口腔拭子和漱口法在口腔癌患者无创唾液基因检测中的应用。
Non-Invasive Saliva-based Detection of Gene Mutations in Oral Cancer Patients by Oral Rub and Rinse Technique.
机构信息
Nitte (Deemed to be University), AB Shetty Memorial Institute of Dental Sciences, Department of Public Health Dentistry, Mangaluru, Karnataka, India.
Nitte (Deemed to be University), Nitte University Centre for Science Education and Research (NUCSER), Division of Molecular Genetics and Cancer, Mangaluru, Karnataka, India.
出版信息
Asian Pac J Cancer Prev. 2021 Oct 1;22(10):3287-3291. doi: 10.31557/APJCP.2021.22.10.3287.
BACKGROUND
Oral Squamous Cell Carcinoma (OSCC) is the most widely reported cancer worldwide. Liquid biopsy, a method that relies on identification of tumor-associated cells and/or cell free nucleic acids from body fluids is becoming increasingly popular in cancer diagnostics. The aim of the study was to evaluate the feasibility of Oral Rub and Rinse (ORR) technique in determining the genetic changes in common biomarkers of oral cancer such as TP53 using DNA obtained from saliva of oral cancer patients.
METHODS
A total of 15 oral cancer patients were recruited in the study and pre-surgical saliva samples were collected using the ORR technique. Tissue samples included in the study were obtained during the surgical excision of the cancerous oral lesion. Genomic DNA was isolated from the salivary cell plug and the tissues and the TP53 gene was amplified by PCR. The PCR products of all the exons of TP53 (Exons 2 to 11) were electrophoresed on agarose gel, purified and sequenced by Sanger method. The obtained sequences were compared with the reference sequence of TP53 gene.
STATISTICAL ANALYSIS USED
Descriptive statistics were used and reported as frequency and percentage.
RESULTS
Capillary sequencing of TP53 gene from tissue DNA revealed the presence of codon 72 c.215C>G (p.Pro72Arg) polymorphism in 10 patients (67%) and a heterozygous mutation at codon 172 c.514 G>T (p.Val172Phe) in 2 patients (13%). Among the 10 samples that showed codon 72 polymorphism, matched salivary DNA was available for 6 samples and 4 out of these showed same genetic change at codon 72. Similarly, of the 3 samples that showed codon 172 mutation, matched salivary DNA was available for 1 sample and the mutation status was identical.
CONCLUSION
The results suggest a potential for clinical applications of ORR technique as an alternative to invasive tissue biopsy for detection of genetic changes in candidate biomarkers in oral cancer.
背景
口腔鳞状细胞癌（OSCC）是全球报道最广泛的癌症。液体活检是一种依赖于从体液中识别肿瘤相关细胞和/或无细胞核酸的方法，在癌症诊断中越来越受欢迎。本研究旨在评估使用口腔擦拭和冲洗（ORR）技术从口腔癌患者唾液中获取 DNA 来确定口腔癌常见生物标志物（如 TP53）的遗传变化的可行性。
方法
本研究共招募了 15 名口腔癌患者，并使用 ORR 技术采集术前唾液样本。研究中纳入的组织样本是在切除癌变口腔病变时获得的。从唾液细胞塞和组织中分离基因组 DNA，并通过 PCR 扩增 TP53 基因。将 TP53 基因的所有外显子（外显子 2 至 11）的 PCR 产物在琼脂糖凝胶上电泳，通过 Sanger 法进行纯化和测序。将获得的序列与 TP53 基因的参考序列进行比较。
统计分析方法
使用描述性统计方法，并以频率和百分比报告。
结果
从组织 DNA 中对 TP53 基因进行毛细管测序显示，10 名患者（67%）存在密码子 72 c.215C>G（p.Pro72Arg）多态性，2 名患者（13%）存在密码子 172 c.514 G>T（p.Val172Phe）杂合突变。在显示密码子 72 多态性的 10 个样本中，有 6 个样本有匹配的唾液 DNA，其中 4 个样本在密码子 72 处显示相同的遗传变化。同样，在显示密码子 172 突变的 3 个样本中，有 1 个样本有匹配的唾液 DNA，突变状态相同。
结论
结果表明，ORR 技术具有临床应用的潜力，可以替代侵袭性组织活检，用于检测口腔癌候选生物标志物中的遗传变化。
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