Clin Transl Oncol DOI 10.1007/s12094-013-1128-8
RESEARCH ARTICLE
Prognostic significance of circulating tumor cells in advanced non-small cell lung cancer patients treated with docetaxel and gemcitabine O. Juan • J. Vidal • R. Gisbert • J. Mun˜oz S. Macia´ • J. Go´mez-Codina
•
Received: 8 July 2013 / Accepted: 22 October 2013 Ó Federacio´n de Sociedades Espan˜olas de Oncologı´a (FESEO) 2013
Abstract Purpose To evaluate the association in the change of circulating tumor cell (CTC) levels and clinical outcomes (PFS and OS) in patients with advanced non-small cell lung cancer (NSCLC) treated homogenously with docetaxel and gemcitabine administered every 2 weeks. Methods We prospectively evaluated 37 patients for CTC levels at baseline and after 2 months of chemotherapy (before third cycle). Detection was carried out with the CellSearch system. Results Nine of the 37 patients (24 %) had C2 CTCs at the baseline determination. Median progression-free survival (PFS) was 4.3 months (95 % CI 2.5–8.3) for patients with CTC 0–1 as compared to 9.4 months (95 % CI 1.2–12.2) for those with CTC C2 (p = 0.3506). Median overall survival (OS) was 8.1 (95 % CI 2.8–16.3) and 12.2 (95 % CI 1.4–12.2) months for patients with 0–1 CTCs and C2 CTCs, respectively (p = 0.7639). Patients with a second CTC quantification were classified as: group 1, CTC = 0–1 at baseline and CTC = 0–1 after second
O. Juan (&) J. Go´mez-Codina Medical Oncology Department, Hospital Universitari I Polite`cnic La Fe, Bulevar Sur s/n, 46026 Valencia, Spain e-mail: [email protected] J. Vidal R. Gisbert Clinical Laboratory, Hospital Arnau de Vilanova, San Clemente 12, 46015 Valencia, Spain J. Mun˜oz Medical Oncology Department, Hospital Dr Peset, Gaspar Aguilar 90, 46017 Valencia, Spain S. Macia´ Medical Oncology Department, Hospital General de Elda, Carretera de Sax 25, 03600 Alicante, Spain
chemotherapy cycle (18 patients); group 2, CTC C2 at baseline and CTC = 0–1 after second determination (5 patients). Median PFS was 7.7 and 9.9 months for group 1 and group 2, respectively (p = 0.4467). Conclusions CTCs C2 at baseline were detected only in 24 % of this group of patients with advanced NSCLC and poor performance status. No significant differences in PFS and OS between patients with or without CTCs at baseline were observed. Keywords Lung cancer Circulating tumor cells Prognosis Non-small cell lung cancer
Introduction Lung cancer is the leading cause of death in developed countries. Less than 15 % of lung cancer patients survive more than 5 years after being diagnosed [1]. Non-small cell lung cancer (NSCLC) is the most frequent histological type and represents 85 % of the cases. The majority of NSCLC patients are diagnosed with locally advanced or metastatic disease, and have a poor prognosis. Platinum-based doublet is considered the standard chemotherapy for advanced NSCLC [2]. However, the risk of adverse effects from this combination is high in patients with a performance status (PS) C2 and elderly patients, and the benefit of platinum-based chemotherapy is questioned. Several studies have suggested the benefit of chemotherapy without platinum in this ‘‘fragile’’ patients group [3–11]. The efficacy of docetaxel plus gemcitabine has been reported to be greater than monotherapy with docetaxel in a phase III trial [10]. Moreover, in a retrospective study, the combination of docetaxel and gemcitabine showed similar efficacy and tolerability in elderly patients (C70 years) and young patients [11].
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Unfortunately, the benefit of chemotherapy in this group of elderly and poor PS NSCLC patients is modest, reduced to a small percentage of patients and associated with toxic effects that impact their quality of life. For these reasons, it would be desirable to identify prognosis factors or early predictors of response to justify the administration of chemotherapy to such patients. Recently, circulating tumor cells (CTC) enumeration has shown to be a potential biomarker of prognosis and serve as early marker of response to chemotherapy [12]. Although, the presence of tumor cells in patients’ circulation is known for over a century, it has only been in the last decade, with the development of new CTC detection and analysis technologies, that interest in research in this field has been renewed. However, the variability of these methods and the lack of reproducibility across different laboratories may present difficulty in the interpretation of the results. Several CTC detection methods have been developed, but the CellSearchTM system (Veridex LLC), an immunomagnetic purification method, is the only validated assay approved by the US Food and Drug Administration (FDA) [13]. An increasing number of studies in breast, colon and prostate cancer have correlated CTCs with clinical outcome parameters, such as progression-free survival (PFS) and overall survival (OS), where changes in the number of CTCs during treatment revealed to be a predictive biomarker of response to treatment [14– 18]. However, fewer studies have explored the clinical value of CTCs in lung cancer, and the meaning of CTCs as prognostic or predictive markers in NSCLC patients is not well-established. Recently, Krebs and colleagues reported data from a study in NSCLC patients using CellSearch for CTC enumeration [19]. Twenty-one of the 101 stage III–IV NSCLC patients included in this prospective study had C2 CTCs at baseline. NSCLC patients with \5 CTCs before chemotherapy had better OS and PFS as compared to patients with C5 CTCs. Similarly, patients with \5 CTCs or those with a reduced number of CTC after one chemotherapy cycle exhibited more favorable clinical outcomes. The purpose of this study is to evaluate the association in the change of CTC number and clinical outcomes (PFS and OS) in patients with advanced NSCLC treated homogenously with docetaxel and gemcitabine administered every 2 weeks. The CellSearch system was used to confirm the feasibility of this method to evaluate the number of CTC in NSCLC patients.
