Fcgammariib Expression As a Prognostic Marker in Chronic Lymphocytic Leukemia

BACKGROUND

The Fcγ receptor IIb (FcγRIIb) is an inhibitory Fcγ receptor that suppresses B-cell activation when coligated with B-cell antigen receptor (BCR). Previous studies from our group indicate that the ability of the FcγRIIb to inhibit BCR signaling after coligation is attenuated in Chronic Lymphocytic Leukemia (CLL). Furthermore, in contrast to what has been described in normal murine B-cells, stimulation of the FcγRIIb alone induces proliferation of CLL cells. However, the correlation between FcγRIIb expression, immunophenotypic characteristics, and clinical variables in patients with CLL has not been studied.

AIM

The aim of this study was to correlate the expression of FcγRIIb on leukemic cells from previously untreated CLL patients and its immunophenotypic features and clinical parameters.

METHODS

The study population included 112 patients with untreated CLL for whom cryopreserved peripheral blood samples were available before treatment. The diagnosis was based on IWCLL 2008 criteria. The median patients’ follow up was 57.92 months (range: 2.23-439.78 months). FcγRIIb expression levels were determined by flow cytometry on CD5+/CD19+ CLL cells using a specific Alexa488-conjugated murine mAb specific for human FcγRIIb. The following combinations were assessed: FcγRIIb/CD38/CD19/CD5, FcγRIIb/CD49d/CD19/CD5, and FcγRIIb/CD69/CD19/CD5. Results were expressed as the ratio between the MFI for FcγRIIb and the MFI for the corresponding isotype (MFIR). FcγRIIb expression levels were correlated with: i) expression of CD49d, CD38 and CD69, ii) clinico-biological characteristics, and iii) clinical outcome. Differences of FcγRIIb expression on dichotomized clinicopathological variables were assessed with Mann Whitney test. Kaplan–Meier survival and Cox regression analysis were performed to evaluate the correlation of FcγRIIb expression with clinical outcome. Best cut-offs for overall survival (OS) and treatment-free survival (TFS) were determined by ROC curves.

RESULTS

All CD5+CD19+ leukemic cells samples expressed FcγRIIb. However, FcγRIIb expression levels markedly varied between patients (median MFIR: 45.8; interquartile range: 14.9-76.6; 5^th-95^th percentile: 17.15-111.4). FcγRIIb expression was significantly higher in patients who had high (≥30%) CD49d expression than in those with low (<30%) CD49d expression (p=0.009). No correlation was observed between FcγRIIb expression and age, disease stage, IGHV mutational status or chromosomal abnormalities analyzed by fluorescence in situ hybridization, ZAP70 and CD38. Furthermore, within individual clones, FcγRIIb expression levels were higher  on CD38+ or CD49d+ cells than on CD38- or CD49d- cells, respectively (median MFIR: 49.05 vs. 36.72, p=0.001 for CD38+ vs. CD38- cells; and 58.87 vs. 35.55, p<0.001 for CD49d+ vs. CD49d- cells).

In  univariate  analysis, low FcγRIIb expression levels (MFIR< 26.67) were associated with shorter OS (HR 4.01, 95%CI 1.15-13.90, p=0.029), together with older age, advanced stage, and expression of CD38 and CD49d. Advanced stage, unmutated IGHV, and CD38, CD49d and ZAP-70 expression  were also associated significantly with shorter TFS. Thus, patients with higher levels of FcγRIIb had better survival than those with lower levels (Log rank test, p= 0.018). A multivariate analysis adjusted for FcγRIIb expression, age, disease stage, CD38, and CD49d identified older age (≥65 yrs) (HR 150.76, 95%CI 5.39-4212.42, p=0.003), low FcγRIIb expression (HR 111.91, 95%CI 6.71-1866.97, p=0.001), advanced stage (B/C) (HR 17.44, 95%CI 1.45-210.24, p=0.024) and CD38 expression (HR 5.02, 95%CI 1.01-25.18, p=0.050) as independent predictors for shorter OS.

CONCLUSIONS

In this study, FcγRIIb expression on leukemic cells from  untreated patients with CLL was found to be an independent prognostic marker for OS, overcoming the prognostic value of CD49d, which  is consistent with the key role of the FcγRIIb in the pathogenesis of CLL. Further analysis aimed at validating this observation and to better understand the functional cooperation of FcγRIIb with other molecules, particularly CD49d, are warranted. These studies could open a new venue in CLL treatment.