O-030. Quantitative PCR for detecting micrometastasis in cervical lymph nodes

Aims: This study was designed to give an accurate diagnosis of micrometastasis in the cervical lymph node (LNs). Methods: We examined the mRNA expression of squamous cell carcinoma antigen (SCCA) and cytokeratin 13 (CK13) in the LNs taken from the patients with oral cancer by means of conventional reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time quantitative PCR (TaqMan PCR). Mutant allele-specific amplification (MASA) was carried out in case with p53 mutation analyzed by direct sequencing. Results: Of 108 historically negative LNs, CK13 and SCCA mRNAs were detected in 9.3 and 7.4% by conventional RT-PCR, and 72.2 and 30.6% by TaqMan PCR. Because the expression of GAPDH varied among patients, relative signal intensity (RSI: CK13/ GAPDH and SCCA/GAPDH) was assessed. CK13 mRNA showed positive RSI in nine of 10 control LNs (Average = 128.4 arbitrary unit: AU), and SCCA mRNA showed no RSI in eight of 10 LNs (Average =5.4 AU) The average RSI of SCCA mRNA in control salivary gland was 11.6 AU. Though TaqMan PCR showed high sensitivity, it also detected low level illegitimate gene expression especially of CK13. Therefore, a definite guideline for cut-off value should be established to give an accurate diagnosis of micrometastasis. MASA method indicated high specificity and sensitivity in some applicable cases in which the primary tumor has genetic alteration. Conclusion: A combination of TaqMan PCR based on SCCA mRNA and MASA amplifying p53 mutation may be a promising method to detect micrometastasis of oral cancer.