Patients and methods Study design Patients with advanced NSCLC and who were included in two multi-institutional phase II trials were eligible for the
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CTC study. These phase II trials were similar, except for the NSCLC population addressed; one of them included elderly patients ([70 years) with PS 0 or 1, while the other included PS 2 patients of any age. The Institutional Review Board approved both protocols and the CTC sub-study, and all patients provided written informed consent. Common inclusion criteria were: cytological or histological diagnosis of advanced NSCLC (stage IIIB with pleural effusion or stage IV); at least one bidimensionally measurable lesion by the RECIST criteria located outside a previously irradiated field; life expectancy C12 weeks; adequate bone marrow [absolute neutrophil count (ANC) C2,000/dL, platelet count C1,00,000/dL, hemoglobin C10 g/dL], renal function (serum creatinine B1.59 upper normal limit), and hepatic function [bilirubin B1.59 upper normal limit, serum glutamate oxaloacetate transaminase (SGOT), and serum glutamate pyruvate transaminase (SGPT) B2.59 upper normal limit]. Patients with treated stable brain metastases were also eligible. Additional informed consent for a CTC analysis was required. In both phase II trials, patients received treatment with: docetaxel at a dose of 50 mg/m2 and a 60-min intravenous (IV) infusion; gemcitabine 2,000 mg/m2, 30-min IV infusion, both treatments on days 1 and 14. Cycles were repeated every 28 days on an outpatient basis. Blood samples were collected for the CTC analysis within 7 days before commencing treatment (baseline) and after two chemotherapy cycles. CTC analysis Before starting chemotherapy (basal) and after 2 months of chemotherapy (before the third cycle), 7.5 mL of whole blood was drawn. Samples were stored in CellSave preservative tubes at room temperature and were analyzed within 72 h after collection. CTC quantification was performed using the semiautomated CellSearch system (Veridex), which has demonstrated its reproducibility across different laboratories and consists in two instruments: the CellTracks AutoPrep system and the CellSpotter analyzer [20]. During the AutoPrep procedure, a special mix of ferrofluids coated with epithelial cell-specific anti-EpCAM antibodies was automatically added to immunomagnetically enriched epithelial cells, separated in a magnetic field. The enriched samples were then stained with fluorescent nuclear 40 ,6diamidino-2-phenylindole (DAPI) and with fluorescent monoclonal antibodies specific for leukocytes (CD45) and epithelial cells (cytokeratins 8, 18, and 19). After a second magnetic separation, the excess staining reagents were aspirated. Finally, cells were resuspended in the Magnest device, which consists in a chamber and two magnets that orient immunomagnetically labeled cells for their
Clin Transl Oncol
subsequent analysis in the CellSpotter analyzer, a fourcolor semi-automated fluorescence microscope. The images obtained by scanning the entire surface were automatically captured and presented in a web-enabled browser to be finally identified and selected by the operator. The criteria used to classify each cell as CTC are: more than 4 lm in diameter; a round or oval morphology; a visible DAPI-positive nucleus present in the CK-positive cytoplasm; and negative staining for CD45. The results are expressed in CTCs/7.5 mL of blood. CTC enumeration was centralized in the Cellular Oncology Laboratory at the Hospital Arnau de Vilanova of Valencia. Statistical analysis A widely accepted C2 CTC in 7.5 mL of blood cut-off was used in this study according to the first CellSearch enumeration study, in which 20 % of patients with metastatic lung cancer had C2 CTCs in 7.5 mL of blood [13]. Analyses were carried out using SPSS version 15.0 (SPSS Inc., Chicago, IL). All the statistical tests were performed two-sided and at the 5 % level of significant difference. Differences of rates between groups were compared with a Chi-square test. Differences between groups with continuous variables were tested by a Student’s t test or by a non-parametric Mann–Whitney U test. The Kaplan–Meier method was used to estimate the probability of PFS and overall survival (OS) over time. PFS was calculated from the baseline blood drawn to any disease progression or the last visit date if there was no progression, and OS was calculated from the baseline blood drawn to death from any cause or to the last followup date if the patient was living at the end of the follow-up. The long-rank test was used to assess the differences between groups in the univariate analysis.
