Prognostic value of CD109+ circulating endothelial cells in recurrent glioblastomas treated with bevacizumab and irinotecan

Abstract

Background: Recent data suggest that circulating endothelial and progenitor cells (CECs and CEPs, respectively) may have predictive potential in cancer patients treated with bevacizumab, the antibody recognizing vascular endothelial growth factor (VEGF). Here we report on CECs and CEPs investigated in 68 patients affected by recurrent glioblastoma (rGBM) treated with bevacizumab and irinotecan and two Independent Datasets of rGBM patients respectively treated with bevacizumab alone (n=32, independent dataset A: IDA) and classical antiblastic chemotherapy (n=14, independent dataset B: IDB).

Methods: rGBM patients with KPS ≥50 were treated until progression, as defined by MRI with RANO criteria. CECs expressing CD109, a marker of tumor endothelial cells, as well as other CEC and CEP subtypes, were investigated by six-color flow cytometry.

Results: A baseline count of CD109+ CEC higher than 41.1/ml (1(st) quartile) was associated with increased progression free survival (PFS; 20 versus 9 weeks, P=0.008) and overall survival (OS; 32 versus 23 weeks, P=0.03). Longer PFS (25 versus 8 weeks, P=0.02) and OS (27 versus 17 weeks, P=0.03) were also confirmed in IDA with CD109+ CECs higher than 41.1/ml but not in IDB. Patients treated with bevacizumab with or without irinotecan that were free from MRI progression after two months of treatment had significant decrease of CD109+ CECs: median PFS was 19 weeks; median OS 29 weeks. The presence of two non-contiguous lesions (distant disease) at baseline was an independent predictor of shorter PFS and OS (P<0.001).

Conclusions: Data encourage further studies on the predictive potential of CD109+ CECs in GBM patients treated with bevacizumab.

Figure 1. CEC evaluation by flow cytometry.

A: Gate used to exclude cell fragments and debris. B: Gate made to identify CD45- cells. C: CD31 expression and Syto16 staining in CD45- cells. D: Negative control for E (CD31+ CD146+, CECs), F (CD31+ CD109+ CECs) and G (CD31-CD140b+, PPCs). E1: Distribution of viable, apoptotic, and necrotic CECs.

Figure 2. Progenitor cell evaluation by flow cytometry.

A: Gate used to exclude cell fragments and debris. B: Gate made to include CD45- and CD45dim cells. C: Gate on Syto16+ 7AAD+ cells. D: Identification of 2 different populations: CD45-CD34+ + and CD133-VEGFR2- (D1), and CD45dimCD34+ and CD133+ cells (D2).

Figure 3. Baseline levels of cell subpopulations in patients treated with bevacizumab and irinotecan and healthy controls.

A: Baseline levels of CECs and CEPs in patients and healthy controls. B: Baseline levels of viable CECs and CD109+ CECs in patients and healthy controls. C: Baseline levels of CD45dimCD34+CD133+ hematopoietic progenitors in patients and healthy controls. D: Baseline levels of CD45dimCD34+ hematopoietic committed progenitors in patients and healthy controls. Boxes: the interquartile range; lines: location of first quartile, median, and third quartile; ○, outliers beyond the standard span. All P values were calculated by the Mann-Witney test. Abbreviations: CECs, circulating endothelial cells; vCECs, viable CECs; CEPs, circulating endothelial progenitors; ctrls, healthy controls; hcp, hematopoietic committed progenitors.

Figure 4. Correlation between baseline CD109+ CECs and PFS/OS in patients treated with bevacizumab+irinotecan or bevacizumab alone.

Patients treated with bevacizumab and irinotecan and showing baseline CD109+ CEC count > 41.1/ml (1^st quartile) had increased PFS and OS (panels A); PFS was significantly increased also in patients belonging to IDA and baseline CD109+ CEC count over the 1^st quartile (panels B).

Figure 5. CECs, CD109+ CECs, CD45dimCD34+VEGFR2+ hp, CD140b+ PPCs before therapy and at 2 months.

A: Counts of CECs, CD109+ CECs, CD45dimCD34+VEGFR2+ hp, CD140b+ PPCs before therapy and 2 months after treatment onset in patients responding to treatment with bevacizumab and irinotecan. B: Counts of CECs, CD109+ CECs, CD45dimCD34+VEGFR2+ hp, CD140b+ PPCs before therapy and 2 months after treatment onset in patients responding to treatment with bevacizumab alone (IDA). Boxes: the interquartile range; lines: location of first quartile, median, and third quartile. ○, outliers beyond the standard span. All P values were calculated by Wilcoxon test. Abbreviations: CECs, circulating endothelial cells; hp, hematopoietic progenitor cells; PPCs, progenitor perivascular cells; VEGFR, vascular endothelial growth factor receptor.