Significance and alterations of p14ARF gene of CDKN2A locus in gastric cancer

AIM:To investigate the significance and changes of p14^ARF gene in gastric cancer.METHODS:The tumors and gastric tissues neighboring carcinoma from 48 patients with gastric cancer were studied. The homozygous deletions, mutations, methylation of the CpG islands, and mRNA expression of p14^ARF gene were assessed by PCR, PCR-SSCP, PCR based methylation assay, and RT-PCR.RESULTS:①The homozygous deletion rate of p14^ARF was 31.3% (15/48), and no homozygous deletions were examined in all the gastric tissues neighboring tumor. ②There were no point mutations of p14^ARF in 33 gastric cancers without homozygous deletion and in the matched gastric tissues adjacent to tumor. ③Methylation rate of the CpG islands of p14^ARF was significantly higher in gastric cancers(47.9%, 23/48) than that in gastric tissues neighboring cancer (4.2%, 2/48)(P＜0.01).④ No expression of p14^ARF mRNA was detected in 45.8%(22/48) of gastric cancers. Moreover, the negative rate (100%, 3/3) of p14^ARF mRNA of gastric cancers with the combined methylation of exons 1β and 2 was significantly higher than that (15%, 3/20) of the sole methylation of exon2(P＜0.05). CONCLUSION:p14^ARF gene is frequently inactivated by homozygous deletion and methylation of the 5' CpG islands in gastric cancer, which may play an important role in the development of gastric cancer.     

Expression of SAL-like 4 protein in human prostate cancer tissue/cell lines and its relationship with clinical outcome

AIM:To evaluate the expression of SAL-like 4 (SALL4) protein in human prostate cancer cell lines and tissues, and to analyze the relationship between SALL4 expression and the clinicopathological parameters. METHODS:Immunofluorescence, RT-PCR and Western blotting were performed to detect the expression of SALL4 at mRNA and protein levels in 3 common prostate cancer cell lines LNCaP, DU145 and PC-3. The normal prostate epithelial cell line RWPE-1 was used for control. The protein levels of SALL4 in the tissues of benign prostate hyperplasia and prostate cancer tissues were determined by the method of immunohistochemistry. RESULTS:The SALL4 protein was predominantly expressed in the cytoplasm of the cells. The protein levels of SALL4 in 3 common prostate cancer cell lines were significantly higher than that in RWPE-1 cells. However, the mRNA level of SALL4 had no obvious difference among the 4 cell lines. Immunohistochemistry results showed that the expression level of SALL4 in the cancerous tissues was significantly higher than that in noncancerous (benign and normal) prostatic tissues. In addition, we found that the expression level of SALL4 in prostate cancer was significantly correlated with the Gleason score, clinical stage, prognosis estimation and tissue prostate-specific antigen (PSA) expression, but not associated with age, the level of serum total PSA, prostate volume and the expression of androgen receptor in the tissues of the patients. CONCLUSION: The over-expression of SALL4 protein may play an important role in the pathogenesis and progression of prostate cancer, and provides some reference indexes for estimating the malignancy, progression and prognosis of prostate cancer.     

Role of androgen receptor in prostate cancer cell proliferation by use of RNAi

AIM: To study the role of androgen receptor (AR) in hormone-dependent and hormone-independent prostate cancer cell proliferation by knocking down AR expression with adenovirus-delivered siRNA. METHODS: Four well-designed siRNAs were synthesized and inserted into the adenovirus plasmid pShuttle-H1-Ri. The recombinant pShuttle-H1-Ri-AR plasmid was then co-transfected with pcDNA-AR to HEK293 cell line and Western blot was used to detect the inhibitory efficiency of different siRNAs on AR expression. Recombinant adenovirus containing more efficient siRNAs were prepared and used to infect three different humane prostate cancer cell lines including LNCap、C4-2B and CWR22Rv1. The efficiency of knocking down AR expression was detected by Western blot. The effect of AR-knocking down on cell proliferation was detected by MTT colorimetric assay. RESULTS: All of the four designed siRNAs could knock down AR expression in transient co-transfection. Infecting with recombinant adenovirus containing more efficient siRNAs in hormone-dependent and hormone-independent prostate cancer cell lines specifically knocked down AR expression with high efficiency. Knocking down AR expression significantly decreased the proliferation rate in all these prostate cancer cells. CONCLUSION: The suppressed expression of AR in prostate cell lines mediated by siRNA could efficiently inhibit the cell proliferation, and these results show that AR plays an important role in the proliferation of hormone-dependent and hormone-independent prostate cancer cells. AR is an important therapeutic target for the treatment of prostate cancer.     