Results Blood samples for CTC enumeration were taken in 37 NSCLC patients treated with docetaxel and gemcitabine administered every 2 weeks participating in two independent clinical trials conducted in ten hospitals from the Mediterranean region in Spain to evaluate chemotherapy combination in fragile patients. One of them included 50 elderly patients (CPNM-ANC-07), and the other 75 patients with PS 2 (CNMP-PS2-07). The CTC sub-study was optional and included only four hospitals. Of the 37 patients enrolled in the CTC study, 20 belonged to the elderly patients study and 17 to the PS 2 study. No statistically significant differences were observed between the
Table 1 Patients’ characteristics and CTCs detection Characteristics
n
%
C2 CTCs
%
0–1 CTCs
%
Age Mean
71 years
Range
44–85
Gender Male
33
89.2
25
89.3
8
88.9
Female
4
10.8
3
10.7
1
11.1
Histology Adenocarcinoma
14
37.8
10
35.7
4
48.0
Squamous cell carcinoma
14
37.8
12
42.9
2
22.2
Poorly differentiated
3
8.1
1
3.6
2
22.6
Other
6
16.2
5
17.9
1
11.1
Response to chemotherapy Partial response
7
18.9
5
17.9
2
22.2
Stable disease Progression disease
14 6
37.8 16.2
12 5
42.9 17.9
2 1
22.2 11.1
No evaluable
10
27
6
21.4
4
44.4
whole population of the studies and the subgroup of patients of the CTC study. The characteristics of the 37 patients assessable for CTC enumeration are summarized in Table 1. Twenty patients (54 %) had PS 0–1 and 17 (46 %) had PS 2. Consistent with other studies carried out in Spain, 37.8 % of all the patients had squamous cell histology. Overall, the presence of C2 CTCs at the baseline determination was detected in 9 of the 37 patients (24 %). Eight patients (21.6 %) had only one CTC and 20 patients had a negative CTC count at baseline. The mean number of CTCs isolated was 1.1 (range 0–9). Presence of C2 CTCs at baseline did not correlate with gender, histological subtype or response to chemotherapy (Table 1). At the time of the analysis, 14 (50 %) of the 28 patients with 0–1 CTCs and 4 (44.5 %) of the nine patients with C2 CTCs had died. PFS and OS were analyzed according to the baseline CTC number. No significant differences in PFS and OS were found in the patients with CTC 0–1 as compared with those with CTC C2. The median PFS was 4.3 months (95 % CI 2.5–8.3) and 9.4 months (95 % CI 1.2–12.2), respectively (log-rank test p = 0.3506, Fig. 1). The median OS was 8.1 (95 % CI 2.8–16.3) and 12.2 (95 % CI 1.4–12.2) months for patients with 0–1 CTCs and C2 CTCs, respectively (log-rank test p = 0.7639, Fig. 2). In 23 patients (62.2 %), a second blood sample was drawn after the second chemotherapy cycle (before the fifth dose of docetaxel and gemcitabine). Patients were
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Clin Transl Oncol Fig. 1 Progression-free survival according to CTC at baseline
Fig. 2 Overall survival according to CTC at baseline
classified according to the baseline and second determinations in two subgroups: group 1, CTC = 0–1 at baseline and CTC = 0–1 after the second chemotherapy cycle (18 patients); group 2, CTC C2 at baseline and CTC = 0–1 after the second determination (5 patients). No differences in age, gender and histologic subtype were observed between groups. In group 1, four patients (22.2 %) obtained a partial response and 11 patients (61 %) showed stable disease (an 83 % disease control rate). In group 2, two patients (40 %) had a partial response and four other patients (40 %) showed stable disease (an 80 % disease control rate). CTC negativization from the baseline to the second determination was better associated with PFS and OS, but there were no statistically significant differences (p value of 0.4467 and 0.4682, respectively). The median PFS was 7.7 and 9.9 months for the patients in group 1 and group 2, respectively (Fig. 3). The median OS for the group 1 patients was 10.1 months, while the median OS for group 2 was not reached at the time of the analysis (Fig. 4).
