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

描述(申请人提供)：拟议研究的长期目标是开发专门针对和摧毁多发性骨髓瘤(MM)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来根除疾病的治疗。尽管有自体外周血干细胞移植(PBSCT)支持的强化化疗，但几乎所有MM患者都会复发，并表达原始的克隆性轻链。我们的假设是残留/持续的克隆型B细胞，它们对所使用的化疗药物具有抵抗力，为复发疾病提供了一个储存库。该方案的主要目的是建立一种鉴定、定量和分离克隆型B细胞的方法，并初步确定用这种方法鉴定的克隆型B细胞的数量是否与大剂量化疗后自体PBSCT的临床结果相关。其具体目的是：1)开发一组独特的单抗作为表型表面标记，使用两步流式细胞术方法，利用PBSC采集样本来鉴定、定量和分离(分类)克隆型B细胞。2)针对特定目的1)FCM鉴定和分离的细胞为克隆型B细胞，方法是a)使用患者等位基因特异性的寡核苷酸引物和探针对CDR3进行实时定量聚合酶链式反应(PCR)分析，证明从每个患者分离的B细胞(可能是克隆型B细胞)中重排的CDR3基因序列与它们的肿瘤浆细胞相同，以及b)表明当分离的B细胞培养在骨髓基质细胞单层上时，可以转化为肿瘤浆细胞。3)初步确定患者PBSC采集标本中克隆型B细胞数量与患者复发时间的关系。

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.