Multiomic Characterization of Clonotypic B Cells in Patients with Monoclonal Gammopathies

Background: Monoclonal gammopathies are defined as plasma cell (PC) neoplasms. However, it is postulated that the cell of origin is a B lymphocyte. A better understanding of this potential discrepancy is paramount for accurate diagnosis and monitoring. Aim: Identify the cell of origin and tumor reservoirs containing driver genetic alterations within the B lineage of patients with Waldenström's macroglobulinemia (WM), light-chain amyloidosis (AL) and multiple myeloma (MM). Methods: 71 individuals were studied: 18 healthy adults older than 58 (range, 58 - 85), 13 WM, 17 AL and 23 MM patients. Single cell RNA and B cell receptor sequencing (scRNA/BCRseq) was performed using 10X in B cell precursors, mature B cells and PC isolated by FACS (n=32). Exome sequencing was performed in sorted CD34 progenitors, B-cell precursors, mature B cells, normal and clonal PC, and T cells as control (n=28). To avoid contamination, normal cell types were isolated before and after transplant in MM patients with negative MRD at 10-6 using NGF. Results: scRNA/BCRseq yielded paired transcriptomes and immunoglobulin gene rearrangements in 129,794, which were classified into B cell precursors (n=7,810), mature B cells (n=84,231) and PC (n=37,753). In healthy adults, clonal expansions were rare and mainly observed in PC. Interestingly, non-tumor clones were significantly increased in mature B cells and PC from WM patients, and in B cell precursors from MM patients. These findings suggest a higher frequency of oligoclonality in the B lineage of patients with monoclonal gammopathies. Next, we investigated the presence of patient-specific tumor BCR in the normal cell compartments of each monoclonal gammopathy. Surprisingly, tumor BCR were detected in 1.7% and 0.3% B-cell precursors from WM and MM patients, as well as in 0.1% and 0.4% mature B cells from AL and MM patients. Phenotypic hallmarks of the B cell differentiation (eg, CD10, CD19, CD20, CD34, PRDM1, or CD138) were similarly expressed in non-tumor vs tumor-related B cell precursors, mature B cells and PC. However, tumor-related cell types from patients showed on average 640 differentially expressed genes when compared to the normal counterpart in healthy adults. These results confirm the immature phenotype of clonotypic cells and uncover altered transcriptomes possibly due to tumor microenvironments. The median numbers of somatic mutations in CD34 progenitors, B-cell precursors, mature B cells and normal PC from healthy adults (>58y) vs patients with monoclonal gammopathies were 97 vs 431 (P = .002), 177 vs 503 (P = .02), 108 vs 564 (P = .004) and 189 vs 573 (P = .009). An average of 31% and 34% of somatic mutations were shared between CD34 progenitors and normal PC from healthy adults and patients, respectively. These data indicates a continuum of mutated cells throughout the B lymphopoiesis, which is increased in monoclonal gammopathies. In AL patients, there were 30%, 34%, 31% and 28% shared mutations between clonal PC and CD34 progenitors, B cell precursors, mature B cells and normal PC. In MM patients, the respective percentages were 20%, 17%, 11% and 17% shared mutations between clonal PC and CD34 progenitors, B cell precursors, mature B cells and normal PC. Similar results were observed before and after transplant. In contrast to WM patients in whom MYD88 mutations were observed in normal cell types (Rodriguez S, Science Ad 2022), driver mutations (eg, in RAS genes) as well as copy number alterations were private in clonal PC, and absent in normal cell types from AL and MM patients. We next hypothesized that clonotypic B cells with a normal phenotype could result in sporadic cases of positive MRD by NGS while negative by NGF. Such cases should have PFS similar to patients with double negative MRD because clonotypic B cells lacked key genetic alterations and therefore cannot drive relapse. Thus, we analyzed PFS in 103 MM patients enrolled in the GEM2012MENOS65 trial who had simultaneous MRD assessment using NGF and NGS. Of the 103 MM patients, 7 were NGF-/NGS+ despite MRD levels above the limit of detection of NGF. After a median follow-up of 80 months, these patients had a median PFS not reached, similarly to those who were NGF-/NGS-. Conclusion: Monoclonal gammopathies stem from a clonal B lymphopoiesis but tumor reservoirs are contained within phenotypically aberrant cells. These results shed light on the cell of origin and have implications on monitoring of patients with WM, AL and MM.