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Discussion The current multi-institutional study has evaluated the prognosis significance of the CTC levels detected by the CellSearch system in 37 patients with advanced NSCLC treated homogenously with the chemotherapy combination of docetaxel and gemcitabine. Our results demonstrate that 24 % of patients had C2 CTCs at the baseline enumeration. The mean number of isolated CTS was 1.1 (range 0–9) and only three patients (8 %) had C5 CTCs before chemotherapy. Surprisingly unlike other studies, a high baseline CTC enumeration was associated with better PFS and OS, although no statistically significant differences were observed. Median PFS and OS were 9.4 vs. 4.3 months (p = 0.3506) and 12.2 vs. 8.1 months (p = 0.7581) for the patients with C2 CTCs and 0–1 CTCs, respectively. However, the group of patients with a high baseline CTC and whose CTC number decreased with chemotherapy to 0–1 CTCs had a better prognosis than those patients with low levels at baseline and after chemotherapy. The median
Clin Transl Oncol Fig. 3 Progression-free survival according to CTC changes along the treatment
Fig. 4 Overall survival according to CTC changes along the treatment
OS for the last group was 10.1 months, but it was not reached for the first group at the time of the analysis. Many different methods have been developed to detect CTCs in the blood of cancer patients and show variable sensitivity and specificity. The two main approaches used are morphologic-based isolation [OncoQuickTM, isolation by size of Epithelial Tumor Cells (ISET)] [21, 22], and immunological approaches (CellSearchTM, CTC-chip) [20, 23]. Nevertheless, only the CellSearch system, a semiautomated immunomagnetic system, has shown sufficient level of robust and reliable validation to be approved by the US FDA. The results from various studies that have used the CellSearch platform have demonstrated the prognosis significance of the CTC level in metastatic breast, colorectal and castrate-resistant prostate cancers [16, 17, 24]. In these studies, different cut-offs have been used to establish the prognosis significance of CTC numbers. Thus, in patients with metastatic breast cancer and castrate-resistant prostate cancer, C5 CTCs per 7.5 mL of blood before
treatment is used as an independent predictor of worse PFS or OS, whereas a cut-off of C3 CTCs is set in metastatic colon cancer. The reasons for using these cut-offs are not clear, but can be explained because the number of patients with C5 CTCs is higher in patients with metastatic breast (26 %) and prostate cancers (41 %) than with colorectal cancer (17 %) [13]. Few studies have been performed in NSCLC patients using the CellSearch system, Krebs et al. investigated the prognosis significance of CTCs in NSCLC patients with stage IIIA, IIIB and IV [19]. In a univariate analysis, patients with \5 CTCs had a higher PFS and OS than those with C5 CTCs: 6.8 and 8.1 months vs. 2.4 and 4.3 months, respectively. In a multivariate analysis, CTC remained as the independent prognosis factor for PFS and OS. Of the total population, 21 % of patients had a positive baseline CTC count (C2 CTCs). The proportion of patients with C2 CTCs was higher for stage IV disease (32 %) than for stages IIIB (7 %) and IIIA (0 %). Our study only included patients with stage IV disease and showed that the
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number of patients with C2 CTCs at baseline is slightly lower (9 of 37 patients, 24 %) than in the study of Krebs et al. [19]. Likewise, the range of CTC number in our study is narrower (0–9) than in the study of Krebs et al. (0–146) [19]. There is no clear explanation for these low CTC levels in our population. Perhaps the patients included in our study had a more advanced stage IV disease and greater tumor burden (PS 2); hence, malignant CTCs lose epithelial cell adhesion molecules (EpCAM) and cytokeratin, which is indicative of an epithelial–mesenchymal transition (EMT). This means the assays which target epithelial cells in blood, such as CellSearch miss the detection of the most invasive tumor cells [25, 26]. It has been recently observed that metastatic breast cancer with unfavorable prognostic factors (triple negative, brain metastases, etc.) had undetectable CTCs, suggesting that an underestimation of CTC number by CellSearch may be due to the EMT of the CTCs [27]. This might explain why the poor prognosis for the NSCLC patients (PS 2 and elderly) included in our study had fewer CTCs, as detected by CellSearch analysis and, unlike other studies, the presence of C2 CTCs did not correlate with a worse prognosis. Variation in the CTC number with chemotherapy was analyzed. CTC negativization from baseline to the second determination was associated with better PFS and OS when comparing patients with negative CTCs at baseline and after chemotherapy, although there were no statistically significant differences. These results agree with other studies in NSCLC and with other tumors in which the persistence of a high CTC number is associated with a poor prognosis [19, 28, 29]. This study has focused on the prognosis significance of CTC enumeration at baseline or on their change with chemotherapy in advanced NSCLC patients. However, several issues remain to be clarified: the EpCAM and CK used to detect tumor cells are not specific and can be expressed aberrantly in other cells; moreover, the loss of EpCAM and CK in tumor cells has been described as an epithelial–mesenchymal transition; and the majority of studies include a heterogeneous population, which makes the comparison among them difficult. In addition to CTC enumeration, the molecular characterization of these cells can provide knowledge on the genotypic and phenotypic characteristics of the tumor without the need of invasive biopsy. The determination of the epidermal growth factor receptor (EGFR) mutational status is particularly interesting, but the small number of patients in whom CTCs are isolated by CellSearch system hampers this molecular characterization. In one study, using CTC-chip, circulating tumor cells were identified in all 27 patients with analyzed NSCLC, and with a median of 74 cells per milliliter (mean 133, range 5–771) [30]. The Scorpion Amplification Refractory Mutation System (SARMS) assay identified
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EGFR mutations in 17 of the 18 CTC specimens (94 %). Among the 12 patients for whom specimens of the primary tumor and CTCs were available for the analysis, genotyping of CTCs gave a sensitivity of 92 % (in 11 of the 12 patients). In addition to the primary activating mutation, T790M-associated resistance to tyrosine kinase inhibitors was detected in CTCs. Serial CTC analysis can allow the molecular characterization of the cancer during the treatment without the need for invasive biopsy. In conclusion, CTC enumeration has demonstrated its clinical usefulness in various tumors, including NSCLC. The use of CellSearch system, complemented by new technologies, opens up the way to understand the metastasis biology and enables the molecular characterization of CTCs without the need of biopsy to improve the therapeutic and clinical management of NSCLC patients. Acknowledgments This work was supported in part by a grant from Sanofi Spain. We thank Elena Aristoy for reviewing the English of the manuscript and Vicente Caran˜ana for helpful assistance in the preparation of the figures. Conflict of interest of interest.
