Improved method for isolating cell-free DNA.
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Bronchoalveolar Lavage Fluid↗
Biomedical subjects
Sabine Weickmann
Publications and source records attributed to Sabine Weickmann.
Quantification of free RNA in serum and bronchial lavage: a new diagnostic tool in lung cancer detection?
Circulating cell-free nucleic acids have been detected in serum. In cancer patients levels of free DNA seem to be higher than in non-tumor controls and the detectable nucleic acids are partly of tumor origin. We asked whether free RNA can be detected as well in cell-free bronchial lavage fluid (BLF) supernatant, and whether quantification of free RNA allows to discriminate between tumor and non-tumor patients. 73 patients with lung cancer (NSCLC n=62, SCLC n=11) and 56 patients with non-malignant lung diseases were included. The RNA was isolated from 1 mL serum and lavage, respectively with the Quiamp MinElute Virus Vacuum Kit (Qiagen, Germany). A real-time quantitative RT-PCR assay was used for transcript quantification of the glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. Intact RNA was detectable in cell-free supernatants of BLF from 126/129 patients and was investigated in all 64 serum samples. RNA levels were higher in the cell-free supernatant of BLF than in serum. RNA concentration in the BLF from tumor patients was higher than in patients with a benign lung disease (p=0.009). In conclusion, quantification of intact RNA isolated from BLF supernatant and from serum might become a valuable tool for differentiation between tumor and non-tumor patients.
Adult↗
TP53 gene in blood plasma DNA of tumor patients.
Tumor-specific TP53 mutations are detectable in the blood plasma of tumor patients. Mutations of the TP53 tumor suppressor gene are risk factors for tumor progression. The objective of this work is to compare the presence of TP53 mutations in plasma-DNA before and after tumor treatment with the status of this gene in the tumor tissue sample. DNA was extracted from plasma samples of 25 patients with gastrointestinal tumors, and from paraffin-embedded tumor tissues from the same patients. Temperature gradient gel electrophoresis (TGGE) was performed for mutation screening of exons 5-8 of GC-clamped polymerase chain reaction products. Mutation-positive and wildtype gel bands from TGGE were cut and reamplified for fluorescence-labeled sequence analysis. The results of several mutation analyses were correlated with analysis of p53 autoantibodies in the same plasma. Mutation frequency (one or several mutations per sample) was 7.1% in blood plasma of tumor-free patients, 87.0% in tumor tissues, 78.6% in plasma before tumor treatment, and 36.8% after treatment. Fifteen of 22 mutations in tumor tissues of 13 patients also were detected in the same exons of plasma before treatment (68.2%). Mutations in plasma after treatment (2-684 days) were the same in 6 of 30 cases of tissue mutations only. Six of seven patients with mutations after treatment in their plasma had metastases. One patient was p53 autoantibody negative, but has a terminator mutation of codon 196 in tissue and in posttreatment plasma as well. Genetic analysis of plasma in tumor patients should be further developed, as it might be of prognostic value.
Autoantibodies↗