1. Efforts in Rhabdomyosarcoma Rhabdomyosarcoma (RMS) is a myogenic cancer that is the most common soft tissue sarcoma of childhood. With the development of multimodal chemotherapy regimens, relapse-free survival rates have improved to 70-80% in patients with localized disease, albeit with significant toxicity. Unfortunately, despite aggressive therapy, the 5-year survival rate for patients with metastatic disease remains only 30%. Therapy assignment is currently based on clinicopathologic features and using these criteria, three distinct subgroups of patients can be identified (low, intermediate and high risk). However, many patients fall into the intermediate risk category (which accounts for about 50% of all patients) and have a heterogeneous clinical outcome. This suggests that some of these children could be treated with less aggressive therapy or alternatively should be considered to have more aggressive disease. In an effort to further characterize the genetic events underlying this tumor type, our group in collaboration with the Children's Oncology Group performed a large sequencing effort using a combination of whole-genome, whole-exome and whole-transcriptome sequencing along with high resolution SNP arrays to characterize the landscape of somatic alterations in 147 tumor/normal pairs. Our findings describe a heterogenous group of genetic events appears to drive RMS most notably the PAX 3/7-FOXO1 fusion in the alveolar subtype and mutation of multiple RAS pathway genes in fusion negative tumors including recurrent genetic lesions in 10 cancer consensus genes (NRAS, KRAS, HRAS, PIK3CA, BCOR, TP53, NF1, FGFR4, FBXW7, CTNNB1). While the majority of these mutations appear to be mutually exclusive, a subset of tumors appears to have coexisting lesions within the same tumor; perhaps indicating a biologically relevant progression in these tumors. Unfortunately, much of the clinical annotation for these cases was incomplete, severely limiting our ability to derive prognostic information from this data set. To overcome this, a more focused retrospective analysis is needed to determine the prognostic significance of the discovered mutations. In collaboration with the Children's Oncology Group, we have sequenced 675 clinically annotated rhabdomyosarcoma cases. In this project, we are using high-throughput capture based sequencing to determine the frequency with which the observed mutations occur and determine whether these mutations could be used as markers of prognosis or response to therapy. The specific objectives of the study are the following: 1. To validate our pilot study and accurately estimate the frequency of NRAS, KRAS, HRAS, PIK3CA, BCOR, TP53, NF1, FGFR4, FBXW7, CTNNB1 mutations in low, intermediate and high-risk embryonal rhabdomyosarcomas. 2. To estimate the frequency of NRAS, KRAS, HRAS, PIK3CA, BCOR, TP53, NF1, FGFR4, FBXW7, CTNNB1 mutations in a small cohort of alveolar histology tumors. 3. To determine the clinical significance of NRAS, KRAS, HRAS, PIK3CA, BCOR, TP53, NF1, FGFR4, FBXW7, CTNNB1 mutations in patients with central pathology confirmed ERMS cases. 4. To develop an accurate and reproducible assay for assessing mutations in NRAS, KRAS, HRAS, PIK3CA, BCOR, TP53, NF1, FGFR4, FBXW7, CTNNB1 that can be used in prospective clinical trials. This year we have validated that our assay can reliably detect mutations even at low frequency and that we can accurately detect gene copy number changes. We have developed a bioinformatics pipeline and visualization portal that enables a user-friendly interface with the generated data. Clinical outcome correlation analysis has been performed in concert with the Childrens Oncology Group and we have demonstrated the clinical impact of the presence or absence of each genetic lesion. Mutations of MYOD1 and TP53 were found to be associated with clinical outcome and these results were presented at the American Society of Clinical Oncology Annual Meeting as well as the Childrens Oncology Group annual meeting. Current efforts are focused on using these results to enlighten prospective clinical trials. This includes implementation of a CLIA certified assay and informatics pipeline to centrally profile all Rhabdomyosarcoma patients enrolled on Childrens Oncology Group trials. 2. Efforts in Malignant Peripheral Nerve Sheath Tumor (MPNST) The goal of this work is to provide further understanding of the genetic and transcriptomic mechanisms of the tumor evolution underlying the transformation of a Neurofibroma to a Malignant Peripheral Nerve Sheath Tumor (MPNST). Leveraging the expertise and patient population within the Pediatric Oncology Branch, in this project we are using preclinical model systems to dissect the genetic and epigenetic changes that occur as NF1 tumors transition from benign precursors to the aggressive MPNST. Specifically, we are studying the effects of mutation or deletion in one of the polycomb complex (PRC2) members SUZ12 or EED as the final step in the transformation to MPNST. The PRC2 complex is a major transcriptional repressor within the cell and recurrent alterations of members of this complex have been discovered in genomic sequencing studies from patient samples. Within the current fiscal year, we have built inducible in vitro model systems and performed ChIPseq, RNAseq and ATACseq to nominate genes whose expression is altered upon loss of these genes. These preclinical tools have allowed us to identify genes downstream of PRC2 loss that are potential therapeutic targets. We further interrogate these genes for functional relevance to MPNST using biochemical, molecular biology and CRISPR technologies.
| Bharathy, Narendra; Svalina, Matthew N; Settelmeyer, Teagan P et al. (2017) Preclinical testing of the glycogen synthase kinase-3? inhibitor tideglusib for rhabdomyosarcoma. Oncotarget 8:62976-62983 |
| Chen, Li; Shern, Jack F; Wei, Jun S et al. (2015) Clonality and evolutionary history of rhabdomyosarcoma. PLoS Genet 11:e1005075 |
