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Updated: Oct 21, 2025
Oncogenic Gene Fusion Detection Using Anchored Multiplex Polymerase Chain Reaction Followed by Next Generation Sequencing
Published on: July 5, 2019
Evaluation of NTRK Gene Fusion by Five Different Platforms in Triple-Negative Breast Carcinoma
Shafei Wu1, Xiaohua Shi1, Xinyu Ren1
1Department of Pathology, State Key Laboratory of Complex Severe and Rare Disease, Molecular Pathology Research Center, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China.
Abstract:
Triple-negative breast carcinoma (TNBC) is an aggressive disease that has a poor prognosis since it lacks effective treatment methods. Neurotrophic tyrosine receptor kinase (NTRK) fusion genes are excellent candidates for targeted RTK inhibitor therapies and there are available targeted therapy drugs for the treatment of TRK fusion-positive tumors in a tumor agnostic pattern. Our study was designed to investigate the NTRK gene fusion status in TNBC patients and to determine whether RTK-targeted therapies are suitable for TNBC patients. A total of 305 TNBC patients were enrolled in our study. IHC was employed as a prescreening method, and IHC positive cases were further submitted for evaluation by FISH, RT-PCR, and NGS methods. NTRK IHC was evaluated successfully in 287 of the 305 cases, and there were 32 (11.15%) positive cases. FISH was carried out in the 32 IHC positive cases. There were 13 FISH-positive cases if the threshold was set as >15% of the 100 counted tumor cells having a split orange and green signal with more than one signal diameter. There were only 2 FISH-positive cases if the cutoff value was defined as >15% of the counted tumor cells having a split signal with more than two signal diameter widths. One of the FISH-positive cases had a separate NTRK3 FISH signal in 88% of the tumor cells, and its IHC result was strong nuclear staining in all the tumor cells. After evaluation of the morphology, it was re-diagnosed as secretory breast carcinoma, and the NGS result confirmed that it had a NTRK3-ETV6 fusion gene. The other FISH-positive cases were all negative for NTRK gene fusion in the NGS or RT-PCR examination. The NTRK gene fusion rate was low in our TNBC cohort. NTRK gene fusion may be a rare event in TNBC. The high false-positive rate of NTRK gene fusion detected by IHC questions its role as a prescreening method in TNBC. More data may be needed to determine a suitable threshold for NTRK FISH in TNBC in the future. More studies are needed to confirm whether RTK-targeted therapies are appropriate treatments for TNBC patients.
Insights
Neurotrophic tyrosine receptor kinase (NTRK) gene fusions are rare in triple-negative breast carcinoma (TNBC). Immunohistochemistry (IHC) showed a high false-positive rate, questioning its use as a prescreening method for NTRK fusions in TNBC.
Area of Science:
- Oncology
- Genetics
- Molecular Biology
Background:
- Triple-negative breast carcinoma (TNBC) presents an aggressive clinical course with limited therapeutic options.
- Neurotrophic tyrosine receptor kinase (NTRK) gene fusions are actionable targets for tyrosine kinase inhibitor (TKI) therapies in various cancers.
- Tumor-agnostic therapies targeting NTRK fusions offer a treatment avenue for fusion-positive malignancies.
Purpose of the Study:
- To investigate the prevalence of NTRK gene fusions in a cohort of TNBC patients.
- To evaluate the potential suitability of NTRK-targeted therapies for TNBC.
- To assess the diagnostic accuracy of immunohistochemistry (IHC) as a prescreening tool for NTRK fusions in TNBC.
Main Methods:
- A cohort of 305 TNBC patients was analyzed.
- Immunohistochemistry (IHC) was used for initial screening of NTRK expression.
- Fluorescence in situ hybridization (FISH), reverse transcription polymerase chain reaction (RT-PCR), and next-generation sequencing (NGS) were employed for confirmation.
Main Results:
- NTRK gene fusions were found to be rare in the studied TNBC cohort.
- Immunohistochemistry (IHC) demonstrated a high false-positive rate for NTRK detection.
- Only one case confirmed by NGS exhibited an NTRK3-ETV6 fusion, re-diagnosed as secretory breast carcinoma.
Conclusions:
- NTRK gene fusions represent a rare event in triple-negative breast carcinoma.
- The utility of IHC as a primary screening method for NTRK fusions in TNBC is questionable due to a high false-positive rate.
- Further research is required to establish optimal diagnostic thresholds for FISH and to determine the efficacy of RTK-targeted therapies in TNBC.
