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1.  Clinicopathologic and prognostic significance of c-MYC copy number gain in lung adenocarcinomas 
British Journal of Cancer  2014;110(11):2688-2699.
Background:
c-MYC copy number gain (c-MYC gain) has been associated with aggressive behaviour in several cancers. However, the role of c-MYC gain has not yet been determined in lung adenocarcinomas classified by genetic alterations in epidermal growth factor receptor (EGFR), KRAS, and anaplastic lymphoma kinase (ALK) genes. We investigated the clinicopathologic and prognostic significance of c-MYC gain for disease-free survival (DFS) and overall survival (OS) according to EGFR, KRAS, and ALK gene status and stages in lung adenocarcinomas.
Methods:
In 255 adenocarcinomas resected in Seoul National University Bundang Hospital from 2003 to 2009, fluorescence in situ hybridisation (FISH) with c-MYC probe and centromeric enumeration probe 8 (CEP8) was analysed using tissue microarray containing single representative core per each case. EGFR (codon 18 to 21) and KRAS (codon 12, 13, and 61) mutations were analysed by polymerase chain reaction and direct sequencing method from formalin-fixed, paraffin-embedded tissue sections. ALK rearrangement was determined by FISH method. c-MYC gain was defined as >2 copies per nucleus, chromosome 8 gain as ⩾3 copies per nucleus, and gain of c-MYC:CEP8 ratio (hereafter, c-MYC amplification) as ⩾2.
Results:
We observed c-MYC gain in 20% (51 out of 255), chromosome 8 gain in 5.5% (14 out of 255), c-MYC amplification in 2.4% (6 out of 255), EGFR mutation in 49.4% (118 out of 239), KRAS mutation in 5.7% (7 out of 123), and ALK rearrangement in 4.9% (10 out of 205) of lung adenocarcinomas. c-MYC gain was observed in 19% (22 out of 118) of patients with lung adenocarcinomas with an EGFR mutation, but not in any patients with a KRAS mutation, or an ALK rearrangement. c-MYC gain (but not chromosome 8 gain or c-MYC amplification) was an independent poor-prognostic factor in the full cohort of lung adenocarcinoma (P=0.022, hazard ratio (HR)=1.71, 95% confidence interval (CI), 1.08–2.69 for DFS; P=0.032, HR=2.04, 95% CI, 1.06–3.91 for OS), as well as in stage I subgroup (P=0.023, HR=4.70, 95% CI, 1.24–17.78 for DFS; P=0.031, HR=4.65, 95% CI, 1.15–18.81 for OS), and in EGFR-mutant subgroup (P=0.022; HR=2.14; 95% CI, 1.11–4.10 for DFS).
Conclusions:
c-MYC gain (but not chromosome 8 gain or c-MYC amplification) was an independent poor-prognostic factor for DFS and OS in lung adenocarcinomas, both in full cohort and stage I cancer, and possibly for DFS in EGFR-mutant adenocarcinomas. Additional studies are required to determine if patients with lung adenocarcinoma with c-MYC gain are candidates for additional first-line treatment to mitigate their increased risk for disease progression and death.
doi:10.1038/bjc.2014.218
PMCID: PMC4037828  PMID: 24809777
lung cancer; adenocarcinoma; c-MYC copy number gain; fluorescence in situ hybridisation; EGFR
2.  Allelotypes of lung adenocarcinomas featuring ALK fusion demonstrate fewer onco- and suppressor gene changes 
BMC Cancer  2013;13:8.
Background
A subset of lung adenocarcinomas harboring an EML4-ALK fusion gene resulting in dominant oncogenic activity has emerged as a target for specific therapy. EML4-ALK fusion confers a characteristic histology and is detected more frequently in never or light smokers and younger patients.
Methods
To gain insights into etiology and carcinogenic mechanisms we conducted analyses to compare allelotypes of 35 ALK fusion-positive and 95 -negative tumours using single nucleotide polymorphism (SNP) arrays and especially designed software which enabled precise global genomic profiling.
Results
Overall aberration numbers (gains + losses) of chromosomal alterations were 8.42 and 9.56 in tumours with and without ALK fusion, respectively, the difference not being statistically significant, although patterns of gain and loss were distinct. Interestingly, among selected genomic regions, oncogene-related examples such as 1p34.3(MYCL1), 7q11.2(EGFR), 7p21.1, 8q24.21(MYC), 16p13.3, 17q12(ERBB2) and 17q25.1 showed significantly less gain. Also, changes in tumour suppressor gene-related regions, such as 9p21.3 (CDKN2A) 9p23-24.1 (PTPRD), 13q14.2 (RB1), were significantly fewer in tumours with ALK fusion.
Conclusion
Global genomic comparison with SNP arrays showed tumours with ALK fusion to have fewer alterations in oncogenes and suppressor genes despite a similar overall aberration frequency, suggesting very strong oncogenic potency of ALK activation by gene fusion.
doi:10.1186/1471-2407-13-8
PMCID: PMC3599044  PMID: 23289484
Lung adenocarcinoma; ALK fusion; SNP array; Allelotype; Copy number
3.  A Comprehensive Characterization of Genome-Wide Copy Number Aberrations in Colorectal Cancer Reveals Novel Oncogenes and Patterns of Alterations 
PLoS ONE  2012;7(7):e42001.
To develop a comprehensive overview of copy number aberrations (CNAs) in stage-II/III colorectal cancer (CRC), we characterized 302 tumors from the PETACC-3 clinical trial. Microsatellite-stable (MSS) samples (n = 269) had 66 minimal common CNA regions, with frequent gains on 20 q (72.5%), 7 (41.8%), 8 q (33.1%) and 13 q (51.0%) and losses on 18 (58.6%), 4 q (26%) and 21 q (21.6%). MSS tumors have significantly more CNAs than microsatellite-instable (MSI) tumors: within the MSI tumors a novel deletion of the tumor suppressor WWOX at 16 q23.1 was identified (p<0.01). Focal aberrations identified by the GISTIC method confirmed amplifications of oncogenes including EGFR, ERBB2, CCND1, MET, and MYC, and deletions of tumor suppressors including TP53, APC, and SMAD4, and gene expression was highly concordant with copy number aberration for these genes. Novel amplicons included putative oncogenes such as WNK1 and HNF4A, which also showed high concordance between copy number and expression. Survival analysis associated a specific patient segment featured by chromosome 20 q gains to an improved overall survival, which might be due to higher expression of genes such as EEF1B2 and PTK6. The CNA clustering also grouped tumors characterized by a poor prognosis BRAF-mutant-like signature derived from mRNA data from this cohort. We further revealed non-random correlation between CNAs among unlinked loci, including positive correlation between 20 q gain and 8 q gain, and 20 q gain and chromosome 18 loss, consistent with co-selection of these CNAs. These results reinforce the non-random nature of somatic CNAs in stage-II/III CRC and highlight loci and genes that may play an important role in driving the development and outcome of this disease.
doi:10.1371/journal.pone.0042001
PMCID: PMC3409212  PMID: 22860045
4.  Relation between smoking history and gene expression profiles in lung adenocarcinomas 
BMC Medical Genomics  2012;5:22.
Background
Lung cancer is the worldwide leading cause of death from cancer. Tobacco usage is the major pathogenic factor, but all lung cancers are not attributable to smoking. Specifically, lung cancer in never-smokers has been suggested to represent a distinct disease entity compared to lung cancer arising in smokers due to differences in etiology, natural history and response to specific treatment regimes. However, the genetic aberrations that differ between smokers and never-smokers’ lung carcinomas remain to a large extent unclear.
Methods
Unsupervised gene expression analysis of 39 primary lung adenocarcinomas was performed using Illumina HT-12 microarrays. Results from unsupervised analysis were validated in six external adenocarcinoma data sets (n=687), and six data sets comprising normal airway epithelial or normal lung tissue specimens (n=467). Supervised gene expression analysis between smokers and never-smokers were performed in seven adenocarcinoma data sets, and results validated in the six normal data sets.
