Search

The PI3K/AKT/mTOR pathway is activated through multiple mechanisms in colorectal carcinoma. Here, the clinicopathologic and molecular features of AKT1 E17K–mutated colorectal carcinoma in comparison with PIK3CA-mutated colorectal carcinoma are described in detail. Interestingly, in comparison with PIK3CA mutants, AKT1 E17K was significantly associated with mucinous morphology and concurrent BRAF V600E mutation. Among PIK3CA mutants, exon 21 mutations were significantly associated with BRAF V600E mutation, MSI-H status, and poor differentiation, while exon 10 mutations were associated with KRAS/NRAS mutations. Three of four AKT1 mutants with data from both primary and metastatic lesions had concordant AKT1 mutation status in both. Both AKT1- and PIK3CA-mutant colorectal carcinoma demonstrated frequent loss of PTEN expression (38% and 34%, respectively) and similar rates of p-PRAS 40 expression (63% and 50%, respectively). Both patients with AKT1 E17K alone had primary resistance to cetuximab, whereas 7 of 8 patients with PIK3CA mutation alone experienced tumor shrinkage or stability with anti-EGFR therapy. These results demonstrate that AKT1 E17K mutation in advanced colorectal carcinoma is associated with mucinous morphology, PIK3CA wild-type status, and concurrent RAS/RAF mutations with similar pattern to PIK3CA exon 21 mutants. Thus, AKT1 E17K mutations contribute to primary resistance to cetuximab and serve as an actionable alteration. Implications: This first systematic study of AKT1 and PIK3CA hotspot mutations and their association with cetuximab resistance and BRAF V600E mutation has important ramifications for the development of personalized medicine, particularly in identifying patient candidates for PI3K or AKT inhibitors. Mol Cancer Res; 13(6); 1003–8. ©2015 AACR.

The identification of specific genetic alterations as key oncogenic drivers and the development of targeted therapies are together transforming clinical oncology and creating a pressing need for increased breadth and throughput of clinical genotyping. Next-generation sequencing assays allow the efficient and unbiased detection of clinically actionable mutations. To enable precision oncology in patients with solid tumors, we developed Memorial Sloan Kettering-Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT), a hybridization capture-based next-generation sequencing assay for targeted deep sequencing of all exons and selected introns of 341 key cancer genes in formalin-fixed, paraffin-embedded tumors. Barcoded libraries from patient-matched tumor and normal samples were captured, sequenced, and subjected to a custom analysis pipeline to identify somatic mutations. Sensitivity, specificity, reproducibility of MSK-IMPACT were assessed through extensive analytical validation. We tested 284 tumor samples with previously known point mutations and insertions/deletions in 47 exons of 19 cancer genes. All known variants were accurately detected, and there was high reproducibility of inter- and intrarun replicates. The detection limit for low-frequency variants was approximately 2% for hotspot mutations and 5% for nonhotspot mutations. Copy number alterations and structural rearrangements were also reliably detected. MSK-IMPACT profiles oncogenic DNA alterations in clinical solid tumor samples with high accuracy and sensitivity. Paired analysis of tumors and patient-matched normal samples enables unambiguous detection of somatic mutations to guide treatment decisions.

Purpose: Genetic alterations affecting the MAPK/ERK pathway are common in lung adenocarcinoma (LAD). Early steps of the signaling pathway are most often affected with EGFR, KRAS, and BRAF mutations encompassing more than 70% of all alterations. Somatic mutations in MEK1, located downstream of BRAF, are rare and remain poorly defined as a distinct molecular subset. Experimental Design: Tumors harboring MEK1 mutations were identified through targeted screening of a large LAD cohort concurrently interrogated for recurrent mutations in MEK1, EGFR, KRAS, BRAF, ERBB2/HER2, NRAS, PIK3CA, and AKT. Additional cases were identified through a search of publically available cancer genomic datasets. Mutations were correlated with patient characteristics and treatment outcomes. Overall survival was compared with stage-matched patients with KRAS- and EGFR-mutant LADs. Results: We identified 36 MEK1-mutated cases among 6,024 LAD (0.6%; 95% confidence interval, 0.42–0.85). The majority of patients were smokers (97%, n = 35/36). There was no association with age, sex, race, or stage. The most common mutations were K57N (64%, 23/36) followed by Q56P (19%, 7/36), all mutually exclusive with other driver mutations in the targeted panel. Transversions G:C>T:A were predominant (89%, 31/35), in keeping with smoking-associated DNA damage. Additional less common somatic mutations were identified in the kinase domain, all of which are predicted to converge into a single interaction area based on in silico 3D modeling. Conclusions: MEK1 mutations define a distinct subset of lung cancers (∼1%) with potential sensitivity to MEK inhibitors. Mutations are predominantly transversions, in keeping with a strong association with smoking. Clin Cancer Res; 21(8); 1935–43. ©2014 AACR.

