MOLECULAR CHARACTERIZATION OF METASTATIC BREAST CELLS

女性转移性乳腺癌目前用多种化疗药物治疗。 治疗方式。既往自体骨髓移植（ABMT） 化疗，虽然可能有效， 很难从肿瘤组织中识别和去除所有转移细胞， 骨髓（BM）。外周血（PB）中的转移细胞也是 很难识别和删除。这个问题的一个重要方面是 以鉴定和表征在转移之前和之后存在的转移细胞， 清洁技术。这些转移性细胞非常罕见， 绝大多数手段都无法察觉这些罕见的转移性细胞 是否与原发病灶有显著差异？让这些 差异被利用，以帮助消除他们的其他技术直接 从外周血和/或骨髓中提取如果成功， 联合化疗将是一种重要而有效的全身治疗方法， 疗法 采用新的特殊高速（大于100，000个细胞/秒）流动 超罕见（少于10-6罕见细胞频率）的细胞计数检测 现在可以检测和分离罕见转移性乳腺癌， BM和PB中的转移性细胞，频率低于每百万个转移性细胞1个 正常细胞将使用特殊的高速分拣技术， 后续分子图像分析和PCR分析 表征单个或少量这些罕见的转移性 细胞将尝试将其从BM中清除。等 这些细胞的免疫细胞化学和分子特征可以 提高对转移性乳腺癌的根本原因的认识， 乳腺癌，并可能提出新的治疗方法。PB中的罕见转移细胞 BM将在多重阳性选择的基础上进行鉴定 包括（但不限于）上皮和乳腺癌细胞的标记物 表面标记PHM-5、2G 3、9 C6、741 F8和/或细胞内标记 GCDFP-15和细胞角蛋白AE 1/AE 3以及多重阴性选择标记。 到目前为止，没有一个单一的标志物被证明是唯一的，无论是积极的， 负选择，但很可能存在这样的组合 阳性和阴性标记物足以执行单细胞 多管分选或不纯分选，随后进一步处理， 用另外的标记物进行图像分析，并克隆用于生长或 对于“饼干切割器”重新分选，用于用一个或多个 分子标记分子特征的比较 转移细胞与原发性病变中的细胞将使用 PCR序列的各种基因经常扩增或过度- 在转移性乳腺癌细胞中表达，包括c-erbB 2、c-myc、int- 2和MTS 1。

Metastatic breast cancer in women is presently treated with a variety of modalities of therapy. Autologous bone marrow transplantation (ABMT) prior to chemotherapy, while potentially effective, suffers from the problem that it is difficult to identify and remove all metastatic cells from the bone marrow (BM). Metastatic cells in peripheral blood (PB) are also difficult to identify and remove. An important aspect of this problem is to identify and characterize the metastatic cells present before and after cleansing techniques. These metastatic cells are so rare that they are undetectable by most means. What properties of the rare metastatic cells significantly differ from those of the primary lesion? Can these differences be exploited to help remove them by other techniques directly from peripheral blood and/or bone marrow? If successful, ABMT when combined with chemotherapy would be an important and effective systemic therapy. Using new special high-speed (greater than 100,000 cells/sec) flow cytometric detection of ultra-rare (less than 10-6 rare cell frequencies) it should now be possible to detect and isolate rare metastatic breast cells in BM and in PB at frequencies below one metastatic cell per million normal cells. Special high-speed sorting techniques will be used with subsequent image analysis and PCR analysis for subsequent molecular characterization of single or small numbers of these rare metastatic cells. Attempts will be made to purge them from BM. Such immunocytochemical and molecular characterizations of these cells could provide improved understanding of the underlying causes of metastatic breast cancer and may suggest new therapies. Rare metastatic cells in PB and BM will be identified on the basis of multiple positive selection markers including (but not limited to) epithelial and breast cancer cell surface markers PHM-5, 2G3, 9C6, 741F8, and/or intracellular markers GCDFP-15 and cytokeratins AE1/AE3 and multiple negative selection markers. No single marker has thus far proved unique either for positive or negative selection, but there may well exist a combination of such positive and negative markers sufficient to perform either a single-cell multi-tube sort or an impure sort with subsequent further processing by image analysis with additional markers and cloning either for growth or for "cookie-cutter" re-sorting for PCR characterization with one or more molecular markers. Comparisons of molecular characterizations of metastatic cells with those in the primary lesion will be performed using PCR sequences for a variety of genes frequently amplified or over- expressed in metastatic breast cancer cells including c-erbB2, c-myc, int- 2 and mts1.