Detection of Micrometastases in Pelvic Lymph Nodes in Patients Undergoing Radical Cystectomy for Focally Invasive Bladder Cancer by Real-time Reverse Transcriptase-PCR for Cytokeratin 19 and Uroplakin II

Abstract

Purpose: The objective of this study was to clarify the significance of micrometastases in pelvic lymph nodes in patients who underwent radical cystectomy for bladder cancer. Experimental Design: We included 40 patients with locally invasive bladder cancer who underwent radical cystectomy and pelvic lymphadenectomy. Expression of cytokeratin 19 (CK19), uroplakin II (UP II), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in 760 lymph nodes were assessed by a fully quantitative real-time reverse transcription-PCR (RT-PCR) assay. The quantification value of CK19 or UP II mRNA was described as each value relative to GAPDH mRNA. In this study, we regarded specimen in which either CK19 or UP II mRNA was positive as ''presence of micrometastasis.'' Results: Routine pathologic examinations detected tumor cells in 29 lymph nodes from six patients. Real-time RT-PCR identified positive expression of CK19 and UP II mRNAs in 49 lymph nodes from 10 patients and 98 lymph nodes from 16 patients, respectively. Of 633 lymph nodes from 34 patients with no pathologic evidence of nodal involvement, 13 nodes from five patients and 58 nodes from 10 patients were diagnosed as positive for CK19 and UP II mRNAs expression, respectively, by real-time RT-PCR. Presence of micrometastases was significantly associated with other conventional prognostic variables, including pathologic stage and microvascular invasion. Disease recurrence was occurred in eight patients, among whom four patients were negative for lymph node metastasis by routine pathologic examination and diagnosed as having micrometastasis by real-time RT-PCR assay. Furthermore, causespecific survival rate in patients without micrometastasis was significantly higher than that in those with micrometastasis, irrespective of the presence of pathologic-positive nodes.Conclusions: Approximately 30% of locally invasive bladder cancer shed cancer cells to pelvic lymph nodes, and disease recurrence after radical cystectomy could be explained, at least in part, by micrometastases in pelvic lymph nodes.

Persistent uroplakin expression in advanced urothelial carcinomas: implications in urothelial tumor progression and clinical outcome

“…Because UPs are rather specific for urothelium and its carcinomas, a positive staining would be highly indicative of a urothelial carcinoma, although a negative UP staining does not rule out such a diagnosis. The relatively moderate (~50%) sensitivity of UP detection could be improved considerably by more sensitive detection methods, such as RT-PCR (37). Even a small number of UP-positive tumor cells inside a lymph node in the case of "micrometastases" could conceivably be detected with this method.…”

A Four-Gene Signature from NCI-60 Cell Line for Survival Prediction in Non–Small Cell Lung Cancer

“…Gene expression was quantified in relation to the expression of the respective control gene with the use of sequence detector software and the relative quantification method (Applied Biosystems). The choice of TBP and GAPDH as the internal control for real-time RT-PCR is based on their invariance of expression in cancer cell lines and clinical cancer specimens (14,15).…”

Quantitative Detection of Micrometastases in Pelvic Lymph Nodes in Patients with Clinically Localized Prostate Cancer by Real-time Reverse Transcriptase-PCR

“…Real-time quantitation was done based on TaqMan assay according to the manufacturer's instruction as described previously (14,15). After the generation of a real-time amplification plot based on normalized fluorescence signals, the threshold cycle (Ct), which is the fractional cycle number at which the amount of amplified target reached a fixed threshold, was determined.…”