Background
Hematogenous spread of cancer cells and subsequent formation of metastases in distant organs is the leading cause of death in cancer patients. A key step in metastasis is intravasation, i.e. the entrance of tumor cells into the hematologic or lymphatic system. Carcinoma-derived tumor cells circulating in the bloodstream, or circulating tumor cells (CTCs), in metastatic breast [
1], prostate [
2], colorectal [
3], and lung [
4,
5] cancer are associated with decreased progression-free survival (PFS) and overall survival (OS), and serial sampling after therapy initiation has also shown a prognostic importance of longitudinal CTC enumeration in metastatic breast cancer [
1,
6‐
9].
Enumeration of CTCs in a liquid biopsy is a non-invasive monitoring that is easy to obtain via a peripheral blood sample and may hold promise for improving cancer prognostication and treatment. The most commonly used enrichment and detection technique for CTCs is the FDA approved CellSearch system (Janssen Diagnostics LLC, Raritan, NJ, USA). Molecular studies of CTCs are accumulating but few studies have thus far described morphological characteristics of CTCs, using either CellSearch-derived CTCs [
10‐
14] or other methods for CTC isolation [
15‐
20].
The malignant potential of CTCs has been suggested to be reflected in their morphological characteristics and these attributes are thus starting to be evaluated in clinical studies and related to outcome. A high fraction of apoptotic CTCs in the blood or apoptotic disseminated tumor cells (DTCs) in the bone-marrow in patients with solid tumors have been reported to be associated with decreased PFS and/or OS [
4,
21‐
24]. The presence of CTC clusters has been reported for patients with metastatic colorectal, renal, prostate, lung and breast cancer [
4,
12,
25‐
29] and the presence of clusters has been correlated to decreased survival in a few studies in small-cell lung cancer [
4] and breast cancer [
12,
14]. Diagnosis of CTC clusters (defined as ≥2 CTCs) have been related to poor outcome in stage III-IV breast cancer using the CellSearch system for CTC enumeration and characterization [
14]. Paoletti et al [
12] defined CTC clusters as ≥3 CTCs in the CellSearch gallery and for definition of apoptotic CTCs they applied M-30 staining as well as morphologic evaluation. They reported on prognostic information obtained by diagnosis of CTC clusters and apoptosis in metastatic triple-negative breast cancer showing that CTC clusters, but not apoptotic CTCs, added prognostic information in FU samples [
12]. To date no consensus has been reached regarding the definitions of these morphologic characteristics using the CellSearch system and if additional biomarkers for diagnosis of apoptosis are needed.
Mixed clusters comprised of CTCs and leukocytes/white blood cells (WBC-CTC) have not been thoroughly investigated, but the complex relationship between CTCs and the immune system is gaining attention [
30]. Generally, interactions between CTCs and the tumor microenvironment are still poorly understood but previous results have shown that specific immune cells have immunosuppressive properties in the peripheral blood, while this effect is absent in these cells in a tumor-associated environment [
31,
32]. Also, association of CTCs with lymphocytes and platelets has been suggested to protect tumor cells against natural-killer (NK) cell-mediated lysis [
33,
34].
We hypothesized that CTC clusters and apoptosis in metastatic breast cancer can provide prognostic information along CTC enumeration in all breast cancer subtypes and we sought to morphologically characterize CTCs in serial blood samples from patients with high risk (≥5 CTCs at base-line (BL)) metastatic breast cancer. All included patients were recently diagnosed with a first metastatic event and about to start first-line therapy in the metastatic setting. We explored whether apoptosis, CTC clusters and WBC-CTCs identified after CellSearch analysis without further staining were related to disease progression and survival, and if morphologic CTC characteristics differ among breast cancer subtypes and during follow-up (FU) from BL to 6 months after first-line systemic therapy. The present study shows that diagnosis of CTC clusters before start of systemic therapy correlate with an aggressive phenotype (triple-negative and HER2-subtype) and that presence of CTC clusters and apoptotic CTCs add prognostic information in FU samples even when adjusting for other prognostic factors.
Discussion
The prognostic information of CTC enumeration in FU blood samples has been shown in a number of studies [
1,
6‐
9] but the added value of CTC characterization in FU samples is largely unknown. Apoptotic CTCs and CTC clusters in metastatic breast cancer has gained recent attention and in the present exploratory study we investigated the significance of these morphologic characteristics using the CellSearch gallery in a homogenous cohort from patients with poor prognosis (≥5 CTCs at base-line (BL)) metastatic breast cancer undergoing first-line systemic therapy including all breast cancer subtypes. We show that the presence of apoptotic CTCs and CTC clusters in FU blood samples at 1–3 and 6 months after treatment initiation indicated poorer prognosis. Moreover, Cox-models with time-dependent covariates confirmed that the presence of apoptotic CTCs and CTC clusters at any time-point during the study was associated with increased mortality independent of other prognostic factors such as CTC numbers and breast cancer subtype.
