Stewart A K, Prince H M, Cappe D, Chu P, Lutzko C, Sutherland D R, Dubé I D
Toronto Hospital Oncology Gene Therapy Program, Ontario, Canada.
Cancer Gene Ther. 1997 May-Jun;4(3):148-56.
One objective of clinical gene marking trials in multiple myeloma (MM) is to determine the extent to which relapse after stem cell transplant is attributable to contamination of the autograft with myeloma cells. A requirement in these studies is ex vivo genetic marking of malignant cells present in autografts which are derived from patients exposed to significant prior chemotherapy. We evaluated gene marking of cloonogenic myeloma cells in marrow aspirates from 14 patients with MM. To effect gene transfer we utilized a long-term marrow culture (LTMC) system previously shown to facilitate gene transfer into a spectrum of hematopoietic progenitor and stem cells. Transduction of cells in LTMC was performed by multiple supernatant exposure. At LTMC initiation and after 21 days of culture malignant cells were assessed by morphology, flow cytometry, and polymerase chain reaction (PCR). The mean number of day 21 LTMC adherent layer-derived granulocyte/macrophage progenitors as a percentage of the original inoculum was within the normal range for this technique. The efficiency of transduction of normal hematopoietic progenitors as determined by the number of colonies positive for proviral DNA by PCR, G418 resistance, and X-gal staining was also within the expected range; 65%, 44% and 23%, respectively. Thus, there was no evidence that prior chemotherapy exposure or malignant cell contamination compromised cell survival or gene transfer efficiency in LTMC. All patients retained plasma cells in LTMCs for the duration of the 21-day culture period. Molecular analysis confirmed the persistence of clonal IgVH gene rearrangements in day 21 LTMC-derived DNA from 6 of 12 informative patients (50%). PCR using allele-specific primers when available confirmed the specificity of IgVH rearrangements for the myeloma clone. In 2 of the 14 patients, expansion of clonogenic cells was demonstrated in LTMC. In both cases there was strong evidence for transfer of reporter genes (neo and LacZ) into the myeloma clone: morphologically abnormal G418-resistant colonies demonstrated intense staining for beta-galactosidase, and cytospin preparations showed 100% plasma cells with monoclonal heavy and light chain restriction. In one patient, individual colonies positive for beta-galactosidase bore a cytogenetic abnormality characteristic of the patient's myeloma clone. PCR of DNA from pooled plasma cell colonies using tumor-specific CDR3 primers was positive. Our results demonstrate the maintenance of myeloma cells in vitro for up to 21 days in LTMC. They further illustrate that these cells can be genetically marked using transduction protocols currently being tested in clinical trials of hematopoietic cell gene transfer.
多发性骨髓瘤（MM）临床基因标记试验的一个目标是确定干细胞移植后复发在多大程度上归因于骨髓瘤细胞对自体移植物的污染。这些研究的一个要求是对来自接受过大量前期化疗患者的自体移植物中存在的恶性细胞进行体外基因标记。我们评估了14例MM患者骨髓抽吸物中克隆性骨髓瘤细胞的基因标记。为实现基因转移，我们利用了一种长期骨髓培养（LTMC）系统，该系统先前已被证明有助于将基因转移到一系列造血祖细胞和干细胞中。通过多次上清液暴露对LTMC中的细胞进行转导。在LTMC开始时以及培养21天后，通过形态学、流式细胞术和聚合酶链反应（PCR）对恶性细胞进行评估。LTMC培养21天的贴壁层来源的粒细胞/巨噬细胞祖细胞的平均数量占原始接种物的百分比在该技术的正常范围内。通过PCR检测前病毒DNA阳性的集落数量、G418抗性和X-gal染色确定的正常造血祖细胞的转导效率也在预期范围内，分别为65%、44%和23%。因此，没有证据表明前期化疗暴露或恶性细胞污染会损害LTMC中的细胞存活或基因转移效率。在21天的培养期内，所有患者的LTMC中均保留有浆细胞。分子分析证实，12例信息充分的患者中有6例（50%）在培养21天的LTMC来源的DNA中克隆性IgVH基因重排持续存在。如有等位基因特异性引物，使用PCR可确认IgVH重排对骨髓瘤克隆的特异性。在14例患者中的2例中，LTMC中显示克隆性细胞扩增。在这两个病例中，都有强有力的证据表明报告基因（neo和LacZ）转移到了骨髓瘤克隆中：形态异常的G418抗性集落显示β-半乳糖苷酶染色强烈，细胞涂片显示100%的浆细胞具有单克隆重链和轻链限制。在1例患者中，β-半乳糖苷酶阳性的单个集落具有该患者骨髓瘤克隆特有的细胞遗传学异常。使用肿瘤特异性CDR3引物对汇集的浆细胞集落的DNA进行PCR检测呈阳性。我们的结果表明，骨髓瘤细胞在LTMC中可在体外维持长达21天。它们进一步说明，使用目前正在造血细胞基因转移临床试验中测试的转导方案可以对这些细胞进行基因标记。
