Analytical and Bioanalytical Chemistry

, Volume 405, Issue 23, pp 7377–7382

Determination of EGFR mutations in single cells microdissected from enriched lung tumor cells in peripheral blood

Research Paper

DOI: 10.1007/s00216-013-7156-y

Cite this article as:
Ran, R., Li, L., Wang, M. et al. Anal Bioanal Chem (2013) 405: 7377. doi:10.1007/s00216-013-7156-y

Abstract

A minimally invasive and repeatable approach for real-time epidermal growth factor receptor (EGFR) mutation surveillance would be highly beneficial for individualized therapy of late stage lung cancer patients whose surgical specimens are often not available. We aim to develop a viable method to detect EGFR mutations in single circulating tumor cells (CTCs). Using a model CTC system of spiked tumor cells in whole blood, we evaluated EGFR mutation determination in single tumor cells enriched from blood. We used magnetic beads labeled with antibody against leukocyte surface antigens to deplete leukocytes and enrich native CTCs independent of epithelial marker expression level. We then used laser cell microdissection (LCM) to isolate individual CTCs, followed by whole-genome amplification of the DNA for exon 19 microdeletion, L858R and T790M mutation detection by PCR sequencing. EGFR mutations were successfully measured in individual spiked tumor cells enriched from 7.5 ml whole blood. Whole-genome amplification provided sufficient DNA for mutation determination at multiple sites. Ninety-five percent of the single CTCs microdissected by LCM (19/20) yielded PCR amplicons for at least one of the three mutation sites. The amplification success rates were 55 % (11/20) for exon 19 deletion, 45 % (9/20) for T790M, and 85 % (17/20) for L858R. Sequencing of the amplicons showed allele dropout in the amplification reactions, but mutations were correctly identified in 80 % of the amplicons. EGFR mutation determination from single captured tumor cells from blood is feasible with the approach described here. However, to overcome allele dropout and to obtain reliable information about the tumor's EGFR status, multiple individual tumor cells should be assayed.

Figure

Captured CTC from patient blood. CTC (green arrow) and WBCs (red arrow) enriched from 7.5 ml peripheral blood of a NSCLC patient were immunostained with anti-Cytokeratin 18 (green) and anti-CD45 (red) antibodies. Cell nuclei were stained with DAPI (blue). Compared with H1975 (Fig. 1), CTC from the NSCLC patient appeared to have reduced CK18 expression

Keywords

EGFR mutations Single cell Circulating tumor cells Laser cell microdissection Enrichment 

Abbreviations

CTC

Circulating tumor cells

EGFR

Epidermal growth factor receptor

LCM

Laser capture microdissection

NSCLC

Non-small cell lung cancer

TKI

Tyrosine kinase inhibitor

WGA

Whole-genome amplification

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  1. 1.Department of Respiratory DiseasesPeking Union Medical College Hospital, Chinese Academy of Medical SciencesBeijingChina
  2. 2.Department of Biochemistry and Molecular Biology, Institute of Basic Medical SciencesChinese Academy of Medical Sciences and School of Basic Medicine, Peking Union Medical CollegeBeijingChina
  3. 3.Cytelligen IncSan DiegoUSA
  4. 4.Department of Medical OncologyChina-Japan Friendship HospitalBeijingChina