Results
Initial unsupervised analysis of 39 adenocarcinomas identified two subgroups of which one harbored all never-smokers. A generated gene expression signature could subsequently identify never-smokers with 79-100% sensitivity in external adenocarcinoma data sets and with 76-88% sensitivity in the normal materials. A notable fraction of current/former smokers were grouped with never-smokers. Intriguingly, supervised analysis of never-smokers versus smokers in seven adenocarcinoma data sets generated similar results. Overlap in classification between the two approaches was high, indicating that both approaches identify a common set of samples from current/former smokers as potential never-smokers. The gene signature from unsupervised analysis included several genes implicated in lung tumorigenesis, immune-response associated pathways, genes previously associated with smoking, as well as marker genes for alveolar type II pneumocytes, while the best classifier from supervised analysis comprised genes strongly associated with proliferation, but also genes previously associated with smoking.
Conclusions
Based on gene expression profiling, we demonstrate that never-smokers can be identified with high sensitivity in both tumor material and normal airway epithelial specimens. Our results indicate that tumors arising in never-smokers, together with a subset of tumors from smokers, represent a distinct entity of lung adenocarcinomas. Taken together, these analyses provide further insight into the transcriptional patterns occurring in lung adenocarcinoma stratified by smoking history.
doi:10.1186/1755-8794-5-22
PMCID: PMC3447685  PMID: 22676229
Lung cancer; Smoking; Gene expression analysis; Adenocarcinoma; EGFR; Never-smokers; Immune response
5.  Abnormalities of the TITF-1 lineage-specific oncogene in NSCLC: Implications in lung cancer pathogenesis and prognosis 
PURPOSE
Emerging evidence suggests that aberrant expression of oncogenes contributes to development of lung malignancy. The thyroid transcription factor 1 (TITF-1) gene functions as a lineage survival gene abnormally expressed in a significant fraction of NSCLCs, in particular lung adenocarcinomas.
EXPERIMENTAL DESIGN
To better characterize TITF-1 abnormality: patterns in NSCLC, we studied TITF-1’s gene copy number using fluorescent in situ hybridization (FISH) and quantitative PCR, as well as its protein expression by immunohistochemistry analysis in a tissue microarray comprised of surgically resected NSCLC (N=321) including 204 adenocarcinomas and 117 squamous cell carcinomas (SCCs). TITF-1 copy number and protein expression were correlated with patients’ clinicopathologic characteristics, and in a subset of adenocarcinomas with EGFR and KRAS mutation status.
RESULTS
We found that increased TITF-1 protein expression was prevalent in lung adenocarcinomas only and was significantly associated with female gender (p<0.001), never smokers (p=0.004), presence of EGFR mutations (p=0.05) and better overall survival (all stages, p=0.0478. stages I and II, p=0.002). TITF-1 copy number gain (CBG) was detected by FISH analysis in both adenocarcinomas (18.9%; high CNG, 8.3%) and SCCs (20.1%; high CNG, 3.0%), and correlated significantly with the protein product (p=0.004) and presence of KRAS mutations (p=0.008) in lung adenocarcinomas. Moreover, multivariate analysis revealed that TITF-1 copy number gain was an independent predictor of poor survival of NSCLC (p=0.039).
CONCLUSIONS
Our integrative study demonstrates that the protein versus genomic expression patterns of TITF-1 have opposing roles in lung cancer prognosis and may occur preferentially in different subsets of NSCLC patients with distinct oncogene mutations.
doi:10.1158/1078-0432.CCR-10-1412
PMCID: PMC3078948  PMID: 21257719
NSCLC; TITF-1; gene copy gain; lineage-specific oncogenes
6.  Family history of lung cancer in never smokers with non-small-cell lung cancer and its association with tumors harboring EGFR mutations 
INTRODUCTION
Inherited susceptibility to lung cancer is understudied. Never smokers are an important subgroup of patients enriched for tumors harboring oncogene aberrations in the EGFR and ALK genes. We aimed to better characterize the incidence of family history of lung cancer among never smokers with NSCLC.
METHODS
Clinicopathologic data, tumor genotype, family history of cancer, and specifically family history of lung cancer from 230 consecutive never smokers was retrospectively compiled and analyzed.
RESULTS
In our cohort, the median age was 56 years, 67% were women, 75% were white, 59% had advanced NSCLC and 87% had adenocarcinoma histology. In these tumors, 98/230 (42%) had an EGFR mutation, 17/155 (11%) had KRAS mutations and 27/127 (21%) had an ALK translocation. Family history of any cancer was common (57%) and specific family history of lung cancer was present in 42/230 cases (18%). The percentage of cases with family history of lung cancer was higher in the EGFR mutated versus EGFR wild-type NSCLCs. Out of the cases with a family history of any cancer, 22/53 (41.5%) EGFR mutated, 1/5 (20%) KRAS mutated and 3/19 (15.5%) ALK translocated cohorts had a family history of lung cancer. The ratio of family history of lung cancer to family history of cancer was significantly higher in the EGFR mutated cohort when compared to the ALK translocated plus KRAS mutated cohorts (p=0.039).
CONCLUSIONS
Family history of lung cancer is common in never smokers with NSCLC, and there seems to be a particular link in families in which the proband has an EGFR mutated tumor when compared to ALK translocated or KRAS mutated tumors. Further study of families with EGFR-mutated NSCLC may yield insights into the pathogenesis of this tumor type.
doi:10.1016/j.lungcan.2012.12.002
PMCID: PMC3566317  PMID: 23273562
lung cancer; non-small-cell lung cancer; family history; never smokers; epidermal growth factor receptor; EGFR; anaplastic lymphoma kinase; ALK; KRAS
7.  Prospective genetic profiling of squamous cell lung cancer and adenosquamous carcinoma in Japanese patients by multitarget assays 
BMC Cancer  2014;14:786.
Background
Despite considerable recent progress in the treatment of lung adenocarcinoma, there has been little progress in the development of efficacious molecular targeted therapies for squamous cell lung cancer. In addition to the recent comprehensive genome-wide characterization of squamous cell lung cancer, it is also important to genotype this form of cancer. We therefore conducted the Shizuoka Lung Cancer Mutation Study to analyze driver mutations in patients with thoracic malignancies. Here we report the results of genotyping in patients with squamous cell lung cancer.
Methods
Based on the biobanking system, in conjunction with the clinic and pathology lab, we developed a genotyping panel designed to assess 24 mutations in 10 genes (EGFR, KRAS, BRAF, PIK3CA, NRAS, MEK1, AKT1, PTEN, HER2 and DDR2), EGFR, MET, PIK3CA, FGFR1 and FGFR2 copy numbers, and EML4-ALK and ROS1 translocations, using pyrosequencing plus capillary electrophoresis, quantitative polymerase chain reaction (PCR) and reverse-transcription PCR, respectively.
Results
A total of 129 patients with squamous cell lung cancer and adenosquamous carcinoma were enrolled in this study between July 2011 and November 2012. We detected genetic alterations in 40% of all cases. Gene alterations included: EGFR mutations, 6%; KRAS mutations, 4%; PIK3CA mutations, 13%; NRAS mutations, 1%; KIF5b-RET fusion gene, 1%; EGFR copy number gain, 5%; PIK3CA copy number gain, 15%; and FGFR1 copy number gain, 5%. Twelve patients (9%) harbored simultaneous genetic alterations. Genetic alterations were detected more frequently in surgically-resected, snap-frozen samples than in formalin-fixed, paraffin-embedded samples (50% vs. 29%). In addition, patients aged ≤70 years old and never-smokers showed high frequencies of genetic alterations.
Conclusions
This study represents one of the largest prospective tumor-genotyping studies to be performed in Asian patients with squamous cell lung cancer. These results suggest that incorporation of genetic profiling into lung cancer clinical practice may facilitate the administration of personalized cancer treatments in patients with squamous cell lung cancer.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2407-14-786) contains supplementary material, which is available to authorized users.
doi:10.1186/1471-2407-14-786
PMCID: PMC4221703  PMID: 25348872
Lung cancer; Squamous cell carcinoma; Adenosquamos carcinoma; Genetic profiling; Driver mutation; PIK3CA mutation; FGFR1 copy number gain
8.  High Resolution Genome-Wide Analysis of Chromosomal Alterations in Burkitt's Lymphoma 
PLoS ONE  2009;4(9):e7089.
