Methods to Detect MDR1/P-Glycoprotein in Clinical Tumor Samples

Abstract

The methods commonly used to detect MDRl/P-glycoprotein (Pgp) in patients’ tumors differ widely in sensitivity and specificity and are not standardized. This seems one reason for the often discrepant results reported on the incidence and clinical relevance of MDRl/Pgp expression in particular tumor types. Recently, an international workshop was conducted which had the goal to standardize MDRl/Pgp detection methods for future clinical studies. Good interlaboratory agreement was found in the detection of high level MDRl/Pgp expression, whereas considerable variation was observed in the detection of low expression levels as typically found in patients’ tumors. Detection was more reliable in leukemias than in solid tumors. RT-PCR showed the highest sensitivity. In leukemias, flow cytometry was more sensitive than immunostaining in detecting Pgp, and analysis of Pgp function was more sensitive than detection of Pgp expression. Major recommendations for future studies were: Assay conditions and reagents need to be properly calibrated. With each assay, the use of appropriate negative and positive controls is crucial. RT-PCR assays need to be performed in the exponential range of amplification. For immunohistochemistry, at least two anti-Pgp antibodies should be used which recognize distinct epitopes. For flow cytometry, antibodies should be preferred which recognize external epitopes, and functional Pgp assays should be performed in the absence versus presence of Pgp-blocking agents. Functional assays should be accompanied by analyses of MDRl/Pgp expression. Multiparameter analyses are encouraged whenever possible.