Serum and Tissue Expression Levels of Leptin and Leptin Receptor Are Putative Markers of Specific Feline Mammary Carcinoma Subtypes

Abstract

Obesity is an established risk factor for breast cancer in post-menopausal women, being associated with elevated serum levels of leptin. Although overweight is a common condition in cat, the role of leptin and its receptor in feline mammary carcinoma remains unsettled. In this study, serum leptin and leptin receptor (ObR) levels were investigated in 58 cats with mammary carcinoma and compared with those of healthy animals, as were the expression levels of leptin and ObR in tumor tissues. The results showed that the Free Leptin Index is significantly decreased in cats with mammary carcinoma (p = 0.0006), particularly in those with luminal B and HER2-positive tumors, and that these animals also present significantly lower serum leptin levels (p < 0.0001 and p < 0.005, respectively). Interestingly, ulcerating tumors (p = 0.0005) and shorter disease-free survival (p = 0.0217) were associated to serum leptin levels above 4.17 pg/mL. In contrast, elevated serum ObR levels were found in all cats with mammary carcinoma (p < 0.0001), with levels above 16.89 ng/mL being associated with smaller tumors (p = 0.0118), estrogen receptor negative status (p = 0.0291) and increased serum levels of CTLA-4 (p = 0.0056), TNF-α (p = 0.0025), PD-1 (p = 0.0023), and PD-L1 (p = 0.0002). In tumor samples, leptin is overexpressed in luminal B and triple-negative carcinomas (p = 0.0046), whereas ObR is found to be overexpressed in luminal B tumors (p = 0.0425). Altogether, our results support the hypothesis that serum levels of leptin and ObR can be used as biomarkers of specific feline mammary carcinoma subtypes, and suggests the use of leptin antagonists as a therapeutic tool, reinforcing the utility of the cat as a cancer model.

Keywords: biomarkers; feline mammary carcinoma; free leptin index; leptin; leptin receptor.

Figure 1

Dot plot diagram showing that the Free Leptin Index (FLI) was significantly elevated in healthy animals than in cats with mammary carcinoma (***p = 0.0006).

Figure 2

Body weight did not influence leptin, neither ObR serum levels in healthy and diseased animals. Correlations were not found between (A) serum leptin (p = 0.0760) or (B) ObR (p = 0.8432) levels and body weight in the control group. Furthermore, evaluating the cancer group, no correlations were detected between (C) serum leptin (p = 0.3294) or (D) ObR (p = 0.9722) levels and feline body weight.

Figure 3

Cats with luminal B and HER2-positive mammary carcinomas showed decreased serum leptin levels, although cats with ulcerated tumors exhibited serum leptin levels above the cut-off value of 4.17 pg/mL, being associated with shorter disease-free survival. (A) Dot plot diagram showing the distribution of serum leptin levels (pg/mL) among healthy animals (control) and cats stratified by the mammary carcinoma subtype. Significant decreased serum levels of leptin were found in cats presenting luminal B or HER2-positive subtypes in comparison to healthy animals (p = 0.0025). (B) The optimal cut-off of serum leptin levels to predict mammary carcinoma was determined to maximize the sum of the sensitivity and specificity (4.17 pg/mL; AUC = 0.7045 ± 0.0757, 95% CI: 0.5561–0.8528, p = 0.0103; sensitivity = 96.9%; specificity = 43.5%). (C) Dot plot diagram showing that serum leptin levels were significantly higher in cats with ulcerated tumors (p = 0.0005). (D) Cats with mammary carcinoma and serum leptin levels higher than 4.17 pg/mL had a lower DFS (p = 0.0217). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 4

Cats with mammary carcinoma showed elevated serum ObR levels, with serum concentrations above 16.89 ng/mL being associated with smaller tumors and an ER-negative status. (A) Dot plot diagram showing the distribution of serum ObR levels (ng/mL) in heathy animals (control) and in cats with mammary carcinoma stratified by the molecular subtype. Significant higher serum levels of ObR were found in all tumor subtypes in comparison to healthy animals (p < 0.0001). (B) The optimal cut-off value of serum ObR levels to predict cats with mammary carcinoma was 16.89 ng/mL with an AUC of 0.9408 ± 0.0288 (95% CI: 0.8842–0.9973, p < 0.0001; sensitivity = 94.8%; specificity = 87.0%). (C) Dot plot diagram showing that serum ObR concentrations were significantly low in tumors larger than 2 cm (p = 0.0118). (D) Dot plot diagram displaying a positive association between higher serum ObR levels and ER-negative status (p = 0.0291). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 5

Serum ObR levels showed a positive correlation with inflammatory mediators, namely (A) serum CTLA-4 levels (p = 0.0056), (B) serum TNF-α levels (p = 0.0025), (C) serum PD-1 levels (p = 0.0023), and (D) serum PD-L1 levels (p = 0.0002).

Figure 6

Final IHC scores for leptin (A) and ObR (B) in cats with mammary carcinoma stratified by the tumor subtype and compared with controls. (A) Leptin expression was significantly higher in luminal B and triple-negative subtypes (p = 0.0046). (B) Expression of ObR was statistically higher in luminal B tumor subtype (p = 0.0425).

Figure 7

Leptin and ObR were overexpressed in luminal B mammary carcinomas. (A) Leptin overexpression in a luminal B mammary carcinoma (IHC score of 1.93) contrasting with (B) a low staining intensity detected in normal mammary tissues (IHC score of 1.34). (C) Luminal B mammary tumors showed a higher staining intensity for ObR (IHC score of 2.50), (D) than normal mammary tissues (IHC score of 1.75) (400× magnification).

Figure 8

Tumor inflammatory cells express leptin and ObR. Luminal B carcinoma subtype showed a (A) positive leptin staining (IHC score of 1.5), which is lower when compared to the (B) ObR immunostaining (IHC score of 2.5) of tumor inflammatory cells. Furthermore, in both samples higher staining intensity was observed in macrophages, when compared to lymphoid cells (IHC score of 2.0 vs. 1.2, respectively for leptin, and IHC score of 3.0 vs. 2.0, respectively for ObR) (400× magnification).

Figure 9

Dot plot diagram showing a negative correlation between serum ObR levels and tumor ObR IHC score (*p = 0.0103).