Analysis of B cell clonal evolution in BM and PBL of AL and MM patients using graphical quantification of lineage trees

The objective of this study was to examine the clonal relationships between B cells in the periphery and the clonal plasma cells in the bone marrow (BM) in AL, Myeloma (MM) and MM with AL (MM + AL) patients, which use the same light chain germline V gene (VL). The clonal light chain variable (V) gene was identified by PCR, cloning and nucleotide sequencing. Our data show that 1/3rd to 1/2 of the peripheral B cell VL gene repertoire is dominated by the clonal light chain gene in AL patients. This dominance is significantly more pronounced in MM patients (with 85-90% of the B cells using the same germline VL gene as the clonal plasma cell in the BM). The clonal relationship between the PBL and BM populations was further confirmed by Ig VH gene analysis. We used a rigorous computer-aided algorithm for extracting the information contained in the lineage trees, using the terms of mathematical graph theory. Lineage tree analysis has allowed us to obtain a high-resolution analysis of nucleotide sequences from these patients in order to determine the process of clonal selection and evolution in AL and MM. This analysis demonstrates the presence of intra-clonal variations within the BM clones, suggesting that these B cells may be seeded into the BM in “waves”. In most patients, the data indicate that the BM clones are derived from the PBL clones, confirming that the clonogenic B cells in the periphery represent a precursor population. The mathematical analysis also reveals that there are significantly more mutations in the VL genes of AL and MM patients compared to normals suggesting either a faster rate of mutation or greater duration of exposure to the mutational process. Additionally, the AL BM sequences show more mutations than the PBL sequences, seeming to suggest lower antigenic selection pressure in the shaping of these trees. In conclusion, lineage tree analysis and the novel mathematical approach of quantifying tree properties provides significant insight into the mechanisms of clonal evolution in the pathogenesis of AL and related plasma cell disorders.