Clin Transl Oncol DOI 10.1007/s12094-014-1223-5
RESEARCH ARTICLE
Quantification of circulating endothelial cells as a predictor of response to chemotherapy with platinum and pemetrexed in patients with advanced non-squamous non-small cell lung carcinoma Alfredo Sa´nchez Herna´ndez • Oscar Jose´ Juan • Jose´ Vidal Martı´nez • Remei Blanco • Sonia Macia´ • Gaspar Esquerdo Galiana • Francisco Aparisi aparisi Javier Garde Noguera • Silvia Catot • Ferran Losa Gaspa´ • Francisco Garcı´a-Pin˜on
•
Received: 27 May 2014 / Accepted: 1 September 2014 Ó Federacio´n de Sociedades Espan˜olas de Oncologı´a (FESEO) 2014
Abstract Introduction Circulating endothelial cells (CEC) play an important role in tumor neovascularization and may have prognostic value in cancer patients. This study was designed to investigate the role of CEC as a marker for predicting platinum plus pemetrexed first-line chemotherapy efficacy in advanced non-squamous non-small cell lung cancer (NSCLC). Methods A prospective study was performed whose main objective was to study whether the numbers of CEC at baseline and prior to the second and third cycle of che-
motherapy were response predictors. Sixty-nine patients received cisplatin plus pemetrexed, and peripheral blood samples were performed at baseline and after second and third cycle. Separation and CEC count were performed using inmunomagnetic separation (CellSearch). Results The CEC count in 4 mL of peripheral blood was obtained prior to the first, second, and third cycle of treatment. Baseline levels and evolution of CEC were correlated with response to treatment according to RECIST criteria after three cycles of treatment. Sixty-nine patients were included: 43 (64.2 %) received cisplatin/pemetrexed and 24 (35.8 %) carboplatin/pemetrexed. Range of baseline CEC: 8–965 (mean of 153 cel/4 mL). The results after 3
On behalf of Grup d’investigacio´ i divulgacio´ en Oncologia (GIDO). A. Sa´nchez Herna´ndez (&) Servicio de Oncologı´a Me´dica, Consorcio Hospitalario Provincial de Castello´n, Avda Dr Clara´ 19, 12003 Castello´n, Spain e-mail: [email protected]
G. Esquerdo Galiana Servicio de Oncologı´a Me´dica, Clinica de Benidorm, Avenida Alfonso Puchades, 8, 03501 Benidorm, Alicante, Spain e-mail: [email protected]
O. Jose´ Juan Servicio de Oncologı´a Me´dica, Hospital Universitari I Polite`cnic la Fe´, Bulevar del Sur, Valencia, Spain e-mail: [email protected]
F. Aparisi aparisi Servicio de Oncologı´a Me´dica, Hospital Virgen de los Lirios Polı´gon Caramanxel s/n, 03804 Alcoy, Alicante, Spain e-mail: [email protected]
J. Vidal Martı´nez Servicio de Ana´lisis Clı´nicos, Hospital Arnau de Vilanova, Calle San Clemente, 12, 46015 Valencia, Spain e-mail: [email protected] R. Blanco Servicio de Oncologı´a Me´dica, Hospital Mu´tua de Terrassa, Placa del Doctor Robert, 5, 08221 Terrassa, Barcelona, Spain e-mail: [email protected]
J. Garde Noguera Servicio de Oncologı´a Me´dica, Hospital Arnau de Vilanova, Calle San Clemente, 12, 46015 Valencia, Spain e-mail: [email protected] S. Catot Servicio de Oncologı´a Me´dica, Clinica Althaia de Manresa, C/ Dr. Joan Soler, 1-3, 08243 Barcelona, Spain e-mail: [email protected]
S. Macia´ Servicio de Oncologı´a Me´dica, Hospital general de Elda, Avda Elda-Sax sn, 03600 Alicante, Spain e-mail: [email protected]
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cycles were: 25 partial responses (36.2 %), 17 cases of stabilization of disease (24.6 %), 16 of progressive disease (23.2 %) and 11 non-evaluables (16 %). No significant relationship between the baseline CEC count and response was found (p value = 0.831). Increase [50 % between the first and second cycle was correlated significantly with progression disease (p = 0.008). Patients who had a baseline CEC count greater than the mean ([153 cells/ 4 mL) showed longer progression-free survival and global survival without statistical significance. Conclusions In this homogeneous group of patients with NSCLC, there is no correlation between response to treatment and CEC baseline levels. The increase in CEC numbers after the first cycle could be a negative predictive factor. Keywords Circulating endothelial cell NSCLC Chemotherapy
Introduction Lung cancer accounts for 12 % of all cancers diagnosed worldwide. Its high incidence with its low survival rate leads it to be the leading cause of cancer death in Western countries [1]. There is a clear predominance of the nonsmall cell histologic type, making up 80 % of cases in Europe [2] and 85 % in the US [3]. Many advances are being made in treatment, despite which the overall prognosis remains poor. Survival at 5 years is directly related to disease stage, being 2 % when it presents as disseminated disease [4]. The study of prognostic and predictive is in constant research. Its aim is to customize treatment and optimize the effectiveness of available agents [5]. In recent years significant progress has been made in both imaging techniques and identification of serological markers. It is important to achieve diagnostic tools to determine at an early stage therapy effectiveness. This would not only optimize costs, but also serve to modify the therapeutic strategy of patients who do not respond and avoid unnecessary side effects.
F. Losa Gaspa´ Servicio de Oncologı´a Me´dica, Hospital Sant joan d’Espı´-Moises Broggi, Carrer de Jacint Verdaguer, 90, 08970 Sant Joan Despı´, Barcelona, Spain e-mail: [email protected] F. Garcı´a-Pin˜on Fundacion de la comunidad Valenciana, Hospital Provincial de Castellon, Avda Dr Clara´ 19, 12003 Castello´n, Spain
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Circulating endothelial cells (CECs) represent a population of mature endothelial cells that are shed from the intima of the blood vessels into the circulation. Their number in healthy individuals is quite low and the increase indicates the presence of vascular endothelium damage [6, 7]. A CEC increase has been observed in various processes, such as infections, cardiovascular diseases, autoimmune diseases and cancer. However, these CEC are heterogeneous and may be apoptotic, necrotic or viable. It has been shown that CEC are more numerous and viable in cancer patients than in healthy individuals [8, 9]. The two main methods for the quantification of CEC are based on flow cytometry or inmunomagnetic separation [10, 11]. To date, several studies have been reported in different types of cancer and disease stage regarding the role of CECs as prognostic and predictive factor. The results show significant differences as both predictive and prognostic factor in terms of response and survival [12–24]. In this study, we investigated whether baseline CEC and evolution during treatment have a prognostic and/or predictive role in patients with non-squamous non-small cell lung cancer (NSCLC) treated in a homogeneous manner with platinum and pemetrexed.
