Contributions
Abstract: PB2343
Type: Publication Only
Background
At present, there is no comprehensive description of aberrations in the TP53 gene in DLBCL. TP53 mutations is the most studied aspect. A comparative analysis of TP53 mutations presented in the special databases shows that their frequency and spectrum can vary for the same type of the tumor depending on the study population. Information about the Russian population in the current version of the IARC TP53 mutation database is not provided.
Aims
The purpose of the present study was to carry out a complex analysis of the frequency and spectrum of mutations in 5-8 exons, the frequency of loss heterozygosity and methylation of the promoter of the TP53 gene in DLBCL.
Methods
Genomic DNA was isolated from formalin-embedded paraffin blocks of lymph nodes and extranodal tumor lesions biopsies of 92 patients with DLBCL by phenol-chloroform extraction method using guanidine. The tissue sections containing at least 70-80% of the tumor cells were taken. Direct Sequencing by Sanger of the TP53 gene was performed according to the IARC protocol. Analysis of the methylation status of the TP53 gene was performed by methyl-specific PCR. Estimation of loss of heterozygosity (LOH) in the gene TP53 was carried out at the microsatellite locus D17S796.
Results
A quarter (24.3%) of patients had mutations in the coding sequences of the gene, which is consistent with the literature data. Four patients had multiple mutations. The distribution of the mutations was as follows: 1 (3%) mutation leading to splicing failure, 11 (33%) intron with an unknown effect, 12 (37%) missense, 6 (18%) - synonymous, 2 (6 %) - nonsense type, 1 (3%) - a mutation leading to a shift in the reading frame in the TP53 gene. 5 (15.6%) of substitutions was GC → AT substitutions in CpG islets.
In 9 tumor samples, rs78378222 was detected, which leads to a violation of the polyadenylation signal and disruption of mRNA translation.
According to the IARC TP53 mutation database, the codons 248, 273, 175, 245, 281, 244, 305, 249 and 297 are the "hot spots" in the TP53 gene in DLBCL. In the samples of patients from Russian cohort, only a mutation in 244 codons was identified (p.G244S). However, mutations p.W146R, p.T155I, p.V272E, p.R213X was verified in two cases of each.
The frequency of methylation of the TP53 gene promoter was 4/69 (5.8%), and the LOH frequency was 10/74 (13.5%).
Conclusion
In total 32.4% of DLBCL samples had TP53 aberrations with proven oncogenic potential. Complex analysis made it possible to clarify that the insufficiency of the function of the TP53 gene in DLBCL can be formed according to the classical "two-stroke" mechanism: almost all cases of LOH were combined either with somatic mutations, germ-line marker rs78378222 or methylation of the TP53 gene promoter.
Session topic: 19. Non-Hodgkin lymphoma Biology & Translational Research
Keyword(s): Diffuse large B cell lymphoma, LOH, Methylation, mutation analysis
Abstract: PB2343
Type: Publication Only
Background
At present, there is no comprehensive description of aberrations in the TP53 gene in DLBCL. TP53 mutations is the most studied aspect. A comparative analysis of TP53 mutations presented in the special databases shows that their frequency and spectrum can vary for the same type of the tumor depending on the study population. Information about the Russian population in the current version of the IARC TP53 mutation database is not provided.
Aims
The purpose of the present study was to carry out a complex analysis of the frequency and spectrum of mutations in 5-8 exons, the frequency of loss heterozygosity and methylation of the promoter of the TP53 gene in DLBCL.
Methods
Genomic DNA was isolated from formalin-embedded paraffin blocks of lymph nodes and extranodal tumor lesions biopsies of 92 patients with DLBCL by phenol-chloroform extraction method using guanidine. The tissue sections containing at least 70-80% of the tumor cells were taken. Direct Sequencing by Sanger of the TP53 gene was performed according to the IARC protocol. Analysis of the methylation status of the TP53 gene was performed by methyl-specific PCR. Estimation of loss of heterozygosity (LOH) in the gene TP53 was carried out at the microsatellite locus D17S796.
Results
A quarter (24.3%) of patients had mutations in the coding sequences of the gene, which is consistent with the literature data. Four patients had multiple mutations. The distribution of the mutations was as follows: 1 (3%) mutation leading to splicing failure, 11 (33%) intron with an unknown effect, 12 (37%) missense, 6 (18%) - synonymous, 2 (6 %) - nonsense type, 1 (3%) - a mutation leading to a shift in the reading frame in the TP53 gene. 5 (15.6%) of substitutions was GC → AT substitutions in CpG islets.
In 9 tumor samples, rs78378222 was detected, which leads to a violation of the polyadenylation signal and disruption of mRNA translation.
According to the IARC TP53 mutation database, the codons 248, 273, 175, 245, 281, 244, 305, 249 and 297 are the "hot spots" in the TP53 gene in DLBCL. In the samples of patients from Russian cohort, only a mutation in 244 codons was identified (p.G244S). However, mutations p.W146R, p.T155I, p.V272E, p.R213X was verified in two cases of each.
The frequency of methylation of the TP53 gene promoter was 4/69 (5.8%), and the LOH frequency was 10/74 (13.5%).
Conclusion
In total 32.4% of DLBCL samples had TP53 aberrations with proven oncogenic potential. Complex analysis made it possible to clarify that the insufficiency of the function of the TP53 gene in DLBCL can be formed according to the classical "two-stroke" mechanism: almost all cases of LOH were combined either with somatic mutations, germ-line marker rs78378222 or methylation of the TP53 gene promoter.
Session topic: 19. Non-Hodgkin lymphoma Biology & Translational Research
Keyword(s): Diffuse large B cell lymphoma, LOH, Methylation, mutation analysis