Tumor molecular profiling is a fundamental component of precision oncology, enabling the identification of genomic alterations in genes and pathways that can be targeted therapeutically. The existence of recurrent targetable alterations across distinct histologically-defined tumor types, coupled with an expanding portfolio of molecularly-targeted therapies, demands flexible and comprehensive approaches to profile clinically significant genes across the full spectrum of cancers. We established a large-scale, prospective clinical sequencing initiative utilizing a comprehensive assay, MSK-IMPACT, through which we have compiled matched tumor and normal sequence data from a unique cohort of more than 10,000 patients with advanced cancer and available pathological and clinical annotations. Using these data, we identified clinically relevant somatic mutations, novel non-coding alterations, and mutational signatures that were shared among common and rare tumor types. Patients were enrolled on genomically matched clinical trials at a rate of 11%. To enable discovery of novel biomarkers and deeper investigation into rare alterations and tumor types, all results are publicly accessible.

BACKGROUND RAS and PIK3CA mutations in metastatic colorectal cancer (mCRC) have been associated with worse survival. We sought to evaluate the impact of RAS and PIK3CA mutations on cumulative incidence of metastasis to potentially curable sites of liver and lung and other sites such as bone and brain. METHODS We performed a computerized search of the electronic medical record of our institution for mCRC cases genotyped for RAS or PIK3CA mutations from 2008 to 2012. Cases were reviewed for patient characteristics, survival, and site-specific metastasis. RESULTS Among the 918 patients identified, 477 cases were RAS wild type, and 441 cases had a RAS mutation (394 at KRAS exon 2, 29 at KRAS exon 3 or 4, and 18 in NRAS). RAS mutation was significantly associated with shorter median overall survival (OS) and on multivariate analysis independently predicted worse OS (HR, 1.6; P

Among gastrointestinal stromal tumors (GISTs) of 10-15% are negative for KIT and PDGFRA, and most of these cases are SDH deficient. Recent studies have provided data on additional molecular alterations such as KRAS in KIT mutant GISTs. We aimed to assess the frequency and spectrum of somatic mutations in common oncogenes as well as copy number variations in GISTs negative for KIT and PDGFRA mutations. GISTs with wild type KIT/PDGFRA were tested via next generation sequencing for somatic mutations in 341 genes. SDHB immunohistochemistry to evaluate for SDH deficiency was also performed. Of 267 GISTs tested for KIT and PDGFRA mutations, 15 were wild type, of which eight cases had material available for further testing. All eight cases had loss of SDHB expression and had various molecular alterations involving ARID1A, TP53, and other genes. One case had a KRAS G12V (c.35G>T) mutation in both the primary gastric tumor and a post-imatinib recurrence. This tumor had anaplastic features and was resistant to multiple tyrosine kinase inhibitors, ultimately resulting in cancer-related mortality within 2 years of diagnosis. In conclusion, KRAS mutations occur in rare GISTs with wild type KIT and PDGFRA. These tumors may display immunohistochemical positivity for KIT and primary resistance to tyrosine kinase inhibitors.

SummaryWe describe the landscape of somatic genomic alterations based on multidimensional and comprehensive characterization of more than 500 glioblastoma tumors (GBMs). We identify several novel mutated genes as well as complex rearrangements of signature receptors, including EGFR and PDGFRA. TERT promoter mutations are shown to correlate with elevated mRNA expression, supporting a role in telomerase reactivation. Correlative analyses confirm that the survival advantage of the proneural subtype is conferred by the G-CIMP phenotype, and MGMT DNA methylation may be a predictive biomarker for treatment response only in classical subtype GBM. Integrative analysis of genomic and proteomic profiles challenges the notion of therapeutic inhibition of a pathway as an alternative to inhibition of the target itself. These data will facilitate the discovery of therapeutic and diagnostic target candidates, the validation of research and clinical observations and the generation of unanticipated hypotheses that can advance our molecular understanding of this lethal cancer.

Although next-generation sequencing (NGS) is a robust technology for comprehensive assessment of EGFR-mutant lung adenocarcinomas with acquired resistance to tyrosine kinase inhibitors, it may not provide sufficiently rapid and sensitive detection of the EGFR T790M mutation, the most clinically relevant resistance biomarker. Here, we describe a digital PCR (dPCR) assay for rapid T790M detection on aliquots of NGS libraries prepared for comprehensive profiling, fully maximizing broad genomic analysis on limited samples. Tumor DNAs from patients with EGFR-mutant lung adenocarcinomas and acquired resistance to epidermal growth factor receptor inhibitors were prepared for Memorial Sloan-Kettering-Integrated Mutation Profiling of Actionable Cancer Targets sequencing, a hybrid capture-based assay interrogating 410 cancer-related genes. Precapture library aliquots were used for rapid EGFR T790M testing by dPCR, and results were compared with NGS and locked nucleic acid-PCR Sanger sequencing (reference high sensitivity method). Seventy resistance samples showed 99% concordance with the reference high sensitivity method in accuracy studies. Input as low as 2.5 ng provided a sensitivity of 1% and improved further with increasing DNA input. dPCR on libraries required less DNA and showed better performance than direct genomic DNA. dPCR on NGS libraries is a robust and rapid approach to EGFR T790M testing, allowing most economical utilization of limited material for comprehensive assessment. The same assay can also be performed directly on any limited DNA source and cell-free DNA.

