Benchmark autostainer

Immunohistochemical analysis for p16 was performed on 4-μm thick tissue sections cut from the FFPE tissue. A monoclonal antibody to p16 (ab108349, Abcam, UK; dilution 1:100) was used with a BenchMark Autostainer (Ventana Medical Systems, Tucson, USA) following the manufacturer’s recommendations. Expression of p16 was considered positive if strong and diffuse nuclear and cytoplasmic staining was detected in ≥70% of the tumor [11 (link)] Two head and neck pathologists reviewed p16 expression and were blinded to the clinical and follow-up data. Discrepancies that occurred between observers were resolved by consensus review.

Histological subtype, tumor grade, LVI (lympovascular invasion), and p16 staining patterns were confirmed by a genitourinary pathologist (PR). For p16 IHC, all samples were tested, and stained on a BenchMark Autostainer (Ventana Medical Systems, Tucson, AZ, USA) as described by the manufacturer’s protocol using a prediluted mouse monoclonal antibody (CINtec^® p16 Histology, clone E6H4, Ventana Medical Systems, Tucson, AZ, USA). p16 staining patterns of 0, 1, 2, and 3 were classified using previously described categories [13 (link),14 (link)]. Based on previously published methods [5 (link),13 (link),14 (link),15 (link)] and our own observations, we created a hybrid system (HS) for analyzing p16 via IHC (Table 1). The HS method used both the percent (>75%) positive staining of the tumor section and the staining pattern. For representative images, slides were scanned using Aperio AT2 total slide scanner (AT2, Leica Biosystems, Vista, CA, USA) and images are at 2X amplification.

Her2/neu testing was carried out at a single pathology laboratory in Modena by immunohistochemistry, and the results were scored as follows: 0, 1 = negative, 2 = indeterminate, and 3 = positive. Patients with HER2 test results reported as “indeterminate” were evaluated by fluorescence in situ hybridization (FISH). ER and PgR testing were conducted with a single report format of “positive” or “negative” test results, as measured by immunohistochemical analysis (clone 6F11, Ventana, for ER; and clone 1E2, Ventana, for PgR) and staining by Ventana Benchmark autostainer. ER and PgR receptor status were tested by evaluating the percentage of nuclear immunoreactivity with respect to all the nuclei in the neoplastic cells, independently of the staining intensity. Nuclear staining 10% of either ER or PgR was considered a positive result. Ki-67 labeling index was determined with the MIB1 monoclonal antibody as nuclear immunoreactivity. The cut-off was equal to 14% to subdivide luminal A and luminal B tumor. Luminal A were tumors with ER/PgR/HER2-/Ki67 < 14%, luminal B were tumors with ER/PgR/HER2-/Ki67 ≥ 14%, luminal/HER2 enriched were tumors with ER/PgR/HER2, triple-negative tumors were ER-/PgR-/HER2-, and HER2 were tumors with ER-/PgR-/HER2.

Two cores (2 mm in diameter) from each specimen were embedded in recipient paraffin blocks using a trephine apparatus (Superbiochips Laboratories, Seoul, Republic of Korea) for TMA construction. IHC staining was conducted on 4-μm-thick TMA sections using the Benchmark autostainer (Ventana, Tucson, AZ) according to the manufacturer’s instructions. IHC staining was conducted using a rabbit monoclonal antibody against pAMPK^T172 (1:100; Cat. #2535; Cell Signaling Technology, Danvers, MA), a rabbit polyclonal antibody against pSMAD2^S467 (1:70; Cat. #ab53100; Abcam, Cambridge, UK), and a mouse monoclonal antibody against SMAD4 (1:100; Cat. #sc-7966; Santa Cruz Biotechnology). One TMA slide was stained without primary anti-pAMPK antibody as a negative control.