Molecular Characterization in Hairy Cell Leukemia (HCL): Rearrangement Analysis of Heavy Chain in Immunoglobulin Genes (IgH).
Authors: Martín-Jiménez Patricia , Alcoceba Miguel , Sarasquete Maria E. , Balanzategui Ana , de Castro David Gonzalez , Chillón Maria C. , Queizán J. A. , Barbón M. , Ortega F. , Peñarrubia M. J. , Borrego D. , Cabezudo M. , Blázquez Lorena , Armellini Adriana , Fernández Carina , García-Sanz Ramón , González Marcos , Miguel J.F. San 
Published: April 2020

Abstract
Introduction: Currently, the origin of the HCL tumor clone is believed to be marginal zone B-cell that has previously contacted the antigen, and has therefore undergone somatic hypermutation (SHM). However, the low frequency of HCL has hamper the investigation of IgH rearrangements in large series of patients and whether preferential uses of specific VDJ segments or non-functional rearrangements exist.
Aims: To characterize IgH gene rearrangements in 25 HCL patients in order to ascertain the frequency and characteristics of both incomplete DJH and complete VDJH rearrangements and to determine their somatic mutations.
Patients and methods: 25 patients with unequivocal diagnosis of HCL (CD19+CD5-CD22+FMC7+CD23-CD103+) were included in the study. Amplification of clonal rearrangements was carried out using the Biomed-2 strategy (Leukemia2003; 17:2257), followed by direct automated sequencing.
Results: All 25 patients displayed a clonal rearrangement, including 21 VDJH rearrangements (84%) and 14 DJH rearrangements (56%). Families VH3 & VH1 were over-represented, while families VH5, VH6 and VH7 were completely absent. The most frequently used VH specific segment was VH3–30 (3/21), by contrast segments used in normal mature B-cells were not found in any of the HLC cases (i.e. VH3–20 & VH3–49). As far as the DH segments is concerned, DH2 family was the most common in both complete and incomplete rearrangements, followed by DH3 in the complete and DH5 in incomplete fusions. Finally, JH6 and JH4 segments were the most frequently observed JH segments in both complete (52 & 42%, respectively) and incomplete (30 & 46%, respectively) rearrangements. Interestingly, JH3 was over-represented in DJH rearrangements three times more frequent than in VDJH rearrangements.
Sequence analysis showed that HLC cases displaying a complete VDJH rearrangement were mutated (76%, average deviation to germline sequence of 4.12%). By contrast, none of the 14 cases with incomplete rearrangements showed SHM, (11 exactly matched the germline sequence and three had some deviation, but always below the 2%, which is the consensus cut-off point to define the presence of SHM).
Conclusions: Although HCL seems to be a homogeneous disease, molecular analysis of IgH rearrangements of tumor cells shows an important biological heterogeneity. Thus, incomplete rearrangements are frequent, and there is a preferential usage in some VH, DH and JH segments. Furthermore, although the majority of cases seem to have a post-follicular origin, a quarter of them may have originated in cells that have not undergone the SHM process. This is only possible if they have not crossed the germinal center or if their SHM machinery is damaged. These observations suggest that the post-follicular origin (marginal zone B-cell) of HCL should be reviewed.