Annals of Oncology Advance Access originally published online on January 22, 2008
Annals of Oncology 2008 19(5):935-938; doi:10.1093/annonc/mdm583
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gastrointestinal tumors |
Circulating tumor cells in colorectal cancer: correlation with clinical and pathological variables
1 Department of Medical Oncology
2 Department of Central Laboratory, Hospital Clínico San Carlos de Madrid, Madrid, Spain
* Correspondence to: Dr J. Sastre, Servicio de Oncología Médica, Hospital Clínico San Carlos, 28040 Madrid, España, Spain. Tel: +34-91-3303546; Fax: +34-91-3303544; E-mail: jsastre.hcsc{at}salud.madrid.org
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Abstract |
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 Top Abstract introductionpatients and methodsresultsdiscussionfundingAcknowledgementsReferences |
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Background: The CellSearch System is a technique to detect circulating tumor cells (CTCs) in patients with cancer. Few data have been published concerning the role of CTCs detection by this method in colorectal cancer. The aim of this study was to correlate the presence of CTCs with the commonest clinical and morphological variables.
Patients and methods: Blood samples were collected from 97 patients and 30 healthy volunteers. Quantification of CTCs in 7.5 ml of blood was carried out with the CellSearch System. The results were expressed as number of CTCs/7.5 ml and the cut-off of 
2 CTCs/7.5 ml was chosen to define the test as positive.
Results: Positive CTCs were detected in 34 of 94 patients (36.2%). Correlation was not found among positive CTCs and location of primary tumor, increased carcinoembryonic antigen level, increased lactate dehydrogenase level or grade of differentiation. Only stage correlated with positive CTCs (20.7% in stage II, 24.1% in stage III and 60.7% in stage IV, P = 0.005).
Conclusions: CTCs detection by CellSearch is a highly reproducible method that correlates with stage but not with other clinical and morphological variables in patients with colorectal cancer. Colon cancer tumor cells are detectable in all stages. Further studies are warranted.
Key words: colorectal cancer, circulating tumor cells, clincal variables
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introduction |
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TopAbstractintroductionpatients and methodsresultsdiscussionfundingAcknowledgementsReferences |
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The CellSearch System (Veridex LLC, Raritan, NJ) has recently been approved by USA Food and Drug Administration for detection of circulating tumor cells (CTCs) in peripheral blood of patients with metastatic breast cancer. Cristofanilli et al. [1] showed in a prospective study that detection of elevated CTCs was a prognostic factor for newly diagnosed metastatic breast cancer patients as well as an accurate indication of rapid progression and mortality [2]. This automated, inmmunomagnetic method for quantification of CTCs has also been validated by three centers in the United States and Europe [3]. Although most current information derives from breast cancer studies, because the test recognizes epithelial cells, it can be suitable for using in different cancers of epithelial origin. In fact, pilot studies in colorectal and prostate cancer found feasible the quantification of CTCs, and preliminary findings indicated its value as prognostic factor [4, 5]. Allard et al. [6], using the CellSearch System in healthy volunteer, patients with nonmalignant diseases and patients with metastatic carcinomas, established that
2 CTCs were only present in malignant epithelial tumors. Taking into account this cut-off, detection of CTCs in advanced colorectal cancer occurred in 30% of patients. The aim of this study was to confirm the feasibility of this method and to correlate the presence of CTCs with the most common clinical and morphological variables.
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patients and methods |
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TopAbstractintroductionpatients and methodsresultsdiscussionfundingAcknowledgementsReferences |
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patients
This prospective single-institution study enrolled 97 consecutive patients with the following criteria: (i) signed inform consent, (ii) newly diagnosed nonmetastatic colon or upper rectum cancer without preoperative chemo or radiotherapy, (iii) newly diagnosed metastatic colorectal cancer, (iv) detection of colorectal cancer relapse in the follow-up and (v) absence of other concomitant or previous malignant disease. A parallel group of 30 healthy volunteers were used as a control group.
