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

In breast cancer, relapses frequently occur many years or even decades after surgical removal of the primary tumor [1]–[3]. During the interval preceding relapse, patients have no clinical or radiological evidence of metastases by the use of standard work-up evaluations. In these patients, recurrence is thought to originate from cells that disseminated from the primary tumor and underwent a period of disease inactivity, termed dormancy, followed by a second period of active growth. These disseminated tumor cells can persist either as solitary dormant cells, which are quiescent, undergoing neither cell division nor apoptosis [4]–[6], or as dormant micrometastases, in which proliferation is balanced by apoptosis [7]. This hypothesis has been supported by mathematical modeling of breast cancer recurrence, showing that dormancy represents a period of cancer quiescence followed by active growth, rather than a phase of linear tumor progression [8]–[10].

The detection of circulating tumor cells (CTCs) in the blood or disseminated tumor cells (DTCs) in the bone marrow in clinically disease-free patients with early breast cancer as well as their association with worse patient prognosis has been well established [11]–[16]. Furthermore, after the completion of adjuvant treatment, a significant percentage of patients still harbor detectable tumor cells in the blood and/or bone marrow, and their presence remains an unfavorable prognostic factor [17]–[23]. In these studies, patients were evaluated shortly after the completion of adjuvant treatment or within 3 years after primary diagnosis [17],[18],[21] when the risk of recurrence is presumably higher [24]. However, in breast cancer, especially for patients with hormone receptor-positive disease undergoing adjuvant hormone therapy for 5 or more years, more than one-half of all recurrences and deaths occur beyond 5 years from diagnosis [3],[25] whereas, from 10 to 20 years, the rate of relapse is relatively steady at about 1.5% yearly [26],[27].

In a report by Meng et al., CTCs were detected in breast cancer patients who had no evidence of disease, 7 to 22 years after mastectomy [28]. In a recent study that included 312 patients with early breast cancer monitored for cytokeratin (CK)-19 mRNA positivity in peripheral blood during follow-up, we reported that 53.8% of patients had detectable CK-19 mRNA CTCs on at least one time point between the third and fifth year of follow-up and that persistence of CK-19 mRNA-positive CTCs during the first 5 years was associated with increased risk for late relapse and death [29]. However, it is evident that despite the presence of CTCs the development of metastases is not universal in all patients; the early recognition of patients who are at increased risk for recurrence remains an unmet need.

Although the precise mechanisms of breast cancer dormancy are still unclear, cellular markers are available to identify dormant CTCs. In experimental models, dormant cells are described as viable cells lacking the expression of both proliferative and apoptotic markers [5],[30]. In addition, evidence exists for a link between tumor dormancy and apoptosis [7], whereas in another report, a dormant tumor population was generated by balanced cell replication and cell death [28]. In the current study, we sought to detect CTCs in dormancy candidate (DC) patients with breast cancer, which were defined as patients who remained free of disease for at least 5 years following surgery, and to characterize their apoptotic and proliferative status by the use of a triple immunofluorescence method. Our results demonstrate that monitoring proliferation and apoptosis in CTCs could serve as a useful tool for the long-term follow-up of primary breast cancer patients.

In the current study we observed that many patients with breast cancer continue to have CTCs beyond 5 years after surgery, despite the absence of clinical evidence of disease recurrence. Herein we provide, to our knowledge for the first time, an insight into the proliferative and apoptotic status of these CTCs; we show that their great majority are neither proliferative nor apoptotic, possibly representing dormant cells. In addition, we demonstrate that these patients also harbor nondormant CTCs and that the dormancy state might be linked to increased apoptosis, whereas escape from dormancy is associated with increased proliferative index in CTCs.

In this trial, we focused in the detection and characterization of CTCs in early breast cancer patients beyond the time frame of the first 5 years of follow up. These patients were characterized as dormancy candidates since they are likely to have CTCs in a dormant state and, at the same time, are still at considerable risk for recurrence and death from their disease. We used a triple immunofluorescence technique in order to detect CK-positive cells on PBMC cytospins and to evaluate their proliferative and apoptotic status after staining with Ki67 and M30 antibodies, respectively [38]. CTCs were detected in 33% of 122 DC, in line with the report by Meng et al. [28] where 36% of 36 dormancy candidates had CTCs detected by immunocytochemistry, 7 to 22 years following surgery. Similarly, Payne et al. [39] reported that measures of minimal residual disease, including CTCs, were evident in patients with primary breast cancer more than 4 years following surgical treatment.

Another objective of this study was the characterization of CTCs in DC according to their proliferative and apoptotic status. It is generally accepted that the majority of disseminated tumor cells found in the bone marrow or the circulation of breast cancer patients are nonproliferative cells [40],[41]. In addition, different rates of apoptosis in DTCs or CTCs have been reported according to the tumor type, the disease stage and/or phase of treatment [42]–[46]. In our study, CTCs lacking staining for the markers Ki67 or M30, which are used to identify proliferative and apoptotic CTCs, respectively, were characterized as dormant [5],[30]. It should be mentioned here that, although it is generally accepted that Ki67 protein expression and cell proliferation are closely linked, the dynamic expression of Ki67 in a cell determined as Ki67 negative, cannot be ruled out. Indeed, it has been suggested that Ki67 may remain undetectable during the G1 phase, therefore, the cell under evaluation could be incorrectly characterized as nonproliferative [47].

