Use of immunoglobulin heavy chain and kappa light chain gene rearrangements by PCR for molecular diagnosis of minimal residual disease in Iranian children suffering from beta-precursor acute lymphoblastic leukemia

Diversity of IgH and IgK molecules is generated during B and T Lymphocyte differentiation through the rearrangement of variable, diversity, junction and constant gene segments. Additionally, random insertion and deletions of nucleotides between gene segments make unique sequences which are cell or clone specific. Similar IgH and IgK genes rearranged in normal cells of lymphoid leukemia cases can be used as a marker of clonality and for evaluation of minimal residual disease [MRD]. The purpose of this study is to evaluate the pattern of IgH chain and IgK gene rearrangements using polymerase chain reaction [PCR] in beta-precursor acute lymphoblastic leukemias [ALL] to follow the MRD at day 14, day 28 [end of remission induction], week 10, 3-6 months and 6-12. month after the initiation of treatment. In our prospective study bone marrow aspirates of 183 children at the mean age of 63.6 months with diagnosis of acute leukemia were collected at admission before any chemotherapy. After reviewing cytomorphology and immunophenotyping, only 140 cases with diagnosis of beta-precursor ALLs were selected for study. Mononuclear cells including leukemic blasts were isolated by density gradient. After DNA extraction, IgH and IgK [V[K] I-IV / Kde] were amplified by consensus primers using PCR. PCR products were analyzed after heteroduplex analysis and polyacrylamide gel electrophoresis [silver stain]. The DNA sequences were compared and aligned with the sequences homologous for IgH and IgK published by Gene Bank. The follow up specimens were collected at day 14, day 28 [end of remission induction], day 45-month 3, and 3-6 months and 6-12 months after initiation of treatment. After routine cytomorphologic analysis, similar PCR was done on follow up extracted DNAs in parallel with diagnosis DNA. MRD was considered to be approved positive if bands similar to those at the time of diagnosis were present. Statistical analysis using SPSS software [version 11.5] was performed. 90.5% of patients had clonal IgH gene rearrangements. Monoclonal, biclonal and oligoclonal patterns were observed in 57.8%, 34.9% and 5.5% of patients with IgH [CDR III] rearrangement, respectively. Clonal patterns of IgK-Kde were detected in 59 [67%; n: 88] of BP-ALLs. According to cytomorphology about 92% of patients were in complete remission. MRD positivity decreased from more than 90% to 20% using different gene rearrangements in defined time points. Four patients who relapsed during follow up were MRD positive using 1-3 rearrangements and all except one were in clinical remission. Clonal rearrangement of IgH had a pattern similar to other populations. IgK was slightly more frequent than previously reported and the VKI [25%] was the most common type. These differences can be explained by different techniques, DNAs and clonality markers. According to the results, these clonal markers can be used in diagnosis and follow up of MRD

pattern of cross lineage T-cell receptor delta/gamma gene rearrangements in B-precursor acute lympho-blastic leukemia of Iranian children using polymerase chain reaction

