Panoramic 250 scanner

Liver samples (seven per group) were fixed with 4% paraformaldehyde, embedded in paraffin and cut into 10 μm thick sections. Microscope slides were stained using Masson’s Trichrome. Liver sections were scanned with a Panoramic scanner 250 from 3dHistech with 40× magnification. The degree of steatosis, hepatocyte ballooning and fibrosis was scored using Masson’s Trichrome-stained liver sections. Each variable was graded from zero (none) to three (severe). ImageJ 1.52 software was used to measure the lipid droplet diameter in each group (mean of 2 representative fields per slide, n = 7 liver sections per group). The image was first converted to 16 bits in black and white, then an automatic threshold was applied with a dark background, and the area of circular objects over 48 μm² was measured automatically. A binary watershed was used to separate touching droplets and objects touching the edge of the image were removed.

Placental explants were fixed in formalin during 24 h at room temperature and embedded in paraffin. Tissue sections (5 μm) were de-waxed using xylene and alcohol and epitope retrieval was carried out using citrate buffer (pH 6) at 95°C during 20 min. Sections were re-hydrated using TBS 0.01% Tween 20 for 5 min and blocked with 2.5% horse serum for 20 min. Immunostainings were performed with the following antibodies: rabbit anti-Cytokeratin-7 (Genetex; 2 μg/mL), mouse anti-Vimentin (Santa-Cruz; 2 μg/mL) and mouse anti-placental alkaline phosphatase (Biolegend; 1 μg/mL). Immunostaining for hCMV was performed as previously described (Benard et al., 2014 (link)), using a mouse monoclonal antibody directed against the hCMV IE antigen (clone CH160, Abcam). Secondary antibody-coupled to biotin was then used prior to Vectastain RTU elite ABC Reagent (Vector laboratories) and staining by diaminobenzidine (DAB). Sections were finally counter-stained with hematoxylin. Image acquisition was performed on a Leica DM4000B microscope or on a Panoramic 250 scanner (3DHISTECH).

Patients who underwent curative surgery for colon cancer, between January 2005 up to and including December 2016 at the LUMC, were retrospectively included in this cohort study. Patients were included when they met the following inclusion criteria: pathological stage II or stage III colon cancer and age ≥ 18 years. The following exclusion criteria were met: rectal cancer, neo-adjuvant treatment, a medical history of cancer 10 years prior to colon cancer (except for basal cell skin cancer or cervical carcinoma in situ) or any colon cancer in history, double tumours, and/or deceased within 3 months after surgery (Supplementary table 1). The H&E-stained slides used for routine diagnostics were collected from the Department of Pathology and the slides were anonymised and scanned with the Panoramic 250 scanner (3DHistech, Hungary) (tissue level pixel size ~ 0.33 µm/pixel) for digital analysis. The observers were blinded for clinical and pathological data and for each other’s results during biomarker scoring.

A Panoramic 250 scanner (3DHistech, Budapest, Hungary) was used to digitally scan the IHC-stained TMA slides at a magnification of 40 ×. Furthermore, we employed QuPath version 0.2.0-m429,30, an open-source image analysis platform (Center for Cancer Research & Cell Biology, University of Edinburgh), to arrange disordered IHC-stained TMAs. All cores were evaluated during the scoring process to manually exclude invalid cores (<10% of the tumor per core or artifacts). A simple, automated, and semi-assisted method using QuPath was used for TMA quantification. After several steps and subsequent validations, the desired threshold for the positive cells was selected for each marker. Staining vectors were automatically analyzed for each scanned TMA slide, followed by total tissue area detection, separation of tumor from non-tumor areas in each core, and automatic cellular detection. Positive cells were assigned using the optical density threshold of the selected cells, tested on each core, and applied to the entire array after validation by an expert pathologist.
The histochemical score (H-score) measures the intensity of staining. The H-score was obtained by calculating the sum of the percentage of staining multiplied by the corresponding intensity, and was used as the expression level.

The cases selected for this study were based on the E-learning module of the UNITED study.⁸ (link) These cases were chosen for training purposes by limiting the number of stroma-low cases (e.g., 10% or 20% stroma cases), in order to increase the number of cases that are difficult to score for pathologists. All patients underwent surgical treatment at the Leiden University Medical Center (LUMC), had stage II or stage III colon cancer, and no patients received neo-adjuvant treatment. The slides were anonymized and scanned with the Panoramic 250 scanner (3DHistech, Hungary) (tissue level pixel size ∼0.33 μm/pixel), or with the IntelliSite Digital pathology slide scanner (Philips, the Netherlands) (tissue level pixel size ∼0.25–0.26 μm/pixel).

The e-Learning module was based on H&E-stained sections of stage II and III colon cancer resection specimens. The cases were randomly selected from the archives of the Pathology Department of Leiden University Medical Centre (LUMC). The number of cases with very low stroma (ie, 10% or 20% stroma area) were limited from the analysis to increase the number of cases that are generally more difficult to score for pathologists and are therefore more suitable for training purposes. In the e-Learning, 55% of the cases were stroma-low (≤50% stroma) and 45% were stroma-high (>50% stroma). None of the patients had received neoadjuvant treatment at the time of sample collection. The sample size was based on a workable amount of cases to maintain quality without the case load being too high.
Slides were scanned using a 20× objective with the Panoramic 250 scanner (3D Histech) or with the IntelliSite Digital pathology slide scanner (Philips).