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
| Autor: | Milena Maule, Giuliana Giribaldi, Oscar Bertetto, Elena Rimini, Franca Mannu, Giorgia Mandili, Luigi Simula, Giovanni Battista Cherchi, Lisa Bonello, Laura Fanchini, Roberta Volpatto, Simone Procida, Daniela Ulliers, Francesco Michelangelo Turrini, Gianruggero Fronda |
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| Rok vydání: | 2006 |
| Předmět: |
Colorectal cancer
Breast Neoplasms Keratin-20 Biology Polymerase Chain Reaction Sensitivity and Specificity Pathology and Forensic Medicine Metastasis Cytokeratin Breast cancer Biomarkers Tumor Tumor Cells Cultured medicine Carcinoma Humans RNA Neoplasm Whole blood Reverse Transcriptase Polymerase Chain Reaction Keratin 20 Cancer medicine.disease Molecular biology Keratins Molecular Medicine Colorectal Neoplasms Regular Articles |
| Zdroj: | The Journal of Molecular Diagnostics. 8:105-112 |
| ISSN: | 1525-1578 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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