David Van Mater

MicroRNA-182 drives metastasis of primary sarcomas by targeting multiple genes

Metastasis causes most cancer deaths, but is incompletely understood. MicroRNAs can regulate metastasis, but it is not known whether a single miRNA can regulate metastasis in primary cancer models in vivo. We compared the expression of miRNAs in metastatic and nonmetastatic primary mouse sarcomas and found that microRNA-182 (miR-182) was markedly overexpressed in some tumors that metastasized to the lungs. By utilizing genetically engineered mice with either deletion of or overexpression of miR-182 in primary sarcomas, we discovered that deletion of miR-182 substantially decreased, while overexpression of miR-182 considerably increased, the rate of lung metastasis after amputation of the tumor-bearing limb. Additionally, deletion of miR-182 decreased circulating tumor cells (CTCs), while overexpression of miR-182 increased CTCs, suggesting that miR-182 regulates intravasation of cancer cells into the circulation. We identified 4 miR-182 targets that inhibit either the migration of tumor cells or the degradation of the extracellular matrix. Notably, restoration of any of these targets in isolation did not alter the metastatic potential of sarcoma cells injected orthotopically, but the simultaneous restoration of all 4 targets together substantially decreased the number of metastases. These results demonstrate that a single miRNA can regulate metastasis of primary tumors in vivo by coordinated regulation of multiple genes.

Distinct and Overlapping Sarcoma Subtypes Initiated from Muscle Stem and Progenitor Cells

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children, whereas undifferentiated pleomorphic sarcoma (UPS) is one of the most common soft tissue sarcomas diagnosed in adults. To investigate the myogenic cell(s) of origin of these sarcomas, we used Pax7-CreER and MyoD-CreER mice to transform Pax7(+) and MyoD(+) myogenic progenitors by expressing oncogenic Kras(G12D) and deleting Trp53 in vivo. Pax7-CreER mice developed RMS and UPS, whereas MyoD-CreER mice developed UPS. Using gene set enrichment analysis, RMS and UPS each clustered specifically within their human counterparts. These results suggest that RMS and UPS have distinct and overlapping cells of origin within the muscle lineage. Taking them together, we have established mouse models of soft tissue sarcoma from muscle stem and progenitor cells.

Acute Tissue Injury Activates Satellite Cells and Promotes Sarcoma Formation via the HGF/c-MET Signaling Pathway

Some patients with soft-tissue sarcoma (STS) report a history of injury at the site of their tumor. Although this phenomenon is widely reported, there are relatively few experimental systems that have directly assessed the role of injury in sarcoma formation. We recently described a mouse model of STS whereby p53 is deleted and oncogenic Kras is activated in muscle satellite cells via a Pax7(CreER) driver following intraperitoneal injection with tamoxifen. Here, we report that after systemic injection of tamoxifen, the vast majority of Pax7-expressing cells remain quiescent despite mutation of p53 and Kras. The fate of these muscle progenitors is dramatically altered by tissue injury, which leads to faster kinetics of sarcoma formation. In adult muscle, quiescent satellite cells will transition into an active state in response to hepatocyte growth factor (HGF). We show that modulating satellite cell quiescence via intramuscular injection of HGF increases the penetrance of sarcoma formation at the site of injection, which is dependent on its cognate receptor c-MET. Unexpectedly, the tumor-promoting effect of tissue injury also requires c-Met. These results reveal a mechanism by which HGF/c-MET signaling promotes tumor formation after tissue injury in a mouse model of primary STS, and they may explain why some patients develop a STS at the site of injury.

Neuroblastoma in a pediatric patient with a microduplication of 2p involving the MYCN locus

Neuroblastoma is the most common solid tumor of infancy, and mutations in several genes have been implicated as playing a role in tumor development. Here, we describe a pediatric patient with a constitutional microduplication of 2p24.3 who developed Stage 4 neuroblastoma at age 11 months. He represents the sixth patient described in the literature with partial trisomy 2p and neuroblastoma. All previous cases had duplication events spanning two genes implicated in neuroblastoma, MYCN and ALK. Our patient is unique because his duplicated region includes the MYCN gene only; the ALK gene is unaffected. These data, combined with the relatively high incidence of neuroblastoma reported in partial trisomy 2p patients, support the notion that MYCN duplication should be added to the growing list of genetic factors associated with an increased risk of neuroblastoma. The mechanism of increased risk is unclear, but the fact that our patient had dramatic amplification of MYCN in his tumor suggests that a germline duplication might predispose to further amplification. Additionally, our patient has several morphologic features common to patients with partial trisomy 2p including high forehead, hypertelorism, postaxial polydactyly, and developmental delay despite having a microduplication spanning approximately 1 Mb and including just three intact genes. This case may therefore help further delineate the genotype–phenotype correlations associated with partial trisomy 2p.

