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1.  Application of next generation sequencing to human gene fusion detection: computational tools, features and perspectives 
Briefings in Bioinformatics  2012;14(4):506-519.
Gene fusions are important genomic events in human cancer because their fusion gene products can drive the development of cancer and thus are potential prognostic tools or therapeutic targets in anti-cancer treatment. Major advancements have been made in computational approaches for fusion gene discovery over the past 3 years due to improvements and widespread applications of high-throughput next generation sequencing (NGS) technologies. To identify fusions from NGS data, existing methods typically leverage the strengths of both sequencing technologies and computational strategies. In this article, we review the NGS and computational features of existing methods for fusion gene detection and suggest directions for future development.
PMCID: PMC3713712  PMID: 22877769
gene fusion; next generation sequencing; cancer; whole genome sequencing; transcriptome sequencing; computational tools
2.  Discovery of a mutant-selective covalent inhibitor of EGFR that overcomes T790M-mediated resistance in NSCLC 
Cancer discovery  2013;3(12):1404-1415.
Non-small cell lung cancer (NSCLC) patients with activating epidermal growth factor receptor (EGFR) mutations initially respond to first generation reversible EGFR tyrosine kinase inhibitors. However, clinical efficacy is limited by acquired resistance, frequently driven by the EGFR T790M mutation. CO-1686 is a novel, irreversible and orally delivered kinase inhibitor that specifically targets the mutant forms of EGFR including T790M while exhibiting minimal activity towards the wild-type (WT) receptor. Oral administration of CO-1686 as single agent induces tumor regression in EGFR mutated NSCLC tumor xenograft and transgenic models. Minimal activity of CO-1686 against the WT EGFR receptor was observed. In NSCLC cells with acquired resistance to CO-1686 in vitro, there was no evidence of additional mutations or amplification of the EGFR gene, but resistant cells exhibited signs of epithelial-mesenchymal transition (EMT) and demonstrated increased sensitivity to AKT inhibitors. These results suggest CO-1686 may offer a novel therapeutic option for patients with mutant EGFR NSCLC.
PMCID: PMC4048995  PMID: 24065731
NSCLC; EGFR; drug resistance; T790M; EMT
3.  Phase II Trial of Gefitinib and Everolimus in Advanced Non-small Cell Lung Cancer 
Concurrent signal transduction inhibition with the epidermal growth factor receptor (EGFR) inhibitor gefitinib and the mammalian target-of-rapamycin inhibitor everolimus has been hypothesized to result in enhanced antitumor activity in patients with non-small cell lung cancer (NSCLC). This phase II trial assessed the efficacy of the combination of gefitinib and everolimus in patients with advanced NSCLC.
Two cohorts of 31 patients with measurable stage IIIB/IV NSCLC were enrolled: (1) no prior chemotherapy and (2) previously treated with cisplatin or carboplatin and docetaxel or pemetrexed. All patients received daily everolimus 5 mg and gefitinib 250 mg. Response was assessed after 1 month and then every 2 months. Pretreatment tumor specimens were collected for mutation testing.
Sixty-two patients were enrolled (median age: 66 years, 50% women, 98% stage IV, all current/former smokers, and 85% adenocarcinoma). Partial responses were seen in 8 of 62 patients (response rate: 13%; 95% confidence interval: 5–21%); five responders had received no prior chemotherapy. Three partial responders had an EGFR mutation. Both patients with a KRAS (G12F) mutation responded. The median time to progression was 4 months. Median overall survival was 12 months, 27 months for no prior chemotherapy patients, and 11 months for patients previously treated with chemotherapy.
The 13% partial response rate observed did not meet the prespecified response threshold to pursue further study of the combination of gefitinib and everolimus. The response rate in patients with non-EGFR mutant tumors was 8%, likely reflecting activity of everolimus. Further investigation of mammalian target-of-rapamycin inhibitors in patients with NSCLC with KRAS G12F-mutated tumors is warranted.
PMCID: PMC4020424  PMID: 20871262
Non-small cell lung cancer; Gefitinib; Everolimus.
4.  Morphologic Features of Adenocarcinoma of the Lung Predictive of Response to the Epidermal Growth Factor Receptor Kinase Inhibitors Erlotinib and Gefitinib 
A subset of lung adenocarcinomas appears preferentially sensitive to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). EGFR-activating mutations and never smoking are associated with response to TKIs.
To describe the morphology of adenocarcinomas responsive to TKIs, compare it to tumors in nonresponding patients, and correlate findings with EGFR mutations, gene copy number, and protein expression.
Material from 52 EGFR TKI-treated patients was studied: 29 responders and 23 nonresponders. Adenocarcinoma subtypes and morphologic features were defined in histologic and cytologic material. EGFR mutations were detected by sequencing, copy number by chromogenic in situ hybridization, and expression by immunohistochemistry.
