Quantitative reverse transcription-polymerase chain reaction detection of cytokeratin 20 in noncolorectal lymph nodes.

Abstract

PURPOSE: Unexpected reverse transcription-PCR detection of cytokeratin 20 (CK20) in samples from healthy individuals and cancer types not expected to express CK20 has cast uncertainty on the role of CK20 as a specific marker of disseminated colorectal cells. We aimed to clarify the specificity of CK20 by examining its expression profile by real-time reverse transcription-PCR. EXPERIMENTAL DESIGN: A quantitative real-time PCR assay on the LightCycler instrument was developed and used to examine CK20 expression in tumors and lymph nodes from subjects with colorectal and breast carcinoma, head and neck and vulval squamous cell carcinoma, and melanoma. To select a method for reproducible quantification, four approaches were evaluated. RESULTS: The developed assay allowed rapid, convenient-to-use, specific, sensitive, and reproducible CK20 quantification amenable to large-scale analysis. For quantity calculation, an efficiency-adjusted relative ratio method was selected that controls for RNA loading and integrity as well as inefficient PCR reactions and provides a platform for standardization across laboratories. Using this assay, we detected CK20 in 41 of 89 (46%) lymph nodes from noncolorectal cancer types. There was a strong association between CK20 detection and lymph node metastasis determined by histology (P < 0.0001). Quantitatively, CK20 expression levels in colorectal cancer lymph nodes significantly exceeded the levels obtained in lymph nodes of extracolonic carcinomas (P < 0.05). Mean CK20 levels in lymph nodes and tumors from subjects with colorectal and breast cancers were similar in a tumor-type specific fashion. CONCLUSIONS: These results characterize low-level, epithelial cell-specific CK20 expression in infiltrated lymph nodes from subjects with noncolorectal cancer types and demonstrate the potential advantages of detecting circulating epithelial cells by quantitative PCR.