Circulating epithelial cells in patients with benign colon diseases.
In the present study, we enrolled 53 patients [20 (37.7%) women and 33 (62.3%) men, mean age, 56.7 years; range, 18 -84 years] with benign colon diseases, including diverticulosis, benign polyps, Crohn disease, ulcerative rectocolitis, colonic endometriosis, and others (Table 1) at the Gastrointestinal Surgery Department, Hospital Saint-Eloi, University Medical Centre, Montpellier, France. None of these patients had a history of solid cancer. Twenty-five healthy control individuals were tested in parallel. The bioethics committee approved the study protocol (biobank no. DC2008830), and all patients provided written informed consent. For the detection of epithelial cells in blood, we used 2 independent CTC assays, both of which are based on the use of CKs for detecting tumor cells. Currently, the CellSearch system is the only technology that has been cleared by the FDA for the detection of CTC in patients with metastatic breast, prostate, or colorectal cancer (5-10). This system is based on positive selection for CTCs with antibodies to EpCAM and the staining of EpCAM-positive cells for CKs with 4',6-diamidino-2-phenylindole and for CD45. Interestingly, isolation and detection of CTCs with immunomagnetic-enrichment methods is critically dependent on the EpCAM clone used (11). More recently, we introduced a new functional test, the epithelial immunospot (EPISPOT) assay, which allows the detection of only viable tumor cells (12-15). Leukocytes in the blood sample are depleted with a cocktail of antibodies (not only anti-CD45, but also anti-CD4, anti-CD8, and anti-CD19), and the [CD45.sup.-] cells are cultured ex vivo. Of this cell subpopulation, only CK19-releasing cells are considered CTCs (16, 17). Even when the first 5 mL of peripheral blood were not discarded, blood analyses of healthy controls previously demonstrated the specificity of both assays (7, 17, 18); however, data for age-matched controls with benign diseases were lacking.
Remarkably, we found circulating epithelial cells in our patient cohort with both the CellSearch system and the CK19-EPISPOT assay, but we found no positive events in the group of healthy volunteers. The diagnosis of positively testing patients was based on the detection of positive events according to the strict criteria defined by both technologies and after comparisons with internal positive controls. The CellSearch system detected CK-positive cells in the blood of 6 (11.3%) of 53 patients, and the CK19-EPISPOT assay found CK19-releasing cells in 10 (18.9%) of 53 patients. Only 1 sample was positive with both CTC assays (see Table 1 in the Data Supplement that accompanies the online version of this brief communication at http://www.clinchem.org/content/vol58/issue5). These results demonstrate that the CellSearch and EPISPOT assays are complementary tools for detecting circulating epithelial cells, a not surprising result given the 2 different capture and detection technologies. Beyond the fact that these circulating cells were both EpCAM positive and pan-CK positive (CK8, CK18, CK19) (Fig. 1), their nuclear morphology was more consistent with a benign gland (Fig. 1, case 1). Concerning the CK19-EPISPOT assay, the fluorescent CK19 immunospots were comparable in size and fluorescence intensity to those obtained with the control HT-29 colon cancer cell line (Fig. 1, case 3). The number of positive cells ranged from 3 to 37 with the CellSearch system and from 1 to 41 with the CK19EPISPOT assay (see Fig. 1 in the online Data Supplement). The probability that these cells were rare blood cells ectopically expressing epithelial CKs is low, because CD45+ cells were excluded by either parallel immunostaining (CellSearch) or immunomagnetic depletion (EPISPOT assay). The numbers of CK-positive cells found in the patients with benign diseases with the 2 technologies were significantly lower than in the cancer group, which was used as a positive control in our study (see Fig. 1 in the online Data Supplement). Finally, after 3 years of follow-up, none of these patients have been diagnosed with colorectal cancer or another epithelial cancer. Of note is that both the CellSearch assay and the EPISPOT assay used in this study use CKs as markers to distinguish epithelial cells from leukocytes, but CKs are expressed in normal and malignant epithelial cells.
[FIGURE 1 OMITTED]
In conclusion, our analysis suggests that epithelial cells from nonmalignant colonic epithelium may enter the bloodstream under certain conditions, such as inflammation. This finding is consistent with the fact that inflammatory cytokines can stimulate the migration of epithelial cells (19). With respect to bowel diseases in particular, the potential background of nonmalignant epithelial cells in blood may be an important confounding factor in cancer patients with very low "CTC" counts and may lead to false-positive findings in CTC diagnostics unless strict morphologic criteria are applied. Unambiguous morphologic identification of each marker-positive cell in a given blood or bone marrow sample is difficult (20), however, and most cells detected in our study met the "tumor cell" criteria of the FDA-cleared CellSearch device.
