Ventana benchmark ultra stainer

FFPE tumor specimens from 311 patients were cut into 4-µm-thick sections, deparaffinized and stained for p16 (INK4A; Roche MTM Laboratories AG, Heidelberg, Germany) using a fully automated Ventana BenchMark ULTRA Stainer (Ventana, Tucson Arizona, USA) according to the manufacturers’ instructions. Binding of peroxidase-coupled antibodies was visualized using 3,3′-diamino-benzidine-tetrahydrochloride (DAB). Slides were counterstained with hematoxylin. P16 immunostained samples were scored independently by two dedicated pathologists (Senada Koljenović, Elisabeth Bloemena). The tumor samples were scored as ‘p16 positive’ when >70 % of the tumor cells showed both nuclear and cytoplasmic staining.
High-risk HPV DNA detection was performed on the p16-positive cases. DNA was extracted from all p16 positive cases using an automated silica-based extraction system, and PCR was performed using the HPV-Risk assay (Self-Screen BV, Amsterdam, the Netherlands) [28 (link)]. The HPV-Risk assay is a novel real-time PCR assay targeting the E7 region of 15 high-risk HPV types (i.e., HPV 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 67 and 68) and provides additional genotype information for HPV 16 and HPV 18. The HPV-Risk assay is clinically validated and meets the cross-sectional clinical and reproducibility criteria of the international guidelines for HPV test requirements.

Twenty-four formalin-fixed, paraffin-embedded TNBC tissue samples were retrieved from the Department of Pathology and Laboratory Medicine, King Hussein Cancer Center (Amman, Jordan). Hematoxylin and eosin staining was performed using standard procedures. The tissue samples were evaluated for the expression of AR by immunohistochemistry and were classified accordingly as AR-positive or -negative, as well as according to their metastasis status. For the immunohistochemistry (IHC), 5 µm thick tissue sections were cut and placed on coated slides with tissue sections of a known positive control placed near the non-frosted edge of the slide. The mouse polyclonal anti-AR antibody (MS-433-R7; Thermo Medical Co., Somerset, NJ, USA) was used for AR staining, which was carried out using Ventana Benchmark Ultrastainer (Ventana Medical Systems, Oro Valley, AZ, USA). The slides were examined by a pathologist who was totally blinded to the reported clinical data. The examined sections were reported as AR-positive when more than 1% of tumor cells had positive nuclear immunostaining. External AR-negative cases were used as a negative control to ensure there were no technical issues.

The following primary antibodies were used: anti-human CD8 (clone C8/144B, Dako, Glostrup, Denmark), anti-human CD68 (clone KP-1, Dako), and anti-human CD163 (clone 10D6,Leica Biosystems Novocastra, Newcastle, UK). Paraffin-embedded tumor specimens were cut into sequential 5 µm thick sections and deparaffinized and stained using a fully automated Ventana BenchMark ULTRA Stainer (Ventana, Tucson Arizona, USA) according to manufacturers' instructions at the pathology department. Binding of peroxidase-coupled antibodies was detected using 3,3′ - diaminobenzidine (DAB) as a substrate and the slides were counterstained with haematoxylin. The specificity of antibodies was checked using isotype-matched controls.

A Ventana BenchMark Ultra stainer (Ventana Medical Systems, Tucson, AZ, USA) was used to perform IHC on tissue sections with a thickness of 2 µm. A variety of antibodies were added, including epidermal growth factor receptor (EGFR; clone 3C6; Ventana Medical Systems), HER2 (clone 4B5; Ventana Medical Systems), HER3 (clone SP71; Abcam, Cambridge, UK), mTOR (clone 49F9; Cell Signaling Technology, Danvers, MA, USA), programmed death-ligand 1 (PD-L1; clone E1L3N; Cell Signaling Technology (until mid-2018; as of mid-2018, Nordic Biosite, Stockholm, Sweden, is using the BSR90 clone)), and PTEN (clone Y184; Abcam).
To assess the immunostaining intensity for the antigens EGFR, mTOR, PDGFRA, PDGFRB, and PTEN, a combinative semi-quantitative score was used. For a comprehensive description of the IHC, we refer to our previous work [9 (link)].