The Role of Multidrug Resistance Proteins and Drug Sensitivity in Relation to Poor Prognosis Identified by ZAP-70 Expression and IgV_H Mutation Status in B-CLL.

A proportion of haematological malignancies over express the multidrug resistance (MDR) efflux proteins - P-glycoprotein (P-gp), multidrug resistance protein (MRP1) and lung resistance proteins (LRP) that confer resistance to a number of structurally unrelated cytotoxic drugs. The clinical significance of the observed over expression of P-gp in B-CLL regardless of stage or treatment of the disease is unclear, although a protective effect on cell survival has been reported. Recent evidence suggest that a sub-group of B-CLL exist with an aggressive clinical course that is often resistant to treatment. B-CLL cells from this subgroup of patients have unmutated immunoglobulin heavy chain variable region (IgV_H) and express zeta-associated protein (ZAP-70). We analysed the association between the expression of MDR proteins and the prognosis indicator ZAP-70 in cells from 46 B-CLL patients (30M/16F, 33 stage A, 7 B, 6 C) for (i) ZAP-70 expression using an antibody (clone 2F3.2)-Alexa Fluor 488 conjugate by flow cytometry and expressed as a ratio of B to T-cell mean cell fluorescence, (ii) the expression of P-gp, MRP1 and LRP protein by flow cytometry using specific antibodies and expressed as MCF ratio to isotype controls, (iii) flow cytometric analysis of functional activity of P-gp expressed as MCF ratio in the presence and absence of verapamil, a known inhibitor of P-gp, (iv) IC₅₀ (μg/ml) for cytotoxic drugs 2-chlorodeoxyadenosine (CdA), chlorambucil (Chl) and fludarabine (FdR) using the cytotoxicity MTT assay and (v) IgV_H mutational status by sequence analysis of FR1/J_H polymerase chain reaction products in 29/46 (63%) of patients. 13/46 (28%) patients were ZAP-70^+ve and the remaining 33/46 (72%) were ZAP-70^−ve. P-gp expression (median 1.59 in both groups p=0.85) and P-gp functional activity (median 2.01 vs 1.84 p=0.77) were not statistically different between ZAP-70^+ve and ZAP-70^−ve groups. There was no significant difference in the expressions of MRP1 (median 2.77 vs 3.48 p=0.31) or LRP (median 4.87 vs 5.18 p=0.56) between ZAP-70^+ve and ZAP-70^−ve patients. The IC₅₀ levels for CdA, Chl and FdR between ZAP^+ve (median 0.04, 6.4 & 0.54mg/ml respectively) and ZAP^−ve groups (median 0.065, 12.15 & 0.72 respectively) were not statistically significant (p=0.67, 0.45, 0.36 respectively). 9 ZAP-70^+ve and 15 ZAP-70^−ve patients were sequentially investigated over a median period of 3 years for expression of P-gp, MRP1, LRP and P-gp function. There were no significant changes in P-gp expression and P-gp function between presentation and follow samples over this period. However, a significant increase in expression of MRP1 (median at presentation 1.65 and follow up 5.23 p= 0.0001) and LRP (median at presentation 3.92 and follow up 5.84 p= 0.0002 respectively) were observed for the ZAP-70^−ve group. IgV_H mutation analysis was concordant with ZAP-70 expression in 6 patients with poor prognosis (ZAP-70^+ve/IgV_H unmutated) and in 16 with good prognosis (ZAP-70^−ve/IgV_H mutated). Statistical analysis of MDR protein expression and IC₅₀ in this smaller cohort of patients provided data similar to that in the main group. MDR proteins, their activity and in vitro drug sensitivity appear not to significantly contribute to the poor prognosis subset of B-CLL identified by their ZAP-70^+ve/IgV_H unmutated status. The significance of increased MRP1 and LRP expression in the good prognosis ZAP^−ve group is unclear.