Diversity in heavy chain immunoglobulin [IgH] and T-cell receptor [TCR] molecules occures during B- and T-lymphocyte differentiation through the rearrangement of variable [V], diversity [D], junction [J] and constant [C] gene segments. Lymphoid leukemia cells are similar to normal precursors and have rearranged IgH, IgK and TCR [cross-lineage rearrangement] genes which can be used as a marker of clonality and evaluation of minimal residual disease [MRD]. The purpose of this study is to evaluate the pattern of TCR- delta/gamma gene rearrangements using Polymerase Chain Reaction [PCR] in B-precursor acute lymphoblastic leukemia [ALL] in Iranian children. In our prospective study, bone marrow aspirates of 183 children with early diagnosis of acute leukemia were collected at admission before any chemotherapy. After reviewing cytomorphology and immunophenotyping, only 140 subjects with diagnosis of B-precursor ALLs were selected for study. Sixteen were excluded from our study due to various reasons including cellular degeneration. Mononuclear cells including leukemic blasts were isolated by density gradient. After DNA extraction, hyper-variable regions TCR-delta [V delta2-D delta3 and D delta2-D delta3] and TCR-gamma [V gamma; V gamma I and V gamma II] were amplified by consensus primers using PCR. PCR products were analyzed after heteroduplex analysis and polyacrylamide gel electrophoresis [silver stain]. The DNA sequences were then compared and aligned to the homologous sequences of Gene Bank for confirmation. T-test, Mann whitney, Fisher exact test and Chi-square were used for data analysis. Clonal rearrangement of TCR-gamma [V gamma] and V gamma l/Il were present in 79.3% and 64.9% of patients respectively and only 5% of cases showed biclonal pattern. The V gamma ll rearrangement was the most common [46.8%] type in TCR-gamma. 47 [45.2%] and 11 [16.6%] of patients had V delta2- D delta3 and D delta2-D delta3 partial gene rearrangements, respectively. Biclonal/oligoclonal patterns were present respectively in 27.7% and 4.3% of cases with Vdelta2-D delta3 rearrangement. Only one patient had biclonal D delta2 D delta3 rearrangement. Clonal rearrangement of TCR-delta [Vdelta2-Ddelta3 and D delta2-D delta3] genes had a pattern similar to other populations. Frequency of TCR- gamma [V gamma I and V gamma II] rearrangements was slightly higher than previous reports, and in contrary to others except for Brazilian report the V gamma II rearrangement was the most common type. We found no significant correlation between presence of different types of rearrangements and quantitative variables. The only significant point was the reduction of Vdelta2Ddelta3 with increase in age. According to preliminary results, these clonal markers can be used in diagnosis and follow up of MRD

Evaluation of bi/oligoclonal rearrangement of immunoglobulin and T-cell receptor genes in Iranian children suffering B-precursor acute lymphoblastic leukemia

Clonal gene rearrangement of immunoglobulin and T cell receptor may have mono, bi or oligoclonal pattern. Significance of these patterns were studied at diagnosis and follow up of MRD in many countries, however, similar studies have not been conducted among Iranian patients. We investigated the bi/oligoclonal pattern and their association with quantitative and qualitative parameters especially MRD in Iranian children suffering from B-precursor acute lymphoblastic leukemia. In our prospective study, bone marrow aspirates of 140 patients with B-precursor ALLs were selected. Mononuclear cells including leukemic blasts isolated by density gradient. Having DNA extracted, hypervariable regions of IgH, IgK, TCR-delta [D delta 2-D delta 3, V delta 2-D delta 3] and TCR-lambda [V lambda, V lambda I, V lambda II] were amplified by consensus primers using PCR. PCR products were analyzed after heteroduplex analysis and polyacrylamide gel electrophoresis [silver stain]. The DNA sequences were compared and aligned to the sequences homologous for IgH and IgK published by Gene Bank. Bone marrow aspirates of days 14, 28 and 45, as well as months 3 and 6 were treated similarly. IgH gene rearrangements were reported in 114 [90.5%] patients using consensus primers for CDR-III and CDR-I regions [monoclonal: 57.8%, biclonal:34.9% and oligoclonal:5.5%]. Clonal pattern of IgK-Kde were present in 59 cases [67%] [biclonal:10%] Clonal rearrangement of TCR-lambda [V lambda] and V lambda I/II were present in 79.3% and 64.9% of patients, respectively, however, only 5% of cases showed biclonal pattern. The V lambda II rearrangement was the most common [46.8%] type in TCR-lambda. 47 [45.2%] and 11 [16.6%] patients had V delta 2-D delta 3 and D delta 2-D delta 3 partial gene rearrangements, respectively. Biclonal/oligoclonal pattern were present in 13 [27.7%] and 2 [4.3%] cases with V delta 2-D delta 3 rearrangement. Only one patient had biclonal D delta 2-D delta 3 rearrangement. No significant difference regarding the quantitative and qualitative parameters and MRD was observed between the two groups. Bi/oligoclonal rearrangement of IgH, IgK, TCR-delta [D delta 2-D delta 3, V delta 2-Ddelta 3] and TCR-lambda [V lambda, V lambda I, Vlambda II] genes had comparable pattern to other populations. Results of MRD study showed no significant differences between the two groups