The authors declare that they have no conflict
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RESEARCH ARTICLE
Prognostic significance of circulating tumor cells in advanced non-small cell lung cancer patients treated with docetaxel and gemcitabine O. Juan • J. Vidal • R. Gisbert • J. Mun˜oz S. Macia´ • J. Go´mez-Codina
•
Received: 8 July 2013 / Accepted: 22 October 2013 Ó Federacio´n de Sociedades Espan˜olas de Oncologı´a (FESEO) 2013
Abstract Purpose To evaluate the association in the change of circulating tumor cell (CTC) levels and clinical outcomes (PFS and OS) in patients with advanced non-small cell lung cancer (NSCLC) treated homogenously with docetaxel and gemcitabine administered every 2 weeks. Methods We prospectively evaluated 37 patients for CTC levels at baseline and after 2 months of chemotherapy (before third cycle). Detection was carried out with the CellSearch system. Results Nine of the 37 patients (24 %) had C2 CTCs at the baseline determination. Median progression-free survival (PFS) was 4.3 months (95 % CI 2.5–8.3) for patients with CTC 0–1 as compared to 9.4 months (95 % CI 1.2–12.2) for those with CTC C2 (p = 0.3506). Median overall survival (OS) was 8.1 (95 % CI 2.8–16.3) and 12.2 (95 % CI 1.4–12.2) months for patients with 0–1 CTCs and C2 CTCs, respectively (p = 0.7639). Patients with a second CTC quantification were classified as: group 1, CTC = 0–1 at baseline and CTC = 0–1 after second
O. Juan (&) J. Go´mez-Codina Medical Oncology Department, Hospital Universitari I Polite`cnic La Fe, Bulevar Sur s/n, 46026 Valencia, Spain e-mail: [email protected] J. Vidal R. Gisbert Clinical Laboratory, Hospital Arnau de Vilanova, San Clemente 12, 46015 Valencia, Spain J. Mun˜oz Medical Oncology Department, Hospital Dr Peset, Gaspar Aguilar 90, 46017 Valencia, Spain S. Macia´ Medical Oncology Department, Hospital General de Elda, Carretera de Sax 25, 03600 Alicante, Spain
chemotherapy cycle (18 patients); group 2, CTC C2 at baseline and CTC = 0–1 after second determination (5 patients). Median PFS was 7.7 and 9.9 months for group 1 and group 2, respectively (p = 0.4467). Conclusions CTCs C2 at baseline were detected only in 24 % of this group of patients with advanced NSCLC and poor performance status. No significant differences in PFS and OS between patients with or without CTCs at baseline were observed. Keywords Lung cancer Circulating tumor cells Prognosis Non-small cell lung cancer
Introduction Lung cancer is the leading cause of death in developed countries. Less than 15 % of lung cancer patients survive more than 5 years after being diagnosed [1]. Non-small cell lung cancer (NSCLC) is the most frequent histological type and represents 85 % of the cases. The majority of NSCLC patients are diagnosed with locally advanced or metastatic disease, and have a poor prognosis. Platinum-based doublet is considered the standard chemotherapy for advanced NSCLC [2]. However, the risk of adverse effects from this combination is high in patients with a performance status (PS) C2 and elderly patients, and the benefit of platinum-based chemotherapy is questioned. Several studies have suggested the benefit of chemotherapy without platinum in this ‘‘fragile’’ patients group [3–11]. The efficacy of docetaxel plus gemcitabine has been reported to be greater than monotherapy with docetaxel in a phase III trial [10]. Moreover, in a retrospective study, the combination of docetaxel and gemcitabine showed similar efficacy and tolerability in elderly patients (C70 years) and young patients [11].
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Unfortunately, the benefit of chemotherapy in this group of elderly and poor PS NSCLC patients is modest, reduced to a small percentage of patients and associated with toxic effects that impact their quality of life. For these reasons, it would be desirable to identify prognosis factors or early predictors of response to justify the administration of chemotherapy to such patients. Recently, circulating tumor cells (CTC) enumeration has shown to be a potential biomarker of prognosis and serve as early marker of response to chemotherapy [12]. Although, the presence of tumor cells in patients’ circulation is known for over a century, it has only been in the last decade, with the development of new CTC detection and analysis technologies, that interest in research in this field has been renewed. However, the variability of these methods and the lack of reproducibility across different laboratories may present difficulty in the interpretation of the results. Several CTC detection methods have been developed, but the CellSearchTM system (Veridex LLC), an immunomagnetic purification method, is the only validated assay approved by the US Food and Drug Administration (FDA) [13]. An increasing number of studies in breast, colon and prostate cancer have correlated CTCs with clinical outcome parameters, such as progression-free survival (PFS) and overall survival (OS), where changes in the number of CTCs during treatment revealed to be a predictive biomarker of response to treatment [14– 18]. However, fewer studies have explored the clinical value of CTCs in lung cancer, and the meaning of CTCs as prognostic or predictive markers in NSCLC patients is not well-established. Recently, Krebs and colleagues reported data from a study in NSCLC patients using CellSearch for CTC enumeration [19]. Twenty-one of the 101 stage III–IV NSCLC patients included in this prospective study had C2 CTCs at baseline. NSCLC patients with \5 CTCs before chemotherapy had better OS and PFS as compared to patients with C5 CTCs. Similarly, patients with \5 CTCs or those with a reduced number of CTC after one chemotherapy cycle exhibited more favorable clinical outcomes. The purpose of this study is to evaluate the association in the change of CTC number and clinical outcomes (PFS and OS) in patients with advanced NSCLC treated homogenously with docetaxel and gemcitabine administered every 2 weeks. The CellSearch system was used to confirm the feasibility of this method to evaluate the number of CTC in NSCLC patients.
