Ig Sequence Analysis in Waldenstrom’s Macroglobulinemia and IgM MGUS.
Authors: Rollett Rebecca A. , Wilkinson Elizabeth , Gonzalez David , Fenton James A.L. , Short Michael , Evans Paul , Owen Roger G. 
Published: October 2019

Abstract
Immunoglobulin heavy chain (IgH) sequence analysis has proved a very effective tool for the investigation of B-cell lymphoproliferative disorders. It provides insight into the cell of origin and in the case of CLL provides very powerful prognostic data. Waldenstrom’s Macroglobulinemia (WM) is a B-cell lymphoproliferative disorder characterised by IgM gammopathy and bone marrow infiltration. Immunophenotypic studies in our laboratory have previously suggested that WM is derived from post germinal centre / marginal zone B-cells. Initial sequencing studies have supported this hypothesis as they have demonstrated that the majority of cases are characterised by mutated Ig genes without intraclonal variation (ICV). This suggests that the clonal cell in WM is derived from a B-cell that has encountered antigen and exited the germinal centre environment without undergoing isotype switch recombination. In order to further clarify this we have sequenced IgH rearrangements in 20 cases of WM. We have also assessed 2 cases of IgM MGUS (the putative precursor lesion in WM) in order to determine whether these cases are associated with intraclonal diversity in a manner analagous to IgG / IgA MGUS and multiple myeloma.
IgM MGUS and WM samples were analysed by PCR using Biomed2 primers (FR1 and JHcons). Gel-purified PCR products were then directly sequenced using Big Dye terminators on the ABI 377 sequencer. The IgM MGUS sequences were then cloned into plasmids and different clones were compared for sequence variation.
WM cases were characterised by high levels of somatic hypermutation, none of the cases had a mutation rate <2%, and only three cases were <5% mutated. The median mutation rate was 6.9% (range 3.6–13.9%). For VH family usage we found predominant use of the VH3 group (13/20), with 4 cases using VH3-23. In contrast to recently published work we found that VH3/JH4 was not a preferential gene combination in WM since this rearrangement was found only once in our study.Two WM cases used the VH4 group and in both cases there was VH4-34 gene usage which has been found to be characteristically absent in multiple myeloma (MM) highlighting a molecular difference between these two conditions. Interestingly both the IgM MGUS samples analysed used a VH3 gene segment, (both VH3-30), with mutation rates of 7% recorded in each case. The IgM MGUS samples were also examined for ICV, the first case showed no ICV in 6 clones but the second case had 3/8 clones displaying the same 1 base pair difference suggesting ongoing somatic hypermutation in this case.
We would conclude that WM is consistently characterised by heavily mutated IgH genes consistent with derivation from a post germinal centre B-cell. IgM MGUS would also appear to be characterised by mutated IgH genes and preliminary data would suggest that some cases may be characterised by ICV. This pattern has also been demonstrated in IgG / IgA MGUS. It remains to be seen whether the presence or absence of ICV in the context of IgM MGUS has any predictive value for progression to WM.