Additional chromosomal abnormalities are currently detected in Burkitt's lymphoma. They play major roles in the progression of BL and in prognosis. The genes involved remain elusive. A whole-genome oligonucleotide array CGH analysis correlated with karyotype and FISH was performed in a set of 27 Burkitt's lymphoma-derived cell lines and primary tumors. More than half of the 145 CNAs<2 Mb were mapped to Mendelian CNVs, including GSTT1, glutathione s-transferase and BIRC6, an anti-apoptotic protein, possibly predisposing to some cancers. Somatic cell line-specific CNVs localized to the IG locus were consistently observed with the 244 K aCGH platform. Among 136 CNAs >2 Mb, gains were found in 1q (12/27), 13q (7/27), 7q (6/27), 8q(4/27), 2p (3/27), 11q (2/27) and 15q (2/27). Losses were found in 3p (5/27), 4p (4/27), 4q (4/27), 9p (4/27), 13q (4/27), 6p (3/27), 17p (3/27), 6q (2/27),11pterp13 (2/27) and 14q12q21.3 (2/27). Twenty one minimal critical regions (MCR), (range 0.04–71.36 Mb), were delineated in tumors and cell lines. Three MCRs were localized to 1q. The proximal one was mapped to 1q21.1q25.2 with a 6.3 Mb amplicon (1q21.1q21.3) harboring BCA2 and PIAS3. In the other 2 MCRs, 1q32.1 and 1q44, MDM4 and AKT3 appeared as possible drivers of these gains respectively. The 13q31.3q32.1 <89.58–96.81> MCR contained an amplicon and ABCC4 might be the driver of this amplicon. The 40 Kb 2p16.1 <60.96–61> MCR was the smallest gained MCR and specifically encompassed the REL oncogene which is already implicated in B cell lymphomas. The most frequently deleted MCR was 3p14.1 <60.43–60.53> that removed the fifth exon of FHIT. Further investigations which combined gene expression and functional studies are essential to understand the lymphomagenesis mechanism and for the development of more effective, targeted therapeutic strategies.
doi:10.1371/journal.pone.0007089
PMCID: PMC2739276  PMID: 19759907
9.  Lung Adenocarcinoma of Never Smokers and Smokers Harbor Differential Regions of Genetic Alteration and Exhibit Different Levels of Genomic Instability 
PLoS ONE  2012;7(3):e33003.
Recent evidence suggests that the observed clinical distinctions between lung tumors in smokers and never smokers (NS) extend beyond specific gene mutations, such as EGFR, EML4-ALK, and KRAS, some of which have been translated into targeted therapies. However, the molecular alterations identified thus far cannot explain all of the clinical and biological disparities observed in lung tumors of NS and smokers. To this end, we performed an unbiased genome-wide, comparative study to identify novel genomic aberrations that differ between smokers and NS.
High resolution whole genome DNA copy number profiling of 69 lung adenocarcinomas from smokers (n = 39) and NS (n = 30) revealed both global and regional disparities in the tumor genomes of these two groups. We found that NS lung tumors had a greater proportion of their genomes altered than those of smokers. Moreover, copy number gains on chromosomes 5q, 7p, and 16p occurred more frequently in NS. We validated our findings in two independently generated public datasets. Our findings provide a novel line of evidence distinguishing genetic differences between smoker and NS lung tumors, namely, that the extent of segmental genomic alterations is greater in NS tumors. Collectively, our findings provide evidence that these lung tumors are globally and genetically different, which implies they are likely driven by distinct molecular mechanisms.
doi:10.1371/journal.pone.0033003
PMCID: PMC3296775  PMID: 22412972
10.  MYC-driven tumorigenesis is inhibited by WRN syndrome gene deficiency 
Molecular cancer research : MCR  2012;10(4):535-545.
MYC-induced DNA damage is exacerbated in WRN deficient cells, leading to replication stress and accelerated cellular senescence. To determine if WRN deficiency impairs MYC driven tumor development, we utilized both xenograft and autochthonous tumor models. Conditional silencing of WRN expression in c-MYC overexpressing non-small cell lung cancer xenografts impaired both tumor establishment and tumor growth. This inhibitory effect of WRN knock-down was accompanied by increased DNA damage, decreased proliferation, and tumor necrosis. In the Eμ-Myc mouse model of B-cell lymphoma, a germline mutation in the helicase domain of Wrn (WrnΔhel/Δhel) resulted in a significant delay in emergence of lethal lymphomas, extending tumor free survival by >30%. Analysis of pre-neoplastic B cells from Eμ-Myc Wrn mutant mice revealed increased DNA damage, elevation of senescence markers, and decreased proliferation in comparison with cells from age-matched Eμ-Myc mice. Immunohistochemical and global gene expression analysis of overt Eμ-Myc WrnΔhel/Δhel lymphomas demonstrated a marked increase in expression of the CDK inhibitor, p16Ink4a, as well as elevation of TAp63, a known mediator of senescence. Collectively, these studies demonstrate that in the context of Myc-associated tumorigenesis, loss of Wrn amplifies the DNA damage response, both in pre-neoplastic and neoplastic tissue, engaging activation of tumor suppressor pathways. This leads to inhibition of tumor growth and prolonged tumor free survival. Targeting WRN or its enzymatic function could prove to be an effective strategy in the treatment of MYC-associated cancers.
doi:10.1158/1541-7786.MCR-11-0508
PMCID: PMC3707802  PMID: 22301954
Werner helicase; therapeutic target; Myc-driven cancer; senescence; tumor suppressors
11.  Promoter methylation of RASSF1A and DAPK and mutations of K-ras, p53, and EGFR in lung tumors from smokers and never-smokers 
BMC Cancer  2007;7:74.
Background
Epidemiological studies indicate that some characteristics of lung cancer among never-smokers significantly differ from those of smokers. Aberrant promoter methylation and mutations in some oncogenes and tumor suppressor genes are frequent in lung tumors from smokers but rare in those from never-smokers. In this study, we analyzed promoter methylation in the ras-association domain isoform A (RASSF1A) and the death-associated protein kinase (DAPK) genes in lung tumors from patients with primarily non-small cell lung cancer (NSCLC) from the Western Pennsylvania region. We compare the results with the smoking status of the patients and the mutation status of the K-ras, p53, and EGFR genes determined previously on these same lung tumors.
Methods
Promoter methylation of the RASSF1A and DAPK genes was analyzed by using a modified two-stage methylation-specific PCR. Data on mutations of K-ras, p53, and EGFR were obtained from our previous studies.
Results
The RASSF1A gene promoter methylation was found in tumors from 46.7% (57/122) of the patients and was not significantly different between smokers and never-smokers, but was associated significantly in multiple variable analysis with tumor histology (p = 0.031) and marginally with tumor stage (p = 0.063). The DAPK gene promoter methylation frequency in these tumors was 32.8% (40/122) and did not differ according to the patients' smoking status, tumor histology, or tumor stage. Multivariate analysis adjusted for age, gender, smoking status, tumor histology and stage showed that the frequency of promoter methylation of the RASSF1A or DAPK genes did not correlate with the frequency of mutations of the K-ras, p53, and EGFR gene.
Conclusion
Our results showed that RASSF1A and DAPK genes' promoter methylation occurred frequently in lung tumors, although the prevalence of this alteration in these genes was not associated with the smoking status of the patients or the occurrence of mutations in the K-ras, p53 and EGFR genes, suggesting each of these events may represent independent event in non-small lung tumorigenesis.
doi:10.1186/1471-2407-7-74
PMCID: PMC1877812  PMID: 17477876
12.  INTEGRATIVE GENOMIC CHARACTERIZATION OF LOWER GRADE GLIOMAS 
Neuro-Oncology  2014;16(Suppl 3):iii3.
