Schoenfeld A, Luqmani Y, Smith D, O'Reilly S, Shousha S, Sinnett H D, Coombes R C
Department of Surgery, Charing Cross Hospital, London, England.
Cancer Res. 1994 Jun 1;54(11):2986-90.
Breast cancer micrometastases in axillary lymph nodes have been detected by serial sectioning and immunohistochemistry, and shown to have prognostic significance. We have used polymerase chain reaction (PCR) to see whether we could further improve the detection rate of micrometastases. Fifty-seven axillary lymph nodes from patients with breast cancer were examined histologically to assess the proportion of tumor involvement. 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 product. Of 39 histologically negative nodes, 4 (10%) gave K19 bands detectable with ethidium staining and a further 10 (28%) gave K19 bands after Southern hybridization. To further increase the detection sensitivity 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 from 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 discrimination between normal nodes and those involved nodes in which K19 expression was only detectable after Southern hybridization. The optimal "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 in patients with breast cancer: sensitivity is limited by the specificity of the tumor marker.
通过连续切片和免疫组织化学已检测到腋窝淋巴结中的乳腺癌微转移，并显示其具有预后意义。我们使用聚合酶链反应（PCR）来观察是否能进一步提高微转移的检测率。对57例乳腺癌患者的腋窝淋巴结进行组织学检查，以评估肿瘤累及的比例。使用抗角蛋白19抗体进行免疫组织化学染色证实了组织学检查结果。然后使用K19引物对提取的RNA进行逆转录PCR，所有18个组织学检查发现有肿瘤累及的淋巴结均产生了预期的460碱基对产物。在39个组织学检查为阴性的淋巴结中，4个（10%）经溴化乙锭染色可检测到K19条带，另外10个（28%）在Southern杂交后出现K19条带。为进一步提高检测灵敏度，使用巢式引物进行了两步扩增，结果在无癌患者的淋巴结以及所有癌症患者的淋巴结中均发现了K19产物。这表明这是内源性mRNA模板的真正低水平表达，而非来自K19假基因的扩增。在两步扩增步骤中减少PCR循环次数无法充分区分正常淋巴结和那些仅在Southern杂交后才能检测到K19表达的受累淋巴结。区分受累淋巴结与正常淋巴结的最佳“临界值”仍为PCR和Southern杂交40个循环的水平。本研究已证明，以K19作为肿瘤标志物的PCR可提高乳腺癌患者腋窝淋巴结微转移的检测率：灵敏度受肿瘤标志物特异性的限制。
