Background
The prognostic value of circulating tumor cell (CTC) enumeration was first shown in patients with metastatic breast cancer (MBC) assessed by the CellSearch system in 2004 [
1]. Since then, several studies have been published in support of these results [
2‐
14] and in 2014 a pooled analysis of data from 1944 patients confirmed that a CTC count of ≥ 5 cells per 7.5 mL blood is an independent predictor of worse progression-free survival (PFS) and overall survival (OS) in patients with MBC [
15]. The authors of the pooled analysis developed a clinicopathological prognostication model that included CTC count in addition to other clinically relevant variables, and concluded that CTC-based survival prognostication models should be considered as optimum prognostic models for counselling of patients [
15]. Recently, a meta-analysis demonstrated that CTC status can be applied in monitoring the effectiveness of systemic therapy for MBC, since a shift in CTC status between two time points was prognostic [
16]. Thus far, most individual studies evaluating CTC count in MBC included patients regardless of prior lines of systemic therapy and baseline CTC was measured in a heterogeneous population of patients on different lines of treatment. The focus of these studies has primarily been on CTC evaluation before starting a new line of treatment or at the first post-treatment evaluation, but no studies have conclusively evaluated long-term monitoring of CTC dynamics. Hence, the presence and dynamics of CTCs during first-line systemic treatment in patients with MBC and its clinical relevance have yet to be fully elucidated. Furthermore, recent studies have shown that detection of CTC clusters in patients with MBC adds prognostic value to CTC enumeration alone [
12,
13,
17], but limited data are available on the prognostic value of CTC clusters in previously untreated patients with MBC before and during treatment [
18].
The aim of this prospective study was to evaluate longitudinal CTC count ≥ 5 cells/7.5 mL blood and CTC clusters using the CellSearch system as a prognostic instrument in women with newly diagnosed MBC from baseline to 6 months, and examine how these relate to progression-free survival (PFS) and overall survival (OS). A secondary aim was to evaluate if early changes in CTC status can predict response at the first radiological evaluation at 3 months.
Discussion
In this study, we showed that longitudinal evaluation of CTC and CTC cluster dynamics for 6 months improves prognostication and treatment monitoring in patients with MBC who are starting first-line systemic therapy. Elevated CTC count ≥ 5 CTCs and detection of CTC clusters were prognostic from all investigated time points, and independently added significant value to a prognostic clinicopathological model at baseline and during follow up. Importantly, changes in CTC count throughout treatment significantly correlated with survival and the prognostic value was more prominent at later time points. To the best of our knowledge, this is the first study to describe the longitudinal dynamics and independent prognostic value of CTCs and CTC clusters within a prospective cohort of patients newly diagnosed with MBC and starting first-line systemic therapy.
The prognostic value of CTC count in patients with MBC has been confirmed at the highest level of evidence [
15] in a pooled analysis; however, previous studies evaluating CTC enumeration in patients with newly diagnosed MBC are sparse and have mainly focused on its prognostic value at baseline [
12] or first follow up [
9]. Few studies have addressed the value added by detection of CTC clusters [
12,
13,
17], and the ones that have, were mostly performed in mixed populations and were not focused on patients starting first-line systemic therapy. Furthermore, the study design was often retrospective [
2,
3,
7] and/or they included only patients fulfilling certain pre-specified criteria such as a specific subtype [
14] or type of systemic therapy [
6].
During first-line systemic therapy in the present study, CTC count decreased rapidly in most patients indicating systemic treatment efficacy. In contrast, patients who switched to second-line systemic therapy more often had ≥ 5 CTCs and did not experience a similar decline in CTC count during treatment (Fig.
1). A change in CTC count from baseline to follow up at 1, 3, and 6 months was prognostic at all time points, and patients with persistent CTCs ≥ 5 had worse PFS and OS compared to patients with ≥ 5 CTCs at baseline but < 5 CTCs in follow-up samples. This is in accordance with previous studies in cohorts that were more heterogeneous [
15,
24] and with a recently published meta-analysis reporting that CTC status predicts treatment response in patients with MBC [
16]. In addition, these results support findings in the recent SWOG S0500 trial suggesting that patients with persistent CTCs ≥ 5 during systemic treatment may harbor cancers that are more resistant to chemotherapy [
11]. However, CTC enumeration alone is not able to elucidate the molecular mechanisms responsible for therapy resistance, nor provide guidance on the selection of systemic therapy. Further molecular characterization of CTCs is important for this purpose, and might provide a basis for modification of future treatment based on CTC molecular subtyping.
The persistent presence of ≥ 5 CTCs at baseline, 1, and 3 months significantly increased the OR for progression at the first-response evaluation. Interestingly, we found that patients who had a decrease in CTCs from ≥ 5 at baseline to < 5 at 1 month had the same probability of progression at first evaluation as those with consistently low CTC count. The usefulness of CTC count in prediction of treatment efficacy has been shown [
16,
25,
26], and our results support the application of CTCs as a marker of therapy response in women with MBC receiving systemic therapy. Our results show that CTC enumeration and CTC clusters are promising candidates for evaluation of therapy efficacy in MBC and provide reliable prognostication during follow up.
