Highlights d Proteomic profiles of extracellular vesicles and particles (EVPs) from 426 human samples d Identification of pan-EVP markers d Characterization of tumor-derived EVP markers in human tissues and plasma d EVP proteins can be useful for cancer detection and determining cancer type

ediatric cancer is rare, with fewer than 10,000 solid tumors diagnosed in children annually in the United States 1. Previous studies interrogating germline predisposition broadly across pediatric cancer types have found heritable germline predisposition in 8-12% of patients. The yield of germline predisposition detected is dependent on the genes included for analysis and variant interpretation as well as the ascertainment biases found in each cohort. Iterative data are required to expand upon the understanding of susceptibility to pediatric cancer and determine the extent to which germline data may translate into clinical practice 2-7. Certain pediatric cancer diagnoses have well-established associations with germline mutations in specific genes and should automatically prompt clinical suspicion of a cancer predisposition, for example, retinoblastoma (RB1), pleuropulmonary blastoma (DICER1), optic pathway glioma (NF1), atypical teratoid/rhabdoid tumors (SMARCB1), small cell hypercalcemic ovarian tumors (SMARCA4), adrenal cortical tumors (TP53) and hypodiploid acute lymphoblastic leukemia (TP53) 8-10. Germline testing can also be critical for distinguishing between conditions like neurofibromatosis type 1 (NF1) and constitutional mismatch repair deficiency (CMMRD), which can be phenocopies of each other. For example, a child presenting with numerous café au lait spots and leukemia may have either of these conditions, but treatment and screening recommendations for the proband and family members will differ depending on the germline diagnosis 11. Besides the known associations of causal germline mutations, broad tumor-normal sequencing has revealed novel associations 9,12. While some of these findings likely represent population detection and do not play a role in the pathogenesis of the cancer in question 13 , other novel associations are likely causal. Population detection

The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase that exists in two complexes (mTORC1 and mTORC2) and integrates extracellular and intracellular signals to act as a master regulator of cell growth, survival, and metabolism. The PI3K/AKT/mTOR pro-survival pathway is often dysregulated in multiple sarcoma subtypes. First-generation allosteric inhibitors of mTORC1 (rapalogues) have been extensively tested with great pre-clinical promise, but have had limited clinical utility. Here we report that MLN0128, a second-generation, ATP-competitive, pan-mTOR kinase inhibitor, acts on both mTORC1 and mTORC2, and has potent in vitro and in vivo anti-tumor activity in multiple sarcoma subtypes. In vitro, MLN0128 inhibits mTORC1/2 targets in a concentration dependent fashion, and shows striking anti-proliferative effect in rhabdomyosarcoma (RMS), Ewing sarcoma (ES), malignant peripheral nerve sheath tumor, synovial sarcoma, osteosarcoma, and liposarcoma. Unlike rapamycin, MLN0128 inhibits phosphorylation of 4EBP1 and NDRG1 as well as prevents the reactivation of pAKT that occurs via negative feedback release with mTORC1 inhibition alone. In xenograft models, MLN0128 treatment results in suppression of tumor growth with two dosing schedules (1 mg/kg daily and 3 mg/kg BID TIW). At the 3 mg/kg dosing schedule, MLN0128 treatment results in significantly better tumor growth suppression than rapamycin in RMS and ES models. Additionally, MLN0128 induces apoptosis in models of RMS both in vitro and in vivo. Results from our study strongly suggest that MLN0128 treatment should be explored further as potential therapy for sarcoma.

The genetic, biologic, and clinical heterogeneity of sarcomas poses a challenge for the identification of therapeutic targets, clinical research, and advancing patient care. Because there are > 100 sarcoma subtypes, in-depth genetic studies have focused on one or a few subtypes. Herein, we report a comparative genetic analysis of 2,138 sarcomas representing 45 pathological entities. This cohort is prospectively analyzed using targeted sequencing to characterize subtype-specific somatic alterations in targetable pathways, rates of whole genome doubling, mutational signatures, and subtype-agnostic genomic clusters. The most common alterations are in cell cycle control and TP53, receptor tyrosine kinases/PI3K/RAS, and epigenetic regulators. Subtype-specific associations include TERT amplification in intimal sarcoma and SWI/SNF alterations in uterine adenosarcoma. Tumor mutational burden, while low compared to other cancers, varies between and within subtypes. This resource will improve sarcoma models, motivate studies of subtype-specific alterations, and inform investigations of genetic factors and their correlations with treatment response.

Purpose: Although multimodal chemotherapy has improved outcomes for patients with osteosarcoma, the prognosis for patients who present with metastatic and/or recurrent disease remains poor. In this study, we sought to define how often clinical genomic sequencing of osteosarcoma samples could identify potentially actionable alterations.Experimental Design: We analyzed genomic data from 71 osteosarcoma samples from 66 pediatric and adult patients sequenced using MSK-IMPACT, a hybridization capture-based large panel next-generation sequencing assay. Potentially actionable genetic events were categorized according to the OncoKB precision oncology knowledge base, of which levels 1 to 3 were considered clinically actionable.Results: We found at least one potentially actionable alteration in 14 of 66 patients (21%), including amplification of CDK4 (n ¼ 9, 14%: level 2B) and/or MDM2 (n ¼ 9, 14%: level 3B), and somatic truncating mutations/deletions in BRCA2 (n ¼ 3, 5%: level 2B) and PTCH1 (n ¼ 1, level 3B). In addition, we observed mutually exclusive patterns of alterations suggesting distinct biological subsets defined by gains at 4q12 and 6p12-21. Specifically, potentially targetable gene amplifications at 4q12 involving KIT, KDR, and PDGFRA were identified in 13 of 66 patients (20%), which showed strong PDGFRA expression by IHC. In another largely nonoverlapping subset of 14 patients (24%) with gains at 6p12-21, VEGFA amplification was identified.Conclusions: We found potentially clinically actionable alterations in approximately 21% of patients with osteosarcoma. In addition, at least 40% of patients have tumors harboring PDGFRA or VEGFA amplification, representing candidate subsets for clinical evaluation of additional therapeutic options. We propose a new genomically based algorithm for directing patients with osteosarcoma to clinical trial options.