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Fusions involving the oncogenic gene RET have been observed in thyroid and lung cancers. Here we report RET gene alterations, including amplification, missense mutations, known fusions, novel fusions, and rearrangements in breast cancer. Their frequency, oncogenic potential, and actionability in breast cancer are described. Two out of eight RET fusions (NCOA4-RET and a novel RASGEF1A-RET fusion) and RET amplification were functionally characterized and shown to activate RET kinase and drive signaling through MAPK and PI3K pathways. These fusions and RET amplification can induce transformation of non-tumorigenic cells, support xenograft tumor formation, and render sensitivity to RET inhibition. An index case of metastatic breast cancer progressing on HER2-targeted therapy was found to have the NCOA4-RET fusion. Subsequent treatment with the RET inhibitor cabozantinib led to a rapid clinical and radiographic response. RET alterations, identified by genomic profiling, are promising therapeutic targets and are present in a subset of breast cancers., Fusions of the gene RET have been described in thyroid and lung cancers. Here, the AUs identify RET gene alterations, including known fusions, novel fusions, and rearrangements in breast cancer (BC) that are involved in the tumorigenic process and show the benefit of RET therapy in a recurrent BC patient carrying the NCOA4-RET fusion.

The protein-protein interaction (PPI) network is crucial for cellular information processing and decision-making. With suitable inputs, PPI networks drive the cells to diverse functional outcomes such as cell proliferation or cell death. Here we characterize the structural controllability of a large directed human PPI network comprised of 6,339 proteins and 34,813 interactions. This allows us to classify proteins as "indispensable", "neutral" or "dispensable", which correlates to increasing, no effect, or decreasing the number of driver nodes in the network upon removal of that protein. We find that 21% of the proteins in the PPI network are indispensable. Interestingly, these indispensable proteins are the primary targets of disease-causing mutations, human viruses, and drugs, suggesting that altering a network's control property is critical for the transition between healthy and disease states. Furthermore, analyzing copy number alterations data from 1,547 cancer patients reveals that 56 genes that are frequently amplified or deleted in nine different cancers are indispensable. Among the 56 genes, 46 of them have not been previously associated with cancer. This suggests that controllability analysis is very useful in identifying novel disease genes and potential drug targets., Comment: 31 pages, 4 figures

Introduction: Patients whose tumors harbor NTRK gene fusions represent an extremely small proportion of the overall cancer population (i.e., ~0.32-1%). While adult and pediatric cancer patients with NTRK fusion-positive solid tumors are rare, they comprise a patient population with a targetable alteration for whom there is currently no accepted standard of care. Comprehensive genomic profiling assays are a critical component in the identification of this sub-population, and herein we describe the clinical validation and analytic performance of FoundationOne CDx™ (F1CDx) as the companion diagnostic for entrectinib, a potent inhibitor of ROS1 and TRK A/B/C kinases. F1CDx and entrectinib were approved in Japan for the respective diagnosis and treatment of adult and pediatric patients with NTRK fusion-positive advanced recurrent solid tumors. Methods: Analytical concordance was determined by comparing F1CDx to a validated RNA NGS fusion panel using 28 NTRK fusion-positive and 53 NTRK fusion-negative samples. Concordance was defined as the overall percent agreement (OPA) between the two assays. Positive and negative percent agreement (PPA, NPA) were calculated using the RNA assay as the reference. Analytical sensitivity was calculated from representative gene fusions based on the 95% probability of detection, and intermediate precision was assessed from NTRK-positive samples run at and slightly above the platform limit of detection. Clinical validation for F1CDx as a companion diagnostic for entrectinib in Japan was estimated based upon patients in the primary efficacy population who had samples remaining for F1CDx testing using multiple imputation and sensitivity analysis of the concordance of F1CDx to the various enrollment assays. Results: OPA between F1CDx and an orthogonally validated RNA NGS fusion panel was 92.6% (95% CI: 84.6-97.2%). PPA and NPA values were 85.7% (95% CI: 67.3-96.0%) and 96.2% (95% CI: 87.0-99.5%), respectively. Platform sensitivity for rearrangements was between 6.4-11.3% tumor purity. Repeatability for NTRK fusions was 100% (95% CI: 73.5-100.0%) and reproducibility was 100% (95% CI: 90.3-100.0%). OPA, PPA and NPA values comparing F1CDx to the multiple enrollment assays utilized for the STARTRK-2 trial were 95.7% (95% CI: 87.8-99.1%), 84.2% (95% CI: 60.4-96.6%), and 100.0% (95% CI: 92.9-100.0%), respectively. The previously reported overall response rate (ORR) for the primary efficacy population in the STARTRK-2 trial was 57.4% (95% CI: 42.3-70.7%)1. Bridging analysis estimated that the ORR for the F1CDx NTRK-positive population was 50.0% (95% CI: 24.7-75.4%), with acceptable balance between co-variates in the F1CDx evaluable and non-evaluable populations. Conclusions: The DNA-based F1CDx assay demonstrated strong concordance compared to an externally validated RNA NGS fusion panel (92.6% OPA) for the detection of NTRK fusions. Furthermore, an analysis of the clinical efficacy demonstrated comparable results between F1CDx NTRK-positive patients (ORR of 50%; 95% CI: 24.7-75.4%) and the overall NTRK fusion-positive population (ORR of 57%; 95% CI: 42.3-70.7%) from the STARTRK-2 clinical trial evaluating entrectinib in patients with solid tumors. 1. ESMO [https://www.esmo.org/Oncology-News/Entrectinib-NTRK-Fusion-Positive-Tumours-Demetri] Citation Format: David Fabrizio, Coren Milbury, Wai-Ki Yip, Bahar Yilmazel, Xiaobo Bai, Juan Liao, David Smith, Christine Burns, Christine Vietz. Clinical and analytic validation of FoundationOne CDx™ for NTRK fusion-positive solid tumors in patients treated with entrectinib [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A028. doi:10.1158/1535-7163.TARG-19-A028