Relationship between mutated k-ras and biological behavior of colorectal cancer

AIM: To investigate mutations of oncogene k-ras in colorectal cancer tissues and the relationship between mutations of k-ras and biological behavior of colorectal carcinoma. METHODS: The specimens of 123 patients with colorectal cancer were collected. Real-time fluorescence quantitative PCR were performed to detect k-ras mutations at codon 12 and codon 13 of exon 1, and the results were analyzed with the corresponding clinical pathological data. RESULTS: Among 123 colorectal cancer cases, point mutations were detected in 53 cases (40.8%), point mutations at codon 12 were found in 42 (34.1%) cases, and 11(8.9%) cases at codon 13. No closely relationship between mutations of k-ras and tumor size, location, invasive depth and differentiation extent was observed. The rate of k-ras mutation in the cases with more invaded lymph nodes was higher than that in the cases without invaded lymph nodes (P<0.05), and the rate of k-ras gene mutation in the cases with hepatic metastases was higher than that in no hepatic metastases (P<0.05). The rate of k-ras gene mutation was higher in TNM staging Ⅲ/Ⅳ than that in Ⅰ/Ⅱ(P<0.05). CONCLUSION: Mutation of oncogene k-ras plays an important role in the carcinogenesis and development of colorectal cancer, and it is closely associated with invaded lymph notes and hepatic metastases, suggesting that mutation of k-ras indicates a poor prognosis.     

Role of androgen receptor in the pathogenesis of human prostate cancer

AIM: To investigate molecular mechanisms associated with the acquisition of androgen-independent growth of human prostate cancer during progression. METHODS: Upon continuously passaging, the androgen-independent LNCaP cell model was established. The expression of AR and PSA proteins in the course of prostate cancer progression was determined by Western blot. RESULTS: Upon continuous passage, the biological behavior of androgen-dependent parental LNCaP C-33 cells (passage number less than 33) was altered. LNCaP C-81 cells (passage number higher than 80) exhibited more aggressive growth and lower androgen-dependence than C-33 and C-51 cells (passage number between 33 and 80). C-81 cells secreted a higher level of PSA and the degree of DHT stimulation on PSA secretion was lower in C-81 cells than C-33 cells. The three LNCaP subclones expressed a similar level of total AR protein, but C-81 cells showed a characteristic loss of expression of the AR-B and increase in expression of the AR-A. CONCLUSION: Multiple factors, including the different expression of AR isoforms, contribute to the development of androgen-independent growth of prostatic carcinoma cells.     

Effect of curcumin on the proliferation and apoptosis in LNCap cells

AIM:To observe the effect of curcumin on the proliferation and apoptosis of prostate cancer cell line LNCap. METHODS:LNCap cells were treated with different doses (10 μmol/L, 20 μmol/L, 30 μmol/L, 40 μmol/L) of curcumin and its effects were analyzed in cell growth and apoptosis by microscope, MTT colorimetric assay and flow cytometry. The expression of prostate specific antigen (PSA) was measured by AXSYMTM system-chemical luciferase methods and expression of androgen receptor (AR) was detected by Western blotting. RESULTS:The results showed that curcumin inhibited the proliferation of LNCap cells. The cell growth was inhibited by curcumin in a dose dependent manner and the optimal dose and time was 40 μmol/L, 24 h. Curcumin induced apoptosis in LNCap cells, the most dramatic dose was 40 μmol/L curcumin, at this dose the apoptosis rate was 9.23%. Curcumin inhibited the expression of PSA in LNCap cells and the most dramatic dose and time was 40 μmol/L, 24 h. The PSA in this group was 20% of the control group. Curcumin inhibited the expression of AR on prostate cancer cells. CONCLUSION:Curcumin decreases proliferation and induces apoptosis in LNCap cells in a dose-dependent manner. Curcumin also inhibites the expression of PSA and AR on LNCap cells.     

