تشخيص مولكولي حداقل بيماري باقيمانده در كودكان ايراني مبتلا به لوسمي لنفوبلاستي حاد با ارزيابي ژن هاي باز آرايي شده زنجيره هاي سنگين و سبك كاپا ايمونوگلوبولين

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 B-precursor acute lymphoblastic leukemia

Background and Objectives 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 B-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 months after the initiation of treatment. Materials and Methods 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 B-precursor ALLs were selected for study. Mononuclear cells including leukemic blasts were isolated by density gradient. After DNA extraction, IgH and IgK ( VK 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. Results 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. Conclusions 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.