Somatic ERBB2 amplification or activating mutations occur in approximately 2-5% of metastatic colorectal adenocarcinomas and are presumed to be oncogenic drivers, but limited evidence exists to suggest these lesions are sensitive to targeted monotherapy in patients. Here we present the case of a patient with advanced CRC with pulmonary metastases, who had progressed on both standard of care cytotoxic chemotherapy and anti-EGFR targeted therapy. Comprehensive genomic profiling (FoundationOne(®)) identified amplification of ERBB2 and a TP53 mutation in the metastatic lesion. Treatment with trastuzumab with a chemotherapy backbone elicited stable disease/minor response in the patient over a one year course of therapy, reducing tumor burden and significantly improving quality of life. This report demonstrates the application of personalized targeted therapy guided by comprehensive genomic profiling in metastatic colorectal adenocarcinoma.

A major objective of systems biology is to organize molecular interactions as networks and to characterize information flow within networks. We describe a computational framework to integrate protein-protein interaction (PPI) networks and genetic screens to predict the 'signs' of interactions (i.e., activation-inhibition relationships). We constructed a Drosophila melanogaster signed PPI network consisting of 6,125 signed PPIs connecting 3,352 proteins that can be used to identify positive and negative regulators of signaling pathways and protein complexes. We identified an unexpected role for the metabolic enzymes enolase and aldo-keto reductase as positive and negative regulators of proteolysis, respectively. Characterization of the activation-inhibition relationships between physically interacting proteins within signaling pathways will affect our understanding of many biological functions, including signal transduction and mechanisms of disease.

Cure rates of children and adults with acute myeloid leukaemia (AML) remain unsatisfactory partly due to chemotherapy resistance. We investigated the genetic basis of AML in 107 primary cases by sequencing 670 genes mutated in haematological malignancies. SETBP1, ASXL1 and RELN mutations were significantly associated with primary chemoresistance. We identified genomic alterations not previously described in AML, together with distinct genes that were significantly overexpressed in therapy-resistant AML. Defined gene mutations were sufficient to explain primary induction failure in only a minority of cases. Thus, additional genetic or molecular mechanisms must cause primary chemoresistance in paediatric and adult AML.

Despite intense efforts, the cure rates of children and adults with AML remain unsatisfactory in large part due to resistance to chemotherapy. Whilst cytogenetic risk stratification proved valuable in identifying causes of therapy failure and disease relapse, cytogenetically normal AML remains the most prevalent disease type, with significant heterogeneity of clinical outcomes, including primary chemoresistance. Using targeted sequencing of 670 genes recurrently mutated in hematologic malignancies, we investigated the genetic basis of primary chemotherapy resistance and remission induction failure of 107 primary cases obtained at diagnosis from children and adults with cytogenetically normal AML. Comparative analysis revealed mutations ofSETBP1, ASXL1andRELNto be significantly enriched at diagnosis in primary induction failure as compared to remission cases. In addition, this analysis revealed novel genomic alterations not previously described in AML, as well as distinct genes that are significantly overexpressed in therapy resistant AML. However, identified gene mutations were sufficient to explain only a minority of cases of primary induction failure. Thus, additional genetic or molecular mechanisms must cause primary chemoresistance in pediatric and adult acute myeloid leukemias.Key PointsTargeted gene sequencing of 670 genes in adult and pediatric AMLProfiling of 107 primary AML samples identifies new genomic alterations for primary chemoresistance

e21629Background: Melanomas are heterogenous tumors that develop on many skin regions and harbor diverse genomic alterations (GA). Lesion subtypes are identifiable by location and/or genomic profil...