Methods to Generate Genetically Engineered Mouse Models of Soft Tissue Sarcoma

We discuss the generation of primary soft tissue sarcomas in mice using the Cre-loxP system to activate conditional mutations in oncogenic Kras and the tumor suppressor p53 (LSL-Kras(G12D/+); p53(flox/flox)). Sarcomas can be generated either by adenoviral delivery of Cre recombinase, activation of transgenic Cre recombinase with tamoxifen, or through transplantation of isolated satellite cells with Cre activation in vitro. Various applications of these models are discussed, including anticancer therapies, metastasis, in vivo imaging, and genetic requirements for tumorigenesis.

Generation and comparison of CRISPR-Cas9 and Cre-mediated genetically engineered mouse models of sarcoma

Genetically engineered mouse models that employ site-specific recombinase technology are important tools for cancer research but can be costly and time-consuming. The CRISPR-Cas9 system has been adapted to generate autochthonous tumours in mice, but how these tumours compare to tumours generated by conventional recombinase technology remains to be fully explored. Here we use CRISPR-Cas9 to generate multiple subtypes of primary sarcomas efficiently in wild type and genetically engineered mice. These data demonstrate that CRISPR-Cas9 can be used to generate multiple subtypes of soft tissue sarcomas in mice. Primary sarcomas generated with CRISPR-Cas9 and Cre recombinase technology had similar histology, growth kinetics, copy number variation and mutational load as assessed by whole exome sequencing. These results show that sarcomas generated with CRISPR-Cas9 technology are similar to sarcomas generated with conventional modelling techniques and suggest that CRISPR-Cas9 can be used to more rapidly generate genotypically and phenotypically similar cancers.

Trilateral Retinoblastoma in a Patient With Peutz–Jeghers Syndrome

Germline loss of function mutations in tumor suppressor genes RB1 and LKB1/STK11 are associated with the autosomal dominant cancer predisposing syndromes familial retinoblastoma and Peutz–Jeghers syndrome (PJS), respectively. We present a rare case of a young woman with trilateral retinoblastoma diagnosed as an infant who survived and was then diagnosed with PJS as a teenager. There was no family history of either disorder. Analysis of the LKB1/STK11 gene sequence identified a germline frameshift mutation (c.107del) leading to a nonsense mutation near the N‐terminus of the protein, confirming a clinical diagnosis of Peutz–Jeghers syndrome. Extensive RB1 gene analysis failed to detect germline mutations or deletions, and immunohistochemical analysis of her ocular tumors demonstrated nuclear staining of immunoreactive pRB. This result suggests that the RB1 gene is intact. We estimate the chance of trilateral retinoblastoma and PJS occurring in the same individual at approximately 1 in 134 billion live births, and we discuss the possibility that this case could be explained by a putative modifier of pRB action that is associated with the LKB1/STK11 pathway.

MLL duplication in a pediatric patient with B-cell lymphoblastic lymphoma.

Lymphoblastic lymphoma is the second most common type of non-Hodgkin lymphoma seen in children. Approximately, 90% of lymphoblastic lymphomas arise from T cells, with the remaining 10% being B-cell-lineage derived. Although T-cell lymphoblastic lymphoma most frequently occurs in the anterior mediastinum (thymus), B-cell lymphoblastic lymphoma (B-LBL) predominates in extranodal sites such as skin and bone. Here, we describe a pediatric B-LBL patient who presented with extensive abdominal involvement and whose lymphoma cells displayed segmental duplication of the mixed lineage leukemia (MLL) gene. MLL duplication/amplification has been described primarily in acute myeloid leukemia and myelodysplastic syndrome with no published reports of discrete MLL duplication/amplification events in B-LBL. The MLL gene duplication noted in this case may represent a novel mechanism for tumorigenesis in B-LBL.

