Angiopoietin-2 Plasma Dosage Predicts Time to First Treatment (TTFT) and Overall Survival (OS) in Chronic Lymphocytic Leukemia.

Angiopoietin-2 (Ang2) is a 75 kDa secreted glycoprotein able to bind to the receptor tyrosine kinase Tie-2 and detectable especially in vascular remodeling sites. High Ang2 levels are observed in highly vascularized tumors and may play a role in tumor angiogenesis. We have recently demonstrated that Ang2 protein secreted by CLL cells increases in vivo and in vitro angiogenesis and that, in a small CLL cohort, higher Ang2 mRNA expression is associated with advanced stage disease and shorter progression-free survival. In order to further investigate potential prognostic role of Ang2 in CLL, we measured Ang2 plasma levels using an ELISA-based method, in a large multicentric series: 42(13.3%) of 316 analyzed patients had previously received treatment and an additional 53(16.8%) required therapy during the study course, with a median TTFT of 40 months. Ang2 dosage ranged from 972 to 17281 pg/ml (median, 2061 pg/ml). The best cut-off point, generated by ROC analysis and Youden's index (state variable, treating), was 2460 pg/ml and divided our cohort in two subsets (high Ang2 and low Ang2) composed by 100 (31.6%) and 216 (68.4%) patients, respectively. The median TTFT resulted significantly shorter (P<.001) in the high Ang2 subgroup (77.5 months) than in the low one (179.2 months). Cox univariate analysis identified Ang2 ≥ 2460 pg/ml as a predictor of reduced TTFT (HR 2.437; 95%CI 1.621 - 3.664, P<.001) as well as advanced Binet stage, unmutated IGHV status, high CD38, ZAP-70 and CD49d expression, intermediate/high cytogenetic risk and high β2 microglobulin (P<.001 in all instances). Multivariate analysis confirmed that high Ang2 levels were an independent prognosticator for TTFT (HR 1.739; 95%CI 1.059 – 2.857; P=.029) together with inter/high FISH risk (P<.001) and unmutated IGHV status (P= .002). Comparing OS between high Ang2 and low Ang2 subgroup, we found that 26% of high Ang2 patients were dead at 10-years from diagnosis, in contrast with 7% of low Ang2 ones (P=.002). In univariate analysis, Ang2 ≥ 2460 pg/ml resulted to be a predictor of poor OS (HR 3.566; 95%CI 1.496 – 8.499; P=.004) as well as most of the other known unfavorable prognosticators. Significant association was found between high Ang2 plasma level and Binet stages B-C (P<.001), high β2 microglobulin (P<.001), unmutated IGHV status (P<.001), high CD38 and ZAP-70 expression (P<.001 and P=.003, respectively) and inter/high cytogenetic risk (P=.005). However, a relevant percentage of patients showed high Ang2 levels in the presence of favorable markers and vice versa: in this cases, defined as discordant, Ang2 helps to refine prognosis identifying CLL subgroup with precocious need for treatment and reduced survival despite characterized by favorable prognostic factors. In order to evaluate if Ang2 dosage could be modulated by treatment, we compare Ang2 level of plasma samples collected before (n=53) and after (n=42) the first therapy and we did not find any statistical difference (P=.6). Finally, we studied two serial plasma samples collected from 36 patients (median interval, 19 months; range, 3-30 months) and we did not find significant changes in Ang2 level between the two measurements, both for the 7 patients treated inside this interval (P=.612) and for the 29 cases untreated (P=.347). These data first demonstrate the prognostic role of Ang2 plasma level for TTFT and OS in CLL, and shows how Ang2 contributes to refine the prognostic assessment of CLL. The measurement of serial plasma samples demonstrates that Ang2 levels are quite stable during the disease course and are not modulated by current treatments: this suggests that higher Ang2 secretion could be a peculiar biological characteristic of more aggressive CLL, already present in the first disease steps. Our study implies a simple ELISA method, with a high reproducibility and reliability of results, that could be used both for clinical practice and for further studies, in order to better understand CLL pathogenesis and identify new target therapies. Since several molecules targeting microenvironment and angiogenesis are developing in clinical trials for CLL, it could be very interesting to evaluate if they are able to modulate Ang2 secretion and modify the disease clinical aggressiveness.