Immunoglobulin (Ig) Variable Gene Usage Identifies a Subset of Unmutated Chronic Lymphocytic Leukemia (CLL) Patients Expressing 51p1-Like alleles and a Particularly Unfavorable Prognostic Outcome.

Great interest has recently focused on a biased use of the immunoglobulin (Ig) heavy chain variable region genes (VH genes) and on a preferential light chain (VL gene) usage in patients with chronic lymphocytic leukemia (CLL). VH / VL bias maybe correlated with a possible response to a common antigen epitope. To further investigate the Ig VH gene usage in CLL, DNA was extracted from peripheral blood mononuclear cells of 137 consecutive unrelated and untreated CLL cases. PCR amplification was performed using VH family specific and consensus JH primers, followed by automated sequencing. The sequences were aligned to Ig sequences from the GenBank and V-BASE databases. The VH family gene usage was as follows: VH1 19.7%, VH2 4.4%, VH3 49.6%, VH4 21.9%, VH5 2.2% and VH6 2.2%. Mutated VH genes (>2% mutations) were found in 58.4% of the cases analyzed and unmutated VH genes (<2% mutations) were detected in 41.6% of samples. Within the VH1 gene family, 88.9% of cases were unmutated and the most frequently expressed alleles were related to 51p1 (a major VH1-69 allele), that accounted for 10.2% of all VH rearranged genes and for 51.9% of rearranged VH1 genes. Among all patients expressing unmutated 51p1-like alleles, a preferential use of certain D segments was observed: D2-2 accounted for 28% of cases, followed by D3-3 and D3-16 that accounted for 21.4% and 14.3%, respectively; D1-20, D3-10, D3-22, D5-18 and D6-6 were less frequently expressed (1 case each). The most frequently used JH gene segment was JH6 (64.3%), whereas JH4 and JH3 accounted for 21.4% and 14.3%, respectively. All patients expressing 51p1-like alleles showed an identical CDR2 region. The CDR3 region was characterized by similarities in a subset of cases: the amino acid motifs DIVVVPAA(I/M) and GGXYDY(I/V)WGSYRPNDAFDI were the most common and accounted each for 14.3% of CLL samples that expressed 51p1-like alleles: 79% of all patients expressing 51p1-like alleles had a long CDR3 region of 19.4 ± 2.9 codons. In contrast to what has been observed in other diseases, e.g. mixed cryoglobulinemia and non-Hodgkin lymphoma, all VH1-69 expressing CLL cases analyzed were not associated with HCV infection. The overall median survival for this subset of patients was 40 months, considerably shorter than the reported median survival of unmutated CLL cases; furthermore, to date 36% of patients have succumbed of their disease. The results of this study confirm, on the one hand, that restricted CDR3 features and a biased VH gene repertoire are suggestive of a role of antigen selection operational in this disease and have allowed, on the other hand, to identify a subset of patients with a particularly unfavorable survival compared to the overall population of unmutated CLL cases.