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

Abstract

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.

Keywords: biomarkers; bitches; extracellular vesicles (EVs); mammary tumors; prognosis.

Figure 1

Photomicrographs of the positive and negative controls of ITIH2, ENO1, and ZEB1. (A)—Positive control of ITIH2 (fallopian tube) and (D)—negative control (tonsil); (B)—Positive control of ENO1 (liver) and (E)—negative control (antibody suppression in dog mammary gland tissue); (C)—Positive control of ZEB1 (lymph node) and (F)—negative control (placenta).

Figure 2

(A) Box plot illustrating age distribution across the malignant, benign, and control groups. p-values were calculated using ANOVA with Bonferroni post hoc tests. The black cross represents the mean age within each group. (B) Proportion representation of breeds within the malignant, benign, and control groups.

Figure 3

Immunohistochemical staining of ITIH2. Control group (A–C); benign tumors (D–F); and malignant tumors (G–I). Photomicrographs at 20 µm showing weak or absent immunostaining in the cytoplasm.

Figure 4

Immunohistochemical staining of ENO1. Control group (A–C); benign tumors (D–F); and malignant tumors (G–I). Photomicrographs at 20 µm showing strong immunostaining in nucleus, cytoplasm, and membrane (I).

Figure 5

Protein expression (HistoScore) of ITIH2 (Inter-Alpha-Trypsin Inhibitor Heavy Chain 2) and ENO1 (Enolase 1). p-values for the Kruskal–Wallis test and Dunn’s post hoc test. The black cross represents the mean.

Figure 6

Immunohistochemical localization of ENO1 across cellular compartments.

Figure 7

Immunohistochemical comparison of ITIH2 (1A) and ZEB1 (1B) staining. The tissues in (1A) and (1B) are identical, differing only by the marker used. Tumor types and grades include carcinoma in mixed tumor grade 2 (A); carcinoma in mixed tumor grade 1 (H–J); tubular carcinoma (C); solid carcinoma grade 3 (E,F); and benign mixed tumor (B,D,G). Metastatic tumors are shown in (A,C,E).

Figure 8

Double immunohistochemical staining showing ENO1 (magenta) and ZEB1 (brown) expression. Tumor types and grades include carcinoma in mixed tumor grade 2 (A); carcinoma in mixed tumor grade 1 (H–J); tubular carcinoma (C); solid carcinoma grade 3 (E,F); and benign mixed tumor (B,D,G).

Figure 9

Roc curves of ITIH2 (Inter-Alpha-Trypsin Inhibitor Heavy Chain 2) and ENO1 (Enolase 1).