Tumors from TKI responders tended to be better-differentiated adenocarcinomas with bronchioloalveolar carcinoma components. Nonresponders showed more heterogeneous morphology, higher grade, and more subtypes, and were more likely to show solid growth. In nonresponders, the only pure bronchioloalveolar carcinoma was mucinous, a subtype known to be negative for EGFR mutations. Using World Health Organization criteria, all tumors in both groups other than pure bronchioloalveolar carcinomas would be classified as adenocarcinomas, mixed subtype, thereby obscuring some of these distinctions. EGFR mutations were significantly more common in responders (22/29 vs 0/23; P < .001). Immunohistochemistry and chromogenic in situ hybridization results were not significantly correlated with EGFR mutations or response to TKIs in this study.
Overall, histologic differences exist between tumors that respond to TKIs and those that do not, although sampling affects classification, and there is significant histologic overlap between the 2 groups. Response is strongly associated with EGFR mutations.
PMCID: PMC4016915  PMID: 19260752
5.  Spectrum of LKB1, EGFR, and KRAS Mutations in Chinese Lung Adenocarcinomas 
Somatic LKB1 mutations are found in lung adenocarcinomas at different frequencies in Caucasian and East Asian (Japanese and Korean) populations. This study was designed to characterize the frequency of LKB1 mutations, their relationship to EGFR and KRAS mutations, and their associated clinicopathologic characteristics in Chinese patients.
Two hundred thirty-nine lung adenocarcinomas consecutively collected from October 2007 to July 2009 were dissected into 3 to 4 small (3 mm) pieces for histopathological analyses of tumor content. Genomic DNA and/or cDNA from 86 samples with more than 70% tumor content were used for sequencing of LKB1 (exons 1–9), EGFR (exons 18–21), and KRAS (exon 2). LKB1 germline mutation status was determined by sequencing of genomic DNA from matched histologically distant lung tissues that are histologically normal.
6.9% of lung adenocarcinomas harbored LKB1 somatic mutations. A total of 10.5% of patients had an LKB1 germline polymorphism, F354L. Interestingly, in two of these patients, tumors displayed loss of heterozygosity at this allele. EGFR kinase domain and KRAS mutations were found in 66.3% and 2.3% of Chinese lung adenocarcinomas, respectively. Concurrent LKB1 and EGFR somatic mutations were observed in one patient. Both KRAS-mutant tumors harbored LKB1 mutations.
These data provide important clinical and molecular characteristics of lung adenocarcinomas from Chinese patients.
PMCID: PMC4009449  PMID: 20559149
Chinese lung adenocarcinoma; LKB1; EGFR; KRAS; Mutation
7.  Somatic mutations of the Parkinson's disease–associated gene PARK2 in glioblastoma and other human malignancies 
Nature genetics  2009;42(1):77-82.
Mutation of the gene PARK2, which encodes an E3 ubiquitin ligase, is the most common cause of early-onset Parkinson's disease1, 2, 3. In a search for multisite tumor suppressors, we identified PARK2 as a frequently targeted gene on chromosome 6q25.2–q27 in cancer. Here we describe inactivating somatic mutations and frequent intragenic deletions of PARK2 in human malignancies. The PARK2 mutations in cancer occur in the same domains, and sometimes at the same residues, as the germline mutations causing familial Parkinson's disease. Cancer-specific mutations abrogate the growth-suppressive effects of the PARK2 protein. PARK2 mutations in cancer decrease PARK2's E3 ligase activity, compromising its ability to ubiquitinate cyclin E and resulting in mitotic instability. These data strongly point to PARK2 as a tumor suppressor on 6q25.2–q27. Thus, PARK2, a gene that causes neuronal dysfunction when mutated in the germline, may instead contribute to oncogenesis when altered in non-neuronal somatic cells.
PMCID: PMC4002225  PMID: 19946270
9.  Analysis of Tumor Specimens at the Time of Acquired Resistance to EGFR TKI therapy in 155 patients with EGFR mutant Lung Cancers 
All patients with EGFR mutant lung cancers eventually develop acquired resistance to EGFR tyrosine kinase inhibitors (TKIs). Smaller series have identified various mechanisms of resistance, but systematic evaluation of a large number of patients to definitively establish the frequency of various mechanisms has not been performed.
Experimental Design
Patients with lung adenocarcinomas and acquired resistance to erlotinib or gefitinib enrolled onto a prospective biopsy protocol and underwent a re-biopsy after the development of acquired resistance. Histology was reviewed. Samples underwent genotyping for mutations in EGFR, AKT1, BRAF, ERBB2, KRAS, MEK1, NRAS and PIK3CA, and FISH for MET and HER2.