Purpose: In contrast to the numerous broad screens for oncogene mutations in adult cancers, few such screens have been conducted in pediatric solid tumors. To identify novel mutations and potential therapeutic targets in pediatric cancers, we conducted a high-throughput Sequenom-based analysis in large sets of several major pediatric solid cancers, including neuroblastoma, Ewing sarcoma, rhabdomyosarcoma (RMS), and desmoplastic small round cell tumor (DSRCT). Experimental Design: We designed a highly multiplexed Sequenom-based assay to interrogate 275 recurrent mutations across 29 genes. Genomic DNA was extracted from 192 neuroblastoma, 75 Ewing sarcoma, 89 RMS, and 24 DSRCT samples. All mutations were verified by Sanger sequencing. Results: Mutations were identified in 13% of neuroblastoma samples, 4% of Ewing sarcoma samples, 21.1% of RMS samples, and no DSRCT samples. ALK mutations were present in 10.4% of neuroblastoma samples. The remainder of neuroblastoma mutations involved the BRAF, RAS, and MAP2K1 genes and were absent in samples harboring ALK mutations. Mutations were more common in embryonal RMS (ERMS) samples (28.3%) than alveolar RMS (3.5%). In addition to previously identified RAS and FGFR4 mutations, we report for the first time PIK3CA and CTNNB1 (β-catenin) mutations in 5% and 3.3% of ERMS, respectively. Conclusions: In ERMS, Ewing sarcoma, and neuroblastoma, we identified novel occurrences of several oncogene mutations recognized as drivers in other cancers. Overall, neuroblastoma and ERMS contain significant subsets of cases with nonoverlapping mutated genes in growth signaling pathways. Tumor profiling can identify a subset of pediatric solid tumor patients as candidates for kinase inhibitors or RAS-targeted therapies. Clin Cancer Res; 18(3); 748–57. ©2011 AACR.

Human cancer cells typically harbour multiple chromosomal aberrations, nucleotide substitutions and epigenetic modifications that drive malignant transformation. The Cancer Genome Atlas ( TCGA) pilot project aims to assess the value of large- scale multi- dimensional analysis of these molecular characteristics in human cancer and to provide the data rapidly to the research community. Here we report the interim integrative analysis of DNA copy number, gene expression and DNA methylation aberrations in 206 glioblastomas - the most common type of primary adult brain cancer - and nucleotide sequence aberrations in 91 of the 206 glioblastomas. This analysis provides new insights into the roles of ERBB2, NF1 and TP53, uncovers frequent mutations of the phosphatidylinositol- 3- OH kinase regulatory subunit gene PIK3R1, and provides a network view of the pathways altered in the development of glioblastoma. Furthermore, integration of mutation, DNA methylation and clinical treatment data reveals a link between MGMT promoter methylation and a hypermutator phenotype consequent to mismatch repair deficiency in treated glioblastomas, an observation with potential clinical implications. Together, these findings establish the feasibility and power of TCGA, demonstrating that it can rapidly expand knowledge of the molecular basis of cancer.

Activation of oncogenes by mechanisms other than genetic aberrations such as mutations, translocations, or amplifications is largely undefined. Here we report a novel isoform of the anaplastic lymphoma kinase (ALK) that is expressed in ∼11% of melanomas and sporadically in other human cancer types, but not in normal tissues. The novel ALK transcript initiates from a de novo alternative transcription initiation (ATI) site in ALK intron 19, and was termed ALK(ATI). In ALK(ATI)-expressing tumours, the ATI site is enriched for H3K4me3 and RNA polymerase II, chromatin marks characteristic of active transcription initiation sites. ALK(ATI) is expressed from both ALK alleles, and no recurrent genetic aberrations are found at the ALK locus, indicating that the transcriptional activation is independent of genetic aberrations at the ALK locus. The ALK(ATI) transcript encodes three proteins with molecular weights of 61.1, 60.8 and 58.7 kilodaltons, consisting primarily of the intracellular tyrosine kinase domain. ALK(ATI) stimulates multiple oncogenic signalling pathways, drives growth-factor-independent cell proliferation in vitro, and promotes tumorigenesis in vivo in mouse models. ALK inhibitors can suppress the kinase activity of ALK(ATI), suggesting that patients with ALK(ATI)-expressing tumours may benefit from ALK inhibitors. Our findings suggest a novel mechanism of oncogene activation in cancer through de novo alternative transcription initiation.