Molecular mechanism of miR-29a inhibiting malignant phenotypes in prostate cancer cells

AIM:To investigate the biological functions of microRNA-29a (miR-29a) in prostate cancer and the molecular mechanism of miR-29a over-expression inhibiting malignant phenotypes of prostate cancer cells. METHODS:The levels of miR-29a expression in the prostate cancer tissues and cells were detected and analyzed using gene microarray and bioinformatics. The expression levels of miR-29a and lysine (K)-specific demethylase 4B (KDM4B) mRNA in prostate cancer tissues, paracarcinomatous tissues, 4 prostate cancer cell lines (PC3, DU145, LNCaP and ArCaP) and normal prostate epithelial cell line (RWPE-1) were measured by real-time PCR. PC3, DU145, LNCaP and ArCaP cells were transfected with pGenesil-1-miR-29a plasmid using transient transfection. The cell viability, colony formation rate and apoptotic rate were analyzed by MTT assay, colony formation assay and flow cytometry with Annexin V-FITC/PI staining, respectively. The protein expression of KDM4B was determined by Western blot. RESULTS:The results of gene microarray and bioinformatic analysis indicated that differential expression of miR-29a was found in the prostate cancer tissues and the paracarcinomatous tissues. The levels of miR-29a in the prostate cancer tissues and prostate cancer cells were significantly decreased, while the mRNA levels of KDM4B were notably increased compared with the paracarcinomatous tissues and RWPE-1 cells, respectively (P<0.05). Compared with negative control (pGenesil-1) group, the cell viability and colony formation rate were significantly decreased, the apoptotic rate was significantly increased, and the protein expression of KDM4B was notably inhibited in the prostate cancer cells with miR-29a over-expression (P<0.05). The cell viability was significantly enhanced, and the apoptosis was significantly inhibited in the prostate cancer cells with KDM4B over-expression (P<0.05). CONCLUSION:Low expression of miR-29a was found in the prostate cancer tissues and cells. miR-29a over-expression inhibits the growth of prostate cancer cells and induces apoptosis. The mechanism may be associated with inhibiting the protein expression of KDM4B.     

Value of endonuclease domain containing 1 in progression of prostate cancer

AIM: To analyze the difference of endonuclease domain containing 1 (ENDOD1) expression between benign prostatic hyperplasia (BPH) tissues and prostate cancer (PCa) tissues and to investigate the effect of ENDOD1 on the biological function of human prostate cancer cells. METHODS: The BPH samples (n=20) and PCa samples (n=21) were processed and analyzed according to the instruction of immunohistochemical (IHC) staining. The mRNA and protein levels of ENDOD1 in the normal prostate epithelial cells and prostate cancer cells were evaluated by RT-qPCR and Western blot, respectively. The recombinant plasmids pCMV-N-Flag-ENDOD1 was constructed and was transfected into the human prostate cancer cells. The proliferation, apoptosis, migration and invasion abilities of the prostate cancer cells were evaluated by MTT assay, flow cytometry, Transwell migration and Matrigel invasion assays, respectively. RESULTS: The analysis of variance of the immunoreactivity score showed that PCa tissues with high Gleason score displayed significantly lower ENDOD1expression than that with low Gleason score and BPH (P<0.05). The expression of ENDOD1 at mRNA and protein levels in PC3 cells and DU145 cells was significantly lower than that in the LNCap cells (P<0.05). The proliferation of DU145 transfected with ENDOD1 was inhibited. The flow cytometry indicated that ENDOD1 over-expression in the DU145 cells resulted in a notable increase in G₀/G₁ phase arrest (P<0.05), but the apoptotic rates showed no statistical difference. The results of Transwell assay showed that migration and invasion abilities of the cells were also inhibited after transfection with over-expressing ENDOD1 plasmid (P<0.05). CONCLUSION: The expression of ENDOD1 significantly decreased in prostate cancer with high Gleaon score. Meanwhile, the ENDOD1 is specifically down-regulated in androgen independent prostate cancer (AIPC) cell lines. Over-expression of ENDOD1 remarkably inhibits the proliferation, migration and invasion abilities of AIPC.     

