Background:AlternativeNTRK1/TrkAsplicing resulting inTrkAIIIexpression, originally discovered in advanced-stage metastatic neuroblastomas, is also pronounced in prostate, medullary thyroid, glioblastoma multiforme, MCPyV-positive Merkel cell, cutaneous malignant melanoma, and pituitary neuroendocrine tumor subsets. In tumor models, TrkAIII exhibits actionable oncogenic activity equivalent to theTrkT3-fused oncogene, and in tumor cell lines, alternativeTrkAIIIsplicing is promoted by hypoxia, nutrient deprivation, endoplasmic reticulum stress, and SV40 large T antigen, implicating tumor microenvironmental conditions and oncogenic polyoma viruses in tumor-associated TrkAIII expression. Collectively, these observations characterize TrkAIII as a potentially frequent, actionable oncogenic alternative toTrkAgene fusion in different tumor types. Currently, therapeutic approval for efficacious Trk inhibitors is restricted toTrk-fusedgene positive tumors and not for tumors potentially driven by TrkAIII.Methods:With the therapeutically relevant aim of improving the identification of tumors potentially driven by TrkAIII, we have developed a TaqMan-based qRT-PCR assay for evaluatingTrkAIIIexpression in tumor cDNAs.Results:This assay, validated using gel-purifiedfs-TrkAandTrkAIIIcDNAs alone and in complex cDNA mixtures, employs primers and probes designed fromfs-TrkAandTrkAIIIsequences, with specificity provided by a TaqMan probe spanning theTrkAIIIexon 5–8 splice junction. It is highly efficient, reproducible, and specific and can detect as few as 10TrkAIIIcopies in complex RNAs extracted from either fresh or FFPE tumor tissues.Conclusions:Inclusion of this assay into precision oncology algorithms, when paired withfs-TrkA qRT-PCR and TrkA immune histochemistry, will make it easier to identify patients with therapy-resistant, advanced-stage metastaticTrk-fusedgene-negative tumors potentially driven by TrkAIII, for whom approval of third-line effective Trk inhibitors could be extended.
背景：NTRK1/TrkA选择性剪接导致TrkAIII表达，最初在晚期转移性神经母细胞瘤中发现，在前列腺癌、甲状腺髓样癌、多形性胶质母细胞瘤、MCPyV阳性默克尔细胞癌、皮肤恶性黑色素瘤及垂体神经内分泌肿瘤亚型中也显著存在。在肿瘤模型中，TrkAIII表现出与Trk T3融合癌基因相当的潜在致癌活性；在肿瘤细胞系中，缺氧、营养剥夺、内质网应激及SV40大T抗原可促进TrkAIII选择性剪接，提示肿瘤微环境条件与致癌性多瘤病毒参与肿瘤相关TrkAIII表达。综合来看，这些发现表明TrkAIII在不同肿瘤类型中可能成为替代TrkA基因融合的常见且可干预的致癌因素。目前，有效Trk抑制剂的治疗批准仅限于Trk融合基因阳性肿瘤，尚未涵盖可能由TrkAIII驱动的肿瘤。 方法：以提高潜在TrkAIII驱动肿瘤的识别能力为治疗相关目标，我们开发了一种基于TaqMan的qRT-PCR检测方法，用于评估肿瘤cDNA中的TrkAIII表达。 结果：该方法通过凝胶纯化的全长TrkA与TrkAIII cDNA单独及复杂cDNA混合物验证，采用基于全长TrkA和TrkAIII序列设计的引物与探针，其特异性由跨越TrkAIII外显子5-8剪接位点的TaqMan探针保障。该检测高效、可重复且特异性强，在新鲜或FFPE肿瘤组织提取的复杂RNA中可检测低至10个TrkAIII拷贝。 结论：将此检测纳入精准肿瘤学流程，结合全长TrkA qRT-PCR与TrkA免疫组化分析，将更易识别可能由TrkAIII驱动、对常规治疗耐药且Trk融合基因阴性的晚期转移性肿瘤患者，从而有望扩展三线有效Trk抑制剂的适用人群。
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