CD49d promotes disease progression in chronic lymphocytic leukemia: new insights from CD49d bimodal expression

Abstract

CD49d is a remarkable prognostic biomarker of chronic lymphocytic leukemia (CLL). The cutoff value for the extensively validated 30% of positive CLL cells is able to separate CLL patients into 2 subgroups with different prognoses, but it does not consider the pattern of CD49d expression. In the present study, we analyzed a cohort of 1630 CLL samples and identified the presence of ∼20% of CLL cases (n = 313) characterized by a bimodal expression of CD49d, that is, concomitant presence of a CD49d+ subpopulation and a CD49d- subpopulation. At variance with the highly stable CD49d expression observed in CLL patients with a homogeneous pattern of CD49d expression, CD49d bimodal CLL showed a higher level of variability in sequential samples, and an increase in the CD49d+ subpopulation over time after therapy. The CD49d+ subpopulation from CD49d bimodal CLL displayed higher levels of proliferation compared with the CD49d- cells; and was more highly represented in the bone marrow compared with peripheral blood (PB), and in PB CLL subsets expressing the CXCR4dim/CD5bright phenotype, known to be enriched in proliferative cells. From a clinical standpoint, CLL patients with CD49d bimodal expression, regardless of whether the CD49d+ subpopulation exceeded the 30% cutoff or not, experienced clinical behavior similar to CD49d+ CLL, both in chemoimmunotherapy (n = 1522) and in ibrutinib (n = 158) settings. Altogether, these results suggest that CD49d can drive disease progression in CLL, and that the pattern of CD49d expression should also be considered to improve the prognostic impact of this biomarker in CLL.

Graphical abstract

Figure 1.

CD49d bimodal and homogeneous expression. (A-B) Histogram plots of CD49d expression (red) in 2 representative CLL cases with CD49d bimodal expression (A) and in 2 representative CLL cases with CD49d homogeneous expression (B). Blue histograms refer to unstained cells. The dark red color corresponds to the overlap of CD49d and unstained histogram plots. (C-D) Relative percentage of frequency of CD49d expression in CLL with CD49d bimodal (C) and CD49d homogeneous (D) expression.

Figure 2.

CD49d expression over time. CD49d expression was evaluated in 2 sequential samples from 94 CLL cases with CD49d bimodal expression either untreated (n = 52; A) or treated once between the samples (n = 42; B). (C) Variation of the amount of CD49d⁺ cells in sequential samples from 13 CD49d bimodal CLL treated with 2 lines of therapy (n = 3; blue symbols), 3 lines of therapy (n = 6; green symbols), or 4 lines of therapy (n = 4; red symbols). Each symbol corresponds to a different case; P values refer to the paired Wilcoxon test (A-B) and to the Bonferroni-corrected Student t test (α = 0.0125).

Figure 3.

Amount of CD49d⁺ cells in different tissue compartments and in intraclonal populations from bimCD49d CLL. (A) Percentage of CD49d⁺ cells in paired PB and BM samples from 13 CLL cases with CD49d bimodal expression. Each symbol corresponds to a different case. (B) Amount of CD49d⁺ cells in intraclonal subpopulations with variable reciprocal densities of CXCR4/CD5 expression (CXCR4^dim/CD5^bright and CXCR4^bright/CD5^dim fractions) from 147 CLL cases with CD49d bimodal expression; P values refer to the paired Wilcoxon test.

Figure 4.

Clinical impact of CD49d bimodal expression in the context of chemoimmunotherapy and ibrutinib treatment. (A) OS Kaplan-Meier curves of all bimCD49d CLL cases split in bimCD49d⁻ (blue curves) and bimCD49d⁺ (red curves) groups according to the 30% cutoff. (B) OS Kaplan-Meier curves of CLL cases with bimodal CD49d expression (bimCD49d, purple curves), homogeneous-negative (homCD49d⁻, blue curves), and homogeneous-positive (homCD49d⁺, red curves) CD49d expression. (C) PFS Kaplan-Meier curves of ibrutinib-treated CLL cases with bimodal CD49d expression (bimCD49d, purple curve), negative (neg; blue curve), and positive (pos; red curve) homogeneous CD49d expression (homCD49d). (D) CD49d expression in 2 sequential samples from 8 CLL cases with CD49d bimodal expression treated with ibrutinib between samplings; P values refer to the log-rank test (A-C) and to the paired Wilcoxon test (D).

Figure 5.

Clinical impact of CD49d expression in the context of CLL-IPI categories. (A) OS Kaplan-Meier curves of CLL cases split in the 4 risk categories of the CLL IPI. (B-E) OS curves of CLL cases with homogeneous-negative CD49d expression (homCD49d⁻, light curve) and the merging of homogeneous-positive (homCD49d⁺) and bimodal CD49d (bimCD49d) expression (dark curve) in the context of the low-risk (B), intermediate-risk (C), high-risk (D) and very-high-risk (E) categories of the CLL-IPI. P values refer to the log-rank test.