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

描述（由申请人提供）：拟议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，从而可能通过比目前可用的措施更温和的方法来根除疾病。尽管在自体外周血干细胞移植（PBSCT）的支持下进行了强化化疗，但几乎所有MM患者都会复发，并伴有原始克隆轻链的表达。我们的假设是，残留的/持续的克隆型b细胞对所用的化疗药物具有耐药性，为复发性疾病提供了一个储存库。本课题的主要目的是建立一种克隆型b细胞的鉴定、定量和分离方法，并初步确定通过该方法鉴定的克隆型b细胞的数量是否与自体PBSCT高剂量化疗后的临床结果相关。具体目标是：1)开发一组独特的单克隆抗体作为表型表面标记，使用两步流式细胞术方法识别，定量和分离（分类）使用PBSC收获样品的克隆型b细胞。2)利用患者等位基因特异性olignucleotides引物和探针对CDR3进行实时定量PCR （real-time quantitative polymerase chain reaction， PCR）分析，证实从每位患者分离的b细胞（假定的克隆型b细胞）及其肿瘤浆细胞中重排的CDR3基因序列是相同的；b)表明分离的b细胞在骨髓（BM）间质细胞单层培养时可以转化为肿瘤浆细胞。3)回顾性初步确定患者PBSC采集标本中克隆型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.