The predictive and prognostic value of circulating endothelial cells in advanced colorectal cancer patients receiving first-line chemotherapy and bevacizumab.

Currently, the use of bevacizumab in combination with fluoropyrimidine-containing chemotherapy is the standard first-line treatment for patients with advanced colorectal cancer (ACC). However, only a subset of ACC patients will respond to treatment, and to date, there is no biological marker that may predict the benefit of this treatment. Recently, Ronzoni et al. reported on the correlation between the baseline levels of both total circulating endothelial cells (CECs) and their subsets and the response to bevacizumab-based first-line treatment in ACC patients. They suggested significant correlations between the CEC baseline levels and the antitumor efficacy [1]. We assessed the prognostic and predictive value of CECs in 473 ACC patients treated in a randomized phase III trial with first-line chemotherapy and targeted therapy including bevacizumab (CAIRO2 trial of the Dutch Colorectal Cancer Group) [2]. Peripheral blood was collected at baseline and at three time points after the start of treatment (1–2, 3–5, and 6–12 weeks). Samples were collected in 10-ml evacuated tubes (CellSave tubes; Veridex LLC, Raritan, NJ) and processed in a central laboratory within 72 hours. The CellSearch System (Veridex LLC) was used for CEC enumeration [3]. The criteria for CEC included round-to-oval morphology, a visible nucleus [4#-6-diamidino-2-phenylindole (DAPI) positive], expression of CD146 as well as CD105, and absence of CD45. The median number of CECs at baseline was 6.8 CEC/ml, which is comparable with that found in other studies using the automated immunomagnetic separation system [3, 4]. In our study population, 17% had a CEC count at baseline above the upper reference limit of 24 CECs/ml, which is comparable with that of Rowand et al. [3]. To define the prognostic value of CECs, the correlation of baseline CEC count and progressionfree survival (PFS) and overall survival (OS) was analyzed. In univariate Cox regression analysis, the hazard ratio (95% confidence interval) of progression was 0.99 (0.90–1.08) and of death 0.95 (0.87–1.05). A significant increase in CECs was observed after 1–2 weeks of treatment compared with baseline (P £ 0.001). Hereafter, no further increase in CEC count was observed. However, we could not predict PFS or OS by changes in CEC levels at any time point during therapy (Table 1). Our results are in contrast to the findings of Ronzoni et al., which are based on a considerable smaller number of patients. Ronzoni et al. [1] only observed a correlation between baseline ‘total’ and ‘resting’ CEC counts and PFS, and no correlation was found regarding activated CECs or circulating progenitor cells and outcome. The lack of correlation of CECs with outcome in our study may be due to the different technique used for CEC enumeration. So far, there is no consensus on the phenotypic characterization of CECs and technique for CEC enumeration, and conflicting results concerning the role of CECs as a marker for prognosis and/or response to treatment have been reported. The most commonly used techniques for CEC evaluation are flow cytometry or microscopic evaluation after immunomagnetic separation. We used the CellSearch System, which is an automated system based on immunomagnetic enrichment targeting the CD146 antigen and detection by fluorescent microscopy of DAPI+, CD105+, CD452 cells, that has shown to be a reproducible and accurate method [3]. Our technique also included morphology in classification of CECs, which allows to discriminate CECs from endothelial microparticles, conglomerates, and/or anuclear cells. In the study by Ronzoni [1], staining with a nucleic acid dye was not included in the definition, which may lead to the inclusion of large platelets rather than endothelial cells into the CEC definition [5]. In conclusion, our study demonstrated that CEC counts at baseline were not prognostic for PFS or OS in a subset of ACC patients. Neither was change in CEC counts at baseline and during treatment predictive for outcome. To our knowledge, this is the first study that investigates the correlation between CECs and bevacizumab-based treatment in a large number of ACC patients. However, different subsets of CECs exist and consensus on phenotypic characterization and enumeration technique is the first step to make comparison of different studies concerning CEC evaluation possible.