Comparison of immunocytochemistry, reverse transcriptase polymerase chain reaction, and nucleic acid sequence-based amplification for the detection of circulating breast cancer cells.

Detection of tumor cells in blood and bone marrow is increasingly used for the staging of patients with breast cancer and to evaluate the presence of tumor cells in peripheral blood progenitor cell collections to be used after high-dose therapy. We evaluated the sensitivity and specificity of three different methods for detection of tumor cells among non-tumor tissue. An immunocytochemical assay using antibodies directed against epitopes of the cytokeratin-19 (CK19) protein and two RNA-based methods: reverse transcriptase polymerase chain reaction (RT-PCR) and Nucleic Acid Sequence-Based Amplification (NASBA) for the same target gene were tested. With all the three methods, false-positive results were observed when peripheral blood mononuclear cells (PBMC) of healthy volunteers were tested. There was no concordance between the RNA-based assays and the immunocytochemical assay. The false-positive results in the RNA-based assays may be due to 'illegitimate expression' of epithelial genes in normal PBMC. The false-positive results in the immunocytochemical assay resulted from background staining of monocytes and granulocytes. This study demonstrates that CK19 is not a suitable target to detect the presence of breast tumor tells in PBMC. To reliably detect circulating tumor cells with RNA methods, the selection of suitable target genes is required, which are highly expressed in tumors but not at all in normal cells of blood and bone marrow. Genes with such characteristics may be identifiable with novel differential display techniques.

Comparative analysis of p53 gene mutations and protein accumulation in human non-small-cell lung cancer.

A series of 54 resected primary non-small-cell lung carcinomas was analyzed for p53 gene mutations and for p53 protein accumulation and the findings were correlated with clinical parameters. Mutations in exons 5 through 8 of the p53 gene were identified by a denaturing gradient gel electrophoresis (DGGE) assay and cycle sequencing, whereas p53 protein accumulation was detected in paraffin-embedded tissue by immunostaining using 2 different murine monoclonal antibodies (MAbs) (BP53-12 and DO7). A p53 gene mutation and/or p53 protein accumulation was found in 37 of 54 tumors. Mis-sense mutations were closely associated with positive immunostaining, which was intense in 15 out of 17 cases with a mutation. In 10 tumors, obvious p53 accumulation was detected in the absence of mutations in exons 5 through 8. Conversely, only one of 8 p53 non-sense mutations led to detectable p53 accumulation. The most frequent single base changes were G --> T transversions and C --> T transitions. The presence of a p53 alteration was not related to age, tumor size, stage or histology. However, we found a significant inverse correlation between p53 alterations and the presence of a K-ras mutation. This was reflected in the overall postoperative survival data: patients with p53 alterations in their tumors tended to have a better prognosis than those without a p53 alteration; however, this difference was lost when cases with a K-ras mutation were omitted from the analysis.