Integrated Oncogenomic Profiling Of Copy Numbers And Gene Expression In Lung Adenocarcinomas Without EGFR Mutations Or ALK Fusion

IntroductionWith the highest mortality rate among all cancers in China and many other countries,lung cancer annually causes approximately 1.38 million deaths worldwide.Lung cancer is histologically sub-classified into four categories:lung adenocarcinoma(LUAD),squamous cell carcinoma(LUSC),large cell carcinoma,and small cell carcinoma of the lung.LUAD,an epithelial cancer of glandular origin,is the most common pathological subtype of non-small cell lung cancer(NSCLC),even for never-smokers.Since LUAD is a heterogeneous tumor with diverse molecular,clinical,and pathological characteristics,the identification of oncogenic drivers has increased understanding of LUAD biology.Recognition of molecular alterations in LUAD has facilitated tailored therapy targeting these alterations and has ushered in the era of "personalized"oncologic medicine.For example,LUAD patients with activating mutations of the.epidermal growth factor receptor gene(EGFR)have a better response to EGFR tyrosine kinase inhibitors(TKIs)than those without EGFR mutations,and rearrangement of the anaplastic lymphoma kinase gene(ALK)is the best predictor of LUAD response to the ALK TKI crizotinib.These facts indicate that therapeutic effectiveness is linked to the presence of specific oncogenomic alterations.However,patients with EGFR mutations or ALK fusion account for only one-third of patients diagnosed with LUAD,which indicates the necessity of understanding of the genetic basis for LUAD without EGFR mutations or ALK fusion.The molecular mechanisms underlying LUAD development are unclear,and the heterogeneous nature of lung cancer makes it difficult to achieve an understanding.It is not known if LUAD patients with EGFR/ALT alterations harbor distinct genetic characteristics compared to those without such alterations.Moreover,little is understood about the correlation of copy numbers and gene expression of top-ranked genes in lung cancer,especially for LUAD without EGFR mutations or ALK fusion.In addition,since the development and progression of LUAD are consequences of gene-gene interactions and regulatory coordination,the identification of therapeutic targets in regulatory pathways can provide insight into the etiology and pathogenesis of LUAD.Whether and how these lung cancer-associated genes are coordinated to affect cellular functions remain largely unexplored.In the present study,we retrospectively analyzed genomic sequencing data for LUAD and LUSC patients.Oncogenomic alterations of 23 top candidate genes were assessed to explore the similarities and differences between these two lung cancer types.The copy numbers of these candidate genes were further evaluated in ten micro-dissected LUADs without EGFR mutations or ALK fusion.For a few genes,further exploration of the associations between copy number and gene expression was conducted.This study revealed a concordant change of PTEN at both genomic DNA and mRNA levels in LUAD without EGFR mutation or ALK fusion.The results provide an approach to validating molecules involved in lung carcinogenesis and a basis for identifying pathways as targets for the treatment of LUAD.Methods and MaterialsSpecimens,including tumor tissue and tumor-distant normal lung tissue from the same patient,were collected from ten patients with LUAD who underwent primary surgery between January 2009 and June 2012 at the First Hospital of Jiamusi University.Informed consent was obtained from all subjects in accordance with the requirements of the Institutional Review Board of the First Hospital of Jiamusi University.Primary LUADs and tumor-distant normal lung tissue specimens were obtained from surgically resected lung tissues,which were preserved as formalin-fixed,paraffin-embedded sections,for biomarker and pathologic analyses.Lung cancer specimens obtained from a clinical molecular diagnostic laboratory were tested for EGFR