Analysis of IgV_H gene mutations in B cell chronic lymphocytic leukaemia according to antigen‐driven selection identifies subgroups with different prognosis and usage of the canonical somatic hypermutation machinery

Abstract

Summary Cases of B‐cell chronic lymphocytic leukaemia (B‐CLL) with mutated (M) IgVH genes have a better prognosis than unmutated (UM) cases. We analysed the IgVH mutational status of B‐CLL according to the features of a canonical somatic hypermutation (SHM) process, correlating this data with survival. In a series of 141 B‐CLLs, 124 cases were examined for IgVH gene per cent mutations and skewing of replacement/silent mutations in the framework/complementarity‐determining regions as evidence of antigen‐driven selection; this identified three B‐CLL subsets: significantly mutated (sM), with evidence of antigen‐driven selection, not significantly mutated (nsM) and UM, without such evidence and IgVH gene per cent mutations above or below the 2% cut‐off. sM B‐CLL patients had longer survival within the good prognosis subgroup that had more than 2% mutations of IgVH genes. sM, nsM and UM B‐CLL were also characterized for the biased usage of IgVH families, intraclonal IgVH gene diversification, preference of mutations to target‐specific nucleotides or hotspots, and for the expression of enzymes involved in SHM (translesion DNA polymerase ζ and η and activation‐induced cytidine deaminase). These findings indicate the activation of a canonical SHM process in nsM and sM B‐CLLs and underscore the role of the antigen in defining the specific clinical and biological features of B‐CLL.

Mutational status of IgVH genes in B-cell chronic lymphocytic leukemia and prognosis: percent mutations or antigen-driven selection?

“…4 Our conclusions may contribute to explain the apparent discrepancies of the reported study by Lane et al 2 with our and other studies on the prognostic value of IgV H mutational status, as investigated by either percent mutations or evidence of antigen-driven selection. 1,4 RNAs extracted from peripheral blood samples of 147 patients affected by typical B-CLL 4 were reverse transcribed and amplified with sense primers annealing either to V H 1-V H 6 leader sequences or to 5 0 -ends of V H 1-V H 6 FR1 and J H -specific antisense primers.…”

“…4 Our conclusions may contribute to explain the apparent discrepancies of the reported study by Lane et al 2 with our and other studies on the prognostic value of IgV H mutational status, as investigated by either percent mutations or evidence of antigen-driven selection. 1,4 RNAs extracted from peripheral blood samples of 147 patients affected by typical B-CLL 4 were reverse transcribed and amplified with sense primers annealing either to V H 1-V H 6 leader sequences or to 5 0 -ends of V H 1-V H 6 FR1 and J H -specific antisense primers. 4 Purified amplicons were cloned and at least 10-20 colonies for each case were sequenced; B-CLL-specific IgV H transcripts, identified by sharing the same CDR3 in multiple clones, were analyzed for percent mutation and antigen-driven selection, exactly as previously reported, 4 by using for sequence analyses the IgBLAST and VBASE directories.…”

“…2 Similarly, in their B-CLL series, percent IgV H mutations split B-CLL cases into two groups with different survivals only by applying a cutoff set at 5% deviation from GL sequences, 2 instead of the standard 2% mismatch usually found appropriate in most of the reports. 1 These results are in contrast with a study of ours, carried out on a wider (124 cases) B-CLL series, 4 in which we demonstrated that analysis of IgV H mutations according to antigen-driven selection, as investigated by applying the same algorithms of Lane et al, 2 was capable of identifying B-CLL subsets with different survivals. In particular, we found that patients affected by B-CLL with evidence of antigen-driven selection (called 'significantly mutated', sM) had longer survivals even within the good prognosis subgroup with more than 2% mutations of IgV H genes, as compared to a subset of B-CLL cases with more than 2% IgV H mutations but lacking evidence of antigen-driven selection (named as 'not significantly mutated', nsM).…”

“…Consistently, survivals of nsM B-CLLs did not significantly differ from those of unmutated (UM, less than 2% IgV H mutations) B-CLLs. 4 From a clinical standpoint, this observation had, in our opinion, a certain importance, since it suggested a caution in the application of the percent mutations as a marker of prognostic value in the absence of evidence of antigen-driven selection.…”

“…1,4 RNAs extracted from peripheral blood samples of 147 patients affected by typical B-CLL 4 were reverse transcribed and amplified with sense primers annealing either to V H 1-V H 6 leader sequences or to 5 0 -ends of V H 1-V H 6 FR1 and J H -specific antisense primers. 4 Purified amplicons were cloned and at least 10-20 colonies for each case were sequenced; B-CLL-specific IgV H transcripts, identified by sharing the same CDR3 in multiple clones, were analyzed for percent mutation and antigen-driven selection, exactly as previously reported, 4 by using for sequence analyses the IgBLAST and VBASE directories. As described, 4,5 alignment of B-CLL-specific IgV H sequences often revealed a certain degree of intraclonal IgV H diversification.…”