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 VH genes, with low levels of intraclonal heterogeneity, while a minor subset have unmutated VH 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 VH and VL tumor-derived genes were evaluated in an expanded series of 38 HCL. From analysis of VH, the VH3 family usage was most common (24/38, 63%), with significant preference of the VH3-30 member in 10/38 cases (26%, p=0,00001), and JH4b 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 VH genes. Analysis of VL 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 Jγ3 segment. Nineteen of 21 cases carried mutated VL genes (94,75%–99.6%) with low levels of intraclonal heterogeneity, while 2 cases carried completely unmutated VL genes, reflecting the status of the VH gene. Strikingly, in-frame functional secondary VL chain rearrangements were observed in the tumor cells of 2 of these cases (Vκ 1 and Vκ 2 in case R1, Vκ 1 and Vκ 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 VH and/or the VL 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 VH or in the VL 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.

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