Breast Cancer Res Treat (2014) 147:345–351 DOI 10.1007/s10549-014-3113-5
CLINICAL TRIAL
Tumor cell dissemination to the bone marrow and blood is associated with poor outcome in patients with metastatic breast cancer Andreas D. Hartkopf • Diana Stefanescu • Markus Wallwiener Markus Hahn • Sven Becker • Erich-Franz Solomayer • Tanja N. Fehm • Sara Y. Brucker • Florin-Andrei Taran
•
Received: 14 August 2014 / Accepted: 18 August 2014 / Published online: 24 August 2014 Ó Springer Science+Business Media New York 2014
Abstract The purpose of this study was to assess the impact of disseminated tumor cells (DTCs) on progressionfree and overall survival (OS) in patients with metastatic breast cancer (MBC) and to compare it to simultaneous detection of circulating tumor cells (CTCs) from the blood in a subgroup. Disseminated tumor cells were identified in bone marrow (BM) aspirates by immunocytochemistry (pancytokeratin antibody A45-B/B3) and cytomorphology prior to the beginning of a new-line therapy. CTCs were enumerated by the CellSearchÒ technology. BM was obtained from 178 patients with MBC; 64/178 (36 %) patients were DTC-positive. Disseminated tumor cells occurred more frequently in patients with visceral metastases (p = 0.020) and C2 lines of therapy (p = 0.017). CTCs were assessed in 33 of these patients and 17/33
(52 %) patients had CTC counts C5 CTCs/7.5 ml blood. There was no significant association between the DTC and CTC status. Univariate analysis revealed DTC detection as a significant predictor of poor OS (p \ 0.001); median OS in DTC-negative versus DTC-positive patients was 52 [95 % confidence interval (CI) 38–67] versus 28 [95 % CI 19–37] months. Moreover, as described previously, patients with C5 CTCs/7.5 ml blood were at an increased risk of disease progression (p = 0.026) and death (p = 0.025). Disseminated tumor cells are predictors of poor prognosis in MBC, highlighting the role of tumor cell dissemination into the BM for breast cancer progression. The absence of a significant association between concurrent DTCs and CTCs suggests they might represent different aspects of systemic BC spread.
A. D. Hartkopf (&) D. Stefanescu M. Hahn S. Y. Brucker F.-A. Taran Department of Obstetrics and Gynecology, University of Tuebingen, Calwerstraße 7, 72076 Tuebingen, Germany e-mail: [email protected]
S. Becker Department of Obstetrics and Gynecology, University of Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany e-mail: [email protected]
D. Stefanescu e-mail: [email protected] M. Hahn e-mail: [email protected] S. Y. Brucker e-mail: [email protected] F.-A. Taran e-mail: [email protected]
E.-F. Solomayer Department of Obstetrics and Gynecology, Saarland University, Kirrberger Straße 100, 66421 Homburg, Germany e-mail: [email protected] T. N. Fehm Department of Obstetrics and Gynecology, University of Duesseldorf, Moorenstraße 5, 40225 Duesseldorf, Germany e-mail: [email protected]
M. Wallwiener Department of Obstetrics and Gynecology, University of Heidelberg, Im Neuenheimer Feld 110, 69120 Heidelberg, Germany e-mail: [email protected]
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Keywords Metastatic breast cancer Disseminated tumor cell Circulating tumor cell Survival Bone marrow Abbreviations BM Bone marrow BC Breast cancer CI Confidence interval CTC Circulating tumor cell DTC Disseminated tumor cell EpCAM Epithelial cell adhesion molecule ER Estrogen receptor HER2 Human epidermal growth factor receptor 2 HR Hormone receptor ISHAGE International Society for Hematotherapy and Graft Engineering MBC Metastatic breast cancer OS Overall survival PR Progesterone receptor
Introduction The dissemination of individual tumor cells is a common phenomenon in solid epithelial cancers. Highly sensitive assays enable the detection of such cells even at early stages of disease. With respect to primary breast cancer (BC), cytokeratin-positive disseminated tumor cells (DTCs) are detected in the bone marrow (BM) in 20–30 % of early-stage patients [1, 2]. Numerous trials have provided high-level evidence that detection of DTCs in the non-metastatic situation is of high and independent prognostic significance [3–8]. Other studies have demonstrated that circulating tumor cells (CTCs) in the peripheral blood are associated with an impaired prognosis in early-stage BC [9, 10]. However, the information from DTCs appears to be of higher prognostic value, probably due to methodological differences between CTC studies and the lower sensitivity of CTC detection [11–13]. To date, only a few small studies have investigated the role of DTCs in metastatic breast cancer (MBC), and results on the prognostic impact of DTC detection are inconsistent [14–16]. Hence, our present knowledge of the role of tumor cell dissemination in patients with advanced BC relies primarily on CTCs. In patients with MBC, CTC counts C5/7.5 ml blood have been clearly demonstrated in large trials to be associated with poor outcome [17, 18]. The current gold standard for CTC detection is the Veridex CellSearchÒ system, which received clearance from the U.S. Food and Drug Administration in 2004 as a diagnostic tool for identifying and counting CTCs of epithelial origin in patients with MBC.