The biological relevance of histological subtyping of ampullary carcinoma into intestinal vs pancreaticobiliary types remains to be determined. In an effort to molecularly profile these subtypes of ampullary carcinomas, we conducted a two-phase study. In the discovery phase, we identified 18 pancreatobiliary-type ampullary carcinomas and 14 intestinal-type ampullary carcinomas using stringent pathologic criteria and performed next-generation sequencing targeting 279 cancer-associated genes on these tumors. Although the results showed overlapping of genomic alterations between the two subtypes, trends including more frequent KRAS alterations in pancreatobiliary-type ampullary carcinoma (61 vs 29%) and more frequent mutations in APC in intestinal-type ampullary carcinoma (43 vs 17%) were observed. Of the entire cohort of 32 tumors, the most frequently mutated gene was TP53 (n = 17); the most frequently amplified gene was ERBB2 (n = 5); and the most frequently deleted gene was CDKN2A (n = 6). In the second phase of the study, we aimed at validating our observation on ERBB2 and assessed ERBB2 amplification and protein overexpression in a series of 100 ampullary carcinomas. We found that (1) gene amplification and immunohistochemical overexpression of ERBB2 occurred in 13% of all ampullary carcinomas, therefore providing a potential target for anti-HER2 therapy in these tumors; (2) amplification and immunohistochemical expression correlated in all cases, thus indicating that immunohistochemistry could be used to screen tumors; and (3) none of the 14 ERBB2-amplified tumors harbored any downstream driver mutations in KRAS/NRAS, whereas 56% of the cases negative for ERBB2 amplification did, an observation clinically pertinent as downstream mutations may cause primary resistance to inhibition of EGFR family members.

To the editor: In a recent paper in Blood , Haroche et al reported dramatic efficacy of vemurafenib in 3 cases of multisystem and refractory Erdheim-Chester disease (ECD) and Langerhans cell histiocytosis harboring the BRAF V600E mutation.[1][1] The findings of an ∼50% prevalence of BRAF

Mutations in the MET exon 14 RNA splice acceptor and donor sites, which lead to exon skipping, deletion of the juxtamembrane domain containing the CBL E3-ubiquitin ligase-binding site, and decreased turnover of the resultant aberrant MET protein, were previously reported to be oncogenic in preclinical models. We now report responses to the MET inhibitors crizotinib and cabozantinib in four patients with stage IV lung adenocarcinomas harboring mutations leading to MET exon 14 skipping, highlighting a new therapeutic strategy for the 4% of lung adenocarcinoma patients whose tumors harbor this previously underappreciated genetic alteration. Significance: Oncogenic mutations in the MET exon 14 splice sites that cause exon 14 skipping occur in 4% of lung adenocarcinomas. We report responses to the MET inhibitors crizotinib and cabozantinib in patients with lung adenocarcinomas harboring MET exon 14 splice site mutations, identifying a new potential therapeutic target in this disease. Cancer Discov; 5(8); 842–9. ©2015 AACR. See related commentary by Ma, p. 802. See related article by Frampton et al., p. 850. This article is highlighted in the In This Issue feature, p. 783

Among gastrointestinal stromal tumors (GISTs) of 10-15% are negative for KIT and PDGFRA, and most of these cases are SDH deficient. Recent studies have provided data on additional molecular alterations such as KRAS in KIT mutant GISTs. We aimed to assess the frequency and spectrum of somatic mutations in common oncogenes as well as copy number variations in GISTs negative for KIT and PDGFRA mutations. GISTs with wild type KIT/PDGFRA were tested via next generation sequencing for somatic mutations in 341 genes. SDHB immunohistochemistry to evaluate for SDH deficiency was also performed. Of 267 GISTs tested for KIT and PDGFRA mutations, 15 were wild type, of which eight cases had material available for further testing. All eight cases had loss of SDHB expression and had various molecular alterations involving ARID1A, TP53, and other genes. One case had a KRAS G12V (c.35GT) mutation in both the primary gastric tumor and a post-imatinib recurrence. This tumor had anaplastic features and was resistant to multiple tyrosine kinase inhibitors, ultimately resulting in cancer-related mortality within 2 years of diagnosis. In conclusion, KRAS mutations occur in rare GISTs with wild type KIT and PDGFRA. These tumors may display immunohistochemical positivity for KIT and primary resistance to tyrosine kinase inhibitors.

