2.2.Immunohistochemical evaluation:
Cytoplasmic and / or membranous staining was considered for the detection of E-cadherin and beta-catenin in tumor cells. Immunoreactive scoring (IRS) was performed based on staining percentage and intensity using a semi-quantitative approach. Staining density was evaluated as follows: 0; no staining, 1; weakly positive, 2; moderately positive, 3; strongly positive. Percentages of tumor cells stained were determined as follows: <5%, no staining, 1: <6-25%, 2; 26-50%, and 3; 51-100% staining. While calculating the IRS score, staining pattern, and intensity scores were multiplied and two groups were formed based on the product of the multiplication as downregulation (≤3), and overexpression (>3) [7].
Percentages of nuclear and cytoplasmic staining in tumor cells for N-cadherin were scored separately based on the percentage of stained cells as follows: 1: 0-20%: 2: 21-40%, and 3: > 40% [8].
For Zeb1, percentage of nuclear staining in tumor cells was scored as follows: 1: 0-9%: 2: 10-50%, and 3:> 50%. Staining intensity was evaluated as follows: 0; no staining, 1; weakly positive, 2; moderately positive, 3; strongly positive. The final score was grouped as negative (≤1) and positive (> 1) based on the product of multiplication between scores of staining percentage and intensity [9].
Zeb1 nuclear positivity in fibroblasts observed in the stroma surrounding the tumor was also scored. The percentage of stained cells was scored as follows: 1:<10%: 2:11-50% and 3:> 50%.
Cytoplasmic and / or membranous staining in fibroblasts located in the surrounding stroma with αSMA was considered and scored as follows: 1: <10%; 2: 11-50% and 3: > 50%.