CTCs detection
Blood samples were always collected from 4 to 12 weeks after surgery and immediately before adjuvant chemotherapy (if indicated) for patients with early stages and before palliative chemotherapy in those with advanced disease. Samples were stored in CellSave Preservative tubes at room temperature for not >72 h. Quantification of CTCs in 7.5 ml of blood was carried out with the semiautomated CellSearch System (Veridex) which enriches the samples for cells expressing specific epithelial cell adhesion molecule antibodies. The isolated cells were fluorescently stained with the nucleic acid dye 4',6-diaminodino-2-phenylindole (DAPI) and labeled with mAbs specific for leukocytes (CD45) and epithelial cells (cytokeratin 8, 18, 19-phycoerythrin). They were analyzed with the CellSpotter Analyzer (VeridexTM). CTCs were defined as nucleated cells lacking CD45 and expressing cytokeratin, and the interpretation has been independently accomplished by five specialists specifically trained and without information about the clinical characteristics of the patients. The results were expressed as number of CTCs/7.5 ml of blood and the cut-off of 2 CTCs/7.5 ml was chosen to define the test as positive for CTCs presence.
statistical analysis
Correlation of positive CTCs with clinical variables such as carcinoembryonic antigen (CEA) level, lactate dehydrogenase (LDH) level, location of primary tumor and morphological data such as tumor–node–metastasis stage and grade of differentiation were done by contingency table analysis using the chi-square test. For patients diagnosed at stage II, correlations between CTCs and prognostic subgroups were analyzed, considering poor prognostic factors one of the following: acute bowel obstruction at diagnosis, tumor perforation, T4, preoperative increased CEA level and <12 lymph nodes removed. In metastatic colorectal cancer patients, the correlation of CTCs and prognostic subgroups according to Köhne classification [7] was explored.
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results |
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TopAbstractintroductionpatients and methodsresultsdiscussionfundingAcknowledgementsReferences |
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Blood samples for CTCs assessment were taken in 97 consecutive patients with colorectal cancer. Three patients were excluded from this analysis because blood samples were spoiled during the process. Clinical and morphological characteristics of the assessable 94 patients are summarized in Table 1. Overall, presence of
2 CTCs was detected in 34 of 94 patients (36.2%). The mean number of CTCs isolated was 3.4 (range 0–61). Correlation was not found among positive CTCs and location of primary tumor, increased CEA level, increased LDH level and grade of differentiation. Only stage correlated with positive CTCs (20.7% in stage II, 24.1% in stage III and 60.7% in stage IV, P = 0.005) (Table 2). When a threshold of 3 CTCs was considered as positive, again stage remained as the only factor associated with CTCs (12% in stage II, 16% in stage III and 64% in stage IV, P = 0.0001).
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There were no statistically significant differences between positive CTCs found in high-risk stage II patients compared with those in the low-risk group (15.4% versus 25%, P = 0.663). Among patients in stage IV, no differences were found among different prognostic subgroups (Table 3). No CTCs were found in the group of healthy volunteers.
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discussion |
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TopAbstractintroductionpatients and methodsresultsdiscussionfundingAcknowledgementsReferences |
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From a clinical perspective, CTCs detection in colorectal cancer may be of prognostic value and help the clinicians regarding therapeutic decisions since the presence of CTCs may represent an early indicator of disseminated disease. RT–PCR and other techniques such as immunochemistry or flow cytometry have been developed to detect the presence of CTCs, but the results reported with these techniques remain controversial. Allen-Mersh et al. [8] suggested a positive prognostic value, pointing out the low probability of recurrence for the subgroup of patients with negative lymph nodes and complete clearance of CTCs after surgery. CTCs determination may be useful to select patients to exclude from receiving adjuvant chemotherapy. On the contrary, other RT–PCR studies failed to find any prognostic value [9, 10]. All these studies are limited by too much of confounding factors to draw any meaningful conclusions: small sample, false positive, messenger RNA released from necrotic or apoptotic CTCs and others that can be shared with the CellSearch technique such as potential of proliferation of CTCs or downregulation of cytokeratin expression in tumor tissue [11]. Reproducibility is also one of the major problems in this field.