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To investigate the role of tumor cell dissemination in MBC, we conducted the present retrospective study, which to the best of our knowledge is the largest BM sampling study in patients with MBC. Our primary objective was to assess the impact of DTC detection on progression-free (PFS) and overall survival (OS). As a secondary objective, we compared DTC and CTC detection in a subset of our study population.
Materials and methods Study population Bone marrow sampling was performed in patients with MBC before the initiation of a new-line therapy at the Department of Obstetrics and Gynecology, Tuebingen University Hospital, Germany, between January 2001 and June 2012. Patients with malignancies other than BC were excluded from this analysis. Patients were followed up by regular clinical and radiologic assessments every 3 months. All patients provided written informed consent for BM aspiration for research purposes. Circulating tumor cell data were collected from the hospital records of the patients who provided BM samples. The clinical cancer registry of the Tuebingen Comprehensive Cancer Center provided additional follow-up data and the survival data. The present analysis was approved by the Ethics Committee of the University of Tuebingen (reference number 560/2012R). Bone marrow DTC studies BM aspirates (10–20 ml) were collected from the anterior iliac crest. Samples were processed within 24 h as described elsewhere [19]. In brief, mononuclear cells were separated by density centrifugation using Ficoll (Biochrom, Berlin, Germany) at a density of 1.077 g/ml, spun down onto a glass slide in a cytocentrifuge (Hettich, Tuttlingen, Germany) and fixed in 4 % formalin. Disseminated tumor cell status was determined by immunostaining using the DAKO Autostainer (Dako, Glostrup, Denmark), the A45B/B3 mouse monoclonal antibody (Micromet, Munich, Germany), which recognizes several cytokeratin epitopes (CK8, CK18, and CK19), and the DAKO-APAA detection kit (Dako, Glostrup, Denmark). Two slides (1 9 106 cells each) per patient were evaluated based on consensus recommendations for standardized tumor cell detection [20, 21]. An additional slide was stained using a non-specific isotype-matched antibody. Moreover, each batch of samples was analyzed together with healthy volunteer leukocytes as a negative control and the human BC cell lines MCF-7 and SKBR-3 as positive controls. To assess the specificity of our DTC detection method, we analyzed BM
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samples from 100 patients without evidence of malignant disease, of whom one was DTC-positive [12].
347 Table 1 Patient characteristics DTC studies DTCpositive n/total (%)
Blood CTC studies Enrichment and enumeration of CTCs using the CellSearchÒ technology (Veridex LLC, Warren, NJ, USA) were essentially performed as described elsewhere [22]. Briefly, 7.5 ml samples of peripheral blood were drawn into CellSave tubes (Veridex LLC, Warren, NJ, USA), kept at room temperature and processed within 72 h. Epithelial cells were enriched immunomagnetically using a ferrofluid with antibodies against the epithelial cell adhesion molecule (EpCAM). Epithelial cell adhesion molecule-positive cells were labeled with the nuclear dye 40 ,6-diamidino-2phenylindole (DAPI) and monoclonal antibodies specific for the leukocyte common antigen CD45. Defined as CD45-negative, cytokeratin-positive cells with an intact nucleus, CTCs were enumerated by trained operators. Blood samples containing C5 CTCs/7.5 ml were considered CTC-positive [17]. Statistical analysis Associations between categorical variables were analyzed using the v2 test. Survival analysis was performed separately for PFS (time from BM aspiration to disease progression) and OS (time from BM aspiration to death from any cause). Data were censored at last follow-up if no event (progression or death) occurred. The influence of DTC status on survival was determined by univariate analysis and expressed as a hazard ratio with a corresponding 95 % confidence interval (CI). Kaplan–Meier curves were constructed and compared by the log-rank test. Multivariate analysis of survival used a Cox proportional regression model. Variables were entered using a stepwise backward method and removed from the model if they failed to reach a significance level of 0.1. The initial model included menopausal status, grading, site of metastasis, number of metastatic sites, line of therapy, receptor status (ER/PR/HER2), and the DTC status. The effect of each variable was assessed using the Wald test and expressed as hazard ratio and 95 % CI. All statistical tests were carried out using PASW Statistics 21 (SPSS Inc., Chicago, IL, USA) and reported two-sided with significance levels set at p \ 0.05.