Our findings agree with a recent study of metastatic triple-negative breast cancer in which presence of CTC clusters diagnosed using the CellSearch gallery in FU blood samples during the first month of treatment was associated to significantly worse PFS [
12]. However, we included patients with all subtypes of breast cancer with CTC number ≥5 at BL and limited inclusion of patients to those about to start first-line systemic therapy, thus the prognostic information yielded by morphologic characteristics is not only related to pretreated patients or any specific subtype of breast cancer. Another recent publication has also suggested that detection of CTC clusters is important when evaluating prognosis in breast cancer [
14]. Both publications [
12,
14] used the CellSearch system for CTC enumeration and characterization, but applied separate definitions for CTC clusters. In the study by Mu et al
. [
14], including patients with breast cancer stage III and IV, the presence of CTC clusters (defined as ≥2 CTCs) at BL was associated to worse prognosis in terms of decreased PFS. A majority of patients (69 out of 115) in this study had inflammatory breast cancer and the authors conclude that these patients had larger clusters; five of seven patients in this study with CTC clusters of ≥3 CTCs had inflammatory breast cancer. The publication by Paoletti et al
. [
12] investigated the importance of CTC clusters and apoptosis in metastatic triple-negative breast cancer. They found that CTC clusters (defined as ≥3 CTCs) but not apoptotic CTCs (defined by any M-30 staining and/or visual characteristics of apoptosis) in FU blood samples during treatment was associated with worse PFS.
In the present study retained CTC clusters added significant prognostic information after 1–3 and 6 months of first line therapy. Also, clusters were found significantly more often in patients with triple-negative breast cancer compared to hormone receptor-positive cancer. Previous studies indicate that CTCs within clusters may represent a more malignant and mesenchymal subpopulation of tumor cells [
20,
28]. Early experiments in animal models indicate that intravenously injected tumor cell clusters have a greater tendency to form metastases than an equal number of injected single CTCs [
39,
40]. Adding to these findings, Aceto et al
. recently reported that CTC clusters in breast cancer have a 23- to 50-fold increased metastatic potential [
20]. Furthermore, no apoptotic CTCs were found within CTC clusters in the blood of patients with lung cancer, suggesting that tumor cells in clusters have a survival advantage compared to solitary CTC [
13]. We confirm these results in breast cancer, as no apoptotic CTCs were found in cell clusters in our patients. Likely, clustered CTCs evade anoikis by retaining cell-to-cell survival signals by expressing proteins responsible for intracellular junctions, exemplified by plakoglobin [
20]. Moreover, characterization of CTCs within clusters supports the hypothesis that CTCs within clusters are different from single CTC—they are non-proliferating as indicated by the absence of Ki67 expression [
4].
In this cohort of patients with metastatic breast cancer, we found no apoptotic CTCs within clusters, but all patients with clusters also had apoptotic single CTCs. The presence of apoptotic CTCs was also related to poor outcome at 1–3 and 6 months, whereas no association to prognosis could be seen at BL. Persistent apoptotic CTCs over time indicates a failure to respond to systemic therapy with retained proliferation and cell turnover in the metastatic lesion and/or primary tumor [
4]. This could be a possible explanation for the dismal prognosis for patients with retained apoptotic CTCs during treatment as found in the present study. Our results are not consistent with previous studies using CellSearch for detection and analysis of apoptotic CTCs [
4,
12]. In contrast to our data, Hou et al
. found that presence of ≥1 apoptotic CTC at BL was associated with significantly worse PFS and OS in lung cancer [
4] (the study did not analyze apoptotic CTCs in FU samples). On the other hand, in metastatic triple-negative breast cancer, Paoletti et al
. found no prognostic effect of apoptotic CTCs either at BL or in FU samples at day 15 and 29 [
12]. Our data suggest that samples taken after several cycles of systemic treatment have a higher significance for prognostic information. In contrast to the studies above, we included no further staining (e.g. M30) after CellSearch analysis and only used morphological criteria for diagnosis of apoptotic CTCs. It is possible that early stage apoptotic CTCs, as detected by additional staining, carry less prognostic information. Also, we found no evidence for prognostic significance by analyzing the fraction of apoptotic CTCs as applied by Paoletti et al. [
12].
