Quantitative IHC Evaluation of Protein Markers

Protein expression of HIF1α, EGFR, pEGFRY1068 and pEGFRY1173 was analysed by IHC using VECTASTATIN® Elite® ABC kit (Vector Laboratories, CA, USA). A detailed protocol is provided in Supplementary Methods, and details of primary antibodies and positive controls are tabulated in Supplementary Table 1. IHC staining was evaluated semi-quantitatively by the pathologists who were blinded to treatment and patient’s outcomes. Expression of HIF1α (nuclear), EGFR (membrane and cytoplasmic), pEGFRY1068 (membrane) and pEGFRY1173 (membrane) was assessed by deriving the H-score (scale: 1–300) using the formula H-score = Σ_Pi (i + 1), where _Pi is the percentage (0–100%) of stained tumour cells at each intensity and i is the intensity: i = 1 (weak), 2 (moderate) and 3 (strong).^{9 (link),15 (link)} Biomarkers were analysed as dichotomised variables. Due to unavailability of consensus regarding H-score cut point to be used for dichotomisation of these biomarkers, the respective median H-score values were used for HIF1α (H-score = 90) and EGFR (membrane, H-score = 100; cytoplasm, H-score = 140).^{26 (link),27 (link)} For pEGFRY1068 and pEGFRY1173, patients with H-score = 0 were categorised as negative and patients with H-score > 0 were positive. IHC staining of HIF1α was independently evaluated by a second pathologist.