All patients
CTCpositive n/total (%)
20/57 (35)
Postmenopausal
44/121 (36)
Grading
0.554 7/12 (58) 10/21 (48)
0.967 42/113 (37)
0.254 15/27 (56)
21/56 (38)
Receptor status
1/4 (25) 0.230
HR-positive/HER2negative
42/111 (38)
HER2-positive HR-negative/HER2negative
7/30 (23) 11/25 (44)
Bone metastasis
0.296 5/11 (46) 1/1 (100) 4/5 (80)
0.816
No
23/62 (37)
Yes
41/116 (35)
Visceral metastasis
0.909 5/10 (50) 12/23 (52) 0.849
0.020
No
20/75 (27)
Yes
44/103 (43)
Number of metastatic sites
9/18 (50) 8/15 (53) 0.148
One site
29/94 (31)
Multiple sites
31/74 (42)
Line of therapy
pvalue
17/33 (52) 0.869
Premenopausal
G3
pvalue
64/178 (36)
Menopausal status
G1–2
CTC studies
0.057 2/9 (22) 6/8 (75) 0.713
0.017
First
27/96 (28)
8/17 (47)
Second or higher
24/50 (48)
7/13 (54)
Bold p values indicate statistical significance HR hormone receptor, HER2 human epithelial growth factor receptor 2
Most patients were postmenopausal (121/178, 68 %) and had grade 1–2 tumors (113/169, 67 %). 111/166 (67 %) patients were hormone receptor (HR)-positive/human epidermal growth factor receptor (HER2)-negative, 30/166 (18 %) were HER2-positive, and 25/166 (15 %) were HRnegative/HER2-negative (triple-negative). Bone metastases and visceral metastases were present in 116/178 (65 %) and 103/178 (58 %) patients, respectively. 94/168 (56 %) patients had metastatic disease at one site and the majority (96/146, 66 %) received first-line therapy.
Results DTC studies Patient characteristics In total, 178 MBC patients were eligible for this study. Table 1 summarizes the patients’ clinical characteristics.
As shown in Table 1, DTCs were detected in the BM of 64/178 (36 %) patients. Disseminated tumor cells were detected significantly more frequently in the BM of
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Table 2 Comparison of blood CTC counts and bone marrow DTC status CTC status Negative (\5 CTCs)
Positive (C 5 CTCs)
Total
DTC status Negative, n (%)
11 (55 %)
9 (45 %)
20 (100 %)
Positive, n (%)
5 (39 %)
8 (61 %)
13 (100 %)
16 (49 %)
17 (52 %)
33 (100 %)
Total, n (%) 2
v test: p = 0.353
patients with visceral metastasis (44/103, 43 %) than in patients without visceral metastasis (20/75, 27 %; p = 0.020). Moreover, patients receiving second or higher line therapy (24/50, 48 %) were significantly more likely to have DTCs in the BM than patients receiving first-line therapy (27/96, 28 %, p = 0.017). CTC studies Table 1 also summarizes the CTC results for a subset of 33 patients. Circulating tumor cell counts ranged from 0 to 198 CTCs/7.5 ml blood with a median of 4 CTCs/7.5 ml. A CTC-positive status, defined as C5 CTCs/7.5 ml, was observed in 17 (52 %) of these patients. Table 2 shows that only 19 (56 %) of the 33 patients in the CTC subgroup were concordant for CTC and DTC status. There was no significant association between the blood and BM results for tumor cell detection (p = 0.353). Survival analysis Follow-up data for PFS and OS were available for 124 and 169 patients, respectively. Median follow-up [95 % CI] was 51.84 [45.00–57.67] months. Univariate analysis as shown in Fig. 1 revealed that the detection of DTC was a significant predictor of poor OS (p \ 0.001) with a median OS [95 % CI] of DTC-negative versus DTC-positive patients of 52 [38–67] versus 28 [19–37] months. DTC status had no significant impact on Fig. 1 Kaplan–Meier plots of progression-free (a) and overall survival (b) by BM DTC status of patients with MBC
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PFS (22 [15–29] versus 21 [12–31] months, p = 0.705). Multivariate analysis as shown in Table 3 identified grading, line of therapy, and HER2-positive disease as predictors of reduced PFS, and DTC and triple-negative disease as predictors of reduced OS Table 3. Figure 2 illustrates the impact of CTCs on survival. CTC-positive patients, i.e., those with counts C5 CTCs/ 7.5 ml blood, were at an increased risk of disease progression (p = 0.026) and death (p = 0.025). Median PFS [95 % CI] in CTC-negative and CTC-positive patients was 49 [0–114] and 15 [0–38] months, and median OS [95 % CI] was 88 [0–180] and 26 [8–44] months, respectively.
Discussion This is, to the best of our knowledge, the largest study to analyze the prognostic significance of DTCs from the BM of patients with MBC. We also compared, in a subset of patients, DTC detection with the CTC status as determined using the CellSearchÒ technology. Our analysis revealed DTC detection to be an independent predictor of poor OS. Although several smaller studies have focused on the impact of DTCs in MBC, the results have been inconsistent [7, 15, 16]. Bidard et al. investigated the presence of DTCs in 138 patients with MBC and reported a higher detection rate of 59 % compared with 36 % in our study. In contrast to our results, however, Bidard and collaborators found no association between DTCs and survival, [15] while Janni et al. reported that DTC detection in patients with distant metastatic disease was a negative predictor of OS [16]. A number of factors may have contributed to these conflicting findings. First, the cited studies differed considerably with respect to patient characteristics. The study by Bidard et al. included more patients receiving first-line therapy [15]. Second, the same study also included a higher proportion of patients with bone metastasis [15]. Finally, the study by Janni et al. comprised only 33 MBC patients [16]. In line with previous CTC enumeration studies in patients with MBC, our findings confirm the prognostic
Breast Cancer Res Treat (2014) 147:345–351 Table 3 Multivariate Cox regression analysis of progression-free survival (PFS) and overall survival (OS)
Parameter
349
PFS
OS
Hazard ratio
95 % CI
p-value
–
NS
Hazard ratio
95 % CI
p-value
1.14–3.32
0.014
–
NS
DTC Status Negative
–
Positive
–
1.00 1.95
Grading G1–2
1.00
G3
2.34
– 1.39–3.96
–
–
–
NS
Line of therapy First
1.00
Second or higher
1.69
1.07–2.67
–
Receptor status Bold p-values indicate statistical significance
HR-pos./HER2-neg. HER2-pos.
