Evaluation of proliferation and apoptosis markers in circulating tumor cells of women with early breast cancer who are candidates for tumor dormancy

Maria Spiliotaki, Dimitris Mavroudis, Kyriaki Kapranou, Harris Markomanolaki, Galatea Kallergi, Filippos Koinis, Kostas Kalbakis, Vassilis Georgoulias, Sofia Agelaki

IntroductionClinical dormancy is frequently observed in breast cancer. In the present study, we aimed to characterize circulating tumor cells (CTCs) in dormancy candidates (DC) with early breast cancer in terms of proliferation and apoptosis.MethodsCytospins of peripheral blood mononuclear cells (PBMCs) were obtained from DC (n =122) who were disease-free for at least five years and from metastatic patients (n =40) who relapsed more than five years after surgery. Sequential samples from eight DC (n =36) who maintained a prolonged disease-free status and from eight DC (n =27) presenting late relapse during follow-up, were also analyzed. PBMCs were triple stained with a pancytokeratin, antibody along with anti-Ki67 and anti-M30 antibodies as proliferation and apoptosis markers, respectively.ResultsCTCs were identified in 40 (33%) of 122 DC and in 15 (37.5%) of 40 metastatic patients. In total 25 (62.5%) DC had exclusively dormant (Ki67(-)/M30(-)), 7 (17.5%) had proliferative Ki67(+)/M30(-), 4 (10%) had apoptotic Ki67(-)/M30(+) and 4 (10%) had both phenotypes of proliferative and apoptotic CTCs. In comparison, 53.4% of CTC-positive metastatic patients had exclusively dormant and 46.6% had proliferative CTCs; none had apoptotic CTCs (P =0.039). Among all CTCs detected in DC patients, 82.4% were dormant, whereas in the non-dormant population, 32.5% were proliferative and 67.5% apoptotic. The respective percentages in metastatic patients were 59.1%, 100% and 0% (P <0.0001). Moreover, apoptotic CTCs prevailed among non-dormant CTCs detected in sequential samples from DC who remained in a prolonged disease-free status compared to those presenting late relapse during follow-up (70.6% versus 43.5% (P =0.0002)).ConclusionsThe apoptotic index of CTCs is increased during clinical dormancy, whereas the proliferation index is increased on relapse. In addition, apoptotic CTCs are more frequently encountered during follow-up in DC patients who remain disease¿free compared to those with subsequent late relapse, suggesting that monitoring proliferation and apoptosis in CTCs during clinical dormancy merits further investigation as a tool for predicting late disease recurrence.