Up to now, few data have been published concerning the role of CTCs detection by the CellSearch System in colorectal cancer. Cohen et al. [4] showed the feasibility of this method in 50 patients with advanced colorectal cancer, 17 of whom had fresh tumor available from surgery for comparison of circulating and in situ tumor cell characteristics. Preliminary results reported by our own group confirmed that CTCs detection by Veridex system is an easy and reproducible technique [12]. Meropol et al. [13], in a multicenter prospective study including 456 patients with metastatic colorectal cancer, demonstrated that CTCs levels before treatment and at subsequent time points were an independent prognostic factor for progression-free survival and overall survival. In their multivariate analysis which included age, Eastern Cooperative Oncology Group (ECOG) performance status, and the line and type of chemotherapy, CTCs levels 3/7.5 ml raised as the strongest predictive factor of outcome. At the time of this analysis, we do not have enough follow-up to confirm the prognostic value of CTCs detection at the same cut-off that will be the object of future analysis. A threshold of 2 CTCs/7.5 ml was chosen in our correlation analysis because 1 CTCs were not found by Allard in healthy volunteers without malignant disease, as we have now confirmed in our healthy volunteers series. The higher sensitivity of this cut-off may be useful to detect positive CTCs in early stages of colorectal cancer if we will be able to demonstrate its prognostic value in future trials. CTCs detection correlates with pathological stage, being significantly more frequent in those patients with advanced disease. In all, 60.7% of stage IV patients included in our series were CTCs positive, in contrast to the 30% previously reported [6, 14]. Correlation of CTCs detected by RT–PCR and the American Joint Committee on Cancer staging were not found when single preoperative samples were taken [10, 15], but significant differences in prevalence have been found when postoperative samples were explored due to a different clearance of CTCs after excision of primary colorectal carcinomas (CTCs prevalence of 16% at 12 weeks for stage I–II versus 54% for stage III and 62% for stage IV, P = 0.0027) [15]. The significantly higher percentage of CTCs in the more advanced stage prompt us to hypothesize whether CTCs detection can be a surrogate marker of tumor burden. No relationship has been found between CTCs and the prognostic subgroups according to the Köhne classification. These subgroups are defined mainly by factors related with tumor burden (ECOG, number of metastatic sites and alkaline phophatase levels) that predict survival. Then, high number of CTCs may be more related with the ability of these cancer cells for migration than a consequence of a high tumor burden.
To our knowledge, no data have been reported in patients with early stages of colorectal cancer with the CellSearch System. In almost a quarter of patients with stage II or III, 2 CTCs/7.5 ml could be detected without significant differences between them. Using multiple blood sampling with two markers for RT–PCR to detect CTCs, >60% of patients had positive CTCs between 6 and 12 weeks after surgery [15]. Follow-up of these patients will be crucial to establish a potential role as prognostic factor for recurrence, as well as future large prospective trials in the adjuvant setting including CTCs determination. CTCs detection would be an easy and reproducible test to select high-risk stage II patient candidates for adjuvant chemotherapy. At the present time, high-risk stage II is defined by clinical/pathological prognostic factors such as T4, perforation, acute bowel obstruction, undifferentiated tumors, high preoperative CEA levels or <12 lymph nodes removed. There is no strong evidence about clear the role of adjuvant chemotherapy even in this subgroup of patients. CTCs detection in this subgroup was not higher than that observed in the low-risk stage II group. In patients in stage III, CTCs detection may be useful to select patients for more or less aggressive approaches.
In conclusion, CTCs detection by CellSearch System (Veridex) is an easy and highly reproducible method that correlates with stage but not with other clinical and morphological variables. Colon cancer tumor cells are detectable in all stages and the clinical significance of that finding deserves future prospective clinical studies.
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Fonolo de Investigaciones Sanitarias (PI 060104); Sanofi-Aventis.
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We thank the study nurses Julio de la Torre, Carmen Seoane and Concha Vila for excellent technical assistance, and Cristina Fernandez for statistical support.
Received for publication September 24, 2007. Revision received November 26, 2007. Accepted for publication November 30, 2007.
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References |
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TopAbstractintroductionpatients and methodsresultsdiscussionfundingAcknowledgementsReferences |
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1. Cristofanilli M, Hayes DF, Budd GT, et al. Circulating tumor cells: a novel prognostic factor for newly diagnosed metastatic breast cancer. J Clin Oncol (2005) 23:1420–1430.
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