Analysis of Non-Expressed IGK Locus Rearrangements in Chronic Lymphocytic Leukemia Indicates a Role for Secondary Rearrangements in Shaping the Expressed Immunoglobulin Repertoire.
Authors: Belessi Chrysoula , Stamatopoulos Kostas , Hatzi Katerina , Hadzidimitriou Anastasia , Marantidou Fotini , Smilevska Tatjana , Stavroyianni Niki , Athanasiadou Anastasia , Kalagiakou Evangelia , Kokkini Garyfallia , Georgopoulos Christos , Paterakis George , Saloum Riad , Fassas Athanasios , Anagnostopoulos Achilles , Laoutaris Nikolaos 
Published: October 2019

Abstract
Immunoglobulin kappa (IGK) locus rearrangements were analyzed in 164 κ-light chain (LC) expressing chronic lymphocytic leukemia (CLL) cases and 95 λ-LC expressing CLL cases. In κ-CLL, 151 IGKV-J transcripts were successfully amplified by RT-PCR; 147/151 were in frame; four out-of-frame IGKV-J transcripts were heavily mutated and contained one or more stop codons, presumably introduced by somatic hypermutation; in all four cases the corresponding expressed transcript was also mutated. DNA-PCR identified 24/164 κ-CLL cases (15%) with double IGKV-J rearrangements; 9/24 non-expressed IGKV-J rearrangements were in-frame; 3/9 were mutated. The most frequently used IGKV genes in expressed IGKV-J rearrangements were: 3–20, 1-39/1D-39, 1–5 and 4-1; the IGKV4-1 gene was by far the most frequent in non-expressed IGKV-J rearrangements (9/24 cases, 33%). In λ-CLL, a total of 65 IGKV-J rearrangements were amplified in 59/95 cases (62.0%); six cases had two different rearrangements. IGVK4-1 was the most frequently used gene (14/65 sequences, 21.5%), followed by IGKV1-16 (8 cases,12.3%), 1-33/1D-33 and 2-30 (7 cases each, 10.8%). IGKV-J transcripts were detected by RT-PCR in 10/59 λ-CLL cases with IGKV-J rearrangements, of which four were in-frame and six out-of-frame. Seven IGKV-J rearrangements in λ-CLL had less than 100% homology to germline; 3/7 were in-frame; 6/7 patients had mutated IGHV and 5/7 had mutated IGLV genes. In κ-CLL, biased usage of the IGKJ1/IGKJ2 genes was observed both in expressed (69%) and non-expressed rearrangements (78%); in contrast, in λ-CLL, downstream IGKJ (IGKJ3-5) usage was observed in 32/59 sequences (53%). Nonproductive rearrangements involving the kappa deleting element (KDE) that render the IGK locus inactive were also analyzed. In κ-CLL, 22/147 cases (15%) carried IGKV to KDE rearrangements, while 24/147 cases (16.5%) carried IGKJ-C- INTRON (JKI) to KDE rearrangements. In λ-CLL, IGKV-KDE rearrangements were amplified in 55/94 cases (59%); JKI-KDE rearrangements were amplified in 52/94 cases (56%). IGKV1D-43 was the most frequent gene in IGKV-KDE joints in κ-CLL (4/22 cases); in contrast, the most frequent genes in IGKV-KDE joints in λ-CLL were IGKV3-20 and IGKV2-30 (9/55 and 7/55 cases, respectively). In conclusion, the present study confirms IGK locus rearrangements in the vast majority of λ-LC expressing CLL cases. Differential usage of IGKJ genes along with significant IGKV repertoire differences in both IGKV-J and IGKV-KDE rearrangements between κ- and λ-CLL allude to prolonged IGK locus recombination before CLL clonogenic cells became λ-producers. The inactivation of productive and potentially functional IGKV-J joints by secondary rearrangements indicates a role for receptor editing in shaping the expressed IG repertoire in CLL. Finally, the identification of mutated, non-expressed IGKV-J rearrangements both in κ- and λ-CLL might be considered as evidence for secondary rearrangements occurring after the onset of somatic hypermutation, at least in a proportion of cases.