Supplementary Material for: The Use of Pan-Tropomyosin Receptor Kinase Immunohistochemistry as a Screening Tool for the Detection of Neurotrophic Tropomyosin-Related Kinase Fusions: Real-World Data from a National Multicentric Retrospective Study

<b><i>Introduction:</i></b> The neurotrophic tropomyosin-related kinase (<i>NTRK</i>) genes encode the tropomyosin receptor kinases (TRKs). Patients with solid tumors harboring an oncogenic <i>NTRK</i> fusion are eligible for treatment with TRK inhibitors. <i>NTRK</i> fusion is often associated with TRK overexpression. Pan-TRK immunohistochemistry (IHC) is used to screen for <i>NTRK</i> fusions, but immunoreactivity patterns are poorly defined. <b><i>Methods:</i></b> Data on pan-TRK immunoreactivity patterns in 2,669 solid tumors (comprising carcinomas, sarcomas, and melanocytic lesions) were retrospectively collected by nine laboratories and comprised tumor type, percentage of pan-TRK-positive tumor cells, staining intensity, cytoplasmic, membrane and/or nuclear staining pattern, and the presence or absence of <i>NTRK</i> fusion. <b><i>Results:</i></b> Overall, 2,457 tumors (92%) were pan-TRK negative and 212 neoplasms (8%) were pan-TRK positive. Twenty-two pan-TRK-positive tumors (0.8%) harbored an <i>NTRK</i> fusion, representing 10% of all pan-TRK-positive tumors. Cytoplasmic immunoreactivity was most often observed, followed by membrane immunoreactivity. Nuclear pan-TRK positivity was least frequent, but was most often (33%) associated with <i>NTRK</i> fusion. <b><i>Conclusion:</i></b> Pan-TRK IHC can be used to screen for <i>NTRK</i> fusions, especially in commonly diagnosed solid tumors with low <i>NTRK</i> fusion prevalence. In case of pan-TRK immunoreactivity, regardless of its intensity and tumor cell percentage, subsequent molecular tests should be performed to formally confirm the presence or absence of <i>NTRK</i> fusions.

**引言：** 神经营养性原肌球蛋白相关激酶（neurotrophic tropomyosin-related kinase，NTRK）基因可编码原肌球蛋白受体激酶（tropomyosin receptor kinases，TRKs）。携带致癌性NTRK融合的实体瘤患者可接受TRK抑制剂治疗。NTRK融合通常与TRK过表达相关。泛TRK免疫组化（immunohistochemistry，IHC）常用于筛查NTRK融合，但免疫反应模式尚缺乏明确定义。
**方法：** 由9家实验室回顾性收集了2669例实体瘤（涵盖癌、肉瘤及黑素细胞病变）的泛TRK免疫反应模式数据，内容包括肿瘤类型、泛TRK阳性肿瘤细胞占比、染色强度、胞质、膜和/或核染色模式，以及是否存在NTRK融合。
**结果：** 总体而言，2457例肿瘤（92%）为泛TRK阴性，212例肿瘤（8%）为泛TRK阳性。其中22例泛TRK阳性肿瘤（0.8%）携带NTRK融合，占所有泛TRK阳性肿瘤的10%。最常见的免疫反应模式为胞质染色，其次为膜染色。核泛TRK阳性最为少见，但该类阳性病例中33%与NTRK融合相关。
**结论：** 泛TRK免疫组化可用于筛查NTRK融合，尤其适用于NTRK融合患病率较低的常见实体瘤。若出现泛TRK免疫反应，无论其强度及阳性肿瘤细胞占比如何，均应开展后续分子检测以正式确认是否存在NTRK融合。