Chapter 4 134 excluded because these contained abundant normal lymphoid tissue (n=2) (Fig. 1). We excluded lymph node resections as these contain PD-1 high-expressing T cells in normal lymphoid tissue, which could potentially lead to false positive results. Lymph node biopsies were included as these are usually targeted biopsies in the tumor region. Figure 1. Study design for the development and validation of the PD-1T signature as biomarker for non-response to PD-1 blockade in NSCLC. Overall workflow for the development of the PD-1T signature using PD-1T IHC high (≥90 per mm2) patients with disease control (DC) at 12 months (n=12) and PD-1T IHC low (<90 per mm2) patients with progressive disease (n=29). An independent cohort of patients was used for validation (n=42). Immunohistochemistry PD-1 and PD-L1 immunostaining of samples from the NKI-AVL cohorts were performed on fresh-cut slides from FFPE blocks using an anti-PD-1 antibody (NAT105, Roche Diagnostics) and anti-PD-L1 antibody (22C3 DAKO, Agilent) on a BenchMark Ultra autostainer Instrument (Ventana Medical Systems) as described previously7.
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