Materials and methods Patients This is a prospective, open, multicenter study involving nine Spanish centers. The main objective was to study whether the numbers of CEC at baseline and prior to the second and third cycle of chemotherapy were response predictors. Secondary endpoints included the quantification of changes in the number of CEC between baseline and before the second and third cycle of chemotherapy, the relationship between decreasing numbers of CEC and treatment, the relationship between decreasing CEC and progression-free survival (PFS) and overall survival (OS), to correlate baseline CEC and survival and, finally, quantify CEC and correlate them with clinicopathological prognostic factors (PS 0 vs. 1 vs. 2, location and number of metastases and comorbidities). Each patient was required to meet the following criteria: age over 18 years, cytologic or histologic diagnosis of stage IV non-squamous NSCLC, ECOG performance status 0–2, not have received prior chemotherapy, measurable disease, adequate renal, liver and bone marrow function, absence of active infection, severe cardiovascular disease, vasculitis, symptomatic brain metastases, absence of other active malignancies in the last 5 years and concomitant systemic immunotherapy or chemotherapy, and a informed consent in writing. The
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study was approved by the ethics committees of the institutions. Therapy schedule To achieve a homogeneous sample, the patients were treated with a platinum salt plus pemetrexed, from 4 up to 6 cycles, according to the following schedules: 75 mg/m2 cisplatin ? pemetrexed 500 mg/m2 every 21 days or carboplatin area under the curve (AUC) 5–6 ? pemetrexed 500 mg/m2 every 21 days. Maintenance therapy with erlotinib or pemetrexed 500 mg/m2 was administered at investigator discretion. Peripheral blood samples were collected at three points of evolution: at baseline and before the second and third cycles of treatment. After third cycle of chemotherapy, a response evaluation was performed according to RECIST 1.1 criteria (response evaluation criteria for solid tumors) [25].
CEC analysis Separation and CEC count were performed at the Hospital Arnau de Vilanova de Valencia using inmunomagnetic separation: CellSearchÒ—Circulating Endothelial Cells Kit (CellTracksAnalyzerÒ). Blood samples were obtained aseptically by venipuncture into a CellSaveÒ preservative tube, after discarding the first blood sample after puncture to avoid interference by the initial trauma of the vascular wall. The tubes were filled until blood flow stopped to ensure the correct proportion of sample and preservative anticoagulant, always ensuring a volume of at least 4 mL for performing analysis. The sample was mixed immediately by gently inverting the tube eight times, preventing clotting. 4 mL was transferred to preservative CellSave blood tubes in the corresponding 15 mL conical tube. Then 10 mL of dilution buffer was added to this conical tube and this was mixed by inverting five times. The tube was centrifuged at 8009g for 10 min. The sample was processed in the CellTracks Autoprep SystemÒ within an hour of sample preparation. Fluorescent reagents used are as follows: anti-CD105-PE/antiCD45-APC (anti-CD105 is specific for that endoglin protein is expressed by endothelial cells, activated monocytes, stromal cells and pre-B cells, and anti-CD45 expression is restricted to leukocytes) and 2-(4-amidinophenyl)-1Hindole-6-carboxamidine (DAPI), which is a fluorescent probe that stains the cell nucleus. The mixture of reagents and sample is dispensed by Autoprep CellTracks SystemÒ equipment in a cartridge that is inserted into a mobile display device MagNestÒ and allowed to incubate in the dark for a minimum of 20 min. The magnetic field strength of the MagNestÒ device makes the cells magnetically marked positive for CD146 (CD146?) move to the surface of the
cartridge. The CellTracks Analyzer IIÒ automatically scans the entire surface of the cartridge, acquires images and displays a gallery format to the user for final classification of magnetically captured cells. An event is classified as a CEC when morphological characteristics are consistent with the cell and phenotypes are displayed correctly, i.e., CD146?, CD105?, and DAPI? and CD45-.
Statistical analysis A sample of 68 patients was estimated to differentiate response to disease from a 45 % change in CEC quantification, with 90 % power and bilateral a = 0.05, considering standard deviation (SD) values for CEC of 832 Kawaishi et al. [17] and assuming losses of 30 % [26]. Overall survival (OS) was calculated from date of enrollment to death or last contact with the patient. The disease-free interval was calculated from the date of enrollment to the date of diagnosis of tumor recurrence, either local or distant. Survival functions were calculated using the Kaplan–Meier method. For comparison of survival curves log-rank test was used. The Cox regression was used to analyze the effect between covariates and survival. ROC curves were used to evaluate the diagnostic performance. The limit of statistical significance was established as a p value of \0.05. Statistical analysis was performed using SPSS (Statistical Package for Social Science) for Windows, version 17.0 (SPSS Inc., Chicago, IL, USA) and R software version 2.15.1.
Results Patient characteristics Between June 2010 and July 2011, 69 patients were included in the study. Table 1 summarizes patients’ characteristics. Median age was 61 years (range 40–82). Of these, 48 were males (69.9 %). Ten patients had PS 0 (14.5 %); 47, PS 1 (68.1 %); and 12, PS 2 (17.4 %). Sixty were adenocarcinomas (87 %) and 45 (64.2 %) had at least two metastatic sites. A total of 251 cycles were administered based on a platinum salt plus pemetrexed. The median was four cycles. Forty-five patients (65.2 %) received the combination of cisplatin/pemetrexed while the remaining 24 (34.8 %) were treated with pemetrexed and carboplatin. For comparative analysis of the CEC count, data from patients from whom not all referred samples were collected were excluded. The total number of patients with three samples was 54.
