The TNF −308G>A Polymorphism Predicts Outcome of Patients with B-Cell Chronic Lymphocytic Leukemia In Relation to the IgVH Mutation Status

Tumor necrosis factor (TNF)–α is an important pro-inflammatory cytokine involved in the modulation of lymphoma development and the balance between cell-mediated and humoral immunity. Deregulated concentrations of TNF–α have been detected in patients with lymphoma and were associated with an adverse prognosis. Evidence that the single-nucleotide polymorphism (SNP) in TNF promoter, TNF −308G>A, could be the susceptibility locus for non-Hodgkin lymphoma (NHL) has been provided by case-control studies. Therefore we tested the hypothesis that TNF −308G>A influences clinical course of B-cell chronic lymphocytic leukemia (CLL).

We genotyped TNF −308G>A (rs1800629) in 278 newly diagnosed patients with CLL and 192 ethnically-matched healthy individuals using the 7900 HT Real-Time (Applied Biosystems, USA). Some randomly selected DNA samples were analysed by direct sequencing using 3130xl Genetic Analyzer (Applied Biosystems, USA). Sequence data were based on the NCI SNP500 website. The IgVH mutation status in CLL patients was performed according to the protocol described by van Dongen et al. Serum samples from the 153 newly diagnosed CLL patients were collected at the time of diagnosis and tested by an enzyme-linked immunosorbent assay (ELISA) kit for human TNF (Quantikine, R&D Systems, USA).

The TNF −308G>A allelic frequencies and distributions were consistent with Hardy-Weinberg equilibrium, and did not differ significantly between CLL patients and the control group. There were no significant differences between TNF allelic or genotype distributions and clinical characteristics of CLL patients at diagnosis, including age, clinical stage according to Rai classification, serum LDH and β2-microglobulin levels, surface CD38 expression, ZAP-70 expression, Döhner's cytogenetic groups, and IgVH mutation status. Neither of assessed TNF polymorphic variants was associated with response to first-line treatment, progression free survival (PFS) nor treatment free survival (TFS) that was measured from time point of diagnosis to first therapy. With a median follow-up of surviving patients of 52 months (range 1–209 months), the group of patients with TNF (−308A) allele (TNF−308AA or TNF−308AG genotypes) had significantly shorter overall survival (OS) compared to those carrying TNF (−308GG) genotype (p=0.01, log–rank test). To further characterize the prognostic impact of genetic variation in TNF on CLL patients survival, we divided the patients according to the IgVH mutation status into a IgVH unmutated (homology ≥98%) and a IgVH mutated (homology <98%) group. We found that the patients carrying TNF (−308A) allele presented significantly shorter OS in the IgVH unmutated group compared to those with TNF (−308GG) genotype (p=0.02). Of note, the TNF −308G>A polymorphism did not influence survival of patients with the IgVH mutated gene. To further investigate this difference, we analyzed the impact of TNF SNP on serum TNF-α levels in CLL patients at the time of diagnosis. We found that high TNF-α levels, greater than the median (16.84 pg/mL) value, correlated with stages III and IV according to Rai classification (p=0.001, chi² test), elevated serum levels of LDH (p=0.01) or β2-microglobulin (p=0.001), CD38 expression ≥30% (p=0.001), ZAP-70 expression >20% (p=0.01) as well as with TNF (−308AA) genotypes (p=0.04). The patients with high TNF-α levels had significantly shorter TFS (p<0.0001) compared to those with low cytokine levels. This association retained its prognostic impact on TFS both in the IgVH unmutated (p=0.004) and mutated (p<0.0001) group. No correlations between serum TNF-α levels and response to first-line treatment or PFS were found. Furthermore, in the group with high TNF-α levels, OS was significantly shorter compared to those in the cytokine low level group (p=0.04). Interestingly, when the patients were stratified according the IgVH mutation status, the high serum TNF-α level retained its prognostic impact on shorter OS only in the IgVH unmutated group compared to the patients with low TNF-α levels (p=0.04).

Our results indicate that the TNF −308G>A polymorphism along with the serum TNF-α level may influence CLL outcome especially in the IgVH unmutated subgroup, which points out the importance of innate immunity genes for CLL variability and prognosis.

No relevant conflicts of interest to declare.