Immunohistochemical fluoro-chromogenic triple staining for accurate detection of PD-L1 and PD-1 in NSCLC
Teppo Haapaniemi (1,2,3), Satu Luhtala (1), Taneli Tani (2), Jorma Isola (1)
1) University of Tampere, Tampere, Finland
2) Päijät-Häme Joint Authority for Health and Wellbeing, Pathology, Lahti, Finland
3) BioSiteHisto Ltd, Tampere, Finland
Background: A novel immunotherapy for non-small cell lung carcinoma (NSCLC) is based on blocking the signaling between the programmed death ligand 1 (PD-L1) and T-cell programmed death receptor 1 (PD-1). Individualized targeted therapy requires demonstration of PD-L1 in malignant tumor cells. A major problem in immunohistochemical detection of PD-L1 expression in pulmonary carcinomas is the expression of PD-L1 in alveolar macrophages, which may lead to false positive staining interpretation. With fluoro-chromogenic immunohistochemical staining, interpretation can be facilitated by exploiting cytokeratin expression of carcinoma cells. Also, ratio of PD-1 positive tumor-infiltrating lymphocytes (TILs) can be analyzed from the same section.
Methods: FFPE-samples of 40 NSCLC were stained using fluoro-chromogenic staining method with PD-L1 (ZR3), PD-1 (BSR1), and cytokeratin (BS5 + BS23) antibodies using LabVision Autostainer platform. PD-L1 and PD-1 were sequentially detected with HRP and AP conjugated polymers and visualized with DAB and Permanent Red. Cytokeratin was demonstrated with Cy2 conjugated IgG. Sensitivity and specificity of ZR3 were compared to performance of 22C3. Samples were scored using virtual slide scans of stacked bright field and fluorescence images by two observers.
Results: Improved accuracy of PD-L1 positive cancer cell count was observed using fluoro-chromogenic staining, especially in tumors with low and/or negative PD-L1 expression and in low grade carcinomas. Staining pattern of PD-L1 antibodies ZR3 and 22C3 was identical. PD-1 positive TILs were typically scattered as single cells in intra-tumoral areas and formed aggregates in peri-tumoral areas.
Conclusion: The accurate and reproducible detection of PD-L1 expression was achieved with the described fluoro-chromogenic method. Stacked virtual slides enable image analysis of PD-L1 and PD-1 and facilitate differentiation between PD-L1 signals localized to carcinoma cells, macrophages and necrotic tissue. Examples of the virtual slides can be found at http://wsiserver.jilab.fi/list/sites/satuteppo/