Geburtshilfe Frauenheilkd 2014; 74 - PO_Onko01_18
DOI: 10.1055/s-0034-1388344

Genomic high resolution profiling of single CK+/CD45- CTCspurified by flow sorting fromclinical CellSearch samples

R Neves 1, K Raba 2, O Schmidt 3, E Honisch 4, F Meier-Stiegen 4, B Behrens 1, B Möhlendick 1, T Fehm 4, H Neubauer 4, CA Klein 3, 5, B Polzer 5, C Sproll 6, JC Fischer 2, D Niederacher 4, N Stoecklein 1
  • 1Department of General, Visceral and Paediatric Surgery, Heinrich-Heine University, Duesseldorf, Germany
  • 2Institute for Transplantation Diagnostics and Cell Therapeutics, Heinrich-Heine University, Duesseldorf, Germany
  • 3Chair of Experimental Medicine and Therapy Research, University of Regensburg, Regensburg, Germany
  • 4Department of Obstetrics and Gynaecology, Heinrich-Heine University, Duesseldorf, Germany
  • 5Fraunhofer Institute for Toxicology and Experimental Medicine, Regensburg, Germany
  • 6Department of Maxillo- and Facial Plastic Surgery, Medical Faculty, University Hospital, Heinrich Heine University, Duesseldorf, Germany

Circulating tumour cells (CTCs) detected by CellSearchTM are important prognostic markers. To learn more about their biology and to use CTCs for clinical applications in sense of a liquid biopsy, we established and tested a workflow for purification of CTCs from CellSearchTM cartridges using a flow sorting approach and that allows their subsequent high-resolution genomic profiling.

34 CellSearchTM pre-processed breast cancer clinical samples were used for single CTCs sorting in a MoFlo sorter. Genome of single CTCs was amplified using an MseI adapter-linker-based PCR. Amplification products were analysed by high-resolution array-based comparative genomic hybridization (aCGH); gene specific real-time PCR based assay to confirm CyclinD1 locus amplification; and genomic sequencing to screen mutations in exon 20 of PIK3CA gene.

A flow sorting protocol was appropriate to the analysis of almost 90% of the cartridges tested. The number of CTCs detected by flow cytometry correlated well with those detected by CellSearchTM. The genome of 72.9% of the sorted single CTCs could be amplified. In over 95% of the cells chromosomal aberrations typically observed in metastatic breast cancers were detected and results for CCND1 locus were validated by real-time PCR. Typical mutations in exon 20 of PIK3CA gene in cells isolated from two patients were detected.

We present a workflow to isolate CTCs from clinical CellSearchTM samples enabling their subsequent genomic profiling using array CGH. Our data give an impression on the genomic makeup of CTCs from metastasized breast cancer patients at high-resolution and might significantly facilitate their further molecular characterization.