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Clin Transl Oncol Table 1 Patient characteristics Patient characteristics
N = 69
Sex Male
48
69.60 %
Female
21
30.40 %
61
(40–82)
0
10
14.50 %
1
47
68.10 %
2
12
17.40 %
Adenocarcinoma Large cell
60 8
87 % 11.60 %
Bronchoalveolar
1
1.40 %
Stage IV (M1b)
67
97.10 %
Stage IV (M1a)
2
2.90 %
0
2
2.90 %
1
22
31.90 %
2
27
39.10 %
3
12
17.40 %
4
4
5.80 %
5
2
2.90 %
Hepatic metastases
15
21.70 %
Brain metastases
11
15.90 %
Adrenal metastases Lung metastases
17 38
24.60 % 55.10 %
Age Median (range) PS-ECOG
Histological type
Fig. 1 CEC count in three determinations
Stage at inclusion
Number of metastatic sites
Site of metastases
Pleural metastases
13
18.80 %
Bone metastases
26
37.70 %
Nodal metastases
8
11.60 %
EGFR mutation No mutation Mutated Not determined
43
62.30 %
1
1.40 %
25
36.30 %
Thirty-eight patients died due to progression disease (57.6 %) and two patients (3 %) due to chemotherapy side effects (one of these patients was receiving carboplatin and the other one received cisplatin). PFS and OS were studied depending on the platin salt that was received without significant differences (p value 0.390 and 0.817, respectively). CEC quantification In 67 patients, baseline studies were performed (the CEC determination was not performed in two patients due to
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clotted sample). CEC range was 8–965 cells/4.0 mL blood (mean: 153, SD: 204). After starting treatment, determinations were obtained from 59 patients before the second cycle and in 54 patients prior to the third cycle. CEC levels were on the rise in these determinations. Prior to the second cycle, range was 10–1,388 cells/4 mL (mean: 195, SD: 263, prior to the third, range was 5–1,857 on average and SD 248,356 (Fig. 1). There were no significant differences in the number of CEC when subgroup analysis was performed according to the characteristics of sex, age, smoking status, PS and number and location of metastases. Mean CEC number at baseline was 152.79, and this value was taken as reference for correlating this result with OS and PFS. Forty-eight patients had a CEC value under the mean; these patients had median OS and PFS results 8 and 5 months, respectively. Rest of the patients (n = 18) had a median OS 11 months and PFS 8 months, with no significant differences (p value = 0.209 for OS and 0.295 for PFS). CEC numbers and objective tumor response to chemotherapy Twenty-five (36.23 %) patients had partial response (PR), 17 (24.64 %) stable disease (SD) and 16 patients (23.19 %) progression disease (PD). Radiological responses obtained were correlated with varying levels of CEC, with no differences obtained between the results for patients who achieved response and/or stability of the disease as compared to those who progressed (Table 2). The analysis by quartiles did not show any significant differences either. However, on studying the relationship between CEC counts and progression and death, it was found that an increase of more than 50 % in the logarithm of CEC count after the first and second cycle as compared to baseline was
Clin Transl Oncol Table 2 Increase in CEC in relation to radiological response Log increase in CEC prior to cycle 2 of treatment
Response assessment PR
SD
PD
No
9
5
9
Yes
14
9
6
29
23
14
15
52
Total Chi square tests
Value
df
p value
2.166
2
0.339
Log increase in CEC prior to cycle 3 of treatment
Total 23
Response assessment PR
SD
No
14
5
6
25
Yes
8
8
10
26
22
13
16
51
Value
df
P value
3.31
2
0.191
Total Chi Square Tests
PD
Total Fig. 2 Progression-free survival according to whether the CEC count was lower or higher than the mean baseline CEC count
statistically correlated with progression in a significant manner (p = 0.008 and p = 0.039), but not with death. This was not found to be the case when analysis was performed between the second and third cycles. CEC counts and survival Median PFS was 5 months (95 % CI 5.47–9.22). Univariate analysis was performed with the mean quantification of all included patients and survival was analyzed depending on whether this level was above or below the same (Table 3). The median PFS of patients with levels above the mean was 8 months versus 5 in patients with lower levels (p = 0.295) (Fig. 2). Median OS for patients with CEC levels above the mean (153) was 11 vs. 8 months for patients with levels below. Despite this trend, there is no statistically significant difference (p = 0.209) (Fig. 3).
Fig. 3 Overall survival according to whether the CEC count was lower or higher than mean baseline CEC count
Relationship between clinicopathologic variables and quantification of CEC The study of baseline measurements of CEC with the following clinical variables: sex, PS, location of metastases and number of metastatic sites, found no statistically
Table 3 Survival according to the baseline CEC count and CEC log above or below the mean Baseline CEC count
N
Mean (months)
95 % confidence interval
Median (months)
95 % confidence interval
Log-rank v2
P value
1.578
0.209
1.098
0.295
Overall survival Less than the mean
48
11.01
(8.35–13.67)
8
(4.21–11.79)
Greater than the mean
18
13.17
(9.25–17.08)
11
(1.22–20.78)
Less than the mean
48
6.71
(4.42–9.01)
5
(3.68–6.32)
Greater than the mean
18
8.18
(5.36–19.99)
8
(2.37–13.63)
Progression-free survival
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significant differences for any of these. The COX regression analysis for specific metastatic sites which were accompanied with poorer prognosis (brain, liver and adrenal) was not found to be significant.
