Analysis of Clonotypic Switch Junctions Reveals Multiple Myeloma Originates from a Single Class Switch Event with Ongoing Mutation in the Isotype Switched Progeny.
Authors: Taylor Brian J. , Kriangkum Jitra , Keats Jonathan J. , Pittman Julie , Reiman Tony , Belch Andrew R. , Pilarski Linda M. 
Published: April 2020

Abstract
Multiple myeloma is a B lineage cancer characterized by an accumulation of malignant plasma cells (PC) in the bone marrow (BM). Tumor cells are defined by a patient specific unique ‘clonotypic’ immunoglobulin heavy chain VDJ rearrangement, and one associated constant region, termed the ‘clinical isotype’ (IgM, IgD, IgG, or IgA). Previously, we have shown that we can detect the full spectrum of clonotypic clinical and non-clinical isotypes to varying degrees at diagnosis, during therapy, and after transplantation in most MM patients (Reiman T et. al., Blood 2001 Nov 1;98(9):2791–9). While the role of these non-clinical isotypes is unclear, expression of clonotypic IgM in MM patients of IgG or IgA clinical isotype was associated with reduced survival and advanced stage of disease at diagnosis. This, coupled with the ability of clonotypic IgM cells to engraft immunodeficient mice suggests a biological role for these cells in MM. One hypothesis that arose from this work was that the MM progenitor may be an IgM expressing B cell that persists throughout the course of disease, undergoing multiple isotype switching events to generate post-switch IgG or IgA expressing PC. To address this we analyzed clonotypic cells of 3 IgA and 12 IgG patients for evidence of multiple switch (S) junctions that may have arisen from IgM progenitors undergoing persistent class switch recombination (CSR) events. For each patient two timepoint samples were tested, usually a diagnostic BM and a second BM or blood sample, 1–4 years later. The 5–7 Kb VDJ-S region of MM patient clonotypic cells was amplified by long distance PCR (LD-PCR) using patient specific CDR2 and S region specific primers. Sequencing was performed on 7/12 IgG patients with an ordered set of 30 primers covering the VDJ-S region, including the S junction. This set also included patient specific CDR2 primers to confirm that the amplified fragments were clonotypic. For all of the patients studied, a single LD-PCR fragment was observed which remained unchanged in size over the timepoints studied. Sequence analysis confirmed that the clonotypic S junctions remained constant in paired samples, suggesting that malignant MM PC studied thus far arise from a single IgM cell that undergoes CSR as opposed to a population of IgM cells undergoing multiple independent CSR events. Interestingly, differences were observed in the DNA sequence of the VDJ-S region of paired timepoint samples: in 5/7 patients, mutations were seen exclusively in the later timepoint samples, and, most notably, 19 new mutations were detected in one patient, including 3 bp and 64 bp deletions immediately downstream of the intronic enhancer. In 4 of these later stage samples, mutations could be found immediately upstream of the intronic enhancer region. The new mutational signature of the VDJ-S regions of later stage timepoint samples represents a kind of intraclonal heterogeneity that has not been previously described in MM. Overall, it appears that switch activity is suppressed in these cells, but mutation is still ongoing. These results suggest that mutations in relapse plasma cells occurred first in clonotypic progenitor B-cells, rather than the malignant plasma cells themselves, because both mutation and recombination activities associated with CSR have been localized to germinal center B-cells. This highlights the potential of post-switch B cells in generating PC progeny throughout the course of malignancy.