Comprehensive NGS profiling to enable detection of ALK gene rearrangements and MET amplifications in non-small cell lung cancer.

Introduction: Next-generation sequencing (NGS) is currently widely used for biomarker studies and molecular profiling to identify concurrent alterations that can lead to the better characterization of a tumor's molecular landscape. However, further evaluation of technical aspects related to the detection of gene rearrangements and copy number alterations is warranted. Methods: There were 12 ALK rearrangement-positive tumor specimens from patients with non-small cell lung cancer (NSCLC) previously detected via fluorescence in situ hybridization (FISH), immunohistochemistry (IHC), and an RNA-based NGS assay, and 26 MET high gene copy number (GCN) cases detected by FISH, selected for this retrospective study. All 38 pre-characterized cases were reassessed utilizing the PGDx™ elio™ tissue complete assay, a 505 gene targeted NGS panel, to evaluate concordance with these conventional diagnostic techniques. Results: The detection of ALK rearrangements using the DNA-based NGS assay demonstrated excellent sensitivity with the added benefit of characterizing gene fusion partners and genomic breakpoints. MET copy number alterations were also detected; however, some discordances were observed likely attributed to differences in algorithm, reporting thresholds and gene copy number state. TMB was also assessed by the assay and correlated to the presence of NSCLC driver alterations and was found to be significantly lower in cases with NGS-confirmed canonical driver mutations compared with those without (p=0.0019). Discussion: Overall, this study validates NGS as an accurate approach for detecting structural variants while also highlighting the need for further optimization to enable harmonization across methodologies for amplifications.

Inhibition of FGF receptor blocks adaptive resistance to RET inhibition in CCDC6-RET-rearranged thyroid cancer.

Genetic alterations in RET lead to activation of ERK and AKT signaling and are associated with hereditary and sporadic thyroid cancer and lung cancer. Highly selective RET inhibitors have recently entered clinical use after demonstrating efficacy in treating patients with diverse tumor types harboring RET gene rearrangements or activating mutations. In order to understand resistance mechanisms arising after treatment with RET inhibitors, we performed a comprehensive molecular and genomic analysis of a patient with RET-rearranged thyroid cancer. Using a combination of drug screening and proteomic and biochemical profiling, we identified an adaptive resistance to RET inhibitors that reactivates ERK signaling within hours of drug exposure. We found that activation of FGFR signaling is a mechanism of adaptive resistance to RET inhibitors that activates ERK signaling. Combined inhibition of FGFR and RET prevented the development of adaptive resistance to RET inhibitors, reduced cell viability, and decreased tumor growth in cellular and animal models of CCDC6-RET-rearranged thyroid cancer.

Validation of a Circulating Tumor DNA-Based Next-Generation Sequencing Assay in a Cohort of Patients with Solid tumors: A Proposed Solution for Decentralized Plasma Testing.

BACKGROUND: Characterization of circulating tumor DNA (ctDNA) has been integrated into clinical practice. Although labs have standardized validation procedures to develop single locus tests, the efficacy of on-site plasma-based next-generation sequencing (NGS) assays still needs to be proved. MATERIALS AND METHODS: In this retrospective study, we profiled DNA from matched tissue and plasma samples from 75 patients with cancer. We applied an NGS test that detects clinically relevant alterations in 33 genes and microsatellite instability (MSI) to analyze plasma cell-free DNA (cfDNA). RESULTS: The concordance between alterations detected in both tissue and plasma samples was higher in patients with metastatic disease. The NGS test detected 77% of sequence alterations, amplifications, and fusions that were found in metastatic samples compared with 45% of those alterations found in the primary tumor samples (p = .00005). There was 87% agreement on MSI status between the NGS test and tumor tissue results. In three patients, MSI-high ctDNA correlated with response to immunotherapy. In addition, the NGS test revealed an FGFR2 amplification that was not detected in tumor tissue from a patient with metastatic gastric cancer, emphasizing the importance of profiling plasma samples in patients with advanced cancer. CONCLUSION: Our validation experience of a plasma-based NGS assay advances current knowledge about translating cfDNA testing into clinical practice and supports the application of plasma assays in the management of oncology patients with metastatic disease. With an in-house method that minimizes the need for invasive procedures, on-site cfDNA testing supplements tissue biopsy to guide precision therapy and is entitled to become a routine practice. IMPLICATIONS FOR PRACTICE: This study proposes a solution for decentralized liquid biopsy testing based on validation of a next-generation sequencing (NGS) test that detects four classes of genomic alterations in blood: sequence mutations (single nucleotide substitutions or insertions and deletions), fusions, amplifications, and microsatellite instability (MSI). Although there are reference labs that perform single-site comprehensive liquid biopsy testing, the targeted assay this study validated can be established locally in any lab with capacity to offer clinical molecular pathology assays. To the authors' knowledge, this is the first report that validates evaluating an on-site plasma-based NGS test that detects the MSI status along with common sequence alterations encountered in solid tumors.

