The Study On Immunoglobulin And T-cell Receptor(Ig/TCR) Gene Rearrangements In Adult Acute Lymphoblastic Leukemia

1. The Pattern of clonal Immunoglobulin and T-cell receptor (Ig/TCR) gene rearrangements in adult acute lymphoblastic leukemia patientsObjective: To study the pattern of clonal Ig/TCR gene rearrangements with adult ALL patients at newly diagnosis, providing a prerequisite for further minimal residual disease (MRD) monitoring using real-time quantitative polymerase chain reaction (RQ-PCR)-based approach. Methods: Mononuclear cells were isolated from bone marrow specimens of forty newly-diagnosed adult patients with B-lineage and T cell acute lymphoblastic leukemia (ALL). Ig and TCR gene rearrangements were detected by using BIOMED-2 primers and protocols. Fifteen multiplex PCR tubes were assigned, which include primer sets for IGH,IGK,IGL,TCRB,TCRG and TCRD gene rearrangement. Clonality was confirmed either by heteroduplex analysis or gene scanning. Results: The total clonality detection rate of IG rearrangements were 96% in adult patients with B-lineage ALL, of which IGH,IGK-Kde,IGL,TCRB,TCRG and TCRD rearrangements were 86%,14%,9%,19%,77%,55%, respectively. While in T-ALL, the total clonality detection rate of TCR rearrangements were 100%, of which IGH,TCRB,TCRG and TCRD rearrangements were 6%,83%,78%,33%. Overall, the detection rates of two or more targets were 91% and 89% in adult patients with B-and T-ALL, respectively. Conclusion: The application of BIOMED-2 primers and protocols can detect virtually all clonal B- and T-cell populations. Several features in the pattern of Ig and TCR gene rearrangements have been noted in these ALL patients. These results are useful for further MRD-RQ-PCR detection and quantification for all patients 2. Assessment of minimal residual disease in adult patients with B-lineage acute lymphoblastic leukemia using rearranged immunoglobulin loci by real-time quantitative PCRObjective: To evaluate the clinical usage of immunoglobulin heavy chain gene (IgH) rearrangement detected by real-time quantitative polymerase chain reaction (RQ-PCR), in hope to provide a helpful method for minimal residual disease (MRD) monitoring in adult B-lineage acute lymphoblastic leukemia (B-ALL) patients. Methods: DNA samples of fifteen adult B-ALL patients at newly diagnosis were obtained. The IgH gene rearrangements were detected by PCR followed by sequencing and subsequent blasting for monoclonal PCR products. Allele-specific oligonuleotides (ASO) were designed based on the sequence of junction regions, using PRIMER5.0 software. MRD targets were detected in 115 bone marrow follow-up samples by RQ-PCR, in which ASO upstream primers in combination with the consensus JH probes and downstream primers were applied. Meanwhile, transcripts copies of BCR/ABL fusion gene were also measured in 7 ALL cases with positive Ph chromosome. Results: The detection sensitivity of ASO-PCR varied between 10-3 and 10-5 leukemia cells in 15 adult ALL patients. The background and nonspecific amplification was detected at a low level. The quantification analysis of monitoring MRD showed that adult ALL patients with high-risk features have a higher MRD level than those of standard-risk at complete remission (CR). Patients with MRD>10-3 have a higher relapse rate and shorter survive time. Beside, the dynamic curves of the quantified level of respective IgH rearrangement were consistent with the expression levels of BCR/ABL fusion genes in seven Ph positive patients during follow-up. Conclusion: The individual quantification of IgH rearrangement by RQ-PCR approach using ASO primers was sensitive, specific and reliable for the accurate evaluation of malignant clones. Meanwhile, these data indicated a close correlation between the level of rearranged IgH and the treatment response and prognosis in adult ALL patients, suggesting a helpful method to monitor MRD in clinical trials.