DETECTION OF BREAST-CANCER MICROMETASTASES IN AXILLARY LYMPH-NODES BYUSING POLYMERASE CHAIN-REACTION

Breast cancer micrometastases in axillary lymph nodes have been detect ed by serial sectioning and immunohistochemistry, and shown to have pr ognostic significance. We have used polymerase chain reaction (PCR) to see whether we could further improve the detection rate of micrometas tases. Fifty-seven axillary lymph nodes from patients with breast canc er were examined histologically to assess the proportion of tumor invo lvement. Immunohistochemical staining with the use of an anti-keratin 19 antibody confirmed the histological findings. Reverse transcription PCR was then performed on extracted RNA by using K19 primers, and all 18 histologically involved nodes yielded the expected 460-base pair p roduct. Of 39 histologically negative nodes, 4 (10%) gave K19 bands de tectable with ethidium staining and a further 10 (28%) gave K19 hands after Southern hybridization. To further increase the detection sensit ivity a two stage amplification was performed by using nested primers, and K19 product was found in lymph nodes from patients without cancer , as well as in all the nodes from cancer patients. This was shown to be genuine low level expression hom endogenous mRNA template, and not derived from amplification of a K19 pseudogene. Reducing the number of PCR cycles in the two amplification steps did not allow sufficient di scrimination between normal nodes and those involved nodes in which K1 9 expression was only detectable after Southern hybridization. The opt imal ''cut-off'' point to distinguish involved nodes from normal nodes remained at the level of 40 cycles of PCR and Southern hybridization. PCR, using K19 as a tumor marker, has been demonstrated in this study to improve the detection of micrometastases in axillary lymph nodes i n patients with breast cancer: sensitivity is limited by the specifici ty of the tumor marker.