We showed that 27.5% and 20% of DC patients harbor proliferative and apoptotic CTCs, respectively. However, most CTCs identified in the whole group were dormant, whereas among the nondormant population, apoptotic CTCs prevailed. Since the half-life of CTCs has been estimated between 1 and 2.4 hours [28], our observations are in line with the hypothesis that, during dormancy, CTCs are most probably derived from undetected micrometastatic deposits where a balance between proliferation and apoptosis, in favor of the latter, exists [4],[7]. Similarly, in the report by Payne et al., apoptosis, as demonstrated by an increase in larger-sized fragments in cell-free DNA, was inversely related to the detection of DTCs in the bone marrow, suggesting that this measure of micrometastatic disease probably emerged from dying micrometastases [39]. In preclinical models, apoptosis, through impaired vascularization or immune-mediated mechanisms, has been considered to be involved in the regulation of dormancy [4],[7],[26]. Interestingly, in a mouse model of primary chemical carcinogenesis, the immune system was shown to restrain the net expansion of ‘dormant’ tumor cells and this was characterized by a combination of increased apoptosis and decreased tumor cell proliferation [48]. However, the simultaneous presence of DTCs prone to mechanisms of cellular dormancy cannot be excluded.

In our study, metastatic patients who recurred after 5.5 to 10 years following surgery, were included as a control group for the evaluation of CTCs in a condition that resembles escape from dormancy. Although a relatively low CTC detection rate was observed among those patients [49], possibly related to the limited number of patients analyzed, it was shown that none had apoptotic CTCs at the time of disease relapse. This is in agreement with the study by Fehm et al., where no apoptotic DTCs were detected in patients with tumor progression after neoadjuvant chemotherapy [42]. In addition, in metastatic patients, dormant CTC counts were decreased and the proliferative index in CTCs was increased as compared to dormancy candidates. Moreover, it is also of interest that subsequent late relapses were recorded in 45.4% out of DC patients with proliferative CTCs compared to only 12% of DC harboring exclusively dormant CTCs. Thus, escape from dormancy and late relapse could be associated with increased proliferation in CTCs. In accordance, studies using preclinical models suggest that escape from dormancy is associated with cellular proliferation driven by microenvironmental signals leading to extracellular signal-related kinase (ERK) activation [50],[51].

We further evaluated the kinetics and the phenotype of CTCs in sequential samples obtained during follow-up from two groups of DC; from one that presented late relapse during follow-up and one that remained in a prolonged disease-free status. This was considered of importance since the fluctuations of these markers over time had not been previously described, and could be informative regarding subsequent disease recurrence.

It was shown that CTCs were intermittently present during the evaluation period albeit at similar frequency among patients in both groups. Interestingly, the group of DC with prolonged disease-free status generally presented lower CTC counts on serial samples compared to the group with late relapse, and this could be related to the maintenance of dormancy. On the other hand, it seems that the repetitive detection of CTCs does not necessarily indicate future relapse and points to the need for further characterization of these CTCs. Thus, the apoptotic index prevailed in the group with prolonged disease-free status (that maintained dormancy). Another interesting finding was that several patients presented, at some point during follow-up, a robust increase in CTC numbers involving both dormant and nondormant CTCs. The balance between proliferation and apoptosis at this time point could be associated with the time to subsequent disease recurrence.

Our study has several limitations that should be considered. First, an overlap was observed in the detection of proliferation and apoptosis markers between the relapse-free DC and those who relapsed, representing a significant limitation for their clinical use, especially when a sample from a single time point is evaluated. Another limitation is the retrospective nature of our study, the small number of patients followed with serial samples, and the lack of matching in terms of patient and disease characteristics between the two groups. Moreover, it could be argued that the actual CTC phenotype could not be reliably depicted in cases with low CTC counts. Nevertheless, this is the real case scenario, since in general, low CTC counts are observed in patients with early disease [14]. Finally, although in breast cancer the immunohistochemical assessment of proliferation using the marker Ki67 is considered important, both in clinical practice and research, substantial interlaboratory variability limits its clinical use [52]. It is conceivable that Ki67 evaluation of isolated cells could be prone to similar drawbacks.

Our data suggest that breast cancer dormancy displays significant differences compared to the overt metastatic state regarding the incidence of dormant and nondormant CTCs as well as the balance between proliferation and apoptosis in CTCs. Moreover, in each patient, the dormancy period seems to be characterized by variations in CTC load, in the shift between dormant and nondormant populations and in the balance between proliferative and apoptotic nondormant CTCs. This balance could be associated with the maintenance of or the escape from clinical dormancy. However, the observed variability in CTC detection rate as well as in the expression of these parameters over time, even among the same individual, precludes firm conclusions to be drawn regarding their use in the prediction of patient prognosis, at least as they stand in the current study. Our findings are rather hypothesis generating and merit further investigation in larger studies designed to evaluate their clinical significance, either alone or as part of a prognostic model, in order to define high-risk patient subgroups that might benefit from extended or secondary adjuvant treatments.