{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T07:37:29Z","timestamp":1782286649750,"version":"3.54.5"},"reference-count":41,"publisher":"MDPI AG","issue":"3","license":[{"start":{"date-parts":[[2025,1,30]],"date-time":"2025-01-30T00:00:00Z","timestamp":1738195200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Department of Biotechnological and Applied Clinical Sciences of the University of L\u2019Aquila","award":["07_DG-2024_01"],"award-info":[{"award-number":["07_DG-2024_01"]}]},{"name":"Department of Biotechnological and Applied Clinical Sciences of the University of L\u2019Aquila","award":["07_DG-2024_06"],"award-info":[{"award-number":["07_DG-2024_06"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cancers"],"abstract":"<jats:p>Background: Alternative NTRK1\/TrkA splicing resulting in TrkAIII expression, 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 the TrkT3-fused oncogene, and in tumor cell lines, alternative TrkAIII splicing 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 to TrkA gene fusion in different tumor types. Currently, therapeutic approval for efficacious Trk inhibitors is restricted to Trk-fused gene 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 evaluating TrkAIII expression in tumor cDNAs. Results: This assay, validated using gel-purified fs-TrkA and TrkAIII cDNAs alone and in complex cDNA mixtures, employs primers and probes designed from fs-TrkA and TrkAIII sequences, with specificity provided by a TaqMan probe spanning the TrkAIII exon 5\u20138 splice junction. It is highly efficient, reproducible, and specific and can detect as few as 10 TrkAIII copies in complex RNAs extracted from either fresh or FFPE tumor tissues. Conclusions: Inclusion of this assay into precision oncology algorithms, when paired with fs-TrkA qRT-PCR and TrkA immune histochemistry, will make it easier to identify patients with therapy-resistant, advanced-stage metastatic Trk-fused gene-negative tumors potentially driven by TrkAIII, for whom approval of third-line effective Trk inhibitors could be extended.<\/jats:p>","DOI":"10.3390\/cancers17030471","type":"journal-article","created":{"date-parts":[[2025,1,30]],"date-time":"2025-01-30T11:08:23Z","timestamp":1738235303000},"page":"471","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["A TaqMan-Based qRT-PCR Assay for Accurate Evaluation of the Oncogenic TrkAIII Splice Variant in Tumor cDNAs"],"prefix":"10.3390","volume":"17","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5770-084X","authenticated-orcid":false,"given":"Maddalena","family":"Sbaffone","sequence":"first","affiliation":[{"name":"Department of Biotechnological and Applied Clinical Sciences, University of L\u2019Aquila, Via Vetoio, 67100 L\u2019Aquila, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0962-6088","authenticated-orcid":false,"given":"Antonietta Rosella","family":"Farina","sequence":"additional","affiliation":[{"name":"Department of Biotechnological and Applied Clinical Sciences, University of L\u2019Aquila, Via Vetoio, 67100 L\u2019Aquila, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0008-6884-4062","authenticated-orcid":false,"given":"Ilaria","family":"Martelli","sequence":"additional","affiliation":[{"name":"Department of Biotechnological and Applied Clinical Sciences, University of L\u2019Aquila, Via Vetoio, 67100 L\u2019Aquila, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-6843-7178","authenticated-orcid":false,"given":"Eugenio","family":"Pontieri","sequence":"additional","affiliation":[{"name":"Department of Biotechnological and Applied Clinical Sciences, University of L\u2019Aquila, Via Vetoio, 67100 L\u2019Aquila, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8525-084X","authenticated-orcid":false,"given":"Stefano","family":"Guadagni","sequence":"additional","affiliation":[{"name":"Department of Biotechnological and Applied Clinical Sciences, University of L\u2019Aquila, Via Vetoio, 67100 L\u2019Aquila, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7096-3759","authenticated-orcid":false,"given":"Andrew Reay","family":"Mackay","sequence":"additional","affiliation":[{"name":"Department of Biotechnological and Applied Clinical Sciences, University of L\u2019Aquila, Via Vetoio, 67100 L\u2019Aquila, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9112-1750","authenticated-orcid":false,"given":"Lucia","family":"Cappabianca","sequence":"additional","affiliation":[{"name":"Department of Biotechnological and Applied Clinical Sciences, University of L\u2019Aquila, Via Vetoio, 67100 L\u2019Aquila, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Veronica","family":"Zelli","sequence":"additional","affiliation":[{"name":"Department of Biotechnological and Applied Clinical Sciences, University of L\u2019Aquila, Via Vetoio, 67100 L\u2019Aquila, Italy"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2025,1,30]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"743","DOI":"10.1038\/319743a0","article-title":"A human oncogene formed by the fusion of truncated tropomyosin and protein tyrosine kinase sequences","volume":"319","author":"Hughes","year":"1986","journal-title":"Nature"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"207","DOI":"10.1007\/1-4020-8107-3_12","article-title":"TRK oncogenes in papillary thyroid carcinoma","volume":"122","author":"Greco","year":"2004","journal-title":"Cancer Treat. 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