We cannot exclude the possibility that some of the false-positive events detected in our study were actually tumor cells already present in some of the benign lesions or in the adjacent colon, even if such cells were not detected by endoscopic visual inspection and/or via routine histopathologic analysis of the resected samples. Because tumor cell dissemination appears to be an early event in tumor progression, CTCs may appear at very early stages of tumor development, and their detection may have potential for use in the early diagnosis of colon cancer. Large-scale epidemiologic studies with long-term follow-up are required to test this provocative hypothesis. Moreover, additional genetic characterization for mutations in oncogenes [e.g., KRAS  (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog)] or tumor suppressor genes [e.g., TP53 (tumor protein p53)], and fluorescence in situ hybridization-based detection of numerical chromosomal aberrations at the single-cell level may add specificity to current CTC assays. Carcinoma cells are genetically heterogeneous, however, a fact that points to the need for complex technologies for multiplexing single CTCs. Moreover, hundreds of patients with benign disease need to be analyzed, because we assume that only a small fraction of CK-positive cells (if any at all) would be tumor cells, given that most of our patients with benign diseases have an excellent prognosis and will not develop cancer or metastasis. Thus, a large effort is required to prove the nature of circulating CK-positive cells in these patients, but the morphology of these cells suggests that most might not be CTC.
Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 3 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; and (c) final approval of the published article.
Authors' Disclosures or Potential Conflicts of Interest: Upon manuscript submission, all authors completed the Disclosures of Potential Conflict of Interest form. Potential conflicts of interest:
Employment or Leadership: None declared.
Consultant or Advisory Role: None declared.
Stock Ownership: None declared.
Honoraria: None declared.
Research Funding: Grant from the European Commission (DISMAL project, contract no. LSHC-CT-2005-018911); K. Pantel, European Research Council (ERC) Advanced Investigator Grant (no. 269081).
Expert Testimony: None declared.
Role of Sponsor: The funding organizations played no role in the design of study, choice of enrolled patients, review and interpretation of data, or preparation or approval of manuscript.
Acknowledgments: We are grateful to Nathalie Pfizster and Delphine Gueroult in Montpellier, as well as to Cornelia Coith and Oliver Mauermann in Hamburg, for their expert technical assistance.
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Klaus Pantel,  Eric Deneve,  David Nocca,  Amandine Coffy,  Jean-Pierre Vendrell,  Thierry Maudelonde,  Sabine Riethdorf,  and Catherine AlixPanabieres [3, 4, 5] *
 Department of Tumor Biology, University Medical Center Hamburg-Eppendorf, Hamburg, Germany;  Department of Digestive Surgery, University Medical Centre, Saint-Eloi Hospital, Montpellier, France;  University Institute of Clinical Research UM1-EA2415-Epidemiology, Biostatistics and Public Health, Montpellier, France; Laboratory of Rare Human Circulating Cells, Institute of Research in Biotherapy, University Medical Centre, Saint-Eloi Hospital, Montpellier, France;  Laboratory of Cell and Hormonal Biology, University Medical Centre, Arnaud de Villeneuve Hospital, Montpellier, France; * address correspondence to this author at: Laboratory of Rare Human Circulating Cells, Institute of Research in Biotherapy, Saint-Eloi Hospital, University Medical Centre, 80 avenue Augustin Fliche, 34295 Montpellier Cedex 5, France. Fax +33-467330113; e-mail email@example.com.
 Nonstandard abbreviations: CTC, circulating tumor cell; FDA, US Food and Drug Administration; EpCAM, epithelial cell adhesion molecule; CK, cytokeratin; EPISPOT, epithelial immunospot (assay).
 Human genes: KRAS, v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog; TP53, tumor protein p53.
Previously published online at DOI: 10.1373/clinchem.2011.175570
Table 1. Distribution of patients with benign colon diseases related to the presence of CTCs and counts of CTCs detected by the EPISPOT and CellSearch assays. EPISPOT assay P [CTC.sup.+] patient 52.5 0.37 age, years (a) [CTC.sup.+] patients, 10/53 (18.9) n/total (%) (b) Sex Female 3/20(15) 0.72 Male 7/33(21.2) Disease type 0.86 Diverticulosis 5/23(21.7) Benign polyps 1/12 (8.3) Crohn disease 2/7 (28.6) Ulcerative rectocolitis 1/5 (20) Other 1/6(18.7) CTC count (c) Total 2.4 (0-41), 0 Per disease Diverticulosis 3.5 (0-41), 0 Benign polyps 0.5 (0-6), 0 Crohn disease 1 (0-6), 0 Ulcerative rectocolitis 6.4 (0-32), 0 Other 0.4 (0-2), 0 CellSearch assay P [CTC.sup.+] patient age, 65.8 0.13 years (a) [CTC.sup.+] patients, 6/53 (11.3) n/total (%) (b) Sex Female 2/20(10) 1.0 Male 4/33 (12.1) Disease type 1.0 Diverticulosis 3/23 (13) Benign polyps 1/12 (8.3) Crohn disease 1/7 (14.3) Ulcerative rectocolitis 0/5 (0) Other 1/6 (18.7) CTC count (c) Total 1.1 (0-37), 0 Per disease Diverticulosis 2 (0-37), 0 Benign polyps 0.2 (0-3), 0 Crohn disease 0.8 (0-5), 0 Ulcerative rectocolitis 0 Other 0.4 (0-3), 0 (a) Data are presented as the mean. (b) Data are presented as the number of CTC+ patients/total number of patients in the group (percent). (c) Data are presented as the mean (range), median.
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|Title Annotation:||Brief Communication|
|Author:||Pantel, Klaus; Deneve, Eric; Nocca, David; Coffy, Amandine; Vendrell, Jean- Pierre; Maudelonde, Thie|
|Date:||May 1, 2012|
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