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Role of Circulating Tumor Cells in Breast Cancer Patients
C H A P T E R
28 Role of Circulating Tumor Cells in Breast Cancer Patients Rita Lampignano1,2, A.M. Sofie Berghuis3, Tanja Fehm1,2 and Hans Neubauer1,2 1
Department of Obstetrics and Gynecology, University Hospital of Duesseldorf, Duesseldorf, Germany 2 Medical Faculty of the Heinrich-Heine, University of Duesseldorf, Duesseldorf, Germany 3 Department of Health Technology and Services Research, Faculty of Behavioural, Management and Social Sciences, Technical Medical Centre, University of Twente, Enschede, The Netherlands
ABBREVIATIONS (m)BC BCSS CTC (D)D/MFS DTC EpCAM Her2/neu MRD OS PFS
(metastatic) breast cancer breast cancer-specific survival circulating tumor cell (distant) disease/metastasisfree survival disseminated tumor cells epithelial cell adhesion molecule human epidermal growth receptor 2 minimal residual disease overall survival progression-free survival
Oncogenomics DOI: https://doi.org/10.1016/B978-0-12-811785-9.00028-4
CELLS RELEASED BY SOLID TUMORS AS “LIQUID BIOPSY” IN BREAST CANCER Despite advances in early detection and identification of optimal treatment strategies, 20% 30% of breast cancer (BC) patients still face long-term systemic relapses, mainly due to the persistence of a minimal residual disease (MRD; Banys-Paluchowski, Krawczyk, MeierStiegen, & Fehm, 2016). The prognostic importance of MRD has been mainly assessed by analyzing patients bone marrow biopsies. In one of the most extensively pooled analyses, Braun and colleagues (2005) reported the
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© 2019 Elsevier Inc. All rights reserved.
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correlation between the presence of disseminated tumor cells (DTCs) in bone marrow and shorter patient survival rates in 4,700 early BC patients. Despite such promising investigations on DTCs, their analysis is often hampered by the invasiveness of the bone marrow aspiration. However, in the past decade, numerous studies have reported a concordant positivity rate of DTCs and circulating tumor cells (CTCs) in the peripheral blood of BC patients (66% 94%, P , .3 to P , .001; Bidard, Proudhon, & Pierga, 2016), thereby indicating CTCs as a “liquid” and easily accessible alternative to DTCs (Pantel & Alix-Panabie`res, 2010). The prognostic role of CTCs has been investigated in numerous tumor entities, and their presence correlates with poor clinical outcomes in breast, colorectal, lung, and prostate cancers (Cabel et al., 2017; Cohen et al., 2008; Cristofanilli et al., 2004; de Bono et al., 2008). To date, BC is the most extensively studied tumor in terms of the clinical role of CTCs, which are observed in 40% 80% of metastatic patients (Banys-Paluchowski et al., 2016). In the discussion regarding the clinical relevance of CTCs as a “liquid biopsy,” two main topics can be distinguished: (1) the clinical validity of CTC enumeration as a predictive and prognostic biomarker, and as a treatment decision tool; and (2) the clinical utility of the CTCs’ phenotypic and molecular characteristics as targetable therapeutic biomarkers.
CLINICAL VALIDITY OF CTC COUNTS AND DYNAMIC CHANGES IN METASTATIC BREAST CANCER The clinical validity for disease prognosis of CTCs in metastatic breast cancer (mBC) was first investigated in a multicenter study by Cristofanilli and colleagues (2004) on 177 patients, blood samples collected before the
beginning of a new therapy (baseline) and at the first follow-up visit. CTCs in the blood were determined with the U.S. Food and Drug Administration-approved EpCAM-based CellSearch system, and the detection of $ 5 CTCs/7.5 mL blood was shown to be prognostic for patients shortened progression-free survival (PFS) and overall survival (OS). On the contrary, patients with ,5 CTCs/7.5 mL blood had longer survival times. The cut-off value of $ 5 CTCs/7.5 mL blood to predict patients poor outcomes was further validated by Hayes and colleagues (2006) on the same cohort of patients, focusing on both baseline and follow-up blood samples. The role of CTC enumeration in the management of mBC patients has been further investigated in more than 20 subsequent studies, mainly by utilizing the CellSearch system (Banys-Paluchowski et al., 2016; Cabel et al., 2017; Khatami et al., 2017). In Table 28.1, major studies evaluating the prognostic and/or predictive role of CTCs in mBC are reported. Interestingly, except for human epidermal growth factor receptor 2 (Her2)/neu-positive mBC patients, the presence of $ 5 CTCs/ 7.5 mL blood was identified as a prognostic factor independently of the molecular subtype of primary tumors (Banys-Paluchowski et al., 2016; Wallwiener et al., 2013). Besides the prognostic validity of CTCs in mBC, their role in monitoring patients treatment responses has been assessed as well. Most of the published studies concluded that dynamic changes in the CTC frequency may be utilized to monitor mBC patients responses to therapies and to predict clinical outcomes (Budd et al., 2006; Cristofanilli et al., 2004; Hayes et al., 2006; Liu et al., 2009). In detail, it was reported that mBC patients with high baseline CTC count but decreased CTC numbers after the first line of chemotherapy have better PFS and OS than patients with persistently high CTC numbers (Cristofanilli et al., 2004; Hayes et al., 2006). Additionally, the presence of persistent CTCs after therapies was compared to
IV. PERSPECTIVES IN BREAST CANCER GENOMICS
CLINICAL VALIDITY OF CTC COUNTS AND DYNAMIC CHANGES IN METASTATIC BREAST CANCER
TABLE 28.1
385
Association of CTC Count and Survival in mBC Patients
Study
Patient Cohort
Association
Bidard et al. (2014)
911
OS, PFS
Budd et al. (2006)
138
OS
Cristofanilli et al. (2004)
177
OS, PFS
Dawood et al. (2008)
185
OS
De Giorgi et al. (2009)
115
OS
Giordano et al. (2012)
517
PFS, OS
Giuliano et al. (2011)
235
PFS, OS
Hayes et al. (2006)
177
PFS, OS
Mu¨ller et al. (2012)
254
OS
Nakamura et al. (2010)
107
OS
Pierga et al. (2012)
267
PFS, OS
Wallwiener et al. (2013)
486
PFS, OS
OS, overall survival; PFS, progression-free survival.
