Apoptotic Circulating Tumor Cells in Early and Metastatic Breast Cancer Patients

Abstract

The detection of circulating tumor cells (CTC) in breast cancer is strongly associated with disease relapse. Since it is unclear whether all CTCs are capable of generating metastasis, we investigated their apoptotic and proliferative status in 56 CTC-positive (29 early and 27 metastatic) patients with breast cancer. Double-staining immunofluorescence experiments were carried out in peripheral blood mononuclear cells (PBMC) cytospins, using the pancytokeratin A45-B/B3 antibody and either M30 (apoptotic marker) or Ki67 (proliferation marker) antibodies. Apoptosis was also evaluated using a polycaspase detection kit. Patients with metastatic disease had significantly lower numbers of apoptotic CTCs compared with patients with early breast cancer (polycaspase kit: 8.1% vs. 47.4% of the total CTC number; P ¼ 0.0001; M30-antibody: 32.1% vs. 76.63%; P ¼ 0.002). The median percentage of apoptotic CTCs per patient was also lower in patients with advanced compared with those with early disease (polycaspase kit: 0% vs. 53.6%; M30-antibody: 15% vs. 80%). Ki67-positive CTCs were identified in 51.7% and 44% of patients with early and metastatic disease, respectively. Adjuvant chemotherapy reduced both the number of CTCs per patient and the number of proliferating CTCs (63.9% vs. 30%). In conclusion, apoptotic CTCs could be detected in patients with breast cancer irrespective of their clinical status, though the incidence of detection is higher in early compared with metastatic patients. The detection of CTCs that survive despite adjuvant therapy implies that CTC elimination should be attempted using agents targeting their distinctive molecular characteristics. Mol Cancer Ther; 12(9); 1886-95. Ó2013 AACR.

Circulating tumor cells in early breast cancer: A connection with vascular invasion

“…In a clinical setting, such a cell-spreading effect is difficult to detect and is probably limited by a number of as yet unknown factors. A CTC population can be composed of dead and/or metastasisinefficient CTCs [34,35]. The substantial reduction (about 70%) in median CTC yield in our CTC+ cases would seem to confirm this and is suggestive of lower metastatic potential.…”

A circulating tumor cell cluster-based model for tumor metastasis (Hypothesis)

“…For instance, normal cells are programmed to die if they are detached from neighboring cells and the extracellular matrix, this is known as anoikis (31). The proportion of non-apoptotic CTCs is essential to the formation of metastases (13). However, tumor cells resist anoikis partially by forming cell clusters (32,33).…”

“…Notably, CTCs can be found in the peripheral blood in the early stages of numerous malignancies (13)(14)(15), while metastasis is considered as concrete evidence of late-stage cancer. This discrepancy suggests that the formation of macrometastasis led by CTC is difficult, even though the critical role of CTCs in tumor metastasis has been widely accepted.…”

The potential for liquid biopsies in the precision medical treatment of breast cancer

“…Furthermore, apoptosis-related DNA strand breaks could be demonstrated by applying the TdT-uridine nick end labeling assay in these cells 117 . The monoclonal antibody targeting the neoepitope M30, revealed by caspase cleavage of CK18 in early apoptosis, has also been shown to be a marker for apoptotic CTCs 118 . Kallergi et al 119 noted the presence of exclusively apoptotic CTCs in a patient may represent a favorable prognostic factor, whereas the preponderance of proliferating cells (determined by Ki-67) could be related to poor patient outcome in breast cancer.…”