Background and objective: Epidermal growth factor receptor (EGFR) is the most important therapeutic target in non-small cell lung cancer (NSCLC). EGFR mutations may predict responsiveness to tyrosine kinase inhibitors (TKIs). These mutations are commonly identified using direct sequencing, which is considered the gold standard. But direct sequencing is time-consuming and hyposensitive. In addition, this method requires a lot of tumor specimens. Denaturing highperformance liquid chromatography (DHPLC) is a rapid automated sensitive and specific method in mutant gene detection. The aim of this study is to evaluate DHPLC as a rapid detection method for EGFR mutations in NSCLC tumor specimens.
Methods: DHPLC was used to evaluate the accuracy and sensitivity of detection the serial dilutions of mutant and wild type EGFR plasma DNA. Frozen tumor specimens of 83 NSCLC patients from various ways had been included, after DNA extraction and polymerase chain reaction (PCR) on EGFR exon 19 and 21, the results from the direct sequencing and DHPLC were compared.
Results: Mutant plasma DNA can be detected in the serial dilution of 1:100 by DHPLC and 1:10 by direct sequencing respectively. The results from DHPLC showed 22 EGFR mutations were detected in 83 NSCLC patients, and only 19 mutation samples had been conformed by direct sequencing. Moreover, the other 61 samples were deemed as wild type by DHPLC and direct sequencing. The sensitivity and specificity of DHPLC was 100% and 95.13% respectively. The detection of the tumor specimens from CT-guided transthoracic needle lung biopsy, lymph node biopsy and surgical resection all showed high sensitivity and specificity. EGFR mutation has strong correlation with gender and pathologic type, but irrelevant to age and smoking status.
Conclusions: DHPLC was a precise rapid preliminary screening method for detection of NSCLC EGFR genotype.
背景与目的: 表皮生长因子受体(epidermal growth factor receptor, EGFR)是最重要的非小细胞肺癌(non-small cell lung cancer, NSCLC)治疗靶点，EGFR突变可预测酪氨酸激酶抑制剂(tyrosine kinase inhibitor, TKI)的疗效。DNA直接测序是检测EGFR突变最常用的方法，同时也作为突变检测的“金标准”，但该方法耗时较长、所需组织量较多且敏感性较低。变性高效液相色谱法是一种快速、自动化、高敏感性的突变检测方法，本研究旨在探讨变性高效液相色谱法(denaturing high-performance liquid chromatography, DHPLC)快速检测NSCLC肿瘤组织EGFR基因突变的诊断价值。
方法: 通过检测已知按不同比例混合的野生型及突变型EGFR质粒DNA，评价DHPLC法的准确性和敏感性。选取经多种途径获取的83例NSCLC患者的冻存肿瘤组织，提取DNA并PCR扩增EGFR外显子19、21，用DHPLC法检测并与直接测序法进行比较。
结果: 当突变型质粒与野生型质粒按1:100混合时，仍可被DHPLC法显著检出，而直接测序法仅可检出1:10水平。83例NSCLC组织标本中，DHPLC法检出22例EGFR突变(突变率26.51%)，3例直接测序法结果为野生型，余19例EGFR突变及61例野生型均与直接测序法结果相符。DHPLC法的敏感度为100%，特异度为95.31%，对经皮细针肺穿刺活检、淋巴结活检以及外科切除等途径获取的肿瘤样本均具有较高的敏感度、特异度。EGFR突变与性别、病理类型显著相关，与吸烟状态、年龄等无相关性。
结论: DHPLC法可作为NSCLC患者EGFR基因型的初筛方法。