Circulating Tumor Cells Count and Morphological Features in Breast, Colorectal and Prostate Cancer Sjoerd T. Ligthart 1 , Frank A. W. Coumans 1 , Francois-Clement Bidard 2 , Lieke H. J. Simkens 3 , Cornelis J. A. Punt 3 , Marco R. de Groot 4 , Gerhardt Attard 5 , Johann S. de Bono 5 , Jean-Yves Pierga 2 , Leon W. M. M. Terstappen 1 * 1 Department of Medical Cell BioPhysics, MIRA Institute, University of Twente, Enschede, The Netherlands, 2 Department of Medical Oncology, Institut Curie, Paris, France, 3 Department of Medical Oncology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands, 4 Department of Internal Medicine, Medisch Spectrum Twente, Enschede, The Netherlands, 5 The Royal Marsden NHS Foundation Trust, Sutton, Surrey, United Kingdom Abstract Background: Presence of circulating tumor cells (CTC) in patients with metastatic breast, colorectal and prostate cancer is indicative for poor prognosis. An automated CTC (aCTC) algorithm developed previously to eliminate the variability in manual counting of CTC (mCTC) was used to extract morphological features. Here we validated the aCTC algorithm on CTC images from prostate, breast and colorectal cancer patients and investigated the role of quantitative morphological parameters. Methodology: Stored images of samples from patients with prostate, breast and colorectal cancer, healthy controls, benign breast and colorectal tumors were obtained using the CellSearch system. Images were analyzed for the presence of aCTC and their morphological parameters measured and correlated with survival. Results: Overall survival hazard ratio was not significantly different for aCTC and mCTC. The number of CTC correlated strongest with survival, whereas CTC size, roundness and apoptosis features reached significance in univariate analysis, but not in multivariate analysis. One aCTC/7.5 ml of blood was found in 7 of 204 healthy controls and 9 of 694 benign tumors. In one patient with benign tumor 2 and another 9 aCTC were detected. Significance of the study: CTC can be identified and morphological features extracted by an algorithm on images stored by the CellSearch system and strongly correlate with clinical outcome in metastatic breast, colorectal and prostate cancer. Citation: Ligthart ST, Coumans FAW, Bidard F-C, Simkens LHJ, Punt CJA, et al. (2013) Circulating Tumor Cells Count and Morphological Features in Breast, Colorectal and Prostate Cancer. PLoS ONE 8(6): e67148. doi:10.1371/journal.pone.0067148 Editor: Masaru Katoh, National Cancer Center, Japan Received March 9, 2013; Accepted May 13, 2013; Published June 27, 2013 Copyright: ß 2013 Ligthart et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by Veridex LLC. GA and JdB are employees of the Section of Medicine at The Institute of Cancer Research which is supported by a Cancer Research UK program grant and an Experimental Cancer Medical Centre (ECMC) grant from Cancer Research UK and the Department of Health (Ref: C51/A7401). GA and JdB also acknowledge NHS funding to the NIHR biomedical research centre. The IC2006-04 study was funded by a public grant from the French government (PHRC AOM06156). The IMMC-01, IMMC-06, and IMMC-38 studies were funded by Veridex LLC. The sponsors of this study had no role in study design, data analysis, data interpretation, or writing of the report. All authors had full access to all the data and the corresponding author had final responsibility for the decision to submit for publication. Competing Interests: GA and JdB are employees of The Institute of Cancer Research, which has a commercial interest in the development of abiraterone acetate and GA is on The Institute of Cancer Research list of rewards to inventors of abiraterone acetate. JdB has served as a paid consultant for Johnson & Johnson and has received research support from Veridex. JYP received research grants and lecture honoraria from Veridex. FCB received lecture honoraria from Veridex. LT is a consultant for Veridex LLC. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: l.w.m.m.terstappen@utwente.nl Introduction Case studies showing the presence of tumor cells in blood of cancer patients have been reported for more than a century [1–4]. These circulating tumor cells (CTC) are extremely rare and technology to reliably enumerate CTC has only become available in recent years [5]. Studies conducted with the validated CellSearch system established the relation between the presence of CTC and poor outcome [6–10]. The ability to assess the presence of treatment targets on CTC demonstrates the potential for a real time liquid biopsy and has certainly spurred the interest in CTC [11–17]. The recent emergence of different technologies to identify CTC has been accompanied with a large range of CTC definitions urging the need for standardization [5,18–31]. Although different CTC characteristics may indeed relate to clinical outcome [32], it is of utmost importance that counting and characterization of CTC can be done accurately, reproducibly, and is validated in controlled clinical studies. Studies have shown that even in the FDA cleared CellSearch system intra-reviewer, inter-reviewer, and inter-laboratory variability is substantial [33,34]. Recently, we developed a CTC detection algorithm that counts CTC in images recorded by the CellSearch system [35]. This algorithm was not developed to copy human reviewers that assign events as CTC, but it used survival data of metastatic prostate cancer patients to arrive at a definition that optimally stratified the patients into groups with favorable and unfavorable survival [35]. This algorithm eliminates reviewer variability, is fast and decreases PLOS ONE | www.plosone.org 1 June 2013 | Volume 8 | Issue 6 | e67148