1.00 0.42
0.23–0.78
0.006
1.00 –
–
NS
PR progesterone receptor, NS non-significant
HR-neg./HER2-neg.
–
–
NS
3.34
1.63–6.84
0.001
Fig. 2 Kaplan–Meier plot of progression-free (a) and overall survival (b) by blood CTC status of patients with MBC
impact of CTC status on PFS and OS [17, 18]. We found no significant association between CTC status and DTC status. Also, there was no correlation of DTC and CTC detection when a threshold of one CTC/7.5 ml blood was used (data not shown). It must, however, be noted that a non-significant result does not exclude an association, especially with respect to the fact that our CTC results are limited by the small subgroup size. Since the antibody A45-B/B3 we used to detect DTCs is different from the antibody to detect CK-positive cells within CellSearch, this could also explain for the observed lack of correlation. When Bidard and collaborators used A45-B/B3 to detect CK-positive cells in both, BM and blood, the authors found a significant association between CTCs and DTCs. [15]. In primary BC, the detection of DTCs has a high and independent adverse prognostic impact [2–8]. A large pooled analysis of over 4,700 patients with early-stage disease published by Braun et al. demonstrated the detection of DTCs to be the strongest independent factor for poor disease-free and OS [1]. However, the clinical use of CTCs in primary disease is challenging because detection rates are low, ranging from 10 to 40 %. In the translational
research program of the German SUCCESS Trial (www. success-studie.de), peripheral blood samples from high-risk BC patients were evaluated for CTCs using the CellSearch technology. Women with C1 CTCs/7.5 ml blood had a significantly reduced disease-free survival [9]. Molloy et al. recently studied 733 patients with primary BC and found that CTC detection by a PCR-based assay was highly associated with DTC status [23]. However, other reports did not confirm these results, probably due to methodological differences between studies [7, 11–13]. Interestingly, we did not find a significant relationship between bone metastasis and the presence of DTCs in the BM. This is in line with results from our previous DTC studies in patients with epithelial cancers of other origin which, in contrast to BC, generally do not metastasize to the skeleton [24, 25]. As the detection rate of DTCs in ovarian, endometrial, and vulvar cancer is comparable to that observed in BC patients, we concluded that the BM might represent only a temporary compartment in the process of hematogenous cancer spread [24, 25]. In the present study, we additionally found a higher prevalence of DTCs in MBC patients with visceral metastasis. These
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results, together with the association of DTC detection with further-line therapies, indicate that DTCs are more frequently detected in patients with more aggressive and more advanced BC. Furthermore, DTCs may have different properties, leading them to metastasize to distinct organs, and their molecular characterization may help to understand the mechanisms of tumor cell homing to particular organs [7, 26]. Recent reports have focused on the clinical value of CTC and DTC detection to monitor the efficacy of systemic therapy [12, 27–29]. Basically, these studies focused on serial CTC enumeration in MBC whereas DTCs were primarily determined during adjuvant therapy for early BC. Both CTC and DTC detection were found to be of prognostic significance during the course of systemic BC therapy, emphasizing their role in treatment monitoring. The importance of changing an ongoing therapy because of elevated CTC levels was recently addressed in the randomized prospective SWOG S0500 trial conducted by the Southwest Oncology Group [29]. While the investigators confirmed that quantification of CTCs during therapy was of high prognostic value, the study participants did not benefit from changing systemic therapy due to elevated CTC counts. Hence, we speculate that phenotyping CTCs and DTCs as opposed to simply enumerating them would probably make more significant contributions toward guiding decisions on the individualized systemic treatment of BC patients [30].
Conclusion Our findings show that DTC detection predicts poor prognosis in patients with MBC, highlighting the role of DTCs for BC progression. As we found no significant association between the concurrent presence of CTCs and DTCs, these cells might represent different aspects of the systemic spread of BC. However, larger trials should simultaneously compare the presence and prognostic impact of CTCs and DTCs in MBC. Ideally, such trials would include phenotyping and molecular characterization studies of single tumor cells in order to better understand disease progression, tailor BC treatment, and to identify new biomarkers as potential therapeutic targets. Acknowledgments We are grateful to Silke Duerr-Sto¨rzer, Ingrid Teufel, Sabine Hofmeister, Angelika Amman, and Brigitte Neth for excellent technical assistance. Conflict of interest of interest.
