Mutated lymphopoiesis and B cell oligoclonality can precede the pathogenesis of multiple myeloma and persist in patients with negative measurable residual disease

Why do patients with multiple myeloma (MM) reaching measurable residual disease (MRD)-negativity still relapse?¹ Could it be due to undetectable MRD inside or outside the bone marrow (BM) or because of so-called cancer stem cells (immature plasma cells [PCs])? Some patients with negative MRD by next generation flow (NGF) are positive by next generation sequencing NGS; NGF focuses on PCs whereas NGS evaluates all B cells and PCs. It is unknown whether immature cells have the same genetic background as MM PCs, so could it be that in some patients clonotypic cells are more immature than PCs?

In order to answer  these questions, at the 61^st American Society of Hematology  Annual Meeting & Exposition, Orlando, FL, US, Sara Rodríguez, Centro de Investigación Médica Aplicada, University of Navarra, Pamplona, ES, reported the results of a study comparing the biological landscape of MM PCs at diagnosis to that of CD34+ progenitors, B cells and normal PCs isolated from patients with undetectable MRD by NGF after treatment.²

Methods and results²

• Samples from transplant eligible patients, enrolled in the GEM2012MENOS65 trial (NCT01916252) collected at baseline, before and after transplant, were assessed by NGF
• At baseline, clonal PCs were isolated from BM, and T cells from peripheral blood (germline control):
  • In the study group (n= 7), to avoid contamination with MM PCs, CD34+ progenitors, B cell precursors, mature B cells, and normal PCs were isolated from patients with undetectable MRD before and after transplant
  • In the control group (n= 14), clonal PCs were isolated from patients with persistent MRD
• Whole-exome sequencing (WES) was performed in a total of 68 BM cell samples isolated from seven patients with MRD-negativity by NGF after induction with bortezomib + lenalidomide + dexamethasone (VRD) and autologous stem cell transplant:
  • Somatic mutations found in MM cells were present in normal cells from 5/7 patients. By contrast, copy number variation (CNV) and MM recurrent mutations present in clonal PCs were never detected in normal PCs
• WES was also performed in matched diagnostic MM PCs and MRD cells persisting after VRD induction in 14 patients as a control group:
  • 40% of mutations and 72% of CNV found at diagnosis were detectable in tumor cells, indicating that few somatic variants present in normal cells were unlikely related to contaminating MRD undetectable by NGF
• The cell fraction of somatic mutations present in normal and clonal PCs was above 25%, whereas somatic mutations present only in clonal PCs had a much higher cell fraction (more than 75%)

Clonal immunoglobulin (Ig) rearrangement

• Deep NGS of B cell receptor immunoglobulin (BcR Ig) gene rearrangements (mean: 69,975 sequences per sample), was performed in 68 mature B cell and normal PC samples:
  • MM clonotypic BcR Ig rearrangements were detectable in normal cells (5/6 patients) but often at a single time point and at very low frequencies. These few somatic variants present in normal cells were unlikely to contaminate MRD below NGF’s limit of detection
  • Regarding patient outcome, MRD reappeared in only one case, and only two patients progressed with extraosseous plasmacytomas and without tumor cells in BM
• Single-cell RNA and BcR Ig sequencing of total BM B cells was performed in six newly-diagnosed (ND) patients with MM to investigate, before treatment, if the clonotypic BcR Ig sequence of MM PCs was detectable in other B cell stages defined by their transcriptome profile:
  • Clonotypic cells were confined mostly but not entirely within PC clusters, and in one patient a completely different clonotype was detectable in mature B cells
• Multidimensional flow cytometry was performed to investigate the frequency of B cell clonality in BM samples from a cohort of 195 patients with NDMM:
  • Of 195 patients, 25 (13%) displayed B cell clonality (median of 0.7% BM clonal B cells)

Conclusions²

• Patients with MM bear somatic mutations in CD34+ progenitors that specifically differentiate into the B cell lineage, likely before the disease onset
• Undetectable MRD < 10^-6 rather than normal cells with a few non-recurrent mutations could be responsible for relapses after MRD-negativity
• This study proposes that the risk of developing B cell and PC oligoclonality, leading to the expansion of MM PCs, may increase because of mutated lymphopoiesis