mutations and ALK fusion.Genomic DNA and total RNA were extracted from formalin-fixed and paraffin-embedded tissue sections.Mutations in EGFR were assessed on genomic DNA,whereas ALK fusions were determined with total RNA.The EGFR mutations were analyzed by fluorescent real-time PCR using Human EGFR Mutation Detection Kits(AmoyDx,Xiamen,China).ALK fusion variants were detected by multiplex One-step RT-PCR using Human ALK Gene Fusions Detection Kits(AmoyDx).RT-PCR was performed on a 7500 Real Time PCR System(Thermo Fisher Scientific,Waltham,MA,USA),and the absence of EGFR mutations and ALK fusion was verified by direct sequencing of PCR and RT-PCR products as previously reported.For analysis of gene copy number and gene expression,cells(5 x 103)were laser-capture micro-dissected from target tissue sections using.the Arcturus PixCell II system(Thermo Fisher Scientific)with an Olympus IX-50 microscope as described previously.RNA was extracted with PicoPure RNA extraction kits(Thermo Fisher Scientific)and amplified by RT-PCR.Genomic DNA was extracted from micro-dissected tissue using PicoPure DNA Isolation kits(Thermo Fisher Scientific).The concentrations were measured with a NanoDrop 2000 spectrophotometer(Thermo Fisher Scientific).Quantitative analysis of copy numbers was conducted by QIAGEN qBiomarker Copy Number PCR assays based on a 7500 Real Time PCR System(Thermo Fisher Scientific)as previously described.Relative gene copy numbers for each specimen were calculated as 2 × Tcopy number(tumor copy number/MRef copy number)/Ncopy number(tumor-distant normal tissue copy number/MRef copy number)from the same patient.Quantitative PCR(qPCR)of gene expression was performed with a 7500 Real Time PCR System using Power SYBR Green Master Mixture(Thermo Fisher Scientific).Reverse transcription was accomplished with random hemaxmer primers and Superscript II Reverse Transcriptase kits(Thermo Fisher Scientific).Fold changes were calculated according to the AACT method.For relative gene expression assays,the endogenous control gene was GAPDH.Retrieval of public genomic datasetsAll datasets used were from publically available sources,including Broad(Broad Institute of MIT and Harvard)and MSKCC(Memorial Sloan Kettering Cancer Center)or from various projects,including TCGA(the Cancer Genome Atlas-Cancer Genome)and TSP(the Tumor Sequencing Project).The whole-genome/exome or targeted sequencing data for tumors from LUAD and LUSC patients and the clinical and demographic information were extracted from these previous studies(all the sequencing data have been deposited online).For example,DNA sequencing data for the tumors and normal controls and the corresponding clinical information from LUAD patients with spirometry data available in the TCGA cohort were downloaded from gdac.broadinstitute.org.A cross-cancer alteration summary for 23 lung cancer-associated genes(selected by QIAGEN within Human Lung Cancer Copy Number PCR Array)was accomplished using c-Bioportal.Data mining was accomplished using cBioPortal for Cancer Genomics(cBioPortal for Cancer Genomics),available at http://www.cbioportal.org,to measure the incidence of conditions that are associated with alterations in these genes.The database query was based on deregulation(amplification,deletion,and mutants)of these genes.Tumordatasets were chosen in accordance with the publication guidelines(last updated on January 17,2014)of TCGA(tcga@mail.nih.gov).Generally,sequence variations were mapped to the corresponding genomic coordinates and inspected using the genome browsers of Ensembl(www.ensembl.org)and NCBI(www.ncbi.nlm.nih.gov).Mutational signature analyses were performed asdescribed previously.Differences of gene copy numbers between paired tumor tissue and tumor-distant normal tissue were tested using the nonparametric Mann-Whitney U test.Differences of gene expression between paired tumor tissue and tumor-distant normal tissue were tested by use of the paired two-sample t test.The associations between DNA