Detection of NTRK fusions in glioblastoma: fluorescent in situ hybridisation is more useful than pan-TRK immunohistochemistry as a screening tool prior to RNA sequencing

Abstract

Glioblastomas are frequent malignant brain tumours with a very poor prognosis and a need for new and efficient therapeutic strategies. With the approval of anti-TRK targeted therapies to treat patients with advanced NTRK-rearranged cancers, independent of the type of cancer, potential new treatment opportunities are available for the 0.5-5% of patients with NTRK-rearranged glioblastomas. Identification of these rare NTRK-rearranged glioblastomas requires efficient diagnostic tools and strategies which are evaluated in this study. We compared the results of NTRK1, NTRK2 and NTRK3 fluorescent in situ hybridisation (FISH) assays to those of pan-TRK immunohistochemistry (IHC) using two EPR17341 and A7H6R clones in a set of 196 patients with glioblastomas. Cases with at least 15% of positive nuclei using FISH analyses were further analysed using RNA sequencing. Above the 15% threshold, seven positive glioblastomas (3.57%) were identified by FISH assays (4 NTRK1, 3 NTRK2, no NTRK3). NTRK rearrangements were confirmed by RNA sequencing analyses in four cases [1 LMNA-NTRK1, 1 PRKAR2A-NTRK2, 1 SPECC1L-NTRK2 and 1 NACC2-NTRK2 fusions, i.e., 4/196 (2%) of NTRK-rearranged tumours in our series] but no rearrangement was detected in three samples with less than 30% of positive tumour nuclei as determined by NTRK1 FISH. Pan-TRK immunostaining showed major discrepancies when using either the EPR17341 or the A7H6R clones for the following criteria: main intensity, H-Score based scoring and homogeneity/heterogeneity of staining (Kappa values <0.2). This led to defining adequate criteria to identify NTRK-rearranged gliomas exhibiting strong and diffuse immunostaining contrasting to the variable and heterogeneous staining in non-NTRK-rearranged gliomas (p<0.0001). As assessing NTRK rearrangements has become crucial for glioma therapy, FISH seems to be a valuable tool to maximise access to TRK testing in patients with glioblastomas. In contrast to other cancers, pan-TRK IHC appears of limited interest in this field because there is no 'on/off' IHC positivity criterion to distinguish between NTRK-rearranged and non-NTRK-rearranged gliomas. RNA sequencing analyses are necessary in FISH positive cases with less than 30% positive nuclei, to avoid false positivity when scoring is close to the detection threshold.

Keywords: NTRK; fluorescent in situ hybridisation; glioblastomas; pan-TRK immunohistochemistry; targeted therapy.