Trisomy 12 chronic lymphocytic leukemia cells exhibit upregulation of integrin signaling that is modulated by NOTCH1 mutations

Abstract

The leukocyte adhesion cascade is important in chronic lymphocytic leukemia (CLL), as it controls migration of malignant cells into the pro-survival lymph node microenvironment. Circulating trisomy 12 CLL cells have increased expression of the integrins CD11a and CD49d, as well as CD38, but the tissue expression of these and other molecules, and the functional and clinical sequelae of these changes have not been described. Here, we demonstrate that circulating trisomy 12 CLL cells also have increased expression of the integrins CD11b, CD18, CD29, and ITGB7, and the adhesion molecule CD323. Notably, there was reduced expression of CD11a, CD11b, and CD18 in trisomy 12 cases with NOTCH1 mutations compared with wild type. Trisomy 12 cells also exhibit upregulation of intracellular integrin signaling molecules CALDAG-GEFI, RAP1B, and Ras-related protein ligand, resulting in enhanced very late antigen-4 [VLA-4] directed adhesion and motility. CD38 expression in CLL has prognostic significance, but the increased CD38 expression in trisomy 12 CLL cells must be taken into account in this subgroup, and the threshold of CD38 positivity should be raised to 40% for this marker to retain its prognostic value. In conclusion, trisomy 12 CLL cells exhibit functional upregulation of integrin signaling, with β2-integrin expression being modulated by NOTCH1 mutation status.

Figure 1

Preserved expression of the integrins CD11a, CD11b, CD18, CD29, CD49d, and ITGB7 on trisomy 12 CLL cells. The expression of the integrins CD11a (A), CD11b (B), CD18 (C), CD29 (D), CD49d (E), and ITGB7 (F) were assessed on PB CLL cells and B cells from healthy age-matched controls. Uniquely among the main cytogenetic categories, CLL cells from patients with trisomy 12 had relatively preserved expression of these integrins, with levels comparable to healthy B cells in some patients. A comparable pattern was observed whether the data were analyzed by “% positive” or by median fluorescence intensity.

Figure 2

The expression of integrins on CLL cells in LNs. The expression of integrins was assessed on nodal CLL cells. In contrast to circulating CLL cells, there was no difference in the expression of CD11a (A), CD18 (B), ITGB7 (C), and CD29 (D) on CLL cells from trisomy 12 and nontrisomy 12 cases.

Figure 3

Increased integrin expression correlates with increased numbers of proliferating B cells in healthy and CLL LNs. (A) Representative images of a secondary follicle in a healthy reactive LN. Proliferating germinal center B cells exhibit higher expression of CD11a, CD18, CD29, and ITGB7 than mantle zone B cells. (B) Across LN biopsies from all cytogenetic groups, the presence of higher numbers of proliferating cells correlated with increased expression of CD11a, CD29, and ITGB7, but not CD18. (C) Representative images of CLL LN biopsies without proliferation centers. Biopsies with high numbers of Ki67⁺ proliferating cells have increased expression of CD11a compared with biopsies with low numbers of Ki67⁺ proliferating cells. The increased expression of CD11a in biopsies with high numbers of Ki67⁺ proliferating cells was due to increased staining of the CD79a⁺ cells.

Figure 4

Impact of NOTCH1 mutations on integrin expression in trisomy 12 CLL. The impact of NOTCH1 mutation status on integrin expression was assessed in a cohort of separate cohort of 15 trisomy 12 CLL patients with known NOTCH1 mutation status. Notably the expression of CD11a (A), CD11b (B), and CD18 (C) was significantly reduced in trisomy 12 CLL cells with a NOTCH1 mutation compared with trisomy 12 CLL cells with wild-type NOTCH1 genes. NOTCH1 mutation status had no impact on the expression of CD29 (D), CD49d (E), or ITGB7 (F).

Figure 5

Implications of the increased expression of CD38 on trisomy 12 CLL cells. (A) The proportion of cells that express CD38 is increased in trisomy 12 cases. (B) NOTCH1 mutation status had no impact on the expression of CD38 in trisomy 12 cases. (C) CD38 is not a surrogate marker of IGVH mutation status in patients with trisomy 12. (D) In contrast, increased expression of ZAP70 retains its association with IGVH mutation status in patients with trisomy 12. Treatment-free survival curves for CLL patients with trisomy 12 with a 40% cutoff for CD38 positivity (E).

Figure 6

Integrin “inside-out” signaling is upregulated in trisomy 12 CLL cells. The expression of molecules involved in integrin signaling was assessed by quantitative RT-PCR in CLL cells with and without trisomy 12 and healthy B cells. (A) The expression of RASGRP2 (CALDAG-GEFI) is increased in trisomy 12 CLL cells comparable to healthy B cells. In contrast, RAP1B (B) and RASSF5 (RAPL) (C) are overexpressed in trisomy 12 CLL cells compared with healthy B cells and nontrisomy 12 CLL cells.

Figure 7

The upregulation of integrin signaling results in increased ligand binding and enhanced adhesion and motility that is predominantly VLA-4 directed. The integrin function of CLL cells and healthy B cells was investigated. (A) The ability of the cells to bind soluble VCAM-1 or ICAM-1 was assessed by flow cytometry after integrin activation by 3 mM MnCl₂. (A) Healthy B cells (n = 4) are able to bind significant amounts after VCAM-1 and ICAM-1 after integrin activation, whereas nontrisomy 12 CLL cells (n = 4) bind comparatively little. Trisomy 12 CLL cells (n = 4) bind an intermediate amount of these ligands consistent with their increased integrin expression. The adhesive ability and nondirectional motility of healthy and malignant B cells on VCAM-1– and ICAM-1–coated plates was examined. (B) The proportion of cells in a “spread” conformation was assessed 30 minutes after stimulation with CXCL12. Trisomy 12 CLL cells exhibit an enhanced ability to adhere to immobilized VCAM-1, but not immobilized ICAM-1. (C) This enhanced adhesion translates into improved motility on VCAM-1, but was not significantly increased on ICAM-1. Error bars in all figures represent standard error of the mean.