Detection and Characterization of Circulating Tumor Associated Cells in Metastatic Breast Cancer

Article
Detection and Characterization of Circulating Tumor Associated Cells in Metastatic Breast Cancer
Zhaomei Mu 1,*, Naoual Benali-Furet 2, Georges Uzan 2, Anaëlle Znaty 2, Zhong Ye 3,
Carmela Paolillo 4, Chun Wang 3, Laura Austin 3, Giovanna Rossi 1, Paolo Fortina 4,5,
Hushan Yang 3 and Massimo Cristofanilli 1,*
1 Department of Medicine-Hematology and Oncology, Robert H Lurie Comprehensive Cancer Center,
Feinberg School of Medicine, Northwestern University, Chicago, IL 60611, USA; giovirossi85@yahoo.it
2 ScreenCell SA, Sarcelles 95200, France; benali@screencell.com (N.B.-F.); guzan@screencell.com (G.U.);
aznaty@screencell.com (A.Z.)
3 Department of Medical Oncology, Sidney Kimmel Cancer Center, Thomas Jefferson University,
Philadelphia, PA 19107, USA; Zhong.Ye@jefferson.edu (Z.Y.); Chun.Wang@jefferson.edu (C.W.);
laustin@gmail.com (L.A.); hushan.yang@jefferson.edu (H.Y.)
4 Department of Cancer Biology, Sidney Kimmel Cancer Center, Thomas Jefferson University,
Philadelphia, PA 19107, USA; carmela.Paolillo@jefferson.edu (C.P.); paolo.Fortina@jefferson.edu (P.F.)
5 Department of Molecular Medicine, University of Rome “Sapienza”, Rome 00185, Italy
* Correspondence: zhaomei.mu@northwestern.edu (Z.M.); massimo.cristofanilli@nm.org (M.C.);
Tel.: +1-312-503-5489 (Z.M.); +1-312-503-5488 (M.C.)
Academic Editor: Dario Marchetti
Received: 5 August 2016; Accepted: 23 September 2016; Published: 30 September 2016
Abstract: The availability of blood-based diagnostic testing using a non-invasive technique holds
promise for real-time monitoring of disease progression and treatment selection. Circulating tumor
cells (CTCs) have been used as a prognostic biomarker for the metastatic breast cancer (MBC).
The molecular characterization of CTCs is fundamental to the phenotypic identification of malignant
cells and description of the relevant genetic alterations that may change according to disease
progression and therapy resistance. However, the molecular characterization of CTCs remains
a challenge because of the rarity and heterogeneity of CTCs and technological difficulties in the
enrichment, isolation and molecular characterization of CTCs. In this pilot study, we evaluated
circulating tumor associated cells in one blood draw by size exclusion technology and cytological
analysis. Among 30 prospectively enrolled MBC patients, CTCs, circulating tumor cell clusters (CTC
clusters), CTCs of epithelial–mesenchymal transition (EMT) and cancer associated macrophage-like
cells (CAMLs) were detected and analyzed. For molecular characterization of CTCs, size-exclusion
method for CTC enrichment was tested in combination with DEPArray™ technology, which allows
the recovery of single CTCs or pools of CTCs as a pure CTC sample for mutation analysis. Genomic
mutations of TP53 and ESR1 were analyzed by targeted sequencing on isolated 7 CTCs from a patient
with MBC. The results of genomic analysis showed heterozygous TP53 R248W mutation from one
single CTC and pools of three CTCs, and homozygous TP53 R248W mutation from one single CTC and
pools of two CTCs. Wild-type ESR1 was detected in the same isolated CTCs. The results of this study
reveal that size-exclusion method can be used to enrich and identify circulating tumor associated
cells, and enriched CTCs were characterized for genetic alterations in MBC patients, respectively.
Keywords: metastatic breast cancer (MBC); circulating tumor associated cells; circulating tumor
cells (CTCs); circulating tumor cell clusters (CTC clusters); epithelial–mesenchymal transition (EMT);
cancer associated macrophage-like cells (CAMLs); size-exclusion technology