Samart Pakakasama

Genetic Polymorphisms and Haplotypes of DNA Repair Genes in Childhood Acute Lymphoblastic Leukemia

Polymorphisms of DNA repair genes can alter protein structure and may impair DNA repair capacity. Defects in repairing damaged DNA lead to genetic instability and carcinogenesis. This study was performed to evaluate the effect of the polymorphisms of DNA repair genes on risk of childhood acute lymphoblastic leukemia (ALL).

Concurrent radiotherapy with temozolomide followed by adjuvant temozolomide and cis-retinoic acid in children with diffuse intrinsic pontine glioma

The prognosis of children with diffuse intrinsic pontine glioma (DIPG) is very poor. Radiotherapy remains the standard treatment for these patients, but the median survival time is only 9 months. Currently, the use of concurrent radiotherapy with temozolomide (TMZ) has become the standard care for adult patients with malignant gliomas. We therefore investigated this approach in 12 children diagnosed with DIPG. The treatment protocol consisted of concurrent radiotherapy at a dose of 55.8-59.4 Gy at the tumor site with TMZ (75 mg/m(2)/day) for 6 weeks followed by TMZ (200 mg/m(2)/day) for 5 days with cis-retinoic acid (100 mg/m(2)/day) for 21 days with a 28-day cycle after concurrent radiotherapy. Ten of the 12 patients had a clinical response after the completion of concurrent radiotherapy. Seven patients had a partial response, four had stable disease, and one had progressive disease. At the time of the report, 9 of the 12 patients had died of tumor progression, one patient was alive with tumor progression, and two patients were alive with continuous partial response and clinical improvement. The median time to progression was 10.2 +/- 3.0 months (95% confidence interval [CI], 4.2-16.1 months). One-year progression-free survival was 41.7% +/- 14.2%. The median survival time was 13.5 +/- 3.6 months (95% CI, 6.4-20.5 months). One-year overall survival was 58% +/- 14.2%. The patients who had a partial response after completion of concurrent radiotherapy had a longer survival time (p = 0.036) than did the other patients (those with stable or progressive disease). We conclude that the regimen of concurrent radiotherapy and TMZ should be considered for further investigation in a larger series of patients.

Generation of CD3+CD56+ cytokine-induced killer cells and their in vitro cytotoxicity against pediatric cancer cells

A certain number of pédiatrie cancer patients still succumb to relapse following conventional treatment of their malignancies. One of the mechanisms of relapse is escape from immunity. Adoptive cellular immunotherapy with effector cells has the potential to overcome this escape. In adults, the CD3+CD56+ cell, a cytokine-induced killer (CIK) cell, appears to be a promising effector cell type with the greatest cytotoxicity. This effector cell type may work in children as well. No similar studies with children have been published. We speculated that expanded CD3+CD56* cells obtained from pédiatrie cancer patients during remission would act similarly against various pédiatrie tumor cell lines; therefore, we undertook the present study to find support for our speculation. This study was undertaken to generate and expand CD3’CD56’ CIK cells from normal peripheral blood mononuclear cells (PBL) obtained from 6 children with cancer (2 with acute lymphoblastic leukemia, 2 with large cell lymphoma, and 2 with osteosarcoma) in remission after intensive chemotherapy and to study the cytotoxic activities of these cells against chronic myeloid leukemia cell line K562 t(9:22), 4 pédiatrie tumor cell lines [infant acute lymphoblastic leukemia RS4 t(4;ll), THL/AML acute lymphoblastic leukemia REH t(12:21), alveolar rhabdomyosarcoma Rh-Cr t(2;13), and Ewing sarcoma EW-Le t(11;22)], and 2 pédiatrie glioblastoma multiforme cultured cell lines (G74 and G77). CIK cells were generated and expanded in culture medium to which interferon y, monoclonal antibody against CD3, and interleukin 2 were added at appropriate times. Cells were counted by flow cytometry. Net lactate dehydrogenase release from target cells incubated with CIK cells was used as an index of CIK cell cytotoxicity against various pédiatrie tumor cell lines. Ihe results show that after 21 days in culture CD3*CD56+ CIK cells derived from the 6 pédiatrie patients accounted for a median of 28.3% of the entire culture (range, 10.7%-36.4%). Before expansion no such cells were found in any of the 6 children. Median lytic activity rates of CIK cells were 45.5% to 64.5%, rates that contrasted drastically to the lytic activity rates of PBL, which were only 8% to 12%. The findings of the present study are encouraging. They provide information for developing adoptive immunotherapy for future clinical trials with pédiatrie cancer patients, particularly those patients with minimal residual disease after intensive chemotherapy or stem cell transplantation (especially nonmyeloablative transplantation procedures).