imaging approaches conventionally utilized for treatment monitoring and was found to accurately reflect patients reactions to therapies (Budd et al., 2006; De Giorgi et al., 2009). However, the clinical role of CTC dynamics as therapy decision tools has not been demonstrated to date, and it is currently being evaluated in clinical trials. Within the SWOG S0500 study, focusing on mBC patients with persistently .5 CTCs/7.5 mL blood after the first line of chemotherapy, Smerage and colleagues (2014) evaluated the effects of an early switch to a second-line chemotherapy on patients outcomes. This treatment switch was hypothesized to greatly improve patients’ OS compared to the standard imaging-based management. However, no survival differences were observed between the two subgroups of patients. The negative results of the SWOG study could be explained by its design and concepts. It is, in fact, unlikely that a secondline chemotherapy may have a significant effect on CTCs which exhibit a primary
resistance to a first-line chemotherapy (AlunniFabbroni, Mu¨ller, Fehm, Janni, & Rack, 2014; Bidard & Pierga, 2015). This is even more true when the type of therapy is not tailored to the genotype or phenotype of the CTCs, thus emphasizing the necessity to identify specific biomarkers in/on CTCs that could be targeted by novel drugs. Nevertheless, detecting persistent CTCs may still serve to identify therapyresistant patients who demand different cures. The role of CTCs in selecting the optimal third-line chemotherapy is being investigated in the ongoing CirCe01 (NCT01349842), which has a similar approach as the SWOG trial (Helissey et al., 2015). Within this study, patients are enrolled before the start of the third line of chemotherapy and are followed throughout the successive lines of chemotherapy in which CTC counts are additionally determined (Helissey et al., 2015). Furthermore, the STIC-CTC trial (NCT01710605) addresses the utility to treat mBC patients with hormone therapy if they
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exhibit a CTC count ,5/7.5 mL blood or with chemotherapy if the CTC count is $ 5/7.5 mL blood (Banys-Paluchowski et al., 2016; Cabel et al., 2017). The trial’s main medical objective is to demonstrate non-inferiority in PFS of the CTC-based strategy compared to the clinicians’ decision, taking into account the criteria usually used in this setting. Last, the DETECT V trial (NCT02344472) is currently investigating CTC dynamics of dual Her2/neu-targeted treatment in combination with chemotherapy or endocrine therapy (Banys-Paluchowski et al., 2016). Despite all these impressive efforts, the actual contribution of CTC changes in selecting appropriate treatments for mBC patients must still be addressed.
CTCs expressing cancer dormancy markers may be involved in the formation of brain metastases (Vishnoi et al., 2015). In addition, mBC patients with $ 100 EpCAM-negative CTCs/mL of blood have a shorter OS (Lustberg et al., 2014). Last, Bulfoni and colleagues (2016) observed worse PFS and OS in mBC patients with CTCs expressing both epithelial and mesenchymal markers (transient phenotype). However, extensive studies on larger cohorts of patients are needed to shed light on the role of the transient/mesenchymal CTCs in the metastatic cascade of different tumor entities.
