下一代测序(NGS)最近在癌症的分子诊断中得到了迅速的应用。但它仍然面临一些障碍。在这项研究中,使用NGS询问665个肺腺癌样本(558个TKI初始样本和107个TKI复发样本),并探索了对适当组织样本进行NGS测试的挑战和可能的解决方案。结果表明,在肿瘤细胞数量<20%的活检样本中观察到的HER2/BRAF/PIK3CA和获得性EGFRT790M突变的频率低于肿瘤细胞数量≥20%的活检样本。但EGFR或KRAS突变频率无显著差异.此外,通过异质性评分(HS)评估肿瘤异质性,通过将肿瘤细胞的突变等位基因频率(MAF)乘以2来计算。在TKI天真的样本中,肿瘤内异质性可能发生在EGFR,KRAS,HER2BRAF,和PIK3CA突变肿瘤,但是程度是可变的。较高的EGFR,但与KRASHS相比,观察到较低的BRAF和PIK3CAHS值。在TKI复发的样本中,对伴随致敏EGFR和T790MMAFs的分析显示,在获得性EGFRT790M突变肿瘤中,瘤内异质性很常见.原发性和转移性肿瘤之间的突变状态通常是一致的,但是KRAS,转移性肿瘤中的HER2和PIK3CAHS显著高于原发性肿瘤。此外,在诊断为模棱两可或多原发肿瘤的多灶性肺腺癌中,突变状态的不一致率很高.一起,我们的发现表明,在组织过程中,全面的质量评估是必要的,以减轻肿瘤细胞性差的挑战,肿瘤异质性,和多灶性克隆非依赖性肿瘤。
Next-generation sequencing (NGS) has recently been rapidly adopted in the molecular diagnosis of cancer, but it still faces some obstacles. In this study, 665 lung adenocarcinoma samples (558 TKI-naive and 107 TKI-relapsed samples) were interrogated using NGS, and the challenges and possible solutions of subjecting appropriate tissue samples to NGS testing were explored. The results showed that lower frequencies of HER2/BRAF/PIK3CA and acquired EGFR T790M mutations were observed in biopsy samples with <20% tumor cellularity than in those with ≥20%, but there were no significant differences in the frequencies of EGFR or KRAS mutations. Moreover, tumor heterogeneity was assessed by heterogeneity score (HS), which was calculated through multiplying by 2 the mutant allele frequency (MAF) of tumor cells. In TKI-naive samples, intratumor heterogeneity could occur in EGFR, KRAS, HER2, BRAF, and PIK3CA mutant tumors, but the degree was variable. Higher EGFR, but lower BRAF and PIK3CA HS values were observed compared with KRAS HS. In TKI-relapsed samples, analysis of concomitant sensitizing EGFR and T790M MAFs showed that intratumor heterogeneity was common in acquired EGFR T790M mutant tumors. The mutational status between primary and metastatic tumors was usually concordant, but KRAS, HER2, and PIK3CA HS were significantly higher in metastatic tumors than in primary tumors. Additionally, the discordance rate of mutational status in multifocal lung adenocarcinomas diagnosed as equivocal or multiple primary tumors was high. Together, our findings demonstrate that a comprehensive quality assessment is necessary during tissue process to mitigate the challenges of poor tumor cellularity, tumor heterogeneity, and multifocal clonally independent tumors.