Ventana virtuoso software

For visual point counting (VPC), photomicrographs of three randomly selected fields were taken at 40X with a background grid and color printed (more fields to achieve the minimal cell count). Each observer counted both the total number tumor cells and the number of Ki-67-positive cells that intersect with first grid line. This process is repeated on every third gridline. All the cells on the slide were counted if three fields could not be obtained however at least 200 total tumor cells were required. For Ki-67 image analysis of the CLIA clinical trial assay, slides were scanned with the iScan Coreo scanner (Ventana). The computer image was reviewed and “Areas of Interest” (AOI) were selected at 4X magnification using the following guidelines: (1) identify the largest AOI of representative clear invasive tumor; (2) exclude DCIS, vessels, lymphocytes; (3) avoid AOIs in peri-necrotic or necrotic areas; (4) identify at least 3 AOIs and a maximum of 10. The image analysis was performed using the FDA cleared VENTANA Companion Algorithm Ki-67 (30-9) and the VENTANA VIRTUOSO software (Roche).

Formalin-fixed, paraffin-embedded 5 μm tissue sections were stained in batches for CD4, CD8, and CD163 in a central laboratory at the Johns Hopkins Hospital according to standard automated protocols. Deparaffinization and rehydration were performed, followed by antigen retrieval and antibody staining. CD4 and CD8IHC was performed using the Ventana Benchmark Ultra autostaining system (Roche) using mouse monoclonal anti-CD8 (C8144B) antibody (Cell marque) and rabbit monoclonal anti-CD4(Sp35) antibody (Roche), followed by detection with the iVIEW DAB Detection Kit (Roche). CD163 IHC was performed on the Leica Bond MAX autostaining system (Leica Biosystems) using anti-CD163 (10D6) antibody (Leica Biosystems) followed by detection with Bond Polymer Refine Detection kit (Leica Biosystems). For tissue section imaging, slides were imaged using a Ventana iScan HT slide scanner (Roche) and processed using the Ventana Virtuoso software (Roche) (Figure S6D).