Analysis of Validated Clonotypic Igh VDJ Sequences from a Large Cohort of MM Patients.
Authors: Taylor Brian J. , Keats Jonathan J. , Strachan Erin R. , Reiman Tony , Belch Andrew R. , Pilarski Linda M. 
Published: April 2020

Abstract
Studies in B cell malignancies such as chronic lymphocytic leukemia have revealed associations between immunoglobulin V gene characteristics and clinical outcomes. Few studies of this kind have been conducted in multiple myeloma (MM), a B lineage cancer characterized by malignant plasma cells that display somatically hypermutated VDJ, clonal homogeneity, and post switch (IgG or IgA) clinical isotypes. In this study we analyzed the clonotypic VDJ sequences for a large cohort of 115 MM patients to define associations with clinical parameters such as staging at diagnosis (e.g. clinical isotype, percent PC, M protein, beta-2-microglobulin) and overall survival. A key feature of this analysis is the rigorous validation of the experimentally derived clonotypic VDJ sequence by confirming its presence in the majority of single MM PC, to exclude any VDJ sequences arising from expanded but unrelated clones of normal PC that are also present in MM bone marrow (BM) samples. This process involves the amplification of patient specific clonotypic VDJ using sets of VH family and constant region primers, direct sequencing of VDJ fragments, and CDR2 / CDR3 patient specific primer design. The proposed VDJ is sequence is confirmed by one of several single cell assays, preferably RT-PCR on CD138+ sorted PC, but also including in-situ RT-PCR and limiting dilution analysis. The set of confirmed clonotypic VDJ sequences were analyzed for several parameters including: VH, DH, and JH gene usage, VH and CDR3 length, percent mutation in the CDR1-CDR3 region, and CDR3 amino acid sequence. We found that V gene usage amongst MM patients was not significantly different from expected values based on the frequency of functional germline alleles: VH3 (52.2%), VH4 (19.1%), VH1 (17.4%), VH2 (7.8%), VH5(3.5%). Frequently used VH alleles included VH3-30 (10.4%), VH1-69 (9.6%), VH3-23 (7.8%). Amongst VH5 alleles, VH5-51 was observed exclusively. VH4-34, which has been associated with autoimmune diseases and is not present in normal plasma cells was observed in a single case. DH and JH usage from most to least frequent was as follows: JH4, JH6, JH3, JH5, JH1, and JH2; DH3, DH2, DH1, DH6, DH5, and DH4. Sequence analysis of the CDR1-CDR3 region revealed 1.8–18.8% mutation with a mean of 9.4%. Using mutation frequency as a guideline, we identified three groups, high (12–18.8%), medium (6–11.9%) and low (1.8–5.9%) frequencies of mutation, and compared their clinical characteristics. In a similar fashion, CDR3 length, selected VH, DH, and JH alleles were also evaluated in the context of clinical parameters. In a preliminary analysis, there were no detectable correlations between any of these V gene characteristics and overall survival. Future studies with this database will include CDR3 sequence analysis to determine if subsets of recurrent amino acid patterns, suggestive of specific antigen involvement are detectable. Unlike other B lineage malignancies, such as B-CLL or Waldenstrom’s macroglobulinemia, the V gene usage of MM appears to be a largely random representation of the normal VH gene repertoire, with no selection for particular VH gene families or individual VH genes, and a broad range of mutation frequencies. This type of analysis has meaning only if the IgH VDJ sequences analyzed are derived from the MM clone itself, making validation of experimentally derived clonotypic sequences a critical priority. Overall, this is the largest analysis to date of rigorously validated IgH VDJ gene sequences for MM.