Accepted Article Preview: Published ahead of advance online publication www.jidonline.org Circulating Melanoma Cell Subpopulations: Their Heterogeneity and Differential Responses to Treatment OPEN Elin S Gray, Anna Reid, Samantha Bowyer, Leslie Calapre, Kelvin Siew, Robert Pearce, Lester Cowell, Markus H Frank, Michael Millward, Mel Ziman Cite this article as: Elin S Gray, Anna Reid, Samantha Bowyer, Leslie Calapre, Kelvin Siew, Robert Pearce, Lester Cowell, Markus H Frank, Michael Millward, Mel Ziman, Circulating Melanoma Cell Subpopulations: Their Heterogeneity and Differential Responses to Treatment, Journal of Investigative Dermatology accepted article preview April 2015; doi: 10.1038/jid.2015.127 This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication NPG are providing this early version of the manuscript as a service to our customers The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply This work is licensed under a Creative Commons Attribution-NonCommercialShareAlike 4.0 International License The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material To view a copy of this license, visit http://creativecommons.org/licenses/by-nc-sa/4.0/ Received 29 September 2014; accepted 19 March 2015; Accepted article preview online April 2015 © 2015 The Society for Investigative Dermatology Title Circulating melanoma cell subpopulations: Their heterogeneity and differential responses to treatment Authors Elin S Gray1, Anna Reid1, Samantha Bowyer2, Leslie Calapre1, Kelvin Siew2, Robert Pearce1, Lester Cowell3, Markus H Frank1,4, Michael Millward2,5, and Mel Ziman1,6 Affiliations School of Medical Sciences, Edith Cowan University, Perth, WA, Australia Department of Medical Oncology, Sir Charles Gairdner Hospital, Nedlands, WA, Australia Level Melanoma Skin Cancer Clinic, Fremantle, WA Transplantation Research Program, Boston Children’s Hospital and Department of Dermatology, Brigham and Women’s Hospital, Harvard Medical School, Boston, USA School of Medicine and Pharmacology, University of Western Australia, Crawley, WA, Australia School of Pathology and Laboratory Medicine, University of Western Australia, Crawley, Australia Corresponding author Elin S Gray, School of Medical Sciences, Edith Cowan University, Perth, WA, Australia 270 Joondalup Drive, Joondalup, Perth, WA 6027 e.gray@ecu.edu.au Short title Circulating melanoma cell subpopulations Abbreviations CTCs: Circulating tumour cells; PFS: progression free survival © 2015 The Society for Investigative Dermatology ABSTRACT Metastatic melanoma is a highly heterogeneous tumour, thus methods to analyse tumour-derived cells circulating in blood should address this diversity Taking this into account, we analysed, using multiparametric flow cytometry, the co-expression of the melanoma markers MCAM and MCSP and the tumour initiating markers ABCB5, CD271 and RANK in individual circulating tumour cells (CTCs) from 40 late stages (III-IV) and 16 early stages (I-II) melanoma patients CTCs were heterogeneous within and between patients, with limited co-expression between the five markers analysed Analysis of patient matched blood and metastatic tumours revealed that ABCB5 and RANK subpopulations are more common amongst CTCs than in the solid tumours, suggesting a preferential selection for these cells in circulation Pairwise comparison of CTC subpopulations longitudinally before and 6-13 weeks after treatment initiation, showed that the percentage of RANK+ CTCs significantly increased in the patients undergoing targeted therapy (N=16, P