Adequate tumor samples for molecular analysis were obtained in 155 patients. Ninety-eight had second-site EGFR T790M mutations (63%, 95% CI 55-70%) and four had small cell transformation (3%, 95% CI 0-6%). MET amplification was seen in 4/75 (5%, 95% CI 1-13%). HER2 amplification was seen in 3/24 (13%, 95% CI 3-32%). We did not detect any acquired mutations in PIK3CA, AKT1, BRAF, ERBB2, KRAS, MEK1, or NRAS. (0/88, 0%, 95% CI 0-4%). Overlap among mechanisms of acquired resistance was seen in 4%.
This is the largest series reporting mechanisms of acquired resistance to EGFR TKI therapy. We identified EGFR T790M as the most common mechanism of acquired resistance, while MET amplification, HER2 amplification, and small cell histologic transformation occur less frequently. More comprehensive methods to characterize molecular alterations in this setting are needed to improve our understanding of acquired resistance to EGFR TKIs.
PMCID: PMC3630270  PMID: 23470965
EGFR mutant lung cancer; lung adenocarcinoma; targeted therapy; acquired resistance; tyrosine kinase inhibitor therapy
10.  Two co-existing germline mutations P53 V157D and PMS2 R20Q promote tumorigenesis in a familial cancer syndrome 
Cancer letters  2013;342(1):36-42.
Germline mutations are responsible for familial cancer syndromes which account for approximately 5–10% of all types of cancers. These mutations mainly occur at tumor suppressor genes or genome stability genes, such as DNA repair genes. Here we have identified a cancer predisposition family, in which eight members were inflicted with a wide spectrum of cancer including one diagnosed with lung cancer at 22 years old. Sequencing analysis of tumor samples as well as histologically normal specimens identified two germline mutations co-existing in the familial cancer syndrome, the mutation of tumor suppressor gene P53 V157D and mismatch repair gene PMS2 R20Q. We further demonstrate that P53 V157D and/or PMS2 R20Q mutant promotes lung cancer cell proliferation. These two mutants are capable of promoting colony formation in soft agar as well as tumor formation in transgenic drosophila system. Collectively, these data have uncovered the important role of co-existing germline P53 and PMS2 mutations in the familial cancer syndrome development.
PMCID: PMC3981830  PMID: 23981578
P53 V157D; PMS2 R20Q; Germline mutation; Familial cancer syndrome; Co-existing
11.  High dose weekly erlotinib achieves therapeutic concentrations in CSF and is effective in leptomeningeal metastases from epidermal growth factor receptor mutant lung cancer 
Journal of neuro-oncology  2010;99(2):283-286.
Leptomeningeal metastases (LM) occur in 5–10% of patients with solid tumors and are associated with a dismal prognosis. We describe LM from lung adenocarcinoma harboring a mutation in the epidermal growth factor receptor (EGFR) gene that confers sensitivity to the EGFR tyrosine kinase inhibitors (EGFR-TKIs) erlotinib and gefitinib. The CSF concentration of EGFR-TKIs achieved by standard daily dosing may be insufficient for therapeutic effect. However, intermittent (pulsatile) high dose administration (1000–1500 mg/week) achieves a higher CSF concentration than standard dosing, and successfully controlled LM in this patient.
PMCID: PMC3973736  PMID: 20146086
Leptomeningeal metastases; EGFR; Lung cancer; Erlotinib
12.  Association of KRAS and EGFR Mutations with Survival in Patients with Advanced Lung Adenocarcinomas 
Cancer  2012;119(2):356-362.
Lung adenocarcinomas can be distinguished by identifying mutated driver oncogenes including EGFR and KRAS. Mutations in EGFR are associated with both an improved survival as well as response to treatment with erlotinib and gefitinib. However, the prognostic significance of KRAS has not been evaluated in large numbers of patients and remains controversial. We examined the association of EGFR and KRAS mutations with survival among patients with advanced lung adenocarcinomas.
We analyzed data from patients with advanced lung adenocarcinomas and known EGFR and KRAS mutation status evaluated between 2002 and 2009. We collected clinical variables including age, gender, Karnofsky Performance Status, smoking history, and treatment history. Overall survival from diagnosis of advanced disease was analyzed using Kaplan-Meier and Cox proportional hazard methods.
We evaluated 1036 patients, including 610 women (59%) and 344 never-smokers (33%). Patients had a median age of 65 (range, 25–92) and the majority (81%) had a KPS ≥80%. In multivariate analysis, EGFR mutations were associated with a longer overall survival (HR= 0.6, p<0.001) and KRAS mutations with a shorter survival (HR=1.21, p=0.048).
KRAS mutations predict shorter survival for patients with advanced lung adenocarcinomas. The presence of EGFR and KRAS mutations define distinct subsets of patients with lung adenocarcinomas, and should be determined in patients upon diagnosis of advanced disease. Clinical trial reports should include EGFR and KRAS mutation status along with other prognostic factors.
PMCID: PMC3966555  PMID: 22810899
non-small cell lung cancer; adenocarcinomas; EGFR; KRAS; survival; prognostic factors
13.  Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor–Resistant Disease 
Journal of Clinical Oncology  2013;31(8):1070-1080.