BACKGROUND: Lower grade gliomas (LGG) are infiltrative brain tumors that include astrocytomas, oligodendrogliomas and oligoastrocytomas, grades II and III. LGGs almost always progress, typically to glioblastoma (GBM, grade IV), and are uniformly fatal. The Cancer Genome Atlas (TCGA) is conducting a comprehensive molecular analysis of LGG, incorporating genetic and genomic alterations, DNA methylation profiles, and RNA and proteomic signatures. The rich, integrated clinical data of TCGA provides an outstanding platform to uncover biomarkers of therapy response and outcome. METHODS: We analyzed 289 lower grade gliomas for exome sequence to uncover somatic mutations; DNA copy number alterations; RNA sequencing for expression and gene fusion; DNA methlyation; microRNA expression; and protein level and phosphorylation. Supervised and unsupervised clustering was performed to segregate LGGs into robust molecular categories. RESULTS: Among 289 LGGs, exome sequencing identified 19 significantly mutated genes, including IDH1, TP53, ATRX, CIC, FUBP1, NOTCH1, PIK3CA, NF1, PIK3R1, ARID1A, PTEN, SMARCA4 and EGFR. Frequent arm-level deletions were 1p, 19q, 13q, 9p, 10q, 10p, 4q and 4p; arm-level gains included 7q, 7p, 19p and 11q. There were 15 significant genomic amplifications, among which were 7p11.2 (EGFR), 12q14.1 (CDK4), 1q32.1 (MDM4), 8q24.21 (MYC), 12p13.32 (CCND2) and 4q12 (PDGFR). Among 28 deletions were 9p21.3 (CDKN2A, CDKN2B), 19q13.42 (TFPT, ZNF331), 2q37.3, 10q26.2 (FGFR2, DUX4) and 14q24.3 (TSHR, GPHN).Global analysis of DNA methylation identified 5 stable clusters, with one showing substantial hypomethylation. Gene expression profiling identified 4 stable clusters. On integrative analysis, IDH1/2 wt LGGs had features of “pre-GBM”: they formed distinct hypomethylation and gene expression clusters; had molecular alterations typical of GBM, such as EGFR amplifications, PTEN mutations, CDKN2A loss and RTK gene fusions; were mostly grade III astrocytomas; and had short survivals. IDH1/2 mutant LGGs were hypermethylated. One subgroup was enriched for 1p/19q co-deletion, CIC and FUBP mutations, and oligodendrogliomas. Another subset was enriched for TP53 and ATRX mutations and astrocytomas. These groups clustered separately on gene expression analysis. A fourth gene expression cluster had elements of these two IDH1/2 mutant subgroups and the longest survival. A cluster-of-cluster analysis of mRNA, copy number, miRNA and methylation analyses reinforced the clear separation of three molecular classes of LGG based on the status of IDH1/2 and 1p/19q. CONCLUSIONS: These data illustrate a potential stratification of LGGs that emphasizes molecular characteristics. IDH wt LGG have molecular alterations and clinical behavior similar to GBMs, whereas IDH mutant LGGs segregate based on 1p/19q status. SECONDARY CATEGORY: Tumor Biology.
doi:10.1093/neuonc/nou206.11
PMCID: PMC4144475
13.  Lung Adenocarcinoma From East Asian Never-Smokers Is a Disease Largely Defined by Targetable Oncogenic Mutant Kinases 
Journal of Clinical Oncology  2010;28(30):4616-4620.
Purpose
To determine the proportion of lung adenocarcinomas from East Asian never-smokers who harbor known oncogenic driver mutations.
Patients and Methods
In this surgical series, 52 resected lung adenocarcinomas from never-smokers (< 100 cigarettes in a lifetime) at a single institution (Fudan University, Shanghai, China) were analyzed concurrently for mutations in EGFR, KRAS, NRAS, HRAS, HER2, BRAF, ALK, PIK3CA, TP53 and LKB1.
Results
Forty-one tumors harbored EGFR mutations, three harbored EML4-ALK fusions, two harbored HER2 insertions, and one harbored a KRAS mutation. All mutations were mutually exclusive. Thus, 90% (47 of 52; 95% CI, 0.7896 to 0.9625) of lung adenocarcinomas from never-smokers were found to harbor well-known oncogenic mutations in just four genes. No BRAF, NRAS, HRAS, or LKB1 mutations were detected, while 15 had TP53 mutations. Four tumors contained PIK3CA mutations, always together with EGFR mutations.
Conclusion
To our knowledge, this study represents the first comprehensive and concurrent analysis of major recurrent oncogenic mutations found in a large cohort of lung adenocarcinomas from East Asian never-smokers. Since drugs are now available that target mutant EGFR, HER2, and ALK, respectively, this result indicates that prospective mutation testing in these patients should successfully assign a targeted therapy in the majority of cases.
doi:10.1200/JCO.2010.29.6038
PMCID: PMC2974342  PMID: 20855837
14.  Spectrum of Oncogenic Driver Mutations in Lung Adenocarcinomas from East Asian Never Smokers 
PLoS ONE  2011;6(11):e28204.
Purpose
We previously showed that 90% (47 of 52; 95% CI, 0.79 to 0.96) of lung adenocarcinomas from East Asian never-smokers harbored well-known oncogenic mutations in just four genes: EGFR, HER2, ALK, and KRAS. Here, we sought to extend these findings to more samples and identify driver alterations in tumors negative for these mutations.
Experimental Design
We have collected and analyzed 202 resected lung adenocarcinomas from never smokers seen at Fudan University Shanghai Cancer Center. Since mutations were mutually exclusive in the first 52 examined, we determined the status of EGFR, KRAS, HER2, ALK, and BRAF in stepwise fashion as previously described. Samples negative for mutations in these 5 genes were subsequently examined for known ROS1 fusions by RT-PCR and direct sequencing.
Results
152 tumors (75.3%) harbored EGFR mutations, 12 (6%) had HER2 mutations, 10 (5%) had ALK fusions all involving EML4 as the 5′ partner, 4 (2%) had KRAS mutations, and 2 (1%) harbored ROS1 fusions. No BRAF mutation were detected.
Conclusion
The vast majority (176 of 202; 87.1%, 95% CI: 0.82 to 0.91) of lung adenocarcinomas from never smokers harbor mutant kinases sensitive to available TKIs. Interestingly, patients with EGFR mutant patients tend to be older than those without EGFR mutations (58.3 Vs 54.3, P = 0.016) and patient without any known oncogenic driver tend to be diagnosed at a younger age (52.3 Vs 57.9, P = 0.013). Collectively, these data indicate that the majority of never smokers with lung adenocarcinoma could benefit from treatment with a specific tyrosine kinase inhibitor.
doi:10.1371/journal.pone.0028204
PMCID: PMC3227646  PMID: 22140546
15.  Finding exclusively deleted or amplified genomic areas in lung adenocarcinomas using a novel chromosomal pattern analysis 
BMC Medical Genomics  2009;2:43.
Background
Genomic copy number alteration (CNA) that are recurrent across multiple samples often harbor critical genes that can drive either the initiation or the progression of cancer disease. Up to now, most researchers investigating recurrent CNAs consider separately the marginal frequencies for copy gain or loss and select the areas of interest based on arbitrary cut-off thresholds of these frequencies. In practice, these analyses ignore the interdependencies between the propensity of being deleted or amplified for a clone. In this context, a joint analysis of the copy number changes across tumor samples may bring new insights about patterns of recurrent CNAs.
Methods
We propose to identify patterns of recurrent CNAs across tumor samples from high-resolution comparative genomic hybridization microarrays. Clustering is achieved by modeling the copy number state (loss, no-change, gain) as a multinomial distribution with probabilities parameterized through a latent class model leading to nine patterns of recurrent CNAs. This model gives us a powerful tool to identify clones with contrasting propensity of being deleted or amplified across tumor samples. We applied this model to a homogeneous series of 65 lung adenocarcinomas.
Results
Our latent class model analysis identified interesting patterns of chromosomal aberrations. Our results showed that about thirty percent of the genomic clones were classified either as "exclusively" deleted or amplified recurrent CNAs and could be considered as non random chromosomal events. Most of the known oncogenes or tumor suppressor genes associated with lung adenocarcinoma were located within these areas. We also describe genomic areas of potential interest and show that an increase of the frequency of amplification in these particular areas is significantly associated with poorer survival.
Conclusion
Analyzing jointly deletions and amplifications through our latent class model analysis allows highlighting specific genomic areas with exclusively amplified or deleted recurrent CNAs which are good candidate for harboring oncogenes or tumor suppressor genes.
doi:10.1186/1755-8794-2-43
PMCID: PMC2718000  PMID: 19594952
16.  Establishment and Characterization of a Singaporean Chinese Lung Adenocarcinoma Cell Line with Four Copies of the Epidermal Growth Factor Receptor Gene 
BioResearch Open Access  2014;3(4):176-182.
