Evaluation of NTRK expression and fusions in a large cohort of early-stage lung cancer

Abstract

Tropomyosin receptor kinases (TRK) are attractive targets for cancer therapy. As TRK-inhibitors are approved for all solid cancers with detectable fusions involving the Neurotrophic tyrosine receptor kinase (NTRK)-genes, there has been an increased interest in optimizing testing regimes. In this project, we wanted to find the prevalence of NTRK fusions in a cohort of various histopathological types of early-stage lung cancer in Norway and to investigate the association between TRK protein expression and specific histopathological types, including their molecular and epidemiological characteristics. We used immunohistochemistry (IHC) as a screening tool for TRK expression, and next-generation sequencing (NGS) and fluorescence in situ hybridization (FISH) as confirmatory tests for underlying NTRK-fusion. Among 940 cases, 43 (4.6%) had positive TRK IHC, but in none of these could a NTRK fusion be confirmed by NGS or FISH. IHC-positive cases showed various staining intensities and patterns including cytoplasmatic or nuclear staining. IHC-positivity was more common in squamous cell carcinoma (LUSC) (10.3%) and adenoid cystic carcinoma (40.0%), where the majority showed heterogeneous staining intensity. In comparison, only 1.1% of the adenocarcinomas were positive. IHC-positivity was also more common in men, but this association could be explained by the dominance of LUSC in TRK IHC-positive cases. Protein expression was not associated with differences in time to relapse or overall survival. Our study indicates that NTRK fusion is rare in early-stage lung cancer. Due to the high level of false positive cases with IHC, Pan-TRK IHC is less suited as a screening tool for NTRK-fusions in LUSC and adenoid cystic carcinoma.

Keywords: Fluorescence in situ hybridization; Immunohistochemistry; Lung cancer; Molecular pathology; NTRK; Next-generation sequencing.

Fig. 1

Flowchart. 972 patients were included in the study. Of these 32 samples had too few non-necrotic tumor cells, and were excluded from further analysis. On the 43 IHC-positive cases, we proceeded with NGS and whole section IHC. In a subgroup of the positive samples, we also performed FISH with both break apart probes and CNV probes. We also performed NGS, FISH-and whole section IHC on a subgroup of negative cases

Fig. 2

Case with strong and focal IHC and negative FISH (a-c). a Case with squamous cell carcinoma. Hematoxylin eosin saffron (HES) staining. b Same case NTRK IHC. Strong, but focal staining. c Fused FISH signals, meaning no evidence of NTRK rearrangement/fusion. Case with negative IHC and high copy number with FISH (d-f). d Case with acinar adenocarcinoma. e Same case with negative TRK IHC. Some unspecific staining in macrophages and necrotic cells. f NTRK1 CNV probe. High NTRK1 copy number (copy number: 8). Picture a, b, d and e: 40 × Roche Ventana DP 200 slide scanner