Targeted deep sequencing reveals clinically relevant subclonal IgHV rearrangements in chronic lymphocytic leukemia

Abstract

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.

IGHV mutational status testing in chronic lymphocytic leukemia

“…In addition, NGS allows for identification of multiple productive clonally unrelated IGHV rearrangements, which were observed in 24% of patients with CLL in one study. 28 …”

“…Identification of more than one clone, where one is mutated while the other is unmutated, also appears to have an intermediate prognosis as compared to mutated alone or unmutated alone. 28,29 In the event of discordant clones, additional prognostic markers can also help guide interpretation and prognostication. For example, concurrent ZAP70 overexpression or 11q deletion by FISH are more likely to be associated with unmutated disease.…”

Molecular pathogenesis of chronic lymphocytic leukaemia

“…Given the importance of IGHV mutational status in CLL, and the labour‐intensive nature of Sanger sequencing, these assays are now becoming utilised more frequently for routine assessment of IGHV mutational status in CLL patient populations. The increased sensitivity of NGS over conventional direct sequencing, has also meant it is now possible to identify small sub‐clones of cells with differing degrees of mutational load (Stamatopoulos et al , ). The detection of these sub‐clones by NGS means the <2% cut‐off in mutational load may need to be reassessed, as these new methodologies become validated and incorporated into routine diagnostic practice.…”

Differential genomic and transcriptomic events associated with high-grade transformation of Chronic Lymphocytic Leukemia

“…Independent cohort FFPE blocks were prepared in 10μm sections, transferred to 1.5 mL tubes (60-100 μm per tube) and in order to prevent RNA degradation stored in -80 degrees C immediately after the preparation. 46…”

“…IgHV mutational status was determined by Sanger and targeted next-generation sequencing as previously described 46 . IgHV analysis was performed on CLL and RS tumor samples in order to determine their clonal relatedness.…”