Detection of micrometastasis by cytokeratin 20 RT-PCR is limited due to stable background transcription in granulocytes

The reverse transcription polymerase chain reaction (RT-PCR) amplification of cytokeratin 20 (CK20) mRNA is considered a promising candidate method for the detection of circulating tumour cells in bone marrow and peripheral blood of cancer patients. In this study we have investigated the diagnostic specificity of the CK20 mRNA detection in samples from healthy donors (HD; n = 33), intensive care units patients (ICU; n = 20) and bone marrow obtained from patients suffering from chronic inflammatory diseases (CID; n = 14). RNAs purified from stabilized lysates showed positive results in 24% of the HD group (8/33), 35% of the ICU group (8/20) and in 40% of the CID group (5/14). The use of Ficoll gradients to separate nucleated cells completely restored the specificity of this CK20 RT-PCR assay. The CK20-expressing cells are positively identified to belong to the granulocyte fraction of leucocytes, which appear to express the gene on a background level. Our results demonstrate for the first time that CK20 mRNA expression is not limited to epithelium. Its occurrence in normal granulocytes has to be considered in tests designed to detect circulating cancer cells or micrometastases. © 1999 Cancer Research Campaig

Detection of cytokeratins 19/20 and guanylyl cyclase C in peripheral blood of colorectal cancer patients

The clinical significance of detecting supposed tumour cell-derived mRNA transcripts in blood using the polymerase chain reaction (PCR) remains unclear. We have used a fully quantitative 5′-nuclease RT-PCR assay to screen for the expression of cytokeratins (ck) 19 and 20 and guanylyl cyclase C (GCC) in the peripheral blood of 21 healthy controls and 27 colorectal cancer patients. Expression of cytokeratin 19 and 20 mRNA was detected in 30% and 100% of samples, respectively, taken from healthy volunteers. There was no apparent difference in ck19 and ck20 mRNA transcription levels between controls and patients, or between patients with different Dukes' stages. While GCC mRNA was detected in only 1/21 control samples, it was expressed in approximately 80% of patients, although again there was no correlation between GCC levels and disease stage. Transcription levels of all three markers varied considerably between samples, even between samples taken from the same person at different times. We conclude that neither ck19 nor ck20 are reliable markers for the detection of colon epithelial cells in peripheral blood and that an evaluation of the usefulness of GCC awaits further longitudinal studies. © 1999 Cancer Research Campaig

The human keratins: biology and pathology

The keratins are the typical intermediate filament proteins of epithelia, showing an outstanding degree of molecular diversity. Heteropolymeric filaments are formed by pairing of type I and type II molecules. In humans 54 functional keratin genes exist. They are expressed in highly specific patterns related to the epithelial type and stage of cellular differentiation. About half of all keratins—including numerous keratins characterized only recently—are restricted to the various compartments of hair follicles. As part of the epithelial cytoskeleton, keratins are important for the mechanical stability and integrity of epithelial cells and tissues. Moreover, some keratins also have regulatory functions and are involved in intracellular signaling pathways, e.g. protection from stress, wound healing, and apoptosis. Applying the new consensus nomenclature, this article summarizes, for all human keratins, their cell type and tissue distribution and their functional significance in relation to transgenic mouse models and human hereditary keratin diseases. Furthermore, since keratins also exhibit characteristic expression patterns in human tumors, several of them (notably K5, K7, K8/K18, K19, and K20) have great importance in immunohistochemical tumor diagnosis of carcinomas, in particular of unclear metastases and in precise classification and subtyping. Future research might open further fields of clinical application for this remarkable protein family