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Expression of multidrug resistance (MDR) proteins and in vitro drug resistance in acute leukemias

Abstract The expression of the multi drug resistance (MDR) proteins may influence the outcome of treatment in patients with acute leukemia. The aim of this study was to determine the IC50 of cytotoxic drugs (cytosine arabinoside, ara-C and daunorubicin, dnr) using the in vitro 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-suljophenyl)2H-tetrazolium, inner salt (MTS) assay method. A total of 82 newly diagnosed acute leukemia cases (43 adult myeloid leukaemia, AML cases and 39 acute lymphoblastic leukaemia, ALL cases) and 16 relapsed cases (8 AML cases and 8 ALL cases) were studied. The MTS assay was performed using two cytotoxic drugs, dnr and ara-C. Cells were incubated with different concentrations of drugs for 4 days and the IC50 was extrapolated from the viability curve. In newly diagnosed cases, we found that childhood ALL samples showed higher IC50 values of dnr (0.040 ± 2.320) compared to adult AML samples (0.021 ± 0.158). In contrast, newly diagnosed adult AML samples showed higher IC50 values of ara-C (0.157 ± 0.529) compared to childhood ALL samples (0.100 ± 2.350). In relapsed cases, two samples of childhood ALL showed IC50 values of dnr (0.910 ± 1.760) and ara-C (1.310 ± 2.390), which was higher compared to childhood AML samples (0.129 ± 0.214 and 0.210 ± 0.003, respectively). However, there was no correlation between IC50 values of these drugs tested with clinical outcome. In conclusion, we found that MTS assay is an easy, rapid and non laborious method to study in vitro drug resistance in acute leukaemia cases.

G6PD deficiency with hemolytic anemia due to a rare gene deletion—A report of the first case in Malaysia

Abstract A 2-year-old Chinese boy was referred to Hospital UKM for investigation of recurrent episodes of dark-coloured urine and pallor since birth. He was born prematurely at 34 weeks gestation and developed severe early-onset neonatal jaundice requiring exchange blood transfusion. Screening at birth showed Glucose-6-phosphate dehydrogenase (G6PD) deficiency. On admission, physical examination revealed pallor, jaundice and mild hepatomegaly. Results of laboratory investigations showed a hemoglobin level of 11.0 g/dl with a hemolytic blood picture, reticulocytosis of 20% and red cell G6PD activity reported as undetectable. The patients DNA was analysed for G6PD mutations by PCR-based techniques and DNA sequencing and results showed a 24 bp deletion of nucleotide 953–976 in the exon 9 of the G6PD gene. DNA analysis was also performed on blood samples of the patients mother and female sibling confirming their heterozygous status, although both showed normal red cell G6PD activity levels. The patient was discharged well and his parents were appropriately advised on the condition and the importance of taking folic acid regularly. This is a first case report in Malaysia of G6PD deficiency causing chronic-hemolytic anemia. The rare 24 bp deletion causes the G6PD Nara variant, previously reported only in two other unrelated males, a Japanese and a Portuguese both with chronic hemolytic anemia.

Unexpected Epithelial Membrane Antigen (EMA) and Cytokeratin Expression in a Case of Infantile Acute Monoblastic Leukaemia.

A previously healthy eleven month old male Malay infant presented with fever, upper respiratory tract infection and right knee swelling. Pallor, bilateral proptosis, hepatosplenomegaly, multiple scalp swellings and a right cheek swelling were observed. Investigations revealed that he had acute monoblastic leukemia or FAB M5a. Immunophenotyping by flow cytometry showed that the blast cells were positive for CD45, CD13, CD33, HLA-DR, CDllc, CD71, EMA, and Cytokeratin. They were negative for CD34, CD19, CD10, CD22, CD2, CD3, CD4, CD7, CD8, CD61, NK, Glycophorin A, and CD14. The monoblasts were used to evaluate anti-EMA and anti-cytokeratin. They were unexpectedly found to be positive. Acute monoblastic leukaemias are well known to show extramedullary infiltration and this may be their primary mode of presentation. Thus, in immunochemostry, when using EMA and cytokeratin expression in the differential diagnosis of neoplastic diseases, it is important to consider that monoblasts may express these markers as illustrated by this case.

Prevalence of UGT1A1 mutations in malay neonates with severe jaundice

Aims Severe hyperbilirubinaemia is associated with permanent brain damage, also known as kernicterus. Many factors have been implicated in its development and more recently a genetic basis to many of these conditions has been established. We looked for the frequency of the UGT1A1 247 (T to C), A(TA) 7 TAA variant and G71R mutation in our Malay population. Methods Cord blood from 98 neonates without hyperbilirubinaemia (controls) was compared with cord blood from 125 neonates (cases) with severe hyperbilirbinaemia (total serum bilirubin >300 µmol/L). Samples were analysed for the nucleotide 247 polymorphism by Taqman single nucleotide polymorphism genotyping assay using Corbett Life Science Rotor-gene 6000 (Corbett Research, Australia) and the A(TA) 7 TAA variant by the Agilent 2100 bioanalyser. Results Of 246 neonates, no mutation for the UGT1A1 247 (T to C) was detected. However, four of 113 neonates with severe hyperbilirubinaemia (3.5%) were found to carry the A(TA) 7 mutation (heterozygous) and one of 87 controls (0.01%) carried the mutation (homozygous). The A(TA) 7 TAA variant was found to have a prevalence of 1.4% within the Malay population. Conclusion We have shown that neonatal jaundice may be contributed by a single dose of the A(TA) 7 TAA variant as an added risk factor to other factors that remain to be studied. We have also successfully established a rapid molecular technique that can be used for the screening of UGT1A1 mutations in neonatal jaundice.