Patients and methods Study design Patients with advanced NSCLC and who were included in two multi-institutional phase II trials were eligible for the
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CTC study. These phase II trials were similar, except for the NSCLC population addressed; one of them included elderly patients ([70 years) with PS 0 or 1, while the other included PS 2 patients of any age. The Institutional Review Board approved both protocols and the CTC sub-study, and all patients provided written informed consent. Common inclusion criteria were: cytological or histological diagnosis of advanced NSCLC (stage IIIB with pleural effusion or stage IV); at least one bidimensionally measurable lesion by the RECIST criteria located outside a previously irradiated field; life expectancy C12 weeks; adequate bone marrow [absolute neutrophil count (ANC) C2,000/dL, platelet count C1,00,000/dL, hemoglobin C10 g/dL], renal function (serum creatinine B1.59 upper normal limit), and hepatic function [bilirubin B1.59 upper normal limit, serum glutamate oxaloacetate transaminase (SGOT), and serum glutamate pyruvate transaminase (SGPT) B2.59 upper normal limit]. Patients with treated stable brain metastases were also eligible. Additional informed consent for a CTC analysis was required. In both phase II trials, patients received treatment with: docetaxel at a dose of 50 mg/m2 and a 60-min intravenous (IV) infusion; gemcitabine 2,000 mg/m2, 30-min IV infusion, both treatments on days 1 and 14. Cycles were repeated every 28 days on an outpatient basis. Blood samples were collected for the CTC analysis within 7 days before commencing treatment (baseline) and after two chemotherapy cycles. CTC analysis Before starting chemotherapy (basal) and after 2 months of chemotherapy (before the third cycle), 7.5 mL of whole blood was drawn. Samples were stored in CellSave preservative tubes at room temperature and were analyzed within 72 h after collection. CTC quantification was performed using the semiautomated CellSearch system (Veridex), which has demonstrated its reproducibility across different laboratories and consists in two instruments: the CellTracks AutoPrep system and the CellSpotter analyzer [20]. During the AutoPrep procedure, a special mix of ferrofluids coated with epithelial cell-specific anti-EpCAM antibodies was automatically added to immunomagnetically enriched epithelial cells, separated in a magnetic field. The enriched samples were then stained with fluorescent nuclear 40 ,6diamidino-2-phenylindole (DAPI) and with fluorescent monoclonal antibodies specific for leukocytes (CD45) and epithelial cells (cytokeratins 8, 18, and 19). After a second magnetic separation, the excess staining reagents were aspirated. Finally, cells were resuspended in the Magnest device, which consists in a chamber and two magnets that orient immunomagnetically labeled cells for their
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subsequent analysis in the CellSpotter analyzer, a fourcolor semi-automated fluorescence microscope. The images obtained by scanning the entire surface were automatically captured and presented in a web-enabled browser to be finally identified and selected by the operator. The criteria used to classify each cell as CTC are: more than 4 lm in diameter; a round or oval morphology; a visible DAPI-positive nucleus present in the CK-positive cytoplasm; and negative staining for CD45. The results are expressed in CTCs/7.5 mL of blood. CTC enumeration was centralized in the Cellular Oncology Laboratory at the Hospital Arnau de Vilanova of Valencia. Statistical analysis A widely accepted C2 CTC in 7.5 mL of blood cut-off was used in this study according to the first CellSearch enumeration study, in which 20 % of patients with metastatic lung cancer had C2 CTCs in 7.5 mL of blood [13]. Analyses were carried out using SPSS version 15.0 (SPSS Inc., Chicago, IL). All the statistical tests were performed two-sided and at the 5 % level of significant difference. Differences of rates between groups were compared with a Chi-square test. Differences between groups with continuous variables were tested by a Student’s t test or by a non-parametric Mann–Whitney U test. The Kaplan–Meier method was used to estimate the probability of PFS and overall survival (OS) over time. PFS was calculated from the baseline blood drawn to any disease progression or the last visit date if there was no progression, and OS was calculated from the baseline blood drawn to death from any cause or to the last followup date if the patient was living at the end of the follow-up. The long-rank test was used to assess the differences between groups in the univariate analysis.