This study is one of the largest to evaluate CTC clusters in patients with MBC on first-line systemic therapy during long-term follow up. The results convincingly showed that the presence of CTC clusters was significantly and independently prognostic at all investigated time points and could identify patients with worse prognosis than those with ≥ 5 CTCs alone. This is in line with our previous results [
17] and other recently published studies showing that CTC clusters add prognostic value to CTC enumeration in women with MBC [
12,
13]. Incorporating CTC counts and CTC clusters into the clinicopathologic prognostication model proposed by Bidard et al. revealed that these factors significantly improved prognostication at all time points for both PFS and OS. Cox regression analysis of OS with time-varying covariates showed that mortality was increased for episodes with concomitant presence of CTCs ≥ 5 and CTC clusters (HRs, 5.7 and 5.1, respectively), thus confirming the poor prognosis over time in patients with high CTC count and presence of CTC clusters. For patients with both high CTC count (CTCs ≥ 5) and presence of CTC clusters, the mortality was 11 times higher than in patients without these factors when adjusting for standard clinicopathological variables. However, presence of ≥ 20 CTCs was strongly associated with the presence of CTC clusters, and thus CTC clusters did not add any significant prognostic information after adjustment for a high CTC count ≥ 20. Notably, half of patients with ≥ 20 CTCs did not have CTC clusters, and some patients with a CTC count < 5 did have CTC clusters. There were few patients with a high CTC count without clusters and a low CTC count and presence of CTC clusters (Additional file
2), therefore it would not have been meaningful to perform survival analyses for these subgroups. These results further support the use of CTC count and CTC cluster presence to improve prognostication in patients with MBC at baseline and during follow-up.
The presence of CTC clusters added significant value independently of CTC count ≥ 5 CTCs, which underlines the importance of future research focusing on the biological significance of CTC clusters in patients with MBC. Previous studies have shown that CTC clusters have a higher metastatic potential than single CTCs [
27‐
30] and CTC cluster-mediated metastasis has emerged as an alternative model of metastatic seeding along with epithelial-mesenchymal transition (EMT) [
18]. Others hypothesize that clusters shed into the circulation as an entity composed of several tumor cells [
27] and sometimes with platelets and/or leukocytes. This is in contrast to the EMT model in which single cells enter the bloodstream after transformation into a mesenchymal phenotype. Clearly, the clusters are tumor cells with an improved capacity to survive in the circulation and can avoid clearance by sheer force. Several studies have shown that none or very few CTCs within clusters are apoptotic, whereas a relatively large number of single CTCs are [
14,
17,
31]. The widely used CellSearch system easily identifies CTC clusters and thus it would be feasible to assess them in all centers that possess this technology.
Survival analysis was performed including patients with 1–4 CTCs (normally considered low risk and grouped with patients with no CTCs) as a separate group to further explore CTC count and CTC cluster detection. This revealed that 6 months after therapy initiation, patients with 1–4 CTCs had PFS and OS closer to patients with ≥ 5 CTCs than to patients with no CTCs, indicating that even a small number of CTCs in addition to the predefined prognostic cutoff ≥ 5 CTCs [
1,
15] might be informative at later follow-up time points. In early breast cancer, a prognostic CTC cutoff ≥ 1 CTC has been proposed [
32], and in a metastatic setting this has also been suggested as an alternative cutoff [
33]. Furthermore, a threshold of ≥20 CTCs was linked to higher incidence of progression and death at all time points, compared to when the predefined cutoff ≥ 5 CTCs was applied. This study was not powered to evaluate cutoffs other than the predefined cutoff ≥ 5 CTCs and our results support the prognostic value of this cutoff. However, our findings also suggest that the threshold for CTC number needs to be interpreted with care (Fig.
3a-h), particularly during follow up. CTC dynamics over time seem to be essential for estimating prognosis, especially in patients with a reduction in CTCs during treatment for whom the estimated prognosis improves.
A strength of this study is the prospective design of serial CTC and CTC cluster evaluation over 6 months in women with newly diagnosed MBC, including sampling before the start of first-line systemic therapy and structured evaluation at pre-specified intervals. The median follow-up time from baseline was 25 (range 7–69) months and the follow-up data were extensive as few patients terminated the study prematurely. Molecular data to determine breast cancer subtype were available from metastasis biopsies from 73% of patients. Furthermore, the treating physicians were blinded to the CTC results, which avoided treatment bias. This study thus enabled investigation of the presence and dynamics of CTCs and CTC clusters during the first 6 months of treatment, and we applied structured monitoring and blood sampling at predefined time points. A potential weakness of this study is the long inclusion period related to the strict inclusion criteria that included only newly diagnosed cases of MBC before the start of first-line therapy and ECOG performance status score between 0 and 2. Moreover, we included patients irrespective of type of systemic therapy and thus we can draw no conclusion on treatment response related to a specific type of therapy.
Acknowledgements
We thank the research nurses Anette Ahlin-Gullers, Jessica Åkesson, Emma Edvik, Lina Zander, and Petra Andersson, and the clinical oncologists at Skåne University Hospital and Halmstad County Hospital for their invaluable work in the inclusion of patients during the study period.
We thank the laboratory technicians Kristina Lövgren, Anna Ebbesson, Sara Baker, and Charlotte Welinder, PhD, for skilled technical assistance in the CellSearch analysis.