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All authors declare that they have no conflicts
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CLINICAL TRIAL
Tumor cell dissemination to the bone marrow and blood is associated with poor outcome in patients with metastatic breast cancer Andreas D. Hartkopf • Diana Stefanescu • Markus Wallwiener Markus Hahn • Sven Becker • Erich-Franz Solomayer • Tanja N. Fehm • Sara Y. Brucker • Florin-Andrei Taran
•
Received: 14 August 2014 / Accepted: 18 August 2014 / Published online: 24 August 2014 Ó Springer Science+Business Media New York 2014
Abstract The purpose of this study was to assess the impact of disseminated tumor cells (DTCs) on progressionfree and overall survival (OS) in patients with metastatic breast cancer (MBC) and to compare it to simultaneous detection of circulating tumor cells (CTCs) from the blood in a subgroup. Disseminated tumor cells were identified in bone marrow (BM) aspirates by immunocytochemistry (pancytokeratin antibody A45-B/B3) and cytomorphology prior to the beginning of a new-line therapy. CTCs were enumerated by the CellSearchÒ technology. BM was obtained from 178 patients with MBC; 64/178 (36 %) patients were DTC-positive. Disseminated tumor cells occurred more frequently in patients with visceral metastases (p = 0.020) and C2 lines of therapy (p = 0.017). CTCs were assessed in 33 of these patients and 17/33
(52 %) patients had CTC counts C5 CTCs/7.5 ml blood. There was no significant association between the DTC and CTC status. Univariate analysis revealed DTC detection as a significant predictor of poor OS (p \ 0.001); median OS in DTC-negative versus DTC-positive patients was 52 [95 % confidence interval (CI) 38–67] versus 28 [95 % CI 19–37] months. Moreover, as described previously, patients with C5 CTCs/7.5 ml blood were at an increased risk of disease progression (p = 0.026) and death (p = 0.025). Disseminated tumor cells are predictors of poor prognosis in MBC, highlighting the role of tumor cell dissemination into the BM for breast cancer progression. The absence of a significant association between concurrent DTCs and CTCs suggests they might represent different aspects of systemic BC spread.
A. D. Hartkopf (&) D. Stefanescu M. Hahn S. Y. Brucker F.-A. Taran Department of Obstetrics and Gynecology, University of Tuebingen, Calwerstraße 7, 72076 Tuebingen, Germany e-mail: [email protected]
S. Becker Department of Obstetrics and Gynecology, University of Frankfurt, Theodor-Stern-Kai 7, 60590 Frankfurt, Germany e-mail: [email protected]
D. Stefanescu e-mail: [email protected] M. Hahn e-mail: [email protected] S. Y. Brucker e-mail: [email protected] F.-A. Taran e-mail: [email protected]
E.-F. Solomayer Department of Obstetrics and Gynecology, Saarland University, Kirrberger Straße 100, 66421 Homburg, Germany e-mail: [email protected] T. N. Fehm Department of Obstetrics and Gynecology, University of Duesseldorf, Moorenstraße 5, 40225 Duesseldorf, Germany e-mail: [email protected]
M. Wallwiener Department of Obstetrics and Gynecology, University of Heidelberg, Im Neuenheimer Feld 110, 69120 Heidelberg, Germany e-mail: [email protected]
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Keywords Metastatic breast cancer Disseminated tumor cell Circulating tumor cell Survival Bone marrow Abbreviations BM Bone marrow BC Breast cancer CI Confidence interval CTC Circulating tumor cell DTC Disseminated tumor cell EpCAM Epithelial cell adhesion molecule ER Estrogen receptor HER2 Human epidermal growth factor receptor 2 HR Hormone receptor ISHAGE International Society for Hematotherapy and Graft Engineering MBC Metastatic breast cancer OS Overall survival PR Progesterone receptor
Introduction The dissemination of individual tumor cells is a common phenomenon in solid epithelial cancers. Highly sensitive assays enable the detection of such cells even at early stages of disease. With respect to primary breast cancer (BC), cytokeratin-positive disseminated tumor cells (DTCs) are detected in the bone marrow (BM) in 20–30 % of early-stage patients [1, 2]. Numerous trials have provided high-level evidence that detection of DTCs in the non-metastatic situation is of high and independent prognostic significance [3–8]. Other studies have demonstrated that circulating tumor cells (CTCs) in the peripheral blood are associated with an impaired prognosis in early-stage BC [9, 10]. However, the information from DTCs appears to be of higher prognostic value, probably due to methodological differences between CTC studies and the lower sensitivity of CTC detection [11–13]. To date, only a few small studies have investigated the role of DTCs in metastatic breast cancer (MBC), and results on the prognostic impact of DTC detection are inconsistent [14–16]. Hence, our present knowledge of the role of tumor cell dissemination in patients with advanced BC relies primarily on CTCs. In patients with MBC, CTC counts C5/7.5 ml blood have been clearly demonstrated in large trials to be associated with poor outcome [17, 18]. The current gold standard for CTC detection is the Veridex CellSearchÒ system, which received clearance from the U.S. Food and Drug Administration in 2004 as a diagnostic tool for identifying and counting CTCs of epithelial origin in patients with MBC.
