Introduction

The reported prevalence of ALK rearrangement in NSCLC ranges from 2%-7%. The primary standard diagnostic method is fluorescence in situ hybridization (FISH). Recently, immunohistochemistry (IHC) has also proven to be a reproducible and sensitive technique. Reverse transcriptase-polymerase chain reaction (RT-PCR) has also been advocated and most recently the advent of targeted Next-Generation Sequencing (NGS) for ALK and other fusions has become possible. This study compares ALK evaluation with all 4 techniques in resected NSCLC from the large ETOP Lungscape cohort.

Methods

96 cases from the ETOP Lungscape iBiobank, with any ALK immunoreactivity were examined by FISH, central RT-PCR and NGS. H-score>120 defines IHC-positivity. RNA was extracted from the same formalin-fixed, paraffin-embedded tissues. For RT-PCR, primers covered the most frequent ALK translocations. For NGS, the Oncomine™ Solid Tumour Fusion Transcript Kit was used. The concordance was assessed using the Cohen’s kappa coefficient (two-sided alpha≤5%).

Results

NGS provided results for 77 out of the 95 cases tested (81.1%), while RT-PCR for 77 out of 96 (80.2%). Concordance occurred in 55 cases out of the 60 cases tested with all 4 methods (43 ALK-negative, 12 ALK-positive). Using ALK co-positivity for IHC and FISH as gold standard, we derive sensitivity for RT-PCR/NGS 70.0%/85.0%, with specificity 87.1%/79.0%. Combining either RT-PCR or NGS with IHC, the sensitivity remains the same, while the specificity increases to 88.7% and 83.9% respectively.

Conclusion

NGS evaluation with the Oncomine™ Solid Tumour Fusion transcript kit and RT-PCR proves to have high sensitivity and specificity advocating their use in routine practice. For maximal sensitivity and specificity, ALK status should be assessed using two techniques and a third one in discordant cases. We therefore propose a customizable testing algorithm. These findings significantly influence existing testing paradigms and have clear clinical and economic impact.

中文翻译：

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NGS和RT-PCR方法对欧洲NSCLC患者ALK重排的评估：ETOP Lungscape项目的结果

介绍

据报道，NSCLC中ALK重排的患病率为2％-7％。主要的标准诊断方法是荧光原位杂交（FISH）。最近，免疫组织化学（IHC）也被证明是一种可重现和敏感的技术。逆转录聚合酶链反应（RT-PCR）也已被提倡，最近，针对ALK和其他融合物的靶向下一代测序（NGS）的问世成为可能。这项研究比较了来自大型ETOP Lungscape队列中切除的NSCLC的ALK评估与所有4种技术的比较。

方法

通过FISH，中央RT-PCR和NGS检查了ETOP Lungscape iBiobank的96例ALK免疫反应性。H得分> 120定义了IHC阳性。从相同的福尔马林固定石蜡包埋组织中提取RNA。对于RT-PCR，引物涵盖了最常见的ALK易位。对于NGS，使用Oncomine™实体肿瘤融合转录试剂盒。使用Cohen卡伯系数（双面alpha≤5％）评估一致性。

结果

NGS为95例受检病例中的77例（81.1％）提供了结果，而RT-PCR为96例中的77例（80.2％）提供了结果。在用全部4种方法测试的60例病例中，有55例出现了一致性（43个ALK阴性，12个ALK阳性）。以IHC和FISH的ALK共阳性作为金标准，我们得出RT-PCR / NGS的敏感性为70.0％/ 85.0％，特异性为87.1％/ 79.0％。将RT-PCR或NGS与IHC结合使用，灵敏度保持不变，而特异性分别提高到88.7％和83.9％。

结论

使用Oncomine™实体肿瘤融合转录本试剂盒和RT-PCR进行的NGS评估具有很高的敏感性和特异性，提倡在常规实践中使用它们。为了获得最大的灵敏度和特异性，应使用两种技术评估ALK的状态，在不一致的情况下应使用第三种技术。因此，我们提出了一种可定制的测试算法。这些发现极大地影响了现有的测试范例，并具有明显的临床和经济影响。