| Objective Telomerase activity is the base of cellular immortality, and it is expressed in about 90% of malignant cells. The assay of telomerase activity may be useful for the diagnosis of malignant tumor. p53 gene is an important tumor suppressor gene, and its mutations are associated with the cancerogenesis and development of malignant tumors. In order to find new molecular biology markers for the differential diagnosis of benign and malignant pleural and peritoneal fluids, the telomerase activity in the exfoliative cells and p53 gene mutations in the supernatants were detected and their diagnostic values were assessed.Methods Forty fresh specimens of pleural and peritoneal effusions were obtained from 40 patients with benign or malignant diseases, including 8 malignant pleural fluids (6 lung cancers, 2 malignant pleural fluids with unknown origin), 15 malignant ascites cases (3 primary hepatic carcinomas, 6 ovarian carcinomas, 3 gastric carcinomas, 3 malignant ascites cases with unknown origin), 9 benign pleural fluids (8 tuberculous pleurisy cases, 1 heart failure), 8 benign ascites cases (6 liver cirrhosis cases, 2 pancreatitis cases). Fluid specimens were spun, and the supematants were collected for the analysis of p53 gene mutations and the residual cells were used for the telomerase activity assay. If the fluids were bloody, distilled water was added to the cell pellets to break the red blood cells. Telomerase activity was determined by telomeric repeat amplification protocol (TRAP). The products were analyzed in polyacrylamide gel electrophoresis with silver staining and the presence of .a ladder with 6 base pairs increments was considered as positive result. SMMC-772 1 cells (a human liver carcinoma cell line) were used as positive control, and the extracts from the positive control cells were heated in 650C for 10 mm as negative control. Genome DNA in the fluid supernatant was conventionally extracted by proteinase K digestion followed by phenol/chloroform extraction. Genome DNA extracted from placenta was used as normal control. The exon 5~8 of p53 gene were amplified by PCR, and their mutations were detected by using single-strand conformation polymorphism (SSCP) with silver staining. The presence of abnormal bands or absence of normal band(s)4was considered as positive. The results of cytological examination and the serum tumor makers assays (AFP, CEA, SF) were collected in all the cases for comparison.Results Telomerase activity was detected in 21 (91.3%) of the 23 malignant fluids, and 9 fluids with negative cytological results all presented positive results. Only 2 (11.8%) fluids were positive for telomerase activity in the 17 benign fluids. The positive rate of telomerase activity assay in malignant fluids was significantly higher than that in benign fluids (P<0.001), and also higher than cytological result (60.9%) (P<0.05). The telomerase activity assay for the diagnosis of malignant fluids was with sensitivity 91.3%, specificity 88.2%, positive predictive value 91.3%, negative predictive value 88.2% and diagnosis accordance rate 90.0%, which were much higher than those of the tumor marker assays (AFP/CEA/SF)(P<0.0%0.0 1). Genome DNA extracted from a little supematant was enough for polymerase chain reaction (PCR) in 17 malignant fluids. Exon 5? of p53 gene in all specimens were successfully amplified by PCR. Silver staining SSCP analysis showed that 3 specimens (47.1%) were positive (3 cases with deletion, 5 cases with abnormal bands) compared with normal control, and mainly occurred in exon 5 and 7. p53 gene mutations were found in 4 of 6 patients with negative cytological result and in I of 2 patients with telomerase activity negative.Conclusions1.Telomerase activity was detected in almost malignant fluids, but not in almost benign fluids, which indicates that telomerase is expressed in exfoliative cells of malignant fluids.2.The assay of telomerase activity for the diagnosis of malignant fluids is sensitive and specific and is usefu... |
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