Clinical Validity of CTCs with Epithelial Mesenchymal Plasticity
The clinical validity of CTCs has been less extensively investigated in nonmetastatic than in metastatic cancers (including breast tumors), mainly due to the lower frequency of CTCs in the nonmetastatic setting. The German SUCCESS trial (NCT02181101) is the first extensive study on early BC patients supporting the prognostic role of CTCs enriched through the CellSearch system (Rack et al., 2014). In 435/2,026 patients enrolled before chemotherapy and in 330/1,493 patients enrolled after chemotherapy, CTCs could be detected and were correlated with short disease-free survival (DFS; P , .0001), distant DFS (DDFS; P , .001), breast cancer-specific survival (BCSS; P , .008), and OS (P , .0002). In this study, numerous predictive CTC cut-off values were investigated (0 vs. $ 1, 0 1 vs. $ 2, and 0 4 vs. $ 5 in 30 mL of blood), and all of them were statistically significant in the correlation with poor clinical outcomes. The worst prognoses were observed in patients with $ 5 CTCs/30 mL blood at baseline. Interestingly, the CTC positivity rate was associated with the nodal status (N 1 19.6% vs. N 22.4%, P , .001) but not with other
In almost all the previously discussed studies, mBC patients prognoses related to the presence of CTCs, which were mainly detected via the EpCAM-based CellSearch system. However, this approach may overlook tumor cells with a plastic epithelial mesenchymal phenotype, resulting from the epithelial-to-mesenchymal transition (Alix-Panabie`res, Mader, & Pantel, 2017). The existence of an EpCAM-low/negative subset of CTCs has been documented for different tumor entities (including breast), and early observations have been published and reviewed elsewhere (Lampignano, Schneck, Neumann, Fehm, & Neubauer, 2017). Even though this fraction of CTCs has not been extensively investigated yet, the first potential correlations with patients clinical outcomes have been reported. Mego and colleagues (2011, 2012) frequently detected brain metastases in mBC patients lacking EpCAM-positive CTCs, suggesting the presence of a highly malignant EpCAM-negative subgroup of tumor cells. In line with these results, EpCAM-negative
CLINICAL VALIDITY OF CTCs IN NONMETASTATIC BREAST CANCER
IV. PERSPECTIVES IN BREAST CANCER GENOMICS
CLINICAL VALIDITY OF CTCS IN NONMETASTATIC BREAST CANCER
TABLE 28.2
387
Association of CTC Count and Survival in Non-mBC Patients
Study
Patient Cohort
Association
Bidard et al. (2010)
115
DDFS, OS
Franken et al. (2012)
404
DDFS, BCSS
Hwang, Bae, Lee, and Kim (2012)
166
OS
Ignatiadis et al. (2007)
444
OS, DFS
a
Janni et al. (2016)
3,173
DFS, DDFS, BCSS, OS
Kuniyoshi et al. (2015)
167
None
Lucci et al. (2012)
302
DFS, OS
Pierga et al. (2017)
152
DFS, OS
Rack et al. (2014)
2,026
DFS, DDFS, BCCS, OS
a
Pooled analysis. BCSS, breast cancer-specific survival; DDFS, distant disease-free survival; DFS, disease-free survival; OS: overall survival; PFS, progression-free survival.
tumor characteristics. This relation between the presence of CTCs in non-mBC patients and poor clinical outcomes could be further confirmed in nearly all the published studies, summarized in Table 28.2 (Banys-Paluchowski et al., 2016; Bidard et al., 2016). The clinical validity of persistent CTCs in non-mBC was mainly investigated in three trials. The correlation between the presence of CTCs after chemotherapy with poor clinical outcomes was reported in the previously mentioned SUCCESS trial (Rack et al., 2014). Subsequently, in the REMAGUS02 study, Bidard and colleagues suggested that the validity of CTCs as therapy monitoring tools in neoadjuvant settings might be restricted to the first years after diagnosis (Bidard et al., 2010; Bidard, Belin, et al., 2013; Bidard, Fehm, et al., 2013). Interestingly, in contrast to those reports, no association between CTC dynamic changes and patientsresponses to treatments could be observed within the GeparQuattro study (Riethdorf et al., 2010), in agreement with previous observations (Pierga et al., 2008). The validity of CTCs as therapy decision tools is currently also being evaluated in non-
mBC in the TREAT CTC trial (NCT01548677). The major aim of this study is to investigate Her2/neu-targeted treatments in patients with initially Her2/neu-negative tumors and detectable Her2/neu-positive CTCs after (neo) adjuvant chemotherapy (Banys-Paluchowski et al., 2016; Cabel et al., 2017). Although the prognostic role of CTCs on non-mBC has been assessed, further research is required given the relatively small patient cohorts compared to the metastatic setting. However, this demand for additional extensive studies is currently limited by the rarity of CTCs in nonmetastatic cancers. One solution to increment the CTC yield may be to increase the analyzed blood volume via appropriate technologies, such as diagnostic leukapheresis, clinically validated in metastatic breast and prostate cancers within the European CTC-Trap consortium (Andree et al., 2017; Fischer et al., 2013).