Abstract
We have established a lung adenocarcinoma cell line, ETCC016, from lung pleural effusion of a male Singaporean Chinese with advanced lung adenocarcinoma. The subject smoked 20 cigarettes per day for more than 30 years. The cell line arose from spontaneous transformation of cells grown in a collagen-coated culture dish. Transformed characteristics of the cell line include the ability to reach high confluency in a culture dish, low cell doubling time, ability to form colonies in soft agar, and ability to form solid tumor in immune-compromised SCID mice. Immunostaining showed that the cells originated from lung epithelial cells. Genomic analysis revealed a large amount of chromosomal aberrations (gain and loss of genetic materials, and loss of heterozygosity [LOH]), indicative of a long history of smoking. The cells have four copies of epidermal growth factor receptor (EGFR) and three copies of MYC, but have lost one copy of the RB1 gene. LOH was detected in TP53 and BRAF genes. There is no anaplastic lymphoma kinase (ALK) gene rearrangement. The ETCC016 lung adenocarcinoma cell line has demonstrated susceptibility towards inhibitors specific for EGFR/HER2 and ALK targets, but resistance to MYC-specific inhibitor. This cell line will be a useful model for further understanding of lung adenocarcinoma.
doi:10.1089/biores.2014.0011
PMCID: PMC4120652  PMID: 25126481
adenocarcinoma; ALK; array CGH; chemosensitivity; EGFR; FISH; lung; mutation
17.  Integrative Genomic Analyses Identify BRF2 as a Novel Lineage-Specific Oncogene in Lung Squamous Cell Carcinoma 
PLoS Medicine  2010;7(7):e1000315.
William Lockwood and colleagues show that the focal amplification of a gene, BRF2, on Chromosome 8p12 plays a key role in squamous cell carcinoma of the lung.
Background
Traditionally, non-small cell lung cancer is treated as a single disease entity in terms of systemic therapy. Emerging evidence suggests the major subtypes—adenocarcinoma (AC) and squamous cell carcinoma (SqCC)—respond differently to therapy. Identification of the molecular differences between these tumor types will have a significant impact in designing novel therapies that can improve the treatment outcome.
Methods and Findings
We used an integrative genomics approach, combing high-resolution comparative genomic hybridization and gene expression microarray profiles, to compare AC and SqCC tumors in order to uncover alterations at the DNA level, with corresponding gene transcription changes, which are selected for during development of lung cancer subtypes. Through the analysis of multiple independent cohorts of clinical tumor samples (>330), normal lung tissues and bronchial epithelial cells obtained by bronchial brushing in smokers without lung cancer, we identified the overexpression of BRF2, a gene on Chromosome 8p12, which is specific for development of SqCC of lung. Genetic activation of BRF2, which encodes a RNA polymerase III (Pol III) transcription initiation factor, was found to be associated with increased expression of small nuclear RNAs (snRNAs) that are involved in processes essential for cell growth, such as RNA splicing. Ectopic expression of BRF2 in human bronchial epithelial cells induced a transformed phenotype and demonstrates downstream oncogenic effects, whereas RNA interference (RNAi)-mediated knockdown suppressed growth and colony formation of SqCC cells overexpressing BRF2, but not AC cells. Frequent activation of BRF2 in >35% preinvasive bronchial carcinoma in situ, as well as in dysplastic lesions, provides evidence that BRF2 expression is an early event in cancer development of this cell lineage.
Conclusions
This is the first study, to our knowledge, to show that the focal amplification of a gene in Chromosome 8p12, plays a key role in squamous cell lineage specificity of the disease. Our data suggest that genetic activation of BRF2 represents a unique mechanism of SqCC lung tumorigenesis through the increase of Pol III-mediated transcription. It can serve as a marker for lung SqCC and may provide a novel target for therapy.
Please see later in the article for the Editors' Summary
Editors' Summary
Background
Lung cancer is the commonest cause of cancer-related death. Every year, 1.3 million people die from this disease, which is mainly caused by smoking. Most cases of lung cancer are “non-small cell lung cancers” (NSCLCs). Like all cancers, NSCLC starts when cells begin to divide uncontrollably and to move round the body (metastasize) because of changes (mutations) in their genes. These mutations are often in “oncogenes,” genes that, when activated, encourage cell division. Oncogenes can be activated by mutations that alter the properties of the proteins they encode or by mutations that increase the amount of protein made from them, such as gene amplification (an increase in the number of copies of a gene). If NSCLC is diagnosed before it has spread from the lungs (stage I disease), it can be surgically removed and many patients with stage I NSCLC survive for more than 5 years after their diagnosis. Unfortunately, in more than half of patients, NSCLC has metastasized before it is diagnosed. This stage IV NSCLC can be treated with chemotherapy (toxic chemicals that kill fast-growing cancer cells) but only 2% of patients with stage IV lung cancer are alive 5 years after diagnosis.
Why Was This Study Done?
Traditionally, NSCLC has been regarded as a single disease in terms of treatment. However, emerging evidence suggests that the two major subtypes of NSCLC—adenocarcinoma and squamous cell carcinoma (SqCC)—respond differently to chemotherapy. Adenocarcinoma and SqCC start in different types of lung cell and experts think that for each cell type in the body, specific combinations of mutations interact with the cell type's own unique characteristics to provide the growth and survival advantage needed for cancer development. If this is true, then identifying the molecular differences between adenocarcinoma and SqCC could provide targets for more effective therapies for these major subtypes of NSCLC. Amplification of a chromosome region called 8p12 is very common in NSCLC, which suggests that an oncogene that drives lung cancer development is present in this chromosome region. In this study, the researchers investigate this possibility by looking for an amplified gene in the 8p12 chromosome region that makes increased amounts of protein in lung SqCC but not in lung adenocarcinoma.
What Did the Researchers Do and Find?
The researchers used a technique called comparative genomic hybridization to show that focal regions of Chromosome 8p are amplified in about 40% of lung SqCCs, but that DNA loss in this region is the most common alteration in lung adenocarcinomas. Ten genes in the 8p12 chromosome region were expressed at higher levels in the SqCC samples that they examined than in adenocarcinoma samples, they report, and overexpression of five of these genes correlated with amplification of the 8p12 region in the SqCC samples. Only one of the genes—BRF2—was more highly expressed in squamous carcinoma cells than in normal bronchial epithelial cells (the cell type that lines the tubes that take air into the lungs and from which SqCC develops). Artificially induced expression of BRF2 in bronchial epithelial cells made these normal cells behave like tumor cells, whereas reduction of BRF2 expression in squamous carcinoma cells made them behave more like normal bronchial epithelial cells. Finally, BRF2 was frequently activated in two early stages of squamous cell carcinoma—bronchial carcinoma in situ and dysplastic lesions.
What Do These Findings Mean?
Together, these findings show that the focal amplification of chromosome region 8p12 plays a role in the development of lung SqCC but not in the development of lung adenocarcinoma, the other major subtype of NSCLC. These findings identify BRF2 (which encodes a RNA polymerase III transcription initiation factor, a protein that is required for the synthesis of RNA molecules that help to control cell growth) as a lung SqCC-specific oncogene and uncover a unique mechanism for lung SqCC development. Most importantly, these findings suggest that genetic activation of BRF2 could be used as a marker for lung SqCC, which might facilitate the early detection of this type of NSCLC and that BRF2 might provide a new target for therapy.
Additional Information
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1000315.
The US National Cancer Institute provides detailed information for patients and professionals about all aspects of lung cancer, including information on non-small cell carcinoma (in English and Spanish)
Cancer Research UK also provides information about lung cancer and information on how cancer starts
MedlinePlus has links to other resources about lung cancer (in English and Spanish)
doi:10.1371/journal.pmed.1000315
PMCID: PMC2910599  PMID: 20668658
18.  Identification of KIF5B-RET and GOPC-ROS1 fusions in lung adenocarcinomas through a comprehensive mRNA-based screen for tyrosine kinase fusions 
Background
The mutually exclusive pattern of the major driver oncogenes in lung cancer suggests that other mutually exclusive oncogenes exist. We performed a systematic search for tyrosine kinase (TK) fusions by screening all TKs for aberrantly high RNA expression levels of the 3′ kinase domain (KD) exons relative to more 5′ exons.
