Abstract
Tumor-derived nucleic acids are frequently present in body fluids (e.g. urine, blood, sputum, stool, bile, and cerebrospinal fluid) that had contact with malignant or premalignant tissues. Detection of this mutant DNA or quantification of aberrant gene expression may offer new noninvasive methods for the diagnosis, staging, and follow-up of patients with cancer. Because only minute amounts of DNA are present in most body fluids, sensitive and reproducible assays are necessary for DNA isolation. We used real-time quantitative PCR to compare several DNA isolation methods. Subsequently, DNA was isolated from serum, cerebrospinal fluid, and bile and tested for the presence of K-ras oncogene mutations. Mutations identical to the tumor were found in serum of colorectal cancer patients with advanced disease. In cerebrospinal fluid of patients with leptomeningeal metastases, K-ras mutations could be detected before cytological confirmation, currently the gold standard. However, bile analysis could not be used for the detection of colorectal liver metastases. Next, accurate quantification of hTERT mRNA, a promising tumor marker, was performed using real-time quantitative RT-PCR. Five different types of tissues associated with body fluids were selected and healthy and tumor tissues were tested for levels of hTERT expression. All tumors expressed hTERT, whereas healthy tissues had lower or no expression. Bladder tumors were selected for a more extensive analysis. A correlation was found with prognosis, defined by stage and grade of the tumor. Moreover, quantification of hTERT mRNA in bladder washings could identify aggressive tumors with high progression risk. In agreement with literature reports our results confirm that aggressive and metastatic tumors frequently shed cells or free DNA in body fluids, whereas small noninvasive tumors can be detected less frequently. Therefore, detection of molecular tumor markers in body fluids offers good prospects for diagnosis of systemic disease
