Incidence of ZAP70 Positivity in Fresh Samples from Unselected Patients Presenting with a B-Cell Lymphoproliferative Disorder.

The expression of ZAP70 has been shown to strongly correlate with unmutated immunoglobulin VH in chronic lymphocytic leukaemia (CLL) cells, which in turn has been shown to distinguish a CLL subgroup of poor prognosis. Retrospective studies have linked ZAP70 expression to a more aggressive clinical course and poorer overall survival in CLL. Previous studies on ZAP70 have largely utilised frozen material. We wanted to avoid the biases of using stored material, potential artefacts of the freeze/thawing process and expand our study beyond CLL. Hence, we analysed ZAP70 by flow cytometry in 184 consecutive cases of B-cell lymphoproliferative disorder (LPD). Only fresh peripheral blood (PB) samples were used. A combination of ZAP 70 (Upstate Biotechnology), CD3 PE and CD56 PE (Coulter Immunotech) antibodies (Ab) were used to exclude the contaminating effects of T and NK cells. CD3 and CD56 staining was followed by fixation and permeabilisation (Dako Intracellular Antigen Kit) and addition of anti-ZAP70 Ab. Indirect labelling of ZAP70 was performed using a secondary conjugate IgG(H+L)-FITC (Southern Biotechnology). Positivity was established at the 20% threshold. 143 cases of CLL were examined for ZAP70 expression: 47 (33%) were positive, with the level of positivity ranging from 20 to 91% (median 41%). Three cases initially ascribed as negative, were subsequently found to be positive upon retesting at a later date. The ZAP70 negative cases of CLL had a median value of 1% (range, 0 – 14%). 41 non CLL cases were examined for ZAP70 expression, mantle cell lymphoma (MCL, 16 cases), hairy cell leukaemia (HCL, 3), diffuse large B-cell lymphoma (2) and 20 cases of B-cell LPD that could not be categorised further. Of these non CLL cases, only 4 were positive for ZAP 70 expression and all of these were cases of MCL (24 – 39% positivity.) The ZAP70 negative cases of MCL had a median value of 0% (range 0–17%). No other non CLL B-cell LPD showed levels of expression >10%. This study of flow cytometric analysis of ZAP70 expression in fresh, presentation PB of all LPDs referred to our laboratory revealed that 33% of CLL and 25% of MCL cases were positive. ZAP70 expression in CLL is bimodal (clearly positive or negative using the 20% threshold) with very few cases in the 10–19% range. No other B-cell LPDs were positive for ZAP70. As ZAP70 testing is now part of our routine LPD panel, ZAP70 positivity provides both diagnostic and prognostic information in CLL, helping to distinguish difficult CLL cases from non CLL (other than MCL). Our 3 cases of changing status may reflect improvements in our protocol or may indicate that ZAP70 status is variable over the course of the disease.