Abstract
Abstract
Background:
Multiple myeloma (MM) is a hematologic malignancy of terminally differentiated plasma cells. While most genomic studies of the disease have focused on characterizing isolated bone marrow plasma cells, few have addressed somatic mutations within the immune microenvironment. We previously demonstrated that somatic mutations are significantly more numerous in unsorted blood compared to bone marrow and that the number of mutations correlated with elevated markers of disease. We also reported that genes implicated in clonal hematopoiesis of indeterminate potential (CHIP) are commonly mutated in the blood from patients with MM.
Aim:
To integrate single-cell DNA mutation analysis and protein expression to map the clonal relationship between circulating plasma cells and non-plasma cells within the peripheral blood of patients with MM.
Methods:
Although bulk DNA sequencing can estimate clonal heterogeneity using analytic deconvolution techniques, it is unable to distinguish which mutations occur in the same clone(s) nor can it correlate genotype with immunophenotype from the same cell. To accurately describe the clonal architecture of the blood, we combined targeted single-cell DNA sequencing (scDNA-seq) coupled with protein expression to define mutations specific to cell phenotypes using the Tapestri Platform (Mission Bio). For this analysis, we developed a custom amplicon panel covering 22 frequently mutated genes in MM and/or CHIP to perform scDNA-seq and 4 antibodies targeting CD138, CD19, CD3, and CD33 for immunophenotyping or plasma cells, B cells, T cells, and myeloid cells respectively.
Results:
Peripheral blood mononuclear cells were isolated from 17 blood samples collected from 12 patients with MM. Three samples were collected at diagnosis before treatment, 8 were collected post-treatment during remission, and 6 were collected at relapse. Serial samples were obtained before and after autologous stem cell transplant from 4 patients. A total of 85,262 cells were sequenced uncovering 154 unique variants in 22 genes after quality filtering. There were 47 somatic variants, defined as those present in less than 40% of cells per sample. There were 52,136 total clones, defined as cells with identical genotypes, within this heterogenous population of cells. The most common non-synonymous somatic mutations were identified in genes SETD2, KMT2C, PPM1D. An immunophenotype was assigned to 82% of cells according to the protein with the most abundant number of reads. Dimensionality reduction of single-cell genotype using t-distributed stochastic neighbor embedding (t-SNE) effectively clustered cells according to sample and by patient in cases where there were multiple samples from the same individual. Patients with active myeloma and known circulating plasma cells detected by clinical flow cytometry were found to have the greatest proportion of CD138+ cells as well as the highest mutational burden. It was rare to detect the same clone in two separate samples collected from the same patient at different time points (median time be collection was 248 days) which may be due to under-sampling or clonal evolution. Surprisingly, all somatic variants detected in plasma cells, were also present in other cell types suggesting they were acquired from a common progenitor cell rather than after differentiation.
Conclusions:
scDNA-seq coupled with immunophenotyping of the peripheral blood of patients with MM reveals somatic variants are shared across multiple hematopoietic cell lineages including plasma cells indicating these mutational are acquired from a common progenitor.
Disclosures
Durruthy-Durruthy: Mission Bio Inc.: Current Employment. Arribas-Layton: Mission Bio, Inc.: Current Employment. Wang: Mission Bio, Inc: Current Employment.