A 75 year-old woman, 50 pack-year ex-smoker, presented with a lung nodule on a routine follow-up chest CT scan for prior colon adenocarcinoma (Figure 1A). A wedge resection revealed an apparently single 1.5 cm nodule on gross examination. Microscopic examination revealed an adenocarcinoma of lung origin, as supported by morphology (Figure 1B) and diffusely positive immunohistochemistry for TTF-1 and Napsin A. The tumor consisted of two geographically-contiguous areas - a 6 mm area with solid and acinar patterns, and a peripheral 9 mm area with a predominantly lepidic pattern (Figure 1C,D). Although the two areas could represent heterogeneous histology in a single adenocarcinoma, the dumbbell shape of the lesion raised the possibility of a collision of two immediately-adjacent separate tumors. The two areas were dissected out and submitted for EGFR and KRAS molecular testing separately. Sequencing revealed a KRAS G12A mutation in the 6 mm area, and a novel KRAS L19F/T20S double-mutation in the 9 mm are (Figure 2A). The latter was confirmed to represent same-allele double-mutation - a complex compound substitution (L19_T20>FS) - by next-generation sequencing (Figure 2B), resulting in potential functional cooperativity based on amino acid modeling (Figure 3). Overall, these molecular data established that the 6 mm and 9 mm areas had distinct KRAS mutations, thus representing two colliding synchronous primaries rather than histologically-heterogeneous areas of a single adenocarcinoma. Figure 1 (A) Chest CT scan: apparently single nodule (arrow) with solid and ground-glass components. (B) Low-power and (C, D) higher power microscopic images of H&E-stained section, revealing two geographically-contiguous areas with distinct growth patterns ... Figure 2 Sanger sequencing electropherograms, revealing a KRAS G12A(c.35G>C) mutation in the 6 mm nodule (A) and KRAS L19F(c.57G>T)/T20S(c.58A>T) double mutation in the 9 mm nodule (B). (C) Split-screen view of aligned sequencing reads ... Figure 3 Amino acid modeling of KRAS L19F/T20S (L19_T20>FS) double mutation. L19F and T20S are highly conserved and belong to the core region of KRAS. L19 is predicted to interact with GTP/GDP-binding residues (N116, A146, and K16 that in turn interact ... This case illustrates a rare phenomenon of collision tumors, in which two distinct tumors co-occur at the same or immediately-adjacent anatomic locations.1 The exact incidence of collision of synchronous lung carcinomas in unknown, but given that 4–20% of patients with lung cancer have multiple primary tumors,2 the odds of two tumors occurring immediately-adjacent to each other are not negligible. As illustrated here, collisions are inapparent on radiologic and gross examination. While microscopic examination is generally effective at identifying collisions of different tumor types, recognition of this phenomenon for synchronous lung adenocarcinomas is particularly challenging because these tumors are at-baseline highly heterogeneous (i.e. composed of a mixture of lepidic, acinar, solid etc patterns), making it difficult to distinguish histologic heterogeneity in a single adenocarcinoma from two colliding, contiguous tumors. Peripheral lepidic pattern is commonly seen in lung adenocarcinomas. In this case, a dumbbell-shaped outline of the lesion raised the possibility of a collision, but a definitive distinction between a single morphologically-heterogeneous adenocarcinoma versus a collision could not be made based on microscopic findings. This case demonstrates of the utility of driver mutation analysis in elucidating tumor clonal relationships in the setting of a possible collision, similar to what has been recently proposed for the utility of molecular profiling for the distinction of synchronous/metachronous primaries from intrapulmonary metastases.3 We also illustrate the use of next-generation sequencing to determine that the two KRAS mutations in the 9 mm nodule resided on same allele, thus excluding the possibility they were a result of yet another unsuspected collision of tumors with distinct KRAS mutations. In the era of predictive molecular testing, collision may be an under-recognized, albeit rare, cause for aberrant driver mutation profiles in lung adenocarcinomas. Specifically, while there is strong evidence that major driver mutations in EGFR and KRAS are distributed homogeneously throughout individual tumors, there is a number of reports to the contrary. One explanation for such findings has been geographic variability in mutant to wild-type allele ratio resulting from variable amplification of the mutant allele and/or variable admixture of non-neoplastic cells, leading to inconsistent detection of mutations (i.e. mutation ‘pseudo-heterogeneity’).4,5 This case illustrates another potential mechanism for mutation ‘pseudo-heterogeneity’. In our case, random microdissection with a presumption that all dissected samples were derived from a single tumor would have resulted in the impression of intra-tumoral heterogeneity of KRAS mutations. Alternatively, molecular testing of the conglomerate nodule in the absence of microdissection would have resulted in ‘pseudo-multiplicity’ of KRAS G12A and L19F/T20S mutations. In contradistinction, the L19F/T20S double-mutation illustrates a rare example of true functionally-significant mutation multiplicity. In summary, this case illustrates unsuspected collision as a previously unreported potential cause of aberrant molecular findings in lung adenocarcinomas, and highlights the importance of careful histologic examination of tissue submitted for molecular testing.

