Department of Pathology and Laboratory Medicine, University of Calgary, Arnie Charbonneau Cancer Institute, Calgary, Alberta, Canada.
Department of Laboratory Medicine and Pathology, University of Alberta, Edmonton, Alberta, Canada; University Health Network, Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario, Canada.
Mod Pathol. 2024 Jan;37(1):100384. doi: 10.1016/j.modpat.2023.100384. Epub 2023 Nov 14.
Tumor-agnostic testing for NTRK1-3 gene rearrangements is required to identify patients who may benefit from TRK inhibitor therapies. The overarching objective of this study was to establish a high-quality pan-TRK immunohistochemistry (IHC) screening assay among 18 large regional pathology laboratories across Canada using pan-TRK monoclonal antibody clone EPR17341 in a ring study design. TRK-fusion positive and negative tumor samples were collected from participating sites, with fusion status confirmed by panel next-generation sequencing assays. Each laboratory received: (1) unstained sections from 30 cases of TRK-fusion-positive or -negative tumors, (2) 2 types of reference standards: TRK calibrator slides and IHC critical assay performance controls (iCAPCs), (3) EPR17341 antibody, and (4) suggestions for developing IHC protocols. Participants were asked to optimize the IHC protocol for their instruments and detection systems by using iCAPCs, to stain the 30 study cases, and to report the percentage scores for membranous, cytoplasmic, and nuclear staining. TRK calibrators were used to assess the analytical sensitivity of IHC protocols developed by using the 2 reference standards. Fifteen of 18 laboratories achieved diagnostic sensitivity of 100% against next-generation sequencing. The diagnostic specificity ranged from 40% to 90%. The results did not differ significantly between positive scores based on the presence of any type of staining vs the presence of overall staining in ≥1% of cells. The median limit of detection measured by TRK calibrators was 76,000 molecules/cell (range 38,000 to >200,000 molecules/cell). Three different patterns of staining were observed in 19 TRK-positive cases, cytoplasmic-only in 7 samples, nuclear and cytoplasmic in 9 samples, and cytoplasmic and membranous in 3 samples. The Canadian multicentric pan-TRK study illustrates a successful strategy to accelerate the multicenter harmonization and implementation of pan-TRK immunohistochemical screening that achieves high diagnostic sensitivity by using laboratory-developed tests where laboratories used centrally developed reference materials. The measurement of analytical sensitivity by using TRK calibrators provided additional insights into IHC protocol performance.
需要进行肿瘤不可知的 NTRK1-3 基因重排检测，以确定可能从 TRK 抑制剂治疗中获益的患者。本研究的总体目标是在加拿大 18 个大型区域病理实验室中使用 pan-TRK 单克隆抗体 EPR17341 建立高质量的 pan-TRK 免疫组织化学（IHC）筛选检测，采用环研究设计。从参与的站点收集 TRK 融合阳性和阴性肿瘤样本，融合状态通过面板下一代测序检测证实。每个实验室收到：（1）30 例 TRK 融合阳性或阴性肿瘤的未染色切片，（2）2 种参考标准：TRK 校准器载玻片和 IHC 关键检测性能对照（iCAPC），（3）EPR17341 抗体，（4）开发 IHC 方案的建议。参与者被要求使用 iCAPC 优化其仪器和检测系统的 IHC 方案，对 30 例研究病例进行染色，并报告膜、细胞质和核染色的百分比评分。使用 2 种参考标准开发的 IHC 方案的分析灵敏度使用 TRK 校准器进行评估。18 个实验室中有 15 个达到了针对下一代测序的 100%诊断敏感性。诊断特异性范围为 40%至 90%。阳性评分基于任何类型的染色与≥1%细胞的总体染色存在的基础上，结果没有显著差异。TRK 校准器测量的检测限中位数为 76,000 个分子/细胞（范围为 38,000 至>200,000 个分子/细胞）。在 19 例 TRK 阳性病例中观察到 3 种不同的染色模式，7 例样本中仅细胞质染色，9 例样本中核细胞质染色，3 例样本中细胞质和膜染色。加拿大多中心 pan-TRK 研究说明了一种成功的策略，可以加速多中心协调和实施 pan-TRK 免疫组织化学筛选，使用实验室开发的测试实现高诊断敏感性，实验室使用中央开发的参考材料。使用 TRK 校准器测量分析灵敏度为 IHC 方案性能提供了更多见解。
