V_H and V_L Genes in Hairy Cell Leukemia Reveal a Dynamic On-Going Modification of the Surface B-Cell Receptor.

Immunoglobulin (Ig) gene analysis delineates critical features of the clonal history of a B-cell tumor. After antigen interaction, mature B-cells undergo somatic mutation of the V-genes and isotype switch recombination, generally in the germinal center (GC). Receptor revision by secondary recombination of the V-genes with re-expression of recombination activating gene (RAG) enzymes rarely occurs at this stage. From a small series, we have reported that most hairy cell leukemias (HCL) carry mutated V_H genes, with low levels of intraclonal heterogeneity, while a minor subset have unmutated V_H genes. Both subsets commonly have ongoing Ig isotype switch events and express activation induced cytidine deaminase (AID). However HCL lack the GC markers CD27 and CD38, and CD23, a chemokine essential for lymph node entry.

In order to probe more fully the differentiation status of the cell of origin, both V_H and V_L tumor-derived genes were evaluated in an expanded series of 38 HCL. From analysis of V_H, the V_H3 family usage was most common (24/38, 63%), with significant preference of the V_H3-30 member in 10/38 cases (26%, p=0,00001), and J_H4b segment utilised in 50% of cases. Most HCL (35/38) carried variable tiers of mutations (87–98.6% homology to germline), with low level of intraclonal heterogeneity also in cases with <2% deviation from germline, while 3/38 (8%) displayed completely unmutated V_H genes. Analysis of V_L genes provided novel insights, when 21/38 HCL were evaluated. The λ light chain was most fequently used (13/21, 62%), to indicate preferential secondary rearrangement to λ chain. All λ cases used J_γ3 segment. Nineteen of 21 cases carried mutated V_L genes (94,75%–99.6%) with low levels of intraclonal heterogeneity, while 2 cases carried completely unmutated V_L genes, reflecting the status of the V_H gene. Strikingly, in-frame functional secondary V_L chain rearrangements were observed in the tumor cells of 2 of these cases (V_κ 1 and V_κ 2 in case R1, V_κ 1 and V_κ 2 in R2). Primary and secondary rearrangements showed mutations (98.1 and 99.6% homology in R1, 97.6 and 99.6% homology in R2). In both cases, RAG1 re-induction was identified by RT-PCR and sequence verification. Both cases expressed AID transcripts and displayed intraclonal mutations in the V_H and/or the V_L genes. These data suggest a dynamic, on-going modification of the B-cell receptor in tumor cells, including receptor revision, which occurs most likely in response to antigenic stimuli. N-glycosylation sites, commonly introduced in the V regions of tumors of the GC by somatic mutation, were not observed in the V_H or in the V_L functional genes, to support the concept that tumor events occur outside the GC.

These data confirm heterogeneity in the cell of origin in terms of mutational status, with a minor subset with unmutated V-genes. Restricted V-gene segment usage, low levels of ongoing mutations with AID activated, and the new observation of receptor revision with re-expression of RAG enzymes indicate that selection by antigen could be a promoting factor in HCL development. Lack of novel glycosylation sites is in favour of interaction with antigenic stimuli occurring at extrafollicular sites.