Melanin-concentrating hormone (MCH) is a cyclic peptide involved in the regulation of food-intake behaviour and stress response in mammals. Expression of the MCH gene predominates in hypothalamic neurons. Mechanisms governing the regulation of expression of MCH gene in established cell lines were not explored yet. Here, we analysed the actions of nerve growth factor (NGF), dexamethasone, forskolin and lithium on MCH mRNA levels in the PC12 pheochromocytoma cell line. We compared them with those observed on tyrosine hydroxylase (TH) mRNA, constitutively expressed in PC12 cells, and neurotensin (NT) mRNA, taken as a control. In untreated cells, MCH RNA species of high molecular weight were found. Exposure of cells at a combination of NGF and lithium resulted in decreased expression of these MCH RNAs and in the transient production of mature MCH mRNA. Strikingly, after short exposure of PC12 cells to NGF, lithium per se elicited a marked increase in MCH mRNA levels whilst it exerted a potent inhibitory action on TH mRNA expression. Detailed investigations revealed that lithium enhanced MCH mRNA expression through post-transcriptional mechanisms whereas it regulated TH gene expression mainly at the level of transcription. These results demonstrate that lithium, an agent widely used for treatment of manic depressive illness, can exert an opposite effect on MCH and TH mRNA production in PC12 cells. The MCH gene system in NGF-treated PC12 cells provides a good opportunity for studying the effect of lithium on gene expression at post-transcriptional levels in a neuron-like cellular model.

Lung squamous cell carcinoma is a common type of lung cancer, causing approximately 400,000 deaths per year worldwide. Genomic alterations in squamous cell lung cancers have not been comprehensively characterized, and no molecularly targeted agents have been specifically developed for its treatment. As part of The Cancer Genome Atlas, here we profile 178 lung squamous cell carcinomas to provide a comprehensive landscape of genomic and epigenomic alterations. We show that the tumour type is characterized by complex genomic alterations, with a mean of 360 exonic mutations, 165 genomic rearrangements, and 323 segments of copy number alteration per tumour. We find statistically recurrent mutations in 11 genes, including mutation of TP53 in nearly all specimens. Previously unreported loss-of-function mutations are seen in the HLA-A class I major histocompatibility gene. Significantly altered pathways included NFE2L2 and KEAP1 in 34%, squamous differentiation genes in 44%, phosphatidylinositol-3-OH kinase pathway genes in 47%, and CDKN2A and RB1 in 72% of tumours. We identified a potential therapeutic target in most tumours, offering new avenues of investigation for the treatment of squamous cell lung cancers., National Institutes of Health (U.S.) (Grant U24 CA126561), National Institutes of Health (U.S.) (Grant U24 CA126551), National Institutes of Health (U.S.) (Grant U24 CA126554), National Institutes of Health (U.S.) (Grant U24 CA126543), National Institutes of Health (U.S.) (Grant U24 CA126546), National Institutes of Health (U.S.) (Grant U24 CA126563), National Institutes of Health (U.S.) (Grant U24 CA126544), National Institutes of Health (U.S.) (Grant U24 CA143845), National Institutes of Health (U.S.) (Grant U24 CA143858), National Institutes of Health (U.S.) (Grant U24 CA144025), National Institutes of Health (U.S.) (Grant U24 CA143882), National Institutes of Health (U.S.) (Grant U24 CA143866), National Institutes of Health (U.S.) (Grant U24 CA143867), National Institutes of Health (U.S.) (Grant U24 CA143848), National Institutes of Health (U.S.) (Grant U24 CA143840), National Institutes of Health (U.S.) (Grant U24 CA143835), National Institutes of Health (U.S.) (Grant U24 CA143799), National Institutes of Health (U.S.) (Grant U24 CA143883), National Institutes of Health (U.S.) (Grant U24 CA143843), National Institutes of Health (U.S.) (Grant U54 HG003067), National Institutes of Health (U.S.) (Grant U54 HG003079), National Institutes of Health (U.S.) (Grant U54 HG003273)

EGFR-mutant lung cancers represent a paradigm for the use of tyrosine kinase inhibitors (TKIs) to treat molecular subsets of cancer, with randomized trials demonstrating the efficacy of first-generation, reversible epidermal growth factor receptor (EGFR) TKIs. However, acquired resistance invariably develops, and rebiopsy of patients with clinical progression has elucidated EGFR T790M mutation as the major resistance mechanism.1 To address this clinical challenge, third-generation pyrimidine-based EGFR TKIs have been designed to have selectivity for EGFR T790M over wild-type EGFR.2 They form a covalent bond with cysteine 797 in the adenosine triphosphate–binding cleft. Early-phase clinical trials have demonstrated their efficacy in patients with double-mutant tumors (EGFR L858R/T790M and ex19del/T790M) and acquired resistance to first-generation EGFR inhibitors.3 Specificity for EGFR T790M may be the result of hydrophobic interactions between the bulky methionine moiety of EGFR T790M and these pyrimidine-based drugs. Mechanisms of resistance to these third-generation EGFR TKIs have been identified in preclinical in vitro models, but none have so far been identified in patients. Mutations at the EGFR C797 codon, located within the kinase-binding site, are a predicted resistance mechanism to irreversible EGFR inhibitors also confirmed in vitro.4 Loss of the potential for covalent bond formation at position 797 by the missense mutation C797S results in a markedly reduced cellular potency of this class of TKIs.5 Interestingly, an analogous cysteine-to-serine mutation is a mechanism of resistance to the irreversible Bruton tyrosine kinase inhibitor ibrutinib in patients with chronic lymphocytic leukemia.6

