1.Analysis of TCR gene rearrangements for diagnosis of T-cell lymphomas
Xinyan PAN ; Changshao YANG ; Guiyun LI ; Julun YANG ; Li WANG
Chinese Journal of Clinical and Experimental Pathology 2015;(4):400-403
Purpose To discuss the TCR gene rearrangements in the diagnosis of T-cell lymphomas. Methods Formalin-fixed and paraffin-embedded samples including 30 cases of T-cell lymphomas and 30 cases of reactive lymphoid hyperplasia were chosen for ex-tracting genomic DNA and PCR amplification using 56 BIOMED-2 primers. PCR products were analyzed by heteroduplex and polyacryl-amide gel electrophoresis. Results In all 30 cases of T-cell lymphomas, 25 cases (83. 3%) showed TCRβ gene monoclonal rear-rangements, 28 cases (93. 3%) of TCRγ gene monoclonal rearrangements, 4 cases (13. 3%) of TCRδ gene monoclonal rearrange-ments. 29 cases (96. 7%) with TCRβ+TCRγ+TCRδ gene monoclonal rearrangements were detected. but no clonal TCR gene rear-rangements were found in 30 cases of reactive lymphoid hyperplasia. Conclusions The detection of TCR gene rearrangements using BIOMED-2 primers is a useful assistant method for the diagnosis of T-cell lymphomas.
2.In vitro Selection of DNA Aptamer to Cyclosporin A by SELEX Method
Weibin LI ; Xiaopeng LAN ; Xiangyue YANG ; Changshao ZHOU
Chinese Journal of Biochemistry and Molecular Biology 2007;23(10):829-834
To obtain the aptamers with high affinity and specificity for cyclosporin A(CsA),a synthesized 78 nt single stranded DNA(ssDNA)random library containing 35 random sequences flanked by invariant primer was subjected to 1 1 rounds of selection against CsA by SELEX protocol.Magnetic beads were used for target immobilization and the biotin-streptavidin-horseradish peroxidase system was employed for determining the binding affinity between the aptamers and CsA. After ten rounds of selection and amplification, with an increasing affinity for each round,the selected aptamers were cloned,sequenced and analyzed for their primaryand secondary structures.The 19 aptamers were divided into five groups based on primary sequence homology.Hairpin loop is the main motif in the predicted secondary structure and is supposed to be the binding part of the aptamers to CsA.The CsA-specific aptamers will be useful for enzyme-linked assays or immunofluorescence asses of CsA.
3.Detection of immunoglobulin and TCR gene rearrangements by PCR using BIOMED-2 multi-plex protocols in T lymphoblastie lymphoma and acute lymphoblastic leukemia
Xinyan PAN ; Qiang FENG ; Guiyun LI ; Changshao YANG ; Julun YANG ; Li WANG
Chinese Journal of Clinical and Experimental Pathology 2015;(10):1131-1134,1139
Purpose To investigate the sensitivity of BIOMED-2 primer system in T lymphoblastic lymphoma ( T-LBL) and acute lym-phoblastic leukemia ( ALL) patients immunoglobulin ( Ig) and T-cell receptor ( TCR) gene rearrangement, and to analyze the co-rear-rangement pattern. Methods Amplification of rearranged Ig and TCR gene was performed in standard PCR in 35 T-LBL/ALL pa-tients. PCR products were analyzed by heteroduplex and polyacrylamide gel electrophoresis. Results 16 cases (45. 7%) of 35 sam-ples were detected to have TCR gene rearrangements, including 6 cases (37. 5%) of TCRβgene monoclonal rearrangements, 4 cases (25. 0%) of TCRγ gene monoclonal rearrangements, 3 cases (18. 8%) of TCRβ and TCRγ gene double rearrangements, 2 cases (12. 5%) of TCRδ gene monoclonal rearrangements and 1 case (6. 3%) of TCRγand TCRδgene double rearrangements were detec-ted. 4 cases (11. 4%) of 35 samples detected to have clonal immunoglobulin and TCR gene rearrangements. 11 cases (39. 3%) of 28 T-LBL patients were detected to have TCR gene rearrangements, 6 cases (85. 7%) of 7 T-ALL have TCR gene rearrangements. Con-clusions BIOMED-2 multiplex PCR analysis strategy is a useful technique in the T-LBL patients.
