Clonotypic B-cell & MM: Surface Marker/Prognostic Impact

描述（由申请方提供）：拟定研究的长期目标是开发特异性靶向和破坏多发性骨髓瘤（MM）中克隆型B细胞的药物，从而可能导致通过比目前可用的措施更温和的措施根除疾病的治疗。尽管在自体外周血干细胞移植（PBSCT）支持下进行了强化化疗，但几乎所有MM患者均复发并表达原始克隆轻链。我们的假设是，残留/持续的克隆型B细胞，这是耐药的化疗药物提供了一个水库复发性疾病。本提案的主要目标是开发一种方法来鉴定、定量和分离克隆型B细胞，并初步确定通过这种方法鉴定的克隆型B细胞的数量是否与使用自体PBSCT进行高剂量化疗后的临床结果相关。具体目标是：1）使用两步流式细胞术方法开发一组独特的单克隆抗体作为表型表面标志物，以使用PBSC收获样品鉴定、定量和分离（分选）克隆型B细胞。2)a）使用患者等位基因特异性寡核苷酸引物和探针对CDR 3进行实时定量聚合酶链反应（PCR）分析，以证明分离的B细胞中重排的CDR 3基因序列（推定的克隆型B细胞）和它们的肿瘤性浆细胞是相同的，和B）显示当在骨髓（BM）基质细胞单层上培养时，分离的B细胞可以转化成肿瘤性浆细胞。3)回顾性分析这些患者外周血干细胞中克隆型B细胞数量与复发时间的关系。

DESCRIPTION (provided by applicant): The long-term objective of the proposed study is to lead to the development of agents to specifically target and destroy clonotypic B-cells in multiple myeloma (MM) thus, possibly leading to therapies for disease eradication by measures less drastic than those currently available. Despite intensive chemotherapy supported by autologous peripheral blood stem cell transplantation (PBSCT), virtually all MM patients relapse with expression of the original clonal light chain. Our hypothesis is that residual/persistent clonotypic B-cells, which are resistant to the chemotherapeutic agents used provide a reservoir for recurrent disease. The main goals of this proposal are to develop a method to identify, quantify and isolate the clonotypic B-cells, and to determine preliminarily if the quantity of clonotypic B-cells identified by such a method correlates with clinical outcome following high dose chemotherapy with autologous PBSCT. The specific aims are to: 1) Develop an unique panel of monoclonal antibodies as phenotypic surface markers using a two step flow cytometric method to identify, quantify and isolate (sort) clonotypic B-cells using PBSC harvest samples. 2) Confirm that the cells identified and isolated by FCM in Specific Aim 1 are clonotypic B-cells by a) performing real-time quantitative polymerase chain reaction (PCR) analysis on CDR3 using patient allele-specific oligonucleotides primers and probes to demonstrate that the rearranged CDR3 gene sequences in the isolated B-cells (putative clonotypic B-cells) from each patient and their neoplastic plasma cells are identical, and b) showing that the isolated B-cells can transform into neoplastic plasma cells when cultured on a bone marrow (BM) stromal cell monolayer. 3) Determine preliminarily the relationship between the quantity of clonotypic B-cells in PBSC harvest samples from these patients and their time to relapse retrospectively.