NTRK gene aberrations in triple‐negative breast cancer: detection challenges using IHC, FISH, RT‐PCR, and NGS

Abstract

Triple‐negative breast cancer (TNBC) is usually an aggressive disease with a poor prognosis and limited treatment options. The neurotrophic tyrosine receptor kinase (NTRK) gene fusions are cancer type‐agnostic emerging biomarkers approved by the Food and Drug Administration (FDA), USA, for the selection of patients for targeted therapy. The main aim of our study was to investigate the frequency of NTRK aberrations, i.e. fusions, gene copy number gain, and amplification, in a series of TNBC using different methods. A total of 83 TNBCs were analyzed using pan‐TRK immunohistochemistry (IHC), fluorescence in situ hybridization (FISH), real‐time polymerase chain reaction (RT‐PCR), and RNA‐based next‐generation sequencing (NGS). Of 83 cases, 16 showed pan‐TRK positivity although no cases had NTRK‐fusions. Indeed, FISH showed four cases carrying an atypical NTRK1 pattern consisting of one fusion signal and one/more single green signals, but all cases were negative for fusion by NGS and RT‐PCR testing. In addition, FISH analysis showed six cases with NTRK1 amplification, one case with NTRK2 copy number gain, and five cases with NTRK3 copy number gain, all negative for pan‐TRK IHC. Our data demonstrate that IHC has a high false‐positive rate for the detection of fusions and molecular testing is mandatory; there is no need to perform additional molecular tests in cases negativity for NTRK by IHC. In conclusion, the NTRK genes are not involved in fusions in TNBC, but both copy number gain and amplification are frequent events, suggesting a possible predictive role for other NTRK aberrations.

From genomic spectrum of NTRK genes to adverse effects of its inhibitors, a comprehensive genome-based and real-world pharmacovigilance analysis

“…The most frequently mutated amino acid sites in NTRK1/2/3 were G169R, A268V, and R153L/Q, respectively. Prior research has indicated that non-fusion NTRK alterations, such as mutations and amplifications, are associated with the response to NTRK inhibitors and their therapeutic efficacy ( Okamura et al, 2018 ; Amatu et al, 2019 ; Zito Marino et al, 2023 ). In particular, mutations at the G595R and G667C sites of NTRK1 , and at G696A and G623R of NTRK3 , have been associated with the acquisition of resistance to first-generation NTRK inhibitors, possibly due to the fact that these point mutations alter the three-dimensional conformation of the kinase’s structural domains, thereby reducing or abolishing binding to the inhibitors ( Russo et al, 2016 ; Drilon et al, 2017 ; Somwar et al, 2020 ).…”

“…Light et al reported that NTRK1/3 were highly expressed in neuroblastoma patients with a better prognosis, whereas NTRK2 was highly expressed in neuroblastoma patients with a poor prognosis ( Light et al, 2012 ). Additionally, in 83 clinical samples of triple-negative breast cancers, NTRK1/2/3 were found to exhibit varying degrees of copy number gain and amplification ( Zito Marino et al, 2023 ). However, these aforementioned studies are limited by focusing on specific cancer types and small sample sizes, highlighting the critical need for comprehensive analyses across multiple tumor types to investigate their functions.…”

Cost-Efficient Detection of NTRK1/2/3 Gene Fusions: Single-Center Analysis of 8075 Tumor Samples

“…TRK expression was detected in 133/898 (15%) NSCLCs; 120 of these carcinomas were available for NGS analysis, with only two instances of NTRK fusions eventually confirmed. Zito Marino et al [ 42 ] revealed pan-TRK IHC positivity in 16 out of 83 triple-negative breast malignancies; however, none of these samples carried translocation upon NGS. Vingiani et al [ 43 ] analyzed the collection composed of several tumor types.…”

Comprehensive Analysis of Clinically Relevant Copy Number Alterations (CNAs) Using a 523-Gene Next-Generation Sequencing Panel and NxClinical Software in Solid Tumors

“…Notably, solid tumors exhibit a heightened prevalence of CNAs, emphasizing their significance in the context of solid tumor biology [3]. While various laboratory-based methods, such as multiplex ligation-dependent probe amplification (MLPA) [4], microarray-based comparative genomic hybridization (aCGH) [5], SNP microarrays [6], RNA sequencing [7], fluorescence in situ hybridization (FISH) [8] and PCR-based approaches [9], facilitate CNA detection, they are often limited by factors like restricted coverage and resolution, tissue consumption, labor intensiveness, low throughput, and high cost [10]. Recent progress in next-generation sequencing (NGS) technologies has facilitated the concurrent identification of targeted CNAs and somatic mutations by utilizing a panel-based NGS approach.…”