EGFR-mutant lung cancer was first described as a new clinical entity in 2004. Here, we present an update on new controversies and conclusions regarding the disease.
This article reviews the clinical implications of EGFR mutations in lung cancer with a focus on epidermal growth factor receptor tyrosine kinase inhibitor resistance.
The discovery of EGFR mutations has altered the ways in which we consider and treat non–small-cell lung cancer (NSCLC). Patients whose metastatic tumors harbor EGFR mutations are expected to live longer than 2 years, more than double the previous survival rates for lung cancer.
The information presented in this review can guide practitioners and help them inform their patients about EGFR mutations and their impact on the treatment of NSCLC. Efforts should now concentrate on making EGFR-mutant lung cancer a chronic rather than fatal disease.
PMCID: PMC3589701  PMID: 23401451
14.  Patterns and processes of somatic mutations in nine major cancers 
BMC Medical Genomics  2014;7:11.
Cancer genomes harbor hundreds to thousands of somatic nonsynonymous mutations. DNA damage and deficiency of DNA repair systems are two major forces to cause somatic mutations, marking cancer genomes with specific somatic mutation patterns. Recently, several pan-cancer genome studies revealed more than 20 mutation signatures across multiple cancer types. However, detailed cancer-type specific mutation signatures and their different features within (intra-) and between (inter-) cancer types remain largely unexplored.
We employed a matrix decomposition algorithm, namely Non-negative Matrix Factorization, to survey the somatic mutations in nine major human cancers, involving a total of ~2100 genomes.
Our results revealed 3-5 independent mutational signatures in each cancer, implying that a range of 3-5 predominant mutational processes likely underlie each cancer genome. Both mutagen exposure (tobacco and sun) and changes in DNA repair systems (APOBEC family, POLE, and MLH1) were found as mutagenesis forces, each of which marks the genome with an evident mutational signature. We studied the features of several signatures and their combinatory patterns within and across cancers. On one hand, we found each signature may influence a cancer genome with different influential magnitudes even in the same cancer type and the signature-specific load reflects intra-cancer heterogeneity (e.g., the smoking-related signature in lung cancer smokers and never smokers). On the other hand, inter-cancer heterogeneity is characterized by combinatory patterns of mutational signatures, where no cancers share the same signature profile, even between two lung cancer subtypes (lung adenocarcinoma and squamous cell lung cancer).
Our work provides a detailed overview of the mutational characteristics in each of nine major cancers and highlights that the mutational signature profile is representative of each cancer.
PMCID: PMC3942057  PMID: 24552141
Somatic mutation; Cancer; Kataegis; Mutation signature; Mutagen; Heterogeneity
15.  Role of DNA Methylation and Epigenetic Silencing of HAND2 in Endometrial Cancer Development 
PLoS Medicine  2013;10(11):e1001551.
TB filled in by Laureen
Please see later in the article for the Editors' Summary
Endometrial cancer incidence is continuing to rise in the wake of the current ageing and obesity epidemics. Much of the risk for endometrial cancer development is influenced by the environment and lifestyle. Accumulating evidence suggests that the epigenome serves as the interface between the genome and the environment and that hypermethylation of stem cell polycomb group target genes is an epigenetic hallmark of cancer. The objective of this study was to determine the functional role of epigenetic factors in endometrial cancer development.
Methods and Findings
Epigenome-wide methylation analysis of >27,000 CpG sites in endometrial cancer tissue samples (n = 64) and control samples (n = 23) revealed that HAND2 (a gene encoding a transcription factor expressed in the endometrial stroma) is one of the most commonly hypermethylated and silenced genes in endometrial cancer. A novel integrative epigenome-transcriptome-interactome analysis further revealed that HAND2 is the hub of the most highly ranked differential methylation hotspot in endometrial cancer. These findings were validated using candidate gene methylation analysis in multiple clinical sample sets of tissue samples from a total of 272 additional women. Increased HAND2 methylation was a feature of premalignant endometrial lesions and was seen to parallel a decrease in RNA and protein levels. Furthermore, women with high endometrial HAND2 methylation in their premalignant lesions were less likely to respond to progesterone treatment. HAND2 methylation analysis of endometrial secretions collected using high vaginal swabs taken from women with postmenopausal bleeding specifically identified those patients with early stage endometrial cancer with both high sensitivity and high specificity (receiver operating characteristics area under the curve = 0.91 for stage 1A and 0.97 for higher than stage 1A). Finally, mice harbouring a Hand2 knock-out specifically in their endometrium were shown to develop precancerous endometrial lesions with increasing age, and these lesions also demonstrated a lack of PTEN expression.
HAND2 methylation is a common and crucial molecular alteration in endometrial cancer that could potentially be employed as a biomarker for early detection of endometrial cancer and as a predictor of treatment response. The true clinical utility of HAND2 DNA methylation, however, requires further validation in prospective studies.
