Gazitt Y, Reading C C, Hoffman R, Wickrema A, Vesole D H, Jagannath S, Condino J, Lee B, Barlogie B, Tricot G
Department of Medicine, University of Arkansas for Medical Sciences, Little Rock, CA 72205, USA.
Blood. 1995 Jul 1;86(1):381-9.
High-dose therapy with autologous marrow or peripheral blood stem cell (PBSC) rescue has been extensively applied in the treatment of multiple myeloma (MM) patients during the past 10 years resulting in improved event-free and overall survival when compared with standard chemotherapy. However, relapses are common and cure is unlikely in the majority of patients. Because both bone marrow and PBSCs are contaminated with myeloma cells it is conceivable that relapse after autotransplantation originates at least in part from autografted tumor cells. In this study, mobilized PBSCs were examined for the presence of myeloma cells based on immunophenotyping and sensitive polymerase chain reaction (PCR)-based techniques. In addition, CD34+ Lin- Thy+ stem cells were purified from mobilized PBSC harvests of 10 MM patients by sequentially using counterflow elutriation centrifugation, treatment with phenylalanine methylester, and flow sorting, using 5-parameter gating (propidium iodide, forward scatter, side scatter, CD34+ v Lin- and CD34+ v Thy+). Virtually all mobilized unsorted PBSC preparations contained myeloma cells in sufficient quantities (range, < 0.01 to > 10%) potentially causing a disease relapse. Stem cell purification led to an overall enrichment by about 50-fold in all 10 patients; approximately 90% of the final cell population expressed CD34+ Lin- Thy+ with no evidence of myeloma cell contamination based on flow cytometric analysis of CD38bright cells (< 0.1%). Quantitative PCR amplification of patient-specific complementarity determining region III (CDRIII) DNA sequences showed depletion of clonal B cells by 2.7 to 7.3 logs, with the highest log reduction noted in the samples initially containing the most tumor cells. Our results show that purification of CD34+ Lin- Thy+ cells depletes myeloma cells to undetectable levels from up to 10% present in unsorted PBSCs, thus offering a tool to investigate whether MM relapse after autotransplantation can be reduced markedly.
在过去10年中，高剂量疗法联合自体骨髓或外周血干细胞（PBSC）解救已广泛应用于多发性骨髓瘤（MM）患者的治疗，与标准化疗相比，可改善无事件生存期和总生存期。然而，复发很常见，大多数患者不太可能治愈。由于骨髓和PBSC均被骨髓瘤细胞污染，可以想象自体移植后的复发至少部分源于自体移植的肿瘤细胞。在本研究中，基于免疫表型分析和灵敏的聚合酶链反应（PCR）技术，检测动员的PBSC中是否存在骨髓瘤细胞。此外，通过依次使用逆流淘析离心、苯丙氨酸甲酯处理和流式分选，利用5参数门控（碘化丙啶、前向散射、侧向散射、CD34⁺ v Lin⁻和CD34⁺ v Thy⁺），从10例MM患者动员的PBSC采集中纯化CD34⁺ Lin⁻ Thy⁺干细胞。实际上，所有未分选的动员PBSC制剂均含有数量足够的骨髓瘤细胞（范围为<0.01%至>10%），可能导致疾病复发。干细胞纯化使所有10例患者的总体富集约50倍；基于CD38亮细胞（<0.1%）的流式细胞术分析，最终细胞群体中约90%表达CD34⁺ Lin⁻ Thy⁺，无骨髓瘤细胞污染证据。患者特异性互补决定区III（CDRIII）DNA序列的定量PCR扩增显示克隆性B细胞减少2.7至7.3个对数，最初含有最多肿瘤细胞的样本中对数减少最高。我们的结果表明，纯化CD34⁺ Lin⁻ Thy⁺细胞可将骨髓瘤细胞从未分选的PBSC中高达10%的水平消耗至检测不到的水平，从而提供了一种工具来研究自体移植后MM复发是否可以显著降低。
