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%.