Molecular and genetic evidence suggests that DNA repair pathways may contribute to lymphoma susceptibility. Several studies have examined the association of DNA repair genes with lymphoma risk, but the findings from these reports have been inconsistent. Here we provide the results of a focused analysis of genetic variation in DNA repair genes and their association with the risk of non-Hodgkin's lymphoma (NHL). With a population of 1,297 NHL cases and 1,946 controls, we have performed a two-stage case/control association analysis of 446 single nucleotide polymorphisms (SNPs) tagging the genetic variation in 81 DNA repair genes. We found the most significant association with NHL risk in the ATM locus for rs227060 (OR = 1.27, 95% CI: 1.13-1.43, p = 6.77×10(-5)), which remained significant after adjustment for multiple testing. In a subtype-specific analysis, associations were also observed for the ATM locus among both diffuse large B-cell lymphomas (DLBCL) and small lymphocytic lymphomas (SLL), however there was no association observed among follicular lymphomas (FL). In addition, our study provides suggestive evidence of an interaction between SNPs in MRE11A and NBS1 associated with NHL risk (OR = 0.51, 95% CI: 0.34-0.77, p = 0.0002). Finally, an imputation analysis using the 1,000 Genomes Project data combined with a functional prediction analysis revealed the presence of biologically relevant variants that correlate with the observed association signals. While the findings generated here warrant independent validation, the results of our large study suggest that ATM may be a novel locus associated with the risk of multiple subtypes of NHL.

e18542 Background: Somatic BAP1 mutations are found in nearly a quarter of MM tumors and germline BAP1 mutations define a new cancer syndrome characterized by familial mesothelioma, uveal melanoma,...

Melanin-concentrating hormone (MCH) mRNA expression is induced by nerve growth factor and lithium in PC12 cells, whereas three large MCH RNA species are found in untreated cells. In this study, we investigated the structures, regulations of expression, and putative functions of these transcripts. Northern blot, rapid amplification of cDNA ends-polymerase chain reaction, reverse transcriptase-polymerase chain reaction, and sequencing experiments demonstrated that they are antisense RNAs complementary to the MCH gene. Two classes of antisense RNAs could be discriminated as follows: 1) non-coding unspliced RNAs that overlap mainly the coding part of the MCH gene; 2) spliced variant mRNAs complementary to the 3'-flanking end of the MCH gene and that encode putative proteins containing DNA/RNA binding domains. We named this new transcriptional unit AROM for antisense-RNA-overlapping-MCH gene. Spliced variant AROM mRNAs are expressed in a broad range of rat organs. Western blot and immunohistochemistry experiments revealed several proteins with cytoplasmic but also nuclear localization in PC12 cells. Time course studies during nerve growth factor and lithium treatment of PC12 cells indicated a reciprocal regulation of the MCH and AROM gene transcripts, reflected also at the level of AROM proteins. The major translational product is a 64-kDa protein (AROM-p64). Recombinant AROM-p64 displayed high binding to single-stranded DNA and poly(A) homopolymers suggesting that this protein could play a role in mRNA maturation/metabolism.

7120 Background: There is a high frequency of activating mutations in proteins of the Ras/Raf/MEK/ERK pathway in melanoma and other solid tumors. The BRAF V600E mutation has been found recently to be highly prevalent in ECD, a rare non-Langerhans cell histiocytosis with poor prognosis, as well as Langerhans cell histiocytosis (LCH). Treatment of patients with ECD and the BRAF V600E mutation with vemurafenib has been associated with unprecedented and dramatic response in a few cases. Methods: We present a 66 year-old man evaluated for several months of cognitive and motor decline. He was found to have multifocal enhancing lesions in the cerebral meninges, multiple masses in the abdomen and sacrum, and abnormal nuclear uptake in the long bones of the legs. Results: Biopsy of a renal mass demonstrated a foamy CD68+/CD1a- histiocytic infiltrate, consistent with ECD. Interrogation of tumor tissue with the Sequenom MassArray system demonstrated absence of the BRAF V600E mutation but presence of the NRAS Q61R mutation. Conclusions: This is, to our knowledge, the first report of an activating NRAS mutation in ECD. The finding of an oncogenic NRAS mutation in ECD further supports the hypothesis that this disease is driven by activation of the Ras/Raf/MEK/ERK pathway. Further investigation into the role of this pathway in ECD and LCH is warranted and may open new opportunities for targeted therapies for these disorders.