Discussion Chemotherapy remains the main therapeutic mainstay in metastatic lung cancer. Despite the progress made in recent years, response rates continue to be around 30–40 % and the median survival is 7–12 months [27–30]. Having early predictors of treatment efficacy can optimize cytostatic treatments, selecting patients who will benefit and avoiding toxicities in those who will not. The results of this study show that the variation in CEC levels before and during treatment is not predictive of response. To our knowledge, this is the first report of CEC in non-squamous NSCLC patients with this therapeutic schema. The results published so far regarding the actual role of the CEC in NSCLC are controversial. Kawaishi et al. [17] investigated the role of CEC as a predictor of chemotherapy response. The series included 31 patients of stages IIIA, IIIB and IV. Patients with PR had significantly decreased number of cells at day 22 of treatment. Patients with PD had smaller changes in the number of cells. In our work, overall in patients the number of CEC detected increased progressively during treatment as compared to baseline measurement, unlike in the study of Kawaishi. When compared with Kawaishi’s work, our study is more powerful, since it was performed in a homogeneous population (patients of the same stage and histology). The larger number of patients in our study also gives it a greater validity. One of the potential explanations why our study did not find a correlation with the response as it in Kawaishi’s study lies in the selected therapeutic regimen. The scheme used for Kawaishi’s group includes paclitaxel, which has a known antiangiogenic mechanism of action. Published data suggest that endothelial cells are more sensitive to paclitaxel than other cell lines [31]. There are doubts regarding whether CEC numbers increase or decrease depending on the therapy used [32– 35]. In a study conducted in China [36], 76 patients were included. Of these, 16 had localized disease and underwent surgery. The rest (60 patients) were randomized to receive chemotherapy alone versus combination chemotherapy with the antiangiogenic agent. The number of CEC decreased significantly after treatment in patients with clinical benefit in both groups, with the benefit being more important in those patients in whom the angiogenic agent was associated with chemotherapy. Increase in CEC was associated with PD. In addition, there was a positive
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correlation between the decrease of endothelial cell levels and the time interval until progression. Other study was published in NSCLC patients with chemotherapy with or without Endostar [24] with different results. Seventy-two patients were randomized to three treatment arms: chemotherapy (cisplatin and vinorelbine scheme) with concomitant Endostar, Endostar with sequential chemotherapy (same scheme) or chemotherapy alone. In this study, coinciding with the results of our project, CEC increased progressively with respect to baseline quantification. The increases were greater after the third cycle in a statistically significant manner. Interestingly, CEC numbers increased both in patients receiving Endostar and in those who did not receive it. CEC changes occurred in both patients with or without PD. As is the case in our study, the rise in the number of CEC was statistically significant only in the group of patients who progressed. The authors propose the changes in CEC as being predictor of non-response, both for the group receiving antiangiogenic agent and in those who did not receive it. In our work, CEC was not correlated with the radiological responses obtained. However, on studying the relationship with progression and mortality, the presence of an increase of more than 50 % in the CEC count logarithm after the first and second cycles of treatment when compared with baseline showed a statistically significant correlation with disease progression (p = 0.008 for the first cycle, p = 0.039 for the second cycle). As in the study of Liu et al., in our study we found no relation between CEC increase and response, except in the case of PD, although the increase must be greater than 50 % to demonstrate statistical significance. One explanation may lie in the fact that, for logistical reasons, for some of the patients it was not possible to collect the three blood samples for determination of CEC. On reducing the total number of patients who had CEC assessed after three cycles of treatment, we had lower statistical power to find significant differences. Despite the lack of correlation between the quantification of CEC and radiological response, the project has the potential to be multicenter, with samples from different geographic locations. Another difference with the results of published studies resides in the determination method. Wang et al. and Liu et al. used flow cytometry, which identifies the CEC whose expression exposure membrane antibody was as follows: CD45-, CD146? and CD105?. CD 105 was used also in the present study, despite there is increasing evidence that it may be correlated with monocytes rather than endothelial cells. Further investigation is required to determine the biological significance of each representative CEC subsets [39, 40]. Finally, there is a Spanish work which also analyzes the role of CEC in NSCLC: Fleitas et al. [23] determined the
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number of CEC in 60 patients with NSCLC by inmunomagnetic separation. No statistically significant association between baseline CEC levels and response was observed. Unlike our series, in this work stages IIIB and IV patients were included, and different chemotherapy regimens were received. This objective has been analyzed in other malignancies and with different treatment regimens, including in some of these antiangiogenic agents and also showing discrepant results [12, 15, 18, 37, 38]. Another analysis undertaken was baseline CEC quantification as a survival predictor. Median OS for patients with CEC levels above the mean (152.79 cells/4 mL) was 11 vs. 8 months for patients with levels below the mean (p = 0.209). The same findings were recorded for PFS. Median PFS for patients with levels above the mean was 8 months vs. 5 in patients with lower levels (p = 0.295). Data found in the literature again demonstrate the existence of controversy of the role of CEC as a prognostic factor. In the previously mentioned work by Fleitas et al. [23], in patients with NSCLC, elevated basal levels of CEC were correlated with poor survival. In contrast, in the study by Kawaishi previously referenced [17], baseline levels above the mean were correlated with increased PFS (244 vs. 69 days), and OS, coinciding with our results. In the study by Liu et al. [24], baseline levels of CEC were not correlated with PFS; the same results were obtained by Wang et al. [31]. No differences were found between baseline levels depending on histology. CEC studies conducted in other malignancies have also investigated the potential prognostic role of baseline CEC levels without definitive conclusions [12–16]. In conclusion, our study results suggest that high baseline levels of CEC may be a predictor of greater diseasefree survival and OS, although the study is limited by insufficient statistical power. Moreover an increase in cell numbers over 50 % between the first and second cycle of chemotherapy and between the first and third confers a statistically significant risk of progression. Further studies are required and probably a better selection of CEC subpopulations would provide some more information. The results do not clarify the role of the CEC as predictor of response in non-squamous NSCLC treated with chemotherapy, but contribute to the initiation of other lines of research. Thus, it would be very interesting to study the quantification of CEC and their variation, and analyze the correlation with other variables for which the present sample of patients was not sufficient. Differences depending on histological subtype are other interesting issue to evaluate. Finally, and given that the mechanism of action of antiangiogenic drugs is to inhibit the pathway of vascular endothelial growth factor (VEGF), study of the
behavior of CEC in NSCLC patients treated with these drugs could also be relevant. Acknowledgments Funding for this study was provided by Eli Lilly and Company; Eli Lilly and Company as such had no role in data collection, analysis or evaluation. CRO Pivotal provided advice to properly handle and submit the paper. Kavita Gandhi has reviewed and edited the manuscript language. Conflict of interest None author has any actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations that could inappropriately influence (bias) their work. Ethical standard Ethics committees have been informed regarding all of this study procedures. Patients were properly informed and signed an informed consent.
