Abstract 1448: Identifying and subtyping circulating tumor cells from breast, prostate, and pancreatic cancer patients based on distinct morphology.
Authors: Adams Daniel , Alpaugh R. Katherine , Cristofanilli Massimo , Martin Stuart , Chumsri Saranya , Charpentier Monica , Bergan Raymond C. , Ogden Irene May , Tsai Susan , Zhu Peixuan , Makarova Olga V. , Li Shuhong , Amstutz Platte T. , Tang Cha-Mei 
Published: August 2014

Abstract
Background: Isolation of circulating tumor cells (CTCs) from peripheral blood based on size exclusion is rapid and straight-forward using precision microfilters. We describe the use of CellSieveTM microfilters to isolate CTCs from the peripheral blood of breast, prostate, and pancreatic cancer patients. It is accepted that CTCs isolated from patient samples represent a highly heterogeneous population with varying degrees of epithelial mesenchymal differentiation. We hypothesized that the CTCs from three different epithelial malignancies can be identified and grouped into distinct subtypes by morphological characterization.
Methods: Prostate, breast, and pancreatic patient blood samples were provided by Northwestern University, Fox Chase Cancer Center, University of Maryland, and Medical College of Wisconsin and analyzed by Creatv MicroTech. The CellSieveTM microfilters have 8 micron diameter pores in a uniform array, with 160,000 pores in a 9 mm diameter area. 7.5 mL of whole blood was diluted in fixative and drawn through a microfilter. CTCs collected by this size exclusion technique were post-fixed, permeabilized, and stained with DAPI, cytokeratin 8, 18 and 19 (FITC), EpCAM (PE), PSMA (Texas Red), and CD45 (Cy5). CTCs were CD45 negative cells identified by their morphology, nuclear profile, and expression of cytokeratin, PSMA, and EpCAM.
Results: Each patient sample was found to have a number of phenotypic CTC subtypes. Distinct morphological patterns emerged in the three malignancies. CTCs from breast cancer patients demonstrated high expression of cytokeratin signal with web-like cytokeratin filamentation. Prostate cancer CTCs had less defined filamentation, but intense PSMA and cytokeratin signal and mottled cytokeratin morphology. Pancreatic CTCs had extremely fine filamentation, with spindle-like morphology and little or no EpCAM expression. Within each cancer, CTCs could be grouped into distinct subtypes. Additional markers, such as vimentin (PE), are used to further analyze the cells after bleaching the original PE.
Conclusions: In addition to enumeration and identification, the phenotypic analysis of CTCs provides new information that can be used to characterize disease status for personalized treatment of cancer patients. We have shown that CTCs can have multiple distinct phenotypes. These phenotypic morphologies may implicate definable traits which can be exploited while tracking site directed treatment of metastatic cancer patients.
Citation Format: Daniel Adams, R. Katherine Alpaugh, Massimo Cristofanilli, Stuart Martin, Saranya Chumsri, Monica Charpentier, Raymond C. Bergan, Irene May Ogden, Susan Tsai, Peixuan Zhu, Olga V. Makarova, Shuhong Li, Platte T. Amstutz, Cha-Mei Tang. Identifying and subtyping circulating tumor cells from breast, prostate, and pancreatic cancer patients based on distinct morphology. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1448. doi:10.1158/1538-7445.AM2013-1448