Treatment of Epstein-Barr virus lymphoproliferative disease after hematopoietic stem-cell transplantation with hydroxyurea and cytotoxic T-cell lymphocytes

Epstein-Barr virus (EBV) lymphoproliferative disease (LPD) is a potentially fatal complication that may follow allogeneic hematopoietic stem-cell transplantation (HSCT). In this article, the authors report a 2-year-old girl with Hurlers syndrome who developed multiple central nervous system (CNS) EBV LPD lesions 1 year after unrelated donor HSCT. Before this CNS occurrence, the patient had a complete response to rituximab treatment for EBV LPD of the spleen and lymph nodes; however, treatment of the CNS disease with rituximab proved ineffective. Because of reported favorable response of primary CNS EBV LPD in two human immunodeficiency virus-positive patients, the authors treated this patient with low-dose oral hydroxyurea. The patient improved clinically, with a decrease in size of multiple EBV LPD brain lesions. Subsequently, the patient received EBV-specific cytotoxic T-cell lymphocytes and remains well. The benefit and limited toxicity of hydroxyurea therapy merit its further consideration as treatment for EBV LPD.

Rationale for an international consortium to study inherited genetic susceptibility to childhood acute lymphoblastic leukemia.

Acute lymphoblastic leukemia is the major pediatric cancer in developed countries. To date most association studies of acute lymphoblastic leukemia have been based on the candidate gene approach and have evaluated a restricted number of polymorphisms. Such studies have served to highlight difficulties in conducting statistically and methodologically rigorous investigations into acute lymphoblastic leukemia risk. Recent genome-wide association studies of childhood acute lymphoblastic leukemia have provided robust evidence that common variation at four genetic loci confers a modest increase in risk. The accumulated experience to date and relative lack of success of initial efforts to identify novel acute lymphoblastic leukemia predisposition loci emphasize the need for alternative study designs and methods. The International Childhood Acute Lymphoblastic Leukaemia Genetics Consortium includes 12 research groups in Europe, Asia, the Middle East and the Americas engaged in studying the genetics of acute lymphoblastic leukemia. The initial goal of this consortium is to identify and characterize low-penetrance susceptibility variants for acute lymphoblastic leukemia through association-based analyses. Efforts to develop genome-wide association studies of acute lymphoblastic leukemia, in terms of both sample size and single nucleotide polymorphism coverage, and to increase the number of single nucleotide polymorphisms taken forward to large-scale replication should lead to the identification of additional novel risk variants for acute lymphoblastic leukemia. Ethnic differences in the risk of acute lymphoblastic leukemia are well recognized and thus in assessing the interplay between inherited and non-genetic risk factors, analyses using different population cohorts with different incidence rates are likely to be highly informative. Given that the frequency of many acute lymphoblastic leukemia subgroups is small, identifying differential effects will realistically only be possible through multi-center pooled analyses. Here, we review the rationale for identifying genetic risk variants for acute lymphoblastic leukemia and our proposed strategy for establishing the International Childhood Acute Lymphoblastic Leukaemia Genetics Consortium.

Hematopoietic stem cell transplantation for homozygous β-thalassemia and β-thalassemia/hemoglobin E patients from haploidentical donors

Thalassemia-free survival after allogeneic stem cell transplantation (SCT) is about 80–90% with either matched-related or -unrelated donors. We explored the use of a mismatched-related (‘haplo- ’) donor. All patients received two courses of pretransplant immunosuppressive therapy (PTIS) with fludarabine (Flu) and dexamethasone (Dxm). After two courses of PTIS, a conditioning regimen of rabbit antithymocyte globulin, Flu and IV busulfan (Bu) was given followed by T-cell-replete peripheral blood progenitor cells. GvHD prophylaxis consisted of cyclophosphamide (Cy) on days SCT +3 and +4 (post-Cy), and on day SCT +5 tacrolimus or sirolimus was started together with a short course of mycophenolate mofetil. Thirty-one patients underwent haplo-SCT. Their median age was 10 years (range, 2–20 years). Twenty-nine patients engrafted with 100% donor chimerism. Two patients suffered primary graft failure. Median time to neutrophil engraftment was 14 days (range, 11–18 days). Five patients developed mild to moderate, reversible veno-occlusive disease, while nine patients developed acute GvHD grade II. Only five patients developed limited-chronic GvHD. Projected overall and event-free survival rates at 2 years are 95% and 94%, respectively. The median follow up time is 12 months (range, 7–33 months).

Variation at 7p12.2 and 10q21.2 influences childhood acute lymphoblastic leukemia risk in the Thai population and may contribute to racial differences in leukemia incidence

Recent genome-wide association (GWA) studies of childhood acute lymphoblastic leukemia (ALL) have identified 7p12.2, 9p21.3, 10q21.2, and 14q11.2 SNPs that confer modest risks of ALL. These studies have been conducted in European populations, and it is unclear whether these observations generalize to other populations with a lower incidence of ALL. To explore the impact of these variants on ALL risk in the Thai population, we genotyped 190 cases of ALL and 182 controls for SNPs rs4132601 (7p12.2), rs3731217 (9p21.3), rs7089424 and rs10821938 (10q21.2), and rs2239633 (14q11.2). Consistent with findings in European populations, rs4132601 genotype was significantly associated with risk of ALL (odds ratio [OR] = 1.57, 95% confidence interval [CI]: 1.01–2.44; p = 0.04), and rs10821938 genotype was significantly associated with B-cell precursor ALL (OR = 0.73, 95% CI: 0.55–0.97; p = 0.03). There were, however, differences in allele frequencies in SNPs observed between Thai and Caucasian populations (e.g. IKZF1, rs4132601; risk allele frequency [RAF] ratio of 0.36 for Thai/Caucasian). These differences, combined with differences in linkage disequilibrium structure between populations or differences in effect size between populations, may contribute to racial differences in ALL incidence.