Please see later in the article for the Editors' Summary
Editors' Summary
Cancer, which is responsible for 13% of global deaths, can develop anywhere in the body, but all cancers are characterized by uncontrolled cell growth and reduced cellular differentiation (the process by which unspecialized cells such as “stem” cells become specialized during development, tissue repair, and normal cell turnover). Genetic alterations—changes in the sequence of nucleotides (DNA's building blocks) in specific genes—are required for this cellular transformation and subsequent cancer development (carcinogenesis). However, recent evidence suggests that epigenetic modifications—reversible, heritable changes in gene function that occur in the absence of nucleotide sequence changes—may also be involved in carcinogenesis. For example, the addition of methyl groups to a set of genes called stem cell polycomb group target genes (PCGTs; polycomb genes control the expression of their target genes by modifying their DNA or associated proteins) is one of the earliest molecular changes in human cancer development, and increasing evidence suggests that hypermethylation of PCGTs is an epigenetic hallmark of cancer.
Why Was This Study Done?
The methylation of PCGTs, which is triggered by age and by environmental factors that are associated with cancer development, reduces cellular differentiation and leads to the accumulation of undifferentiated cells that are susceptible to cancer development. It is unclear, however, whether epigenetic modifications have a causal role in carcinogenesis. Here, the researchers investigate the involvement of epigenetic factors in the development of endometrial (womb) cancer. The risk of endometrial cancer (which affects nearly 50,000 women annually in the United States) is largely determined by environmental and lifestyle factors. Specifically, the risk of this cancer is increased in women in whom estrogen (a hormone that drives cell proliferation in the endometrium) is functionally dominant over progesterone (a hormone that inhibits endometrial proliferation and causes cell differentiation); obese women and women who have taken estrogen-only hormone replacement therapies fall into this category. Thus, endometrial cancer is an ideal model in which to study whether epigenetic mechanisms underlie carcinogenesis.
What Did the Researchers Do and Find?
The researchers collected data on genome-wide DNA methylation at cytosine- and guanine-rich sites in endometrial cancers and normal endometrium and integrated this information with the human interactome and transcriptome (all the physical interactions between proteins and all the genes expressed, respectively, in a cell) using an algorithm called Functional Epigenetic Modules (FEM). This analysis identified HAND2 as the hub of the most highly ranked differential methylation hotspot in endometrial cancer. HAND2 is a progesterone-regulated stem cell PCGT. It encodes a transcription factor that is expressed in the endometrial stroma (the connective tissue that lies below the epithelial cells in which most endometrial cancers develop) and that suppresses the production of the growth factors that mediate the growth-inducing effects of estrogen on the endometrial epithelium. The researchers hypothesized, therefore, that epigenetic deregulation of HAND2 could be a key step in endometrial cancer development. In support of this hypothesis, the researchers report that HAND2 methylation was increased in premalignant endometrial lesions (cancer-prone, abnormal-looking tissue) compared to normal endometrium, and was associated with suppression of HAND2 expression. Moreover, a high level of endometrial HAND2 methylation in premalignant lesions predicted a poor response to progesterone treatment (which stops the growth of some endometrial cancers), and analysis of HAND2 methylation in endometrial secretions collected from women with postmenopausal bleeding (a symptom of endometrial cancer) accurately identified individuals with early stage endometrial cancer. Finally, mice in which the Hand2 gene was specifically deleted in the endometrium developed precancerous endometrial lesions with age.
What Do These Findings Mean?
These and other findings identify HAND2 methylation as a common, key molecular alteration in endometrial cancer. These findings need to be confirmed in more women, and studies are needed to determine the immediate molecular and cellular consequences of HAND2 silencing in endometrial stromal cells. Nevertheless, these results suggest that HAND2 methylation could potentially be used as a biomarker for the early detection of endometrial cancer and for predicting treatment response. More generally, these findings support the idea that methylation of HAND2 (and, by extension, the methylation of other PCGTs) is not a passive epigenetic feature of cancer but is functionally involved in cancer development, and provide a framework for identifying other genes that are epigenetically regulated and functionally important in carcinogenesis.
Additional Information
Please access these websites via the online version of this summary at
The US National Cancer Institute provides information on all aspects of cancer and has detailed information about endometrial cancer for patients and professionals (in English and Spanish)
The not-for-profit organization American Cancer Society provides information on cancer and how it develops and specific information on endometrial cancer (in several languages)
The UK National Health Service Choices website includes an introduction to cancer, a page on endometrial cancer, and a personal story about endometrial cancer
The not-for-profit organization Cancer Research UK provides general information about cancer and specific information about endometrial cancer
Wikipedia has a page on cancer epigenetics (note: Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)
The Eve Appeal charity that supported this research provides useful information on gynecological cancers
PMCID: PMC3825654  PMID: 24265601
16.  Characteristics of Lung Cancers Harboring NRAS Mutations 
We sought to determine the frequency and clinical characteristics of patients with lung cancer harboring NRAS mutations. We used preclinical models to identify targeted therapies likely to be of benefit against NRAS mutant lung cancer cells.
