High Expression of LAG3, LPL and ZAP-70 Genes in B-CLL Strongly Correlates with Unmutated IgVH and Early Therapy Requirement.
Authors: Kotaskova Jana , Mraz Marek , Tichy Boris , Kabathova Jitka , Malcikova Jitka , Trbusek Martin , Francova Hana , Doubek Michael , Brychtova Yvona , Mayer Jiri , Pospisilova Sarka 
Published: April 2020

Abstract
Mutational status of immunoglobulin genes coding for heavy chain variable region (IgVH) stratifies patients with B-cell chronic lymphocytic leukemia (CLL) into two distinct prognostic groups. Expression level of specific genes such as LPL, ZAP-70, ADAM29 or CLLU1 has previously been shown to distinguish these two subgroups, although their exact role in CLL pathogenesis remains unclear. In our study we performed microarray and Real-time PCR analyses of gene expression in B-CLL cells with different IgVH mutational status in an attempt to further identify potential discriminating genes. Heparinised peripheral blood samples were collected from 92 previously untreated B-CLL patients (40 IgVH unmutated vs. 52 IgVH mutated) with a median age of 62 years (ranging from 40 to 81 years). B-cells were separated using cocktails of selected bispecific antibodies directed against cell surface antigens of hematopoietic cells and glycophorin A (RosetteSep® B Cell Enrichment Kit, StemCell Technologies). Great attention was paid to T-cell depletion using Human CD3 Depletion Cocktail. Samples with lower percentage of B-CLL cells were further processed by immunomagnetic separation (EasySep® FITC Selection Cocktail, anti-CD5FITC) and only samples with B-CLL cell content higher than 95 % (assessed using flow cytometry) were used for the gene expression analysis. Microarray gene expression analysis of 20 B-CLL patients (10 with mutated and 10 with unmutated IgVH) was performed using Agilent Human 1A Arrays and obtained data were statistically analysed using SAM (Significance Analysis of Microarrays). Among approx. 50 diferentially expressed genes, high expression of LPL and LAG3 (Lymphocyte- Activation Gene 3) was the most prominently associated with unmutated IgVH subtype. The mRNA levels of selected genes (LAG3, LPL, ZAP-70, AICDA, BCL2, CLLU1, TCF7, SEPT10, ADAM29) were further tested on a cohort of 92 B-CLL patients using quantitative RT-PCR (TaqMan® Gene Expression Assay, Applied Biosystems). Among them, LAG3, LPL and ZAP-70 reached the best statistical significance (p-value &lt; 0.001; Wilcoxon’s rank-sum test). LAG3 expression level can predict unmutated IgVH with 100% sensitivity and could be used as a novel potential CLL prognostic marker. LAG3 plays role in T-cell dependent B-cell activation during affinity maturation and its over-expression in cells with unmutated IgVH may be a consequence of improper signalling in this process. Combination of expression data of three genes LAG3, LPL and ZAP-70 reliably assigned 95% of CLL samples into a correct IgVH subtype and seems to be better predictor of IgVH status than either of these genes itself. Moreover, we have analysed the correlation between expression of those three IgVH surrogate markers and necessity of therapeutical intervention. The patients manifesting high expression of LAG3, LPL and ZAP-70 required therapy significantly earlier than patients with low expression of those genes as calculated by Kaplan-Meier estimator (median time to the first therapy was 797 days in patients with high expression of surrogate markers, median was not reached in patients with low expression of surrogate markers). Differences of time to the first therapy between patients with differential expression of three IgVH surrogate markers (p=4.4*10−8, log-rank test) and differences between patients with different IgVH status (p=7.5*10−8, log-rank test) showed high significance. These results suggest that IgVH status could be precisely assessed using expression analysis of three genes LAG3, LPL and ZAP-70. The expression data of those surrogate markers provide even better statistical concordance with time to requirement of the first therapy than IgVH mutational status itself and possibly could be used in the diagnostics.