Cancer Diagnosis Program | Biorepositories and Biospecimen Research Branch

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Detection of circulating tumor cells in peripheral blood of patients with metastatic breast cancer: a validation study of the CellSearch system.

Author(s): Riethdorf S, Fritsche H, Müller V, Rau T, Schindlbeck C, Rack B, Janni W, Coith C, Beck K, Jänicke F, Jackson S, Gornet T, Cristofanilli M, Pantel K

Publication: Clin Cancer Res, 2007, Vol. 13, Page 920-8

PubMed ID: 17289886 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated intra- and interassay variability, inter-instrument variability, sensitivity, and specificity in the detection of CTCs in peripheral blood using the CellSearch system and also examined the effects of shipping and storage duration at room temperature or 4°C on cell stability. Blood (10 mL) was collected in CellSave tubes at two collection sites from 92 patients with metastatic breast cancer and seven healthy controls. To avoid contamination of epithelial skin cells, one site collected blood in the Cell Save tube after a first specimen was collected. Specimens were stored at room temperature or 4°C, processed within 72 h using the CellSearch Epithelial Cell kit, and CTCs were enumerated by visualization using a semiautomated fluorescence microscope. Assay sensitivity was also assessed in peripheral blood from healthy volunteers that was spiked with a known number of SK-BR-3 cells (a breast cancer cell line). To assess the effects of storage and shipment on cell stability, blood was collected from 20 metastatic breast cancer patients in two to four CellSave tubes and processed immediately or stored for 24, 48, 72, or 96 h at room temperature or 4°C until processing or after 24-96 h of shipping (details not provided). Inter-instrument variability was assessed by shipping duplicate specimens from Hamburg, Germany to a third testing site in Munich, Germany.

   Summary of Findings:

   Sensitivity of the CellSearch assay was comparable between the two collection sites for CTC detection in peripheral blood specimens from metastatic breast cancer patients for 1 CTC/7.5 mL of blood detected (71.4% and 65.1%), ≥5 CTCs/7.5 mL blood (38.8% and 34.9%), ≥50 CTCs/7.5 mL blood (five and eight patients), and zero CTCs detected (28.6% and 34.9%). A lack of difference in CTC detection between sites that differed in sequential collection suggests any impact of specimen contamination with epithelial cells is nonsignificant. Assay specificity was also similar between the two sites, as CTCs were not detectable in blood collected from seven healthy volunteers at either site. Recovery rates were also similar between the two sites, with 80% and 82% of SK-BR-3 cells recoverable after 4-12 cells (per 7.5 mL blood) were spiked into plasma from healthy donors (recovery ranges 30-200% and 60-90%). Intra-assay variability was low when a single run of eight CellSearch controls containing a defined number of cells (low=50 or high=1,000) were analyzed at both sites with 21-80 cells detected for low controls (CV=26% and CV=18.4%) and 791-1,157 for high controls (CV=2.1% and CV=11.6%). Inter-assay variability, as determined by running one CellSearch control every day for 20 days and by testing two controls every morning and afternoon of the same day, was less than 5% for both sites. Inter-instrument variability was also low as duplicate specimens tested at different sites within 24-48 h did not differ significantly in the number of CTCs (P=0.119), and displayed 78.6% concordance between sites (4/14 cases CTC negative at both sites, 7/14 cases CTC positive for 1-4 CTCs at both sites, three cases CTC positive at only one site). There were no statistically significant differences in the number of detected CTCs following specimen storage for up to 72 h at room temperature or 4°C for either site, but variation in CTC counts was higher when specimens were stored at 4°C. There was also no significant difference in CTC numbers from specimens analyzed after storage at room temperature and those shipped for 24 to 96 h (P = 0.209).

   Biospecimens

   • Bodily Fluid - Blood

   Preservative Types

   • Other Preservative

   Diagnoses:

   • Neoplastic - Carcinoma

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Fluorescent microscopy

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Aliquots and Components	Cell number	≥5 ≥50 0
   Storage	Time at room temperature	0 h 24 h 48 h 72 h
   Storage	Storage duration	0 h 24 h 48 h 72 h
   Storage	Specimen transport duration/condition	24-96 h
   Biospecimen Aliquots and Components	Aliquot sequential collection	1st collection 2nd collection

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