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To investigate the role of tumor cell dissemination in MBC, we conducted the present retrospective study, which to the best of our knowledge is the largest BM sampling study in patients with MBC. Our primary objective was to assess the impact of DTC detection on progression-free (PFS) and overall survival (OS). As a secondary objective, we compared DTC and CTC detection in a subset of our study population.
Materials and methods Study population Bone marrow sampling was performed in patients with MBC before the initiation of a new-line therapy at the Department of Obstetrics and Gynecology, Tuebingen University Hospital, Germany, between January 2001 and June 2012. Patients with malignancies other than BC were excluded from this analysis. Patients were followed up by regular clinical and radiologic assessments every 3 months. All patients provided written informed consent for BM aspiration for research purposes. Circulating tumor cell data were collected from the hospital records of the patients who provided BM samples. The clinical cancer registry of the Tuebingen Comprehensive Cancer Center provided additional follow-up data and the survival data. The present analysis was approved by the Ethics Committee of the University of Tuebingen (reference number 560/2012R). Bone marrow DTC studies BM aspirates (10–20 ml) were collected from the anterior iliac crest. Samples were processed within 24 h as described elsewhere [19]. In brief, mononuclear cells were separated by density centrifugation using Ficoll (Biochrom, Berlin, Germany) at a density of 1.077 g/ml, spun down onto a glass slide in a cytocentrifuge (Hettich, Tuttlingen, Germany) and fixed in 4 % formalin. Disseminated tumor cell status was determined by immunostaining using the DAKO Autostainer (Dako, Glostrup, Denmark), the A45B/B3 mouse monoclonal antibody (Micromet, Munich, Germany), which recognizes several cytokeratin epitopes (CK8, CK18, and CK19), and the DAKO-APAA detection kit (Dako, Glostrup, Denmark). Two slides (1 9 106 cells each) per patient were evaluated based on consensus recommendations for standardized tumor cell detection [20, 21]. An additional slide was stained using a non-specific isotype-matched antibody. Moreover, each batch of samples was analyzed together with healthy volunteer leukocytes as a negative control and the human BC cell lines MCF-7 and SKBR-3 as positive controls. To assess the specificity of our DTC detection method, we analyzed BM
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samples from 100 patients without evidence of malignant disease, of whom one was DTC-positive [12].
347 Table 1 Patient characteristics DTC studies DTCpositive n/total (%)
Blood CTC studies Enrichment and enumeration of CTCs using the CellSearchÒ technology (Veridex LLC, Warren, NJ, USA) were essentially performed as described elsewhere [22]. Briefly, 7.5 ml samples of peripheral blood were drawn into CellSave tubes (Veridex LLC, Warren, NJ, USA), kept at room temperature and processed within 72 h. Epithelial cells were enriched immunomagnetically using a ferrofluid with antibodies against the epithelial cell adhesion molecule (EpCAM). Epithelial cell adhesion molecule-positive cells were labeled with the nuclear dye 40 ,6-diamidino-2phenylindole (DAPI) and monoclonal antibodies specific for the leukocyte common antigen CD45. Defined as CD45-negative, cytokeratin-positive cells with an intact nucleus, CTCs were enumerated by trained operators. Blood samples containing C5 CTCs/7.5 ml were considered CTC-positive [17]. Statistical analysis Associations between categorical variables were analyzed using the v2 test. Survival analysis was performed separately for PFS (time from BM aspiration to disease progression) and OS (time from BM aspiration to death from any cause). Data were censored at last follow-up if no event (progression or death) occurred. The influence of DTC status on survival was determined by univariate analysis and expressed as a hazard ratio with a corresponding 95 % confidence interval (CI). Kaplan–Meier curves were constructed and compared by the log-rank test. Multivariate analysis of survival used a Cox proportional regression model. Variables were entered using a stepwise backward method and removed from the model if they failed to reach a significance level of 0.1. The initial model included menopausal status, grading, site of metastasis, number of metastatic sites, line of therapy, receptor status (ER/PR/HER2), and the DTC status. The effect of each variable was assessed using the Wald test and expressed as hazard ratio and 95 % CI. All statistical tests were carried out using PASW Statistics 21 (SPSS Inc., Chicago, IL, USA) and reported two-sided with significance levels set at p \ 0.05.
