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Background: Immunohistochemical staining of programmed death-ligand 1 (PD-L1) is used to determine which patients with non-small cell lung cancer (NSCLC) may benefit most from immunotherapy. Therapeutic management of many patients with NSCLC is based on cytology instead of histology. In this study, concordance of PD-L1 immunostaining between cytology cell blocks and their histologic counterparts was analyzed. Furthermore, the effect of various fixatives and fixation times on PD-L1 immunoreactivity was studied., Methods: Paired histologic and cytologic samples from 67 patients with NSCLC were collected by performing fine-needle aspiration on pneumonectomy/lobectomy specimens. Formalin-fixed, agar-based or CytoLyt/PreservCyt-fixed Cellient cell blocks were prepared. Sections from cell blocks and tissue blocks were stained with SP263 (standardized assay) and 22C3 (laboratory-developed test) antibodies. PD-L1 scores were compared between histology and cytology. In addition, immunostaining was compared between PD-L1-expressing human cell lines fixed in various fixatives at increasing increments in fixation duration., Results: Agar cell blocks and tissue blocks showed substantial agreement (κ = 0.70 and κ = 0.67, respectively), whereas fair-to-moderate agreement was found between Cellient cell blocks and histology (κ = 0.28 and κ = 0.49, respectively). Cell lines fixed in various alcohol-based fixatives showed less PD-L1 immunoreactivity compared with those fixed in formalin. In contrast to SP263, additional formalin fixation after alcohol fixation resulted in preserved staining intensity using the 22C3 laboratory-developed test and the 22C3 pharmDx assay., Conclusions: Performing PD-L1 staining on cytologic specimens fixed in alcohol-based fixatives could result in false-negative immunostaining results, whereas fixation in formalin leads to higher and more histology-concordant PD-L1 immunostaining. The deleterious effect of alcohol fixation could be reversed to some degree by postfixation in formalin., (© 2020 The Authors. Cancer Cytopathology published by Wiley Periodicals LLC on behalf of American Cancer Society.)

Purpose: Pretreatment selection of patients with non-small cell lung cancer (NSCLC) who would derive clinical benefit from treatment with immune checkpoint inhibitors (CPIs) would fulfill an unmet clinical need by reducing unnecessary toxicities from treatment and result in substantial health care savings., Experimental Design: In a retrospective study, mass spectrometry (MS)-based proteomic analysis was performed on pretreatment sera derived from patients with advanced NSCLC treated with nivolumab as part of routine clinical care ( n = 289). Machine learning combined spectral and clinical data to stratify patients into three groups with good ("sensitive"), intermediate, and poor ("resistant") outcomes following treatment in the second-line setting. The test was applied to three independent patient cohorts and its biology was investigated using protein set enrichment analyses (PSEA)., Results: A signature consisting of 274 MS features derived from a development set of 116 patients was associated with progression-free survival (PFS) and overall survival (OS) across two validation cohorts ( N = 98 and N = 75). In pooled analysis, significantly better OS was demonstrated for "sensitive" relative to "not sensitive" patients treated with nivolumab; HR, 0.58 (95% confidence interval, 0.38-0-87; P = 0.009). There was no significant association with clinical factors including PD-L1 expression, available from 133 of 289 patients. The test demonstrated no significant association with PFS or OS in a historical cohort ( n = 68) of second-line NSCLC patients treated with docetaxel. PSEA revealed proteomic classification to be significantly associated with complement and wound-healing cascades., Conclusions: This serum-derived protein signature successfully stratified outcomes in cohorts of patients with advanced NSCLC treated with second-line PD-1 CPIs and deserves further prospective study., (©2020 American Association for Cancer Research.)

