Competing Influences of Genetic and Leukaemia-Specific Factors on P-Glycoprotein Expression in Blasts from 817 Patients Entered into the NCRN AML14 and 15 Trials.

Background. P-glycoprotein (pgp), expressed on acute myeloid leukaemia (AML) blasts, is associated with failure to respond to chemotherapy in AML. This study aimed to determine whether expression and function of pgp may be linked to polymorphisms of the encoding gene (MDR1, also known as ABCB1) and whether leukaemia-specific changes in cell biology may override genetic factors in predicting pgp expression. G1199A, G2677T and C3435T polymorphisms in MDR1 (RFLP analysis) as well as pgp protein expression (using MRK-16) and function (modulation of R123 accumulation by PSC 833) were studied in leukaemic blast samples from 817 patients with AML entered into the NCRN AML14 and AML15 clinical trials.

Age, low white blood cell count (WBC), high bcl2, secondary AML and MDS and adverse cytogenetics all correlated with high pgp expression. However, ABCB1 3435TT homozygosity had a negative impact on pgp. Overall, pgp expression was high in 41% of the cohort, but in elderly patients high pgp expression was found in 54% with the 3435C allele versus 32% in the 3435TT group (P=0.009). This is similar to the frequency of pgp in younger patients (34%) and indicates that the 3435TT polymorphism affects pgp expression in elderly patients by preventing the upregulation of pgp protein normally seen in this group. No association was found between genetic factors and pgp function. In a subset of 341 patients, pgp protein levels were analysed on a quantitative scale in relation to C3435T and G2677T gene polymorphisms. The lowest protein expression occurred in the variant TT group for both polymorphisms. Differences were even more marked in the elderly subset (using Mann-Whitney test, P = 0.003 and 0.002 respectively). Linkage disequilibrium occurs between positions 2677 and 3435, and there was a highly significant association between the haplotype and pgp protein expression (P=0.001). ABCB1 mRNA was also measured in 81 patients and was found to be high in patients with the G1199A allele (median expression 0.36 ABCB1/b2M ratio v 0.06 in the GG group, P=0.04), suggesting that the 1199A polymorphism affects ABCB1. However no further correlations were found between the 1199A polymorphism and pgp protein or function. The association between pgp and a low white WBC, (P<0.001) was further examined by cell cycle analysis in 40 patients confirming that pgp is associated with a low proportion of cycling cells - median % cycling cells: pgp negative/low 4.0%, pgp intermediate/high 1.0% (P=0.02).

Conclusions. We conclude that there is an extended MDR phenotype of indolent, pgp positive, bcl2-high cells in AML, particularly in the elderly, and that pgp is affected by an interaction between genetic factors and acquired, leukaemia-specific factors.