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PURPOSE Mitogen-activated protein kinase pathway–activating mutations occur in the majority of colorectal cancer (CRC) cases and show mutual exclusivity. We identified 47 epidermal growth factor receptor/BRAF inhibitor-naive CRC patients with dual RAS hotspot/ BRAF V600E mutations (CRC-DD) from a cohort of 4,561 CRC patients with clinical next-generation sequencing results. We aimed to define the molecular phenotypes of the CRC-DD and to test if the dual RAS hotspot/BRAF V600E mutations coexist within the same cell. MATERIALS AND METHODS We developed a single-cell genotyping method with a mutation detection rate of 96.3% and a genotype prediction accuracy of 92.1%. Mutations in the CRC-DD cohort were analyzed for clonality, allelic imbalance, copy number, and overall survival. RESULTS Application of single-cell genotyping to four CRC-DD revealed the co-occurrence of both mutations in the following percentages of cells per case: NRAS G13D/ KRAS G12C, 95%; KRAS G12D/ NRAS G12V, 48%; BRAF V600E/ KRAS G12D, 44%; and KRAS G12D/ NRAS G13V, 14%, respectively. Allelic imbalance favoring the oncogenic allele was less frequent in CRC-DD (24 of 76, 31.5%, somatic mutations) compared with a curated cohort of CRC with a single-driver mutation (CRC-SD; 119 of 232 mutations, 51.3%; P = .013). Microsatellite instability–high status was enriched in CRC-DD compared with CRC-SD (23% v 11.4%, P = .028). Of the seven CRC-DD cases with multiregional sequencing, five retained both driver mutations throughout all sequenced tumor sites. Both CRC-DD cases with discordant multiregional sequencing were microsatellite instability–high. CONCLUSION Our findings indicate that dual-driver mutations occur in a rare subset of CRC, often within the same tumor cells and across multiple tumor sites. Their presence and a lower rate of allelic imbalance may be related to dose-dependent signaling within the mitogen-activated protein kinase pathway.

Purpose:Invasive mucinous adenocarcinoma (IMA) is a unique subtype of lung adenocarcinoma, characterized genomically by frequent KRAS mutations or specific gene fusions, most commonly involving NRG1. Comprehensive analysis of a large series of IMAs using broad DNA- and RNA-sequencing methods is still lacking, and it remains unclear whether molecular subtypes of IMA differ clinicopathologically.Experimental Design:A total of 200 IMAs were analyzed by 410-gene DNA next-generation sequencing (MSK-IMPACT; n = 136) or hotspot 8-oncogene genotyping (n = 64). Driver-negative cases were further analyzed by 62-gene RNA sequencing (MSK-Fusion) and those lacking fusions were further tested by whole-exome sequencing and whole-transcriptome sequencing (WTS).Results:Combined MSK-IMPACT and MSK-Fusion testing identified mutually exclusive driver alterations in 96% of IMAs, including KRAS mutations (76%), NRG1 fusions (7%), ERBB2 alterations (6%), and other less common events. In addition, WTS identified a novel NRG2 fusion (F11R–NRG2). Overall, targetable gene fusions were identified in 51% of KRAS wild-type IMAs, leading to durable responses to targeted therapy in some patients. Compared with KRAS-mutant IMAs, NRG1-rearranged tumors exhibited several more aggressive characteristics, including worse recurrence-free survival (P < 0.0001).Conclusions:This is the largest molecular study of IMAs to date, where we demonstrate the presence of a major oncogenic driver in nearly all cases. This study is the first to document more aggressive characteristics of NRG1-rearranged IMAs, ERBB2 as the third most common alteration, and a novel NRG2 fusion in these tumors. Comprehensive molecular testing of KRAS wild-type IMAs that includes fusion testing is essential, given the high prevalence of alterations with established and investigational targeted therapies in this subset.

Background: Lung cancer is a major cause of death in HIV-infected (HIV+) persons. In this study, we compared the prevalence of tumour EGFR and KRAS mutations in a cohort of lung adenocarcinoma patients by HIV status. Methods: We collected data from 55 HIV+ patients with lung adenocarcinoma matched to 136 uninfected comparators. We compared the prevalence of EGFR and KRAS mutations by HIV status. We then compared survival by HIV status and by cancer mutation status among HIV+ subjects. Results: Presence of KRAS and EGFR genetic alterations did not vary by HIV status (all P>0.1). There was no difference in overall survival by HIV status or by mutation status among HIV+ subjects. Conclusions: We found no major differences in the prevalence of EGFR or KRAS lung adenocarcinoma mutations by HIV status, suggesting that mutational testing should be conducted similarly regardless of the HIV status.

Introduction Half of inflammatory myofibroblastic tumors (IMTs) regardless of anatomic location harbor anaplastic lymphoma kinase gene (ALK) rearrangements and overexpress anaplastic lymphoma kinase protein. The wide application of next-generation sequencing and the clinical benefit to tyrosine kinase inhibitors have opened new opportunities for investigation of ALK-negative IMTs. Methods In this study, we have investigated a series of pediatric and adult thoracic IMTs for abnormalities in a wide spectrum of actionable kinases by applying a variety of molecular and next-generation sequencing techniques, including fluorescence in situ hybridization (FISH), targeted RNA sequencing, and NanoString assay. Results There were 33 patients with thoracic IMTs, including five children; their mean age was 37. The tumors showed a monomorphic spindle cell phenotype, except for one with an epithelioid morphologic pattern and moderate to severe atypia. By immunohistochemistry, 24 tumors were ALK positive, and 19 of the 24 showed ALK rearrangements and one ret proto-oncogene gene (RET) rearrangement by FISH. RNA sequencing was performed in the remaining four cases lacking ALK abnormalities by FISH, revealing ALK fusions involving tropomyosin 4 gene (TMP4) and echinoderm microtubule associated protein like 4 gene (EML4) as partner in three cases. NanoString assay was performed in the remaining case, revealing ALK alternative transcription initiation (ALKATI). Nine cases lacking ALK abnormalities were further tested by FISH or targeted RNA sequencing, revealing ROS1 rearrangement in six cases and ETS variant 6 gene (ETV6)–neurotrophic receptor tyrosine kinase 3 gene (NTRK3) fusion in three cases, respectively. Conclusions By using a battery of complementary molecular techniques, we have shown that all the thoracic IMTs harbored a tyrosine kinase abnormality, with 30% involving a kinase gene other than ALK, including ROS1, NTRK3, and RET gene fusions. We have also described for the first time ALKATI-induced ALK oncogenic activation in IMTs.

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.

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.

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.

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)