Biomarker Assessment in Tumor Samples

PD-L1 expression was assessed in the tumor cells and immune cells using SP142 antibody (Ventana Medical Systems, Oro Valley, AZ). PD-L1 expression was considered positive if either tumor cell or immune cells exhibited any membranous or cytoplasmic staining.^{11 (link),14 (link)} AR (clone 441, Leica Biosystems, Buffalo Grove, IL) was analyzed using a 10% or higher threshold for nuclear positivity.^{12 (link)–14 (link)} The ARv7 splice variant was explored at the protein level by immunohistochemistry (EPR15656; Abcam) and at the messenger RNA level using anchored multiplex polymerase chain reaction for targeted RNA sequencing (ArcherDX).^{9 (link),11 (link)} Topoisomerase 1 (Topo1) expression (clone 1D6; Leica Biosystems, Nussloch, Germany) was scored as 0+, 1+, 2+, or 3+ depending on the staining intensity, and the percent tumor stained was also recorded. The threshold for Topo1 overexpression was a staining intensity of 2+ or higher in 30% or more of the cancer cells.^{15 (link)}