Ventana system

To determine the HER2 amplification status and to assess chromosome 17 copy number status, SISH was performed on all 44 tumor samples using the INFORM HER2 Dual colour SISH DNA probe cocktail (Roche Diagnostics GmbH) in combination with the VENTANA system (Roche Diagnostics GmbH) according to the manufacturers protocol. Twenty nuclei were counted independently by 2 individuals. The mean from these 2 nuclei counts was calculated. Interpretation was as follows: a ratio (HER2 probe/centromere probe) of <1.8 represented no amplification; a ratio of 1.8–2.2 was equivocal and a ratio of >2.2 represented amplification.

Tumor samples were fixed in 4% formaldehyde for 24 h. Subsequently, they were dehydrated, embedded in paraffin, and sectioned at 4 µm. Hematoxylin and Eosin (H&E) staining was performed using standard protocols. Immunohistochemical stainings were done using a Ventana System (Roche) according to manufacturers’ specifications. The antibodies rabbit anti-Ki67 (Abcam #ab16667, 1:200) and mouse anti-BAF47 (BD Biosciences #612110, 1:50) were used for Ki67 and SMARCB1 stainings. For 19 patients, the mitotic figures of ten high power fields (HPF) were counted for each primary tumor and related recurrence with subsequent comparison of the averages. Ki67-positive nuclei of 16 cases were counted and the frequency was compared between primary and recurrent AT/RT. Significance was determined using a t test for paired data. SMARCB1-deficiency was identified by immunohistochemical staining using anti-SNF5/SMARCB1 antibody (Abcam #ab88589, 1:50). For SMARCA4-deficient cases, anti-BRG1 antibody (Abcam #ab110641, 1:25) was used.

To establish the HER2 expression status, IHC was performed on all 44 tumor samples using the Ventana anti-Her2/neu(4B5) (Roche Diagnostics GmbH, Mannheim, Germany) in combination with the VENTANA system (Roche Diagnostics GmbH) according to the manufacturers protocol. IHC 0 and 1+ were considered to represent normal expression, 2+ was considered borderline with regard to expression and 3+ was considered to represent overexpression.

Heart tissues from each mouse were fixed in 10% formalin, paraffin-embedded, and sectioned in 7 μm sections for hematoxylin-eosin staining following standard protocols, as previously described [50 (link)]. After scanning the slides with GTX450 (Leica biosystems) and conducting the initial histologic evaluation, histochemistry with Masson’s trichrome was performed (Ventana system; Roche). Histochemistry was performed following the manufacturer’s instructions. Fibrosis area was determined using ImageJ version 1.54. An expert pathologist performed the histopathological analyses of the heart valves following a blinded protocol.

Cells were detached using trypsin-EDTA and then washed and centrifuged. The cell pellets were fixed in formalin and embedded in paraffin. Tumor sections were prepared, and immunohistochemical studies were carried out for the mesothelial markers calretinin, WT1, CK5/6, and mesothelin and BAP1 using specific antibodies (Santa Cruz Biotechnology, TX). All immunostaining was carried out using an automated Ventana system (Ventana Medical Systems, AZ) using their UltraView polymer based detection system. IHC staining was scored semiquantitatively as follows: negative (less than 5% of cells stained), 1+ positive (5- 50%), and 2+ positive (50-100%).

Multiplex immunohistochemistry (IHC) was performed using the automated Ventana system (Ventana Medical Systems, Oro Valley, AZ, USA). The Opal^TM detection system (Akoya Biosciences, Marlborough, MA, USA) was used as it allows for repeated cycles of staining and stripping with antibodies of the same species without cross-reactivity, which enables simultaneous staining of five targets in the same tissue section. The staining protocol involved initial deparaffinisation and epitope retrieval at pH 8.5 followed by multiple cycles of incubation with primary antibody, secondary antibody and Opal^TM detection. The cycles were separated by a short denaturation at pH 6. UltraMap anti-rabbit/mouse HRP conjugated secondary antibodies were used (Roche, Basel, Switzerland). Antibody and Opal^TM concentrations are listed in Supplementary Table S1. After the automated protocol the slides were manually washed by three 5 min cycles of a 1:10 dilution of EZ preparation (Ventana Medical Systems) before being counter-stained with a 1:120,000 dilution of DAPI (ThermoFisher, MA, USA) for five minutes. The slides were then cover-slipped with ProLong^TM Gold Antifade Mountant (ThermoFisher).