Mutated IGHV 1-69 B-CLL Patients Have Indolent Disease and Short CDR3 Sequences.
Authors: Galligan Leeona , Catherwood Mark A. , Morris Treen C. , Alexander H. Denis 
Published: October 2019

Abstract
Background B-CLL is a heterogenous disorder with a highly variable clinical course. Although certain acquired cytogenetic aberrations, unmutated IGVH genes, ZAP-70 positivity and an atypical phenotype have been linked with more aggressive disease and a poorer outcome, the search for further markers of poor prognosis is ongoing is ongoing. IGHV 3–48, 3–53 and 3–21 gene usages have previously been associated with more aggressive disease and shorter survival times. Furthermore, IGHV 1–69 is expressed at a high frequency in B-CLL suggesting a role for antigenic selection in disease pathogenesis. It has been suggested that comparison to germline immunoglobulin sequence using the NCBI/Ig blast only may result in over estimation of mutated IGHV frequency associate with IGHV 1–69 rearrangements in B-CLL. Thus the clinical significance of IGHV 1–69 gene usage remains to be clarified. Aims The aims of this study were to determine the frequency of IGVH1–69 gene usage in B-CLL, to assess the structural features of the CDR3 region, mutational status of the IGHV 1–69 gene using the up-dated IgBLAST and V-QUEST databases and to and to determine if patients with IGHV1–69 rearrangements differ in clinical course to those patients with alternative IGHV gene usage.
Methods Two hundred and seventy patients were recruited for this study. IGHV gene usage was determined using multiplex BIOMED-2 primers (InVivoScribe Technologies) and protocol. IGHV mutational status was determined by sequence analysis using BigDye chemistry and homology comparison with the up-dated NCBI/IgBLAST and IMGT/V-QUEST databases. Interphase FISH analysis was performed to screen for common cytogenetic aberrations and serum thymidine kinase (TK) levels were measured using a radioenzyme assay.
Results We identified IGHV1–69 gene usage in 31/270 CLL patients (11.5%), 30 of which were characterised further. We found that it was associated with unmutated IGHV status (80% of cases) and a preponderance of males (73%). Poor/intermediate prognosis cytogenetic abnormalities were detected in 10 of 15 (67%) cases analysed. Although 21 patients (70%) were found to have Binet stage A disease upon presentation, the majority have since progressed to stage B or C. The median TK level was 16.1 U/L (>8.5 U/L is associated with progressive disease). IGHV somatic hypermutation was found in 6 cases (20%) and was associated with shorter CDR3 regions, IGHJ 4 gene usage and lower TK levels (median of 6.8 U/L). These latter patients have early stage, apparently indolent disease, with no evidence so far of progression.
Summary/conclusions We have found that IGHV1–69 is over-expressed in B-CLL, is associated with advanced disease and may have potential as an independent prognostic marker of progressive disease. However, this remains to be determined as its strong association with IGHV status may be the determining factor. We present evidence that IGHV1–69 B-CLL cases can be divided into mutated (n=6) and unmutated (n=24) sub-groups and that mutated cases are defined by short CDR3 regions, biased IGHJ 4 gene usage and indolent disease. We are currently investigating the possible common antigenic stimuli which may be involved in leukaemogenesis in this sub-group.