Methods
We studied 69 patients (including 5 never smokers and 64 current or former smokers) with lung adenocarcinoma negative for all major mutations in KRAS, EGFR, BRAF, MEK1, and HER2, and for ALK fusions (termed “pan-negative”). A NanoString-based assay was designed to query the transcripts of 90 TKs at two points: 5′ to the KD and within the KD or 3′ to it. Tumor RNAs were hybridized to the NanoString probes and analyzed for outlier 3′ to 5′ expression ratios. Presumed novel fusion events were studied by rapid amplification of cDNA ends (RACE) and confirmatory RT-PCR and FISH.
Results
We identified 1 case each of aberrant 3′ to 5′ ratios in ROS1 and RET. RACE isolated a GOPC-ROS1 (FIG-ROS1) fusion in the former and a KIF5B-RET fusion in the latter, both confirmed by RT-PCR. The RET rearrangement was also confirmed by FISH. The KIF5B-RET patient was one of only 5 never smokers in this cohort.
Conclusion
The KIF5B-RET fusion defines an additional subset of lung cancer with a potentially targetable driver oncogene enriched in never smokers with “pan-negative” lung adenocarcinomas. We also report for the first time in lung cancer the GOPC-ROS1 fusion previously characterized in glioma.
doi:10.1158/1078-0432.CCR-12-0838
PMCID: PMC4234119  PMID: 23052255
lung cancer; kinase; gene fusion; RET; ROS1; ALK
19.  Evidence that synthetic lethality underlies the mutual exclusivity of oncogenic KRAS and EGFR mutations in lung adenocarcinoma 
eLife  null;4:e06907.
Human lung adenocarcinomas (LUAD) contain mutations in EGFR in ∼15% of cases and in KRAS in ∼30%, yet no individual adenocarcinoma appears to carry activating mutations in both genes, a finding we have confirmed by re-analysis of data from over 600 LUAD. Here we provide evidence that co-occurrence of mutations in these two genes is deleterious. In transgenic mice programmed to express both mutant oncogenes in the lung epithelium, the resulting tumors express only one oncogene. We also show that forced expression of a second oncogene in human cancer cell lines with an endogenous mutated oncogene is deleterious. The most prominent features accompanying loss of cell viability were vacuolization, other changes in cell morphology, and increased macropinocytosis. Activation of ERK, p38 and JNK in the dying cells suggests that an overly active MAPK signaling pathway may mediate the phenotype. Together, our findings indicate that mutual exclusivity of oncogenic mutations may reveal unexpected vulnerabilities and therapeutic possibilities.
DOI: http://dx.doi.org/10.7554/eLife.06907.001
eLife digest
A person develops cancer when changes in a cell's DNA (called mutations) allow the cell to grow rapidly and spread around the body. The mutated genes are often involved in controlling the growth of cells, such as two genes called EGFR and KRAS, which are associated with forms of lung cancer.
In a type of lung cancer called adenocarcinoma, the KRAS gene is mutated in about one-third of tumors and the EGFR gene is mutated in about 15%. However, the two mutations rarely or never occur in the same tumor. This could be because the effects of the mutations overlap, so that cells with both mutations have no advantages over cells with just one. Alternatively, it is possible that having both mutations may be harmful to tumor cells.
Here, Unni, Lockwood et al. analyzed genetic data from over 600 lung tumors and confirmed that none of them have cancer-causing mutations in both KRAS and EGFR. Then, Unni, Lockwood et al. carried out experiments using genetically engineered mice with mutated forms of both KRAS and EGFR that are activated by a drug called doxycycline. As expected, the mice developed lung tumors when exposed to the drug, but these tumors didn't grow any faster than mouse tumors that had mutations in only one of the genes. In the mice with both mutant genes, only one of the two genes was actually active in most of the tumor cells.
Unni, Lockwood et al. manipulated human lung tumor cells in the laboratory so that the cells had mutated versions of both genes. These cells developed serious abnormalities and died, which may be due to the over-activation of a communication pathway within the cells called MAPK signaling. The next challenges are to understand why the combination of these two mutant genes kills these cancer cells and to look for other combinations of mutations that can be toxic to cancer cells. In the future, it might be possible to develop drugs that can mimic the effects of these gene mutations to treat cancers.
DOI: http://dx.doi.org/10.7554/eLife.06907.002
doi:10.7554/eLife.06907
PMCID: PMC4478584  PMID: 26047463
lung cancer; oncogenes; synthetic lethality; human; mouse
20.  Network modeling of the transcriptional effects of copy number aberrations in glioblastoma 
DNA copy number aberrations (CNAs) are a characteristic feature of cancer genomes. In this work, Rebecka Jörnsten, Sven Nelander and colleagues combine network modeling and experimental methods to analyze the systems-level effects of CNAs in glioblastoma.
We introduce a modeling approach termed EPoC (Endogenous Perturbation analysis of Cancer), enabling the construction of global, gene-level models that causally connect gene copy number with expression in glioblastoma.On the basis of the resulting model, we predict genes that are likely to be disease-driving and validate selected predictions experimentally. We also demonstrate that further analysis of the network model by sparse singular value decomposition allows stratification of patients with glioblastoma into short-term and long-term survivors, introducing decomposed network models as a useful principle for biomarker discovery.Finally, in systematic comparisons, we demonstrate that EPoC is computationally efficient and yields more consistent results than mRNA-only methods, standard eQTL methods, and two recent multivariate methods for genotype–mRNA coupling.
Gains and losses of chromosomal material (DNA copy number aberrations; CNAs) are a characteristic feature of cancer genomes. At the level of a single locus, it is well known that increased copy number (gene amplification) typically leads to increased gene expression, whereas decreased copy number (gene deletion) leads to decreased gene expression (Pollack et al, 2002; Lee et al, 2008; Nilsson et al, 2008). However, CNAs also affect the expression of genes located outside the amplified/deleted region itself via indirect mechanisms. To fully understand the action of CNAs, it is therefore necessary to analyze their action in a network context. Toward this goal, improved computational approaches will be important, if not essential.
To determine the global effects on transcription of CNAs in the brain tumor glioblastoma, we develop EPoC (Endogenous Perturbation analysis of Cancer), a computational technique capable of inferring sparse, causal network models by combining genome-wide, paired CNA- and mRNA-level data. EPoC aims to detect disease-driving copy number aberrations and their effect on target mRNA expression, and stratify patients into long-term and short-term survivors. Technically, EPoC relates CNA perturbations to mRNA responses by matrix equations, derived from a steady-state approximation of the transcriptional network. Patient prognostic scores are obtained from singular value decompositions of the network matrix. The models are constructed by solving a large-scale, regularized regression problem.
We apply EPoC to glioblastoma data from The Cancer Genome Atlas (TCGA) consortium (186 patients). The identified CNA-driven network comprises 10 672 genes, and contains a number of copy number-altered genes that control multiple downstream genes. Highly connected hub genes include well-known oncogenes and tumor supressor genes that are frequently deleted or amplified in glioblastoma, including EGFR, PDGFRA, CDKN2A and CDKN2B, confirming a clear association between these aberrations and transcriptional variability of these brain tumors. In addition, we identify a number of hub genes that have previously not been associated with glioblastoma, including interferon alpha 1 (IFNA1), myeloid/lymphoid or mixed-lineage leukemia translocated to 10 (MLLT10, a well-known leukemia gene), glutamate decarboxylase 2 GAD2, a postulated glutamate receptor GPR158 and Necdin (NDN). Furthermore, we demonstrate that the network model contains useful information on downstream target genes (including stem cell regulators), and possible drug targets.
We proceed to explore the validity of a small network region experimentally. Introducing experimental perturbations of NDN and other targets in four glioblastoma cell lines (T98G, U-87MG, U-343MG and U-373MG), we confirm several predicted mechanisms. We also demonstrate that the TCGA glioblastoma patients can be stratified into long-term and short-term survivors, using our proposed prognostic scores derived from a singular vector decomposition of the network model. Finally, we compare EPoC to existing methods for mRNA networks analysis and expression quantitative locus methods, and demonstrate that EPoC produces more consistent models between technically independent glioblastoma data sets, and that the EPoC models exhibit better overlap with known protein–protein interaction networks and pathway maps.