The prognostic importance of WBC-CTCs in patients with metastatic breast cancer has, to our knowledge, not been previously investigated. Interestingly, univariable analysis in this cohort indicated worse prognosis for patients with WBC-CTCs present whereas adjustment for other prognostic factors such as CTC number, age, and time to recurrence lowered HR to less than 1, suggesting that WBC-CTCs could be favorable for survival. Possibly further characterization of CTC-associated leukocytes may provide prognostic information [
41], but the CellSearch methodology only specifies leukocyte presence by the phenotype CK-/CD45+/DAPI+. Thus, no detailed information on leukocyte types associated with CTC is available within the present study.
As enumeration of CTCs was prognostic in the presented cohort and we found an association between CTC number and all morphologic characteristics, we also accounted for the fraction of CTCs with the respective morphological characteristic in relation to the total CTC count in each patient. The fraction of CTC clusters added prognostic information in FU samples, supporting that CTC clusters add important prognostic information to enumeration of CTCs. The fraction of apoptotic CTCs and WBC-CTCs was not significantly related to prognosis in contrast to the presence of the respective morphological trait.
A limitation of the study is that we included only 52 patients with metastatic breast cancer in this exploratory analysis and the statistical power was consequently limited. Although we selected patients based on cut-point for CTC enumeration, the median CTC count at BL was 45 for all included patients as an indication of a group of patients with dismal prognosis. Another limitation of the study is that patients within triple-negative and HER2-positive subgroups were underrepresented but diagnosed significantly more often with CTC clusters at BL. Four patients with HER2-positive subtype were included, three of which cleared their CTCs after 6 months of FU and one with progressive disease after 5 months (Additional file
4). The patient with disease progression had increased number of CTCs and also presented with clusters at 6 months. The most efficient eradication of CTCs and CTC clusters occurred in a patient treated with HER2-directed double blockade diagnosed with 311 CTC at BL and four clusters. The finding indicates that dynamic changes of CTCs evolving under the pressure of systemic therapy may be predictive for treatment success. In contrast, all patients in the difficult to treat triple-negative subgroup (
N = 4) had a constant presence of apoptotic CTCs during the study (Table
2), both at BL and during chemotherapy. A higher cell turn-over rate in patients with triple-negative subtype may be one explanation for the dismal prognosis in this breast cancer subtype.
This exploratory study was performed by evaluating CTCs captured with the FDA approved CellSearch system without any downstream staining and our study can be repeated in more patients in clinical studies using CTCs as surrogate marker. CTC apoptosis, clusters and WBC-CTCs were assessed morphologically directly in the CellSearch gallery following assessment guidelines proposed in previous publications. Even if the CTC galleries provided by the CellTracks Analyzer II system only offers pictures with 10 x magnifications, CellSearch is the most used and well documented CTC isolation system available. Thus, being able to extract putative prognostic information by including basic and easy-to-assess morphologic characteristics in addition to today’s enumeration, an even more powerful prognostic tool may lie ahead for patients presenting with ≥5 CTC/7.5 ml blood at BL. Confirmation that apoptotic CTCs and CTC clusters observed in CellSearch analyses are not a result of artifacts but indeed true morphologic characteristics have been presented previously [
4,
12,
42]. We applied a published definition of a CTC cluster to enable comparison with previous studies [
12,
13] and the definition of apoptosis was according to criteria used in clinical pathology [
13,
36] without the need of further staining after CellSearch analysis. Further validation of morphological assessment of CTCs in independent and larger cohorts is warranted.
Abbreviations
BL, base-line; CI, confidence interval; CK, cytokeratin; CTC, circulating tumor cell; EpCAM, epithelial cell adhesion molecule; ER, estrogen receptor; FU, follow-up; HER2, human epidermal growth factor receptor 2; HR, Hazard ratio; KM, Kaplan-Meier; OS, overall survival; PFS, progression-free survival; PgR, progesterone receptor; WBC, white blood cell, leukocyte
Acknowledgements
We thank Dr. David Gisselsson Nord for support and guidance for setting up the CTC apoptosis criteria and research nurses Anette Ahlin Gullers, Jessica Åkesson, Emma Edvik, Lina Zander and Petra Andersson for collection of patient blood samples. We thank research engineer Sara Baker for preparing the database of background patient characteristics and for CellSearch analyses, and laboratory assistant Kristina Lövgren for CellSearch analyses.