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Purpose: The small molecule inhibitors larotrectinib and entrectinib have recently been approved as cancer agnostic drugs in patients with tumours harbouring a rearrangement of the neurotrophic tropomyosin receptor kinase (NTRK). These oncogenic fusions are estimated to occur in 0.1-3 % of non-small cell lung cancers (NSCLC). Although molecular techniques are most reliable for fusion detection, immunohistochemical analysis is considered valuable for screening. Therefore, we evaluated the newly introduced diagnostic immunohistochemical assay (clone EPR17341) on a representative NSCLC cohort.

Methods: Cancer tissue from 688 clinically and molecularly extensively annotated NSCLC patients were comprised on tissue microarrays and stained with the pan-TRK antibody clone EPR17341. Positive cases were further analysed with the TruSight Tumor 170 RNA assay (Illumina). Selected cases were also tested with a NanoString NTRK fusion assay. For 199 cases, NTRK RNA expression data were available from previous RNA sequencing analysis.

Results: Altogether, staining patterns for 617 NSCLC cases were evaluable. Of these, four cases (0.6 %) demonstrated a strong diffuse cytoplasmic and membranous staining, and seven cases a moderate staining (1.1 %). NanoString or TST170-analysis could not confirm an NTRK fusion in any of the IHC positive cases, or any of the cases with high mRNA levels. In the four cases with strong staining intensity in the tissue microarray, whole section staining revealed marked heterogeneity of NTRK protein expression.

Conclusion: The presence of NTRK fusion genes in non-small cell lung cancer is exceedingly rare. The use of the immunohistochemical NTRK assay will result in a small number of false positive cases. This should be considered when the assay is applied as a screening tool in clinical diagnostics.

Immunohistochemistry (IHC) is influenced by several factors such as cold ischemia time, fixative, fixation time, paraffin, storage time, antibody, antigen retrieval technique and detection systems. In the setting of post-mortem tissue, not only post-mortem delay, but also agonal state is of interest. Here, we assessed an additional variable, i.e., the thickness of the section, and noted that this variable also influenced the IHC outcome. This is of significance when the extent of labelling is a parameter to be assessed, for example when assigning a stage or grade of a disease. Furthermore, when assessing brain tissue with neurons, soma measuring from 4 to 100 µm, various cellular compartments composed of different proteins are localised in sections measuring 4 or 7 µm. Thus, what is seen in a 7-µm-thick section might be lacking in a 4-µm-thick section. Lack of information regarding the molecular size of commercial antibodies is also disturbing as this parameter might influence the distribution of the molecule in the three-dimensional section. The choice of antibody to be used and the staining methodology have been acknowledged being of significance for IHC outcome; however, neither sections thickness or the molecular weight has been discussed sufficiently. IHC has been shown to be an unpredictable technique used for assessment of tissue. This emphasises the need for detailed methodological descriptions in publications, the need to acknowledge and to harmonize all eventual pitfalls related to this methodology.

Antibody-based proteomics provides a powerful approach for the functional study of the human proteome involving the systematic generation of protein-specific affinity reagents. We used this strategy to construct a comprehensive, antibody-based protein atlas for expression and localization profiles in 48 normal human tissues and 20 different cancers. Here we report a new publicly available database containing, in the first version, ∼400,000 high resolution images corresponding to more than 700 antibodies toward human proteins. Each image has been annotated by a certified pathologist to provide a knowledge base for functional studies and to allow queries about protein profiles in normal and disease tissues. Our results suggest it should be possible to extend this analysis to the majority of all human proteins thus providing a valuable tool for medical and biological research.