Kasimir-Bauer Sabine, Otterbach Friedrich, Oberhoff Carsten, Schmid Kurt Werner, Kimmig Rainer, Seeber Siegfried
Department of Internal Medicine (Cancer Research), West German Cancer Center, University of Essen, Hufelandstrasse 55, 45122 Essen, Germany.
Int J Mol Med. 2003 Dec;12(6):969-75.
Occult disseminated tumor cells are the major cause of relapse in patients with primary operable breast cancer but detection and characterization of these few cells is difficult. Applying immunohistochemistry, an immunomagnetic enrichment technique (IET) and immunocytochemistry (IC), we studied 58 breast cancer patients without overt metastases for the frequency of cytokeratin-positive (CK+) bone marrow (BM) cells coexpressing the epithelial adhesion molecule 17-1A (EpCAM) and c-erbB-2 and analyzed the primary tumor for these antigens as a strategy for additional immunotherapy. The primary tumors were analyzed for the target antigens by a pathologist. Dissemination of CK+ cells was studied in 4-6 x 10(6) BM cells by IC alone. For characterization of CK+ cells, 10-15 x 10(6) BM cells were incubated with microbeads coupled to antibodies detecting the target antigens, labelled cells were separated on selection columns and the positively (BM cells carrying the target antigen) and negatively (BM cells without target antigen) selected fractions were stained for CK+ cells. The effectiveness of these methods was confirmed in cell culture models. 17-1A was detected in all primary tumors and c-erbB-2 overexpression (2+, 3+) was found in 25/58 tissue samples. In total, analyzing 15-20 x 10(6) BM cells in each patient, the detection rate for CK+ cells in the BM was 69% (40/58 patients). Interestingly, analysis of the positive and negative enrichment fractions showed that the 17-1A antigen was coexpressed on CK+ cells in only 6 patients and c-erbB-2/CK+ cells were found in only one patient. Although 17-1A and c-erbB-2 were frequently detected in the primary tumor, these antigens were rarely expressed on CK+ BM cells. Whether the applied IET is not able to detect low amounts of these target antigens has to be clarified. Nevertheless, applying cell-cycle independent protocols in clinical trials requires careful elucidation of those patients who might benefit from these therapies.
隐匿性播散肿瘤细胞是原发性可手术乳腺癌患者复发的主要原因，但检测和鉴定这些少量细胞却很困难。我们应用免疫组织化学、免疫磁珠富集技术（IET）和免疫细胞化学（IC），研究了58例无明显转移的乳腺癌患者细胞角蛋白阳性（CK+）骨髓（BM）细胞共表达上皮黏附分子17-1A（EpCAM）和c-erbB-2的频率，并分析原发性肿瘤中的这些抗原，作为额外免疫治疗的策略。由病理学家分析原发性肿瘤中的靶抗原。仅通过IC在4 - 6×10⁶个BM细胞中研究CK+细胞的播散情况。为鉴定CK+细胞，将10 - 15×10⁶个BM细胞与偶联检测靶抗原抗体的微珠孵育，标记细胞在选择柱上分离，对阳性（携带靶抗原的BM细胞）和阴性（无靶抗原的BM细胞）选择组分进行CK+细胞染色。这些方法的有效性在细胞培养模型中得到证实。在所有原发性肿瘤中均检测到17-1A，在25/58个组织样本中发现c-erbB-2过表达（2+、3+）。总共分析每位患者15 - 20×10⁶个BM细胞，BM中CK+细胞的检出率为69%（40/58例患者）。有趣的是，对阳性和阴性富集组分的分析表明，仅6例患者的CK+细胞共表达17-1A抗原，仅1例患者发现c-erbB-2/CK+细胞。尽管在原发性肿瘤中经常检测到17-1A和c-erbB-2，但这些抗原在CK+ BM细胞上很少表达。所应用的IET是否无法检测到这些靶抗原的低含量还有待阐明。然而，在临床试验中应用细胞周期非依赖性方案需要仔细阐明哪些患者可能从这些治疗中获益。
