ALBERT

Description: Background: Polymorphisms at the thiopurine S-methyltransferase coding gene (TPMT) determine enzyme activity and consequently, the development of toxicity secondary to thiopurines. Methods: A total of 108 DNA samples from volunteer donors and 39 from patients with acute lymphoblastic leukemia (ALL) were analyzed. Genomic DNA from peripheral blood leukocytes was isolated by standard methods. Known (wild-type and polymorphic) sequenced polymerase chain reaction (PCR) fragments of the TPMT gene were used as controls. TPMT gene fragments were amplified for exons 5, 7 and 10. Thereafter, PCR products were analyzed by denaturating high performance liquid chromatography (DHPLC) for the most frequent mutant TPMT alleles, according to the method developed by Schaeffeler et al. on an analysis system from Transgenomics. Results: No elution profiles on DHPLC analysis, different from those previously reported, were documented. Frequency of allele polymorphisms was 27.3% in healthy and 19.7% in ALL population. However, only 17.6% of all polymorphisms found are considered functional, the most frequent being *3A (n = 13; 4.4%), followed by *3B (n = 5; 1.7%), *3C (n = 5; 1.7%), and *2 (n = 3; 1.0%). From 39 ALL patients, 22 were treated with thiopurines, and particularly one patient, who was referred to our Hospital to investigate relapse, after developing pancytopenia while on treatment with thiopurine, had a TPMT*3C allele. He required broad spectrum antibiotics, platelets transfusions, as well as G-CSF. This example illustrates that TPMT polymorphisms must be determined before thiopurines are indicated. Conclusions: DHPLC is a highly sensitive, rapid and efficient method to identify relevant TPMT gene mutations which allows the screening for genetic variability in the TPMT gene. This is the first analysis of the polymorphisms at this gene in Mexican population. The frequency of known polymorphisms was similar to those reported in other regions worldwide. Since a direct relation has been documented within functional polymorphisms and enzyme activity, and this methology is feasible to realize in any phase during the treatment of ALL patients, the routine typing of TPMT polymorphisms in the patients with ALL has been set in our Institution. Table 1. Frequency of TPMT polymorphisms in mexican population Population TPMT*1 TPMT*1S TPMT*2 TPMT*3A TPMT*3B TPMT*3C * Acute Lymphoblastic Leukemia Healthy n(%) 50 (72.7) 42 (19.4) 2 (0.92) 7 (3.24) 5 (2.3) 3 (1.4) ALL* n(%) 24 (80.3) 5 (6.9) 2 (2.7) 6 (7.6) -- 2 (2.5)

Description: Introduction Antibody-dependent cellular cytotoxicity (ADCC) is an important mechanism in the clinical activity of rituximab for the treatment of B-cell malignancies. The NK cells, through the activating receptor FcγRIIIa, play a major role in the rituximab-mediated ADCC. We have studied the effect of NK stimulators such IL-15 and CpG oligodeoxynucleotides (ODN) in the enhancement of rituximab-mediated ADCC against B-cell lymphoma. Methods ADCC was performed by standard 51Cr release assays using peripheral blood mononuclear cells (PBMC) purified from 4 normal volunteers by Ficoll-Hypaque gradient centrifugation. Cells were cultured for 18 hours in the presence of human recombinant interleukin 15 (rhIL-15; 10 ng/ml) or CpG ODN (2,5 μg/ml). The B-lymphoma cell line Raji was used as target in the presence of rituximab (10 μg/ml) or human IgG1 as control (10 μg/ml) for 30 min at room temperature. Target cells and effector cells were incubated at different ratios ranging from 1:1 to 30:1, for 4 hours at 37°C. The polymorphism at amino acid 158 of the activating FcγRIIIa receptor (FcγRIIIa V→F) was analyzed in effector cells from normal donors with a polymerase chain reaction (PCR)-based allele-specific restriction analysis assay. Results Lysis of B lymphoma cells treated with rituximab alone was 40% ± 5% (mean ± SD) at the highest ratio, as compared to 12% ± 7% (p