Standardization of TP53 gene mutations analysis on oral squamous cell carcinoma from paraffin-embedded tissues

Jornal Brasileiro de Patologia e Medicina Laboratorial

José De Assis Silva Júnior¹  Danielle Resende Camisasca¹  Débora Dos Santos Tavares¹  Paulo Antônio Silvestre De Faria¹  Fernando Luiz Dias¹  Georgina Severo Ribeiro¹  Lídia Maria Da Fonte De Amorim¹  Simone De Queiroz Chaves Lourenço¹ 

关键词: TP53 gene;    squamous cell carcinoma;    oral cancer;    DNA sequencing;    methodology;    gene TP53;    carcinoma de células escamosas;    câncer da boca;    inclusão em parafina;    sequenciamento de DNA;    metodologia;   

DOI  :  10.5935/1676-2444.20140009

来源: SciELO

【 摘 要 】

Introduction:The tumor protein p53 gene (TP53) is a constant target of investigation in cancer pathogenesis. Analysis by immunohistochemistry provides limited data about p53 in oral carcinogenesis, and TP53sequencing can contribute to this analysis. However, obtaining high-quality and contamination-free deoxyribonucleic acid (DNA) for a proper amplification can be a difficult task when using paraffin-embedded tissues.Objective:Standardize DNA extraction, polymerase chain reaction (PCR) amplification and DNA sequencing techniques for TP53 mutation analysis.Material and methods:Thirty-nine cases of oral squamous cell carcinoma (OSCC) were selected from the Pathology Division of Instituto Nacional de Câncer (Inca). The DNA extraction method used was the QIAamp® DNA minikit® system. After DNA quantification by spectrophotometry, 250 ng of genetic material obtained from TP53 gene were amplified by PCR for exon 2 and by nested PCR for exon 6. Out of the total sample, 11 cases were selected for exon 2 sequencing. Results: The DNA samples presented mean concentration of 119.74 ± 88.86 ng/µl (28.9-556.4) and purity of 1.69 ± 0.18 (1-1.9). Thirty-three (84.6%) samples were amplified for exon 2, and all samples for exon 6 (39/100%). Readable sequencing data were obtained in 10 (90.9%) cases.Conclusion:Optimization of conditions for TP53 sequencing was obtained, and this will facilitate the analysis of mutations in paraffin-embedded tissues, allowing molecular retrospective studies.

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