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30. Paz-Ares L, de Marinis F, Dediu M, Thomas M, Pujol JL, Bidoli P, et al. Maintenance therapy with pemetrexed plus best supportive care versus placebo plus best supportive care after induction therapy with pemetrexed plus cisplatin for advanced non-squamous non-smallcell lung cancer (PARAMOUNT): a double-blind, phase 3, randomised controlled trial. Lancet Oncol. 2012;13: 247–55. 31. Wang J, Lou P, Lesniewski R, Henkin J. Paclitaxel at ultra low concentrations inhibits angiogenesis without affecting cellular microtubule assembly. Anticancer Drugs. 2003;14:13–9. 32. Beaudry P, Force J, Naumov GN, Wang A, Baker CH, Ryan A, et al. Differential effects of vascular endothelial growth factor receptor-2 inhibitor ZD6474 on circulating endothelial progenitors and mature circulating endothelial cells: implications for use as a surrogate marker of antiangiogenic activity. Clin Cancer Res. 2005;11(9):3514–22. 33. Patterson TA, Lobenhofer EK, Fulmer-Smentek SB, Collins PJ, Chu TM, Bao W, et al. Performance comparison of one-color and two-color platforms within microarray Quality Control (MAQC) project. Nat Biotechnol. 2006;24(9): 1140–50. 34. Li H, Raia V, Bertolini F, Price DK, Figg WD. Circulating endothelial cells as a therapeutic marker for thalidomide in combined therapy with chemotherapy drugs in a human prostate cancer model. BJU Int. 2008;101(7):884–8. 35. Shaked Y, Henke E, Roodhart JM, Mancuso P, Langenberg MH, Colleoni M, et al. Rapid chemotherapy-induced acute endothelial progenitor cell mobilization: implications for antiangiogenic drugs as chemosensitizing agents. Cancer Cell. 2008;14(3):263–73. 36. Wang L, Wei J, Qian X, Yin H, Zhao Y, Yu L, et al. ERCC1 and BRCA1 mRNA expression levels in metastatic malignant effusions is associated with chemosensitivity to cisplatin and/or docetaxel. BMC Cancer. 2008;8:97. 37. Strijbos MH, Gratama JW, Schmitz PI, Rao C, Onstenk W, Doyle GV, et al. Circulating endothelial cells, circulating tumour cells, tissue factor, endothelin-1 and overall survival in prostate cancer patients treated with docetaxel. Eur J Cancer. 2010;46(11):2027–35. 38. Stubbe J. Ribonucleotide reductases in the twenty-first Century. Proc Natl Acad Sci USA. 1998;95:2723–4. 39. Gruenwald V, Beutel G, Schuch-Jantsch S, Reuter C, Ivanyi P, Ganser A, et al. Circulating endothelial cells are an early predictor in renal cell carcinoma for tumor response to sunitinib. BMC Cancer. 2010;31(10):695. 40. Yu HK, Lee HJ, Choi HN, Ahn JH, Choi JY, Song HS, et al. Characterization of CD45-/CD31?/CD105? circulating cells in the peripheral blood of patients with gynecologic malignancies. Clin Cancer Res. 2013;19(19):5340–50.
RESEARCH ARTICLE
Quantification of circulating endothelial cells as a predictor of response to chemotherapy with platinum and pemetrexed in patients with advanced non-squamous non-small cell lung carcinoma Alfredo Sa´nchez Herna´ndez • Oscar Jose´ Juan • Jose´ Vidal Martı´nez • Remei Blanco • Sonia Macia´ • Gaspar Esquerdo Galiana • Francisco Aparisi aparisi Javier Garde Noguera • Silvia Catot • Ferran Losa Gaspa´ • Francisco Garcı´a-Pin˜on
•
Received: 27 May 2014 / Accepted: 1 September 2014 Ó Federacio´n de Sociedades Espan˜olas de Oncologı´a (FESEO) 2014
Abstract Introduction Circulating endothelial cells (CEC) play an important role in tumor neovascularization and may have prognostic value in cancer patients. This study was designed to investigate the role of CEC as a marker for predicting platinum plus pemetrexed first-line chemotherapy efficacy in advanced non-squamous non-small cell lung cancer (NSCLC). Methods A prospective study was performed whose main objective was to study whether the numbers of CEC at baseline and prior to the second and third cycle of che-
motherapy were response predictors. Sixty-nine patients received cisplatin plus pemetrexed, and peripheral blood samples were performed at baseline and after second and third cycle. Separation and CEC count were performed using inmunomagnetic separation (CellSearch). Results The CEC count in 4 mL of peripheral blood was obtained prior to the first, second, and third cycle of treatment. Baseline levels and evolution of CEC were correlated with response to treatment according to RECIST criteria after three cycles of treatment. Sixty-nine patients were included: 43 (64.2 %) received cisplatin/pemetrexed and 24 (35.8 %) carboplatin/pemetrexed. Range of baseline CEC: 8–965 (mean of 153 cel/4 mL). The results after 3
On behalf of Grup d’investigacio´ i divulgacio´ en Oncologia (GIDO). A. Sa´nchez Herna´ndez (&) Servicio de Oncologı´a Me´dica, Consorcio Hospitalario Provincial de Castello´n, Avda Dr Clara´ 19, 12003 Castello´n, Spain e-mail: [email protected]
G. Esquerdo Galiana Servicio de Oncologı´a Me´dica, Clinica de Benidorm, Avenida Alfonso Puchades, 8, 03501 Benidorm, Alicante, Spain e-mail: [email protected]
O. Jose´ Juan Servicio de Oncologı´a Me´dica, Hospital Universitari I Polite`cnic la Fe´, Bulevar del Sur, Valencia, Spain e-mail: [email protected]
F. Aparisi aparisi Servicio de Oncologı´a Me´dica, Hospital Virgen de los Lirios Polı´gon Caramanxel s/n, 03804 Alcoy, Alicante, Spain e-mail: [email protected]
J. Vidal Martı´nez Servicio de Ana´lisis Clı´nicos, Hospital Arnau de Vilanova, Calle San Clemente, 12, 46015 Valencia, Spain e-mail: [email protected] R. Blanco Servicio de Oncologı´a Me´dica, Hospital Mu´tua de Terrassa, Placa del Doctor Robert, 5, 08221 Terrassa, Barcelona, Spain e-mail: [email protected]
J. Garde Noguera Servicio de Oncologı´a Me´dica, Hospital Arnau de Vilanova, Calle San Clemente, 12, 46015 Valencia, Spain e-mail: [email protected] S. Catot Servicio de Oncologı´a Me´dica, Clinica Althaia de Manresa, C/ Dr. Joan Soler, 1-3, 08243 Barcelona, Spain e-mail: [email protected]
S. Macia´ Servicio de Oncologı´a Me´dica, Hospital general de Elda, Avda Elda-Sax sn, 03600 Alicante, Spain e-mail: [email protected]
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cycles were: 25 partial responses (36.2 %), 17 cases of stabilization of disease (24.6 %), 16 of progressive disease (23.2 %) and 11 non-evaluables (16 %). No significant relationship between the baseline CEC count and response was found (p value = 0.831). Increase [50 % between the first and second cycle was correlated significantly with progression disease (p = 0.008). Patients who had a baseline CEC count greater than the mean ([153 cells/ 4 mL) showed longer progression-free survival and global survival without statistical significance. Conclusions In this homogeneous group of patients with NSCLC, there is no correlation between response to treatment and CEC baseline levels. The increase in CEC numbers after the first cycle could be a negative predictive factor. Keywords Circulating endothelial cell NSCLC Chemotherapy
Introduction Lung cancer accounts for 12 % of all cancers diagnosed worldwide. Its high incidence with its low survival rate leads it to be the leading cause of cancer death in Western countries [1]. There is a clear predominance of the nonsmall cell histologic type, making up 80 % of cases in Europe [2] and 85 % in the US [3]. Many advances are being made in treatment, despite which the overall prognosis remains poor. Survival at 5 years is directly related to disease stage, being 2 % when it presents as disseminated disease [4]. The study of prognostic and predictive is in constant research. Its aim is to customize treatment and optimize the effectiveness of available agents [5]. In recent years significant progress has been made in both imaging techniques and identification of serological markers. It is important to achieve diagnostic tools to determine at an early stage therapy effectiveness. This would not only optimize costs, but also serve to modify the therapeutic strategy of patients who do not respond and avoid unnecessary side effects.
