Importance Understanding molecular mechanisms of response and resistance to anticancer therapies requires prospective patient follow-up and clinical and functional validation of both common and low-frequency mutations. We describe a whole-exome sequencing (WES) precision medicine trial focused on patients with advanced cancer. Objective To understand how WES data affect therapeutic decision making in patients with advanced cancer and to identify novel biomarkers of response. Design, Setting, and Patients Patients with metastatic and treatment-resistant cancer were prospectively enrolled at a single academic center for paired metastatic tumor and normal tissue WES during a 19-month period (February 2013 through September 2014). A comprehensive computational pipeline was used to detect point mutations, indels, and copy number alterations. Mutations were categorized as category 1, 2, or 3 on the basis of actionability; clinical reports were generated and discussed in precision tumor board. Patients were observed for 7 to 25 months for correlation of molecular information with clinical response. Main Outcomes and Measures Feasibility, use of WES for decision making, and identification of novel biomarkers. Results A total of 154 tumor-normal pairs from 97 patients with a range of metastatic cancers were sequenced, with a mean coverage of 95× and 16 somatic alterations detected per patient. In total, 16 mutations were category 1 (targeted therapy available), 98 were category 2 (biologically relevant), and 1474 were category 3 (unknown significance). Overall, WES provided informative results in 91 cases (94%), including alterations for which there is an approved drug, there are therapies in clinical or preclinical development, or they are considered drivers and potentially actionable (category 1-2); however, treatment was guided in only 5 patients (5%) on the basis of these recommendations because of access to clinical trials and/or off-label use of drugs. Among unexpected findings, a patient with prostate cancer with exceptional response to treatment was identified who harbored a somatic hemizygous deletion of the DNA repair gene FANCA and putative partial loss of function of the second allele through germline missense variant. Follow-up experiments established that loss of FANCA function was associated with platinum hypersensitivity both in vitro and in patient-derived xenografts, thus providing biologic rationale and functional evidence for his extreme clinical response. Conclusions and Relevance The majority of advanced, treatment-resistant tumors across tumor types harbor biologically informative alterations. The establishment of a clinical trial for WES of metastatic tumors with prospective follow-up of patients can help identify candidate predictive biomarkers of response.

Secreted frizzled related proteins (sFRPs) have emerged as key regulators of a wide range of developmental and disease processes, with virtually all known functions of mammalian sFRPs attributed to their ability to antagonize Wnt signaling. Recently however, the Xenopus and zebrafish sFRP, Sizzled, was shown to function as an antagonist of Chordin processing by Tolloid-like metalloproteinases, leading to the proposal that sFRPs may function as evolutionarily-conserved antagonists of the chordinase activities of this class of proteinases. Herein, in contrast to this proposal, we show that the mammalian sFRP, sFRP2, does not affect Chordin processing, but instead can serve as a direct enhancer of the procollagen C-proteinase activity of Tolloid-like metalloproteinases. We further show that the level of fibrosis, in which procollagen processing by Tolloid-like proteinases plays a rate-limiting role, is markedly reduced in sFRP2-null mice subjected to myocardial infarction. Importantly, this reduced level of fibrosis is accompanied by significantly improved cardiac function. This study thus uncovers a novel function for sFRP2 and a potential therapeutic application for sFRP2 antagonism in controlling fibrosis in the infarcted heart.

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We report that Id knockout mouse embryos display multiple cardiac defects but mid-gestation lethality is rescued by injection of 15 wild type ES cells into mutant blastocysts. Myocardial markers altered in Id mutant cells are restored to normal throughout the chimeric myocardium. Intraperitoneal injection of ES cells into female mice prior to conception also partially rescues the cardiac phenotype with no incorporation of ES cells. IGF1, a long-range secreted factor, in combination with WNT5a, a locally secreted factor likely account for complete reversion of the cardiac phenotype. Thus, ES cells have the potential to reverse congenital defects through Id-dependent local and long-range effects in a mammalian embryo.