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CLINICAL UTILITY OF CTC CHARACTERIZATION During the past decade, the development of high-resolution technologies for single cell analysis permitted numerous investigations on phenotypes and genotypes of CTCs, which are of utmost importance to understand the invasion metastasis cascade. Despite all the efforts, there are still no clinical trials focusing on genomic or transcriptomic analysis of these tumor cells. On the contrary, the clinical utility of analyzing the expression of selected biomarkers on CTCs is currently being evaluated. The multicenter trials NCT00820924 and DETECT III (NCT01619111; Bidard et al., 2013; Schramm et al., 2016) have investigated or are still investigating the efficacy of anti-Her2/neu treatments in mBC patients with Her2/neu-negative primary tumors but with CTCs expressing the biomarker Her2/neu (NCT00820924 [Pestrin et al., 2012], DETECT III) or epidermal growth factor receptor (NCT00820924; Stebbing et al., 2013). However, no clear responses to therapy were observed in both parts of the NCT00820924 trial, thereby demonstrating that these types of investigations are still in their infancy and require increased patient cohorts in future studies. There are several reasons potentially underlying the lack of evidence on clinical utility for CTCs that can be addressed by gaining insight into the developmental stage in which research on CTCs is currently ongoing. In the development of new technologies, several developmental phases can be distinguished, ranging from technical validation to identification, clinical validation, and clinical utility. For such development, it is expected that the amount of research performed in each of these phases follows some sort of trend over time, with decreasing numbers of studies as the technology proceeds in the developmental process. In classifying CTCs according to these developmental phases, it is
expected that the amount of research performed would follow a similar trend over time, in which a substantial number of studies are in the early developmental phases, and the number of studies is expected to decrease the further CTCs proceed in this developmental process. However, this trend does not exist for CTCs in general in mBC. The number of studies investigating these biomarkers increases in the first phases of the developmental process until the phase in which the ability to detect biomarkers in blood is addressed. A substantial number of studies focus on basic predictive, prognostic, and observational research, whereas only a few studies focus on technical and clinical validation. This may be explained by the fact that most studies have small sample or population sizes, resulting in potentially underpowered studies that might complicate further progression in the development process. Most studies on CTCs focus on enumerating or characterizing CTCs and try to link specific concentrations of CTCs or genetic abnormalities to potential outcome measures in terms of survival (Berghuis, Koffijberg, Prakash, Terstappen, & IJzerman, 2017). In summary, a substantial amount of studies investigate CTCs for their ability to predict survival, whereas technical and prognostic validation still receive insufficient attention. Furthermore, there still is a lack of studies focusing on addressing the clinical utility of CTCs, by which the additional clinical value still seems to be limited according to the investigated evidence (Berghuis et al., 2017). However, although the clinical utility is not yet sufficiently addressed, it may not be realistic or feasible to investigate all potential CTC applications in clinical studies because technology is very rapidly developing. For several applications for CTCs, it might be initially interesting to address their potential in modelbased analyses.
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REFERENCES
CONCLUSIONS AND FUTURE PERSPECTIVES Although the prognostic validity of CTCs has been confirmed in both metastatic and nonmetastatic BC, their enumeration has not been implemented in routine clinical practice, mainly due to the unaddressed utility of these tumor cells. However, before the clinical utility of using CTCs can be further addressed in clinical trials, extensive studies are required to investigate the role of CTCs as therapy decision tools. To this end, it is highly important to initially solve some basic challenges regarding the research on CTCs: improving the sensitivity and specificity of CTC detection technologies, thereby enabling the enumeration and isolation of all CTC subtypes (i.e., epithelial, mesenchymal, with epithelial mesenchymal plasticity, etc.); improving bio-banking methods for these tumor cells; developing appropriate high-resolution approaches for molecular analysis on single CTCs necessary to investigate the cancer heterogeneity; and improving the in vitro culture of these tumor cells to establish CTC-derived cell lines that could support the identification of future therapy targets. Although the clinical utility can only be addressed after other challenges in detecting CTCs are solved, their potential clinical value can already be addressed within model-based analyses.
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breast cancer patients: The DETECT study. Breast Cancer Research, 14, R118. Available from https://doi.org/ 10.1186/bcr3243. Nakamura, S., Yagata, H., Ohno, S., Yamaguchi, H., Iwata, H., Tsunoda, N., . . . Suzuki, E. (2010). Multi-center study evaluating circulating tumor cells as a surrogate for response to treatment and overall survival in metastatic breast cancer. Breast Cancer, 17(3), 199 204. Available from https://doi.org/10.1007/s12282-009-0139-3. Pantel, K., & Alix-Panabie`res, C. (2010). Circulating tumour cells in cancer patients: Challenges and perspectives. Trends in Molecular Medicine, 16(9), 398 406. Available from https://doi.org/10.1016/j.molmed.2010.07.001. Pestrin, M., Bessi, S., Puglisi, F., Minisini, A. M., Masci, G., Battelli, N., . . . Leo, A. D. (2012). Final results of a multicenter phase II clinical trial evaluating the activity of single-agent lapatinib in patients with HER2-negative metastatic breast cancer and HER2-positive circulating tumor cells: A proof-of-concept study. Breast Cancer Research and Treatment, 134(1), 283 289. Available from https://doi.org/10.1007/s10549-012-2045-1. Pierga, J.-Y., Bidard, F.-C., Autret, A., Petit, T., Andre, F., Dalenc, F., . . . Viens, P. (2017). Circulating tumour cells and pathological complete response: Independent prognostic factors in inflammatory breast cancer in a pooled analysis of two multicentre phase II trials (BEVERLY-1 and -2) of neoadjuvant chemotherapy combined with bevacizumab. Annals of Oncology, 28(1), 103 109. Available from https://doi.org/10.1093/annonc/ mdw535. Pierga, J.-Y., Bidard, F.-C., Mathiot, C., Brain, E., Delaloge, S., Giachetti, S., . . . Marty, M. (2008). Circulating tumor cell detection predicts early metastatic relapse after neoadjuvant chemotherapy in large operable and locally advanced breast cancer in a phase II randomized trial. Clinical Cancer Research, 14(21), 7004 7010. Available from https://doi.org/10.1158/1078-0432.CCR-08-0030. Pierga, J.-Y., Hajage, D., Bachelot, T., Delaloge, S., Brain, E., Campone, M., . . . Bidard, F.-C. (2012). High independent prognostic and predictive value of circulating tumor cells compared with serum tumor markers in a large prospective trial in first-line chemotherapy for metastatic breast cancer patients. Annals of Oncology, 23 (3), 618 624. Available from https://doi.org/10.1093/ annonc/mdr263. Rack, B., Schindlbeck, C., Ju¨ckstock, J., Andergassen, U., Hepp, P., Zwingers, T., . . . Janni, W. (2014). Circulating tumor cells predict survival in early average-to-high risk breast cancer patients. Journal of the National Cancer Institute, 106(5). Available from https://doi.org/ 10.1093/jnci/dju066. Riethdorf, S., Mu¨ller, V., Zhang, L., Rau, T., Loibl, S., Komor, M., . . . Pantel, K. (2010). Detection and HER2 expression of circulating tumor cells: Prospective
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monitoring in breast cancer patients treated in the neoadjuvant geparquattro trial. Clinical Cancer Research, 16(9), 2634 2645. Available from https://doi.org/ 10.1158/1078-0432.CCR-09-2042. Schramm, A., Friedl, T. W., Schochter, F., Scholz, C., de Gregorio, N., Huober, J., & Fehm, T. (2016). Therapeutic intervention based on circulating tumor cell phenotype in metastatic breast cancer: Concept of the DETECT study program. Archives of Gynecology and Obstetrics, 293 (2), 271 281. Available from https://doi.org/10.1007/ s00404-015-3879-7. Smerage, J. B., Barlow, W. E., Hortobagyi, G. N., Winer, E. P., Leyland-Jones, B., & Srkalovic, G. (2014). Circulating tumor cells and response to chemotherapy in metastatic breast cancer: SWOG S0500. Journal of Clinical Oncology, 32, 3483 3489. Available from https://doi.org/10.1200/jco.2014.56.2561.
