Monoclonal B-Cell Lymphocytosis (MBL) and CLL Show Intraclonal Variation: Cases Classified as “Unmutated” Have the Greatest Clonal Diversity.
Authors: Rawstron Andy C. , Fenton James A.L. , Plummer Marieth , King Harry O. , Bennett Fiona L. , Jack Andrew S. , Hillmen Peter 
Published: October 2019

Abstract
A monoclonal B-cell lymphocytosis (MBL) is detectable in approximately 3% of the general adult population. In most cases the abnormal cells have a phenotype and genotype that is indistinguishable from indolent CLL. Individuals presenting with an MBL count over 500 cells per microlitre progress to CLL requiring treatment at a rate of approximately 1% per year. The relationship between MBL and CLL therefore appears to be similar to that of MGUS and myeloma. Intraclonal variation (ICV) in the immunoglobulin heavy chain gene (IgVH) gene occurs in approximately half of MGUS patients but is not present in myeloma. The majority MGUS patients that progress to myeloma lack intraclonal variation at the MGUS stage suggesting that clonal selection is a critical pathway for disease progression in myeloma. The aim of this study was to compare the rate of intraclonal variation in MBL and CLL and determine whether low rates of ICV are associated with progressive disease. DNA was extracted from ammonium-chloride lysed blood samples from individuals with CLL-phenotype MBL (n=20) and CLL with progressive disease (n=10). IgVH and BCL6 PCR products were directly sequenced and also cloned and at least 10 clones sequenced. Intraclonal variation was defined as the number of unique sequences as a percentage of total clones sequenced. Unmutated CLL was defined as having <2% difference in the predominant IgVH clone sequence from germline. The median IgVH mutation rate in the MBL group was 7.4% (range 1.3–13.7%) and in the progressive CLL group 0.6% (range 0–11.3%). Intraclonal variation was observed in both groups of patients: the median number of unique clones was 2/10 (range 0–7/10) in MBL patients and 3/10 (range 0–5/10) in CLL patients. Intraclonal variation was generally restricted to 1 or 2 point mutations in each sequence and for the VH gene the replacement/silent (R/S) ratio of mutations was 1.7 in the framework regions and 3.3 in the complementarity-determining regions. Independent of disease category, unmutated CLL/MBL had a higher degree of intraclonal variation than mutated CLL with a median ICV for unmutated cases of 31% and for mutated cases of 20%. The proportion of patients showing complete clonal homogeneity was lower for unmutated CLL/MBL (14%) than mutated CLL/MBL (27%). The BCL6 gene, which is also mutated during the canonical somatic hypermutation process, showed similar results with a greater degree of intraclonal variation in unmutated CLL/MBL. The results demonstrate that intraclonal variation is a frequent occurrence in both MBL and CLL particularly when non-immunoglobulin genes are also considered. Clonal heterogeneity is either independent of, or inversely related to, the immunoglobulin mutation status demonstrating that both mutated and unmutated CLL have undergone (or are continuing to undergo) somatic hypermutation. The mechanisms of disease progression in MBL/CLL are clearly biologically distinct from MGUS/Myeloma and this data provides strong evidence for an antigen-driven selection process in CLL. Disease progression in CLL may therefore be the result of continued activity of the somatic hypermutation machinery, demonstrated by AID expression in some patients. Coupled with a selective pressure to maintain the immunoglobulin gene this process may result in the selection of CLL clones with transforming mutations in non-immunoglobulin genes.