The knowledge about specific mechanisms generating TP53 dysfunction in diffuse large B-cell lymphoma is limited. The aim of the current study was to comprehensively explore TP53 gene variability resulting from somatic mutations, promoter methylation, and allelic imbalance in tumorous tissue of diffuse large B-cell lymphoma (DLBCL). DNA samples from 74 patients with DLBCL were used. Genomic DNA was isolated from paraffin blocks of lymph nodes or from extranodal biopsies of tumors by the phenol–chloroform extraction method with guanidine. Analysis of coding sequences of the TP53 gene was based on Sanger’s direct sequencing method. The methylation status of the TP53 promoter was analyzed using by methylation-specific PCR on bisulfite-converted DNA. Assessment of the detected mutations was carried out in the IARC TP53 Database and the TP53 UMD mutation database of human cancer. The mutations in regions coding for the DNA-binding domain were prevalent (95%). In the analyzed sample of patients, codons 275, 155, 272, and 212 were hotspots of mutations in the TP53 gene. In addition, functionally significant intron mutations (IVS6-36G > C and IVS5 + 43G > T) were detected. Instances of TP53 promoter methylation were observed only in a few samples of diffuse large B-cell lymphoma tissue. Furthermore, loss of heterozygosity was revealed only in the subgroup of patients with altered status of the gene (mutations were detected in five patients and promoter methylation in one case). Thus, the results suggest that there are two sequential events in the formation of diffuse large B-cell lymphoma in at least some cases. The first event is mutation or methylation of the TP53 promoter, leading to appearance of a cell with increased risk of malignant transformation. The second event is the loss of an intact allele of the gene; this change is necessary for tumorigenesis. We identified TP53 mutation patterns in a Russian cohort of patients with de novo DLBCL who were treated with R-CHOP and R-CHOP-like regimens and confirmed that TP53 mutation status is a valuable prognostic biomarker.

The regulation of oncogenes by microRNA is a focus of medical research. hsa-miR-203, hsa-mir-129, hsa-miR-34a, hsa-miR-34b and hsa-miR-34c are oncosuppressive microRNAs that mediate the antitumor activity of p53. We seek to evaluate the frequencies, co-occurrence and clinical significance of the methylation of the MIR-203, MIR-129-2, MIR-34A and MIR-34B/C genes in the tumor tissue of diffuse large B-cell lymphoma (DLBCL). The methylation was assessed in 73 samples of DLBCL and in 11 samples of lymph nodes of reactive follicular hyperplasia by Methyl-Specific Polymerase Chain Reaction (MS-PCR) and Methylation-Sensitive High-Resolution-Melting (MS-HRM) methods. All four studied genes were not methylated in the tissue of reactive lymphatic nodes. The methylation frequencies of the MIR-129-2, MIR-203, MIR-34A and MIR-34B/C genes in lymphoma tissue were 67%, 66%, 27% and 62%, respectively. Co-occurrence of MIR-203, MIR-129-2 and MIR-34B/C genes methylation, as well as the methylation of MIR-34B/C and MIR-34A pair genes were detected. The MIR-34A gene methylation was associated with increased International Prognostic Index (IPI) (p = 0.002), whereas the MIR-34B/C (p = 0.026) and MIR-203 (p = 0.011) genes’ methylation was connected with Ki-67 expression level in tumor tissue at more than 45%. We found an increasing frequency of detection of MIR-34A gene methylation in the group of patients with the Germinal-Center B-cell like (GCB-like) subtype of DLBCL (p = 0.046). There was a trend towards a decrease in the remission frequency after the first line of therapy (p = 0.060) and deterioration in overall survival (OS) (p = 0.162) in patients with DLBCL with methylation of the MIR-34A promoter. The methylation of the MIR-34A, MIR-34B/C, MIR-129-2 and MIR-203 genes in DLBCL is tumor-specific and occurs in combination. The methylation of the studied genes may be a potential differential diagnostic biomarker to distinguish between lymphoma and reactive lymph nodes, while its independent predictive value has not been confirmed yet.

A comparative analysis of oncogene mutations shows that variations in their frequency, spectrum, and hot-spot locations depends on the type of tumor and the ethnic origin of the population studied. The current version of the IARC TP53 Mutation Database lacks information about the frequency and spectrum of TP53 mutations in patients with DLBCL in Russia. The aim of this study was to assess the frequency and functional significance of TP53 mutations in patients with DLBCL in Novosibirsk. The TP53 coding sequence and the adjacent intron regions were analyzed by direct sequencing in the tumor material from 74 patients with DLBCL. Mutations of the TP53 coding sequence were found in 18 (24.3%) patients. These data are consistent with the frequency of TP53 mutations observed in other studies. The spectrum of nucleotide substitutions found in DLBCL specimens corresponded to that described in the IARC TP53 Mutation Database. According to bioinformatic data and to reported experiments in vitro, most of the mutations detected result in the production of functionally inactive p53. Our results show that DLBCL progression is accompanied by the functional selection for mutations in TP53 exons 5–8.

The association of the rs1042522, rs17878362, and rs1625895 polymorphisms of TP53 with risk of non-Hodgkin’s lymphoma (NHL) has not been studied in full detail. NHL has many variants, and each individual polymorphism exerts only a minor effect, requiring the polymorphisms to be studied for particular histological subtypes of lymophomas and as components of haplotype groups. The objective of this work was to analyze the frequencies and linkage disequilibrium for rs1042522, rs1625895, and rs17878362 and their combined haplotype in patients with diffuse large B-cell lymphoma (DLBCL) and control subjects. Differences in linkage disequilibrium structure were observed for rs17878362, rs1042522, and rs1625895 in the Siberian population. The haplotype proved to be more informative than the individual TP53 polymorphisms in a case-control association analysis in DLBCL. Haplotype wArgG was associated with predisposition to DLBCL, while haplotypes wProG and dupProG were found to exert a protective effect. The effect of the haplotype at three TP53 polymorphisms was observed to vary depending on their homozygous or heterozygous state.