The tuberous sclerosis complex (TSC) family of tumor suppressors, TSC1 and TSC2, function together in an evolutionarily conserved protein complex that is a point of convergence for major cell signaling pathways that regulate mTOR complex 1 (mTORC1). Mutation or aberrant inhibition of the TSC complex is common in various human tumor syndromes and cancers. The discovery of novel therapeutic strategies to selectively target cells with functional loss of this complex is therefore of clinical relevance to patients with nonmalignant TSC and those with sporadic cancers. We developed a CRISPR-based method to generate homogeneous mutant Drosophila cell lines. By combining TSC1 or TSC2 mutant cell lines with RNAi screens against all kinases and phosphatases, we identified synthetic interactions with TSC1 and TSC2. Individual knockdown of three candidate genes (mRNA-cap, Pitslre, and CycT; orthologs of RNGTT, CDK11, and CCNT1 in humans) reduced the population growth rate of Drosophila cells lacking either TSC1 or TSC2 but not that of wild-type cells. Moreover, individual knockdown of these three genes had similar growth-inhibiting effects in mammalian TSC2-deficient cell lines, including human tumor-derived cells, illustrating the power of this cross-species screening strategy to identify potential drug targets.

Despite intense efforts, the cure rates of children and adults with AML remain unsatisfactory. Resistance to cytotoxic chemotherapy is the dominant cause of treatment failure. To investigate the molecular basis of primary chemoresistance, we used targeted gene sequencing using the Foundation One Heme platform to profile 405 genes in DNA and 265 genes in RNA known to be somatically mutated in hematologic malignancies in 107 primary specimens obtained at diagnosis from children and adults with cytogenetically normal AML. Comparative analysis revealed mutations of SETBP1 and ASXL1 to be enriched in patients with failure of induction chemotherapy as compared to those who achieved remission (8 out of 50 versus 1 out of 57, p = 0.01). However, the low prevalence of these alterations indicated that other genomic or functional mechanisms must mediate chemoresistance. To identify these mechanisms, we mapped kinase signaling cascades activated in chemoresistant AML cells using functional mass spectrometry proteomics. Phosphopeptides were enriched in primary leukemic blasts isolated from cytogenetically normal AML patients, with relative abundances in each patient determined from iTRAQ 8-plex reporter ions in the associated MS/MS fragment ion spectra. This analysis identified phosphorylation of the leukemogenic transcription factor MEF2C at S222 in the majority of cases. We found MEF2C to be overexpressed in cytogenetically normal AML, as well as MLL-rearranged leukemias, and to be associated with increased risk of relapse (Laszlo et al, ASH submitted). Using reporter assays, we found that phosphorylation of MEF2C S222 was both necessary and sufficient to fully transactivate MEF2C promoter elements (Fig. 1). Expression of mutant S222A MEF2C that cannot be phosphorylated, but not phosphomimetic S222D, induced mitochondrial apoptosis in human AML and genetically-engineered MLL-AF9 mouse leukemia but not in normal mouse hematopoietic progenitor cells. In syngeneic mouse transplant models, we found that MEF2C phosphorylation was required for the maintenance of MLL-AF9 leukemias in vivo. Transcriptome and proteome analysis of gene expression changes and composition of MEF2C transcriptional complexes induced by phosphorylation mutants showed that aberrant leukemia cell survival is caused at least in part by alterations in the expression of apoptotic regulators. Using a recombinant kinase screen, we identified MARK kinases as specific enzymes that phosphorylate MEF2C S222. Treatment of primary patient specimens and human AML cell lines with the MARK kinase inhibitor MRT199665 induced MEF2C downregulation and apoptosis in cells with MEF2C but not those lacking MEF2C expression (Fig. 2). Thus, aberrant MEF2C phosphorylation is associated with primary chemoresistance and induction failure, is required for enhanced leukemia cell survival, and confers susceptibility to MARK inhibition therapy. In addition, genome and proteome profiles should provide additional biomarkers and targeted therapeutic strategies to overcome chemoresistance in AML. Figure 1. MEF2C phosphorylation is required for transactivation of MEF2C promoter elements Figure 1. MEF2C phosphorylation is required for transactivation of MEF2C promoter elements Figure 2. Treatment of AML cells with MRT199665 Figure 2. Treatment of AML cells with MRT199665 Disclosures He: Foundation Medicine, Inc.: Employment, Equity Ownership. Balasubramanian:Foundation Medicine, Inc.: Employment. Zhong:Foundation Medicine, Inc.: Employment, Equity Ownership. Pavlick:Foundation Medicine, Inc.: Employment. Yilmazel:Foundation Medicine, Inc.: Employment. Stone:Merck: Consultancy; AROG: Consultancy; Abbvie: Consultancy; Juno: Consultancy; Amgen: Consultancy; Celator: Consultancy; Agios: Consultancy; Karyopharm: Consultancy; Pfizer: Consultancy; Roche/Genetech: Consultancy; Sunesis: Consultancy, Other: DSMB for clinical trial; Celgene: Consultancy; Novartis: Research Funding. Byrd:Acerta Pharma BV: Research Funding. Levine:CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Foundation Medicine: Consultancy; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees. Armstrong:Epizyme, Inc: Consultancy.