All patients
CTCpositive n/total (%)
20/57 (35)
Postmenopausal
44/121 (36)
Grading
0.554 7/12 (58) 10/21 (48)
0.967 42/113 (37)
0.254 15/27 (56)
21/56 (38)
Receptor status
1/4 (25) 0.230
HR-positive/HER2negative
42/111 (38)
HER2-positive HR-negative/HER2negative
7/30 (23) 11/25 (44)
Bone metastasis
0.296 5/11 (46) 1/1 (100) 4/5 (80)
0.816
No
23/62 (37)
Yes
41/116 (35)
Visceral metastasis
0.909 5/10 (50) 12/23 (52) 0.849
0.020
No
20/75 (27)
Yes
44/103 (43)
Number of metastatic sites
9/18 (50) 8/15 (53) 0.148
One site
29/94 (31)
Multiple sites
31/74 (42)
Line of therapy
pvalue
17/33 (52) 0.869
Premenopausal
G3
pvalue
64/178 (36)
Menopausal status
G1–2
CTC studies
0.057 2/9 (22) 6/8 (75) 0.713
0.017
First
27/96 (28)
8/17 (47)
Second or higher
24/50 (48)
7/13 (54)
Bold p values indicate statistical significance HR hormone receptor, HER2 human epithelial growth factor receptor 2
Most patients were postmenopausal (121/178, 68 %) and had grade 1–2 tumors (113/169, 67 %). 111/166 (67 %) patients were hormone receptor (HR)-positive/human epidermal growth factor receptor (HER2)-negative, 30/166 (18 %) were HER2-positive, and 25/166 (15 %) were HRnegative/HER2-negative (triple-negative). Bone metastases and visceral metastases were present in 116/178 (65 %) and 103/178 (58 %) patients, respectively. 94/168 (56 %) patients had metastatic disease at one site and the majority (96/146, 66 %) received first-line therapy.
Results DTC studies Patient characteristics In total, 178 MBC patients were eligible for this study. Table 1 summarizes the patients’ clinical characteristics.
As shown in Table 1, DTCs were detected in the BM of 64/178 (36 %) patients. Disseminated tumor cells were detected significantly more frequently in the BM of
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Table 2 Comparison of blood CTC counts and bone marrow DTC status CTC status Negative (\5 CTCs)
Positive (C 5 CTCs)
Total
DTC status Negative, n (%)
11 (55 %)
9 (45 %)
20 (100 %)
Positive, n (%)
5 (39 %)
8 (61 %)
13 (100 %)
16 (49 %)
17 (52 %)
33 (100 %)
Total, n (%) 2
v test: p = 0.353
patients with visceral metastasis (44/103, 43 %) than in patients without visceral metastasis (20/75, 27 %; p = 0.020). Moreover, patients receiving second or higher line therapy (24/50, 48 %) were significantly more likely to have DTCs in the BM than patients receiving first-line therapy (27/96, 28 %, p = 0.017). CTC studies Table 1 also summarizes the CTC results for a subset of 33 patients. Circulating tumor cell counts ranged from 0 to 198 CTCs/7.5 ml blood with a median of 4 CTCs/7.5 ml. A CTC-positive status, defined as C5 CTCs/7.5 ml, was observed in 17 (52 %) of these patients. Table 2 shows that only 19 (56 %) of the 33 patients in the CTC subgroup were concordant for CTC and DTC status. There was no significant association between the blood and BM results for tumor cell detection (p = 0.353). Survival analysis Follow-up data for PFS and OS were available for 124 and 169 patients, respectively. Median follow-up [95 % CI] was 51.84 [45.00–57.67] months. Univariate analysis as shown in Fig. 1 revealed that the detection of DTC was a significant predictor of poor OS (p \ 0.001) with a median OS [95 % CI] of DTC-negative versus DTC-positive patients of 52 [38–67] versus 28 [19–37] months. DTC status had no significant impact on Fig. 1 Kaplan–Meier plots of progression-free (a) and overall survival (b) by BM DTC status of patients with MBC
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PFS (22 [15–29] versus 21 [12–31] months, p = 0.705). Multivariate analysis as shown in Table 3 identified grading, line of therapy, and HER2-positive disease as predictors of reduced PFS, and DTC and triple-negative disease as predictors of reduced OS Table 3. Figure 2 illustrates the impact of CTCs on survival. CTC-positive patients, i.e., those with counts C5 CTCs/ 7.5 ml blood, were at an increased risk of disease progression (p = 0.026) and death (p = 0.025). Median PFS [95 % CI] in CTC-negative and CTC-positive patients was 49 [0–114] and 15 [0–38] months, and median OS [95 % CI] was 88 [0–180] and 26 [8–44] months, respectively.
Discussion This is, to the best of our knowledge, the largest study to analyze the prognostic significance of DTCs from the BM of patients with MBC. We also compared, in a subset of patients, DTC detection with the CTC status as determined using the CellSearchÒ technology. Our analysis revealed DTC detection to be an independent predictor of poor OS. Although several smaller studies have focused on the impact of DTCs in MBC, the results have been inconsistent [7, 15, 16]. Bidard et al. investigated the presence of DTCs in 138 patients with MBC and reported a higher detection rate of 59 % compared with 36 % in our study. In contrast to our results, however, Bidard and collaborators found no association between DTCs and survival, [15] while Janni et al. reported that DTC detection in patients with distant metastatic disease was a negative predictor of OS [16]. A number of factors may have contributed to these conflicting findings. First, the cited studies differed considerably with respect to patient characteristics. The study by Bidard et al. included more patients receiving first-line therapy [15]. Second, the same study also included a higher proportion of patients with bone metastasis [15]. Finally, the study by Janni et al. comprised only 33 MBC patients [16]. In line with previous CTC enumeration studies in patients with MBC, our findings confirm the prognostic
Breast Cancer Res Treat (2014) 147:345–351 Table 3 Multivariate Cox regression analysis of progression-free survival (PFS) and overall survival (OS)
Parameter
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PFS
OS
Hazard ratio
95 % CI
p-value
–
NS
Hazard ratio
95 % CI
p-value
1.14–3.32
0.014
–
NS
DTC Status Negative
–
Positive
–
1.00 1.95
Grading G1–2
1.00
G3
2.34
– 1.39–3.96
–
–
–
NS
Line of therapy First
1.00
Second or higher
1.69
1.07–2.67
–
Receptor status Bold p-values indicate statistical significance
HR-pos./HER2-neg. HER2-pos.
