High expression of lymphocyte-activation gene 3 (LAG3) in chronic lymphocytic leukemia cells is associated with unmutated immunoglobulin variable heavy chain region (IGHV) gene and reduced treatment-free survival

Abstract

Chronic lymphocytic leukemia (CLL) is characterized by a monoclonal expansion of mature B-lymphocytes. Mutational status of the immunoglobulin variable heavy chain region (IGHV) gene stratifies CLL patients into two prognostic groups. We performed microarray analysis of CLL cells using the Agilent platform to detect the most important gene expression differences regarding IGHV status in CLL cells. We analyzed a cohort of 118 CLL patients with different IGHV mutational status and completely characterized all described prognostic markers using expression microarrays and quantitative real-time RT-PCR (reverse transcription PCR). We detected lymphocyte-activation gene 3 (LAG3) as a novel prognostic marker: LAG3 high expression in CLL cells correlates with unmutated IGHV (P < 0.0001) and reduced treatment-free survival (P = 0.0087). Furthermore, quantitative real-time RT-PCR analysis identified a gene-set (LAG3, LPL, ZAP70) whose overexpression is assigned to unmutated IGHV with 90% specificity (P < 0.0001). Moreover, high expression of tested gene-set and unmutated IGHV equally correlated with reduced treatment-free survival (P = 7.7 * 10(-11) vs. P = 1.8 * 10(-11)). Our results suggest that IGHV status can be precisely assessed using the expression analysis of LAG3, LPL, and ZAP70 genes. Expression data of tested markers provides a similar statistical concordance with treatment-free survival as that of the IGHV status itself. Our findings contribute to the elucidation of CLL pathogenesis and provide novel prognostic markers for possible application in routine diagnostics.

Figure 1

Significance Analysis of Microarrays supervised analysis of differently expressed genes. Expression profiles obtained using microarray analysis were divided into groups according to IGHV mutational status (M, mutated IGHV; n = 10 and U, unmutated IGHV; n = 10). Significant genes were selected with Significance Analysis of Microarrays and grouped into clusters. Gene coloring is based on normalized patient-to-reference RNA log2 ratios as shown at the top of the figure. Genes selected for further validation using quantitative real time RT-PCR are marked in colors on the side.

Figure 2

Kaplan-Meier curves for treatment-free survival (TFS) from diagnosis to onset of CLL-related therapy according to IGHV mutational status (A) and expression of tested genes (B, C, D, E, and F). Statistical significance was calculated using a log-rank test. A: Patients were grouped according to IGHV mutational status; the TFS median was not reached vs. 13 months in mutated and in unmutated arms, respectively (P < 0.001). Solid line corresponds to mutated IGHV; dotted line corresponds to unmutated IGHV. B: Patients were grouped according to LPL gene expression; the TFS median was 157 vs. 17 months in low and high expression arms, respectively (P < 0.001). Solid line corresponds to low expression; dotted line corresponds to high expression of LPL. C: Patients were grouped according to LAG3 gene expression; the TFS median was not reached vs. 50 months in low and high expression arms, respectively (P < 0.0089). Solid line corresponds to low expression; dotted line corresponds to high expression of LAG3. D: Patients were grouped according to ZAP70 gene expression; the TFS median was not reached vs. 33 months in low and high expression arms, respectively (P < 0.001). Solid line corresponds to low expression; dotted line corresponds to high expression of ZAP70. E: Patients were grouped according to combined 3-gene set expression (LPL + LAG3 + ZAP70); the TFS median was 157 vs. 15 months in low and high expression arms, respectively (P < 0.001). Solid line corresponds to low expression and dotted line corresponds to high expression of gene set. F: Patients were grouped according to combined 3-gene set expression (LPL + LAG3 + ZAP70) considering number of up-regulated genes; the TFS median was: (a) not reached in arm with all 3 genes down-regulated (n = 16), dash-and-dot line, P < 0.001, as compared with (d); (b) not reached in arm with 1 gene up-regulated (n = 23), dashed line, P < 0.001, as compared with (d); (c) 157 months in arm with 2 genes up-regulated (n = 22), solid line, P < 0.001, as compared with (d); and (d) 15 months in arm with all three genes up-regulated (n = 57), dotted line.