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

[Prevalence of Helicobacter pylori infection in children with chronic immune [idiopathic] thrombocytopenic purpura and evaluation of Helicobacter pylori eradication on platelet count]

Recently, the high prevalence of Helicobacter pylori infection has been reported in adult patients with chronic immune [idiopathic] thrombocytopenic purpura. Furthermore, after Helicobacter pylori eradication therapy in such patients, their platelet counts have been observed to increase, suggesting that Helicobacter pylori may be a causative agent of adults' chronic idiopathic thrombocytopenic purpura. However, there have been only a few reports of this subject in children with chronic thrombocytopenic purpura. The purpose of this study is to determine prevalence of Helicobacter pylori infection in Iranian children with chronic thrombocytopenic purpura and role of Helicobacter pylori eradication in rising platelet count of these patients. This descriptive-clinical trial study was performed in 31 children under 14 years old with chronic thrombocytopenic purpura who attended hematology ward of Mofid paediatric hospital. After determining platelet count, and filling the results patients referred to gastrointestinal ward of the hospital to perform urea breath test for evaluation of Helicobacter pylori infection, then Helicobacter pylori-infected patients who were diagnosed by this test, received eradication therapy using triple therapy regimen [containing Omeperasole, Amoxicillin and Clarithromycin] for 2 weeks and their platelet counts were recorded during the follow up period. Mean age of the patients was 8.9 +/- 3.2 years old ranging from 3.5 to 14 years old. They were 17 [54.9%] girls and 14 [45.1%] boys. Mean platelet count of the patients was 51.4 +/- 34.3x10[9] / L ranging form 125x10[9]/ L to 8x10[9] / L. Mean duration of disease in the patients was 27.7 +/- 20.2 months ranging from 7 to 96 months. Helicobacter pylori infection was found in only 4 children [12.9%] and Helicobacter pylori eradication therapy was not effective in rising platelet count to achieve complete or partial remission. Comparing Helicobacter pylori-positive and negative patients, there were no significant differences regarding their age, platelet count and duration of disease. This study shows that prevalence of Helicobacter pylori infection in children with chronic immune thrombocytopenic purpura is less than that is in adults. Furthermore, we have found that platelet count in Helicobacter pylori-positive children have not been risen after eradication therapy. We suggest that more studies in different gender groups and different zones in the world with more number of samples should be performed, especially in children in order to determine both the exact role of Helicobacter pylori's pathogenesis in developing the chronic idiopathic thrombocytopenic purpura and the effectiveness of eradication therapy in rising platelet count in these patients

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