Visser Mike, Jiwa Mehdi, Horstman Anja, Brink Antoinette A T P, Pol Rene P, van Diest Paul, Snijders Peter J F, Meijer Chris J L M
Department of Pathology, VU Medical Center Amsterdam, The Netherlands.
Int J Cancer. 2008 Jun 1;122(11):2562-7. doi: 10.1002/ijc.23451.
Staging by sentinel node (SN) biopsy is the standard procedure for clinically node-negative breast cancer patients. Intra-operative analysis of the SN allows immediate axillary lymph node (ALN) dissection in SN positive patients, but a quick, reliable and reproducible method is lacking. We tested the suitability of a quantitative cytokeratin 19 (CK19) mRNA one step nucleic acid amplification (OSNA#) technique (OSNA-CK19) for intra-operative SN analysis. OSNA-CK19 involves a short manual sample preparation step and subsequent fully automated amplification of CK19 mRNA based on reverse transcription loop-mediated isothermal amplification, with results available within 30-40 min. OSNA-CK19 was compared to histological staining (Hematoxylin&Eosin and CAM5.2 and CK19 immunostaining) of 346 frozen ALNs from 32 breast cancer patients, using half of the lymph node for each method. 267 samples were negative and 61 positive by both methods. Three samples were histology positive and OSNA-CK19 negative. Fifteen samples were histology negative and OSNA-CK19 positive, 11 of which had copy numbers close to the cut-off level of OSNA-CK19. Seven of these 15 samples were RT-PCR positive for epithelial markers and/or showed CK19 protein expression by Western blot suggesting the presence of tumor deposits in the lymph node part investigated by OSNA-CK19. Concordance with histology was 94.8%, and 96.8% after exclusion of the latter 7 discordant cases. Sensitivity was 95.3% and specificity was 94.7% before and 97.1% after discordant case investigation. Our results indicate that OSNA-CK19 can potentially be useful in an intra-operative clinical setting to detect SN tumor involvement in breast cancer patients.
前哨淋巴结（SN）活检分期是临床腋窝淋巴结阴性乳腺癌患者的标准程序。术中对前哨淋巴结进行分析可使前哨淋巴结阳性患者立即进行腋窝淋巴结清扫，但目前仍缺乏一种快速、可靠且可重复的方法。我们测试了定量细胞角蛋白19（CK19）mRNA一步核酸扩增（OSNA#）技术（OSNA-CK19）用于术中前哨淋巴结分析的适用性。OSNA-CK19包括一个简短的手工样本制备步骤，随后基于逆转录环介导等温扩增对CK19 mRNA进行全自动扩增，30 - 40分钟内即可获得结果。将OSNA-CK19与32例乳腺癌患者的346个冷冻腋窝淋巴结的组织学染色（苏木精和伊红染色、CAM5.2和CK19免疫染色）进行比较，每种方法使用一半淋巴结。两种方法检测的267个样本为阴性，61个样本为阳性。3个样本组织学阳性但OSNA-CK19阴性。15个样本组织学阴性但OSNA-CK19阳性，其中11个样本的拷贝数接近OSNA-CK19的临界值。这15个样本中有7个通过逆转录聚合酶链反应检测上皮标志物呈阳性和/或通过蛋白质印迹显示CK19蛋白表达，提示在OSNA-CK19检测的淋巴结部分存在肿瘤沉积物。与组织学的一致性为94.8%，排除后7例不一致病例后为96.8%。在调查不一致病例之前，敏感性为95.3%，特异性为94.7%，之后敏感性为97.1%。我们的结果表明，OSNA-CK19在术中临床环境中可能有助于检测乳腺癌患者前哨淋巴结的肿瘤累及情况。
