NIH, National Cancer Institute, Division of Cancer Treatment and Diagnosis (DCTD) NIH - National Institutes of Health National Cancer Institute DCTD - Division of Cancer Treatment and Diagnosis

Analysis of circulating tumor cells in patients with non-small cell lung cancer using epithelial marker-dependent and -independent approaches.

Author(s): Krebs MG, Hou JM, Sloane R, Lancashire L, Priest L, Nonaka D, Ward TH, Backen A, Clack G, Hughes A, Ranson M, Blackhall FH, Dive C

Publication: J Thorac Oncol, 2012, Vol. 7, Page 306-15

PubMed ID: 22173704 PubMed Review Paper? No

Purpose of Paper

This paper compared the sensitivities of two different analytical platforms, CellSearch and the isolation by size of epithelial tumor cells” (ISET) method, for the detection of circulating tumor cells (CTCs) in blood collected from patients diagnosed with non-small cell lung cancer (NSCLC). Molecular characteristics of CTCs and circulating tumor microemboli (CTM) isolated by the ISET method were also examined.

Conclusion of Paper

When both platforms were considered, CTCs were detected in 85% of all patients but when platforms were considered separately, ISET was associated with a higher percentage of patients with detectable CTCs (80% compared to 23%) and a higher number of mean CTCs than when analyzed by CellSearch. ISET also resulted in CTCs with a greater range of cell sizes, less contamination of circulating endothelial cells, and recovery of CTMs. Molecular analysis of ISET spots revealed heterogeneity among patient specimens when individual cells, as opposed to CTMs, were analyzed for epithelial (cytokeratin and EGFR) and proliferation (Ki67) markers, as all CTMs were cytokeratin and EGFR positive and Ki67 negative.


  1. Study Purpose

    This study compared the sensitivities of two CTC detection platforms that differ in their approach.  CTC detection by CellSearch is based on enrichment of cells expressing epithelial cell adhesion molecule (EpCAM) while ISET enriches for CTCs based upon cell size.  CTCs were quantified in peripheral blood collected from 40 patients diagnosed with non-small cell lung cancer (NSCLC)up to one hour before an initial chemotherapy cycle. Blood (10 ml) was collected in a CellSave tube, stored at room temperature, and processed according to standard protocols within 96 hours of collection. Specimens were considered positive if ≥ 2 CTCs per 7.5 ml of blood were detected. An additional 8 - 10 ml blood were collected in an EDTA tube, stored at 4°C, and processed by ISET within 4 hours of collection. One ml aliquots were diluted with red cell lysis buffer which contained 0.8% formaldehyde, placed into an ISET filter module (8 µm pore diameter), and filtered using gentle suction. Membranes were air dried at room temperature and then stored at -20°C until morphological analysis, immunohistochemical staining (allophycocyanin, phycoerythrin, CD45, cytokeratin, EpCAM, EGFR, Ki67, and VE-cadherin), and counterstaining (DAPI).

    Summary of Findings:

    When results of both platforms were combined, CTCs were detected in 85% of all patients, but when platforms were considered separately, ISET was associated with a higher percentage of patients with CTCs (80% compared to 23%) and a wider range of cell sizes (12 - 30 µm compared to 4 - 18 µm) in comparison to the CellSearch method. CTCs were detected only by ISET in 25 patients, only by CellSearch in two patients, and detected by both ISET and CellSearch in seven patients. The mean number of CTCs isolated by ISET was also higher than CellSearch (71 versus 4), and there was no statistical concordance between the two techniques (P<0.001, r = 0.988). Contamination with circulating endothelial cells (typically observed in cells isolated by the CellSearch assay) was rarely observed among ISET-isolated cells. CTMs were detectable in 38% of patients using ISET and displayed a similar morphology to clusters of cells within the primary tumor as determined by examination of biopsy and/or cytology tumor specimens; however, CTMs were not detected by CellSearch. To determine if CTCs from NSCLC patients can be EpCAM negative, blood specimens from nine patients were analyzed by CellSearch and ISET using filter spots and all were determined to be EpCAM negative. Of these 9 specimens, six specimens (67%) were negative for CTCs by CellSearch but yielded between 12-188 CTCs per specimen by ISET.  While both platforms detected CTCs in the remaining three specimens, a higher number of CTCs were detected by ISET (17–84 CTCs/7.5 ml of blood) than by CellSearch (3-18 CTCs/7.5 ml of blood). Immunohistochemical analysis of filter spots from blood specimens with ≤ 15 CTCs per 7.5 ml of blood detected by ISET revealed that all CTMs were positive for cytokeratins and EGFR but negative for Ki67. In contrast, a broad range in the percentage of immunopositive cells per patient was observed when individual CTCs were analyzed (0-90% cytokeratin positive, 0-85% EGFR positive, and 0-62% Ki67 positive).

    Preservative Types
    • Other Preservative
    • Neoplastic - Carcinoma
    AnalyteTechnology Platform
    Morphology Light microscopy
    Protein Immunohistochemistry
    Cell count/volume Light microscopy
    Pre-analytical Factors:
    ClassificationPre-analytical FactorValue(s)
    Immunohistochemistry Specific Targeted peptide/protein CD45
    Biospecimen Aliquots and Components Cell capture method CellSearch
    Isolation by size of epithelial tumor cells (ISET)

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