Mutations of K-ras oncogene in primary colorectal tumors,related liver metastases and portal vein blood of patients with colorectal cancer

AIM: To evaluate the concordance of K-ras oncogene mutations in primary colorectal tumors, liver metastases and portal vein blood of the patients with colorectal cancer, and to find out the relationship between mutated K-ras oncogene and liver metastases in colorectal cancer.METHODS: Fifty-nine patients with colorectal cancer were screened for the mutations of K-ras oncogene in the tissue samples of primary tumors, portal vein blood and liver metastases (only 15 cases of the 59 patients) by real-time fluorescence quantitative PCR and DNA sequencing. The results were also analyzed with the clinical data of the patients.RESULTS: Point mutations of K-ras were found in the primary tumors in 20 (33.9%) of the 59 patients with colorectal cancer, and 18 (30.5%) of the 59 patients in their portal vein blood. K-ras mutations in 8 (53.3%) of 15 liver metastases were also detected. No significant difference among the rates of K-ras mutation in primary tumor tissues, portal vein blood and related liver metastases was observed (P>0.05). Eighteen cases with mutated K-ras gene in portal vein blood showed the mutations in primary tumor tissues. The patients without mutated K-ras gene in primary tumor tissue also showed negative mutation of K-ras in the portal vein blood. The mutated K-ras gene in both liver metastase and portal vein blood were detected in 8 of the 15 cases with liver metastases, and no mutated K-ras gene was detected in the others with liver metastases. The main types of K-ras mutations found in primary tumors, liver metastases (5 simultaneous, 2 metachronous) and portal vein blood were GGT to GAT and GGT to GTT at codon 12. A K-ras mutation at codon 13 (GGC to GAC) was found in one case with metachronous liver metastases. The rate of concordance of K-ras status between primary tumors and portal vein blood was 96.6%. Detection of K-ras mutations in liver metastases was accordant with that in portal vein blood, but the type of K-ras mutation was partially discordant.CONCLUSION: The K-ras mutations in primary tumors, liver metastases and portal vein blood of patients with colorectal cancer are concordant, and mutated K-ras detected in both cancer tissue and portal vein blood may indicate liver micrometastases from colorectal cancer.     

Analysis of gene structure and diversity of new alternative spliced variants of prostate specific membrane antigen

AIM:To find out the gene structure and diversity of protate specific membrane antigen (PSMA) alternative spliced variants, and probe into the pathogenesis of prostate cancer. METHODS:5-RACE and 3-RACE methods were used to amplify the 5 and 3end of alternative spliced variant and then those viariants were sequenced for analyzing the gene stucture and diversity of PSMA alternative spliced variants of prostate cancer tissues. RESULTS:Four new alternative spliced variants of PSMA were discovered from prostate cancer tissues. Compared with reported PSMA alternative spliced variants, different insertions and deletions existed in different sites of those new variants. CONCLUSION:The discovery of the new variants confirms the diversity of PSMA spliced variants and provides the clues for seeking the target of diagnosis and therapy of prostate cancer.     

Clinical significance of a new alternatively spliced variant of prostate specific membrane antigen

AIM:To discuss the relationship between prostate specific membrane antigen (PSMA) and prostate cancer and to seek a target for diagnosis and therapy of prostate cancer. METHODS:A pair of primers was designed according to the published PSMA mRNA sequence. Total RNA was extracted from prostate cancer tissues and was reversely transcribed into cDNA, which was used as a template for PCR to amplify the PSMA gene. The recombinant was sequenced and the result was analyzed by BLAST. The PSMA5 gene specific primers were designed to identify its expression in different cells and prostate tissues. RESULTS:A new alternatively spliced variant of PSMA named PSMA5 was discovered when sequencing the recombinant. PSMA5 showed well pathological tissue-specificity, and its expression rate in prostate cancer, benign prostatic hyperplasia of prostate, and normal prostate tissue were 92.6%, 78.8% and 10.0%, respectively. It expressed specifically in Pca cell line LNCaP, not in cell lines of PC3, bladder carcinoma, renal carcinoma, or hepatoma. CONCLUSION:A new alternative spliced variant of PSMA named PSMA5 was discovered, which was well correlated with prostate cancer and benign prostatic hyperplasia. This finding may give a new clue to the evolution of prostate cancer and may provide a target for the diagnosis and therapy of prostate cancer.     

