[ARMS-PCR combined with capillary electrophoresis can be a sensitive and quantitative method for detection of MYD88-L265P mutation in lymphoma].

OBJECTIVE: To investigate the feasibility of sensitive and quantitative detection of MYD88 gene L265P mutation in lymphoma patients by using ARMS-PCR combined with capillary electrophoresis. METHODS: ARMS-PCR amplified MYD88 gene was analyzed by capillary electrophoresis in ABI 3730 sequencer; Exon 5 of the same gene was sequenced bi-directionally as reported. RESULTS: The sensitivity of detection L265P mutations by the ARMS-PCR combined with capillary electrophoresis and direct sequencing was 0.2% and 5%, respectively, according to the detection of the gradient-diluted plasmid standards. The detection rate of 184 patients was 13.59% and 8.28%, respectively (p<0.001). Moreover, the former method can successfully detect the mutation ratio(R2=0.979), and the repeatabilities (CV=2.86%, 1.94%, 5.49%) are acceptable. CONCLUSION: ARMS-PCR combined with capillary electrophoresis can quantitatively detect the MYD88 gene L265P mutation, and the detection sensitivity is significantly higher than sanger sequencing. As a supplement to the latter, it can effectively lead to the earlier diagnose and monitoring of minimal residual disease.

[Frequently ABL kinase domain G:C→A:T mutation and uracil DNA glycosylase abnormal expression in TKI-resistant acute lymphoblastic leukemia of Chinese population].

Most Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph(+) ALL) patients often show rapid recurrence and development of ABL kinase domain (KD) mutation after tyrosine kinase inhibitor (TKI) treatment. To further investigate the mechanism of Ph(+) ALL fast relapse after TKI treatment, ABL KD mutation in 35 Chinese Ph(+) ALL with TKI resistance was detected by direct sequencing. The results showed that 77.1% (27/35) Ph(+) ALL patients with TKI resistance had ABL KD mutation and 55.6% (15/27) Ph(+) ALL patients with ABL KD mutation had T315I. Interestingly, 77.8% (21/27) Ph(+)ALL showed ABL mutation G: C→A:T, including T315I, E255K and E459K. Furthermore, all the Ph(+) ALL patients with two or more ABL KD mutations collaborated with complex chromosome abnormality and all the TKI-resistant Ph(+) ALL patients, whose karyotype progressed from simple t (9;22) into complex, developed ABL KD mutation. Moreover, the expression level of uracil-DNA glycosylase UNG2, which inhibits G:C→A:T transition in genomic DNA, decreased in Ph(+) ALL with TKI-resistance compared to that in newly diagnosis Ph(+) ALL. It is concluded that there is a high frequent ABL KD G:C→A:T mutation and a high genomic instability in Chinese TKI-resistant Ph(+) ALL. In addition, the decreased UNG2 expression in TKI-resistant Ph(+) ALL probably contributes to their high rate of ABL KD G:C→A:T mutation.

Patterns of T-cell reconstitution by assessment of T-cell receptor excision circle and T-cell receptor clonal repertoire after allogeneic hematopoietic stem cell transplantation in leukemia patients--a study in Chinese patients.

OBJECTIVE: Successful allogeneic hematopoietic stem cell transplantation (HSCT) requires reconstitution normal T-cell immunity. Measurement of T-cell receptor excision circles (TRECs) and T-cell receptor beta (TCRBV) CDR3 repertoire is a means of quantifying recent thymic T-cell production and reflecting antigen-specific T-cell clones proliferation. METHODS: We used real-time quantitative PCR to detect TRECs from 43 Chinese patients who underwent three kind of allo-HSCT without T-cell depletion. RT-PCR was performed to amplify 24 subfamily genes of TCRBV in 24 patients of them. RESULTS: For haploidentical-D group, the TRECs numbers were lower up to 24 months. For matched-sibling donor (MSD) group, the recovery of TRECs was faster than those of other two groups. TRECs values in matched-unrelated donor (MUD) were in the middle. During 2-19 months after transplantation, there were 6-16 BV subfamilies expressed and 33-48% of them were polyclones. The usage rate of TCRBV and percentage of polyclones in haploidentical-D were less than those of other two groups. Twenty-three CDR3 molecules were obtained from nine patients who were potentially associated with GVHD or CMV infection. CONCLUSIONS: Analyzing the changes of TCRBV repertoire and measuring TRECs during immune reconstitution would be useful to determine the host's current immune status and ability of T-cell immune reconstitution and also to find antigen-specific T-cell clones in the three kinds of HSCT.