Patients and Methods
We reviewed clinical data from patients whose lung cancers were identified at 6 institutions or reported in the Catalogue of Somatic Mutations in Cancer (COSMIC) to harbor NRAS mutations. 6 NRAS mutant cell lines were screened for sensitivity against inhibitors of multiple kinases (i.e. EGFR, ALK, MET, IGF-1R, BRAF, PI3K and MEK).
Among 4562 patients with lung cancers tested, NRAS mutations were present in 30 (0.7%; 95% confidence interval, 0.45% to 0.94%); 28 of these had no other driver mutations. 83% had adenocarcinoma histology with no significant differences in gender. While 95% of patients were former or current smokers, smoking-related G:C>T:A transversions were significantly less frequent in NRAS mutated lung tumors compared to KRAS-mutant NSCLCs (NRAS: 13% (4/30), KRAS: 66% (1772/2733), p<0.00000001). 5 of 6 NRAS mutant cell lines were sensitive to the MEK inhibitors, selumetinib and trametinib, but not to other inhibitors tested.
NRAS mutations define a distinct subset of lung cancers (~1%) with potential sensitivity to MEK inhibitors. While NRAS mutations are more common in current/former smokers, the types of mutations are not those classically associated with smoking.
PMCID: PMC3643999  PMID: 23515407
NRAS mutation; EGFR mutation; KRAS mutation; lung cancer; non-small cell lung cancer; driver mutation; MEK inhibitor; erlotinib; gefitinib; crizotinib
17.  Detecting somatic point mutations in cancer genome sequencing data: a comparison of mutation callers 
Genome Medicine  2013;5(10):91.
Driven by high throughput next generation sequencing technologies and the pressing need to decipher cancer genomes, computational approaches for detecting somatic single nucleotide variants (sSNVs) have undergone dramatic improvements during the past 2 years. The recently developed tools typically compare a tumor sample directly with a matched normal sample at each variant locus in order to increase the accuracy of sSNV calling. These programs also address the detection of sSNVs at low allele frequencies, allowing for the study of tumor heterogeneity, cancer subclones, and mutation evolution in cancer development.
We used whole genome sequencing (Illumina Genome Analyzer IIx platform) of a melanoma sample and matched blood, whole exome sequencing (Illumina HiSeq 2000 platform) of 18 lung tumor-normal pairs and seven lung cancer cell lines to evaluate six tools for sSNV detection: EBCall, JointSNVMix, MuTect, SomaticSniper, Strelka, and VarScan 2, with a focus on MuTect and VarScan 2, two widely used publicly available software tools. Default/suggested parameters were used to run these tools. The missense sSNVs detected in these samples were validated through PCR and direct sequencing of genomic DNA from the samples. We also simulated 10 tumor-normal pairs to explore the ability of these programs to detect low allelic-frequency sSNVs.
Out of the 237 sSNVs successfully validated in our cancer samples, VarScan 2 and MuTect detected the most of any tools (that is, 204 and 192, respectively). MuTect identified 11 more low-coverage validated sSNVs than VarScan 2, but missed 11 more sSNVs with alternate alleles in normal samples than VarScan 2. When examining the false calls of each tool using 169 invalidated sSNVs, we observed >63% false calls detected in the lung cancer cell lines had alternate alleles in normal samples. Additionally, from our simulation data, VarScan 2 identified more sSNVs than other tools, while MuTect characterized most low allelic-fraction sSNVs.
Our study explored the typical false-positive and false-negative detections that arise from the use of sSNV-calling tools. Our results suggest that despite recent progress, these tools have significant room for improvement, especially in the discrimination of low coverage/allelic-frequency sSNVs and sSNVs with alternate alleles in normal samples.
PMCID: PMC3971343  PMID: 24112718
18.  HER2 amplification: a potential mechanism of acquired resistance to EGFR inhibition in EGFR mutant lung cancers that lack the second-site EGFR T790M mutation 
Cancer discovery  2012;2(10):922-933.
EGFR-mutant lung cancers eventually become resistant to treatment with EGFR tyrosine kinase inhibitors (TKIs). The combination of EGFR-TKI afatinib and anti-EGFR antibody cetuximab can overcome acquired resistance in mouse models and human patients. Since afatinib is also a potent HER2 inhibitor, we investigated the role of HER2 in EGFR-mutant tumor cells. We show in vitro and in vivo that afatinib plus cetuximab significantly inhibits HER2 phosphorylation. HER2 overexpression or knockdown confers resistance or sensitivity, respectively, in all studied cell line models. Fluorescent in situ hybridization analysis revealed that HER2 was amplified in 12% of tumors with acquired resistance versus only 1% of untreated lung adenocarcinomas. Notably, HER2 amplification and EGFR T790M were mutually exclusive. Collectively, these results reveal a previously unrecognized mechanism of resistance to EGFR TKIs and provide a rationale to assess the status and possibly target HER2 in EGFR mutant tumors with acquired resistance to EGFR TKIs.
