Prognostic Potential of Galectin-9 mRNA Expression in Chronic Lymphocytic Leukemia

Abstract

Galectin-9 (Gal-9), very poorly characterized in chronic lymphocytic leukemia (CLL), was chosen in our study to examine its potential role as a CLL biomarker. The relation of Gal-9 expression in malignant B-cells and other routinely measured CLL markers, as well as its clinical relevance are poorly understood. Gal-9 mRNA expression was quantified with RT-qPCR in purified CD19+ B-cells of 100 CLL patients and analyzed in the context of existing clinical data. Our results revealed the upregulation of Gal-9 mRNA in CLL cells. High Gal-9 mRNA expression was closely associated with unfavorable prognostic markers. In addition, Gal-9 expression in leukemic cells was significantly elevated in CLL patients who did not respond to the first-line therapy compared to those who did respond. This suggests its potential predictive value. Importantly, Gal-9 was an independent predictor for the time to treatment parameters. Thus, we can suggest an adverse role of Gal-9 expression in CLL. Interestingly, it is possible that Gal-9 expression is induced in B-cells by EBV infection, so we determined the patients' EBV status. Our suggestion is that EBV coinfection could worsen prognosis in CLL, partly due to Gal-9 expression upregulation caused by EBV.

Keywords: CLL; EBV; Galectin-9; biomarker; prognostic markers.

Figure 1

Level of Gal-9 mRNA in CLL cells and B-lymphocytes from healthy volunteers (HV) (a). Gal-9 mRNA expression at various Rai stages (b). The median is represented through the central line. “Whiskers” indicate the IQR (interquartile range). ** p < 0.01, *** p < 0.001; ns, not significant.

Figure 2

Gal-9 mRNA expression in malignant B-cells from ZAP-70-positive and ZAP-70-negative (a) CLL patients and from CD38-positive and CD38-negative groups (b). CLL patients with different IGHV mutation status (c). The individual values and median with IQR (interquartile range) are displayed. * p < 0.05; ns, not significant. IGHV, immunoglobulin heavy chain variable gene; M-CLL, mutated IGHV; U-CLL, unmutated IGHV; ZAP-70, zeta-chain-associated protein kinase 70.

Figure 3

Level of Gal-9 mRNA in patients with cytogenetic changes (17p-, 11q-, or +12 in isolation or in conjunction with 13q deletion) (a). Gal-9 mRNA expression in a group of CLL patients with sole 13q deletion type (b). The individual values and median with IQR are displayed. ** p < 0.01, *** p < 0.001; ns, not significant; IQR, interquartile range.

Figure 4

Gal-9 mRNA expression in leukemic B-cells in EBV-negative (n = 62) and EBV-positive (n = 33) CLL patients. Individual values and median with IQR are shown. * p < 0.05. EBV, Epstein–Barr virus; IQR, interquartile range.

Figure 5

Patients with CLL who needed therapy and those who did not require therapy were compared according to Gal-9 mRNA expression levels (a). The expression levels of Gal-9 mRNA in CLL patients who respond to treatment and those with progressive disease (b). The individual values and median with IQR are displayed. ** p < 0.01, * p < 0.05; ns, not significant; CR, complete response; PR, partial response; SD, stable disease; PD, disease progression; IQR, interquartile range.

Figure 6

Receiver operating characteristic curve (ROC) was used to determine the optimal cut-off for Gal-9 mRNA expression in malignant B-cells. The calculation included the cut-off value (3.389), AUC (0.831), sensitivity (89%), specificity (73%), and Youden index (0.62) (a). Kaplan–Meier survival curves for Gal-9^low (<3.389) and Gal-9^high (≥3.389) groups comparing TTT (b). TTT, time to treatment; AUC, area under the ROC curve.

Figure 7

Ki-67 (a) and PCNA (b) mRNA expressions were correlated with Gal-9 mRNA expression determined by RT-qPCR. The strength of the association between variables was measured using Spearman’s rank correlation coefficient (r). PCNA, proliferating cell nuclear antigen.

Figure 8

Ki-67 (a) and PCNA (b) mRNA expressions in Gal-9^low (<3.389) and Gal-9^high groups (≥3.389). Individual values and median with IQR are shown. ** p < 0.001, **** p < 0.0001. PCNA, proliferating cell nuclear antigen; IQR, interquartile range.

Figure 9

Comparison of Ki-67 (a) and PCNA (b) mRNA expression between four groups: EBV(−)Gal-9^low (n = 32), EBV(+)Gal-9^high (n = 21), EBV(+)Gal-9^low (n = 12), EBV(−)Gal-9^high (n = 27). Individual values and median with IQR are shown. * p < 0.05, ** p < 0.01, *** p < 0.0001, ns, not significant. PCNA, proliferating cell nuclear antigen; IQR, interquartile range.