Neurotrophic tropomyosin receptor kinase (NTRK) gene fusions are established, druggable drivers in thyroid cancer. Standard detection in many centers relies on DNA-based or targeted gene panel assays performed on surgical or advanced-disease specimens. However, some fusion configurations may be missed by routine panel testing. This study aimed to identify patients with NTRK fusions who could be overlooked by panel testing and to evaluate the utility of combining pan-TRK immunohistochemistry (IHC) with RNA sequencing in this context.
The investigators analyzed two distinct patient cohorts. Group A comprised 100 patients with advanced thyroid cancer who underwent gene panel testing as part of their molecular workup. Group B included 76 surgical cases of papillary thyroid carcinoma that were screened by immunohistochemistry.
For Group A, pan-TRK IHC was performed on available material, and RNA sequencing was added for cases that lacked mitogen-activated protein kinase (MAPK) pathway alterations on panel testing. For Group B, surgical specimens were screened by IHC both for BRAF mutation and for pan-TRK expression; RNA sequencing was then performed on cases that were BRAF-negative and pan-TRK-positive.
The source reports use of routine gene panel testing to detect driver alterations affecting the MAPK pathway (including BRAF mutation, RET fusions, and RAS mutations) as well as NTRK fusions when present on the panel. Pan-TRK immunostaining was applied to identify TRK protein expression as a screening step. RNA sequencing was used as an orthogonal method to identify or confirm fusion transcripts in selected cases—specifically in panel-negative cases without MAPK pathway alterations in Group A and in BRAF-negative, pan-TRK-positive cases in Group B.
Among 100 advanced thyroid cancer cases in Group A, gene panel testing identified MAPK pathway alterations in 88 cases. The distribution of alterations detected by panel was: BRAF mutation in 72 cases, RET fusion in 8 cases, RAS mutation in 7 cases, and an NTRK fusion in 1 case.
Pan-TRK immunostaining was performed across this cohort, and RNA sequencing was subsequently applied to eight cases that lacked MAPK alterations on the panel. RNA sequencing on these eight cases did not detect pathological NTRK fusions according to the data reported.
In the 76 surgical papillary thyroid carcinoma cases of Group B, immunohistochemical screening identified two cases harboring NTRK fusions. One case carried a TFG-NTRK1 fusion, which was detected by panel testing. The other case harbored a SQSTM1-NTRK3 fusion that was not identified by the gene panel and was discovered through the IHC screening followed by RNA sequencing.
These findings indicate that panel testing detected some NTRK fusions but missed at least one clinically relevant fusion that was revealed by combining pan-TRK IHC and RNA sequencing.
Immunostaining intensity with the pan-TRK antibody varied by fusion partner and gene. The authors report that NTRK1 fusion-positive tumors showed relatively strong pan-TRK staining. By contrast, NTRK3 fusion-positive tumors demonstrated weak staining in at least one case, and notably one case with an ETV6-NTRK3 fusion showed no detectable pan-TRK staining on IHC. These observations underscore variable sensitivity of pan-TRK IHC across different fusion types.
The study identified at least one thyroid carcinoma case with an NTRK fusion that was overlooked by conventional gene panel testing. Based on these results, the authors recommend active searching for NTRK fusions in thyroid cancers that lack MAPK pathway abnormalities on panel testing or that show pan-TRK positivity on immunohistochemistry. The combined use of pan-TRK IHC as a screening tool and RNA sequencing as a confirmatory method can detect fusion events missed by panels and may therefore identify additional patients eligible for targeted therapies.
The report emphasizes that immunohistochemical detection of TRK protein can vary by fusion partner, with weaker or absent staining for some NTRK3 fusions; this variability should be considered when triaging cases for RNA-based testing. The source does not provide additional methodological detail, broader prevalence estimates beyond the described cohorts, or outcome data for patients treated based on the detected fusions.