Abstract 131: Concordance of mRNA expression (nCounter) and protein expression (IHC) for the detection of PD-L1 in patients with advanced non-small cell lung cancer (NSCLC)
Authors: Teixido Cristina , Marin Elba , Aguado Cristina , Pare Laia , Gimenez-Capitan Ana , Lopez-Prades Sandra , Cardona Andres Felipe , Cabrera Carlos , Gonzalvo Elena , Lopez Laura , Roman Ruth , Martinez Daniel , Sullivan Ivana , Jares Pedro , Prat Aleix , Molina-Vila Miguel Angel , Reguart Noemi 
Published: August 2019

Abstract
Background: PD-L1 immunohistochemistry (IHC) staining is currently accepted as the gold-standard biomarker for immune therapy in advanced non-small cell lung cancer (NSCLC). However, the use of various antibodies and cut-offs as well as certain degree of subjectivity in pathological evaluation has overshadowed the clear-cut predictive performance of PD-L1 expression. Multiplexed technologies can be of help in this setting providing an objective measurement of PD-L1 levels. On the other hand, gene expression signatures incorporating not only PD-L1 but also other components of the stroma might better capture the immune-context of the molecular heterogeneity of NSCLC tumors. nCounter gene expression technology is an alternative method to measure PD-L1 gene expression by digital counting proving a direct measurement of mRNA levels.
Methods: A 7-gene ‘immune signature’ comprising CD4, CD8, programmed cell death-1 (PD-1), programmed death-ligand 1 (PD-L1), interferon gamma (IFNG), granzyme M (GZMM) and forkhead box P3 (FOXP3) were included in a customized nCounter panel (NanoString Technologies), used in our institution on a routine basis to simultaneously screen for relevant oncogenic-drivers (ALK, ROS1, RET, NTRK1 gene fusions and METΔ14 mutation). Total RNA obtained from formalin-fixed paraffin embedded (FFPE) samples was used for PD-L1 digital counting (nCounter) which was normalised with six housekeeping genes (ACTB, MRPL19, PSMC4, RPLP0, SF3A1, GAPDH) and compared with PD-L1 protein IHC evaluation using whole tissue section with 22C3 monoclonal mouse anti-PD-L1 antibody measured on tumor cells.
Results: A total of 425 FFPE samples from advanced NSCLC were analyzed with the nCounter panel. Among them, 25 samples were not evaluable (5.9%). PD-L1 IHC was available for 163 FFPE samples and were compared with nCounter PD-L1 expression results. By IHC, 63/163 samples (38.65%) were scored as negative for PD-L1 protein expression, whereas 100/163 (61.35%) were evaluated as positive. Among positive, 62 (38.04%) and 38 (23.31%) presented a moderate (≥ 1-49%) and high PD-L1 staining (≥50%) respectively. Using an appropriate cut-off value (IHC≥1%), PD-L1 mRNA expression levels correlated with PD-L1 IHC evaluation with a 76% of concordance and a 0.755 Cohen’s kappa (confidence interval 95% 0.651- 0.858). Unsupervised clustering across of mRNA expression data from 395 samples using the seven-immune-related genes and correlations between each immune gene were performed and a high correlation was found between PD-1 and FOXP3 (r=0.9) and PD-1 with GZMM (r=0.8).
Conclusions: PD-L1 mRNA gene expression shows promising in predicting PD-L1 protein expression in NSCLC. Further clinical validation is ongoing to confirm if PD-L1 gene expression by nCounter can be an alternative to IHC to select patients’ candidates for immune check-point inhibitors.
Citation Format: Cristina Teixido, Elba Marin, Cristina Aguado, Laia Pare, Ana Gimenez-Capitan, Sandra Lopez-Prades, Andres Felipe Cardona, Carlos Cabrera, Elena Gonzalvo, Laura Lopez, Ruth Roman, Daniel Martinez, Ivana Sullivan, Pedro Jares, Aleix Prat, Miguel Angel Molina-Vila, Noemi Reguart. Concordance of mRNA expression (nCounter) and protein expression (IHC) for the detection of PD-L1 in patients with advanced non-small cell lung cancer (NSCLC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 131.