Molecular Quantification of Pre-Treatment Bone Marrow Clonal Cells in Multiple Myeloma Is a Novel Predictive Indicator of Remission Length.
Authors: Thulien Kyle J. , Reiman Tony , Lai Jocelyn , Belch Andrew R. , Pilarski Linda M. 
Published: October 2019

Abstract
Multiple myeloma (MM) is a clonal B-cell malignancy with an accumulation of plasma cells in the bone marrow (BM). Newer treatments continue to increase remission occurrence and decrease side effects, however, relapse invariably occurs. Current diagnostic and monitoring criteria such as bone marrow plasmacytosis (BMPC) and serum M-protein levels (Mpr) fail to predict the length of remission for a given patient. The clonal IgH VDJ gene rearrangement provides a molecular method of measuring the MM disease burden. This unique biomarker can identify and quantify the number of malignant cells through analysis of genomic DNA with quantitative PCR. Here we use SYBR Green real-time quantitative PCR (rqPCR) to measure the level of clonal cells, termed VDJ%, in 91 previously untreated and 51 relapsed pre-treatment BM aspirates from MM patients. Kaplan-Meier analysis showed that VDJ%s lower than the median were associated with longer event free survivals (EFSs) (p=0.0077, HR=0.57). These patients were treated with a variety of different regimens (including VAD, ASCT, Melphalan, Prednisone, Thalidomide, Dexamethasone, Velcade or Revlimid). A modest correlation between the VDJ% and the BMPC was observed (p=0.0003, r2=0.099) but, the BMPC was not able to stratify these patients into groups with significantly different EFSs (p=0.1153, HR=0.6521) as was the VDJ%. This result confirms the BMPC’s poor association with outcome. The Mpr was not significantly correlated with VDJ% (p=2312, r2=0.012). The IMWG response subcategories correlated well with EFS where the poorer the response the shorter the EFS. However, there was no significant correlation between the pre-treatment VDJ% and the response subcategories. In addition to the pre-treatment BM samples, the tumor burden in 30 remission BM samples was quantified. To date, neither the remission VDJ% nor the reduction in VDJ% relative to the pre-treatment VDJ% was associated with EFS in a Kaplan-Meier analysis. The remission VDJ% had a significant yet modest correlation with the remission BMPC (p=0.014, r2=0.27) and nadir Mpr (p=0.0001, r2=0.61). Additionally, the IMWG response subcategory correlated with the proportionate reduction of the remission BM (p=0.036). Taken together these results indicate that VDJ% in pre-treatment BMs, but not the matched BMPC or Mpr, is a significant measure of outcome. The percent of clonal cells as measured in BM aspirates with rqPCR could be developed as a novel tool for predicting outcome by placing patients into a high risk or low risk category for EFS. Patients with a higher than median VDJ% have a shorter EFS than patients with a VDJ% below the median. RqPCR enumerates all cells containing the clonal sequence and thus may include relevant cells types not counted in the BMPC or reflected in the protein electrophoresis and not proportionally represented in remission BMs. The predictive value of pre-treatment VDJ% occurred despite varying types of therapy and tumor response, suggesting that these factors might provide independent prognostic information.