Folate pathway genetic polymorphisms and susceptibility of central nervous system tumors in Thai children

BACKGROUND Folate is an important micronutrient molecule participating in DNA synthesis, methylation and repair mechanisms. Genetic polymorphisms in folate pathway related enzymes including methylenetetrahydrofolate reductase (MTHFR) C677T and A1298C, methionine synthase (MTR) A2756G, thymidylate synthase (TS) 28-bp tandem repeat, and reduced folate carrier (RFC) G80A have been shown to be associated with increased susceptibility for several cancers. The aim of the present study was to evaluate whether single nucleotide polymorphisms in the genes encoding enzymes of the folate pathway predispose to any CNS tumors in Thai children. METHODS In the present case-control study, we investigated these polymorphisms in genomic DNA from peripheral blood mononuclear cells in 73 Thai children with various types of central nervous system tumors and in 205 age and sex matched controls. RESULTS Thirty-one out of 73 patients were diagnosed with glial tumors (astrocytoma, oigodendroglioma and ependymoma), 28 with embryonal CNS tumors (medulloblastoma, pinealoblastoma and primitive neuroectodermal tumor), 13 with germ cell tumors and 1 with meningioma. We found that the homozygous CC allele of MTHFR A1298C conferred an increased risk of embryonal CNS tumors (OR: 3.9; 95% CI: 1.3-11.4, p=0.02). CONCLUSION Our findings thus suggest that folate metabolism may play a role in the pathogenesis of certain specific subtypes of pediatric brain tumor in Thai children, especially embryonal CNS tumors.

Genetic polymorphisms of folate metabolic enzymes and toxicities of high dose methotrexate in children with acute lymphoblastic leukemia

Dear Editor,Methotrexate (MTX) inhibits several enzymes in folatemetabolic pathway, including dihydrofolate reductase,thymidylate synthase (TYMS), and methylenetetrahydrofo-late reductase (MTHFR), causing defects in DNA and RNAsyntheses and methylation process [1]. Genetic polymor-phisms of genes encoding proteins in folate metabolismaffect their structures and functions. MTHFR C677T andA1298C are associated with decreased enzyme activity andhyperhomocysteinemia [1]. There are 28 nucleotide-tandemrepeat variations in 5’untranslated region of the TYMSgene. The most common variations of this polymorphismare two and three repeats (2R and 3R). TYMS gene with3R polymorphism has a higher expression level than theone with 2R [1, 2]. Reduced folate carrier (RFC) is anessential carrier protein for folate and MTX. RFC G80Apolymorphism affects MTX level in patients treated withhigh dose of MTX. MTX level has been found to besignificantly higher in patients with RFC 80 AA genotypethan other genotypes [1, 3].Our institute normally uses two courses of high-doseMTX (1.5 g/m

Simple multiplex RT-PCR for identifying common fusion transcripts in childhood acute leukemia

Nonrandom gene rearrangements have been demonstrated in leukemic cells at diagnosis. These genetic abnormalities are associated with specific types, clinical characteristics, and prognosis of acute leukemia. Common fusion transcripts in childhood acute lymphoblastic leukemia (ALL) are TEL‐AML1, E2A‐PBX, MLL‐AF4, and BCR‐ABL (p190) and in acute nonlymphoblastic leukemia (ANLL) are AML‐ETO, PML‐RARA, and CBFB‐MYH11. Reverse transcription‐polymerase chain reaction (RT‐PCR) for detection of each individual fusion transcript is impractical and time consuming. The purpose of this study was to develop simple RT‐PCR methods to identify common fusion transcripts of newly diagnosed acute leukemia in children. Total RNA was extracted from bone marrow samples of children diagnosed with acute leukemia. Multiplex RT‐PCR panel A (ALL) included primers for TEL‐AML1, E2A‐PBX, MLL‐AF4, and BCR‐ABL (p190) whereas panel B (ANLL) composed of primers for AML‐ETO, PML‐RARA, and CBFB‐MYH11. Known leukemic cell lines were used to serve as positive controls. Eighty three children diagnosed with ALL (n = 63) and ANLL (n = 20) were included in this study. Fusion transcripts could be identified using multiplex RT‐PCR panel A for ALL and panel B for ANLL in 26/83 (31.3%) cases. In ALL samples, we found TEL‐AML1 = 16/63 (25.4%), E2A‐PBX = 3/63 (4.8%), MLL‐AF4 = 1/63 (1.6%), and BCR‐ABL = 1/63 (1.6%). Four cases of AML1‐ETO (20%) and one PML‐RARA (5%) were found in ANLL samples. In conclusion, our simple multiplex RT‐PCR for detection of fusion transcripts in childhood acute leukemia was found to be a rapid, accurate, and effective method.