Stebbing, J., Payne, R., Reise, J., Frampton, A. E., Avery, M., Woodley, L., . . . Coombes, R. C. (2013). The efficacy of lapatinib in metastatic breast cancer with HER2 non-amplified primary tumors and EGFR positive circulating tumor cells: A proof-of-concept study. PLoS One, 8(5), e62543. Available from https://doi. org/10.1371/journal.pone.0062543. Vishnoi, M., Peddibhotla, S., Yin, W., Scamardo, A. T., George, G. C., Hong, D. S., & Marchetti, D. (2015). The isolation and characterization of CTC subsets related to breast cancer dormancy. Scientific Reports, 5, 17533. Available from https://doi.org/10.1038/srep17533. Wallwiener, M., Hartkopf, A. D., Baccelli, I., Riethdorf, S., Schott, S., Pantel, K., . . . Fehm, T. N. (2013). The prognostic impact of circulating tumor cells in subtypes of metastatic breast cancer. Breast Cancer Research and Treatment, 137(2), 503 510. Available from https://doi. org/10.1007/s10549-012-2382-0.
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28 Role of Circulating Tumor Cells in Breast Cancer Patients Rita Lampignano1,2, A.M. Sofie Berghuis3, Tanja Fehm1,2 and Hans Neubauer1,2 1
Department of Obstetrics and Gynecology, University Hospital of Duesseldorf, Duesseldorf, Germany 2 Medical Faculty of the Heinrich-Heine, University of Duesseldorf, Duesseldorf, Germany 3 Department of Health Technology and Services Research, Faculty of Behavioural, Management and Social Sciences, Technical Medical Centre, University of Twente, Enschede, The Netherlands
ABBREVIATIONS (m)BC BCSS CTC (D)D/MFS DTC EpCAM Her2/neu MRD OS PFS
(metastatic) breast cancer breast cancer-specific survival circulating tumor cell (distant) disease/metastasisfree survival disseminated tumor cells epithelial cell adhesion molecule human epidermal growth receptor 2 minimal residual disease overall survival progression-free survival
Oncogenomics DOI: https://doi.org/10.1016/B978-0-12-811785-9.00028-4
CELLS RELEASED BY SOLID TUMORS AS “LIQUID BIOPSY” IN BREAST CANCER Despite advances in early detection and identification of optimal treatment strategies, 20% 30% of breast cancer (BC) patients still face long-term systemic relapses, mainly due to the persistence of a minimal residual disease (MRD; Banys-Paluchowski, Krawczyk, MeierStiegen, & Fehm, 2016). The prognostic importance of MRD has been mainly assessed by analyzing patients bone marrow biopsies. In one of the most extensively pooled analyses, Braun and colleagues (2005) reported the
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28. ROLE OF CIRCULATING TUMOR CELLS IN BREAST CANCER PATIENTS
correlation between the presence of disseminated tumor cells (DTCs) in bone marrow and shorter patient survival rates in 4,700 early BC patients. Despite such promising investigations on DTCs, their analysis is often hampered by the invasiveness of the bone marrow aspiration. However, in the past decade, numerous studies have reported a concordant positivity rate of DTCs and circulating tumor cells (CTCs) in the peripheral blood of BC patients (66% 94%, P , .3 to P , .001; Bidard, Proudhon, & Pierga, 2016), thereby indicating CTCs as a “liquid” and easily accessible alternative to DTCs (Pantel & Alix-Panabie`res, 2010). The prognostic role of CTCs has been investigated in numerous tumor entities, and their presence correlates with poor clinical outcomes in breast, colorectal, lung, and prostate cancers (Cabel et al., 2017; Cohen et al., 2008; Cristofanilli et al., 2004; de Bono et al., 2008). To date, BC is the most extensively studied tumor in terms of the clinical role of CTCs, which are observed in 40% 80% of metastatic patients (Banys-Paluchowski et al., 2016). In the discussion regarding the clinical relevance of CTCs as a “liquid biopsy,” two main topics can be distinguished: (1) the clinical validity of CTC enumeration as a predictive and prognostic biomarker, and as a treatment decision tool; and (2) the clinical utility of the CTCs’ phenotypic and molecular characteristics as targetable therapeutic biomarkers.
