Background: Targeted next-generation sequencing (NGS) panels are increasingly being utilized to identify actionable gene amplifications (copy number > 4) among solid tumors. Methods: This study validated the analytical performance of two amplicon-based NGS assays, the Oncomine Comprehensive Panel (OCAv3) and the Oncomine Focus Assay (OFA), for detecting gene amplification in formalin-fixed paraffin-embedded (FFPE) tumors of varying cellularity. OCAv3 was assessed for amplification detection in 756 FFPE samples comprising various tumor types. Results: We demonstrated that with standardized quality control metrics, including median absolute pairwise difference score, these assays can achieve a near-perfect positive predictive value, although their sensitivity for detecting amplifications significantly decreased in tumors with cellularity below 30%. Stratifying tumor cellularity into 10–30%, 31–60%, and 61–95% groups revealed significantly higher gene amplification detection rates in the 31–60% and 61–95% groupsversusthe 10–30% group (20.6% and 26.7% vs. 9.2%,p< 0.0001). When considering all detected gene amplifications, the average amplification calling per sample was nearly five-fold lower in the 10–30% groupversusthe 61–95% group (0.11 vs. 0.52;p< 0.0001). To further investigate the analytic performance of OCAv3 in detectingERBB2amplification, we analyzed a cohort of 121 uterine carcinomas with confirmedERBB2status by HER2 IHC or FISH, in which a threshold incorporating amplifications and tumor cellularity achieved 79% sensitivity and 100% specificity, potentially eliminating the need for FISH analysis in 34% of equivocal cases. In a separate validation cohort, similar analytical performance was observed, with the threshold demonstrating consistent sensitivity and specificity. Conclusions: This study highlights the strengths and limitations of amplicon-based NGS assays in detecting amplifications using real-world data.
背景：靶向二代测序（NGS）检测组合正日益广泛地应用于实体瘤中可操作基因扩增（拷贝数>4）的识别。方法：本研究验证了两种基于扩增子的NGS检测方法——Oncomine综合检测组合（OCAv3）和Oncomine聚焦检测（OFA）——在不同细胞丰度的福尔马林固定石蜡包埋（FFPE）肿瘤组织中检测基因扩增的分析性能。研究评估了OCAv3在756份涵盖多种肿瘤类型的FFPE样本中的扩增检测能力。结果：研究表明，在采用包括中位数绝对配对差异评分在内的标准化质量控制指标后，这些检测方法可实现近乎完美的阳性预测值，但其检测扩增的灵敏度在细胞丰度低于30%的肿瘤中显著下降。将肿瘤细胞丰度分为10–30%、31–60%和61–95%三组后发现，31–60%组和61–95%组的基因扩增检出率显著高于10–30%组（分别为20.6%、26.7%对比9.2%，p<0.0001）。当考虑所有检测到的基因扩增时，10–30%组每样本的平均扩增检出数较61–95%组降低近五倍（0.11对比0.52；p<0.0001）。为深入探究OCAv3检测ERBB2扩增的分析性能，我们分析了121例子宫癌队列，这些病例均通过HER2免疫组化或荧光原位杂交确认ERBB2状态。结果显示，结合扩增水平与肿瘤细胞丰度的阈值实现了79%的敏感性和100%的特异性，可能使34%的不确定病例无需再进行FISH检测。在另一独立验证队列中观察到相似的分析性能，该阈值展现出稳定的敏感性与特异性。结论：本研究基于真实世界数据，揭示了基于扩增子的NGS检测方法在扩增检测中的优势与局限性。
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