The B-Cell Receptor Signaling Inhibitor Molecules CD305 and CD307b Are Markers of Favorable Prognosis in Chronic Lymphocytic Leukemia with Both Mutated and Unmutated IGHV Gene Status

Background. The regulation of the B cell receptor (BCR)-mediated response to antigenic stimulation in chronic lymphocytic leukemia (CLL) is balanced by competing activating and inhibitory signals derived from specific co-receptors. In particular, the BCR inhibitory molecules Leukocyte associated Ig like receptor 1 (LAIR-1)/CD305 and human Fc receptor-like 2 (FCRL2)/CD307b have been independently reported as molecules associated with longer time to first treatment in CLL, although their synergic functional role and combined prognostic relevance remains to be explored. Aim. To assess the functional role and prognostic value of CD305 and CD307b as OS predictors in CLL. Methods. The study included 467 CLL cases all characterized at diagnosis for Rai stage (stages 0-I: 355 cases), CD49d expression (CD49d- CLL, <30% of positive cells by flow cytometry: 266), IGHV mutational status (mutated, M: 270), karyotype abnormalities according to the hierarchical stratification (normal/13q-/+12: 365). Median follow-up of patients was 70 months with 82 deaths. Immunophenotypic analysis was performed using a combination of anti-CD5 FITC, -CD19PE-Cy7, -CD305PerCPCy5.5, CD307bAPC mAbs. For functional assays, mouse anti-human IgM, and agonistic mouse anti-human CD305 and CD307b were used. Fab goat anti-mouse was used as cross linker. Phosphorylation state of ERK (pERK) and ATK (pATK) were evaluated by either flow cytometry or western blotting. Calcium flux was analysed by flow cytometry. Results.A significantly higher (p<0.0001) CD305 and CD307b expression in M versus UM CLL was documented, with mean % of expression of 58% vs. 33% (CD305), and 88% vs. 74% (CD307b). The best cut-off levels for OS, calculated using a ROC analysis, were 10% for CD305 and 85% for CD307b. Using these cut-offs, 331 (70.9%) and 296 (63.4%) were classified CD305⁺ and CD307b^bright, respectively. The clinical impact of both markers as OS predictors was confirmed in both univariate (hazard ratio/confidence interval (HR/CI)= 0.39/0.25-0.60; p<0.0001 for CD305; HR/CI= 0.26/0.16-0.41; p<0.0001 for CD307b), and multivariate (HR/CI=0.41/0.26-0.63; p=0.0001 for CD305;HR/CI=0.27/0.17-0.42; p<0.0001 for CD307b) analyses. Therefore, we dichotomized CLL cases according to the expression of 2 markers (n=220) versus 0/1 markers (n=247). The prognostic impact of this combined markers expression was tested in univariate analysis (HR/CI=0.23/0.13-0.40; p<0.0001 ) and in a multivariate model including: IGHV mutational status, CD49d expression, Rai stage (stage 0-I versus stages II-IV), karyotype abnormalities (normal/del13/+12 versus del11/del17). The combined markers expression retained its prognostic impact (HR/CI=0.37/0.21-0.68; p=0.0013), along with the UM IGHV (HR/CI=2.54/1.51-4.26; p=0.0004), CD49d expression (HR/CI=1.83/1.13-2.97; p=0.015) del11/del17 (HR/CI=1.86/1.14-3.05; p=0.014). Consistently, expression of 2 markers identified subsets with longer OS in the context of both M (p=0.009) and UM CLL (p=0.004; see Figure). To functionally explain the peculiar clinical behavior of CLL expressing these molecules, we evaluated CLL cell response to BCR triggering in CD305⁺/CD307b^bright CLL, with either a M (n=9) or a UM (n=7) IGHV gene status. A significant increase of anti-IgM response was found in 24-hour cultures compared to 2-hour cultures, irrespective of the IGHV mutational status: p-ERK, 41.2% vs 20% (p=0.006); pAKT, 18% vs 7% (p=0.037); calcium flux, mean AUC 2.0x10⁶ vs 1.6x10⁶ (p=0.01). This increased BCR response was paralleled by a significant down-regulation of both CD305 and CD307b after 24-hour culture compared to 2-hour culture (mean MFI 40 vs 92.5 for CD305, p=0.0009; 95.5 vs 414 for CD307b, p=0.001) with no difference between M and UM cases. We next tested the effects on BCR signaling of anti-CD305 and anti-CD307b ligation (n=4). While BCR engagement induced pERK (mean fold increase compared to the control=12), concomitant engagement of BCR and CD305 or CD307b reduced the pERK level to 68% or 40%, respectively. Moreover, simultaneous engagement of both CD305 and CD307b further inhibited pERK level to 28%. Conclusions.A CD305⁺/ CD307b^bright phenotype predicts longer OS in CLL, in both M and UM IGHV CLL. The synergic effect of the B-cell receptor signaling inhibitors CD305 and CD307b may functionally explain this peculiar clinical behavior.

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