非小细胞肺癌小活检标本质量与EGFR基因突变检出率关系分析

Analysis of the Relationship Between the Quality of Small Biopsy Specimens of Non-small Cell Lung Cancer and the Mutation Rate of EGFR Gene

背景与目的表皮生长因子受体(epidermal growth factor receptor, EGFR)是非小细胞肺癌(non-small celllungcancer,NSCLC)患者中突变率最高的基因,准确检测出其突变类型有助于指导患者接受靶向药物治疗从而延长患者生存期。为了得到准确的检查结果,基因检测平台对标本质量有一定要求。已有文献报道标本的肿瘤细胞数量及其比例等会影响EGFR基因突变检出率,本研究旨在分析NSCLC小活检标本质量与突变扩增系统(amplification refractory mutation system, ARMS)法检测EGFR基因突变检出率的关系。方法收集299例小活检肺腺癌病例的临床特征、在显微镜下评估切片的肿瘤细胞数量及比例、标本提取的DNA浓度、EGFR基因突变检测结果,分析标本质量与EGFR基因突变检出率的关系。结果肿瘤细胞数≤500组与>500组EGFR基因突变阳性率分别为40.7%(11/27)、43.8%(119/272),两者间无统计学差异(P=0.764)。DNA浓度≤20.4 ng/μL组及>20.4 ng/μL组的基因突变阳性率分别为42.7%(64/150)、44.3%(66/149),两者间无统计学差异(P=0.776)。肿瘤细胞比例≤30%及>30%组的阳性率分别为29.4%(20/68)、47.6%(110/231),两者间存在统计学差异(P=0.008)。多因素Logistic分析显示男性、甲状腺转录因子1(thyroid transcription factor-1, TTF-1)阴性、有吸烟史及肿瘤细胞比例<30%是影响EGFR基因突变低检出率的主要因素。结论在达到检测的最低要求后,肿瘤细胞比例仍可影响EGFR基因突变检出率。有必要在基因检测切片后再次评估最后一张切片的肿瘤细胞比例,对于肿瘤细胞比例过低的标本,建议通过显微切割等方法富集肿瘤细胞,提高其肿瘤细胞比例,得出更准确的检测结果。对于无法进行肿瘤细胞富集的标本,可行循环肿瘤DNA(circulating tumor DNA, ct DNA)检测作为补充,若结果仍为阴性才需考虑再次活检获取足够的肿瘤标本进行分子检测。

Background and objective Epidermal growth factor receptor(EGFR) is the gene with the highest mutation rate in non-small cell lung cancer(NSCLC) patients, and the accurate evaluation of its mutational status can facilitate patients receiving targeted drug therapy and thereby prolong patients’ survival. The gene testing platform has adequacy requirements for the specimen quality in order to obtain accurate examination results. It has been reported that the number and proportion of tumor cells in samples will affect the detection rate of EGFR gene mutation. The present study aims to analyze the relationship between the quality of small biopsy specimens of NSCLC and the mutation rate of EGFR gene with amplification refractory mutation system(ARSM) test. Methods After collecting the clinical characteristics of 299 cases small biopsy of lung adenocarcinoma, DNA concentration of the specimens and the mutational status of EGFR gene, the number and proportion of tumor cells in HE stained sections evaluated using light microscopy, the relationship between specimen quality and the mutation rate of EGFR gene were analyzed. Results The mutation rates of EGFR for the groups with tumor cell number ≤500 and >500 were 40.7%(11/27) and 43.8%(119/272) respectively, without significant difference(P=0.764). The mutation rates for the groups with DNA concentration ≤20.4 ng/μL and >20.4 ng/μL were 42.7%(64/150) and 44.3%(66/149) respectively, without significant difference(P=0.776). The mutation rates for the groups with tumor cells proportion ≤30% and >30% were 29.4%(20/68) and 47.6%(110/231) respectively, demonstrating significant difference(P=0.008). Multivariate Logistic analysis showed that male, thyroid transcription factor-1(TTF-1) negative, smoking history and tumor cell proportion less than 30% were main factors that contributes to the low detection rate of EGFR gene mutation. Conclusion After meeting the minimum requirements for detection, the EGFR mutation rate is affected by the proportion of tumor cells in the sample. Therefore, it is necessary to re-evaluate the tumor cell proportion in the last section after the genetic test section. For samples with lower tumor cell proportion, enriching tumor cells through microdissection and other methods is recommended for a more accurate detection result. For specimens that cannot be enriched with tumor cells, circulating tumor DNA(ct DNA) test can be performed as a supplement. If the result is still negative, another biopsy should be considered to obtain enough tumor specimens for molecular testing.