Evaluation Of Multidrug Resistance (MDR) Functional Activity and Expression Levels Of P-Glycoprotein and MDR Related Protein-1 For The Prediction Of Treatment Failure In Chronic Myeloid Leukemia Patients
Authors: Park Sang Hyuk , Park Chan-Jeoung , Kim Dae-Young , Lee Bo-Ra , Kim Young Jin , Cho Young-Uk , Jang Seongsoo , Chi Hyun-Sook 
Published: October 2019

Abstract

Background
One of the resistance mechanisms of chronic myeloid leukemia (CML) is multidrug resistance (MDR) mediated by overexpression of P-glycoprotein (P-gp) and MDR related protein-1 (MRP1). The flow cytometric functional assay using efflux inhibitor cyclosporine and Rhodamine-123 (Rho-123 efflux assay) detects MDR functional activity. In this study, we performed Rho-123 efflux assay as well as MRP1 and P-gp expression assays by flow cytometry, and evaluated the performance of each test for the prediction of treatment failure in CML patients.

Methods
A total of 229 peripheral blood samples (19 diagnosis and 210 follow-up) from 94 CML patients treated with TKIs were enrolled. These samples were categorized into three subgroups [optimal response (N=120), suboptimal response (N=54), treatment failure (N=36)] defined by the response criteria to treatment with TKIs in CML patients (Best Pract Res Clin Haematol 2009;22:331-41), and ABL1 mutation analysis was performed in some patients (N=18) with treatment failure. In addition, cutoff values (mean + 1.65SD) for each test were determined from 35 normal controls. The Rho-123 efflux assay, MRP1 and P-gp expression assays were performed separately with all samples. In the Rho-123 efflux assay, the MDR functional activity was measured as the ratio of mean fluorescence intensity (mean channel fluorescence, mcf) with and without cyclosporine. In the P-gp and MRP1 expression assays, MRP1 and P-gp expression levels were expressed as the positivity (%) with each representative monoclonal antibody. The results were compared between patients with treatment response (optimal and suboptimal) and failure. The sensitivity/specificity/negative predictive values (NPV)/positive predictive values (PPV) of 3 test results were calculated using estimated cutoff values and compared.

Results
The cutoff values of Rhodamine-123 efflux assay, MRP1, and P-gp expression assays were calculated to be > 1.68, > 4.18%, and > 5.07%, respectively. The patients with treatment failure showed significantly higher MRP1 expression [mean 14.08% vs. 4.48%, P = 0.038 (diagnosis); 5.24% vs. 3.54%, P = 0.006 (follow-up)] and P-gp expression [12.27% vs. 3.41%, P = 0.027 (diagnosis); 5.25% vs. 3.48%, P = 0.005(follow-up)] than those with treatment response. However, there were no significant differences in the Rhodamine-123 efflux assay (P = 0.769 and 0.199 at diagnosis and follow-up) between 2 groups. Subsequent analysis revealed that 58.7% of follow-up patients with treatment failure show positive results in at least one test, and 53.3% of these patients with negative for the ABL1 kinase domain mutation also show positive results in at least one test. The sensitivity/specificity/NPV/PPV of 3 test results for the prediction of treatment failure were estimated as 22.9%/85.1%/84.6%/23.5% (Rhodamine-123 efflux assay), 37.1%/69.7%/83.9%/20.6% (MRP1 expression assay), and 31.4%/78.8%/84.4%/23.9% (P-gp expression assay).

Conclusion
MRP1 and P-gp expression were significantly higher in follow-up CML patients with treatment failure than those with treatment response. The patients who experienced treatment failure, had shown significantly higher MRP1 and P-gp expression at diagnosis than those who did not. Low sensitivity and PPV are inevitable for assays measuring MDR, because MDR is only one of mechanism that causes treatment failure. Our study revealed relatively high specificity and NPV in both MRP-1 and P-gp expression assay, and at least half of ABL1 mutation negative patients with treatment failure possessed positivity in MDR tests. Therefore, it can be speculated that the estimation of MRP-1 and P-gp expression levels can provide useful informations for the prediction of treatment failure in CML patients.

Disclosures:
No relevant conflicts of interest to declare.