Establishment and application of a MassARRAY platform-based method to detect multiplex genetic mutations in lung cancer / 中国肿瘤临床

Objective:To establish a method based on the iPLEX analysis of MassARRAY mass spectrometry platform to detect multiplex genetic mutations among Chinese lung cancer patients. Methods:We reviewed the related literature and data of lung cancer treatments. We also determined 99 mutation hot spots in 13 target genes, namely, EGFR, KRAS, ALK, FGFR1, FGFR2, FGFR3, PIK3CA, BRAF, PTEN, MET, ERBB2, AKT1, and STK11, which are closely related to the pathogenesis, drug resistance, and metastasis of lung cancer and are associated with relevant transduction pathways. A total of 297 primers comprising 99 paired forward and reverse amplification primers and 99 matched extension primers were designed by using Assay Design in accordance with the mutation label and format requirements of the MassARRAY platform. The detection method was established by analyzing eight cell lines and six lung cancer specimens;the proposed method was then validated through comparisons with a LungCarta kit. The sensitivity and specificity of the proposed method were evaluated by directly sequencing EGFR and KRAS genes in 100 lung cancer cases. Results:The proposed method could detect multiplex genetic mutations in the lung cancer cell lines, and this finding is consistent with that observed using previously reported methods. The proposed method could also detect such mutations in clinical lung cancer specimens;this result is also consistent with that observed by using the LungCarta kit. However, an FGFR2 mutation was detected only by using the proposed method. The measured sensitivity and specificity were 100%and 96.3%, respectively. Conclusion:The proposed MassARRAY technology-based method could detect multiplex genetic mutations among Chinese lung cancer patients. Indeed, the proposed method can be potentially applied to detect mutations in cancer cells.

Diversity of EML4-ALK fusion variants in non-small cell lung cancer / 中华检验医学杂志

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.

Expression of reconstructed BCR-ABL-pIRES-SEA plasmids in the skeletal muscles of BALB/c mice / 生物医学工程学杂志

This paper is aimed to investigate the transcription and expression of BCR-ABL-pIRES-SEA fusion gene vaccines in vivo in mice. The reconstructed plasmids (BCR-ABL-pIRES-SEA) which were developed previously in our laboratory were injected into the skeletal muscles of BALB/c mice at 14d intervals for three cycles. The transcription and expression of BCR-ABL and staphylococcal enterotoxin A (SEA) in injection site were detected using RT-PCR and immunohistological methods. The BCR-ABL/SEA mRNA and protein could be identified in the injection site of BCR-ABL-pIRES-SEA vaccinated mice. The reconstructed BCR-ABL-pIRES-SEA plasmids can effectively express gene production in the skeletal muscles of mice and have the common features of DNA vaccine.

Effect of cimetidine on clonal expansion of TCR V? subfamily of T cells in cord blood stimulated by K562 cells / 第三军医大学学报

Objective To investigate the effect of cimetidine on the clonal expansion of TCR V? subfamily of T cells in cord blood after the stimulation by K562 cells in vitro.Methods Cimetidine(1?10-5 mol/L) or K562 cells(1?106/ml) or both of them were respectively cultured with mononuclear cells(MNC) isolated from normal human cord blood for 2 weeks.After the induction,specific cytotoxicity of the proliferated T cells were detected with K562 cells as the target cells.The selective usages and clonal expansion of TCR V? subfa-mily of T cells were analyzed by RT-PCR and genescan technique.Results After induction for 2 weeks,the 3 groups showed the increased cell proliferation,in which specific cytotoxicity of T cells induced by both cimetidine and K562 cells against K562 cells was enhanced significantly compared with the other 2 groups(P

A Clinical Study of 2788 Newborns Screened for Hearing and Gene / 听力学及言语疾病杂志

G mutation were intervened and avoided the occurrence of deafness,1 babies with 235delC homozygote was confirmed severe sensorineural hearing loss in the hearing screening.Conclusion Newborn gene screening make up the defects of missed diagnosis in simple hearing screening in finding the newborn babies with late-onset deafness or the high risk as well as the pathogenic carriers.So the hearing and gene screening were necessary in the current situation,and this screening strategy would be developed further in Henan province.