Nanozoomer series digital slide scanner

Stained slides were digitalized using NanoZoomer-Series Digital slide scanner (Hamamatsu Photonics). DAB-positive areas were quantified using HALO® platform from Indica Labs. Hematoxylin was used as nuclear counter stain. Gross tumor areas were annotated by a pathology expert and image analysis was performed with the same predefined settings for all slides. The percentage of positive or negative cells of all cells within the region was used for downstream analysis.

A tissue microarray (TMA) containing samples from the patient cohort mentioned above was established, comprising at least two cores of primary tumor and surrounding liver tissue, as well as of relapse tumors, lymph node and distant metastases, and tumor thrombi if available. After antigen retrieval, tissue microarray slides were stained with the respective antibodies (see below). Staining was done using an automated staining system (DAKO Autostainer plus, Agilent Technologies, Santa Clara, CA, USA) and the Dako EnVision FLEX staining system (Agilent Technologies, Santa Clara, CA, USA) in accordance with the manufacturer’s instructions. Prior to image analysis, TMA slides were digitalized using the NanoZoomer-Series Digital slide scanner (Hamamatsu Photonics, Hamamatsu, Japan). Immunoreactivity was either scored semiquantitatively according to Remmele et al. [18 (link)], or in the case of Ki67 and CD34, digital image analysis was performed using the HALO platform from Indica Labs (Corrales, NM, USA), including the TMA module and the CytoNuclear v1.6 module. Missing or erroneous cores (e.g., those with extensive tumor necrosis) were excluded from the analysis. In the case of Ki67, positive nuclei were counted; in the case of CD34, the positively stained area was quantified. The antibodies, dilutions, and antigen retrieval methods used are summarized in Appendix A.