Monitoring apoptosis and Bcl-2 on circulating tumor cells in patients with metastatic breast cancer

Abstract

Background: Enumeration of circulating tumor cells (CTC) from whole blood permits monitoring of patients with breast carcinoma. Analysis of apoptosis & Bcl-2 expression in CTC might add additional prognostic and predictive information. We estimated the degree of these markers in CTC from patients being treated for metastatic breast cancer.

Methods: Eighty-three evaluable patients initiating a new therapy for metastatic breast cancer were enrolled. Whole blood was collected at baseline, at one of three short term time windows (24, 48, or 72 h) after initiating treatment, and at first follow-up (3-5 weeks). CTC were isolated, enumerated, and expression of M30 and Bcl2 was determined using the CellSearch(®) System.

Results: At baseline, window, and 3-5 weeks post-treatment, 41/80 (51%), 40/80 (50%) and 21/75 (28%) patients had ≥5 CTC, respectively. At baseline, the proportion of CTC-apoptosis (M30) was inversely correlated with CTC number, and modestly inversely correlated with CTC-Bcl-2. As expected, higher CTC levels at baseline or first follow-up were associated with worse prognosis. Surprisingly, in patients with elevated CTC, higher levels of CTC-apoptosis were associated with worse prognosis, while higher CTC-Bcl-2 levels correlated with better outcomes.

Conclusions: CTC apoptosis and expression of Bcl-2 can be analytically determined in patients with metastatic breast cancer and may have biological and clinical implications. Characterization of CTC for these and other markers could further increase the utility of CTC monitoring patients in clinical investigations of new anti-neoplastic agents.

Figure 1

REMARK diagram of patient flow in the clinical study.

Figure 2

Composite figures of CTC analysis by CellSearch® System, illustrating the appearance of M30 (A) and Bcl‐2 (B) negative and positive CTC within a single blood specimen drawn from a patient with metastatic breast cancer. Check marks beside the composite represent cytokeratin positive, CD45 negative, nucleated cells, representing CTC. Check marks beside the composite for M30 and Bcl‐2 represent CTC that are positive for M30 (Figure 2A) or Bcl‐2 (Figure 2B). Smudged events in the unchecked frames represent cytokeratin positive, CD45 negative, nucleated cells that are negative for the respective markers.

Figure 3

PFS according to CTC enumeration as determined in apoptosis aliquot at 1st follow‐up (∼3–5 weeks after the initiation of therapy) in 73 metastatic breast cancer patients who had blood drawn prior to evidence of progression. (solid line: CTC <5/7.5 ml whole blood; dotted line: ≥5 CTC/7.5 ml whole blood).

Figure 4

Association of CTC phenotypes and CTC levels at baseline and with each other. A. Association of apoptosis, analyzed by M30 expression, and number of CTC/7.5 ml whole blood in baseline blood draw of patients with ≥1 CTC at baseline. B. Association of Bcl‐2 and number of CTC/7.5 ml whole blood in baseline blood draw of patients with ≥1 CTC at baseline. C. Association of Bcl‐2 and apoptosis (analyzed by M30 expression) on CTC in baseline blood draw of patients with an average between aliquot tubes of ≥5 CTC at baseline.

Figure 5

PFS according to CTC enumeration as determined at baseline and 1st follow‐up (∼3–5 weeks after the initiation of therapy). M30 and Bcl‐2 positivity was only considered in patients with ≥5 CTC. A cutoff of ≥50% CTC positive for apoptosis (5A) or Bcl‐2 (5B) was used to distinguish positive from negative for construction of the Kaplan–Meier curves. A. PFS according to CTC‐apoptosis (as determined by M30 staining). (solid line: CTC <5/7.5 ml whole blood; dotted line: ≥5 CTC/7.5 ml whole blood and M30 negative; dashed line: ≥5 CTC/7.5 ml whole blood and M30 positive). B. PFS according to CTC‐Bcl‐2. (solid line: CTC <5/7.5 ml whole blood; dotted line: ≥5 CTC/7.5 ml whole blood and Bcl‐2 negative; dashed line: ≥5 CTC/7.5 ml whole blood and Bcl‐2 positive).