1.00 0.42
0.23–0.78
0.006
1.00 –
–
NS
PR progesterone receptor, NS non-significant
HR-neg./HER2-neg.
–
–
NS
3.34
1.63–6.84
0.001
Fig. 2 Kaplan–Meier plot of progression-free (a) and overall survival (b) by blood CTC status of patients with MBC
impact of CTC status on PFS and OS [17, 18]. We found no significant association between CTC status and DTC status. Also, there was no correlation of DTC and CTC detection when a threshold of one CTC/7.5 ml blood was used (data not shown). It must, however, be noted that a non-significant result does not exclude an association, especially with respect to the fact that our CTC results are limited by the small subgroup size. Since the antibody A45-B/B3 we used to detect DTCs is different from the antibody to detect CK-positive cells within CellSearch, this could also explain for the observed lack of correlation. When Bidard and collaborators used A45-B/B3 to detect CK-positive cells in both, BM and blood, the authors found a significant association between CTCs and DTCs. [15]. In primary BC, the detection of DTCs has a high and independent adverse prognostic impact [2–8]. A large pooled analysis of over 4,700 patients with early-stage disease published by Braun et al. demonstrated the detection of DTCs to be the strongest independent factor for poor disease-free and OS [1]. However, the clinical use of CTCs in primary disease is challenging because detection rates are low, ranging from 10 to 40 %. In the translational
research program of the German SUCCESS Trial (www. success-studie.de), peripheral blood samples from high-risk BC patients were evaluated for CTCs using the CellSearch technology. Women with C1 CTCs/7.5 ml blood had a significantly reduced disease-free survival [9]. Molloy et al. recently studied 733 patients with primary BC and found that CTC detection by a PCR-based assay was highly associated with DTC status [23]. However, other reports did not confirm these results, probably due to methodological differences between studies [7, 11–13]. Interestingly, we did not find a significant relationship between bone metastasis and the presence of DTCs in the BM. This is in line with results from our previous DTC studies in patients with epithelial cancers of other origin which, in contrast to BC, generally do not metastasize to the skeleton [24, 25]. As the detection rate of DTCs in ovarian, endometrial, and vulvar cancer is comparable to that observed in BC patients, we concluded that the BM might represent only a temporary compartment in the process of hematogenous cancer spread [24, 25]. In the present study, we additionally found a higher prevalence of DTCs in MBC patients with visceral metastasis. These
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results, together with the association of DTC detection with further-line therapies, indicate that DTCs are more frequently detected in patients with more aggressive and more advanced BC. Furthermore, DTCs may have different properties, leading them to metastasize to distinct organs, and their molecular characterization may help to understand the mechanisms of tumor cell homing to particular organs [7, 26]. Recent reports have focused on the clinical value of CTC and DTC detection to monitor the efficacy of systemic therapy [12, 27–29]. Basically, these studies focused on serial CTC enumeration in MBC whereas DTCs were primarily determined during adjuvant therapy for early BC. Both CTC and DTC detection were found to be of prognostic significance during the course of systemic BC therapy, emphasizing their role in treatment monitoring. The importance of changing an ongoing therapy because of elevated CTC levels was recently addressed in the randomized prospective SWOG S0500 trial conducted by the Southwest Oncology Group [29]. While the investigators confirmed that quantification of CTCs during therapy was of high prognostic value, the study participants did not benefit from changing systemic therapy due to elevated CTC counts. Hence, we speculate that phenotyping CTCs and DTCs as opposed to simply enumerating them would probably make more significant contributions toward guiding decisions on the individualized systemic treatment of BC patients [30].
Conclusion Our findings show that DTC detection predicts poor prognosis in patients with MBC, highlighting the role of DTCs for BC progression. As we found no significant association between the concurrent presence of CTCs and DTCs, these cells might represent different aspects of the systemic spread of BC. However, larger trials should simultaneously compare the presence and prognostic impact of CTCs and DTCs in MBC. Ideally, such trials would include phenotyping and molecular characterization studies of single tumor cells in order to better understand disease progression, tailor BC treatment, and to identify new biomarkers as potential therapeutic targets. Acknowledgments We are grateful to Silke Duerr-Sto¨rzer, Ingrid Teufel, Sabine Hofmeister, Angelika Amman, and Brigitte Neth for excellent technical assistance. Conflict of interest of interest.
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All authors declare that they have no conflicts
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