Rüdiger Rüger

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

Identification of occult tumor cells in node negative lymph nodes of colorectal cancer patients by cytokeratin 20 gene and protein expression

Background and aimsEvaluation of cytokeratin 20 (CK20) specific quantitative reverse transcriptase polymerase chain reaction (QRT-PCR) and immunohistochemistry (IHC) for detection of occult tumor cells in lymph nodes of 72 patients with colorectal carcinoma (UICC stage I and II).MethodsSerial sections of formalin-fixed, paraffin-embedded lymph nodes (mean 14.3/case) were used for microdissection, RNA isolation and QRT-PCR and for CK20 IHC using routine protocols. Results of QRT-PCR and IHC were compared and correlated to the CK20 expression pattern of the primary tumors and clinical follow-up.ResultsIHC revealed CK20-positive tumor cells in lymph nodes of 14.5% (10/69) and 0% (0/3) cases with a CK20-positive and CK20-negative primary tumor, respectively. CK20 mRNA was detected in the lymph nodes of 36.8% (7/19) cases by QRT-PCR with all 7 cases also expressing CK20 mRNA in the primary tumor. CK20 mRNA (QRT-PCR) and protein (IHC) detection in serial sections did not agree in 25% (5/20) of cases. A trend was seen towards a worse disease course for patients with CK20-positive lymph nodes by IHC (incidence of recurrent disease) and QRT-PCR (disease-free survival, incidence of recurrent disease).ConclusionCK20-specific IHC and QRT-PCR are supportive tools to conventional histology for detection of occult tumor cells in archival tissues, with the restriction that a laborious QRT-PCR procedure is necessary to achieve appropriate specificity. A prognostic value of CK20 IHC or QRT-PCR for stratification of UICC stage I and II patients into those likely to develop recurrent disease was not evident.