418 chapter 8 Tissue sections were deparaffinized and rehydrated. Next, GLUT1, GLUT3, HIF1a, CA-IX, and Ki-67 staining was performed using a semi-automated immunostainer (Thermo Scientific, LabvisionTM 488) following a standardized protocol according to manufacturer instructions. GLUT4, HK1, HK2, MCT4, VEGF, and NIS staining was performed manually. Detailed immunohistochemistry procedures are provided in the Supplementary data. In summary, antigen retrieval was performed, nonspecific immunoreactivity was blocked, and slides were subsequently incubated with primary and secondary antibodies. Slides were incubated using 3,3-diaminobenzidine (DAB) as chromogen. Negative control samples were processed without primary antibodies. Positive control tissues were used as recommended by the manufacturer. Finally, slides were counterstained with haematoxylin and dehydrated. All slides were reviewed to quantify the expression of the immunohistochemical markers by evaluating the DAB staining by two members of the research team (EK and AE), who were blinded to the clinical data and [18F]FDG-PET/CT results (Figure 1). The staining was assessed using the Remmele and Stegner immunoreactive score (IRS) by multiplying the staining intensity (score 0-3: negative, weak, intermediate, strong) with the proportion of stain-positive cells in the index nodule (score 0-4: 0%, 1-10%, 11-50%, 51-80%, >80%) (Supplementary Table 1) [639]. Consequently, the minimum IRS of 0 represents absent staining and the maximum IRS of 12 represents strong staining in a majority of cells in the lesion. For the Ki-67 stain, the proliferation index was assessed as the percentage of cells with positive nuclear staining (score 1-3: <3%, 3-5%, >5%) among at least four representative fields of 100 tumour cells. Statistical analysis Baseline characteristics were compared between the allocated groups using Pearson’s chi-squared for categorical data and one-way ANOVA or Kruskall-Wallis tests for continuous data, where appropriate. Unsupervised cluster analysis with ordered leaves was performed using the IRS of all stains and Ki-67 proliferation index, and visualized in a dendrogram with heat map. The IRS of all stains were correlated with each other, the SUVmax, SUVpeak, and SUVmax-ratio using the Spearman’s rank correlation coefficient with its degrees of freedom (rs[df]). Next, the IRS of all immunomarkers were compared between TN, FP, and TP groups using Kruskall-Wallis (omnibus) tests and visualized using violin plots. In case the Kruskall-Wallis test indicated statistical significance, Dunn’s post hoc tests were used for each pair of groups and p values are presented. Counteracting for multiple comparisons was performed using Bonferroni correction; statistical significance after correction is indicated by an asterisk (*). A p value of 0.05 or less was considered statistically significant. Statistical analysis was performed using IBM SPSS Statistics version 27 (IBM Corp., Armonk, NY, USA) and Orange: Data Mining Toolbox version 3.30.2 (Bioinformatics Lab, University of Ljubljana, Slovenia) [640].
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