Preliminary analysis of decreased expression of NAP1 gene in nasopharyngeal carcinoma specimens

AIM:To analyze the mechanism of decreased expression of NAP1 gene in nasopharyngeal carcinoma (NPC) specimens. METHODS:H-E staining and immunohistochemistry were performed on NPC and nasopharynx (NP) tissue sections. Point mutations and other aberrations in the coding region of NAP1 gene were analyzed by PCR-SSCP. Possible CpG island in 5’ non-coding region of NAP1 was analyzed in NAP1 gene genomic region. RESULTS:By HE staining and immunohistochemistry, no CD35+ FDCs of the germinal center was observed in NPC nests. However, it was observed around NPC nests, but the number of CD35+ FDCs around NPC nests was much less than that in NP stroma. The ratio of germinal center numbers (per 1.77 mm2 in 40 NPC over 20 NP specimens) was 0.33 (0.4/1.2). In addition, immunohistochemistry demonstrated that expression level of NAP1 was directly related to CD35+ FDCs numbers of the germinal center in NP and NPC stroma. We did not find point mutations and other aberrations in the coding region of NAP1 gene by PCR-SSCP. In addition, analysis of NAP1 gene genomic region did not identify possible CpG island in 5’ non-coding region of NAP1. CONCLUSION:Decreased expression of NAP1 in NPC compared with NP represents merely a physiopathological fluctuation in the number and function of FDC that populate various NPC biopsies.      

Androgen responsive element decoy DNA inhibits the promoter of prostate specific antigen and induces apoptosis of LNCaP cells

AIM:To observe the effect of exogenous androgen responsive element decoy on the promoter of prostate specific antigen (PSA) and the growth of LNCaP cells for searching the possibility of gene therapy for prostate cancer. METHODS:Firstly, pGL3-PSA luciferase expression vector containing 640bp-promoter fragment of PSA gene was constructed. Then, a 23-mer phosphorothioated ARE decoy based on the deduced ARE sequence at the promoter region of PSA gene was synthesized. pGL3-PSA and ARE decoy DNA were cotransfected into PC3-M cell by lipofectamineTM 2000. Through detecting the activity of luciferase, the effect of ARE decoy on the promoter of PSA was studied. Then the ARE decoy DNA was transfected into LNCaP cells. The effect of decoy DNA on the proliferation of LNCaP cells was examined by using MTT assay. The effects of apoptosis were detected by phase contrast microscopy, DNA agrose gel electrophoresis and flow cytometry. Meanwhile, the nuclear extract was prepared from LNCaP cells and DNA-protein interactions were examined by electrophoretic mobility shift assay (EMSA). RESULTS:The reporter assay showed that the activity of luciferase was significantly reduced in the ARE decoy-transfected cells, bnt not in the cells transfected with the control decoy. EMSA demonstrated specific binding of the ARE decoy to androgen receptor. The growth of LNCaP was remarkably inhibited and apoptotic morphological changes as well as DNA fragmentation were observed in the ARE decoy-transfected cells. The rate of apoptosis was 22.4% detected by FCM. CONCLUSION:The ARE decoy is capable of inhibiting the promoter of PSA gene and inducing the apoptosis in prostate cancer cells. It may become a potential therapeutic tool for prostate cancers.     

Cholesterol-sensitive action sites in promoter of prohibitin gene

AIM: To verify the cholesterol-sensitive sites in the promoter of prohibitin (PHB) gene. METHODS: A series of successive PHB promoter truncated segments were amplified by PCR and cloned into pGL3-Basic luciferase reporter vector. Human prostate cancer PC-3 cells were transfected with the recombinant plasmids and then cultured in normal medium (NM) or cholesterol-depleted medium (CDM). The activity of PHB promoter was detected by luciferase activity analysis to find the cholesterol-sensitive region of approximately 200 bp in PHB promoter. Point mutations in sterol regulatory element (SRE) homologous loci of this 200　bp region were made to confirm the cholesterol-sensitive role of this SRE site. RESULTS: Plasmids with PHB promoter segments were successfully constructed. Luciferase activity in PC-3 cells transfected with PHB promoter segment pPHB-179 (-35/+138) plasmid was significantly decreased compared with the cells transfected with the whole PHB promoter pPHB-1192 plasmid (P<0.05). After point mutation was made in the SRE homologous locus located at -117 to -108 bp and this plasmid was transfected into PC-3 cells, the luciferase activity in these cells was significant decreased compared with the cells transfected with pPHB-1192 plasmid (P<0.05). CONCLUSION: Cholesterol regulates the activity of PHB promoter in human prostate cancer PC-3 cells. The cholesterol-sensitive site in PHB promoter is located at -117 to -108 bp of SRE homologous locus. PHB may become a new target of gene therapy for prostate cancer.     

