Abstract：
Tropomyosin receptor kinase (TRK) inhibitors have been approved for metastatic solid tumors harboring NTRK fusions, but the detection of NTRK fusions is challenging. International guidelines recommend pan-TRK immunohistochemistry (IHC) screening followed by next generation sequencing (NGS) in tumor types with low prevalence of NTRK fusions, including metastatic colorectal cancer (mCRC). RNA-based NGS is preferred, but is expensive, time-consuming, and extracting good-quality RNA from FFPE tissue is challenging. Alternatives in daily clinical practice are warranted. We assessed the diagnostic performance of RNA-NGS, FFPE-targeted locus capture (FFPE-TLC), fluorescence in situ hybridization (FISH), and the 5\'/3\' imbalance quantitative RT-PCR (qRT-PCR) after IHC screening in 268 patients with microsatellite-instability-high mCRC, the subgroup in which NTRK fusions are most prevalent (1-5%). A consensus result was determined after review of all assay results. In 16 IHC positive tumors, 10 NTRK fusions were detected. In 33 IHC negative samples, no additional transcribed NTRK fusions were found, underscoring the high sensitivity of IHC. Sensitivity of RNA-NGS, FFPE-TLC, FISH, and qRT-PCR was 90%, 90%, 78%, and 100%, respectively. Specificity was 100% for all assays. Robustness, defined as the percentage of samples that provided an interpretable result in the first run, was 100% for FFPE-TLC, yet more limited for RNA-NGS (85%), FISH (70%), and qRT-PCR (70%). Overall, we do not recommend FISH for the detection of NTRK fusions in mCRC due to its low sensitivity and limited robustness. We conclude that RNA-NGS, FFPE-TLC, and qRT-PCR are appropriate assays for NTRK fusion detection, after enrichment with pan-TRK IHC, in routine clinical practice.
摘要：
原肌球蛋白受体激酶（TRK）抑制剂已被批准用于携带NTRK融合的转移性实体瘤，但是NTRK融合的检测具有挑战性。国际指南建议泛TRK免疫组织化学（IHC）筛查，然后在NTRK融合患病率低的肿瘤类型中进行下一代测序（NGS）。包括转移性结直肠癌（mCRC）。基于RNA的NGS是优选的，但是很贵，耗时,从FFPE组织中提取高质量的RNA具有挑战性。日常临床实践中的替代方案是必要的。我们评估了RNA-NGS的诊断性能，FFPE靶向基因座捕获（FFPE-TLC），荧光原位杂交（FISH），和5'/3'不平衡定量RT-PCR（qRT-PCR）的IHC筛查后的268例微卫星不稳定性高mCRC患者，NTRK融合最普遍的亚组(1-5%)。在审查所有测定结果后确定一致结果。在16个IHC阳性肿瘤中，检测到10个NTRK融合。在33个IHC阴性样本中，没有发现其他转录的NTRK融合体，强调了IHC的高灵敏度。RNA-NGS的敏感性，FFPE-TLC,FISH,qRT-PCR为90%，90%,78%,100%,分别。所有测定的特异性为100%。鲁棒性，定义为在第一次运行中提供可解释结果的样本百分比，FFPE-TLC为100%，RNA-NGS（85%）更有限，鱼（70%），和qRT-PCR(70%)。总的来说,我们不推荐FISH用于mCRC中NTRK融合的检测,因为其灵敏度低,鲁棒性有限.我们得出结论，RNA-NGS，FFPE-TLC,qRT-PCR是NTRK融合检测的合适方法，用泛TRKIHC富集后，在常规临床实践中。
