Detection of Micrometastasis in Lymph Nodes of Gastric Cancer Patients by CD44-Specific Reverse Transcriptase-Polymerase Chain Reaction

ABSTRACT

PURPOSE: Lymph-node metastasis is one of the most useful prognostic factors in patients with gastric carcinomas. However, micrometastatic carcinoma cells are frequently missed by conventional histo pathologic examination. Therefore, efforts have been made to detect micrometastasis in lymph nodes at the molecular level. In this study, we used a well-known stomach-cancer target molecule, a CD44 variant, to detect micrometastasis in lymph nodes. METHODS: The authors used the reverse transcriptase-polymerase chain reaction (RT-PCR) to detect tumour- specific splice variants of the transcript of the CD44 gene from 50 histologically metastasis negative lymph nodes in 11 gastric-cancer patients. Oligonucleotide primers as 5'-TCCAGACGAAGACAGT CCCGGAT-3' and 5'-CACTGGGGTGGAATGTGTCTTGGTC-3' were used. Southern blotting with a 40-mer oligonucleotide probe (5'-CCGACAGCACAGACAGAATCCCC- TGCTAC-3') corresponding to a sequence found in all known CD44 transcript isoforms was used to enhance the sensitivity. RESULTS: 1) CD44E could be detected at a concentration of as low as ten tumor cells per 104 peripheral blood lymphocytes in RT-PCR in serial 10-fold dilutions of SNU-16 gastric carcinoma cells with normal peripheral lymphocytes. Southern blotting enhanced the sensitivity to ten tumor cells per 106 peripheral blood lymphocytes. Normal blood did not show overexpression of CD44 splice variant. 2) Gel electrophoresis of reverse transcriptase-polymerase chain reaction products from histologically metastasis-negative lymph nodes of patients with stomach cancer identified a band of 479 base pairs corresponding to the CD44E form in 6 (12%) lymph nodes. 3) Southern blotting using a pan CD44 probe increased the sensitivity of the detection. Of the 50 lymph node samples, ten (20%) showed abnormal CD44 gene activity indicating the presence of micrometastasis. CONCLUSION: The findings of this study suggest that the CD44 RT-PCR/Southern blot hybridization is useful for the detection micrometastasis in lymph nodes. This method would be of practical value in selecting patients at high risk for relapse from those who have no histologically postitive lymph nodes.