# Immunohistochemical Analysis of Inter-Alpha-Trypsin Inhibitor Heavy Chain 2 and Enolase 1 in Canine Mammary Tumors: Associations with Tumor Aggressiveness and Prognostic Significance

## TL;DR

This study examines ITIH2 and ENO1 in canine mammary tumors to determine their potential as biomarkers for tumor aggressiveness and prognosis.

## Contribution

The study investigates the immunohistochemical expression of ITIH2 and ENO1 in canine mammary tumors and their association with tumor progression.

## Key findings

- ITIH2 and ENO1 showed distinct tissue and subcellular localization patterns in canine mammary tumors.
- Neither ITIH2 nor ENO1 demonstrated strong prognostic value based on histoscore and ROC curve analysis.
- ZEB1 expression tended to be higher in malignant tumors and associated with ENO1.

## Abstract

Mammary tumors in dogs are commonly seen in middle-aged and older females, particularly in those that are not spayed. These tumors share many similarities with human breast cancer, making dogs a useful model for studying the disease. This study focuses on two potential biomarkers—Inter-Alpha-Trypsin Inhibitor Heavy Chain 2 (ITIH2) and Enolase 1 (ENO1)—which have been identified in previous research as relevant to canine mammary tumors. We used immunohistochemistry to analyze tissue samples from healthy dogs and those with benign or malignant tumors. The results showed specific patterns of expression for both ITIH2 and ENO1, suggesting that these proteins may play important roles in tumor development. This study highlights their potential as biomarkers for diagnosing and treating mammary tumors, both in dogs and, possibly, in human breast cancer.

Mammary neoplasms in dogs are a common clinical concern, especially in middle-aged and older intact females. These tumors share similarities with human breast cancer in terms of histology, disease progression, and risk factors, making dogs a relevant model for breast cancer research. The search for biomarkers in canine mammary tumors is essential to understand tumor progression and identify potential therapeutic targets. This study investigated the expression of two potential biomarkers—Inter-Alpha-Trypsin Inhibitor Heavy Chain 2 (ITIH2) and Enolase 1 (ENO1)—in the mammary glands of healthy and tumor-bearing dogs using immunohistochemistry. Both proteins were identified in previous proteomic analyses of extracellular vesicles derived from the plasma of healthy and tumor-bearing dogs. A total of fifty-one canine mammary tissue samples were analyzed and categorized into three groups: (i) the control group, composed of five samples of normal mammary tissue without neoplasia; (ii) benign tumors, composed of nineteen samples of benign mixed tumors; and (iii) malignant tumors, which included six carcinomas in grade 1 mixed tumors, five carcinomas in grade 2 mixed tumors, thirteen solid carcinomas of grade 3, one papillary carcinoma, and two tubular carcinomas. Regarding the intensity of staining, quantified by histoscore, there were no significant differences in the comparison between the groups; for ITIH2, the p-value was 0.33, and for ENO1, the p-value was 0.57. Regarding the predictive potential of their respective ROC curves, the proteins demonstrated low predictive power in canine mammary tumors. These findings indicate that neither ITIH2 nor ENO1 demonstrated strong prognostic value in this setting, as demonstrated by their moderate AUC values, wide confidence intervals, and lack of statistical significance. However, this study found distinct tissue localization patterns for ITIH2 and subcellular localization for ENO1. As an additional way to examine possible associations of these proteins with epithelial–mesenchymal transition, the ZEB1 antibody was tested by both single and double immunohistochemistry, demonstrating a tendency to be more intensely expressed in the malignant group and tending to be associated with ENO1 in canine mammary tumors.