Molecular profiling of single circulating tumor cells with diagnostic intention

EMBO Molecular Medicine

Bernhard Polzer⁶  Gianni Medoro⁵  Sophie Pasch²  Francesca Fontana⁵  Laura Zorzino³  Aurelia Pestka⁸  Ulrich Andergassen⁸  Franziska Meier-Stiegen¹  Zbigniew T Czyz⁶  Barbara Alberter⁶  Steffi Treitschke⁶  Thomas Schamberger²  Maximilian Sergio⁵  Giulia Bregola⁵  Anna Doffini⁵  Stefano Gianni⁵  Alex Calanca⁵  Giulio Signorini⁵  Chiara Bolognesi⁵  Arndt Hartmann⁴  Peter A Fasching⁷  Maria T Sandri³  Brigitte Rack⁸  Tanja Fehm¹  Giuseppe Giorgini⁵  Nicolò Manaresi⁵ 

[1] Department of Gynecology and Obstetrics, University of Düsseldorf, Düsseldorf, Germany;Experimental Medicine and Therapy Research, University of Regensburg, Regensburg, Germany;Division of Laboratory Medicine, European Institute of Oncology, Milan, Italy;Department of Pathology, University Erlangen, Erlangen, Germany;Silicon Biosystems S.p.A., Bologna, Italy;Project Group “Personalized Tumor Therapy”, Fraunhofer Institute for Toxicology und Experimental Medicine, Regensburg, Germany;Department of Gynecology and Obstetrics, University Erlangen, Erlangen, Germany;Department of Gynecology and Obstetrics, University Munich, Munich, Germany

关键词: breast cancer;    circulating tumor cells;    metastasis;    single cell analysis;   

DOI  :  10.15252/emmm.201404033

来源: Wiley

【 摘 要 】

Abstract

Several hundred clinical trials currently explore the role of circulating tumor cell (CTC) analysis for therapy decisions, but assays are lacking for comprehensive molecular characterization of CTCs with diagnostic precision. We therefore combined a workflow for enrichment and isolation of pure CTCs with a non-random whole genome amplification method for single cells and applied it to 510 single CTCs and 189 leukocytes of 66 CTC-positive breast cancer patients. We defined a genome integrity index (GII) to identify single cells suited for molecular characterization by different molecular assays, such as diagnostic profiling of point mutations, gene amplifications and whole genomes of single cells. The reliability of > 90% for successful molecular analysis of high-quality clinical samples selected by the GII enabled assessing the molecular heterogeneity of single CTCs of metastatic breast cancer patients. We readily identified genomic disparity of potentially high relevance between primary tumors and CTCs. Microheterogeneity analysis among individual CTCs uncovered pre-existing cells resistant to ERBB2-targeted therapies suggesting ongoing microevolution at late-stage disease whose exploration may provide essential information for personalized treatment decisions and shed light into mechanisms of acquired drug resistance.

Synopsis

A novel workflow enabling detection, isolation and characterization of single circulating tumors cells (CTCs) from blood suggests that CTCs may harbor genetic alterations undetectable in the primary tumor and associated with therapy resistance.

• Single circulating tumor cells (CTCs) are analyzed by a semi-automated workflow combining CellSearch® enrichment, DEPArrayTM isolation and Ampli1TM whole genome amplification (WGA).
• The WGA quality of single CTCs is assessed by a genome integrity index (GII).
• The GII predicts outcome of downstream sequence-based molecular assays.
• Single cell analysis reveals the existence of rare potential therapy escape variants.
• The diagnostic precision of the workflow enables molecular monitoring of CTCs under iatrogenic selection.

【 授权许可】

CC BY   
© 2014 The Authors. Published under the terms of the CC BY 4.0 license

Creative Commons Attribution 4.0 License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.