In summary, we conclude that large-scale integrative modeling reveals mechanistically and prognostically informative networks in human glioblastoma. Our approach operates at the gene level and our data support that individual hub genes can be identified in practice. Very large aberrations, however, cannot be fully resolved by the current modeling strategy.
DNA copy number aberrations (CNAs) are a hallmark of cancer genomes. However, little is known about how such changes affect global gene expression. We develop a modeling framework, EPoC (Endogenous Perturbation analysis of Cancer), to (1) detect disease-driving CNAs and their effect on target mRNA expression, and to (2) stratify cancer patients into long- and short-term survivors. Our method constructs causal network models of gene expression by combining genome-wide DNA- and RNA-level data. Prognostic scores are obtained from a singular value decomposition of the networks. By applying EPoC to glioblastoma data from The Cancer Genome Atlas consortium, we demonstrate that the resulting network models contain known disease-relevant hub genes, reveal interesting candidate hubs, and uncover predictors of patient survival. Targeted validations in four glioblastoma cell lines support selected predictions, and implicate the p53-interacting protein Necdin in suppressing glioblastoma cell growth. We conclude that large-scale network modeling of the effects of CNAs on gene expression may provide insights into the biology of human cancer. Free software in MATLAB and R is provided.
doi:10.1038/msb.2011.17
PMCID: PMC3101951  PMID: 21525872
cancer biology; cancer genomics; glioblastoma
21.  Correlation between familial cancer history and epidermal growth factor receptor mutations in Taiwanese never smokers with non-small cell lung cancer: a case-control study 
Journal of Thoracic Disease  2015;7(3):281-287.
Background
Lung cancer is a leading cause of cancer deaths in the world. Cigarette smoking remains a prominent risk factor, but lung cancer incidence has been increasing in never smokers. Genetic abnormalities including epidermal growth factor receptor (EGFR) mutations predominate in never smoking lung cancer patients. Furthermore, familial aggregations of patients with these mutations reflect heritable susceptibility to lung cancer. The correlation between familial cancer history and EGFR mutations in never smokers with lung cancer requires investigation.
Methods
This was a retrospective case-control study that evaluated the prevalence of EGFR mutations in lung cancer patients with familial cancer history. Never smokers with lung cancer treated at a hospital in Taiwan between April 2012 and May 2014 were evaluated. Inclusion criteria were never smokers with non-small cell lung cancer (NSCLC). Exclusion criteria involved patients without records of familial cancer history or tumor genotype.
Results
This study included 246 never smokers with lung cancer. The study population mainly involved never smoking women with a mean age of 60 years, and the predominant tumor histology was adenocarcinoma. Lung cancer patients with familial cancer history had an increased prevalence of EGFR mutations compared to patients without family history [odds ratio (OR): 5.9; 95% confidence interval (CI): 3.3-10.6; P<0.001]. Specifically, 57 out of 85 cancer patients (67%) with familial cancer history had these mutations, while 41 out of 161 patients (25%) without family history harbored mutations. Subgroup analysis also revealed that patients with familial lung cancer history had stronger association with EGFR mutations (OR: 7.5; 95% CI: 3.4-16.3; P<0.001) compared to patients with family history of non-pulmonary cancers (OR: 5.0; 95% CI: 2.5-10.0; P<0.001).
Conclusions
The study demonstrated an increased prevalence of EGFR mutations in Taiwanese never smoking lung cancer patients with familial cancer history. Moreover, a sizable proportion of never smoking cancer patients harbored these mutations. These observations have implications for the treatment of lung cancer in never smokers.
doi:10.3978/j.issn.2072-1439.2015.02.03
PMCID: PMC4387390  PMID: 25922704
Epidermal growth factor receptor (EGFR); family; lung neoplasms; non-small cell lung cancer (NSCLC); risk factors
22.  Molecular epidemiology of lung cancer and geographic variations with special reference to EGFR mutations 
Lung cancer is a leading cause of cancer-related mortality in many countries. Although recent advances in targeted therapy against driver oncogenes have significantly improved patient outcome, cure of this disease is still exceptional. Although tobacco is a known cause of lung cancer, not all smokers develop lung cancer, and conversely many patients, especially Asian female patients with lung cancer, are lifetime never-smokers. Therefore, efforts to understand the basis for different susceptibilities to lung cancer among individuals with different genetic, biologic, ethnic, and social backgrounds are important to help develop effective preventive measures. Lung cancer in never-smokers has many different characteristics to lung cancer in smokers, such as adenocarcinoma predominance and high frequency of epidermal growth factor receptor (EGFR) mutation yet low number of genetic changes. Epidemiologic studies suggest that East Asians are more susceptible to smoking-unrelated lung cancer but less susceptible to smoking-related lung cancer compared with Caucasians. Mutations in the EGFR gene are more common in Asian females and never-smokers. Our case-control study suggests that EGFR mutation occurs independent of smoking, and that the apparent low frequency of EGFR mutations in smokers may be the result of dilution by smoking-related lung cancer. The frequencies of three EGFR gene polymorphisms associated with increased protein expression are significantly different between East Asians and Caucasians, favoring lower protein expression in East Asians. Although these may be associated with preferred expression of the EGFR mutant allele, it is difficult to explain the frequent EGFR mutation in Asian patients. Genome wide association studies (GWAS) revealed several loci related to lung cancer susceptibility. In the future, GWAS may identify loci that are specifically related to EGFR-targeted carcinogenesis, leading to identification of carcinogens that induce EGFR mutations and effective prevention measures.
doi:10.3978/j.issn.2218-6751.2014.08.04
PMCID: PMC4367697  PMID: 25806302
Susceptibility; tobacco smoke; never smokers; ethnic difference; adenocarcinoma; genome wide association studies (GWAS)
23.  Amplification and Overexpression of Hsa-miR-30b, Hsa-miR-30d and KHDRBS3 at 8q24.22-q24.23 in Medulloblastoma 
PLoS ONE  2009;4(7):e6159.
Background
Medulloblastoma is the most common malignant brain tumour of childhood. The identification of critical genes involved in its pathogenesis will be central to advances in our understanding of its molecular basis, and the development of improved therapeutic approaches.
Methodology/Principal Findings
We performed a SNP-array based genome-wide copy number analysis in medulloblastoma cell lines, to identify regions of genomic amplification and homozygous deletion, which may harbour critical disease genes. A series of novel and established medulloblastoma defects were detected (MYC amplification (n = 4), 17q21.31 high-level gain (n = 1); 9p21.1–p21.3 (n = 1) and 6q23.1 (n = 1) homozygous deletion). Most notably, a novel recurrent region of genomic amplification at 8q24.22–q24.23 was identified (n = 2), and selected for further investigation. Additional analysis by interphase fluorescence in situ hybridisation (iFISH), PCR-based mapping and SNP-array revealed this novel amplification at 8q24.22–q24.23 is independent of MYC amplification at 8q24.21, and is unique to medulloblastoma in over 800 cancer cell lines assessed from different tumour types, suggesting it contains key genes specifically involved in medulloblastoma development. Detailed mapping identified a 3Mb common minimal region of amplification harbouring 3 coding genes (ZFAT1, LOC286094, KHDRBS3) and two genes encoding micro-RNAs (hsa-miR-30b, hsa-miR-30d). Of these, only expression of hsa-miR-30b, hsa-miR-30d and KHDRBS3 correlated with copy number status, and all three of these transcripts also displayed evidence of elevated expression in sub-sets of primary medulloblastomas, measured relative to the normal cerebellum.
Conclusions/Significance
These data implicate hsa-miR-30b, hsa-miR-30d and KHDRBS3 as putative oncogenic target(s) of a novel recurrent medulloblastoma amplicon at 8q24.22–q24.23. Our findings suggest critical roles for these genes in medulloblastoma development, and further support the contribution of micro-RNA species to medulloblastoma pathogenesis.
doi:10.1371/journal.pone.0006159
PMCID: PMC2702821  PMID: 19584924
24.  Smoking status and self-reported race affect the frequency of clinically-relevant oncogenic alterations in non-small-cell lung cancers at a United States-based academic medical practice 
Introduction
The identification of somatic genomic aberrations in non-small-cell lung cancer (NSCLC) is part of evidence-based practice guidelines for care of patients with NSCLC. We sought to establish the frequency and correlates of these changes in routine patient-tumor sample pairs.
