38 Chapter 3 Morphometry The method of nuclear size quantitation was performed on haematoxylin- eosin stained sections as described before46. The measuring system was a commercially available interactive video overlay-based system (Q-PRODIT; Leica, Cambridge, UK). The microscopic image (100x objective) was recorded by a video camera and shown on a computer screen (final magnification 3000x). Biopsy area was selected by pathologist (E.T.) and nuclear area measured with manual delineation. At least 25 nuclei of neoplastic cells, and nuclei of type II pneumocytes, if present, were measured. The mean, median, 75th and 90th percentile of the nuclear size was estimated in all patients. Immunohistochemistry The paraffin blocks of the preoperative biopsies contained too few residual tumour tissue to perform the additional immunohistochemical analysis for MIB-1 and PD-L1. For this reason, immunohistochemistry analysis was only performed on the resection specimen. In short, immunohistochemistry for Ki67 (clone MIB-1clone (Dako/Agilent, Glostrup, Denmark) was performed in the Ventana Benchmark Ultra (Tucson, USA) diluted 1/50 and incubated for 32 minutes after antigen retrieval with Cell Conditioning Buffer 1 (CC1) 24 minutes at 100°C detection with Optiview DAB Detection Kit. The proliferation fraction of the residual tumour in the resection specimen was estimated by scoring the overall number of positive staining tumour cells of one whole tumour section in one of three categories: <20%, 20-50% and >50%47,48. PD-L1 immunohistochemistry with the 22C3 clone was performed in a laboratory developed test (LDT) as previously described49. The tumour proportion score (TPS) of PDL-1 positive tumour cells was assigned to one of 3 categories: <1% (negative), 1-49% (weak positivity) and ≥ 50% (strong positivity)50. Statistics Statistical analyses were carried out by SPSS for Windows and Mac version 26 (IBM Corp., Armonk, NY, USA). Kaplan-Meier curves with log-rank test were used to compare overall survival (OS) and disease-free survival (DFS) between the different categories of MIB1 and PD-L1 status. OS was defined as time from diagnosis to time of death, DFS was defined as time from diagnosis to date of recurrence. Patients alive or without recurrence were censored at their last date of follow-up. Nuclear measurements in biopsies were compared to those in the resection specimens within patients using by the Wilcoxon sign rank test. Nuclear measurements in the resection specimen and normal tissue in the resection specimen from patients with and without recurrent disease were compared, using the Mann-Whitney U test. Data are described by median and range. A p-value < 0.05 was considered significant.
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