Results Blood samples for CTC enumeration were taken in 37 NSCLC patients treated with docetaxel and gemcitabine administered every 2 weeks participating in two independent clinical trials conducted in ten hospitals from the Mediterranean region in Spain to evaluate chemotherapy combination in fragile patients. One of them included 50 elderly patients (CPNM-ANC-07), and the other 75 patients with PS 2 (CNMP-PS2-07). The CTC sub-study was optional and included only four hospitals. Of the 37 patients enrolled in the CTC study, 20 belonged to the elderly patients study and 17 to the PS 2 study. No statistically significant differences were observed between the
Table 1 Patients’ characteristics and CTCs detection Characteristics
n
%
C2 CTCs
%
0–1 CTCs
%
Age Mean
71 years
Range
44–85
Gender Male
33
89.2
25
89.3
8
88.9
Female
4
10.8
3
10.7
1
11.1
Histology Adenocarcinoma
14
37.8
10
35.7
4
48.0
Squamous cell carcinoma
14
37.8
12
42.9
2
22.2
Poorly differentiated
3
8.1
1
3.6
2
22.6
Other
6
16.2
5
17.9
1
11.1
Response to chemotherapy Partial response
7
18.9
5
17.9
2
22.2
Stable disease Progression disease
14 6
37.8 16.2
12 5
42.9 17.9
2 1
22.2 11.1
No evaluable
10
27
6
21.4
4
44.4
whole population of the studies and the subgroup of patients of the CTC study. The characteristics of the 37 patients assessable for CTC enumeration are summarized in Table 1. Twenty patients (54 %) had PS 0–1 and 17 (46 %) had PS 2. Consistent with other studies carried out in Spain, 37.8 % of all the patients had squamous cell histology. Overall, the presence of C2 CTCs at the baseline determination was detected in 9 of the 37 patients (24 %). Eight patients (21.6 %) had only one CTC and 20 patients had a negative CTC count at baseline. The mean number of CTCs isolated was 1.1 (range 0–9). Presence of C2 CTCs at baseline did not correlate with gender, histological subtype or response to chemotherapy (Table 1). At the time of the analysis, 14 (50 %) of the 28 patients with 0–1 CTCs and 4 (44.5 %) of the nine patients with C2 CTCs had died. PFS and OS were analyzed according to the baseline CTC number. No significant differences in PFS and OS were found in the patients with CTC 0–1 as compared with those with CTC C2. The median PFS was 4.3 months (95 % CI 2.5–8.3) and 9.4 months (95 % CI 1.2–12.2), respectively (log-rank test p = 0.3506, Fig. 1). The median OS was 8.1 (95 % CI 2.8–16.3) and 12.2 (95 % CI 1.4–12.2) months for patients with 0–1 CTCs and C2 CTCs, respectively (log-rank test p = 0.7639, Fig. 2). In 23 patients (62.2 %), a second blood sample was drawn after the second chemotherapy cycle (before the fifth dose of docetaxel and gemcitabine). Patients were
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Clin Transl Oncol Fig. 1 Progression-free survival according to CTC at baseline
Fig. 2 Overall survival according to CTC at baseline
classified according to the baseline and second determinations in two subgroups: group 1, CTC = 0–1 at baseline and CTC = 0–1 after the second chemotherapy cycle (18 patients); group 2, CTC C2 at baseline and CTC = 0–1 after the second determination (5 patients). No differences in age, gender and histologic subtype were observed between groups. In group 1, four patients (22.2 %) obtained a partial response and 11 patients (61 %) showed stable disease (an 83 % disease control rate). In group 2, two patients (40 %) had a partial response and four other patients (40 %) showed stable disease (an 80 % disease control rate). CTC negativization from the baseline to the second determination was better associated with PFS and OS, but there were no statistically significant differences (p value of 0.4467 and 0.4682, respectively). The median PFS was 7.7 and 9.9 months for the patients in group 1 and group 2, respectively (Fig. 3). The median OS for the group 1 patients was 10.1 months, while the median OS for group 2 was not reached at the time of the analysis (Fig. 4).
