Stereotyped Patterns of Somatic Hypermutation (SHM) in Subsets of Patients with Chronic Lymphocytic Leukemia (CLL): Implications for the Role of Antigen Selection in Leukemogenesis.
Authors: Murray Fiona , Darzentas Nikos , Hadzidimitriou Anastasia , Tobin Gerard , Boudjogra Myriam , Scielzo Cristina , Laoutaris Nikos , Karlsson Karin , Baran-Marzsak Fanny , Tsaftaris Athanasios , Moreno Carol , Anagnostopoulos Achilles , Caligaris-Cappio Federico , Vaur Dominique , Ouzounis Christos , Belessi Chrysoula , Ghia Paolo , Davi Frederic , Rosenquist Richard , Stamatopoulos Kostas 
Published: October 2019

Abstract
We examined SHM features in 1967 IGH rearrangements from 1939 patients with CLL. The sequences were divided into four “identity groups”; “truly unmutated” (100% identity to germline; 677 sequences), “minimally mutated” (99–99.9% identity; 133 sequences), “borderline mutated” (98–98.9% identity; 93 sequences) and “mutated” (<98% identity; 1064 sequences). At the cohort level, SHM patterns were typical of a canonical SHM process. However, important differences emerged on analysis of subgroups of sequences. In particular, the IGHV repertoire of the four “identity groups” differed considerably, with the IGHV1-69 and IGHV1-2 genes predominating among “truly unmutated” and “minimally mutated” sequences, respectively. In contrast, other genes were mostly used in “mutated” rearrangements (eg, IGHV4-34/3-23/3-7). Of note, IGHV3-21 and IGHV3-48 had the highest proportion of “borderline mutated” rearrangements. In selected groups of sequences, a remarkable preservation of the germline configuration was observed in superantigenic binding motifs, prompting speculation that subsets of CLL cells could also receive stimulation signals by superantigenic-like interactions. In detail, the vast majority of IGHV4-34 sequences retained germline conformation at the four FR1 positions of the IGHV4-34-specific I/i binding motif. Similarly, IGHV3-21 sequences displayed remarkably few alterations of the IGHV3-specific staphylococcal protein A binding motif. Following established criteria, we identified 530/1967 sequences (27%) as belonging to 113 different subsets with stereotyped HCDR3. The distribution of sequences among subsets differed significantly according to mutational status: 43% of “truly unmutated” sequences belonged to a subset, compared to only 16% of the “mutated” group (p<0.001). Of note, among certain IGHV genes (ie, IGHV1-2/3-21/4-34/4-4), shared “stereotyped” amino acid (AA) changes (i.e. the same AA replacement at the same position) occurred across the entire IGHV sequence significantly more frequently in cases with stereotyped vs. heterogeneous HCDR3s and therefore, could be considered as “subset-biased”. Stereotyped AA changes were also observed in subsets of minimally mutated cases, indicating that even a low level of mutations may be functionally relevant. Comparison to public-database non-CLL sequences revealed that certain stereotyped AA changes were over-represented in CLL and thus could also be considered as “CLL-biased”. The very precise targeting and distinctive features of SHM in subgroups of CLL patients provide further evidence for the important role of selection by specific antigen(s) in CLL leukemogenesis.