Relationship between the point mutation of ABCB4 gene and intrahepatic cholestasis of pregnancy

AIM:To investigate the relationship between the point mutation of ABCB4 gene and intrahepatic cholestasis of pregnancy (ICP). METHODS:Using the method of polymerase chain reaction single strand conformation polymorphism (PCR-SSCP), the point mutation of the ABCB4 gene exon 6 and exon 14 in 31 women with ICP were detected. The PCR products of suspected cases with the point mutation were further DNA sequenced so as to determine its mutation characters. RESULTS:The target band of ABCB4 gene exon 6 and exon 14 was found in all blood samples from 31 cases with ICP. No ABCB4 gene exon 6 and exon 14 deletion and point mutation were detected by PCR and PCR-SSCP, respectively. To check the finding, 7 cases amongst the 31 ICP cases were selected randomly for DNA sequencing. No point mutation of ABCB4 gene exon 6 and exon 14 were detected also. CONCLUSION:The finding suggests that there may be no relationship between the two mutation hot spots and the ICP pathogenesis. Further investigation of other mutation hot spots of ABCB4 gene and enlargement sample size are needed to testify the relationship between the point mutation of ABCB4 gene and the pathogenesis of ICP.     

Progress of c-FLIP overexpression in prostatic carcinoma

Cellular FLICE-inhibitory protein (c-FLIP) is a key anti-apoptotic regulator that inhibits death receptor (DR) signal pathway mediated by suppressing procaspase-8 activation. c-FLIP has been found to be elevated in prostatic carcinoma (PCa), and there is a strong correlation between the overexpression of c-FLIP and PCa. In the present paper, we review the links beteween c-FLIP and androgen receptor (AR) signal pathway, which regulate and promote the survival of androgen-independent prostate cancer (AIPC) and describe the possibility of c-FLIP as a therapeutic target of PCa.     

Construction and sequence analysis of eukaryotic expression vector of Chinese prostate-specific membrane antigen

AIM: To obtain eukaryotic expression vector of Chinese prostate-specific membrane antigen. METHODS: Chinese prostate-specific membrane antigen (PSMA) cDNA was amplified by RT-PCR from prostate cancer tissues, then cloned into eukaryotic expression vector pcDNA3.0 and sequenced. RESULTS: Seven bases in Chinese PSMA cDNA sequence were found different from those reported by Israeli, which lead to two different amino acids. CONCLUSION: We have obtained the PSMA cDNA, and the recombinant eukaryotic expression vector was successfully constructed. The study lays foundation for DCs vaccine modified by PSMA gene for the treatment of prostate neoplasms.     

Detection of EML4-ALK fusion gene and analysis of its clinical features in NSCLC patients with EGFR mutation

AIM: To detect the frequency of EML4-ALK fusion gene in non-small-cell lung cancer (NSCLC) patients who had epidermal growth factor receptor (EGFR) mutation, and to analyze the relationship between EML4-ALK fusion gene and clinical features. METHODS: One hundred and two Chinese patients with NSCLC were selected on the basis of one or more of the following characteristics: female, never/light smoking history and adenocarcinoma histology. The EML4-ALK fusion gene was identified by single-tube multiplex RT-PCR. In EML4-ALK-positive samples, exon 18 to 21 EGFR mutations and exon 1 and 2 KRAS (Kirsten rat sarcoma) mutations were detected by DNA direct sequencing after PCR amplification. RESULTS: Eight specimens (7.8%) were identified with EML4-ALK fusion genes from 102 NSCLC tissues. Of all positive cases, 7 were variant 1 (V1) and 1 was variant 2 (V2). In 8 EML4-ALK-positive samples, exon 18 to 21 EGFR and exon 1 and 2 KRAS were wild-types. Among 8 EML4-ALK-positive cases, 5 cases (5/8, 62.5%) were younger than the mean age. Six cases (6/8, 75%) were female and 7 cases (7/8, 87.5%) were non-smokers. Five cases (5/8, 62.5%) had adenocarcinoma histology. CONCLUSION: EML4-ALK fusion gene defines a new molecular subset of NSCLC with distinct characteristics. The EML4-ALK fusion gene is mutually exclusive to EGFR and KRAS mutations.