10524 Background: As more therapies targeting genomic alterations become available, genotyping (e.g., by Sequenom) is increasingly performed in tumor types where mutational status may drive treatment choice. Next-generation sequencing (NGS) technology can expand on genotyping of individual base pairs because it can detect mutations across entire exons, copy changes and fusion genes. However, for NGS to be clinically viable, it must be made compatible with FFPE tissues and shown concordant with best current diagnostic methods. Methods: To explore a potential clinical role for NGS, we selected 120 FFPE specimens (68 NSCLC, 32 CRC, 20 melanoma) previously tested for 97 oncogenic mutations in 8 oncogenes by Sequenom, and sequenced all exons of 182 cancer genes. Genotyping and sequencing were both performed in CLIA compliant labs. DNA was extracted from 4x 10μ unstained sections from the diagnostic FFPE block, followed by library construction and hybridization capture of 3230 exons and 37 commonly rearranged introns. Average coverage of >900X uniquely-mapping reads was obtained. Sequence data were analyzed for all genomic alterations and examined for potentially actionable mutations. Results: High concordance was noted between Sequenom and NGS: 103 and 105 mutations were called by the two technologies, respectively, at mutually tested sites, with 97 mutation calls in common. Notably, mutant allele frequencies in concordant calls ranged as low as 2% by NGS, highlighting the sensitivity of detection enabled by both approaches. Furthermore, in 45/120 (38%) specimens, NGS revealed additional alterations that may confer sensitivity or resistance to approved or experimental targeted therapies and thus plausibly influence treatment decisions. These included 39 copy changes or loss-of-function variants best assayed by an NGS approach. Conclusions: Our results demonstrate technical feasibility and highlight potential benefits of comprehensive cancer gene characterization through next-generation sequencing of clinical FFPE specimens. As NGS is the most practical means to detect all classes of somatic alteration in a small, clinically relevant sample, we suggest that this type of testing will become an essential component of patient care.

As genomic data accumulate at an ever-increasing rate, it is becoming evident that a comprehensive description of molecular aberrations that define individual patients’ tumors will prove useful to determine optimal therapeutic strategies. Several platforms are currently in use for clinical molecular diagnostics testing, including PCR, Sanger sequencing, restriction fragment length polymorphism analysis, and mass spectrometric genotyping (Sequenom). Massively parallel sequencing (MPS) technology has the potential to expand upon Sequenom genotyping because it allows for the identification of mutations across entire exons (rather than single base pairs) as well as copy gains, losses and gene fusions. However, for MPS to be considered a viable clinical strategy, it must first be shown to be compatible with formalin-fixed, paraffin embedded (FFPE) tissue across a range of tumor types, sizes, and cellularities. Also, it must exhibit high concordance with mutation profiles derived using the current best diagnostic methods available. To explore the clinical utility of MPS, we selected 71 surgically resected FFPE tumors that had previously been tested for approximately 100 oncogenic mutations in 8 oncogenes by Sequenom genotyping and subjected them to targeted DNA sequencing of 189 cancer-related genes. DNA was extracted from four 10-micron unstained sections from the diagnostic FFPE block (yielding a minimum of 250 nanograms per case), followed by sequencing library construction and hybridization-based capture of 3230 exons and 37 intronic intervals. Deep sequencing was performed, yielding an average coverage of >750X for uniquely-mapping reads. Sequence data were analyzed for single nucleotide variants and small insertions and deletions. High concordance was noted between Sequenom and MPS: 62 and 65 mutations were called by the two technologies, respectively, at mutually tested sites, with 60 mutation calls in common. Notably, mutant allele frequencies in these concordant calls ranged as low as 4% by MPS, highlighting the sensitivity of detection enabled by both approaches. The few discordant mutation calls exhibited no or weak evidence in the other dataset, possibly due to local tumor heterogeneity. Further, MPS revealed 73 sequence variants at additional sites of known recurrent somatic mutations and 30 loss-of-function variants in key tumor suppressor genes not tested by Sequenom. Many of these variants represent plausibly actionable mutations that could influence treatment decisions. Thus, we conclude that: (1) MPS exhibits high concordance with current best methods and is a viable strategy for clinical diagnostics, and (2) MPS captures additional variants not typically interrogated in the current clinical setting but with potential implications for the selection of approved and/or experimental targeted therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 965. doi:1538-7445.AM2012-965

8033 Background: KRAS mutations define the largest subset of oncogene-driven lung adenocarcinomas (25-30%). Among patients with KRAS mutations, their clinical course during treatment is heterogeneo...