ABC transporters are predictors of treatment failure in acute myeloid leukaemia.

INTRODUCTION: To date, no chemoresistance predictors are included in acute myeloid leukaemia (AML) prognostic scoring systems to distinguish responding and refractory AML patients prior to chemotherapy. ABC transporters have been described as altering AML chemosensitivity; however, a relevant study investigating their role at various molecular levels was lacking. METHODS: Gene expression, genetic variants, methylation and activity of ABCA2, ABCA5, ABCB1, ABCB6, ABCC1, ABCC3 and ABCG2 were analysed in AML blasts and healthy myeloblasts. Differences between responding and refractory AML in a cohort of 113 patients treated with 3 + 7 induction therapy were explored. RESULTS: ABCC3 variant rs2301837 (p = 0.049), ABCG2 variant rs11736552 (p = 0.044), higher ABCA2 (p = 0.021), ABCC1 (p = 0.017), and ABCG2 expression (p = 0.023) and a higher number of concurrently overexpressed transporters (p = 0.002) were predictive of treatment failure by multivariate analysis. Expression of ABCA5 (p = 0.003), ABCB6 (p = 0.001) and ABCC3 (p < 0.0001) increased significantly after chemotherapy. Higher ABCG2 promoter methylation correlated with lower ABCG2 expression (p = 0.0001). ABCC1 was identified as the most active transporter in AML blasts by functional analysis. CONCLUSIONS: ABC transporters, especially ABCC1 seem to contribute substantially to AML chemoresistance. A detailed understanding of chemoresistance mechanisms and the clinical implications of chemosensitivity predictors may lead to alternative therapeutic approaches for AML patients with unveiled chemoresistance signatures.

Validation of a single tube 3-colour immature red blood cell screening assay for the detection and enumeration of small, medium and large paroxysmal nocturnal haemoglobinuria clones by flow cytometry.

INTRODUCTION: The reliable diagnosis of paroxysmal nocturnal haemoglobinuria (PNH) by flow cytometry is based on mandatory analysis of the erythroid, neutrophilic and monocytic lineages. In this study, we have evaluated the performance characteristics of a recently published immature red blood cell (iRBC) assay as a potential screening test for PNH by flow cytometry. METHODS: Intra- and inter-assay imprecision were determined in five replicates of small, medium and large PNH iRBC clones. Analytical and functional sensitivity was assessed by performing spiking tests for five replicates. Thirty healthy donors and 441 PNH patients were tested for evaluation of clinical specificity, sensitivity, positive and negative predictive values. RESULTS: Coefficients of variation (CV) for intra-/inter-assay imprecision analyses were 1.31/1.50, 3.19/2.61 and 3.99/1.58 for the big, medium and small clone sizes, respectively. Absolute values (100%) were found for both clinical specificity and sensitivity as well as for both positive and negative predictive values. The CV from 5 replicate results for 10 clustered events was 15.7%. The coefficient of determination (r2 ), Pearson's correlation coefficient (r) and Bland-Altman mean bias were 0.9436/0.9234/1.7 for PNH iRBC compared to PNH neutrophils and 0.9553/0.9387/2.1 for PNH iRBCs compared to PNH monocytes. CONCLUSION: Our results confirm very good performance characteristics, high analytical and functional sensitivity, absolute clinical specificity and sensitivity as well as favourable correlation between PNH iRBCs and both PNH neutrophils and monocytes, suggesting that this cost-effective 3-colour iRBC assay can be used as a reliable screening test for evaluation of small, medium and large PNH clones by flow cytometry.

Performance characteristics of consensus approaches for small and minor paroxysmal nocturnal hemoglobinuria clone determination by flow cytometry.

BACKGROUND: Evaluation of paroxysmal nocturnal hemoglobinuria (PNH) clones by flow cytometry (FCM) is not standardized and is associated with consistent inter-laboratory variability. METHODS: In order to rule out the influence of particular approach in generating final results, we analyzed the performance characteristics of individual consensus strategies for small to intermediate (1%-20%) and minor (<1%) PNH clones within the white blood cell (WBC) and red blood cell (RBC) compartments with sensitivity up to 0.1%. RESULTS: Coefficient of variation (CV) for precision/reproducibility analysis ranged from 0.67%/1.49% to 2.56%/3.09% for granulocytes, from 0.93%/3.09% to 7.76%/12.06% for monocytes and from 0.41%/4.73% to 6.53%/5.1% for RBCs. Coefficient of determination (r2) for linear regression analysis ranged from 0.95 to 0.99, Wilcoxon ranks test showed no statistically significant differences (p>0.05), Bland-Altman analysis demonstrated performance agreement with mean bias ranging from -0.18 to 1.24. CONCLUSIONS: Our results confirmed very good performance characteristics for precision and reproducibility analysis, excellent correlation and favorable agreement between strategies, suggesting that reported inter-laboratory variability is related mainly to incorrect performance and/or insufficient experience with PNH testing by flow cytometry, rather than to relevant limitations of any particular approach.