressive complex (PRC) that is mutated or translocated in human cancers. Here we report on the identification of somatic internal tandem duplications (ITDs) clustering in the C terminus of BCOR in 23 of 27 (85%) pediatric clear cell sarcomas of the kidney (CCSK) from two independent cohorts. We profile CCSK tumours using a combination of whole-exome, transcriptome and targeted sequencing. Identical ITD mutations are found in primary and relapsed tumour pairs but not in adjacent normal kidney or blood. Mutant BCOR transcripts and proteins are markedly upregulated in ITD-positive tumours. Transcriptome analysis of ITD-positive CCSKs reveals enrichment for PRC2-regulated genes and similarity to undifferentiated sarcomas harbouring BCOR-CCNB3 fusions. The discovery of recurrent BCOR ITDs defines a major oncogenic event in this childhood sarcoma with significant implications for diagnostic and therapeutic approaches to this tumour.
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http://dx.doi.org/10.1038/ncomms9891DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4660214PMC
November 2015

Clinical Application of Prognostic Gene Expression Signature in Fusion Gene-Negative Rhabdomyosarcoma: A Report from the Children's Oncology Group.

Clin Cancer Res 2015 Oct;21(20):4733-9

Pauline Allen Gill Center for Cancer and Blood Disorders, Children's Medical Center, Dallas, Texas. Department of Pediatrics, Division of Hematology/Oncology, University of Texas Southwestern Medical Center, Dallas, Texas.

Purpose: Pediatric rhabdomyosarcoma (RMS) has two common histologic subtypes: embryonal (ERMS) and alveolar (ARMS). PAX-FOXO1 fusion gene status is a more reliable prognostic marker than alveolar histology, whereas fusion gene-negative (FN) ARMS patients are clinically similar to ERMS patients. A five-gene expression signature (MG5) previously identified two diverse risk groups within the fusion gene-negative RMS (FN-RMS) patients, but this has not been independently validated. The goal of this study was to test whether expression of the MG5 metagene, measured using a technical platform that can be applied to routine pathology material, would correlate with outcome in a new cohort of patients with FN-RMS.

Experimental Design: Cases were taken from the Children's Oncology Group (COG) D9803 study of children with intermediate-risk RMS, and gene expression profiling for the MG5 genes was performed using the nCounter assay. The MG5 score was correlated with clinical and pathologic characteristics as well as overall and event-free survival.

Results: MG5 standardized score showed no significant association with any of the available clinicopathologic variables. The MG5 signature score showed a significant correlation with overall (N = 57; HR, 7.3; 95% CI, 1.9-27.0; P = 0.003) and failure-free survival (N = 57; HR, 6.1; 95% CI, 1.9-19.7; P = 0.002).

Conclusions: This represents the first, validated molecular prognostic signature for children with FN-RMS who otherwise have intermediate-risk disease. The capacity to measure the expression of a small number of genes in routine pathology material and apply a simple mathematical formula to calculate the MG5 metagene score provides a clear path toward better risk stratification in future prospective clinical trials.
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http://dx.doi.org/10.1158/1078-0432.CCR-14-3326DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4610152PMC
October 2015
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