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.
| Conway, E Joseph; Wen, Jianguo; Feng, Yongdong et al. (2009) Phenotyping studies of clonotypic B lymphocytes from patients with multiple myeloma by flow cytometry. Arch Pathol Lab Med 133:1594-9 |
