Relative Prognostic Significance of ZAP-70 and IGHV1-69 Expression in Chronic Lymphocytic Leukemia,
Authors: Rassenti Laura Z. , Ghia Emanuela M. , Werner Lillian , Neuberg Donna , Widhopf George F. , Kipps Thomas J. 
Published: October 2019

Abstract
Abstract 3891
The leukemia B cells of patients (pts) with chronic lymphocytic leukemia (CLL) express a restricted repertoire of immunoglobulin heavy chain variable region (IGHV) genes. Moreover, certain IGHV genes appear over-represented in this repertoire. Among these, IGHV1-69 was first identified as most frequently expressed, being used by the CLL cells of nearly 20% of all pts (PNAS, 86:5913–7, 1989). CLL cells most frequently express IGHV1-69 without somatic mutations and often with restricted D and JH segment use, providing for stereotypic motifs in the heavy chain third complementarity determining region (HCDR3). We addressed whether there were peculiar biologic or clinical features of pts with CLL that used IGHV1-69. For this, we studied 452 pts identified in a cohort of 2,866 followed by the CLL Research Consortium (CRC) found to have CLL cells that express IGHV1-69. This accounted for 16% of all pts. We found that 420 of 452 CLL samples (93%) express IGHV1-69 without somatic mutation (≥98% sequence homology with germline IGHV1-69), which is in significant contrast to the frequency use of UM IGHV among CLL samples that do not use IGHV1-69. As noted for CLL pts in general, there is a strong association between mutation status and clinical behavior. Among pts that use IGHV1-69, the 32 of 452 pts that use MU IGHV1-69 had a highly indolent clinical course, with a median time from diagnosis to initial treatment (TFS) of 17.3 years. This was significantly longer than the median TFS of 2.8 yrs for the 452 pts that used UM IGHV1-69 (p<0.0001). Among the pts that used UM IGHV1-69 we identified a stereotypic HDCR3 motif shared by more than one patient in 249/452 (55%) of the cases. Multivariate analysis failed to discriminate any significant differences in the median TFS of pts that used IGHV1-69 that had a stereotypic CDR3 motif versus pts who had an idiosyncratic HCDR3 (2.9 yrs vs 3.0 yrs, respectively p=0.14). Interphase FISH for common cytogenetic aberrations in CLL were available for 281 of the 452 cases. Among these, 58% of the cases had deletions at 17p (18%), 11q (23%), or trisomy 12 (17%). The remaining cases had no detectable chromosomal abnormalities (24%) or isolated deletion of 13q (18%).
The CLL cells of all 452 pts were examined for ZAP-70. There was a strong association between the expression of ZAP-70 and use of UM IGHV1-69. However, the association between ZAP-70 expression and use of UM IGHV1-69 was not absolute. Only 70% with UM IGHV1-69 were ZAP-70 positive, as were 12.5% that used M IGHV1-69. Of all 452 pts that expressed IGHV1-69, 300 (66%) had ZAP-70 positive CLL cells. These pts had a median TFS of 2.3 yrs, which was significantly shorter than that of the remaining 152 pts with ZAP-70-negative CLL, who had a median TFS of 4.3 yrs (p<0.0001). Moreover, of the 420 pts that used UM IGHV1-69, 296 (70%) had CLL cells that expressed ZAP-70; these pts had a median TFS of 2.3 yrs. This was significantly shorter than the median TFS of pts with CLL cells that express UM IGHV1-69, but were ZAP-70 negative 4.1 yrs (p<0.0001).
A Cox regression model revealed that although the presence of detectable chromosomal aberrations was associated to a shorter median TFS, ZAP-70 was a stronger predictor of short TFS (HR for 13q =1.1 p =0.03, HR for trisomy 12 =1.2 p =0.03, HR for 11q =1.6 p =0.03, HR for 17p =1.8, p =0.03) (HR for ZAP-70 positive = 1.8, p=0.0004).
The Cox regression model was used to assess the associations of ZAP-70, and the use or not of the UM IGHV1-69 gene with TFS (p-value <0.05 were considered as significant). We investigated these associations using a previously published cohort of characterized 705 CLL pts (Blood.2008;112:1923). The HR associated with the expression of ZAP-70 (HR=3.2) (p<0.0001) was significantly higher than if either the UM IGHV1-69 or the cases with UM IGHV other than IGHV1-69 were incorporated into the model (HR=1.9 and HR=1.6 respectively, p=0.001).
We conclude that cases that use IGHV1-69 are peculiar in that they more frequently use UM IGHV and appear to have a higher frequency of adverse cytogenetic features than CLL cases at large. In addition, we found that CLL-cell expression of ZAP-70 can segregate pts that use UM IGHV1-69 into subgroups with disparate clinical behavior, despite the fact that all patients use the same IGHV gene. Moreover, multivariable analyses revealed that ZAP-70 was strongest predictor of short TFS among all other considered prognostic parameters in this distinctive cohort of pts.

Disclosures:
Kipps: Igenica: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Research Funding; Abbott Industries: Research Funding; Genentech: Research Funding; GSK: Research Funding; Gilead Sciences: Consultancy, Research Funding; Amgen: Research Funding.