Urinary cytokeratin 20 mRNA expression has the potential to predict recurrence in superficial transitional cell carcinoma of the bladder.

Higher levels of cytokeratin 20 (CK 20) mRNA are expressed in malignant urothelial tissue compared to normal tissue. We determined the CK 20 mRNA expression in urine from patients with transitional cell carcinoma (TCC) of the bladder and assessed the biological behavior of such tumors in a 5-year follow-up. Second voided urine was preoperatively collected from 56 patients with bladder carcinoma, from 20 patients with nonmalignant urological diseases and from 40 healthy volunteers. RNA extraction from exfoliated urothelial cells was followed by quantitative real-time RT-PCR with the Light Cycler. Patients in the superficial TCC group had a median expression of 8226AU (arbitrary units) with and 1523AU without tumor recurrence (P=0.023). No such correlation was detected in the group with muscle-invasive tumors. Kaplan-Meier analysis revealed a significant difference between recurrent and nonrecurrent disease (P=0.019) in superficial but not in muscle-invasive TCC (P=0.84). CK 20 mRNA expression in urine has the potential to identify patients at risk for recurrence of noninvasive papillary urothelial tumors. It helps to categorize patients prior to TUR-B, so that the cystoscopy interval during follow-up may be extended in those with low-risk superficial TCC.

Real-time PCR complements immunohistochemistry in the determination of HER-2/neu status in breast cancer.

BACKGROUND: The clinical benefit of determining the status of HER-2/neu amplification in breast cancer patients is well accepted. Although immunohistochemistry (IHC) is the most frequently used method to assess the over-expression of HER-2 protein, fluorescent in-situ hybridization (FISH) is recognized as the "gold standard" for the determining of HER-2/neu status. The greatest discordance between the two methods occurs among breast tumors that receive an indeterminate IHC score of 2+. More recently, a real-time polymerase chain reaction (PCR) assay using the LightCycler(R) has been developed for quantifying HER-2/neu gene amplification. In this study, we evaluated the sensitivity and specificity of a commercially available LightCycler assay as it compares to FISH. To determine whether this assay provides an accurate alternative for the determination of HER-2/neu status, we focused primarily on tumors that were deemed indeterminate or borderline status by IHC. METHODS: Thirty-nine breast tumors receiving an IHC score of 2+ were evaluated by both FISH and LightCycler(R) technologies in order to determine whether quantitative real-time PCR provides an accurate alternative for the determination of HER-2/neu status. RESULTS: We found a high concordance (92%) between FISH and real-time PCR results. We also observed that 10% of these tumors were positive for gene amplification by both FISH and real-time PCR. CONCLUSION: The data show that the results obtained for the gene amplification of HER-2/neu by real-time PCR on the LightCycler instrument is comparable to results obtained by FISH. These results therefore suggest that real-time PCR analysis, using the LightCycler, is a viable alternative to FISH for reassessing breast tumors which receive an IHC score of 2+, and that a combined IHC and real-time PCR approach for the determination of HER-2 status in breast cancer patients may be an effective and efficient strategy.

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.

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.