Superior detection of NTRK fusions across Chinese solid tumors using amplicon-based DNA and RNA NGS co-detection compared with hybrid-capture DNA-NGS

Background: NTRK fusions are clinically actionable drivers across many solid tumors, with several tumor-agnostic targeted therapies approved. Traditional DNA-based assays often lack sensitivity for NTRK rearrangements. Although RNA-NGS improves fusion detection, the relative performance of amplicon-based DNA+RNA NGS co-detection versus hybrid-capture DNA-NGS for identifying NTRK fusions in pan-solid tumors remains unclear. Methods: This study analyzed NTRK fusion detection across 16,546 tumors using a 35-gene amplicon-based DNA+RNA NGS co-detection and 4,709 tumors using hybrid-capture DNA-NGS. Tumor types included BRCA, COAD, HNSC, melanoma, NSCLC, PAAD, SARC, THCA, BTC, and STAD. Specifically, the co-detection cohort consisted of BRCA (27), COAD (3,839), HNSC (39), melanoma (111), NSCLC (11,242), PAAD (89), SARC (61), THCA (62), BTC (310), and STAD (766), whereas the DNA-NGS cohort included BRCA (126), COAD (857), HNSC (79), melanoma (42), NSCLC (2,504), PAAD (184), SARC (67), THCA (62), BTC (398), and STAD (446). Results: Amplicon-based DNA+RNA NGS co-detection demonstrated higher NTRK fusion positivity across multiple solid tumors, including BRCA (2/27, 7.41%), COAD (7/3,839, 0.19%), HNSC (1/39, 2.56%), melanoma (1/111, 0.90%), NSCLC (18/11,242, 0.16%), PAAD (2/89, 2.25%), SARC (1/61, 1.64%), and THCA (1/62, 1.61%), with no NTRK fusions detected in BTC or STAD. All 33 fusions detected by Amplicon-based DNA+RNA NGS were classical 5′-3′ events, with predominant fusion partners including TPM3-NTRK1(42.42%), SQSTM1-NTRK3(12.12%), CD74-NTRK1(9.09%), and several rare forms involving LMNA-NTRK1(3.03%), KIF5B-NTRK2(3.03%), ETV6-NTRK3(21.21%), AKAP13-NTRK3(3.03%), CD74-NTRK3(3.03%), EML4-NTRK3(3.03%). In contrast, hybrid-capture DNA-NGS showed generally lower detection rates across the same tumor types, identifying only sporadic NTRK-positive cases in COAD (1/857, 0.12%), NSCLC (4/2,504, 0.16%), BTC (1/398, 0.25%, NTRK1), and STAD (1/446, 0.22%), and none in BRCA, HNSC, melanoma, PAAD, SARC, or THCA. Among the fusions detected by hybrid-capture DNA-NGS, all but one were classical 5′-3′ rearrangements; a single NSCLC case harbored a non-canonical 5′-5′ ETV6-NTRK3 fusion. Fusion partners detected by this method included PIGR-NTRK1, NTRK1-RHBG, NTRK1-HAPLN2, NTRK1-S100A12, NTRK2-TMPRSS2, and ETV6-NTRK3. Collectively, co-detection demonstrated a markedly broader fusion spectrum and substantially improved sensitivity, particularly in BRCA, COAD, HNSC, and PAAD. Conclusion: Amplicon-based DNA+RNA NGS outperformed hybrid-capture DNA-NGS in sensitivity and fusion-partner breadth, highlighting the importance of RNA analysis in routine testing for NTRK-guided tumor-agnostic therapy.