Optimal Markers for Real-Time Quantitative Reverse Transcription PCR Detection of Circulating Tumor Cells from Melanoma, Breast, Colon, Esophageal, Head and Neck, and Lung Cancers Liqiang Xi, 1 Daniel G. Nicastri, 1 Talal El-Hefnawy, 2 Steven J. Hughes, 2 James D. Luketich, 2,3 and Tony E. Godfrey 1* Background: The detection of circulating tumor cells (CTCs) may prove useful for screening, prognostication, and monitoring of response to therapy. However, given the large background of circulating cells, it is probably necessary to detect 1 cancer cell in >10 6 leukocytes. Although reverse transcription (RT)-PCR is potentially sensitive and specific enough to achieve this goal, suc- cess will require the use of appropriate mRNA markers. The goal of this study was to identify optimal marker combinations for detection of CTCs. Methods: An extensive literature and internet database survey was conducted to identify potential markers. We then used real-time quantitative RT-PCR to test for expression of selected potential markers in tissue sam- ples from primary tumors of breast, colon, esophagus, head and neck, lung, and melanoma and normal blood samples. Markers with high expression in tumors and a median 1000-fold lower expression in normal blood were considered potentially useful for CTC detection and were tested further in an expanded sample set. Results: A total of 52 potential markers were screened, and 3– 8 potentially useful markers were identified for each tumor type. The mRNAs for all but 2 markers were found in normal blood. Marker combinations were identified for each tumor type that had a minimum 1000-fold higher expression in tumors than in normal blood. Conclusions: Several mRNA markers may be useful for RT-PCR– based detection of CTCs from each of 6 cancer types. Quantification of these mRNAs is essential to distinguish normal expression in blood from that due to the presence of CTCs. Few markers provide adequate sensitivity individually, but combinations of markers may produce good sensitivity for detection of the pres- ence of these 6 neoplasms. © 2007 American Association for Clinical Chemistry Despite surgical resection and adjuvant therapy, the path of many cancer patients unfortunately ends with distant metastatic disease. The prognosis of patients with distant metastases in cancers such as breast, colon, esophagus, head and neck, lung, and melanoma is extremely poor. Five-year survival probability for stage IV patients, for example in lung cancer, is typically 10%, although some reports suggest survival of 30%–35% for selected patients after complete metastectomy (1, 2). Presumably, distant metastases are the result of hematogenous dissemination of tumor cells, and in this context, it is assumed that stage IV patients, and some earlier-stage patients, have circu- lating tumor cells (CTCs) 4 even before clinical manifesta- tion of distant metastasis. Reproducible detection of these rare CTCs is extremely challenging but could potentially be useful for diagnosis, risk stratification, recurrence prediction, development of novel therapies, and treat- 1 Mount Sinai School of Medicine, New York, NY. 2 Department of Surgery, University of Pittsburgh, Pittsburgh, PA. 3 Heart, Lung, and Esophageal Surgery Institute, Pittsburgh, PA. * Address correspondence to this author at: Mount Sinai School of Medi- cine, Box 1079, 1 Gustave L. Levy Pl., New York, NY 10029. Fax 212-241-5432; e-mail: tony.godfrey@mssm.edu. Received October 16, 2006; accepted May 3, 2007. Previously published online at DOI: 10.1373/clinchem.2006.081828 4 Nonstandard abbreviations: CTC, circulating tumor cell; RT, reverse transcription; QPCR, quantitative PCR; C T , threshold cycle; qRT-PCR, quanti- tative RT-PCR. Clinical Chemistry 53:7 1206 –1215 (2007) Molecular Diagnostics and Genetics 1206