The Earliest Activation Marker CD69 Is An Independent and Strong Progression Indicator in B-Cell Chronic Lymphocytic Leukemia.

B-cell chronic lymphocytic leukemia (B-CLL) exhibits features of activated and antigen-experienced B-lymphocytes and CD69 overexpression resembles B cells at an earlier and greater state of activation (Damle, 2002 and 2007). Moreover, the recent in vitro generation of anti-CD69 monoclonal antibodies able to inhibit either tumor growth or enhance NK cell function (Esplugues, 2005) prompt us to extensively evaluate CD69 antigen in our B-CLL cases. The primary endpoints of our research were: 1) to determine progression-free survival (PFS) and overall survival (OS) upon CD69; 2) to assess the additive prognostic value of CD69 and ZAP-70 and finally 3) to confirm CD69 as an independent prognostic factor. We investigated 401 patients (pts), median age 65 years, 219 males and 182 females. With regard to modified Rai stages, 120 pts had a low stage, 263 an intermediate stage and 18 a high stage. ZAP-70 and CD69 were determined by multicolor flow cytometry, fixing the cut-off values > 20% and >30%, respectively. ZAP-70+ and CD69+ pts were 166/401 (41%) and 108/401 (27%), respectively. CD69 lower than 30% was significantly associated with low Rai stage (105/120; P<0.0001), lymphocyte doubling time >12 months (258/332; P=0.00001), beta-2 microglobulin (B-2M) <2.2 mg/dl (181/223; P=0.00004) and soluble CD23 <70 U/ml (196/240; P<0.00001). There were significant correlations between lower CD69 and IgVH gene mutated status (283 total cases, 150/210; P=0.0001) or low risk (normal or 13q-) FISH cytogenetics (296 total cases, 149/213; P<0.00001). Equally, a strict association was found between lower CD69 and lower ZAP-70 (185/293; P=0.0003). With regard to clinical outcome, both a shorter PFS and OS were observed in ZAP-70+ pts (6% vs 56% at 12 years and 30% vs 95% at 16 years; P<0.00001) as well as in CD69+ pts (9% vs 49% at 14 years, P<0.00001 and 49% vs 75% at 16 years; P=0.00002). Noteworthy, ZAP-70 and CD69 showed additive prognostic properties, since ZAP-70 <20% plus CD69 <30% identified a B-CLL subset at better prognosis with regard to PFS (65% vs 2% at 12 years; P<0.00001, Figure) and OS (97% vs 35% at 14 years; P<0.00001). The two discordant subsets (CD69+ZAP-70 negative and CD69-ZAP-70+) showed an intermediate outcome (Figure). In multivariate analysis of PFS, CD69 (P=0.001) and ZAP-70 (P=0.005) together with cytogenetics and B-2M were confirmed to be independent prognostic factors. On the other hand, with regard to multivariate analysis of OS, age > or < 60 years resulted to be the most significant prognostic factor (P=0.004) followed by CD38 (P=0.01), IgVH status (P=0.02) and ZAP-70 (P=0.04). In conclusion, CD69 antigen, determined by flow cytometry, should be considered a novel important prognostic parameter in B-CLL and its easy and rapid laboratory determination may allow us to identify early progressive pts in order to take timely therapeutic decisions.

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No relevant conflicts of interest to declare.