p-STAT3 staining

The biomarker protocol for the evaluation of p-STAT3 phosphorylation in association with the clinical outcome was approved by the Institutional Review Board of Hadassah Medical Center and the BIG2-98 Study Steering Committee. From the 2,887 patients randomised in the BIG 2–98 trial, 2,173 cases had tumour blocks that were centrally evaluated. A TMA was constructed from 950 blocks.

Paraffin blocks were submitted to the coordinating center and 4 cores from each tumour were collected and placed in 2 different TMAs; each TMA contains 2 cores of the same tumour. Two laboratories performed independent staining for p-STAT3 [immunohistochemistry (IHC) and immunofluorescence (IF)]: Each laboratory received a set of available BIG2-98 specimens in TMAs. The BIG 2-98 TMA set contained 19 slides with ~170 tissue cores per slide. Two slides containing ER-negative samples were of low quality, and although stained, could not be annotated. In total, 610 and 585 ER-positive samples were interpretable for p-STAT3 by IHC and IF, respectively.

IHC was performed experimentally. The tissue micro-array sections slides were deparaffinised with xylene rinses and then transferred through two changes of 100% ethanol. Endogenous peroxidase activity was blocked by a 5-min incubation in a 3% hydrogen peroxide buffer. Antigen retrieval was performed using Tris-EDTA buffer (10 mM Tris Base, 1 mM EDTA solution). Following antigen retrieval, the slides were incubated with blocking buffers at room temperature to reduce non-specific background staining and then incubated with primary antibody at 4˚C overnight (1:100 dilution of p-STAT3 antibody; cat. no. 9145-D3A7-XP; Cell Signaling Technology, Beverly, MA, USA) followed by 30 min of incubation with the secondary anti-rabbit antibody (Histofine^® Simple Stain™ MAX PO (R), cat. no. 414141F; Nichirei Biosciences Inc., Tokyo, Japan). Staining was visualised using DAB.

The IHC nuclear p-STAT3 staining was determined and scored separately for each spot and specimen by two pathologists (R.S. and G.V.E.) who were blinded to the clinical pathological data and reached an agreed score for each spot. The staining was analysed according to the H-score (range, 0–300; intensity 1–3 × percentage of positive cells 1–100). For each spot, separate scores where provided for the tumour and stromal parts. For specimens that were uninterpretable, a score of 'not applicable' (N/A) was assigned. To define tumours as p-STAT3-positive, a cut-off point of >0 H score was selected, as well as analysing the data as a continuous variable. Specificity/sensitivity, performance and reproducibility tests of the p-STAT3 IHC were performed. Full slides from representative positive (n=9) and negative (n=8) TMAs were stained processed, and evaluated similarly as the experimental slides, for the indication of reproducibility and performance of the TMAs. In total, 8/9 positive TMAs were found to be positive on the full slides and 8/8 of the negative TMAs were found to be negative on the full slides, demonstrating 100% sensitivity and 89% specificity. As the controls for our procedure, we used HeLa cells without treatment that served as a negative control and serum-starved HeLa cells prepared with interferon-α (IFN-α) treatment that served as a positive control (all control slides were purchased from Cell Signaling Technology). The control slides were used for qualifying the procedure and not as an interpretation reference. The presence of a brown reaction product at the cell nucleus was indicative of positive reactivity.

In parallel, TMAs were stained using IF at the laboratory of J.F.B. For IF staining, the TMAs were deparaffinised and processed using the automated Ventana deparaffinization solution (Ventana Medical Systems, Inc., Tucson, AZ, USA) and CC1 antigen retrieval. The tissue sections were blocked for 30 min in 10% normal goat serum, 2% BSA in phosphate-buffered saline (PBS). The incubation with the p-STAT3 antibody was carried for 2 h, followed by 16 min of biotinylated goat anti-rabbit IgG (cat. no. PK6101; Vector Laboratories, Inc., Burlingame, CA, USA) at a 1:200 dilution (=7.5 µg/ml). Streptavidin-HRP (Ventana Medical Systems, Inc.) was applied for 12 min followed by incubation with Tyramide-Alexa Fluor 488 (cat. no. {"type":"entrez-nucleotide","attrs":{"text":"T20922","term_id":"2756841","term_text":"T20922"}}T20922; Invitrogen, Carlsbad, CA, USA) for 16 min. p-STAT3 antibody was from Cell Signaling Technology (cat. no. 9145-D3A7-XP) a rabbit monoclonal has been purchased in large amounts (5 ml), mixed and re-aliquoted to avoid batch-batch variation. All slides were in the same run.

The IF results were analysed using a semi-supervised free-scoring tool by a digital imaging system. Specifically, each slide was scanned using Pannoramic Flash (3DHistech Ltd., Budapest, Hungary) and each tumour/stromal section was exported into a tif. image by Pannoramic Viewer (3DHistech Ltd.). Image analysis was performed using Metamorph (Molecular Devices, Sunnyvale CA, USA) in which the number of nuceli was counted using the DAPI channel, and the level of green fluorescence (p-STAT3)/nuclei was quantified.

Tissue microarray construction, the determination of the proteomic status, patient selection, assay performance and data analysis are reported according to the Recommendations for Tumour Marker Prognostic Studies (REMARK) criteria (25).