Chapter 2 34 PD-L1 scoring Tumor PD-L1 expression was assessed according to the instruction manual of the qualitative, clinical grade LDT IHC assay (22C3 pharmDx, Dako) as used in routine clinical practice at NKI-AVL. As high concordance between the 22C3 and 22-8 PD-L1 antibodies has been reported18,19, the 22C3 clone was also used to assess the predictive value of PD-L1 for nivolumab. PD-L1 expression levels were manually scored by a trained MD (K.H.) under the supervision of an experienced pathologist (K.M.) blinded for clinical outcome. The PD-L1 Tumor Proportion Score (TPS) was determined by calculating the percentage of PD-L1+ tumor cells of total viable tumor cells (Table S2). PD-L1 positivity was defined as tumor cells showing circumferential and/or partial linear expression (at any intensity) of PD-L1 on the plasma cell membrane. A CD68 staining was manually evaluated and compared with PD-L1 stained slides to avoid false positive results due to PD-L1 expressing macrophages in between tumor cells. PD-L1 IC was manually scored as the proportion of tumor area that is occupied by PD-L1+ immune cells (ICs) of any intensity (IC0: <1%, IC1: ≥1% and <5%, IC2: ≥5% and <10% and IC3: ≥10%) as described20,21. Scoring of tertiary lymphoid structures A CD20 (yellow)/CD3 (purple) double staining was used to identify tertiary lymphoid structures (TLS). CD20-CD3 IHC images were scanned and analyzed using HALO™. Lymphoid niches were manually identified based on the presence of B cell (CD20+) clusters and T cell (CD3+) zones as described22,23. Next, areas were measured in HALOTM and assigned as TLS (>60,000 µm2) or lymphoid aggregate (LA) (10,000-60,000 µm2)16. Finally, tumor areas were digitally annotated as described above and the number of TLS per mm2 and the combined number of TLS and LA (TLS+LA) per mm2 tumor area were determined (Table S2). CD20 quantification by digital image analysis The Area Quantification v1.0 module of the HALOTM software was used to generate an analysis algorithm to measure the total area with CD20 expression on the CD20/ CD3 images. The total CD20 positive area was selected because the dense clustering of CD20+ cells in TLS precluded the setup of a reliable algorithm to quantify cell numbers. Tumor areas were digitally annotated as described above and the CD20positive area was normalized per mm2 tumor area (Table S2). Statistical analysis Patient characteristics were descriptively reported using mean ±s.d., interquartile range (IQR) or frequencies (percentages). Differences in patient and sample characteristics between cohorts (training and validation), between outcome groups (disease control vs PD) and between groups created by the biomarker were assessed
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