Ibrutinib Unmasks Critical Role of Bruton Tyrosine Kinase in Primary CNS Lymphoma.

Bruton tyrosine kinase (BTK) links the B-cell antigen receptor (BCR) and Toll-like receptors with NF-κB. The role of BTK in primary central nervous system (CNS) lymphoma (PCNSL) is unknown. We performed a phase I clinical trial with ibrutinib, the first-in-class BTK inhibitor, for patients with relapsed or refractory CNS lymphoma. Clinical responses to ibrutinib occurred in 10 of 13 (77%) patients with PCNSL, including five complete responses. The only PCNSL with complete ibrutinib resistance harbored a mutation within the coiled-coil domain of CARD11, a known ibrutinib resistance mechanism. Incomplete tumor responses were associated with mutations in the B-cell antigen receptor-associated protein CD79B. CD79B-mutant PCNSLs showed enrichment of mammalian target of rapamycin (mTOR)-related gene sets and increased staining with PI3K/mTOR activation markers. Inhibition of the PI3K isoforms p110α/p110δ or mTOR synergized with ibrutinib to induce cell death in CD79B-mutant PCNSL cells.Significance: Ibrutinib has substantial activity in patients with relapsed or refractory B-cell lymphoma of the CNS. Response rates in PCNSL were considerably higher than reported for diffuse large B-cell lymphoma outside the CNS, suggesting a divergent molecular pathogenesis. Combined inhibition of BTK and PI3K/mTOR may augment the ibrutinib response in CD79B-mutant human PCNSLs. Cancer Discov; 7(9); 1018-29. ©2017 AACR.See related commentary by Lakshmanan and Byrd, p. 940This article is highlighted in the In This Issue feature, p. 920.

Trkb signaling in pericytes is required for cardiac microvessel stabilization.

Pericyte and vascular smooth muscle cell (SMC) recruitment to the developing vasculature is an important step in blood vessel maturation. Brain-derived neurotrophic factor (BDNF), expressed by endothelial cells, activates the receptor tyrosine kinase TrkB to stabilize the cardiac microvasculature in the perinatal period. However, the effects of the BDNF/TrkB signaling on pericytes/SMCs and the mechanisms downstream of TrkB that promote vessel maturation are unknown. To confirm the involvement of TrkB in vessel maturation, we evaluated TrkB deficient (trkb (-/-)) embryos and observed severe cardiac vascular abnormalities leading to lethality in late gestation to early prenatal life. Ultrastructural analysis demonstrates that trkb(-/-) embryos exhibit defects in endothelial cell integrity and perivascular edema. As TrkB is selectively expressed by pericytes and SMCs in the developing cardiac vasculature, we generated mice deficient in TrkB in these cells. Mice with TrkB deficiency in perivascular cells exhibit reduced pericyte/SMC coverage of the cardiac microvasculature, abnormal endothelial cell ultrastructure, and increased vascular permeability. To dissect biological actions and the signaling pathways downstream of TrkB in pericytes/SMCs, human umbilical SMCs were treated with BDNF. This induced membranous protrusions and cell migration, events dependent on myosin light chain phosphorylation. Moreover, inhibition of Rho GTPase and the Rho-associated protein kinase (ROCK) prevented membrane protrusion and myosin light chain phosphorylation in response to BDNF. These results suggest an important role for BDNF in regulating migration of TrkB-expressing pericytes/SMCs to promote cardiac blood vessel ensheathment and functional integrity during development.

EGFL7: a unique angiogenic signaling factor in vascular development and disease.

EGFL7 is a secreted angiogenic factor that is highly conserved in vertebrates. Most secreted angiogenic signaling molecules, including VEGF and fibroblast growth factor-2, are mainly expressed by non-endothelial cell types such as fibroblasts. In contrast, EGFL7 is unique because it is almost exclusively expressed by and acts on endothelial cells. Egfl7 expression is highest when the endothelium is in an active, proliferating state. This factor acts as a chemoattractant for endothelial cells and binds to components of the extracellular matrix. In vivo, Egfl7 is important for regulating tubulogenesis in zebrafish and for controlling vascular patterning and integrity in mice. Its function in blood vessel development is mediated, at least in part, through modulation of Notch signaling. In this review, we summarize the findings that support a role for Egfl7 in developmental and postnatal angiogenesis and describe the EGFL7-signaling pathways that underlie these processes. In addition, we discuss a potential role for EGFL7 in vascular repair and its possible use as a therapeutic target for treatment of hypoxia-induced injury. Finally, we consider EGFL7 action during tumorigenesis and its potential as an antiangiogenic agent.

Impaired angiogenesis and altered Notch signaling in mice overexpressing endothelial Egfl7.