CLINICAL VALIDITY OF CTC COUNTS AND DYNAMIC CHANGES IN METASTATIC BREAST CANCER The clinical validity for disease prognosis of CTCs in metastatic breast cancer (mBC) was first investigated in a multicenter study by Cristofanilli and colleagues (2004) on 177 patients, blood samples collected before the
beginning of a new therapy (baseline) and at the first follow-up visit. CTCs in the blood were determined with the U.S. Food and Drug Administration-approved EpCAM-based CellSearch system, and the detection of $ 5 CTCs/7.5 mL blood was shown to be prognostic for patients shortened progression-free survival (PFS) and overall survival (OS). On the contrary, patients with ,5 CTCs/7.5 mL blood had longer survival times. The cut-off value of $ 5 CTCs/7.5 mL blood to predict patients poor outcomes was further validated by Hayes and colleagues (2006) on the same cohort of patients, focusing on both baseline and follow-up blood samples. The role of CTC enumeration in the management of mBC patients has been further investigated in more than 20 subsequent studies, mainly by utilizing the CellSearch system (Banys-Paluchowski et al., 2016; Cabel et al., 2017; Khatami et al., 2017). In Table 28.1, major studies evaluating the prognostic and/or predictive role of CTCs in mBC are reported. Interestingly, except for human epidermal growth factor receptor 2 (Her2)/neu-positive mBC patients, the presence of $ 5 CTCs/ 7.5 mL blood was identified as a prognostic factor independently of the molecular subtype of primary tumors (Banys-Paluchowski et al., 2016; Wallwiener et al., 2013). Besides the prognostic validity of CTCs in mBC, their role in monitoring patients treatment responses has been assessed as well. Most of the published studies concluded that dynamic changes in the CTC frequency may be utilized to monitor mBC patients responses to therapies and to predict clinical outcomes (Budd et al., 2006; Cristofanilli et al., 2004; Hayes et al., 2006; Liu et al., 2009). In detail, it was reported that mBC patients with high baseline CTC count but decreased CTC numbers after the first line of chemotherapy have better PFS and OS than patients with persistently high CTC numbers (Cristofanilli et al., 2004; Hayes et al., 2006). Additionally, the presence of persistent CTCs after therapies was compared to
IV. PERSPECTIVES IN BREAST CANCER GENOMICS
CLINICAL VALIDITY OF CTC COUNTS AND DYNAMIC CHANGES IN METASTATIC BREAST CANCER
TABLE 28.1
385
Association of CTC Count and Survival in mBC Patients
Study
Patient Cohort
Association
Bidard et al. (2014)
911
OS, PFS
Budd et al. (2006)
138
OS
Cristofanilli et al. (2004)
177
OS, PFS
Dawood et al. (2008)
185
OS
De Giorgi et al. (2009)
115
OS
Giordano et al. (2012)
517
PFS, OS
Giuliano et al. (2011)
235
PFS, OS
Hayes et al. (2006)
177
PFS, OS
Mu¨ller et al. (2012)
254
OS
Nakamura et al. (2010)
107
OS
Pierga et al. (2012)
267
PFS, OS
Wallwiener et al. (2013)
486
PFS, OS
OS, overall survival; PFS, progression-free survival.
imaging approaches conventionally utilized for treatment monitoring and was found to accurately reflect patients reactions to therapies (Budd et al., 2006; De Giorgi et al., 2009). However, the clinical role of CTC dynamics as therapy decision tools has not been demonstrated to date, and it is currently being evaluated in clinical trials. Within the SWOG S0500 study, focusing on mBC patients with persistently .5 CTCs/7.5 mL blood after the first line of chemotherapy, Smerage and colleagues (2014) evaluated the effects of an early switch to a second-line chemotherapy on patients outcomes. This treatment switch was hypothesized to greatly improve patients’ OS compared to the standard imaging-based management. However, no survival differences were observed between the two subgroups of patients. The negative results of the SWOG study could be explained by its design and concepts. It is, in fact, unlikely that a secondline chemotherapy may have a significant effect on CTCs which exhibit a primary
resistance to a first-line chemotherapy (AlunniFabbroni, Mu¨ller, Fehm, Janni, & Rack, 2014; Bidard & Pierga, 2015). This is even more true when the type of therapy is not tailored to the genotype or phenotype of the CTCs, thus emphasizing the necessity to identify specific biomarkers in/on CTCs that could be targeted by novel drugs. Nevertheless, detecting persistent CTCs may still serve to identify therapyresistant patients who demand different cures. The role of CTCs in selecting the optimal third-line chemotherapy is being investigated in the ongoing CirCe01 (NCT01349842), which has a similar approach as the SWOG trial (Helissey et al., 2015). Within this study, patients are enrolled before the start of the third line of chemotherapy and are followed throughout the successive lines of chemotherapy in which CTC counts are additionally determined (Helissey et al., 2015). Furthermore, the STIC-CTC trial (NCT01710605) addresses the utility to treat mBC patients with hormone therapy if they
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28. ROLE OF CIRCULATING TUMOR CELLS IN BREAST CANCER PATIENTS
exhibit a CTC count ,5/7.5 mL blood or with chemotherapy if the CTC count is $ 5/7.5 mL blood (Banys-Paluchowski et al., 2016; Cabel et al., 2017). The trial’s main medical objective is to demonstrate non-inferiority in PFS of the CTC-based strategy compared to the clinicians’ decision, taking into account the criteria usually used in this setting. Last, the DETECT V trial (NCT02344472) is currently investigating CTC dynamics of dual Her2/neu-targeted treatment in combination with chemotherapy or endocrine therapy (Banys-Paluchowski et al., 2016). Despite all these impressive efforts, the actual contribution of CTC changes in selecting appropriate treatments for mBC patients must still be addressed.
