The immunoglobulin heavy-chain variable region gene (IgHV) mutational status is considered the gold standard of prognostication in chronic lymphocytic leukemia (CLL) and is currently determined by Sanger sequencing that allows the analysis of the major clone. Using next-generation sequencing (NGS), we sequenced the IgHV gene from two independent cohorts: (A) 270 consecutive patient samples obtained at diagnosis and (B) 227 patients from the UK ARCTIC-AdMIRe clinical trials. Using complementary DNA from purified CD19+CD5+ cells, we demonstrate the presence of multiple rearrangements in independent experiments and showed that 24.4% of CLL patients express multiple productive clonally unrelated IgHV rearrangements. On the basis of IgHV-NGS subclonal profiles, we defined five different categories: patients with (a) multiple hypermutated (M) clones, (b) 1 M clone, (c) a mix of M-unmutated (UM) clones, (d) 1 UM clone and (e) multiple UM clones. In population A, IgHV-NGS classification stratified patients into five different subgroups with median treatment-free survival (TFS) of >280(a), 131(b), 94(c), 29(d), 15(e) months (P<0.0001) and a median OS of >397(a), 292(b), 196(c), 137(d) and 100(e) months (P<0.0001). In population B, the poor prognosis of multiple UM patients was confirmed with a median TFS of 2 months (P=0.0038). In conclusion, IgHV-NGS highlighted one quarter of CLL patients with multiple productive IgHV subclones and improves disease stratification and raises important questions concerning the pre-leukemic cellular origin of CLL.
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This research was supported by the 'Wallonie-Bruxelles International World' (WBI World), the IRIS-Recherche fund, and the Bekales Foundation and 'Les Amis de l’Institut Bordet'. BS is supported by a Télévie grant (FRS-FNRS—Fonds de la Recherche Scientifique—FNRS). AS, PR, AB, DV, PA and JM are supported by the NIHR Biomedical Research Centre (Oxford).
BS and AT were the principal investigators and took primary responsibility for the paper; BS and AT performed research and wrote the manuscript. DB, HD and JM provided help for the next-generation sequencing experiments. DV, LL and PA performed stereotyped receptor analysis, coverage checking and verified the statistics. TS performed the bioinformatics validation. RC, PR and AB participated to the scientific discussion and revised the manuscript; AS supervised and revised the manuscript and the research.
The views expressed are those of the authors and not necessarily those of the Department of Health.
The authors declare no conflict of interest.
Supplementary Information accompanies this paper on the Leukemia website
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Stamatopoulos, B., Timbs, A., Bruce, D. et al. Targeted deep sequencing reveals clinically relevant subclonal IgHV rearrangements in chronic lymphocytic leukemia. Leukemia 31, 837–845 (2017). https://doi.org/10.1038/leu.2016.307
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