PMCID: PMC3473100  PMID: 22956644
EGFR mutations; lung cancer; EGFR tyrosine kinase inhibitors; erlotinib; afatinib; cetuximab; HER2 amplification; EGFR T790M; acquired resistance
19.  Impact on disease-free survival of adjuvant erlotinib or gefitinib in patients with resected lung adenocarcinomas that harbor epidermal growth factor receptor (EGFR) mutations 
Patients with stage IV lung adenocarcinoma and EGFR mutation derive clinical benefit from treatment with EGFR tyrosine kinase inhibitors (TKI). Whether treatment with TKI improves outcomes in patients with resected lung adenocarcinoma and EGFR mutation is unknown.
Data were analyzed from a surgical database of patients with resected lung adenocarcinoma harboring EGFR exon 19 or 21 mutations. In a multivariate analysis, we evaluated the impact of treatment with adjuvant TKI.
The cohort consists of 167 patients with completely resected stage I–III lung adenocarcinoma. 93 patients (56%) had exon 19 del, 74 patients (44%) had exon 21 mutations, 56 patients (33%) received perioperative TKI. In a multivariate analysis controlling for sex, stage, type of surgery and adjuvant platinum chemotherapy, the 2-year DFS was 89% for patients treated with adjuvant TKI compared with 72% in control group (hazard ratio [HR] = 0.53; 95% confidence interval [CI] 0.28 to 1.03; p = 0.06). The 2-year OS was 96% with adjuvant EGFR TKI and 90% in the group that did not receive TKI (HR 0.62; 95% CI 0.26 to 1.51; p = 0.296).
Compared to patients who did not receive adjuvant TKI, we observed a trend toward improvement in disease free survival among individuals with resected stages I–III lung adenocarcinomas harboring mutations in EGFR exons 19 or 21 who received these agents as adjuvant therapy. Based on these data, 320 patients are needed for a randomized trial to prospectively validate this DFS benefit.
PMCID: PMC3778680  PMID: 21150674
20.  Mapping the hallmarks of lung adenocarcinoma with massively parallel sequencing 
Cell  2012;150(6):1107-1120.
Lung adenocarcinoma, the most common subtype of non-small cell lung cancer, is responsible for over 500,000 deaths per year worldwide. Here, we report exome and genome sequences of 183 lung adenocarcinoma tumor/normal DNA pairs. These analyses revealed a mean exonic somatic mutation rate of 12.0 events/megabase and identified the majority of genes previously reported as significantly mutated in lung adenocarcinoma. In addition, we identified statistically recurrent somatic mutations in the splicing factor gene U2AF1 and truncating mutations affecting RBM10 and ARID1A. Analysis of nucleotide context-specific mutation signatures grouped the sample set into distinct clusters that correlated with smoking history and alterations of reported lung adenocarcinoma genes. Whole genome sequence analysis revealed frequent structural re-arrangements, including in-frame exonic alterations within EGFR and SIK2 kinases. The candidate genes identified in this study are attractive targets for biological characterization and therapeutic targeting of lung adenocarcinoma.
PMCID: PMC3557932  PMID: 22980975
21.  BRAF L597 mutations in melanoma are associated with sensitivity to MEK inhibitors 
Cancer discovery  2012;2(9):791-797.
Kinase inhibitors are accepted treatment for metastatic melanomas that harbor specific driver mutations in BRAF or KIT, but only 40–50% of cases are positive. To uncover other potential targetable mutations, we performed whole-genome sequencing of a highly aggressive BRAF (V600) and KIT (W557, V559, L576, K642, D816) wildtype melanoma. Surprisingly, we found a somatic BRAF L597R mutation in exon 15. Analysis of BRAF exon 15 in 49 tumors negative for BRAF V600 mutations as well as driver mutations in KIT, NRAS, GNAQ, and GNA11, showed that 2 (4%) harbored L597 mutations and another 2 involved BRAF D594 and K601 mutations. In vitro signaling induced by L597R/S/Q mutants was suppressed by MEK inhibition. A patient with BRAF L597S mutant metastatic melanoma responded significantly to treatment with the MEK inhibitor, TAK-733. Collectively, these data demonstrate clinical significance to BRAF L597 mutations in melanoma.
PMCID: PMC3449158  PMID: 22798288
melanoma; BRAF L597; whole genome sequencing; BRAF inhibitor; MEK inhibitor; TAK-733
22.  Effects of Pharmacokinetic Processes and Varied Dosing Schedules on the Dynamics of Acquired Resistance to Erlotinib in EGFR-Mutant Lung Cancer 
Erlotinib (Tarceva) is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, which effectively targets EGFR-mutant driven non–small-cell lung cancer. However, the evolution of acquired resistance because of a second-site mutation (T790M) within EGFR remains an obstacle to successful treatment.