copy numbers and gene expression levels were evaluated by Pearson's correlation test.Standard false discovery rate(FDR)and Bonferroni corrections were applied for the analysis of oncogenomic alterations.A P value<0.05 was considered as statistically significant,and all statistical tests were two-sided.The analyses were performed using SPSS 24,R package,and GraphPad Prism 6.0.Results1.Analysis of somatic alterations for cancer-associated genes in lungcancersAlmost half of the checked genes,including CCND1 CSMD1,EGFR,EMSY,MYC,PDGFRA,PIK3CA,PTPRD,RBI,REL,and ZNF217,displayed multiple alterations(amplification,deletion,and mutation occurring simultaneously).Although there were multiple alterations in the MYC gene only in LUSC,various alterations for genes CCND1,EMSY,PIK3CA,and ZNF217 were present in LUAD.LUAD harbored higher frequencies of amplification of CCND1,PIK3CA,FGFR1 REL,and ZNF217 than LUSC,whereas LUSC harbored higher frequencies of mutations of EMSY,PDGFRA,and REL.In LUAD,the PTEN gene,exhibited higher incidences of mutation and deletion.The VEGFA gene had mutations only in LUSC.The genomic alteration patterns varied among the genes.BCL2L1,CCND1,CCNE1,CDK4,ERBB2,FGFR1,MYC,PIK3CA,REL,VEGFA,and ZNF217 predominated in gene amplifications;CSMD1EGFR,MET,PDGFRA,PRDM1,PTPRD,and RB1 largely exhibited gene mutations;other genes(CDKN2A,FHIT,and PTEN)had more deletions.2.Clinicopathological characteristics of the patients with LUAD without EGFR mutations or ALK fusionThese patients included five men and five women who had a median age of 55 years(range:46-72 years).Six patients were never smokers and four were smokers.All tumors from these.patients were negative for EGFR mutations and ALK fusion.The pathological stages were "Tumor Stage ?"(n=7)and "Tumor Stage ?"(n=3),with "Moderate" tumor grade predominating(n=5).Six patients had tumor invasion of local lymph nodes.3.Comparative quantification of gene copy numbers in LUAD tumors and corresponding normal lung tissuesMost of the 23 genes did not show significant changes in CNAs,compared with those for normal lung tissues.Three tumor suppressor genes(PTEN,RBI,and PTPRD)and one oncogene(HMGA2)had lower copy numbers(all P<0.05)in LUAD tissues.4.Correlations of copy numbers and mRNA levels for genes with lower copy numbers in LUAD tumorsPTEN and RBI were significantly lower(both P<0.05)based on comparisons of mRNA levels in tumor and normal tissues.However,for the tumor suppressor gene PTPRD and the oncogene HMGA2,there were no significant changes in gene expression in LUAD tumors relative to normal tissues(n=6,P=0.506 for PTPRD and n=8,P=0.462 for HMGA2).For PTEN,there was a correlation between these two parameters(R2=0.827,P=0.003).5.Interactions among lung cancer-associated genes based on copy numbers in LUAD without EGFR mutations or ALK fusionThe correlations of EMSY/CCND1EMSY/PIK3CA,CCND1/CDKN2A,and CCND1/PIK3CA were strong(all P<0.001).The correlations of EMSY/CCND1,EMSY/PIK3CA,and CCND1/PIK3CA were all positive(all matrix values>0.9),as these genes alter cell cycle progression,promote cellular growth,and contribute to tumorigenesis.CDKN2A,which functions as a tumor suppressor gene,correlated negatively with CCND1(matrix value=-0.927,P<0.001).ConclusionsWe conducted an analysis of public datasets to assess the genomic alterations of 23 lung cancer-associated genes and found clues to the etiology and pathogenesis of LUAD and LUSC.We also assessed the oncogenomic profiles of surgically resected tumors without EGFR mutations or ALK fusion and tumor-distant normal lung tissues from LUAD patients and evaluated possible associations between these candidate genes.Our findings have implications for the molecular stratification and therapeutic targeting of LUAD without EGFR mutations or ALK fusion.This information also advances understanding of lung carcinogenesis as it relates to oncogenomic CNAs and gene expression and facilitates the identification of personal therapeutic strategies for patients with LUAD.