Methods
Clinicopathologic data and tumor genotype were retrospectively compiled and analyzed from an overall cohort of 381 patient-tumor samples.
Results
Of these patients, 75.9% self-reported White race, 13.1% Asian, 6.5% Black, 27.8% were never-smokers, 54.9% former-smokers and 17.3% current-smokers. The frequency of EGFR mutations was 23.9%(86/359), KRAS mutations 34.2%(71/207) and ALK FISH positivity 9.1%(23/252) in tumor samples, and almost all had mutually exclusive results for these oncogenes. In tumors from White, Black and Asian patients, the frequencies of EGFR mutations were 18.4%, 18.2% and 62%, respectively; of ALK FISH positivity 7.81%, 0% and 14.8%, respectively; and of KRAS mutations 41.6%, 20% and 0%. These patterns changed significant with increasing pack-year history of smoking. In White patients, the frequencies of EGFR mutations and ALK FISH positivity decreased with increasing pack-year cohorts; while the frequencies of KRAS mutations increased. Interestingly, in Asian patients the frequencies of EGFR mutations were similar in never smokers and in the cohorts with less then 45pack-year histories of smoking and only decreased in the 45pack-year plus cohort.
Conclusions
The frequencies of somatic EGFR, KRAS, and ALK gene abnormalities using routine lung cancer tissue samples from our United States-based academic medical practice reflect the diverse ethnicity (with a higher frequency of EGFR mutations in Asian patients) and smoking patterns (with an inverse correlation between EGFR mutation and ALK rearrangement) of our tested population. These results may help other medical practices appreciate the expected results from introduction of routine tumor genotyping techniques into their day-to-day care of NSCLC.
doi:10.1016/j.lungcan.2013.07.013
PMCID: PMC3800098  PMID: 23932486
lung cancer; non-small-cell lung cancer; never smokers; epidermal growth factor receptor; EGFR; anaplastic lymphoma kinase; ALK; KRAS; tumor genotype; ethnicipty; Asian; White; Black
25.  A Genome-Wide Screen for Promoter Methylation in Lung Cancer Identifies Novel Methylation Markers for Multiple Malignancies  
PLoS Medicine  2006;3(12):e486.
Background
Promoter hypermethylation coupled with loss of heterozygosity at the same locus results in loss of gene function in many tumor cells. The “rules” governing which genes are methylated during the pathogenesis of individual cancers, how specific methylation profiles are initially established, or what determines tumor type-specific methylation are unknown. However, DNA methylation markers that are highly specific and sensitive for common tumors would be useful for the early detection of cancer, and those required for the malignant phenotype would identify pathways important as therapeutic targets.
Methods and Findings
In an effort to identify new cancer-specific methylation markers, we employed a high-throughput global expression profiling approach in lung cancer cells. We identified 132 genes that have 5′ CpG islands, are induced from undetectable levels by 5-aza-2′-deoxycytidine in multiple non-small cell lung cancer cell lines, and are expressed in immortalized human bronchial epithelial cells. As expected, these genes were also expressed in normal lung, but often not in companion primary lung cancers. Methylation analysis of a subset (45/132) of these promoter regions in primary lung cancer (n = 20) and adjacent nonmalignant tissue (n = 20) showed that 31 genes had acquired methylation in the tumors, but did not show methylation in normal lung or peripheral blood cells. We studied the eight most frequently and specifically methylated genes from our lung cancer dataset in breast cancer (n = 37), colon cancer (n = 24), and prostate cancer (n = 24) along with counterpart nonmalignant tissues. We found that seven loci were frequently methylated in both breast and lung cancers, with four showing extensive methylation in all four epithelial tumors.
Conclusions
By using a systematic biological screen we identified multiple genes that are methylated with high penetrance in primary lung, breast, colon, and prostate cancers. The cross-tumor methylation pattern we observed for these novel markers suggests that we have identified a partial promoter hypermethylation signature for these common malignancies. These data suggest that while tumors in different tissues vary substantially with respect to gene expression, there may be commonalities in their promoter methylation profiles that represent targets for early detection screening or therapeutic intervention.
John Minna and colleagues report that a group of genes are commonly methylated in primary lung, breast, colon, and prostate cancer.
Editors' Summary
Background.
Tumors or cancers contain cells that have lost many of the control mechanisms that normally regulate their behavior. Unlike normal cells, which only divide to repair damaged tissues, cancer cells divide uncontrollably. They also gain the ability to move round the body and start metastases in secondary locations. These changes in behavior result from alterations in their genetic material. For example, mutations (permanent changes in the sequence of nucleotides in the cell's DNA) in genes known as oncogenes stimulate cells to divide constantly. Mutations in another group of genes—tumor suppressor genes—disable their ability to restrain cell growth. Key tumor suppressor genes are often completely lost in cancer cells. But not all the genetic changes in cancer cells are mutations. Some are “epigenetic” changes—chemical modifications of genes that affect the amount of protein made from them. In cancer cells, methyl groups are often added to CG-rich regions—this is called hypermethylation. These “CpG islands” lie near gene promoters—sequences that control the transcription of DNA into RNA, the template for protein production—and their methylation switches off the promoter. Methylation of the promoter of one copy of a tumor suppressor gene, which often coincides with the loss of the other copy of the gene, is thought to be involved in cancer development.
Why Was This Study Done?
The rules that govern which genes are hypermethylated during the development of different cancer types are not known, but it would be useful to identify any DNA methylation events that occur regularly in common cancers for two reasons. First, specific DNA methylation markers might be useful for the early detection of cancer. Second, identifying these epigenetic changes might reveal cellular pathways that are changed during cancer development and so identify new therapeutic targets. In this study, the researchers have used a systematic biological screen to identify genes that are methylated in many lung, breast, colon, and prostate cancers—all cancers that form in “epithelial” tissues.
What Did the Researchers Do and Find?
The researchers used microarray expression profiling to examine gene expression patterns in several lung cancer and normal lung cell lines. In this technique, labeled RNA molecules isolated from cells are applied to a “chip” carrying an array of gene fragments. Here, they stick to the fragment that represents the gene from which they were made, which allows the genes that the cells express to be catalogued. By comparing the expression profiles of lung cancer cells and normal lung cells before and after treatment with a chemical that inhibits DNA methylation, the researchers identified genes that were methylated in the cancer cells—that is, genes that were expressed in normal cells but not in cancer cells unless methylation was inhibited. 132 of these genes contained CpG islands. The researchers examined the promoters of 45 of these genes in lung cancer cells taken straight from patients and found that 31 of the promoters were methylated in tumor tissues but not in adjacent normal tissues. Finally, the researchers looked at promoter methylation of the eight genes most frequently and specifically methylated in the lung cancer samples in breast, colon, and prostate cancers. Seven of the genes were frequently methylated in both lung and breast cancers; four were extensively methylated in all the tumor types.
What Do These Findings Mean?
These results identify several new genes that are often methylated in four types of epithelial tumor. The observation that these genes are methylated in multiple independent tumors strongly suggests, but does not prove, that loss of expression of the proteins that they encode helps to convert normal cells into cancer cells. The frequency and diverse patterning of promoter methylation in different tumor types also indicates that methylation is not a random event, although what controls the patterns of methylation is not yet known. The identification of these genes is a step toward building a promoter hypermethylation profile for the early detection of human cancer. Furthermore, although tumors in different tissues vary greatly with respect to gene expression patterns, the similarities seen in this study in promoter methylation profiles might help to identify new therapeutic targets common to several cancer types.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030486.
US National Cancer Institute, information for patients on understanding cancer
CancerQuest, information provided by Emory University about how cancer develops
Cancer Research UK, information for patients on cancer biology
Wikipedia pages on epigenetics (note that Wikipedia is a free online encyclopedia that anyone can edit)
The Epigenome Network of Excellence, background information and latest news about epigenetics
doi:10.1371/journal.pmed.0030486
PMCID: PMC1716188  PMID: 17194187

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