CTCs expressing cancer dormancy markers may be involved in the formation of brain metastases (Vishnoi et al., 2015). In addition, mBC patients with $ 100 EpCAM-negative CTCs/mL of blood have a shorter OS (Lustberg et al., 2014). Last, Bulfoni and colleagues (2016) observed worse PFS and OS in mBC patients with CTCs expressing both epithelial and mesenchymal markers (transient phenotype). However, extensive studies on larger cohorts of patients are needed to shed light on the role of the transient/mesenchymal CTCs in the metastatic cascade of different tumor entities.
Clinical Validity of CTCs with Epithelial Mesenchymal Plasticity
The clinical validity of CTCs has been less extensively investigated in nonmetastatic than in metastatic cancers (including breast tumors), mainly due to the lower frequency of CTCs in the nonmetastatic setting. The German SUCCESS trial (NCT02181101) is the first extensive study on early BC patients supporting the prognostic role of CTCs enriched through the CellSearch system (Rack et al., 2014). In 435/2,026 patients enrolled before chemotherapy and in 330/1,493 patients enrolled after chemotherapy, CTCs could be detected and were correlated with short disease-free survival (DFS; P , .0001), distant DFS (DDFS; P , .001), breast cancer-specific survival (BCSS; P , .008), and OS (P , .0002). In this study, numerous predictive CTC cut-off values were investigated (0 vs. $ 1, 0 1 vs. $ 2, and 0 4 vs. $ 5 in 30 mL of blood), and all of them were statistically significant in the correlation with poor clinical outcomes. The worst prognoses were observed in patients with $ 5 CTCs/30 mL blood at baseline. Interestingly, the CTC positivity rate was associated with the nodal status (N 1 19.6% vs. N 22.4%, P , .001) but not with other
In almost all the previously discussed studies, mBC patients prognoses related to the presence of CTCs, which were mainly detected via the EpCAM-based CellSearch system. However, this approach may overlook tumor cells with a plastic epithelial mesenchymal phenotype, resulting from the epithelial-to-mesenchymal transition (Alix-Panabie`res, Mader, & Pantel, 2017). The existence of an EpCAM-low/negative subset of CTCs has been documented for different tumor entities (including breast), and early observations have been published and reviewed elsewhere (Lampignano, Schneck, Neumann, Fehm, & Neubauer, 2017). Even though this fraction of CTCs has not been extensively investigated yet, the first potential correlations with patients clinical outcomes have been reported. Mego and colleagues (2011, 2012) frequently detected brain metastases in mBC patients lacking EpCAM-positive CTCs, suggesting the presence of a highly malignant EpCAM-negative subgroup of tumor cells. In line with these results, EpCAM-negative
CLINICAL VALIDITY OF CTCs IN NONMETASTATIC BREAST CANCER
IV. PERSPECTIVES IN BREAST CANCER GENOMICS
CLINICAL VALIDITY OF CTCS IN NONMETASTATIC BREAST CANCER
TABLE 28.2
387
Association of CTC Count and Survival in Non-mBC Patients
Study
Patient Cohort
Association
Bidard et al. (2010)
115
DDFS, OS
Franken et al. (2012)
404
DDFS, BCSS
Hwang, Bae, Lee, and Kim (2012)
166
OS
Ignatiadis et al. (2007)
444
OS, DFS
a
Janni et al. (2016)
3,173
DFS, DDFS, BCSS, OS
Kuniyoshi et al. (2015)
167
None
Lucci et al. (2012)
302
DFS, OS
Pierga et al. (2017)
152
DFS, OS
Rack et al. (2014)
2,026
DFS, DDFS, BCCS, OS
a
Pooled analysis. BCSS, breast cancer-specific survival; DDFS, distant disease-free survival; DFS, disease-free survival; OS: overall survival; PFS, progression-free survival.