We used mathematical modeling and available clinical trial data to predict how different pharmacokinetic parameters (fast versus slow metabolism) and dosing schedules (low dose versus high dose; missed doses with and without make-up doses) might affect the evolution of T790M-mediated resistance in mixed populations of tumor cells.
We found that high-dose pulses with low-dose continuous therapy impede the development of resistance to the maximum extent, both pre- and post-emergence of resistance. The probability of resistance is greater in fast versus slow drug metabolizers, suggesting a potential mechanism, unappreciated to date, influencing acquired resistance in patients. In case of required dose modifications because of toxicity, little difference is observed in terms of efficacy and resistance dynamics between the standard daily dose (150 mg/d) and 150 mg/d alternating with 100 mg/d. Missed doses are expected to lead to resistance faster, even if make-up doses are attempted.
For existing and new kinase inhibitors, this novel framework can be used to rationally and rapidly design optimal dosing strategies to minimize the development of acquired resistance.
PMCID: PMC3693219  PMID: 22982659
EGFR-mutant lung cancer; Erlotinib; Evolutionary cancer modeling; Pharmacokinetic modeling; Acquired resistance; EGFR T790M mutation
23.  Core Needle Lung Biopsy Specimens: Adequacy for EGFR and KRAS Mutational Analysis 
The purpose of this study was to prospectively compare the adequacy of core needle biopsy specimens with the adequacy of specimens from resected tissue, the histologic reference standard, for mutational analysis of malignant tumors of the lung.
The first 18 patients enrolled in a phase 2 study of gefitinib for lung cancer in July 2004 through August 2005 underwent CT- or fluoroscopy-guided lung biopsy before the start of gefitinib therapy. Three weeks after gefitinib therapy, the patients underwent lung tumor resection. The results of EGFR and KRAS mutational analysis of the core needle biopsy specimens were compared with those of EGFR and KRAS mutational analysis of the surgical specimens.
Two specimens were unsatisfactory for mutational analysis. The results of mutational assay results of the other 16 specimens were the same as those of analysis of the surgical specimens obtained an average of 31 days after biopsy.
Biopsy with small (18- to 20-gauge) core needles can yield sufficient and reliable samples for mutational analysis. This technique is likely to become an important tool with the increasing use of pharmacotherapy based on the genetics of specific tumors in individual patients.
PMCID: PMC3676673  PMID: 20028932
biopsy; lung cancer; molecular typing; personalized medicine; targeted therapy
24.  Brief report: Screening for germline EGFR T790M mutations through lung cancer genotyping 
Journal of Thoracic Oncology  2012;7(6):1049-1052.
The study of patients carrying germline EGFR mutations, which have been found in cases of familial lung adenocarcinoma, could provide unique insight into lung cancer risk and carcinogenesis in non-smokers. However, investigations into the biology of germline EGFR mutations have been hampered by the lack of an effective strategy for screening for carriers. We hypothesized that patients with lung cancers found to harbor the EGFR T790M resistance mutation prior to treatment, an uncommon occurrence, would be likely to carry underlying germline T790M mutations.
Eleven unrelated patients were identified with lung cancer harboring an EGFR T790M mutation from a 7-year institutional experience with tumor genotyping. Ten patients had benign tissue available, which was anonymously tested for presence of germline EGFR mutations.
Five of 10 cases carried a germline T790M mutation (50%, CI 27%–73%). One patient’s cancer exhibited a distinctive indolent growth which has also been described in preclinical studies of T790M-mutant cancers. A second patient underwent resection of 6 separate primary lung adenocarcinomas, each carrying different sensitizing EGFR mutations as well as T790M.
Genotyping of lung cancers, now commonly performed to predict benefit from treatment with EGFR tyrosine kinase inhibitors, can also be used as a screening tool to identify patients at risk of carrying germline EGFR mutations. Once identified, these patients and their families can be studied prospectively in order to explore appropriate screening strategies. Further studies using existing oncogenomic data to provide insight into underlying germline genetics are warranted.
PMCID: PMC3354706  PMID: 22588155
Familial lung cancer; EGFR mutations; genetic susceptibility; T790M
25.  Complications of Targeted Drug Therapies for Solid Malignancies: Manifestations and Mechanisms 
This article reviews important complications of targeted drug therapies for solid malignancies that can be identified on diagnostic imaging. Wherever possible, known or proposed mechanistic explanations for drug complications are emphasized.
Familiarity with the toxicity profiles of different targeted cancer therapies is important for identifying drug-related complications and for differentiating drug effects from disease progression. A mechanistic understanding may be useful for associating individual drugs with their complications and for predicting the complications of emerging agents.
PMCID: PMC3652983  PMID: 23436834
bevacizumab; drug toxicities; imatinib; ipilimumab; targeted agents; temsirolimus; vemurafenib

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