Analysis of B-CLL with Discordant ZAP-70 Expression and IgVH Mutational Status.
Authors: Letestu Remi , Le Garff-Tavernier Magali , Vaur Dominique , Ticchioni Michel , Baran-Marszak Fanny , Davi Frederic , Salaun Veronique , Genevieve Franck , Eclache Virginie , Naguib Dina , N’Guyen Florence , Troussard Xavier , Merle-Beral Helene , Ajchenbaum-Cymbalista Florence 
Published: October 2019

Abstract
The clinical course of CLL is heterogeneous and presence or absence of IgVH somatic mutations has been correlated with stable and evolutive disease respectively. ZAP-70 protein expression was shown to be associated with unmutated IgVH genes in CLL and was proposed as a surrogate for mutational status. Recent studies of large number of cases have shown various percentage of discrepancy with respect to ZAP-70 expression and IgVH mutational status. As aggressive disease is not always associated with unmutated IgVH genes we aimed at better characterize the ZAP-70 discordant cases by determining the other prognostic factors such as cytogenetics, expression of CD38 and proliferation markers (thymidine kinase and sCD23). We investigated 292 patients with previously untreated B-CLL. Although several antiZAP-70 antibodies adapted to flow cytometry (FCM) are commercially available, staining procedure and interpretation of the results still remain controversial. Therefore, ZAP-70 expression was determined in the four participating centers by the same FCM method using the 2F3.2 mAb with indirect staining, and expression of the results was standardized. Moreover, ZAP-70 expression was investigated by RQ-PCR in 62 cases and by Western blot in 61 further cases on isolated B cells. The results obtained with these techniques were correlated with FCM data. ZAP-70 was found expressed in 141 cases, among which 38 cases (27%) exhibited ≥2% somatic mutations. Conversely, among the 151 ZAP-70 negative cases, only 6 cases (4%) were found unmutated (≥ 98% VH homology). We focused on the characteristics of the mutated ZAP-70 positive cases. Only 26/38 were in stage A at diagnosis. Incidence of CD38 expression >10% B cells was low (7/28 cases). Analysis of VH sequences pointed to the frequency of VH3-21 usage in 8/38 cases (21%) as compared to an expected 3% frequency in the French population. FISH analysis identified one case with del11q22.3, one case with del17p and two cases with trisomy 12. Del13q14 was present in half of cases. Proliferation markers were significantly higher in these cases than in ZAP-70 negative mutated cases, even among stage A patients. Follow-up of these patients is still too short for significant event free survival as compared with other groups of patients but the number of evolutive Binet stage A patients and advanced B and C stages was already higher than among the ZAP negative mutated cases. In conclusion, in our hands, ZAP-70 was almost always expressed in unmutated cases (103/109). Among mutated cases, ZAP-70 expression was present in 38/183 cases (21%), and brought prognostic information, independently of CD38 or chromosomal alterations. The correlation between ZAP-70 and proliferation markers suggests that ZAP-70 expression may result in a survival advantage of the malignant cells independently of the mutational status.