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One of the greatest challenges in studying the genomic basis of human cancer is distinguishing the few mutations that directly contribute to tumorigenesis (“drivers”) from the many biologically neutral mutations (“passengers”). Existing methods can identify highly recurrent individual mutations or mutated genes, but capturing functional yet infrequent “long-tail” mutations remains challenging. While individually uncommon, these long-tail mutations are present in as many as one-third of cancer patients, and for many of them targeted therapies currently exist. Therefore, improved approaches to identify individual driver mutations in the long tail will facilitate our understanding of their potentially diverse biological and clinical significance. Some of these mutations can be identified as hotspots based on their recurrence on or around the same residue. Others may occur in different regions but are actually close in protein three-dimensional structures. We have developed a novel method that identifies mutations that significantly cluster together within three-dimensional proximity in protein structures, or 3D hotspots. We applied this method to a combined collection of mutation data of 21,000 sequenced tumor samples in 74 cancers. Our analysis confirmed many well-studied 3D hotspots of functional mutations in cancer genes such as KRAS, BRAF, IDH2, SMAD4, FBXW7, SPOP, RHOA, and PTPN11. Most of these genes have well known individual hotspots, but our analysis suggests that additional mutations in other residues of the protein are adjacent in the folded protein structure and may also be oncogenic. We also identified novel 3D hotspots in known cancer genes such as EP300, MAP2K1, and KDR, as well as several other genes of unknown significance that harbor 3D hotspots (e.g., DCC). Most of these hotspots are present in multiple cancers. To explore the functional consequences and potential translational significance of long-tail low-incidence mutations identified by our method, we assessed several MAP2K1 mutations in vitro. MAP2K1 (MEK1) is a critical effector of MAPK signaling, harbors conventional single-codon hotspots in several cancer types, and a number of selective MAPK pathway inhibitors are either FDA-approved or are being investigated in early-phase clinical trials. Our experiments confirmed that these mutations activate MAP2K1 and confer sensitivity to MAP2K1 inhibition. We have provided an implementation of the algorithm via an interactive web resource connecting to cBioPortal for Cancer Genomics (http://cbioportal.org/) for easy interpretation of cancer genomics data in the context of three-dimensional structures. By adding to cBioPortal's already powerful capacity of clinical decision support, our method and analysis provide a useful approach of interpretation, prioritization and extension of biologically significant and clinically actionable mutations in cancer. Citation Format: Jianjiong Gao, Matthew T. Chang, Brooke E. Sylvester, Hannah C. Johnsen, Sizhi P. Gao, S. Onur Sumer, David B. Solit, Barry S. Taylor, Nikolaus Schultz, Chris Sander. Identification of oncogenic mutation hotspots via three-dimensional proximity. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3606.

Amplification of HER2 (ERBB2) has been well characterized as an oncogenic driver, but only recently have mutations in ERBB2 also been shown to have activating and tumorigenic potential. We sought to characterize a large number of ERBB2 mutations seen in sequencing data from patients at MSKCC as well as the cBioPortal for Cancer Genomics, and determine the therapeutic response of these mutations to HER2 targeted therapies. HER2 is a transmembrane tyrosine kinase that is responsible for regulating cell growth and proliferation, among other cell processes. The ERBB2 gene is amplified in approximately 25% of breast cancers and in this disease, HER2 directed therapies are now a component of standard care. More recently, ERBB2 mutations were identified in a variety of cancers, most commonly cancers of the urinary tract but also at lower frequency in cancers of the breast, stomach, colon and lung, among others. Recent data has shown that select ERBB2 mutations activate HER kinase signaling and promote anchorage independent growth in vitro and tumor formation in mice. However, only a small number of ERBB2 mutations have been reported and biologically characterized as oncogenic in preclinical models. Moreover, the therapeutic impact of ERBB2 mutations has not been validated. To screen for activating potential of a large number of ERBB2 mutations, cells were transiently transfected to express ERBB2 mutants generated using site-directed mutagenesis. Expression of several mutations led to increased receptor activation and downstream signaling, as determined by levels of phosphorylated HER2, AKT, ERK, and S6 protein, whereas other mutations had little impact on activity compared to a wild type HER2 control. Stable cell lines were then generated to express the activating mutations and tested for transforming ability in soft agar. Mutations promoted colony formation, with certain mutations producing more colonies. Cells were also grown in the presence of the dual EGFR/HER2 inhibitor, neratinib. While certain mutations were more sensitive to lower doses of neratinib, addition of neratinib at higher doses abrogated colony formation in soft agar for all mutations. While a number of ERRB2 mutations have been shown to have tumorigenic potential, not all mutations respond similarly to HER2 targeted therapies. We hope to create an annotated database of alterations so that in the future, patients can be screened for specific mutations to minimize ineffective and unnecessary treatment options. Citation Format: Hannah Johnsen, Aphrothiti Hanrahan, Alexis Jones, David Solit. Functional characterization of ERBB2 mutations and response to targeted therapies. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 36.

