Mutational Status of IgVH Genes in B-Cell Chronic Lymphocytic Leukemia (B-CLL) and Prognosis: Percent Mutations or Evaluation of Antigen-Driven Selection?
Authors: Gattei Valter , Dal Michele , Zucchetto Antonella , Benedetti Dania , Zaina Eva , Nanni Paola , Sonego Paolo , Poeta Giovanni Del , Degan Massimo , Bomben Riccardo 
Published: October 2019

Abstract
Quantitative evaluation of IgVH genes mutations is widely considered a reliable prognosticator in B-CLL. Conversely, conflicting results have been reported regarding the prognostic impact of IgVH gene mutations when evidence of Ag-driven selection is investigated. To address this issue, the mutational status of IgVH genes was analysed in peripheral blood samples of 147 B-CLL patients, all with survivals, by a strategy of RT-PCR and cloning; B-CLL specific IgVH transcripts were analysed for both % mutation and Ag-driven selection by applying a multinomial statistical model evaluating the excess/scarcity of replacement/silent mutations in FR/CDR sequences of IgVH genes. Maximally selected log-rank statistics, applied for IgVH gene % mutations, estimated the most appropriate cutoffs capable to separate B-CLL patients into two subgroups with different survival; this approach identified an absolute peak at 0.2% IgVH mutations and two relative peaks at about 2% and 4% IgVH mutations. We therefore tested the impact of Ag-driven selection on B-CLL patient survival in the context of the cutoffs of 0.2, 2 and 4% IgVH mutations. For each cutoff, B-CLL cases were identified as mutated (M) or unmutated (UM) if the % IgVH mutations were above or below the chosen cutoff, respectively; M B-CLLs with evidence of Ag selection were named significantly mutated (sM), while cases lacking such an evidence were reported as not-sM (nsM). 1) 0.2% IgVH cutoff - The 133 B-CLLs with >0.2% mutations had longer survivals as compared to 14 cases with <0.2% mutations (p=2.6x10-5); regarding the prognostic impact of Ag selection within the 133 M B-CLLs, 78 sM B-CLL patients had longer survivals than the 55 nsM B-CLLs (p=2.7x10-2). This latter group maintained a better prognosis as compared to UM B-CLLs (p=8.8x10-3); 2) 2% IgVH cutoff - Using the standard cutoff, 99 M B-CLL cases, displaying >2% mutations, had longer survivals as compared to 48 UM cases with <2% mutations (p=1.4x10-4); again, within the M B-CLL group, 65 sM B-CLL patients had longer survivals than 34 nsM B-CLLs (p=1.4x10-2); nsM and UM B-CLL patients had similar survivals (p = 0.1); 3) 4% IgVH cutoff - According to this cutoff, 75 B-CLLs were M B-CLLs, while 72 cases were UM B-CLLs. Only percent of IgVH mutations split B-CLLs into two subgroups with different survivals (p=4.9x10-4); sM and nsM B-CLLs with >4% mutations had similar survivals; 4) sM vs. nsM (all cases) - In the absence of any cutoff, the 78 patients with sM B-CLLs had longer survival as compared to the 69 affected by nsM B-CLLs (p=9.6x10-4). By taking together this data, it appears that the use of % IgVH mutations remains the gold-standard for the definition of prognosis in B-CLL; however, at least when the canonical 2% cutoff is applied, a certain caution should be used by predicting patient survival in the absence of information regarding the affinity maturation of B-CLL clones. The expression by B-CLL cells of IgVH segments with evidence of affinity maturation seems to lose its positive prognostic impact when a cutoff is set at 4% IgVH mutations. In addition to its obvious clinical impact, these observations provide a putative explanation of the apparent discrepancies found in the scientific literature about this matter.