Additional file 2: Figure S1. Receiver operating characteristic curve for determining PD-L1 threshold. (A) Example images of high powered (20x) ROIs, InForm pathology view (showing only PD-L1 expression by mIF) counterstained with DAPI. Green is used here to label random cells that could be visually classified as PD-L1 positive versus negative by the reading pathologist, and used to ascertain an appropriate QIF cutoff for PD-L1 positivity. (B) Histogram of the distribution average per-cell QIF PD-L1 levels of 55,108 cells pooled from 24 ROI across 4 patients). (C) ROC curve illustrating sensitivity and specificity for given PD-L1 QIF thresholds. A threshold of ≥2.6 was selected, corresponding with sensitivity of 91%, specificity of 99%, AUC of 0.97, and accuracy of 95%. ROI: region of interest; PD-L1: programmed death ligand 1; mIF: multispectral immunofluorescence; DAPI: 4′,6-diamidino-2-phenylindole; QIF: quantitative immunofluorescence; ROC: receiver operating characteristic; AUC: area under the curve.