F. Losa Gaspa´ Servicio de Oncologı´a Me´dica, Hospital Sant joan d’Espı´-Moises Broggi, Carrer de Jacint Verdaguer, 90, 08970 Sant Joan Despı´, Barcelona, Spain e-mail: [email protected] F. Garcı´a-Pin˜on Fundacion de la comunidad Valenciana, Hospital Provincial de Castellon, Avda Dr Clara´ 19, 12003 Castello´n, Spain
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Circulating endothelial cells (CECs) represent a population of mature endothelial cells that are shed from the intima of the blood vessels into the circulation. Their number in healthy individuals is quite low and the increase indicates the presence of vascular endothelium damage [6, 7]. A CEC increase has been observed in various processes, such as infections, cardiovascular diseases, autoimmune diseases and cancer. However, these CEC are heterogeneous and may be apoptotic, necrotic or viable. It has been shown that CEC are more numerous and viable in cancer patients than in healthy individuals [8, 9]. The two main methods for the quantification of CEC are based on flow cytometry or inmunomagnetic separation [10, 11]. To date, several studies have been reported in different types of cancer and disease stage regarding the role of CECs as prognostic and predictive factor. The results show significant differences as both predictive and prognostic factor in terms of response and survival [12–24]. In this study, we investigated whether baseline CEC and evolution during treatment have a prognostic and/or predictive role in patients with non-squamous non-small cell lung cancer (NSCLC) treated in a homogeneous manner with platinum and pemetrexed.
Materials and methods Patients This is a prospective, open, multicenter study involving nine Spanish centers. The main objective was to study whether the numbers of CEC at baseline and prior to the second and third cycle of chemotherapy were response predictors. Secondary endpoints included the quantification of changes in the number of CEC between baseline and before the second and third cycle of chemotherapy, the relationship between decreasing numbers of CEC and treatment, the relationship between decreasing CEC and progression-free survival (PFS) and overall survival (OS), to correlate baseline CEC and survival and, finally, quantify CEC and correlate them with clinicopathological prognostic factors (PS 0 vs. 1 vs. 2, location and number of metastases and comorbidities). Each patient was required to meet the following criteria: age over 18 years, cytologic or histologic diagnosis of stage IV non-squamous NSCLC, ECOG performance status 0–2, not have received prior chemotherapy, measurable disease, adequate renal, liver and bone marrow function, absence of active infection, severe cardiovascular disease, vasculitis, symptomatic brain metastases, absence of other active malignancies in the last 5 years and concomitant systemic immunotherapy or chemotherapy, and a informed consent in writing. The
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study was approved by the ethics committees of the institutions. Therapy schedule To achieve a homogeneous sample, the patients were treated with a platinum salt plus pemetrexed, from 4 up to 6 cycles, according to the following schedules: 75 mg/m2 cisplatin ? pemetrexed 500 mg/m2 every 21 days or carboplatin area under the curve (AUC) 5–6 ? pemetrexed 500 mg/m2 every 21 days. Maintenance therapy with erlotinib or pemetrexed 500 mg/m2 was administered at investigator discretion. Peripheral blood samples were collected at three points of evolution: at baseline and before the second and third cycles of treatment. After third cycle of chemotherapy, a response evaluation was performed according to RECIST 1.1 criteria (response evaluation criteria for solid tumors) [25].
CEC analysis Separation and CEC count were performed at the Hospital Arnau de Vilanova de Valencia using inmunomagnetic separation: CellSearchÒ—Circulating Endothelial Cells Kit (CellTracksAnalyzerÒ). Blood samples were obtained aseptically by venipuncture into a CellSaveÒ preservative tube, after discarding the first blood sample after puncture to avoid interference by the initial trauma of the vascular wall. The tubes were filled until blood flow stopped to ensure the correct proportion of sample and preservative anticoagulant, always ensuring a volume of at least 4 mL for performing analysis. The sample was mixed immediately by gently inverting the tube eight times, preventing clotting. 4 mL was transferred to preservative CellSave blood tubes in the corresponding 15 mL conical tube. Then 10 mL of dilution buffer was added to this conical tube and this was mixed by inverting five times. The tube was centrifuged at 8009g for 10 min. The sample was processed in the CellTracks Autoprep SystemÒ within an hour of sample preparation. Fluorescent reagents used are as follows: anti-CD105-PE/antiCD45-APC (anti-CD105 is specific for that endoglin protein is expressed by endothelial cells, activated monocytes, stromal cells and pre-B cells, and anti-CD45 expression is restricted to leukocytes) and 2-(4-amidinophenyl)-1Hindole-6-carboxamidine (DAPI), which is a fluorescent probe that stains the cell nucleus. The mixture of reagents and sample is dispensed by Autoprep CellTracks SystemÒ equipment in a cartridge that is inserted into a mobile display device MagNestÒ and allowed to incubate in the dark for a minimum of 20 min. The magnetic field strength of the MagNestÒ device makes the cells magnetically marked positive for CD146 (CD146?) move to the surface of the
cartridge. The CellTracks Analyzer IIÒ automatically scans the entire surface of the cartridge, acquires images and displays a gallery format to the user for final classification of magnetically captured cells. An event is classified as a CEC when morphological characteristics are consistent with the cell and phenotypes are displayed correctly, i.e., CD146?, CD105?, and DAPI? and CD45-.
Statistical analysis A sample of 68 patients was estimated to differentiate response to disease from a 45 % change in CEC quantification, with 90 % power and bilateral a = 0.05, considering standard deviation (SD) values for CEC of 832 Kawaishi et al. [17] and assuming losses of 30 % [26]. Overall survival (OS) was calculated from date of enrollment to death or last contact with the patient. The disease-free interval was calculated from the date of enrollment to the date of diagnosis of tumor recurrence, either local or distant. Survival functions were calculated using the Kaplan–Meier method. For comparison of survival curves log-rank test was used. The Cox regression was used to analyze the effect between covariates and survival. ROC curves were used to evaluate the diagnostic performance. The limit of statistical significance was established as a p value of \0.05. Statistical analysis was performed using SPSS (Statistical Package for Social Science) for Windows, version 17.0 (SPSS Inc., Chicago, IL, USA) and R software version 2.15.1.