tumor characteristics. This relation between the presence of CTCs in non-mBC patients and poor clinical outcomes could be further confirmed in nearly all the published studies, summarized in Table 28.2 (Banys-Paluchowski et al., 2016; Bidard et al., 2016). The clinical validity of persistent CTCs in non-mBC was mainly investigated in three trials. The correlation between the presence of CTCs after chemotherapy with poor clinical outcomes was reported in the previously mentioned SUCCESS trial (Rack et al., 2014). Subsequently, in the REMAGUS02 study, Bidard and colleagues suggested that the validity of CTCs as therapy monitoring tools in neoadjuvant settings might be restricted to the first years after diagnosis (Bidard et al., 2010; Bidard, Belin, et al., 2013; Bidard, Fehm, et al., 2013). Interestingly, in contrast to those reports, no association between CTC dynamic changes and patientsresponses to treatments could be observed within the GeparQuattro study (Riethdorf et al., 2010), in agreement with previous observations (Pierga et al., 2008). The validity of CTCs as therapy decision tools is currently also being evaluated in non-
mBC in the TREAT CTC trial (NCT01548677). The major aim of this study is to investigate Her2/neu-targeted treatments in patients with initially Her2/neu-negative tumors and detectable Her2/neu-positive CTCs after (neo) adjuvant chemotherapy (Banys-Paluchowski et al., 2016; Cabel et al., 2017). Although the prognostic role of CTCs on non-mBC has been assessed, further research is required given the relatively small patient cohorts compared to the metastatic setting. However, this demand for additional extensive studies is currently limited by the rarity of CTCs in nonmetastatic cancers. One solution to increment the CTC yield may be to increase the analyzed blood volume via appropriate technologies, such as diagnostic leukapheresis, clinically validated in metastatic breast and prostate cancers within the European CTC-Trap consortium (Andree et al., 2017; Fischer et al., 2013).
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CLINICAL UTILITY OF CTC CHARACTERIZATION During the past decade, the development of high-resolution technologies for single cell analysis permitted numerous investigations on phenotypes and genotypes of CTCs, which are of utmost importance to understand the invasion metastasis cascade. Despite all the efforts, there are still no clinical trials focusing on genomic or transcriptomic analysis of these tumor cells. On the contrary, the clinical utility of analyzing the expression of selected biomarkers on CTCs is currently being evaluated. The multicenter trials NCT00820924 and DETECT III (NCT01619111; Bidard et al., 2013; Schramm et al., 2016) have investigated or are still investigating the efficacy of anti-Her2/neu treatments in mBC patients with Her2/neu-negative primary tumors but with CTCs expressing the biomarker Her2/neu (NCT00820924 [Pestrin et al., 2012], DETECT III) or epidermal growth factor receptor (NCT00820924; Stebbing et al., 2013). However, no clear responses to therapy were observed in both parts of the NCT00820924 trial, thereby demonstrating that these types of investigations are still in their infancy and require increased patient cohorts in future studies. There are several reasons potentially underlying the lack of evidence on clinical utility for CTCs that can be addressed by gaining insight into the developmental stage in which research on CTCs is currently ongoing. In the development of new technologies, several developmental phases can be distinguished, ranging from technical validation to identification, clinical validation, and clinical utility. For such development, it is expected that the amount of research performed in each of these phases follows some sort of trend over time, with decreasing numbers of studies as the technology proceeds in the developmental process. In classifying CTCs according to these developmental phases, it is
expected that the amount of research performed would follow a similar trend over time, in which a substantial number of studies are in the early developmental phases, and the number of studies is expected to decrease the further CTCs proceed in this developmental process. However, this trend does not exist for CTCs in general in mBC. The number of studies investigating these biomarkers increases in the first phases of the developmental process until the phase in which the ability to detect biomarkers in blood is addressed. A substantial number of studies focus on basic predictive, prognostic, and observational research, whereas only a few studies focus on technical and clinical validation. This may be explained by the fact that most studies have small sample or population sizes, resulting in potentially underpowered studies that might complicate further progression in the development process. Most studies on CTCs focus on enumerating or characterizing CTCs and try to link specific concentrations of CTCs or genetic abnormalities to potential outcome measures in terms of survival (Berghuis, Koffijberg, Prakash, Terstappen, & IJzerman, 2017). In summary, a substantial amount of studies investigate CTCs for their ability to predict survival, whereas technical and prognostic validation still receive insufficient attention. Furthermore, there still is a lack of studies focusing on addressing the clinical utility of CTCs, by which the additional clinical value still seems to be limited according to the investigated evidence (Berghuis et al., 2017). However, although the clinical utility is not yet sufficiently addressed, it may not be realistic or feasible to investigate all potential CTC applications in clinical studies because technology is very rapidly developing. For several applications for CTCs, it might be initially interesting to address their potential in modelbased analyses.
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REFERENCES
CONCLUSIONS AND FUTURE PERSPECTIVES Although the prognostic validity of CTCs has been confirmed in both metastatic and nonmetastatic BC, their enumeration has not been implemented in routine clinical practice, mainly due to the unaddressed utility of these tumor cells. However, before the clinical utility of using CTCs can be further addressed in clinical trials, extensive studies are required to investigate the role of CTCs as therapy decision tools. To this end, it is highly important to initially solve some basic challenges regarding the research on CTCs: improving the sensitivity and specificity of CTC detection technologies, thereby enabling the enumeration and isolation of all CTC subtypes (i.e., epithelial, mesenchymal, with epithelial mesenchymal plasticity, etc.); improving bio-banking methods for these tumor cells; developing appropriate high-resolution approaches for molecular analysis on single CTCs necessary to investigate the cancer heterogeneity; and improving the in vitro culture of these tumor cells to establish CTC-derived cell lines that could support the identification of future therapy targets. Although the clinical utility can only be addressed after other challenges in detecting CTCs are solved, their potential clinical value can already be addressed within model-based analyses.
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