Molecular characterization of circulating tumor cells in large quantities of contaminating leukocytes by a multiplex real-time PCR.
Detection of circulating tumor cells (CTCs) in whole blood from metastatic cancer patients by the CellSearch CTC Test (Veridex LLC, Warren, NJ, USA) has been shown to have clinical relevance. In addition to enumeration, there is great interest in molecular characterization of these CTCs. We aimed to establish a robust method to perform mRNA expression analysis of multiple genes by a real-time reverse transcriptase (RT)-PCR on small numbers of CTCs enriched from whole blood by the CellSearch system. Despite the 4 log depletion of leukocytes after CellSearch enrichment, the CTC-enriched fractions still contained leukocytes, in particular B-lymphocytes, which severely interfered with our CTC-specific gene expression profiling. After extensive washing and leukocyte-specific depletion by anti-CD45 coated magnetic beads prior to CellSearch enrichment, the number of leukocytes present in the enriched fraction was still high (range 60-929). However, by using a set of genes with no or minor expression by leukocytes, we succeeded to perform quantitative gene expression profiling specific for as little as one breast cancer CTC present in a CTC-enriched environment typically containing over 800 contaminating leukocytes. Our method allows molecular characterization specific for as little as one CTC, and can be used to expand the understanding of the biology of metastasis and, potentially, to improve patient management.
Sieuwerts
Anieta M
AM
Department of Medical Oncology, Josephine Nefkens Institute and Cancer Genomics Centre, Erasmus MC, Room BE-400, Dr. Molewaterplein 50, 3015 GE, Rotterdam, The Netherlands. a.sieuwerts@erasmusmc.nl
Kraan
Jaco
J
Bolt-de Vries
Joan
J
van der Spoel
Petra
P
Mostert
Bianca
B
Martens
John W M
JW
Gratama
Jan-Willem
JW
Sleijfer
Stefan
S
Foekens
John A
JA
D014408:Biomarkers, Tumor / Q000097:blood / Q000235:genetics; D001943:Breast Neoplasms / Q000097:blood* / Q000235:genetics*; D002469:Cell Separation / Q000379:methods*; D005260:Female; D005434:Flow Cytometry; D020869:Gene Expression Profiling / Q000379:methods*; D006801:Humans; D007962:Leukocytes / Q000166:cytology*; D009360:Neoplastic Cells, Circulating; D020133:Reverse Transcriptase Polymerase Chain Reaction
D014408:Biomarkers, Tumor