Clonotype-Naïve Detection of Clonality in Patients Suspected of Having Multiple Myeloma or Monoclonal Gammopathy Using Peripheral Blood Cell-Free RNA (cfRNA)

Introduction: Detection of B-cell clonality in tissue and in cell-free DNA (cfDNA) has been used in various types of lymphoma and multiple myeloma (MM). However, there is no data on using cell-free RNA (cfRNA) in detecting clonality. Since plasma cells contain unproportional quantities of immunoglobulin RNA for the synthesis and secretion of immunoglobulin, using RNA in the detection of B-cell clonality is particularly relevant. This higher sensitivity in testing is needed when there is no prior knowledge of the specific clonotype expressed in multiple myeloma. Furthermore, using peripheral blood cfRNA in screening for B-cell clonality has the potential of replacing the need for bone marrow biopsies for both the diagnosis and the monitoring of patients with multiple myeloma. We explored the potential of using peripheral blood cfRNA in detecting heavy chain and light chain clonality using next generation sequencing (NGS). We analyzed peripheral blood samples from healthy normal control, individuals with clonal hematopoiesis of indeterminate potential (CHIP), and patients clinically suspected of having MM, Waldenstrom macroglobulinemia (WM) or monoclonal gammopathy of uncertain significance (MGUS).

Methods: We isolated cfRNA from the peripheral blood of 80 patients with MM (N=53), MGUS (N=18), WM (N=9), normal individuals (N=430), and individuals with CHIP (N=502). The cfRNA was sequenced using a 1500-gene targeted RNA next generation sequencing (NGS) panel that included all immunoglobulin heavy chain (IgH), Kappa (IgK), and Lambda (IgL) genes, and all T-cell receptors alpha (TRA), beta (TRB), and gamma (TRG) genes. Sequencing was performed using the Illumina NovaSeq 6000 instrument. More than 80 million reads and a percentage of spliced reads above 20% were required for acceptable evaluation.

Results: Based on testing 430 normal control, a cut-off point for determining clonality was established at a clonotype ratio of tenfold above the next highest clonotype. Using this stringent criterion, 44 (9%) of the 502 patients with CHIP showed evidence of clonality in peripheral blood, likely reflecting undiagnosed MGUS or monoclonal B-cell lymphocytosis (MBL). Of the 80 tested patients suspected of MM, MGUS, or WM, 35 (44%) showed clonality in either heavy chain or light chain or both. Of these, 6 patients showed clonality in light chain only without demonstratable clonality in heavy chain. B-cell clonality was detected in 7 cases (8%) clinically referred as WM, 23 cases (43%) referred as MM, and 5 cases referred as MGUS (28%). Sixty-four patients showed mutations in the tested cfRNA that are typically seen in lymphoid neoplasms including MYD88, KRAS, and NRAS. Of the 80 cases, 16 (20%) showed either no mutations at all or mutations consistent with CHIP. However, 14 of these 16 cases (88%) showed B-cell clonality. The overall confirmation of the presence of either clonal B-cell abnormality or mutations associated with MM or WM (MYD88) was 98% in this patient population. None of the 80 tested cases showed evidence of clonality in T-cell receptors.

Conclusions: In the absence of prior tissue-based information on clonotype, liquid biopsy testing of cfRNA by combining B-cell clonality evaluation and mutation detection is reliable in evaluating patients suspected of having MGUS, MM, or WM. This approach may replace the need for bone marrow biopsy as a screening test. The sensitivity of such an approach is increased significantly when the clonotype and mutations are known (tumor-informed) (data not presented), which can be used for minimal residual monitoring. Further studies are needed to confirm the clinical utility of this approach in monitoring minimal residual disease.