Peptide nucleic acid clamp PCR: a novel K-ras mutation detection assay for colorectal cancer micrometastases in lymph nodes.

Abstract

Inaccurate staging of colorectal cancer (CRC) has been attributed to the failure to detect lymph node metastases by conventional pathology. We have previously reported the use of lymphatic mapping to accurately identify those lymph nodes most likely to harbor micrometastatic disease and permit focused pathologic examination. Mutation of K-ras allele at codons 12 or 13 occurs frequently in early stages of CRC development. The purpose of our study was to assess sentinel lymph nodes (SLN) for occult CRC micrometastases using a unique peptide nucleic acid (PNA) clamp PCR assay specific for K-ras mutations. Seventy-two paraffin-embedded primary CRC and paired SLN were evaluated by PNA clamp PCR for K-ras mutations. Thirty primary tumors (42%) were positive for K-ras mutations, and in 5 of these cases the SLN were positive for metastases by Hematoxylin and Eosin staining. PNA clamp PCR identified occult metastases in an additional 6 patients, upstaging 24% of K-ras positive primary CRCs (p = 0.014). No K-ras mutations were detected among the 20 noncancer lymph nodes assessed. This study demonstrates the utility, specificity and sensitivity of PNA clamp PCR assay in identifying occult micrometastases in the SLN of CRC patients by single-base mutation analysis.