Mitotic rate

A blinded histopathology scoring study was performed by a single scorer, under the guidance of a veterinary pathologist. Twenty-eight A1d1a CTVT histology samples were scored, together with 31 CTVT_HT1 and CTVT_HT2 CTVT controls. CTVT_HT1 and CTVT_HT2 controls were phylogenetically matched with A1d1a tumours. The 28 A1d1a CTVTs belonged to 8 horizontal transfer groups (HT3, 2 tumours; HT8, 6 tumours; HT9, 13 tumours; HT10, 1 tumour; HT13, 1 tumour; HT14, 1 tumour; HT16, 1 tumour; HT17, 3 tumours). Histology slides were scanned using a Hamamatsu Nanozoomer 2.0-HT slide scanner (C9600) before scoring, and were viewed using the NDP.view2 software (Hamamatsu Photonics, Hamamatsu, Japan). Each field was defined to consist of at least half CTVT parenchyma, avoiding areas of artefactual distortion, ulceration or haemorrhage. The number of mitotic figures (including bizarre mitotic figures) per 10 high-power fields (40×, a mitotic box of area 0.0325 cm², perimeter 722 µm on NDP.view2) was assessed. Statistical significance was tested using an unpaired two-tailed Student’s t test implemented in R⁴¹.