Evaluation of ZAP-70 Expression Using Real-Time Quantitative RT-PCR (RQ-PCR) in Chronic Lymphocytic Leukemia (CLL), a Reference Method for Flow Cytometry (FCM) Harmonization?.
Authors: Letestu Remi , Baran-Marszak Fanny , Lefebvre Valerie , Salmon Celine , Deglesne Pierre-Antoine , Clavereau Isabelle , Roger Claudine , Ajchenbaum-Cymbalista Florence 
Published: April 2020

Abstract
Mechanisms underlying the heterogeneity of the clinical course of CLL patients are not completely understood and the identification of high-risk patients at diagnosis remains problematic. Somatic mutation status of IgVH genes is a major prognostic factor in CLL but its determination remains labor intensive and costly. ZAP-70 expression was shown by genomic studies to be correlated with IgVH mutational status and proposed as a surrogate. Furthermore, its own prognostic value was demonstrated. Among the different techniques used to evaluate ZAP-70 expression, FCM appeared promising. As different commercial antibodies against ZAP-70 are readily available and as various FCM procedures have been described with different positivity thresholds, a harmonization is warranted. Therefore, for this process, a reference method would be highly valuable. The aim of this study was to evaluate RQ-PCR as a reference method, test different modes of expression for flow cytometry results and correlate to IgVH mutational status. RQ-PCR assays used TaqMan® technology and were run on ABI 7000® system (Applied Biosystems). FCM assays used indirect labeling with 2F3.2 antiZAP-70 antibody, data were acquired on FACSCanto® flow cytometer (Becton Dickinson). The positivity thresholds for RQ-PCR and FCM were established using purified T or B-lymphocytes (Ly) from 15 healthy donors and mononuclear cells from a series of 45 normal controls. Impact of T cell carryover on PCR results was tested by dilution experiments. 71 fully clinically annotated CLL cases were studied for IgVH mutational status and ZAP-70 expression by RQ-PCR and FCM. RQ-PCR was run on purified B-cells isolated by negative selection and only 2 cases (2.8%) were inconclusive. Different formulations of the FCM data were evaluated: % of positive B cells according to isotype control or T cell population and ZAP-70 MFI ratios (R1 = T-Ly/CLL cells, R2 = CLL cells/normal B-Ly). The various FCM parameters showed an applicability ranging from 93 to 94.4% and results were globally well correlated with RQ-PCR. When considering MFI ratios R1 and R2, we observed respectively 2 and 1 (3 and 1.5%) discrepant cases with PCR data. Conversely when using a threshold on T-Ly, discrepancy reached 8.9%, and 25% when isotype control was used. When comparing ZAP-70 expression and mutational status, 17 cases were discordant: 13 were mutated ZAP-70 positive and 4 were unmutated ZAP-70 negative. Among these cases, both RQ-PCR and FCM were discordant with mutational status.
In conclusion, our results show that RQ-PCR on purified B cells is an accurate method for the evaluation of ZAP-70 expression in CLL and corroborate FCM results when expressed as MFI ratio. We propose to use RQ-PCR on purified B cells as a reference method for harmonization of FCM technique and expression of results.