TPMT*3C as a Predictor of 6-Mercaptopurine-Induced Myelotoxicity in Thai Children with Acute Lymphoblastic Leukemia.

The response to 6-mercaptopurine (6-MP) can be altered by genetic polymorphisms in genes encoding drug-metabolizing enzymes and drug transporters. The purpose of this study was to investigate the association between genetic polymorphisms of drug-metabolizing enzymes (TPMT 719A > G (*3C), ITPA 94C > A and ITPA 123G > A) and drug transporters (MRP4 912C > A and MRP4 2269G > A) with 6-MP-related myelotoxicity and hepatotoxicity in Thai children with acute lymphoblastic leukemia (ALL). The prescribed dosage of 6-MP and its adverse effects were assessed from medical records during the first 8 weeks and 9-24 weeks of maintenance therapy. Children with the TPMT*1/*3C genotype had a higher risk of leukopenia with an odds ratio (OR) of 4.10 (95% confidence interval (CI) of 1.06-15.94; p = 0.033) compared to wild type (TPMT*1/*1) patients. Heterozygous TPMT*3C was significantly associated with severe neutropenia with an increased risk (OR, 4.17; 95% CI, 1.25-13.90); p = 0.014) during the first 8 weeks. No association was found among ITPA94C > A, ITPA123G > A, MRP4 912C > A, and MRP4 2269G > A with myelotoxicity and hepatotoxicity. The evidence that TPMT heterozygotes confer risks of 6-MP-induced myelotoxicity also supports the convincing need to genotype this pharmacogenetic marker before the initiation of 6-MP therapy.

NUDT15 genetic variants are related to thiopurine-induced neutropenia in Thai children with acute lymphoblastic leukemia.

Aim: 6-Mercaptopurine (6MP) is key to the treatment of acute lymphoblastic leukemia (ALL) as part of maintenance therapy. NUDT15 was identified as a novel thiopurine regulator conferring 6MP sensitivity. The aim of this study was to evaluate the influence of NUDT15 variants on 6MP-induced neutropenia in Thai children with ALL. Materials & methodology: Genotyping of NUDT15 (c.415C>T; rs116855232) and c.36_37insGGAGTC; rs554405994) was performed by Sanger sequencing in 100 patients with ALL. Patients were classified into wild-type (group 1), heterozygous variant (group 2) and homozygous variant (group 3). Clinical and laboratory features during the first 6 months of maintenance therapy were investigated. Therapy-induced neutropenia was observed in 31 patients during the weeks 1-8 (early myelotoxicity), while therapy-induced neutropenia was observed in 47 patients during the weeks 9-24 (late myelotoxicity). Results: There were 85 wild-type patients, 14 heterozygous variant patients and one homozygous variant patient. NUDT15 variants were associated with neutropenia as compared with wild-type (odds ratio: 17.862; 95% CI: 4.198-75.992, padj = 9.5 × 10-5). Multivariate analysis showed that the low-risk group was associated with neutropenia (p = 0.014) in the first 8 weeks of 6MP therapy. Group 2 and group 3 patients had significantly lower absolute neutrophil counts compared with group 1. The adjusted dose during the first 6 months of maintenance therapy with NUDT15 genotype group 1, 2 and 3 were 50, 36.6 and 12.5 mg/m2/day, respectively. Conclusion: Taken together, our results indicate NUDT15 variants may cause neutropenia, and the 6MP dosage should be considered in patients according to the NUDT15 variants to inform personalized 6MP therapy.

Development and validation of a reliable method for thiopurine methyltransferase (TPMT) enzyme activity in human whole blood by LC-MS/MS: An application for phenotypic and genotypic correlations.

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed for the determination of thiopurine methyltransferase (TPMT) activity in human whole blood lysate, based on conversion of 6-mercaptopurine (6-MP) by TPMT to 6-methylmercaptopurine (6-MMP) using S-adenosyl-l-methionine (SAM) as the methyl donor. This method was improved from the previous laborious method for washing of red cell lysate preparation to develop whole blood EDTA lysate. In addition, the TPMT incubation was optimized and the chromatography was performed in a short runtime of 7min on a C18-column by detection via triple quadrupole mass spectrometry. The MS/MS was optimally tuned to monitor mass to charge a ratio (m/z) for 6-MMP 167.2→151.9 and the isotope 6-MMP-d3 with m/z of 170.5→152.2 were applied as an internal standard. The calibration curve covered the range of 2.5-360ng/ml and the correlation coefficient was greater than 0.999. The accuracy of this method was determined in four concentrations of control of quality that ranged between 99.33 and 106.33%. The intra-assay coefficient of variation (CV) was less than 4.41% and the inter-assay was less than 5.43%. This method developed for measuring TPMT by LC-MS/MS is a reliable, safe, and simple with a small volume requirement (100µl of whole blood EDTA). The assay was used to study TPMT activity in 132 Thai children with a range from 29.0 to 89.1nmol 6-MMP/g Hb/h with means and median values of TPMT activity 55.9±12.47nmol 6-MMP/g Hb/h and 54.2nmol 6-MMP/g Hb/h. The genotype-phenotype association of TPMT was evaluated for common ethnic Thai single nucleotide polymorphisms (SNP) in 30 samples and demonstrated good concordance.

The prevalence of anti-hepatitis E in occupational risk groups.

The seroprevalence of anti-hepatitis E virus (HEV) IgG was investigated by using ELISA commercial anti HEV test kit in 408 healthy adults who lived in central part of Thailand, 168 of which were swine workers, 102 were poultry farmers and 138 were government officers. The overall rate of seroprevalence of IgG anti-HEV was 23.3 % (range 16.7-27.9%). The prevalence of anti-HEV antibodies in government officers was 16.7 % and in subjects from swine workers and poultry farmers who worked in farms for more than 2 years were 27.9 % and 24.5%, respectively. Although there was no difference in anti-HEV prevalence according to three job categories (p = 0.06) and to age groups (p = 0.4), but seroreactivity of anti-HEV in swine and poultry farmers were statistically significantly higher than those in officers (p < 0.01). From this preliminary study, HEV is supposed to be circulating in the central area of Thailand. It appeared that the probability of exposure and reinfection to HEV are higher in farmers than that in government officers. Poor environmental conditions in farms, occupation and low socioeconomic status might be risk factors in HEV infection.

Distribution of CD38 molecules on CD3+ and CD8+ T- lymphocyte in adulthood HIV-1-uninfected Thais.

The expression of CD38 on CD8+ T-lymphocyte is a significant predictive value in disease progression of HIV infected individuals and in monitoring a response to therapy. CD38 molecules expressing on CD3+ and CD8+ T-cells were measured quantitatively by flow cytometry in 30 healthy Thai adults. In each experiment, the known amount of fluorochrome in CD38 antibodies bound per cell of QuantiBRITE PE beads was plotted, and set a regression line. With this line, the amount of CD38 molecules bound to CD3 and CD8 target cells was estimated. The aim of this study was to determine the reference value of CD38 molecules on CD8+ T-lymphocyte, which is the baseline in comparison to the CD38 molecule expressing on CD8+ T-lymphocyte in HIV-infected individuals. The present results showed that the amount of CD38 expressions on CD8+ T-lymphocyte in HIV negative Thai adults was about 2 times higher than those from Caucasian's lymphocyte. The reference range of CD38 molecules in the present study would best be used as baseline in prognosis and drug monitoring of HIV-1 infection in Thailand.