Karim Tabiti

Quantification of CK20 gene and protein expression in colorectal cancer by RT-PCR and immunohistochemistry reveals inter- and intratumour heterogeneity.

Cytokeratin 20 (CK20) is an epithelial protein expressed almost exclusively in the gastrointestinal (GI) tract and is widely used as immunohistochemical marker for routine diagnosis. In contrast, CK20 gene expression is not an established marker for the classification of tumours and the detection of disseminated cancer cells in colorectal cancer. Recently, real‐time reverse transcriptase polymerase chain reaction (RT‐PCR) has provided the means for reproducible and quantitative investigation of molecular markers. This report directly compares CK20 mRNA and protein expression in serial sections of archival, formalin‐fixed, paraffin‐embedded (FFPE) colorectal adenocarcinomas. CK20 expression was detected by immunohistochemistry (IHC) in 60/63 (95.2%) cases, by conventional RT‐PCR in 58/60 (96.7%) and by quantitative RT‐PCR using the LightCycler® (LightCycler® is a trademark of a Member of the Roche Group) System in 29/32 (90.6%) microdissected cases, one case yielding variable results. Despite the high detection rate of all three techniques, marked heterogeneity of CK20 expression was seen between different cases and also within individual cases. CK20 expression profiles were not related to particular histopathological features of the tumours. A good correlation (r = 0.8964) was found between CK20 mRNA and protein expression by comparing quantitative RT‐PCR with IHC in 32 cases. This was also true for selected heterogeneous tumour cells within individual cases. Both RT‐PCR and IHC are therefore valuable tools for CK20 detection in colorectal adenocarcinoma, with real‐time RT‐PCR providing supplementary quantitative information. This suggests a promising supportive role for quantitative RT‐PCR in molecular pathology. Copyright

Effect of blood sample handling and reverse transcriptase-polymerase chain reaction assay sensitivity on detection of CK20 expression in healthy donor blood.

Data concerning the specificity of cytokeratin 20 (CK20) as a reverse transcriptase–polymerase chain reaction analysis (RT–PCR) marker to detect disseminated tumor cells in blood are conflicting. Underlying causes for these discrepancies need to be determined to clarify the significance of CK20 detection. Because differences in RT–PCR assays and blood sample handling may be important, their influence on CK20 detection was studied. Using a series of healthy donor blood samples spiked with colon tumor cells, the authors compared the sensitivities of two conventional PCRs with different primer sets and a quantitative LightCycler PCR (Roche Diagnostics GmbH, Penzberg, Germany). Additionally, the influence of sample collection and preparation on assay specificity was studied by examining CK20 expression in the mononuclear cell fraction (MNC) of the first and the second aliquot of blood drawn from healthy donors and in the granulocyte cell fraction. At the concentration of one spiked tumor cell/mL blood, the CK20 detection frequency varied from 17% and 67% for the conventional to 78% for the LightCycler PCR. In the unspiked samples, CK20 was detected in 0% and 8% of the conventional and in 11% of the LightCycler PCR tests. Quantitative analysis revealed that CK20 was expressed at a high level in the granulocyte samples. The results demonstrate that differences in assay sensitivity and sample handling influence CK20 detection in blood.