Aims: Investigate the impact of interlaboratory- and interobserver variability of immunohistochemistry on the assessment of programmed death ligand 1 (PD-L1) in non-small cell lung cancer (NSCLC)., Methods: Two tissue microarrays (TMAs) were constructed from 50 (TMA-A) and 51 (TMA-B) resected NSCLC cases, and distributed among eight centres. Immunostaining for PD-L1 was performed using Agilent's 22C3 pharmDx Assay (pharmDx) and/or a 22C3 laboratory developed test (LDT). The interlaboratory variability of staining- and interobserver variability of scoring for PD-L1 were assessed in selected critical samples (samples at the cut-off of positivity) and non-critical samples. Also, PD-L1 epitope deterioration in time in stored unstained slides was analysed. Krippendorff's alpha values (0=maximal, 1=no variability) were calculated as measure for variability., Results: For interlaboratory variability of immunostaining, the percentage of PD-L1 positive cases among centres ranged 40%-51% (1% cut-off) and 23%-30% (50% cut-off). Alpha values at 1% cut-off were 0.88 (pharmDx) and 0.87 (LDT) and at 50% cut-off 0.82 (pharmDx) and 0.95 (LDT). Interobserver variability of scoring resulted in PD-L1 positive cases ranging 29%-55% (1% cut-off) and 14%-30% (50% cut-off) among pathologists. Alpha values were at 1% cut-off 0.83 (TMA-A) and 0.66 (TMA-B), and at 50% cut-off 0.77 (TMA-A) and 0.78 (TMA-B). Interlaboratory variability of staining was higher (p<0.001) in critical samples than in non-critical samples at 50% cut-off. Furthermore, PD-L1 epitope deterioration in unstained slides was observed after 12 weeks., Conclusions: The results provide insight in factors contributing to variability of immunohistochemical assessment of PD-L1, and contribute to more reliable predictive testing for PD-L1., Competing Interests: Competing interests: RB: none. N‘tH: MSD (unrestricted grant), Pfizer (personal fee). KM: Roche (research grant, personal fee), MSD (research grant, personal fee), Astra Zeneca (research grant, personal fee), Pfizer (personal fee), Benecke (personal fee), BMS (personal fee), Abbvie (personal fee), Diaceutics (personal fee). E-JS: MSD (research grant, personal fee), BMS (research grant, personal fee), Novartis (research grant), AstraZeneca (research grant), AbbVie (personal fee), Bayer (personal fee), Roche (personal fee). ET: HistoGeneX (personal fee), Roche Diagnostics (personal fee). JHVdT: Astellas (research grant), BMS (research grant, personal fee), AbbVie (personal fee), Astra Zeneca (personal fee), Boehringer-Ingelheim (personal fee), BMS (personal fee), Eli Lilly (personal fee), MSD (personal fee), Pfizer (personal fee), Roche (personal fee). WT: MSD (personal fee), Roche-Ventana (personal fee), Pfizer (personal fee), Astra Zeneca (personal fee), GSK (personal fee), Chiesi (personal fee), Dutch Asthma Fund (research grant), Biotest (personal fee), Novartis (personal fee), Lilly Oncology (personal fee), Boehringer Ingelheim (personal fee). MJvdV: MSD (research grant), Roche (personal fee)., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

PD-L1, as assessed by immunohistochemistry, is a predictive biomarker for immuno-oncology treatment in lung cancer. Different scoring methods have been used to assess its status, resulting in a wide range of positivity rates. We use the European Thoracic Oncology Platform Lungscape non-small cell lung carcinoma cohort to explore this issue. PD-L1 expression was assessed via immunohistochemistry on tissue microarrays (up to four cores per case), using the DAKO 28-8 immunohistochemistry assay, following a two-round external quality assessment procedure. All samples were analyzed under the same protocol. Cross-validation of scoring between tissue microarray and whole sections was performed in 10% randomly selected samples. Cutoff points considered: ≥1, 50 (primarily), and 25%. At the two external quality assessment rounds, tissue microarray scoring agreement rates between pathologists were: 73% and 81%. There were 2008 cases with valid immunohistochemistry tissue microarray results (50% all cores evaluable). Concordant cases at 1, 25, and 50% were: 85, 91, and 93%. Tissue microarray core results were identical for 70% of cases. Sensitivity of the tissue microarray method for 1, 25, and 50% was: 80, 78, and 79% (specificity: 90, 95, 98%). Complete agreement between tissue microarrays and whole sections was achieved for 60% of the cases. Highest sensitivity rates for 1% and 50% cutoffs were detected for higher number of cores. Underestimation of PD-L1 expression on small samples is more common than overestimation. We demonstrated that classification of PD-L1 on small biopsy samples does not represent the overall expression of PD-L1 in all non-small cell cancer carcinoma cases, although the majority of cases are 'correctly' classified. In future studies, sampling more and larger biopsies, recording the biopsy size and tumor load may permit further refinement, increasing predictive accuracy.

