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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.

Programmed cell death ligand 1 (PD-L1) expression within the same lung cancer tissue is variable. In this study we evaluated if the PD-L1 expression on small biopsy specimens represent the PD-L1 status of the corresponding resection specimen. Our results indicate a relative good agreement between biopsy and surgical specimens, with a discordance in approximately 10% of the cases. Background: The immunohistochemical analysis of programmed cell death ligand 1 (PD-L1) expression in tumor tissue of non-small-cell lung cancer patients has now been integrated in the diagnostic workup. Analysis is commonly done on small tissue biopsy samples representing a minimal fraction of the whole tumor. The aim of the study was to evaluate the correlation of PD-L1 expression on biopsy specimens with corresponding resection specimens. Materials and Methods: In total, 58 consecutive cases with preoperative biopsy and resected tumor specimens were selected. From each resection specimen 2 tumor cores were compiled into a tissue microarray (TMA). Immunohistochemical staining with the antibody SP263 was performed on biopsy specimens, resection specimens (whole sections), as well as on the TMA. Results: The proportion of PD-L1-positive stainings were comparable between the resection specimens (48% and 19%), the biopsies (43% and 17%), and the TMAs (47% and 14%), using cutoffs of 1% and 50%, respectively (P > .39 all comparisons). When the resection specimens were considered as reference, PD-L1 status differed in 16%/5% for biopsies and in 9%/9% for TMAs (1%/50% cutoff). The sensitivity of the biopsy analysis was 79%/82% and the specificity was 90%/98% at the 1%/50% cutoff. The Cohens kappa value for the agreement between biopsy and tumor. was 0.70 at the 1% cutoff and 0.83 at the 50% cutoff. Conclusion: The results indicate a moderate concordance between the analysis of biopsy and whole tumor tissue, resulting in misclassification of samples in particular when the lower 1% cutoff was used. Clinicians should be aware of this uncertainty when interpreting PD-L1 reports for treatment decisions.

Context.—The immune microenvironment is involved in fundamental aspects of tumorigenesis, and immune scores are now being developed for clinical diagnostics. Objective.—To evaluate how well small diagnostic biopsies and tissue microarrays (TMAs) reflect immune cell infiltration compared to the whole tumor slide, in tissue from patients with non–small cell lung cancer. Design.—A TMA was constructed comprising tissue from surgical resection specimens of 58 patients with non–small cell lung cancer, with available preoperative biopsy material. Whole sections, biopsies, and TMA were stained for the pan-T lymphocyte marker CD3 to determine densities of tumor-infiltrating lymphocytes. Immune cell infiltration was assessed semiquantitatively as well as objectively with a microscopic grid count. For 19 of the cases, RNA sequencing data were available. Results.—The semiquantitative comparison of immune cell infiltration between the whole section and the biopsy displayed fair agreement (intraclass correlation coefficient [ICC], 0.29; P ¼ .01; CI, 0.03–0.51). In contrast, the TMA showed substantial agreement compared with the whole slide (ICC, 0.64; P , .001; CI, 0.39–0.79). The grid-based method did not enhance the agreement between the different tissue materials. The comparison of CD3 RNA sequencing data with CD3 cell annotations confirmed the poor representativity of biopsies as well as the stronger correlation for the TMA cores. Conclusions.—Although overall lymphocyte infiltration is relatively well represented on TMAs, the representativity in diagnostic lung cancer biopsies is poor. This finding challenges the concept of using biopsies to establish immune scores as prognostic or predictive biomarkers for diagnostic applications.

Introduction

Tertiary lymphoid structures (TLS) are lymphocyte aggregates resembling secondary lymphoid organs and are pivotal in cancer immunity. The ambiguous morphological definition of TLS makes it challenging to ascertain their clinical impact on patient survival and response to immunotherapy.

Objectives

This study aimed to characterize TLS in hematoxylin-eosin tissue sections from lung cancer patients, assessing their occurrence in relation to the local immune environment, mutational background, and patient outcome.

Methods

Two pathologists evaluated one whole tissue section from resection specimens of 680 NSCLC patients. TLS were spatially quantified within the tumor area or periphery and further categorized based on the presence of germinal centers (mature TLS). Metrics were integrated with immune cell counts, genomic and transcriptomic data, and correlated with clinical parameters.

Results

TLS were present in 86% of 536 evaluable cases, predominantly in the tumor periphery, with a median of eight TLS per case. Mature TLS were found in 24% of cases. TLS presence correlated positively with increased plasma cell (CD138+) and lymphocytic cell (CD3+, CD8+, FOXP3+) infiltration. Tumors with higher tumor mutational burden exhibited higher numbers of peripheral TLS. The overall TLS quantity was independently associated with improved patient survival, irrespective of TLS maturation status. This prognostic association held true for peripheral TLS but not for tumor TLS.

Conclusion

TLS in NSCLC is common and their correlation with a specific immune phenotype suggests biological relevance in the local immune reaction. The prognostic significance of this scoring system on routine hematoxylin-eosin sections has the potential to augment diagnostic algorithms for NSCLC patients.