title: Epithelial tumour cell detection and the unsolved problems of nested RT-PCR: a new sensitive one step method without false positive results.
abstract: Sensitive detection of circulating epithelial cancer cells might have important therapeutic and prognostic implications in patients with breast cancer (BC) receiving high-dose chemotherapy and PBSC support. We have compared the specificity and sensitivity of the recently developed 'one tube' reverse transcriptase PCR (RT-PCR) assay with the more widely used nested RT-PCR method for detection of cytokeratin 19 (CK19)-positive cells. The analysis of 30 control samples provides evidence that one tube RT-PCR is highly specific in contrast to the nested method which showed 23% false positive results. The sensitivity of both techniques to detect tumour contamination was 10(-6). PBSC harvests from 45 BC patients were tested with both RT-PCR methods and the results were compared with immunocytochemistry (ICC). The five samples found positive by ICC were also positive by one tube RT-PCR; in addition, 11 more samples were positive by one tube RT-PCR analysis. The greater number of PBSC found positive by one tube RT-PCR might be due to the larger number of cells analysed. We conclude that one tube RT-PCR is sensitive and reveals no false positive results. This method is less time consuming than the nested one, technically simpler and should be considered for tumour cell detection.
authors.affiliation: Division of Medical Oncology, IRCCS S Maugeri Foundation, Pavia, Italy.
authors.forename: M
authors.initials: M
authors.lastname: Battaglia
authors.forename: P
authors.initials: P
authors.lastname: Pedrazzoli
authors.forename: B
authors.initials: B
authors.lastname: Palermo
authors.forename: A
authors.initials: A
authors.lastname: Lanza
authors.forename: F
authors.initials: F
authors.lastname: Bertolini
authors.forename: N
authors.initials: N
authors.lastname: Gibelli
authors.forename: G A
authors.initials: GA
authors.lastname: Da Prada
authors.forename: A
authors.initials: A
authors.lastname: Zambelli
authors.forename: C
authors.initials: C
authors.lastname: Perotti
authors.forename: G
authors.initials: G
authors.lastname: Robustelli della Cuna
mesh_terms: D014408:Biomarkers, Tumor; D001943:Breast Neoplasms / Q000378:metabolism / Q000473:pathology*; D002277:Carcinoma / Q000378:metabolism / Q000473:pathology*; D005189:False Positive Reactions; D005260:Female; D006801:Humans; D007633:Keratins / Q000032:analysis*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
chemical_list: D014408:Biomarkers, Tumor; D007633:Keratins