Background: In non-small cell lung cancer (NSCLC), PD-L1 expression on either tumor cells (TC) or both TC and tumor-infiltrating immune cells (IC) is currently the most used biomarker in cancer immunotherapy. However, the mechanisms involved in PD-L1 regulation are not fully understood. To provide better insight in these mechanisms, a multiangular analysis approach was used to combine protein and mRNA expression with several clinicopathological characteristics., Patients and Methods: Archival tissues from 640 early stage, resected NSCLC patients were analyzed with immunohistochemistry for expression of PD-L1 and CD8 infiltration. In addition, mutational status and expression of a selection of immune genes involved in the PD-L1/PD-1 axis and T-cell response was determined., Results: Tumors with high PD-L1 expression on TC or on IC represent two subsets of NSCLC with minimal overlap. We observed that PD-L1 expression on IC irrespective of expression on TC is a good marker for inflammation within tumors. In the tumors with the highest IC expression and absent TC expression an association with reduced IFNγ downstream signaling in tumor cells was observed., Conclusions: These results show that PD-L1 expression on TC and IC are both independent hallmarks of the inflamed phenotype in NSCLC, and TC-negative/IC-high tumors can also be categorized as inflamed. The lack of correlation between PD-L1 TC and IC expression in this subgroup may be caused by impaired IFNγ signaling in tumor cells. These findings may bring a better understanding of the tumor-immune system interaction and the clinical relevance of PD-L1 expression on IC irrespective of PD-L1 expression on TC., Competing Interests: Willemijn SME Theelen: none. Thomas Kuilman: none. Katja Schulze: Genentech employee and holder of Roche stock. Wei Zou: Genentech employee and holder of Roche stock. Oscar Krijgsman: none. Dennis DGC Peters: none. Sten Cornelissen: none. Kim Monkhorst: personal fees from Roche, Pfizer, BMS, Abbvie, AstraZeneca, grants from AstraZeneca, non -financial support from Roche, non-financial support from Genentech outside the submitted work. Pranamee Sarma: Genentech employee and holder of Roche stock. Teiko Sumiyoshi: Genentech employee and holder of Roche stock Lukas C Amler: Genentech employee and holder of Roche stock. Stefan M Willems: medical advisor for and/or receives research grants from Roche, Pfizer, MDS, AstraZeneca, Cergentis, Merck Hans LG Blaauwgeers: none. Carel JM van Noesel: none. Daniel S Peeper: none. Michel M van den Heuvel: none Marcin Kowanetz: Genentech employee and holder of Roche stock. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Introduction: The PD-L1 biomarker is an important factor in selecting patients with non-small cell lung cancer for immunotherapy. While several reports suggest that PD-L1 positivity is linked to a poor prognosis, others suggest that PD-L1 positive status portends a good prognosis., Methods: PD-L1 positivity prevalence, assessed via immunohistochemistry (IHC) on tissue microarrays (TMAs), and its association with clinicopathological characteristics, molecular profiles and patient outcome- Relapse-free Survival (RFS), Time-to-Relapse (TTR) and Overall Survival (OS)- is explored in the ETOP Lungscape cohort of stage I-III non-small cell lung cancer (NSCLC). Tumors are considered positive if they have ≥1/5/25/50% neoplastic cell membrane staining., Results: PD-L1 expression was assessed in 2182 NSCLC cases (2008 evaluable, median follow-up 4.8 years, 54.6% still alive), from 15 ETOP centers. Adenocarcinomas represent 50.9% of the cohort (squamous cell: 42.4%). Former smokers are 53.7% (current: 31.6%, never: 10.5%). PD-L1 positivity prevalence is present in more than one third of the Lungscape cohort (1%/5% cut-offs). It doesn't differ between adenocarcinomas and squamous cell histologies, but is more frequently detected in higher stages, never smokers, larger tumors (1/5/25% cut-offs). With ≥1% cut-off it is significantly associated with IHC MET overexpression, expression of PTEN, EGFR and KRAS mutation (only for adenocarcinoma). Results for 5%, 25% and 50% cut-offs were similar, with MET being significantly associated with PD-L1 positivity both for AC (p < 0.001, 5%/25%/50% cut-offs) and SCC (p < 0.001, 5% & 50% cut-offs and p = 0.0017 for 25%). When adjusting for clinicopathological characteristics, a significant prognostic effect was identified in adenocarcinomas (adjusted p-values: 0.024/0.064/0.063 for RFS/TTR/OS 1% cut-off, analogous for 5%/25%, but not for 50%). Similar results obtained for the model including all histologies, but no effect was found for the squamous cell carcinomas., Conclusion: PD-L1 positivity, when adjusted for clinicopathological characteristics, is associated with a better prognosis for non-metastatic adenocarcinoma patients., (Copyright © 2019 Elsevier B.V. All rights reserved.)