4.Mutation status of epidermal growth factor receptor and KRAS gene in non-small cell lung cancers at Xuanwei regions of Yunnan Province
Changshao YANG ; Xinyan PAN ; Qiang FENG ; Yuanyuan WANG ; Wenmang XU ; Ting JIANG ; Li WANG ; Julun YANG ; Li WANG
Chinese Journal of Pathology 2016;45(4):226-230
Objective To investigate the mutation status of epidermal growth factor receptor ( EGFR) and KRAS gene in patients with non-small cell lung cancer ( NSCLC) in Xuanwei, Yunnan and to correlate the mutation status with clinicopathologic features.Methods Mutation status of exons 18, 19, 20 and 21 of EGFR, and codons 12, 13 of KRAS in 63 cases of NSCLC were analyzed by gene sequencing and ARMS-Taqman probe method.Correlation with patients′clinicopathological characteristics was performed. Results EGFR and KRAS mutations were present in 55.6% (35/63) and 6.3% (4/63), respectively. EGFR gene mutations were present, including exon 18 G719X in 14.3% (5/35), exon 19 in 14.3%(5/35), exon 20 S768I and T790M in 20.0% (7/35), exon 21 L858R in 31.4% (11/35), exon 18 G719X and exon 20 S768I double mutation in 17.1%(6/35), and exon 20 T790M and exon 21 L858R double mutation in 2.9%(1/35).KRAS mutations were seen in codon 12 in 3 of 4 cases, and codon 13 in 1 of 4 cases.EGFR mutations were mutually exclusive with KRAS mutations.According to statistic analysis, EGFR mutations were associated with the histological types of NSCLC ( P<0.05 ) , but without correlation with patient′s gender, age, smoking status and lymph node metastasis ( P >0.05 ) .KRAS mutations in NSCLC had no correlation with the clinical pathologic characteristics of the patients.Conclusions A higher frequency of EGFR exon 18 G719X and 20 exon S768I mutations are found in the patients in Xuanwei, Yunnan.EGFR mutations are associated with histologic types of NSCLC, but without correlation with patient′s gender, age, smoking status and lymph node metastasis.KRAS mutation in NSCLC has no correlation with the clinicopathologic characteristics of the patients.
5.Analysis of epidermal growth factor receptor gene T790M mutation in plasmatic ctDNA samples of pa-tients with non-small cell lung cancer
Yaxi DU ; Yuhui MA ; Changshao YANG ; Lianhua YE ; Xiaojie DING ; Quan LI ; Yinjin GUO ; Junxi LIU ; Yunchao HUANG ; Yongchun ZHOU
The Journal of Practical Medicine 2017;33(19):3228-3232
Objective To investigate epidermal growth factor receptor(EGFR)gene T790M mutation in plasmatic ctDNA samples from 171 patients with non-small cell lung cancer and analyze the relationship between EGFR T790M mutation and the clinical factors. Methods The EGFR T790M mutation was detected in 171 cases by super amplification refractory mutation system(Super ARMS)in this paper. Rusults The EGFR gene T790M mutation was identified in 7.60%(13/171)plasmatic ctDNA samples which mostly came from patients withⅢb~Ⅳstages of lung cancer. The EGFR T790M mutation rate was identified in 2.05%(3/146)plasmatic samples of pa-tients who did not received treatment of EGFR-TKIs,which was lower than 40.00%(10/25,P<0.05)plasmatic samples of patients who received treatment of first generational EGFR-TKIs. The EGFR T790M mutation rate was identified in 75.00%(3/4) and 60.00%(6/10) plasmatic samples of patients who have received TKI for 6 to 10 months and more than 10 months,which was higher than 9.10%(1/11,P < 0.05)plasmatic samples of patients who have received TKIs for less than 6 months. Conclusions This article demonstrated that EGFRT790M muta-tion was more common in lately NSCLC patients who have received TKIs treatmentover 6 months,meanwhile the EGFR T790M mutation dynamical detective technology will effectively guide the clinic treatment.