Tumor Budding Quantification in Digital TMA

All ngTMA slides were scanned (Pannoramic P250, 3DHistech, Hungary, 20× objective lens) and evaluated using Scorenado, a TMA analysis tool for digital TMA slides, as described previously (19 (link)). Each tumor punch contained an area of 0.785 mm². Only Pan-CK staining was used to evaluated tumor budding in the present study. Tumor buds were defined as single cells or cell cluster of up to 4 tumor cells according to the ITBCC guidelines (13 (link)). One experienced pathologist (A.L.) evaluated the number of tumor buds on H&E and Pan-CK staining at the tumor front (PTB and PMB) as well as intratumoral (ITB and IMB). Representative examples for PMB and IMB are given in Figure 1.