Disease Progression in Monoclonal B-Cell Lymphocytosis Is Independent of VH Mutation Status.
Authors: Bennett Fiona L. , Fenton James A.L. , O’Connor Sheila J.M. , Hillmen Peter , Rawstron Andy C. 
Published: October 2019

Abstract
A monoclonal B-cell Lymphocytosis (MBL) is detected in the peripheral blood of around 3% of otherwise healthy adults, the majority of these have a CLL immunophenotype. We have previously demonstrated that the cells in MBL are indistinguishable from good risk CLL, sharing the same immunophenotypic profile, genetic aberrations and IgVH gene usage. MBL exerts an increasing burden on haematology clinics with over 100 new patients diagnosed per annum in our regional haemato-oncology laboratory. This is largely as a result of the increased tendency to investigate patients with a mild lymphocyosis although <1% of patients show progression to CLL per year. Immunoglobulin VH gene mutational status is the gold standard marker for predicting disease progression in early stage A CLL. The aim of this study was to determine if IgVH mutational status predicts outcome in MBL. DNA was extracted from 72 presentation MGG stained blood films from patients diagnosed with MBL between 1995 and 2000 (37 male, 35 female median age 71 years) using a modified version of the QIAamp mini kit protocol for dried blood spots (Qiagen GmbH). In addition DNA was extracted from leucocytes prepared from ammonium-chloride lysed whole blood (<24hrs old) from MBL patients (n=20). DNA was then amplified using BIOMED-2 IgH primers for FR1 or FR2. The products of the PCR were purified from 2% agarose gels and used for direct sequencing of the IgH genes. DNA was successfully amplified from 67/72 archived samples and 20/20 fresh samples. Direct sequencing has been performed on 32/67 archive cases and 20/20 fresh cases to date. Rearranged VH segments were identified in a total of 47/52 cases (27 archive, 20 fresh) using IgBlast (www.ncbi.nlm.nih.gov/BLAST/IgBlast). Rearrangements with more than 2% deviation from the germline sequence were considered somatically hypermutated (SHM). Gene usage in fresh MBL reflected the bias seen in indolent CLL with a predominance of VH4-34 and VH3-7 and no evidence of VH1-69. This was mirrored in archived material with 11/27 cases utilising VH4-34 (n=6) or VH3-7 (n=5) and no cases using VH1-69. The median level of SHM in fresh MBL samples was 5.7% (range 1.3–13.7%). All archived samples analysed showed deviation from germline sequences of greater than 2% (median 6.5% range 2.7–3.2%). Follow-up data from archived samples revealed that 7 patients showed resolution of their lymphocytosis, with persistent but decreasing levels of CLL-phenotype cells. 10 patients had stable lymphocyte counts, with CLL phenotype cells present at levels between 5,000 and 10,000/μL. 9 patients had persistently increasing lymphocyte counts, in all cases CLL cells were present at levels >10,000/μL and included 3 patients who progressed to a clinical stage requiring treatment. VH gene usage was different in each of these 3 patients was with VH3-7, VH4-34 and VH5-51 identified, the levels of SHM were 5.8, 4.0 and 5.3% respectively. Follow-up data shows that a deletion of ATM was detected at progression in one of these patients. In conclusion, we show that MBL is predominantly mutated and that progression to CLL is independent of mutational status. Other prognostic markers, particularly assessment of chromosomal aberrations, may be informative but this would probably require cell selection to increase sample purity. Our data suggests that current prognostic markers are not effective at predicting outcome in MBL and periodic monitoring is required.