4.2. Immunohistochemistry for c-MYC and Staining Evaluation Score

Formalin-fixed, paraffin embedded sections (3 μm thick) were mounted on positive charged glass slides. For antigen retrieval to detect c-MYC protein, deparaffinized and rehydrated sections were boiled in TRIS-EDTA buffer solution (pH 9; for Abcam antibody) for 20 min or microwaved for 8–15 min in 10 mM sodium citrate (pH 6.0; for Santa Cruz Biotechnology antibody). The slides were cooled, and endogenous peroxidase were blocked with peroxidase block buffer (citric acid 0.04 M, Na₂HPO4 × 2H₂O 0.12 M, NaN3 0.03 M and H₂O₂ at 1.5% v/v) for 15 min at room temperature. Then, the sections were blocked with 1% BSA in PBS for 20 min and incubated for 72 h at 4 °C with rabbit monoclonal antibody anti-c-MYC (clone Y69; 1:100 diluition, Abcam, Cambridge, UK).

The primary antibodies were visualized using the avidin-biotin-peroxidase complex method (UltraTek HRP Anti-polyvalent, ScyTek, Logan, UT, USA) according to the instruction manual. 3,3′-diaminobenzidine was used as the enzyme substrate to observe the specific antibody localization, and Mayer hematoxylin was used as a nuclear counterstain.

The staining intensity of tissue slides was evaluated independently by 2 observers (L.M.L and M.M.) who were blinded toward the patients’ characteristics and survival. Cases with disagreement were discussed using a multiheaded microscope until agreement was achieved. To assess differences in staining intensity, an immunoreactivity scoring system was applied. c-MYC expression in each specimen was scored according to the extent (percent of stained cells) and intensity of nuclear expression staining. The score for the extent of the immunohistochemistry (IHC) stained area was scaled as 0 for no IHC signal at all, 1 for 1–30%, 2 for 31–70% and 3 for 71–100% of tumor cells stained. The score for IHC intensity was also scaled as 0 for no IHC signal, 1 for weak, 2 for moderate, and 3 for strong IHC signals. The final score used in the analysis was calculated by multiplying the extent score and intensity score, with a maximum score equal to 9.

The selection of cutoff scores for c-MYC expression in the prediction of anti-EGFR therapy response were based on ROC analysis Receiver Operating Characteristics (ROC) curve analysis. At each score, the sensitivity and specificity values were plotted thus generating a ROC curve. The score located closest to the point with both maximum sensitivity and specificity on the curve (0.0, 1.0), was selected as the cutoff score leading to the greatest number of tumors which were correctly classified as having or not having the outcome. Area under the ROC curves summarize the discriminatory power of c-MYC expression for the outcome with values of 0.5 indicating low power and those closer to 1.0 higher power. We compare three different immunohistochemical score for low and high expression of c-MYC: 0–1 versus 2–9; 0–3 versus 4–9; 0–6 versus 7–9. The immunohistochemical score 0–3 and 4–9, discriminating samples with low or high expression c-MYC, respectively, was identified as the best cutoff score in the prediction of anti-EGFR therapy response (p < 0.001; AUC = 0.899; 95% CI from 0.832 to 0.946; Youden index J = 0.6204; Sensitivity 96%; Specificity 66%; Figure S4). c-MYC staining that was confined to some nuclei of scattered cells at the bases of crypts (<5% positive cells) was considered as a normal mucosa staining pattern (internal positive control; 1–2 score). Negative controls were tumor sections stained in the absence of the primary antibody. Positive controls were Burkitt lymphoma samples. The c-MYC expression was also evaluated on a cohort of 40 normal colonic mucosae samples, without pathological features, belonging to the same mCRC cohort. All samples were stained more than once, and the results were highly reproducible [34].