Results Patient characteristics Between June 2010 and July 2011, 69 patients were included in the study. Table 1 summarizes patients’ characteristics. Median age was 61 years (range 40–82). Of these, 48 were males (69.9 %). Ten patients had PS 0 (14.5 %); 47, PS 1 (68.1 %); and 12, PS 2 (17.4 %). Sixty were adenocarcinomas (87 %) and 45 (64.2 %) had at least two metastatic sites. A total of 251 cycles were administered based on a platinum salt plus pemetrexed. The median was four cycles. Forty-five patients (65.2 %) received the combination of cisplatin/pemetrexed while the remaining 24 (34.8 %) were treated with pemetrexed and carboplatin. For comparative analysis of the CEC count, data from patients from whom not all referred samples were collected were excluded. The total number of patients with three samples was 54.
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Clin Transl Oncol Table 1 Patient characteristics Patient characteristics
N = 69
Sex Male
48
69.60 %
Female
21
30.40 %
61
(40–82)
0
10
14.50 %
1
47
68.10 %
2
12
17.40 %
Adenocarcinoma Large cell
60 8
87 % 11.60 %
Bronchoalveolar
1
1.40 %
Stage IV (M1b)
67
97.10 %
Stage IV (M1a)
2
2.90 %
0
2
2.90 %
1
22
31.90 %
2
27
39.10 %
3
12
17.40 %
4
4
5.80 %
5
2
2.90 %
Hepatic metastases
15
21.70 %
Brain metastases
11
15.90 %
Adrenal metastases Lung metastases
17 38
24.60 % 55.10 %
Age Median (range) PS-ECOG
Histological type
Fig. 1 CEC count in three determinations
Stage at inclusion
Number of metastatic sites
Site of metastases
Pleural metastases
13
18.80 %
Bone metastases
26
37.70 %
Nodal metastases
8
11.60 %
EGFR mutation No mutation Mutated Not determined
43
62.30 %
1
1.40 %
25
36.30 %
Thirty-eight patients died due to progression disease (57.6 %) and two patients (3 %) due to chemotherapy side effects (one of these patients was receiving carboplatin and the other one received cisplatin). PFS and OS were studied depending on the platin salt that was received without significant differences (p value 0.390 and 0.817, respectively). CEC quantification In 67 patients, baseline studies were performed (the CEC determination was not performed in two patients due to
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clotted sample). CEC range was 8–965 cells/4.0 mL blood (mean: 153, SD: 204). After starting treatment, determinations were obtained from 59 patients before the second cycle and in 54 patients prior to the third cycle. CEC levels were on the rise in these determinations. Prior to the second cycle, range was 10–1,388 cells/4 mL (mean: 195, SD: 263, prior to the third, range was 5–1,857 on average and SD 248,356 (Fig. 1). There were no significant differences in the number of CEC when subgroup analysis was performed according to the characteristics of sex, age, smoking status, PS and number and location of metastases. Mean CEC number at baseline was 152.79, and this value was taken as reference for correlating this result with OS and PFS. Forty-eight patients had a CEC value under the mean; these patients had median OS and PFS results 8 and 5 months, respectively. Rest of the patients (n = 18) had a median OS 11 months and PFS 8 months, with no significant differences (p value = 0.209 for OS and 0.295 for PFS). CEC numbers and objective tumor response to chemotherapy Twenty-five (36.23 %) patients had partial response (PR), 17 (24.64 %) stable disease (SD) and 16 patients (23.19 %) progression disease (PD). Radiological responses obtained were correlated with varying levels of CEC, with no differences obtained between the results for patients who achieved response and/or stability of the disease as compared to those who progressed (Table 2). The analysis by quartiles did not show any significant differences either. However, on studying the relationship between CEC counts and progression and death, it was found that an increase of more than 50 % in the logarithm of CEC count after the first and second cycle as compared to baseline was
Clin Transl Oncol Table 2 Increase in CEC in relation to radiological response Log increase in CEC prior to cycle 2 of treatment
Response assessment PR
SD
PD
No
9
5
9
Yes
14
9
6
29
23
14
15
52
Total Chi square tests
Value
df
p value
2.166
2
0.339
Log increase in CEC prior to cycle 3 of treatment
Total 23
Response assessment PR
SD
No
14
5
6
25
Yes
8
8
10
26
22
13
16
51
Value
df
P value
3.31
2
0.191
Total Chi Square Tests
PD
Total Fig. 2 Progression-free survival according to whether the CEC count was lower or higher than the mean baseline CEC count
statistically correlated with progression in a significant manner (p = 0.008 and p = 0.039), but not with death. This was not found to be the case when analysis was performed between the second and third cycles. CEC counts and survival Median PFS was 5 months (95 % CI 5.47–9.22). Univariate analysis was performed with the mean quantification of all included patients and survival was analyzed depending on whether this level was above or below the same (Table 3). The median PFS of patients with levels above the mean was 8 months versus 5 in patients with lower levels (p = 0.295) (Fig. 2). Median OS for patients with CEC levels above the mean (153) was 11 vs. 8 months for patients with levels below. Despite this trend, there is no statistically significant difference (p = 0.209) (Fig. 3).
Fig. 3 Overall survival according to whether the CEC count was lower or higher than mean baseline CEC count
Relationship between clinicopathologic variables and quantification of CEC The study of baseline measurements of CEC with the following clinical variables: sex, PS, location of metastases and number of metastatic sites, found no statistically
Table 3 Survival according to the baseline CEC count and CEC log above or below the mean Baseline CEC count
N
Mean (months)
95 % confidence interval
Median (months)
95 % confidence interval
Log-rank v2
P value
1.578
0.209
1.098
0.295
Overall survival Less than the mean
48
11.01
(8.35–13.67)
8
(4.21–11.79)
Greater than the mean
18
13.17
(9.25–17.08)
11
(1.22–20.78)
Less than the mean
48
6.71
(4.42–9.01)
5
(3.68–6.32)
Greater than the mean
18
8.18
(5.36–19.99)
8
(2.37–13.63)
Progression-free survival
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significant differences for any of these. The COX regression analysis for specific metastatic sites which were accompanied with poorer prognosis (brain, liver and adrenal) was not found to be significant.