Favorable response to nivolumab in a young adult patient with metastatic histiocytic sarcoma

To the Editor: Histiocytic sarcoma (HS) is a rare hematopoietic neoplasm consisting of a malignant proliferation of cells that resembles mature tissue histiocytes.1 Prognosis for HS is poor; a recent survey of the Surveillance, Epidemiology, and End Results (SEER) database demonstrated a median overall survival of 6 months among a cohort of 158 patients.2 There is not a universally accepted treatment for HS, though responses have been seenwith chemotherapy, surgery, or radiation therapy.3,4 Recent reports have demonstrated a high rate of PDL1 expression in histiocytic disorders, suggesting a potential therapeutic target.5,6 Here we report a case of HS in a young adult patient who had a favorable response to nivolumab. A 17-year-old female presented to her primary care provider with intermittent left upper leg pain, fatigue, fevers, unintentional weight loss, and swollen lymphnodes. Initial physical examinationwas notable for thin body habitus and prominent cervical and supraclavicular adenopathy. She was referred to pediatric hematology-oncology for evaluation. An 18F-fluorodeoxyglucose (FDG) PET-CT revealed multifocal, hypermetabolic axial and appendicular skeletal metastases, extensive hypermetabolic lymphadenopathy, and multiple pulmonary nodules (Supporting Information Figure 1A). No evidence of intracranial diseasewas noted on brainMRI. Bonemarrow biopsy and aspirate were negative. Biopsy of the right iliac mass revealed atypical cell nodules with a histiocytic immunophenotype including CD68+, CD163+, lysozyme+,

Abstract A17: Generation and comparison of CRISPR/Cas9 and Cre-mediated genetically engineered mouse models of sarcoma

Genetically engineered mouse models (GEMMs) that employ site-specific recombinase (SSR) technology are important tools for cancer research, and recently the CRISPR/Cas9 system has been increasingly utilized to model cancer in mice. Here, we used CRISPR/Cas9 to generate two primary mouse models of sarcoma, undifferentiated pleomorphic sarcoma (UPS) in a GEMM, and malignant peripheral nerve sheath tumor (MPNST) in wild-type mice, to demonstrate the versatility of the system to generate multiple soft-tissue sarcoma subtypes. Because CRISPR technology is becoming more prevalent in cancer modeling, it is critical to thoroughly evaluate if these models are indeed comparable as tools to study cancer biology compared to conventional GEMMs initiated by recombinase technology. We used two Kras-driven sarcoma models of UPS generated with either Cre recombinase technology or CRISPR/Cas9 technology and compared the mutational profiles, histology, and growth kinetics of these models. KrasLSL-G12D/+; Rosa26LSL-Cas9-EGFP/+ (KC) mice received intramuscular delivery of an adenovirus expressing Cre recombinase and a single guide RNA (sgRNA) targeting Trp53. Cre-mediated expression of oncogenic Kras and Cas9, in combination with CRISPR/Cas9-mediated knockout of Trp53, was sufficient to generate primary soft-tissue sarcomas. Compared to the Cre/loxP model, we determined that sarcomas generated with CRISPR/Cas9 had similar growth kinetics, histology, copy number variation, and mutational load as assessed by whole-exome sequencing. We also demonstrated that off-target mutations in the sarcomas initiated by the Cas9 endonuclease were rare in tumors. Finally, we analyzed the Cas9-mediated indels present in tumors as genetic barcodes, which will enable future studies of tumor heterogeneity and clonality. These results show that sarcomas generated with CRISPR/Cas9 technology are similar to sarcomas generated with conventional modeling techniques. Ultimately this work corroborates CRISPR/Cas9-generated mouse models with traditional GEMMs phenotypically and genotypically, and expands the range of sarcoma mouse models available for research. Citation Format: Jianguo Huang, Mark Chen, Melodi Javid Whitley, Hsuan-Cheng Kuo, Eric S. Xu, Andrea Walens, Yvonne M. Mowery, David Van Mater, William C. Eward, Diana M. Cardona, Lixia Luo, Yan Ma, Omar M. Lopez, Christopher E. Nelson, Jacqueline N. Robinson-Hamm, Anupama Reddy, Sandeep S. Dave, Charles A. Gersbach, Rebecca D. Dodd, David G. Kirsch. Generation and comparison of CRISPR/Cas9 and Cre-mediated genetically engineered mouse models of sarcoma [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 A17.