CD69 is independently prognostic in chronic lymphocytic leukemia: a comprehensive clinical and biological profiling study

Abstract

Background: CD69 is expressed in several hemopoietic cells and is an early activation marker in chronic lymphocytic leukemia. Chronic lymphocytic leukemia is a clinically heterogeneous disease which needs novel prognostic parameters which can be easily and efficiently managed.

Design and methods: We investigated CD69 by flow cytometry in a series of 417 patients affected by chronic lymphocytic leukemia and compared this to other biological and clinical prognosticators.

Results: CD69 was associated with Rai stages (P=0.00002), β(2)-microglobulin (P=0.0005) and soluble CD23 (P<0.0001). CD69 and ZAP-70 (P=0.018) or CD38 (P=0.00015) or immunoglobulin variable heavy chain gene mutations (P=0.0005) were also significantly correlated. Clinically, CD69 positive chronic lymphocytic leukemias received chemotherapy more frequently (74%; P<0.0001), and presented a shorter duration of response after fludarabine plus rituximab (P=0.010) as well as shorter progression free survival and overall survival (P<0.0001). CD69 demonstrated true additive prognostic properties, since the CD69(+) plus ZAP-70(+) or CD38(+) or immunoglobulin variable heavy chain gene unmutated patients had the worst progression free survival and overall survival (P<0.0001). Interestingly, low CD69 expression was necessary to correctly prognosticate the longer progression free survival of patients with a low tumor burden of β(2)-microglobulin (P=0.002), of soluble CD23 (P=0.020), or of Rai stages 0-I (P=0.005). CD69 was confirmed to be an independent prognostic factor in multivariate analysis of progression free survival (P=0.017) and overall survival (P=0.039).

Conclusions: Our data indicate that CD69 is significantly correlated with poor clinical and biological prognostic factors and is confirmed to be an independent disease prognosticator. This supports its introduction in a routine laboratory assessment and, possibly, in a prognostic scoring system for chronic lymphocytic leukemia, after an adequate standardization process.

Figure 1.

Characteristics of CD69 expression. (A) Forward and side scatter of a representative PB sample is shown with a region (R1) drawn around the lymphocytes. Lymphocytes were R1 gated and then B-CLL (CD5⁺CD19⁺) cells (the R2 region in plot (B) were selected according to their phenotype. (C) Expression of mouse IgG1 isotypic controls after lymphocyte (R1) gating. (D–F) Flow cytometric dot-plots of CD19-APC versus CD69-PE expression on PB samples of 3 representative CLL cases. Reported percentage of CD69-expressing cells are calculated on the gated CD5⁺CD19⁺ CLL population (R2). In each case, mouse IgG1 isotypic antibody is used as control for CD69 positivity.

Figure 2.

Bi-variant plots for CD69 and CD38 or ZAP-70 or CD49d or IGHV. y axis reports values for CD69 and x axis values for (A) CD38, (B) ZAP-70, (C) CD49d and (D) IGHV mutations, measured as percentage of positive cells (CD69, CD38, ZAP-70, CD49d) or percentage mutations (IGHV). Number of cases expressing concordant and discordant profiles are reported within each quadrant of the plots.

Figure 3.

Duration of response after fludarabine and rituximab in relation to CD69 expression. Progression free survival after treatment was significantly longer within CLL subgroup showing CD69 lower than 30% (60% vs. 40% at six years; P=0.010).

Figure 4.

Progression-free survival (PFS) and overall survival (OS) curves based on CD69 expression. Kaplan-Meier plot comparing (A) PFS and (B) OS based on the detection of greater than 30% (CD69⁺) or lower than 30% CD69⁺ B cells (CD69⁻). CD69⁻ patients both experienced a longer PFS and OS (P<0.0001).

Figure 5.

PFS and OS curves in relation to combined CD69 and CD38 expression or CD69 and CD49d or CD69 and ZAP-70 or CD69 and IGHV mutational status. PFS and OS were significantly longer (A, E) within the CD69-CD38- subgroup, or (B, F) within CD69-CD49d- patients or (C, G) within CD69-ZAP-70 negative cases. (D, H) Equally, CD69-IGHV mutated (M) patients experienced longer PFS and OS. (A–H) Conversely, double positive (CD69⁺CD38⁺ or CD69⁺CD49d⁺ or CD69⁺ZAP⁻70⁺) or CD69⁺IGHV unmutated patients demonstrated worse PFS and OS. Discordant patients showed intermediate outcomes.

Figure 6.

PFS of CD69 in combination with markers of tumor burden. Kaplan-Meier curves obtained by associating CD69 expression to 3 tumor burden markers, i.e. (A) β₂M, (B) sCD23 serum levels and (C) Rai stage risk groups. For each Kaplan-Meier analysis, 4 groups were compared: patients lacking both negative prognosticators, patients with presence of both negative prognosticators, patients with presence of either one unfavorable prognosticator.