Afami-cel RNA in situ hybridization and CD3 IHC
HEAT
RNA in situ hybridization for MAGE-A4 coding SPEAR (Advanced Cell Diagnostics) was performed on the Ventana Discovery Ultra automation platform (Roche Diagnostics) using the RNAscope 2.5 LS Red kit (Advanced Cell Diagnostics, 322150) according to the manufacturer’s instructions. In brief, 4-μm formalin-fixed, paraffin-embedded (FFPE) tissue sections were pre-treated with heat and protease before hybridization with the target oligo probes. Pre-amplifier, amplifier and alkaline phosphatase-labeled oligos were then hybridized sequentially. RNAscope assay was followed by CD3 chromogenic precipitate IHC (anti-CD3 (2GV6) rabbit monoclonal primary antibody, 790-4341, CONFIRM, Roche Diagnostics) using the DISCOVERY TEAL HRP detection kit (Roche Diagnostics, 08254338001). Each sample was quality controlled for RNA integrity with an RNAscope probe specific to peptidylpropyl isomerase B (PPIB) RNA (Hs-PPIB, 313909). Specific RNA staining signal was identified as red punctate dots, and CD3 was identified by the teal signal. Samples were counterstained with hematoxylin.The whole slides were scanned using the AxioScan.Z1 microscope slide scanner and analyzed using HALO image analysis software (Indica Labs). Images were annotated and analyzed using Indica Labs-ISH version 3.3.9 algorithm, with CD3 staining as the nuclear stain, resulting in CD3