Establishment and Characterization of an Ig Lambda Secreting Cell Line from a Patient with AL Amyloidosis.

Abstract

Immunoglobulin (Ig) light chain (LC)-associated amyloidosis (AL) is a fatal plasma cell (PC) disorder characterized by the overproduction of Ig light chains that deposit in an abnormal conformation as amyloid throughout the body. Lambda LC are involved in amyloid deposition 2–3 times more often than kappa LC, and certain LC Ig variable genes are more frequently involved than others and influence clinical presentation and outcome. AL is a potential complication of any immunoglobulin clonal process, but is most often pathologically associated with minimal clonal expansion of PC, as seen in monoclonal gammopathy of undetermined significance. It is much less commonly observed in patients with multiple myeloma (MM). Because of this, and to our knowledge, there are no cell lines that have been established from AL patients, an experimental tool that would be of great value in studying amyloid formation and the biology of amyloid producing PC. In this study, we have established two cell lines from a 50 yr old female initially diagnosed with AL. Upon initial diagnosis, the BM aspirate consisted of 27% IgG λPC with a PC labeling index (PCLI) of 1.9% and a κ to λratio of <0.1. Amyloid was present in periosteal vessel walls and in the fat aspirate. The first cell line, ALMC-1, was established from BM mononuclear cells isolated from the diagnostic aspirate. The patient received a peripheral blood stem cell transplant (PBSCT) 2 months later, but relapsed within 100 days post-PBSCT with symptomatic myeloma. At relapse, the patient’s BM aspirate consisted of 70% λ+ PC and a PCLI of 20%; the second cell line, ALMC-2 was established from this aspirate. IgVL and IgVH analysis revealed that both cell lines expressed identical sequences and used IgVλ 6–57*01, IgλJ3*02, and IgλC3*03: both used the IgVH VH3–21 gene and the extent of somatic mutation was approximately 4%. Both cell lines produce significant FLC, and studies are currently underway to characterize in vitro amyloid production. We next used fluorescence in situ hybridization (FISH) to identify the genetic defects in this patient’s tumor population before and after transition to symptomatic MM. The initial BM aspirate at time of diagnosis of AL revealed approximately 30% of the PC had clear evidence of c-MYC gene amplification. In the subsequent draws as well as in both cell lines, 100% c-MYC amplification was identified, consistent with clonal selection. FISH analysis also revealed that 40% of cells had p53 deletion upon initial diagnosis, whereas the subsequent samples and cell lines were 100% for p53 deletion. All cells also had an IgH translocation that did not involve any commonly observed chromosome partners. Lastly, we have characterized the cytokine responsiveness of both cell lines. Although some differences are observed between ALMC-1 and ALMC-2, IL-6 and IGF-1 stimulated growth in both cell lines to varying degrees and both lines expressed autocrine IGF-I. In summary, our initial characterization of ALMC-1 and ALMC-2 predicts that these unique cell lines will prove to be an invaluable tool to better understand AL, from the combined perspectives of amyloidogenic protein structure and amyloid formation, genetics, and cell biology. Insight gained from this model system may eventually have an impact in the clinical arena by providing a better understanding of this incurable disease.