Thiopurine Methyl Transferase (TPMT) Activity and Erythrocyte Thioguanine Nucleotide Levels Are Reliable Markers of Tolerability to 6-Mercaptopurine in Adult Acute Lymphoblastic Leukemia

Introduction: Dosing of 6-mercaptopurine (6MP) has remained empirical in acute lymphoblastic leukemia (ALL) with leucocyte count and TPMT genotype guiding clinical decisions. A majority of our patients do not tolerate standard doses of 6MP and require frequent dose interruptions or modifications. There is marked inter-individual variability in the tolerability of 6MP and TPMT genotype alone cannot account for all the variability observed in clinical practice. Better predictors of toxicity are needed to guide hemato-oncologists in dose selection and dose optimization of 6MP to achieve maximum anti-leukemic activity. The present study aims to identify factors that influence tolerability of 6MP, including pharmacokinetic parameters of 6MP and its metabolites, so as to enable the development of predictive models of 6MP toxicity in the future.

Methods: Adult ALL patients aged ≥18 years receiving 6MP during interim maintenance (IM) or maintenance (M) phase of MCP841or BFM-90 protocol were enrolled after informed consent. All patients received 6MP at the protocol specified dose of 75mg/m². Complete blood counts and liver function tests were done at baseline, day 8 and 22 of IM, before each cycle of M Phase and as clinically indicated. Serial blood samples were collected at 0, 1, 2, 4 and 6 hours after 6MP dose on day 1 of IM and M phase and areas under the concentration-time curve (AUC) was estimated by non-compartmental analysis. RBC levels of 6-methylmercaptopurine (6mMP) and 6-thioguanine nucleotide (6TGN) were measured on day8 and day22 using a validated HPLC method. TPMT activity was measured using a validated HPLC method at the time of enrollment. TPMT genotyping was done using Sequenom MassARRAY platform. Dose interruptions, cumulative duration of interruption and dose reduction were recorded as measures of intolerance to 6MP.

Statistical analysis: Descriptive data is either expressed as mean±SD or median(range). Difference in TPMT activity or 6TGN levels between groups was compared using Mann-Whitney test. Proportions were compared using Chi-Square/Fischer Exact test. Spearman's correlation was used to study the correlation between two continuous variables.

Results: Forty five patients (males-36, females-9) were enrolled on the study from November 2012 to November 2014. The median age was 28(range:16-50) years. The baseline hemoglobin concentration and absolute neutrophil count (ANC) was 8.75±1.2 g/dl and 3.24±2.57X10³ cells/µl respectively.Dose was interrupted in 36 patients (80%) patients, median number of interruptions being 2(1-6). The cumulative duration of dose interruption was 23 (4-67) days. Twenty patients (44%) required dose reduction due to grade 4 cytopenias, median number of dose reductions being 1(1-2). TPMT genotype was available for 33 patients (28 WT, 5 heterozygotes). The average TPMT activity was 1.44±1.35units/ml RBCs.

No correlation was observed between TPMT genotype and TPMT activity. Genotype was not a significant determinant of dose reduction, dose interruption or cumulative duration of interruption. On the other hand, marked difference in TPMT activity was observed between those who required dose reduction versus those who did not (0.92±0.65 vs. 2.16±1.77, P <0.05). Similarly, markedly higher TPMT activity was observed in patients who did not require dose interruption compared to those who did (3.56±2.22 vs. 1.36±1.17) although the difference was not statistically significant. An inverse correlation was observed between TPMT activity and cumulative duration of dose interruption (Rho = -0.356, P=0.06). Similarly, erythrocyte 6TGN concentration on day 22 was markedly higher in patients who did not experience dose reduction as compared to those who did (0.52±0.28 ng/8x10⁸ RBC vs. 0.11±0.10 ng/8x10⁸ RBC, P <0.05). AUC of 6MP on day1 did not influence the tolerability of the drug nor did baseline covariates such as hemoglobin and ANC.

Conclusions: TPMT activity and erythrocyte 6TGN concentration on day 22 were major determinants of tolerability of 6MP. TPMT genotype did not show up as a reliable marker of tolerability to 6MP. The ease of determining TPMT activity as well as 6TGN concentration by HPLC makes them attractive markers for routine clinical use. A mathematical model to predict 6MP toxicity based on these parameters is being developed.