Epidermal growth factor-like domain 7 (Egfl7) is important for regulating tubulogenesis in zebrafish, but its role in mammals remains unresolved. We show here that endothelial overexpression of Egfl7 in transgenic mice leads to partial lethality, hemorrhaging, and altered cardiac morphogenesis. These defects are accompanied by abnormal vascular patterning and remodeling in both the embryonic and postnatal vasculature. Egfl7 overexpression in the neonatal retina results in a hyperangiogenic response, and EGFL7 knockdown in human primary endothelial cells suppresses endothelial cell proliferation, sprouting, and migration. These phenotypes are reminiscent of Notch inhibition. In addition, our results show that EGFL7 and endothelial-specific NOTCH physically interact in vivo and strongly suggest that Egfl7 antagonizes Notch in both the postnatal retina and in primary endothelial cells. Specifically, Egfl7 inhibits Notch reporter activity and down-regulates the level of Notch target genes when overexpressed. In conclusion, we have uncovered a critical role for Egfl7 in vascular development and have shown that some of these functions are mediated through modulation of Notch signaling.

Elevated expression of the metabolic regulator receptor-interacting protein 140 results in cardiac hypertrophy and impaired cardiac function.

AIMS: Receptor-interacting protein 140 (RIP140) is a ligand-dependent cofactor for nuclear receptors that regulate networks of genes involved in cellular processes, including metabolism. An important role for RIP140 in metabolic control has been identified in RIP140 null mice, whose phenotypes include derepression of genes involved in energy mobilization or catabolism in adipocytes and a switch to more oxidative fibres in skeletal muscle. We hypothesized that ubiquitous expression of RIP140 would suppress metabolic processes, leading to defects in development or cellular function. METHODS AND RESULTS: The primary effect of exogenous expression of RIP140 mRNA (real-time PCR) and protein (western blotting) in transgenic mice is impaired postnatal heart function. There was rapid onset of cardiac hypertrophy and ventricular fibrosis, detected microscopically, in male RIP140 transgenic mice from 4 weeks of age, resulting in 25% mortality by 5 months. RIP140 exogenous expression in the heart leads to decreased mitochondria state III and state IV membrane potential and oxygen consumption. Quantitative PCR showed more than 50% reduced expression of genes involved in mitochondrial activity and fatty acid metabolism, including mitochondrial transcription factor A, cytochrome oxidase VIIa, cytochrome XII, CD36, medium-chain acyl dehydrogenase, and fatty acid transport protein, many of which are known targets for nuclear receptors, including peroxisome proliferator-activated receptors PPARalpha and PPARdelta and oestrogen-related receptors ERRalpha and ERRgamma. CONCLUSION: This study demonstrates that RIP140 is an important cofactor in postnatal cardiac function and that inhibition of the action of RIP140 may provide a model system to investigate specific interventions designed to prevent or delay the onset of cardiac disease.

The transcriptional corepressor RIP140 regulates oxidative metabolism in skeletal muscle.

Nuclear receptor signaling plays an important role in energy metabolism. In this study we demonstrate that the nuclear receptor corepressor RIP140 is a key regulator of metabolism in skeletal muscle. RIP140 is expressed in a fiber type-specific manner, and manipulation of its levels in null, heterozygous, and transgenic mice demonstrate that low levels promote while increased expression suppresses the formation of oxidative fibers. Expression profiling reveals global changes in the expression of genes implicated in both myofiber phenotype and metabolic functions. Genes involved in fatty-acid oxidation, oxidative phosphorylation, and mitochondrial biogenesis are upregulated in the absence of RIP140. Analysis of cultured myofibers demonstrates that the changes in expression are intrinsic to muscle cells and that nuclear receptor-regulated genes are direct targets for repression by RIP140. Therefore RIP140 is an important signaling factor in the regulation of skeletal muscle function and physiology.

RIP140 expression is stimulated by estrogen-related receptor alpha during adipogenesis.

RIP140 is a corepressor for nuclear receptors that regulates energy expenditure in adipose tissue by suppressing the expression of clusters of metabolic genes involved in glucose and lipid metabolism. The gene encoding RIP140/Nrip1 contains only one coding exon but has multiple promoters and 5' non-coding exons that are subject to alternative splicing. In adipocytes we have defined a promoter, referred to as P2, that is preferentially utilized and activated during adipogenesis. Expression studies and chromatin immunoprecipitation experiments indicate that estrogen-related receptor alpha (ERRalpha), the level of which increases during adipogenesis in parallel with RIP140, stimulates transcription from the P2 promoter. Further analysis indicates that ERRalpha is capable of activating RIP140 gene transcription by two mechanisms, directly by binding to an estrogen receptor element/ERR element at -650/-633 and indirectly through Sp1 binding sites in the proximal promoter. Thus, the up-regulation of RIP140 by ERRalpha during adipogenesis may provide an inhibitory feedback mechanism to control the expression of many nuclear receptor target genes.