Carney complex (CNC) is a familial multiple neoplasia syndrome characterized by cardiac and extracardiac myxomas in the setting of spotty skin pigmentation and endocrinopathy. We previously identified PRKAR1A (regulatory subunit 1α of protein kinase A) mutations in CNC. Mutational analyses of the PRKAR1A gene in 51 unrelated CNC probands now detect mutations in 65%. All mutations, except for one unique missense mutation, lead to PRKAR1A haploinsufficiency. Therefore, we studied the consequences of prkar1a haploinsufficiency in mice. Although we did not observe cardiac myxomas or altered pigmentation in prkar1a +/– mice, we did observe some phenotypes similar to CNC, including altered heart rate variability. Moreover, prkar1a +/– mice exhibited a marked propensity for extracardiac tumorigenesis. They developed sarcomas and hepatocellular carcinomas. Sarcomas were frequently associated with myxomatous differentiation. Tumors from prkar1a +/– mice did not exhibit prkar1a loss of heterozygosity. Thus, we conclude that although PRKAR1A haploinsufficiency does predispose to tumorigenesis, distinct secondary genetic events are required for tumor formation.

Congenital heart disease is the most common form of human birth defects, yet much remains to be learned about its underlying causes. Here we report that mice lacking functional ADAM19 (mnemonic for a disintegrin and metalloprotease 19) exhibit severe defects in cardiac morphogenesis, including a ventricular septal defect (VSD), abnormal formation of the aortic and pulmonic valves, leading to valvular stenosis, and abnormalities of the cardiac vasculature. During mouse development, ADAM19 is highly expressed in the conotruncus and the endocardial cushion, structures that give rise to the affected heart valves and the membranous ventricular septum. ADAM19 is also highly expressed in osteoblast-like cells in the bone, yet it does not appear to be essential for bone growth and skeletal development. Most adam19 ؊/؊ animals die perinatally, likely as a result of their cardiac defects. These findings raise the possibility that mutations in ADAM19 may contribute to human congenital heart valve and septal defects.ADAMs (mnemonic for a disintegrin and metalloprotease) are membrane-anchored glycoproteins with key roles in fertilization, neurogenesis, angiogenesis, Alzheimer's disease, and the release of proteins such as epidermal growth factor (EGF) receptor ligands and tumor necrosis factor family members from the plasma membrane (3,4,17,37,39,41). ADAM19 (also referred to as meltrin ␤) was initially identified in muscle cells and was later found to be expressed in several other tissues, most prominently in heart, lung, and bone (18, 27, 52), during dendritic cell differentiation (13) and Notch-induced T-cell maturation (9). The catalytic activity of ADAM19 towards candidate substrates has been explored by overexpression in cells and by purifying recombinantly expressed soluble forms of the entire ectodomain or the pro-and metalloprotease domains (7,42,49,53). Overexpressed ADAM19 enhances ectodomain shedding of two of several splice variants of neuregulin I-␤ (42), a ligand for the ErbB family of receptor tyrosine kinases (11). Furthermore, overexpression of ADAM19 increases ectodomain release of tumor necrosis factor-related activation-induced cytokine (TRANCE, also referred to as osteoprotegerin-ligand [OPGL]) (7), a protein with important roles in osteoclast differentiation, dendritic cell survival, and mammary gland development (12,25,28).In light of the high expression of ADAM19 in heart and bone and its ability to cleave TRANCE as well as splice variants of neuregulin I-␤, we were interested in evaluating the function of ADAM19 in mice, with an emphasis on its role in heart and bone development. Here we present an analysis of mice lacking functional ADAM19 (adam19 Ϫ/Ϫ mice). MATERIALS AND METHODS Generation of adam19؊/؊ mice. adam19 ϩ/Ϫ mice were generated by the SloanKettering Institute transgenic facility by following standard procedures using stem cells with a secretory gene trap insertion in ADAM19 (30). All mice evaluated in this study were of mixed genetic background (129Sv/C57BL6), and morphological and hist...

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