title: Specific detection of cytokeratin 20-positive cells in blood of colorectal and breast cancer patients by a high sensitivity real-time reverse transcriptase-polymerase chain reaction method.
abstract: A real-time reverse transcriptase-polymerase chain reaction (RT-PCR) method for detection of cytokeratin 20-positive cells in blood characterized by two novel features was developed and tested on 99 patients with colorectal cancer, 110 with breast cancer, and 150 healthy subjects. To optimize the specificity and sensitivity of the method, two novel features were used. First, a primer overlapping two adjacent exons was generated to inhibit nonspecific amplification both in healthy donors and cancer patients; second, a non-end-point first-round amplification was used to increase sensitivity. The number of first-round cycles was chosen to reach the highest level of sensitivity while conserving quantitative characteristics. PCR efficiency increased from 88.9% in single-round RT-PCR to 99.0% in nested real-time RT-PCR. To establish sensitivity and specificity of the method, HT29 cells were serially diluted with normal blood. Detection limit improved from 100 HT29 cells (single-round RT-PCR) to 1 to 10 cells (nested real-time RT-PCR) per 3 ml of whole blood. None of the healthy subjects was positive, whereas 22 and 29% of all colorectal and breast cancer patients, respectively, had cytokeratin 20 cell equivalents in blood. The association between cytokeratin 20 cell equivalents and metastasis was statistically significant for breast (P = 0.026) but not colorectal cancer patients (P = 0.361). Negativity of all 150 healthy controls examined confers diagnostic potential to the method.
authors.affiliation: Università di Torino, Dipartimento di Genetica, Biologia e Biochimica, Via Santena 5 bis, 10126 Torino, Italy. giuliana.giribaldi@unito.it
authors.forename: Giuliana
authors.initials: G
authors.lastname: Giribaldi
authors.forename: Simone
authors.initials: S
authors.lastname: Procida
authors.forename: Daniela
authors.initials: D
authors.lastname: Ulliers
authors.forename: Franca
authors.initials: F
authors.lastname: Mannu
authors.forename: Roberta
authors.initials: R
authors.lastname: Volpatto
authors.forename: Giorgia
authors.initials: G
authors.lastname: Mandili
authors.forename: Laura
authors.initials: L
authors.lastname: Fanchini
authors.forename: Oscar
authors.initials: O
authors.lastname: Bertetto
authors.forename: Gianruggero
authors.initials: G
authors.lastname: Fronda
authors.forename: Luigi
authors.initials: L
authors.lastname: Simula
authors.forename: Elena
authors.initials: E
authors.lastname: Rimini
authors.forename: Giovanni
authors.initials: G
authors.lastname: Cherchi
authors.forename: Lisa
authors.initials: L
authors.lastname: Bonello
authors.forename: Milena Maria
authors.initials: MM
authors.lastname: Maule
authors.forename: Francesco
authors.initials: F
authors.lastname: Turrini
mesh_terms: D014408:Biomarkers, Tumor / Q000032:analysis*; D001943:Breast Neoplasms / Q000175:diagnosis / Q000378:metabolism*; D002277:Carcinoma / Q000378:metabolism*; D015179:Colorectal Neoplasms / Q000175:diagnosis / Q000378:metabolism*; D006801:Humans; D053554:Keratin-20; D007633:Keratins / Q000097:blood*; D016133:Polymerase Chain Reaction; D012334:RNA, Neoplasm / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
chemical_list: D014408:Biomarkers, Tumor; C507499:KRT20 protein, human; D053554:Keratin-20; D012334:RNA, Neoplasm; D007633:Keratins