Selective kinase inhibitors, such as erolotinib and lapatinib, and monoclonal antibodies, such as cetuximab, trastuzumab and pertuzumab, targeting EGFR and ERBB2 have elicited significant response rates in lung cancer patients harboring mutant EGFR alleles and breast cancer patients whose tumors harbor gene amplification of ERBB2. To date, only a small number of ERBB2 and ERBB3 mutations have been reported and biologically characterized. Moreover, the therapeutic impact of ERBB2 or ERBB3 mutant alleles and their value as an actionable target in patients has not been validated. While ERBB2 itself cannot bind ligand, it is a potent receptor that integrates growth signals via homo- or heterodimerization with other ERBB family members. Genomic alterations increase ERBB2 kinase activity by constitutively activating the kinase domain, enabling ligand-independent receptor activation, by promoting enhanced dimerization or other mechanisms that have yet to be elucidated. Although ERBB3 has limited inherent kinase activity and cannot homodimerize, ligand stimulation promotes dimerization with active kinases like ERBB2 and EGFR that can phosphorylate ERBB2 which then promotes transformation. Thus genomic alterations in ERBB3 may be a key means of promoting oncogenic signaling despite the protein lacking robust enzymatic activity. We performed an analysis of ∼100 large-scale next generation sequencing datasets newly generated by our group (bladder), our institution (MSKCC-IMPACT), or found in recently published repositories (The Cancer Genome Atlas (TCGA), Broad, Genentech, Sanger, etc) to characterize the distribution and spectrum of mutations in the ERBB2 and ERBB3 genes. Data generated suggests that ERBB2 and ERBB3 mutations are highly prevalent in bladder cancer (each ∼10%), and recurrent, but with less frequency ( Citation Format: Aphrothiti J. Hanrahan, David M. Hyman, John Sfakianos, Alexis Jones, Ricardo Ramirez, Hannah Johnsen, Gopakumar Iyer, Hikmat A. Al-Ahmadie, Dean Bajorin, Bernard H. Bochner, Jonathan A. Coleman, Jonathan E. Rosenberg, Michael F. Berger, Sarat Chandarlapaty, Jose Baselga, David B. Solit. Functional genomics of HER2 and HER3 mutations and response to neratinib. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1101. doi:10.1158/1538-7445.AM2015-1101

Many mutations in cancer are of unknown functional significance. Standard methods use statistically significant recurrence of mutations in tumor samples as an indicator of functional impact. We extend such analyses into the long tail of rare mutations by considering recurrence of mutations in clusters of spatially close residues in protein structures. Analyzing 10,000 tumor exomes, we identify more than 3000 rarely mutated residues in proteins as potentially functional and experimentally validate several in RAC1 and MAP2K1. These potential driver mutations (web resources: 3dhotspots.org and cBioPortal.org) can extend the scope of genomically informed clinical trials and of personalized choice of therapy. Electronic supplementary material The online version of this article (doi:10.1186/s13073-016-0393-x) contains supplementary material, which is available to authorized users.