Motonari Daito

Combination Analysis of a Whole Lymph Node by One-Step Nucleic Acid Amplification and Histology for Intraoperative Detection of Micrometastasis

Objective: The aim was to develop a more efficient molecular detection system than histological examination (HE) for lymph node (LN) metastasis. Methods: Cytokeratin (CK) 19 mRNA copy numbers of 5 colon carcinoma cell lines (Lovo, DLD1, WiDr, Colo201 and Colo320) were calculated and compared by one-step nucleic acid amplification (OSNA) and conventional real-time reverse-transcription polymerase chain reaction (RT-PCR). Then, 91 LN submitted for HE from 6 patients with advanced colorectal adenocarcinoma and 64 LN submitted for frozen diagnosis from 47 patients with different malignancies were examined by OSNA and HE. Results: CK19 mRNA copy numbers of all but Colo320 cells detected by OSNA were within double of those detected by RT-PCR. The least cell count of Lovo cells detected at one reaction (2 µl) by OSNA was calculated as 0.8 cells. Carcinoma metastasis showing either HE+ or OSNA+ was detected in 7.9% of the LN from advanced colorectal adenocarcinomas and in 30.0% of the LN for frozen diagnosis from different malignancies; HE–/OSNA+ metastasis was detected in 4.8 and 4.0%, respectively. OSNA analysis of 1 LN could be completed within 40 min. Conclusion: A combined analysis of LN by HE and OSNA could increase the sensitivity for detecting micrometastasis during surgery.

An Optimal mRNA Marker for OSNA (One-step Nucleic Acid Amplification) Based Lymph Node Metastasis Detection in Colorectal Cancer Patients

BACKGROUND We previously reported that the one-step nucleic acid amplification assay is effective for lymph node metastasis detection in breast cancer patients. This paper describes the identification of CK19 mRNA as an optimal marker and its cut-off value for use in the detection of one-step nucleic acid amplification-based lymph node metastasis in colorectal cancer patients. METHODS Candidate mRNA markers selected from the genome-wide expressed sequence tag database were evaluated by quantitative RT-PCR using a mixture of metastasis-positive and another mixture of metastasis-negative lymph nodes (n = 5 each), followed by quantitative RT-PCR using metastasis-positive and -negative lymph nodes (n = 10 each) from 20 patients. The one-step nucleic acid amplification assay for mRNA markers selected above was examined using 28 positive lymph nodes from 19 patients and 38 negative lymph nodes from the 11 pN0 patients. RESULTS Quantitative RT-PCR analyses of the 98 mRNAs selected from the genome-wide expressed sequence tag database and the subsequent quantitative RT-PCR analyses of the nine mRNAs selected above indicated that CK19 and CEA mRNAs have the highest capability for distinguishing between positive and negative lymph nodes. CK19, CEA and CK20 mRNAs were evaluated by the one-step nucleic acid amplification assay. An area under a receiver-operating-characteristic curve for CK19 mRNA (0.999) was slightly larger than that for CEA mRNA (0.946; P = 0.062) and significantly larger that than for CK20 mRNA (0.875; P = 0.006). CONCLUSION We found that CK19 mRNA has the best diagnostic performance and its cut-off value for discriminating positive from negative lymph nodes can be set in the range of 75-500 copies/µl with 96.4% sensitivity and 100% specificity.