Critical Appraisal of Programmed Death Ligand 1 Reflex Diagnostic Testing: Current Standards and Future Opportunities Matthew P. Humphries, PhD, Stephen.

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Critical Appraisal of Programmed Death Ligand 1 Reflex Diagnostic Testing: Current Standards and Future Opportunities Matthew P. Humphries, PhD, Stephen McQuaid, PhD, Stephanie G. Craig, PhD, Victoria Bingham, MSc, Perry Maxwell, PhD, Manisha Maurya, PhD, Fiona McLean, BSc, James Sampson, MBChB, Patricia Higgins, BSc, Christine Greene, BSc, Jacqueline James, PhD, Manuel Salto-Tellez, MBChB Journal of Thoracic Oncology Volume 14, Issue 1, Pages 45-53 (January 2019) DOI: 10.1016/j.jtho.2018.09.025 Copyright © 2018 International Association for the Study of Lung Cancer Terms and Conditions

Figure 1 Summary of the validation on tissue microarrays of the programmed death ligand 1 (PD-L1) SP263 clone on Ventana BenchMark against the Dako PD-L1 22C3 clone on the Dako Autostainer Link 48 in (A) lung adenocarcinomas and (B) squamous cell carcinomas. A very high level of concordance was observed. For adenocarcinomas, there were 111 of 113 tissue microarray (TMA) cores (98%); and for squamous cell carcinomas, there were 100 of 103 TMA cores (97%). (C) For (i) 22C3 and (ii) SP263 there is no PD-L1 expression in lung adenocarcinoma, whereas (iii) 22C3 and (iv) SP263 show comparable expression of more than 50% on tumor epithelium in a squamous cell carcinoma. And in (v) 22C3, two or three tumor epithelial cells are expressing PD-L1, but this expression was not observed in a serial core with the SP263 clone (vi). Journal of Thoracic Oncology 2019 14, 45-53DOI: (10.1016/j.jtho.2018.09.025) Copyright © 2018 International Association for the Study of Lung Cancer Terms and Conditions

Figure 2 Comparable categorical distribution of programmed death ligand 1 (PD-L1) expression. (A) Within all 564 clinical cases. (B) In adenocarcinomas. (C) In squamous cell carcinomas. Inadequate cases were defined as those which had insufficient tumor content (<100 malignant cells) available for analysis. (D, E) Left-to-right display representative images of (i) hematoxylin and eosin staining (H&E), (ii) negative PD-L1 expression, (iii) 1% to 49% PD-L1 expression, and (iv) more than 50% positive PD-L1 expression for adenocarcinoma and squamous cells carcinoma, respectively. Journal of Thoracic Oncology 2019 14, 45-53DOI: (10.1016/j.jtho.2018.09.025) Copyright © 2018 International Association for the Study of Lung Cancer Terms and Conditions

Figure 3 Range of sample types tested, including tumor content available for programmed death ligand 1 (PD-L1) testing. (A) The range of samples types received for testing, resection, biopsy and cell block (CB). (B) Confirms that tumor content availability did not significantly affect the PD-L1 category determined. (C) PD-L1 expression according to sample type. p value is determined by the chi-square test. (D) PD-L1 expression within the cases of differing EGFR status. Journal of Thoracic Oncology 2019 14, 45-53DOI: (10.1016/j.jtho.2018.09.025) Copyright © 2018 International Association for the Study of Lung Cancer Terms and Conditions

Figure 4 Challenges with programmed death ligand 1 (PD-L1) assessments. (A) PD-L1 expressing tumor cells in a cell block which are confirmed by TTF1 expression a highly specific marker for primary lung adenocarcinomas. The case was reported as more than 50% PD-L1 expression in tumor cells. (B) PD-L1 expression in foci of cells in a lung cell block which, when phenotyped with macrophage and epithelial markers, were identified as macrophages. The case was reported as PD-L1–negative. (C) Resection of adenocarcinoma in which there is strong expression of PD-L1 on lymphocytes which “hug” the tumor. (D) Resection sample from squamous cell carcinoma shows distinct heterogeneity of PD-L1 expression ranging from absent to more than 50% expression in various fields across the tumor bed. TTF-1, thyroid transcription factor 1; HE, hematoxylin and eosin. Journal of Thoracic Oncology 2019 14, 45-53DOI: (10.1016/j.jtho.2018.09.025) Copyright © 2018 International Association for the Study of Lung Cancer Terms and Conditions

Supplemental Figure 1 Journal of Thoracic Oncology 2019 14, 45-53DOI: (10.1016/j.jtho.2018.09.025) Copyright © 2018 International Association for the Study of Lung Cancer Terms and Conditions

Supplemental Figure 2 Journal of Thoracic Oncology 2019 14, 45-53DOI: (10.1016/j.jtho.2018.09.025) Copyright © 2018 International Association for the Study of Lung Cancer Terms and Conditions

Supplemental Figure 3 Journal of Thoracic Oncology 2019 14, 45-53DOI: (10.1016/j.jtho.2018.09.025) Copyright © 2018 International Association for the Study of Lung Cancer Terms and Conditions