Discussion Chemotherapy remains the main therapeutic mainstay in metastatic lung cancer. Despite the progress made in recent years, response rates continue to be around 30–40 % and the median survival is 7–12 months [27–30]. Having early predictors of treatment efficacy can optimize cytostatic treatments, selecting patients who will benefit and avoiding toxicities in those who will not. The results of this study show that the variation in CEC levels before and during treatment is not predictive of response. To our knowledge, this is the first report of CEC in non-squamous NSCLC patients with this therapeutic schema. The results published so far regarding the actual role of the CEC in NSCLC are controversial. Kawaishi et al. [17] investigated the role of CEC as a predictor of chemotherapy response. The series included 31 patients of stages IIIA, IIIB and IV. Patients with PR had significantly decreased number of cells at day 22 of treatment. Patients with PD had smaller changes in the number of cells. In our work, overall in patients the number of CEC detected increased progressively during treatment as compared to baseline measurement, unlike in the study of Kawaishi. When compared with Kawaishi’s work, our study is more powerful, since it was performed in a homogeneous population (patients of the same stage and histology). The larger number of patients in our study also gives it a greater validity. One of the potential explanations why our study did not find a correlation with the response as it in Kawaishi’s study lies in the selected therapeutic regimen. The scheme used for Kawaishi’s group includes paclitaxel, which has a known antiangiogenic mechanism of action. Published data suggest that endothelial cells are more sensitive to paclitaxel than other cell lines [31]. There are doubts regarding whether CEC numbers increase or decrease depending on the therapy used [32– 35]. In a study conducted in China [36], 76 patients were included. Of these, 16 had localized disease and underwent surgery. The rest (60 patients) were randomized to receive chemotherapy alone versus combination chemotherapy with the antiangiogenic agent. The number of CEC decreased significantly after treatment in patients with clinical benefit in both groups, with the benefit being more important in those patients in whom the angiogenic agent was associated with chemotherapy. Increase in CEC was associated with PD. In addition, there was a positive
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correlation between the decrease of endothelial cell levels and the time interval until progression. Other study was published in NSCLC patients with chemotherapy with or without Endostar [24] with different results. Seventy-two patients were randomized to three treatment arms: chemotherapy (cisplatin and vinorelbine scheme) with concomitant Endostar, Endostar with sequential chemotherapy (same scheme) or chemotherapy alone. In this study, coinciding with the results of our project, CEC increased progressively with respect to baseline quantification. The increases were greater after the third cycle in a statistically significant manner. Interestingly, CEC numbers increased both in patients receiving Endostar and in those who did not receive it. CEC changes occurred in both patients with or without PD. As is the case in our study, the rise in the number of CEC was statistically significant only in the group of patients who progressed. The authors propose the changes in CEC as being predictor of non-response, both for the group receiving antiangiogenic agent and in those who did not receive it. In our work, CEC was not correlated with the radiological responses obtained. However, on studying the relationship with progression and mortality, the presence of an increase of more than 50 % in the CEC count logarithm after the first and second cycles of treatment when compared with baseline showed a statistically significant correlation with disease progression (p = 0.008 for the first cycle, p = 0.039 for the second cycle). As in the study of Liu et al., in our study we found no relation between CEC increase and response, except in the case of PD, although the increase must be greater than 50 % to demonstrate statistical significance. One explanation may lie in the fact that, for logistical reasons, for some of the patients it was not possible to collect the three blood samples for determination of CEC. On reducing the total number of patients who had CEC assessed after three cycles of treatment, we had lower statistical power to find significant differences. Despite the lack of correlation between the quantification of CEC and radiological response, the project has the potential to be multicenter, with samples from different geographic locations. Another difference with the results of published studies resides in the determination method. Wang et al. and Liu et al. used flow cytometry, which identifies the CEC whose expression exposure membrane antibody was as follows: CD45-, CD146? and CD105?. CD 105 was used also in the present study, despite there is increasing evidence that it may be correlated with monocytes rather than endothelial cells. Further investigation is required to determine the biological significance of each representative CEC subsets [39, 40]. Finally, there is a Spanish work which also analyzes the role of CEC in NSCLC: Fleitas et al. [23] determined the
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number of CEC in 60 patients with NSCLC by inmunomagnetic separation. No statistically significant association between baseline CEC levels and response was observed. Unlike our series, in this work stages IIIB and IV patients were included, and different chemotherapy regimens were received. This objective has been analyzed in other malignancies and with different treatment regimens, including in some of these antiangiogenic agents and also showing discrepant results [12, 15, 18, 37, 38]. Another analysis undertaken was baseline CEC quantification as a survival predictor. Median OS for patients with CEC levels above the mean (152.79 cells/4 mL) was 11 vs. 8 months for patients with levels below the mean (p = 0.209). The same findings were recorded for PFS. Median PFS for patients with levels above the mean was 8 months vs. 5 in patients with lower levels (p = 0.295). Data found in the literature again demonstrate the existence of controversy of the role of CEC as a prognostic factor. In the previously mentioned work by Fleitas et al. [23], in patients with NSCLC, elevated basal levels of CEC were correlated with poor survival. In contrast, in the study by Kawaishi previously referenced [17], baseline levels above the mean were correlated with increased PFS (244 vs. 69 days), and OS, coinciding with our results. In the study by Liu et al. [24], baseline levels of CEC were not correlated with PFS; the same results were obtained by Wang et al. [31]. No differences were found between baseline levels depending on histology. CEC studies conducted in other malignancies have also investigated the potential prognostic role of baseline CEC levels without definitive conclusions [12–16]. In conclusion, our study results suggest that high baseline levels of CEC may be a predictor of greater diseasefree survival and OS, although the study is limited by insufficient statistical power. Moreover an increase in cell numbers over 50 % between the first and second cycle of chemotherapy and between the first and third confers a statistically significant risk of progression. Further studies are required and probably a better selection of CEC subpopulations would provide some more information. The results do not clarify the role of the CEC as predictor of response in non-squamous NSCLC treated with chemotherapy, but contribute to the initiation of other lines of research. Thus, it would be very interesting to study the quantification of CEC and their variation, and analyze the correlation with other variables for which the present sample of patients was not sufficient. Differences depending on histological subtype are other interesting issue to evaluate. Finally, and given that the mechanism of action of antiangiogenic drugs is to inhibit the pathway of vascular endothelial growth factor (VEGF), study of the
behavior of CEC in NSCLC patients treated with these drugs could also be relevant. Acknowledgments Funding for this study was provided by Eli Lilly and Company; Eli Lilly and Company as such had no role in data collection, analysis or evaluation. CRO Pivotal provided advice to properly handle and submit the paper. Kavita Gandhi has reviewed and edited the manuscript language. Conflict of interest None author has any actual or potential conflict of interest including any financial, personal or other relationships with other people or organizations that could inappropriately influence (bias) their work. Ethical standard Ethics committees have been informed regarding all of this study procedures. Patients were properly informed and signed an informed consent.
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