ABSTRACT
ObjectiveTo investigate the fusion sequence complexity of EML4-ALK in non-small cell lung cancer (NSCLC) patients,and the potential mutation in tyrosine kinase ( TK ) domain of ALK gene.MethodsIn routine practice,a novel echinoderm microtubule-associated protein-like4 and anaplastic lymphoma kinase (EML4-ALK) V3c variant was detected by rapid amplification of cDNA ends-polymerase chain reaction ( RACE-PCR )-sequencing technology in a patient with NSCLC.The further consecutive 39 cases( total of 40 cases)were screened by use of reverse transcription (RT)-PCR for EML4-ALK fusion.Positive PCR products were purified and cloned into T vectors,transformed into DH5a germ cells and colony picked up and sequenced for sequence complexity analysis.Tyrosine kinase domain of ALK was amplified by RT-PCR and sequenced.ResultsThree out of 40 cases had EML4-ALK fusion.One case had six novel variants of EML4-ALK co-existing,termed as V3c ( 64.6％ ),V3d ( 25.0％ ),V3e ( 2.1％ ),V3f (4.2％ ),V3g(2.1％ )and V3h(2.1％ ) variants,whereas without common V3a and V3b variants.In other two positive cases,one was V1 variant,another was concurrent V2,V3a and V3b variants.No mutations were detected in the TK domain of EML4-ALK in any case.ConclusionsSeveral EML-ALK variants could co-exist in a given lung cancer tissue,which suggest that the diversity and sequence complexity of EML4-ALK fusion are exist.Attentions should be paid to screen all the variants in clinic to improve the pick-up rate.
ABSTRACT
Objective To establish a HRM assay to screen for KRAS mutations in clinical colorectal cancer patients.Methods The sensitivity of HRM was analyzed by detecting somatic mutations in exon 2,notably codons 12 and 13 of the KRAS gene in the serial plasmid mixture samples which were mixed using the different proportions mutation plasmid and wide type plasmid of KRAS.HRM analysis was performed for KRAS on DNA insolated from a panel of 60 colorectal cancer samples derived from fresh tissues.The results were compared with the direct sequencing data.Results After the PCR amplification,the mutation results could be available by performing HRM analysis in the same tube on a real time PCR machine with HRM capability.HRM detection could identify KRAS mutation in a proportion of 10% of mutation plasmid DNA.All 60 samples identified the KRAS mutation by HRM and sequencing.17 samples were positive(28.3%) by HRM for KRAS exon 2 mutations,and 15 samples were confirmed the presence of codon 12 or 13 mutations(25.0%) and the other 2 samples were wild type by sequencing.The 60 samples detected by HRM were given 100% sensitivity with 96% specificity.Conclusions HRM is a sensitive intube methodology to screen for mutations in clinical samples.HRM will enable high-throughput screening to gene mutations to allow appropriate therapeutic choices for patients and accelerate research aimed at identifying novel mutations in human cancer.
ABSTRACT
Objective To investigate the expression changes of translation elongation factor 1?1 in anti-BPDE transformed and carcinoma 16 HBEs. Methods Suppression subtractive hybridization (SSH), bioinformatics and semi-quantitative RT-PCR were applied. The cDNA of anti-BPDE transformed and carcinoma 16 HBE cells were used as tester respectively, and the cDNA of normal 16 HBE was used as driver, the library of subtractive hybridization were profiled and inverted into TA cloning vector after two times of hybridization and two times of PCR. After the screening, sequencing and analysis of sequences, semi-quantitative RT-PCR was performed accompanying to the inner reference of ?-actin. Results 9 differentially expressed fragments were consistent with translation elongation factor 1?1 in different regions in Genbank, and the expressions were up regulated in BPDE transformed and carcinoma 16 HBE cells. Conclusion Translation elongation factor 1?1 may be related to the transforming effect and carcinogenesis of anti-BPDE.