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Discussion The current multi-institutional study has evaluated the prognosis significance of the CTC levels detected by the CellSearch system in 37 patients with advanced NSCLC treated homogenously with the chemotherapy combination of docetaxel and gemcitabine. Our results demonstrate that 24 % of patients had C2 CTCs at the baseline enumeration. The mean number of isolated CTS was 1.1 (range 0–9) and only three patients (8 %) had C5 CTCs before chemotherapy. Surprisingly unlike other studies, a high baseline CTC enumeration was associated with better PFS and OS, although no statistically significant differences were observed. Median PFS and OS were 9.4 vs. 4.3 months (p = 0.3506) and 12.2 vs. 8.1 months (p = 0.7581) for the patients with C2 CTCs and 0–1 CTCs, respectively. However, the group of patients with a high baseline CTC and whose CTC number decreased with chemotherapy to 0–1 CTCs had a better prognosis than those patients with low levels at baseline and after chemotherapy. The median
Clin Transl Oncol Fig. 3 Progression-free survival according to CTC changes along the treatment
Fig. 4 Overall survival according to CTC changes along the treatment
OS for the last group was 10.1 months, but it was not reached for the first group at the time of the analysis. Many different methods have been developed to detect CTCs in the blood of cancer patients and show variable sensitivity and specificity. The two main approaches used are morphologic-based isolation [OncoQuickTM, isolation by size of Epithelial Tumor Cells (ISET)] [21, 22], and immunological approaches (CellSearchTM, CTC-chip) [20, 23]. Nevertheless, only the CellSearch system, a semiautomated immunomagnetic system, has shown sufficient level of robust and reliable validation to be approved by the US FDA. The results from various studies that have used the CellSearch platform have demonstrated the prognosis significance of the CTC level in metastatic breast, colorectal and castrate-resistant prostate cancers [16, 17, 24]. In these studies, different cut-offs have been used to establish the prognosis significance of CTC numbers. Thus, in patients with metastatic breast cancer and castrate-resistant prostate cancer, C5 CTCs per 7.5 mL of blood before
treatment is used as an independent predictor of worse PFS or OS, whereas a cut-off of C3 CTCs is set in metastatic colon cancer. The reasons for using these cut-offs are not clear, but can be explained because the number of patients with C5 CTCs is higher in patients with metastatic breast (26 %) and prostate cancers (41 %) than with colorectal cancer (17 %) [13]. Few studies have been performed in NSCLC patients using the CellSearch system, Krebs et al. investigated the prognosis significance of CTCs in NSCLC patients with stage IIIA, IIIB and IV [19]. In a univariate analysis, patients with \5 CTCs had a higher PFS and OS than those with C5 CTCs: 6.8 and 8.1 months vs. 2.4 and 4.3 months, respectively. In a multivariate analysis, CTC remained as the independent prognosis factor for PFS and OS. Of the total population, 21 % of patients had a positive baseline CTC count (C2 CTCs). The proportion of patients with C2 CTCs was higher for stage IV disease (32 %) than for stages IIIB (7 %) and IIIA (0 %). Our study only included patients with stage IV disease and showed that the
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number of patients with C2 CTCs at baseline is slightly lower (9 of 37 patients, 24 %) than in the study of Krebs et al. [19]. Likewise, the range of CTC number in our study is narrower (0–9) than in the study of Krebs et al. (0–146) [19]. There is no clear explanation for these low CTC levels in our population. Perhaps the patients included in our study had a more advanced stage IV disease and greater tumor burden (PS 2); hence, malignant CTCs lose epithelial cell adhesion molecules (EpCAM) and cytokeratin, which is indicative of an epithelial–mesenchymal transition (EMT). This means the assays which target epithelial cells in blood, such as CellSearch miss the detection of the most invasive tumor cells [25, 26]. It has been recently observed that metastatic breast cancer with unfavorable prognostic factors (triple negative, brain metastases, etc.) had undetectable CTCs, suggesting that an underestimation of CTC number by CellSearch may be due to the EMT of the CTCs [27]. This might explain why the poor prognosis for the NSCLC patients (PS 2 and elderly) included in our study had fewer CTCs, as detected by CellSearch analysis and, unlike other studies, the presence of C2 CTCs did not correlate with a worse prognosis. Variation in the CTC number with chemotherapy was analyzed. CTC negativization from baseline to the second determination was associated with better PFS and OS when comparing patients with negative CTCs at baseline and after chemotherapy, although there were no statistically significant differences. These results agree with other studies in NSCLC and with other tumors in which the persistence of a high CTC number is associated with a poor prognosis [19, 28, 29]. This study has focused on the prognosis significance of CTC enumeration at baseline or on their change with chemotherapy in advanced NSCLC patients. However, several issues remain to be clarified: the EpCAM and CK used to detect tumor cells are not specific and can be expressed aberrantly in other cells; moreover, the loss of EpCAM and CK in tumor cells has been described as an epithelial–mesenchymal transition; and the majority of studies include a heterogeneous population, which makes the comparison among them difficult. In addition to CTC enumeration, the molecular characterization of these cells can provide knowledge on the genotypic and phenotypic characteristics of the tumor without the need of invasive biopsy. The determination of the epidermal growth factor receptor (EGFR) mutational status is particularly interesting, but the small number of patients in whom CTCs are isolated by CellSearch system hampers this molecular characterization. In one study, using CTC-chip, circulating tumor cells were identified in all 27 patients with analyzed NSCLC, and with a median of 74 cells per milliliter (mean 133, range 5–771) [30]. The Scorpion Amplification Refractory Mutation System (SARMS) assay identified
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EGFR mutations in 17 of the 18 CTC specimens (94 %). Among the 12 patients for whom specimens of the primary tumor and CTCs were available for the analysis, genotyping of CTCs gave a sensitivity of 92 % (in 11 of the 12 patients). In addition to the primary activating mutation, T790M-associated resistance to tyrosine kinase inhibitors was detected in CTCs. Serial CTC analysis can allow the molecular characterization of the cancer during the treatment without the need for invasive biopsy. In conclusion, CTC enumeration has demonstrated its clinical usefulness in various tumors, including NSCLC. The use of CellSearch system, complemented by new technologies, opens up the way to understand the metastasis biology and enables the molecular characterization of CTCs without the need of biopsy to improve the therapeutic and clinical management of NSCLC patients. Acknowledgments This work was supported in part by a grant from Sanofi Spain. We thank Elena Aristoy for reviewing the English of the manuscript and Vicente Caran˜ana for helpful assistance in the preparation of the figures. Conflict of interest of interest.
The authors declare that they have no conflict
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