Reliable single cell array CGH for clinical samples.
BACKGROUND
Disseminated cancer cells (DCCs) and circulating tumor cells (CTCs) are extremely rare, but comprise the precursors cells of distant metastases or therapy resistant cells. The detailed molecular analysis of these cells may help to identify key events of cancer cell dissemination, metastatic colony formation and systemic therapy escape.


RESULTS
Using the Ampli1™ whole genome amplification (WGA) technology and high-resolution oligonucleotide aCGH microarrays we optimized conditions for the analysis of structural copy number changes. The protocol presented here enables reliable detection of numerical genomic alterations as small as 0.1 Mb in a single cell. Analysis of single cells from well-characterized cell lines and single normal cells confirmed the stringent quantitative nature of the amplification and hybridization protocol. Importantly, fixation and staining procedures used to detect DCCs showed no significant impact on the outcome of the analysis, proving the clinical usability of our method. In a proof-of-principle study we tracked the chromosomal changes of single DCCs over a full course of high-dose chemotherapy treatment by isolating and analyzing DCCs of an individual breast cancer patient at four different time points.


CONCLUSIONS
The protocol enables detailed genome analysis of DCCs and thereby assessment of the clonal evolution during the natural course of the disease and under selection pressures. The results from an exemplary patient provide evidence that DCCs surviving selective therapeutic conditions may be recruited from a pool of genomically less advanced cells, which display a stable subset of specific genomic alterations.
Experimental Medicine and Therapy Research, University of Regensburg, Regensburg, Germany ; Project Group Personalized Tumor Therapy, Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, Regensburg, Germany.
Zbigniew T
ZT
Czyż
Project Group Personalized Tumor Therapy, Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, Regensburg, Germany.
Martin
M
Hoffmann
Hospital Augsburg, II. Medical Center, Augsburg, Germany.
Günter
G
Schlimok
Project Group Personalized Tumor Therapy, Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, Regensburg, Germany.
Bernhard
B
Polzer
Experimental Medicine and Therapy Research, University of Regensburg, Regensburg, Germany ; Project Group Personalized Tumor Therapy, Fraunhofer Institute for Toxicology and Experimental Medicine ITEM, Regensburg, Germany.
Christoph A
CA
Klein
D045744:Cell Line, Tumor; D055028:Comparative Genomic Hybridization; D056915:DNA Copy Number Variations / Q000235:genetics; D017931:DNA Primers / Q000378:metabolism; D006801:Humans; D009369:Neoplasms / Q000473:pathology; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D059010:Single-Cell Analysis; D013194:Staining and Labeling
D017931:DNA Primers