Serum levels and tumour expression of leptin and leptin receptor as promising clinical biomarkers of specific feline mammary carcinoma subtypes

Obesity is a risk factor for human breast cancer, being associated with increased serum levels of leptin. In cat, although obesity is a common nutritional disorder, the role of leptin and its receptor in mammary carcinoma is unknown. In this study, serum levels of leptin and leptin receptor (ObR) were evaluated in 58 cats with mammary carcinoma and compared with healthy controls by ELISA, as tumour expression by immunohistochemistry. Results showed that the free leptin index is decreased in cats with mammary carcinoma (p=0.0006), particularly for those with luminal B and HER2-positive disease that showed significantly lower serum leptin levels (p<0.0001 and p<0.005, respectively). Serum leptin levels above 4.17 pg/mL were associated with ulcerating tumours (p=0.0005) and shorter DFS (p=0.0217). 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 tumours (p=0.0118), ER-negative status in HER2-positive tumours (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 tumour samples, leptin was overexpressed in luminal B and triple-negative carcinomas (p=0.0046), whereas ObR was found overexpressed in luminal B samples (p=0.0425). Altogether, our results reinforce the importance of feline mammary carcinoma in comparative oncology.


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The feline mammary carcinoma (FMC) is a high prevalence disease (12 to 40% of all tumours in 28 cat) that shows similar clinicopathological features to human breast cancer [1], supporting its use in 29 comparative oncology studies [2] [3]. Likewise, obesity is a common nutritional disorder in cat, with 30 higher prevalence in indoor and sterilized animals above three years of age [4]. In humans, obesity 31 induces a chronic inflammatory status, being a risk factor for breast cancer [5] [6] [7]. correlation was found between leptin overexpression in the tumour microenvironment and oestrogen receptor (ER) positive breast cancer, and with a human epidermal growth factor receptor 48 2 (HER 2)-positive status frequently related to a more invasive tumour phenotype [14].

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In parallel, the leptin receptor (ObR, was found to be involved in innate and 50 adaptive immunity [15], being expressed in several organs, including breast and peripheral tissues, 51 as well as in adipocytes [16] [17] and immune cells. ObR is constituted by an extracellular N-terminus 52 domain, a transmembrane domain, and a cytoplasmic C-terminus domain. Upon leptin ligation, ObR 53 homodimerizes and the associated JAK monomer is auto phosphorylated to activate the downstream 54 signalling pathways [8]. The soluble ObR form is a 146 kDa protein [18] that could be generated by 55 cellular apoptosis or by the proteolytic cleavage of the extracellular anchored protein domain, with 56 this shedding being more frequent in shorter intracellular isoforms. In serum, the ObR modulates the 57 leptin bioavailability, being decreased in obese humans [8]. In breast cancer patients, ObR is 58 overexpressed independently of the ER status [6], being correlated with low overall survival (OS) [9].

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Furthermore, the ratio between leptin/ObR serum levels (free leptin index -FLI) is considered an 60 useful predictor of leptin activity, reflecting the individual metabolic status [19] and when increased, 61 is an important risk factor for breast cancer development [20]. In parallel, studies in breast cancer

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To the best of our knowledge, this study is the first to evaluate the leptin and ObR serum levels 81 and tissue expression in cats with mammary carcinoma. The main goals of this study were as follows: 82 1) to compare the serum leptin and ObR levels between cats with mammary carcinoma stratified by 83 molecular subtype and healthy controls, using ELISA; 2) to investigate the leptin and ObR expression 84 in mammary carcinoma samples and compare with normal mammary tissues, using 85 immunohistochemistry (IHC); 3) to search for associations between serum leptin/ObR levels and 86 leptin/ObR IHC scores in tumour mammary tissues; 4) to test for associations between serum 87 leptin/ObR levels and several clinicopathological features, in order to evaluate the utility of leptin 88 and ObR as diagnostic and/or prognosis biomarkers or as promising drug targets for feline mammary 89 carcinoma.
Tumour tissue and serum samples were collected from 58 female cats with fully documented 93 history of FMC that underwent mastectomy and 24 serum samples from healthy cats presented for 94 elective ovariohysterectomy, at the Teaching Hospital of the Faculty of Veterinary Medicine, were collected with no interference in animal well-being, with the procedures involving 97 manipulation of animals being consented by the owners. Briefly, all tissue samples were embedded 98 in paraffin after fixation in 10% buffered neutralized formalin (pH 7.2), during 24-48 hours, while 99 serum samples were separated from clotted blood by centrifugation (1500g, 10min, 4ºC) and stored 100 at -80ºC until further use. All samples that showed haemolysis were discarded, as recommended [1] 101 [33].

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For each animal enrolled in the study, the clinicopathological data were recorded, including age, 103 breed, body weight, reproductive status and contraceptive administration, treatment (none, 104 mastectomy or mastectomy plus chemotherapy), number, location and size of tumoral lesions, 105 histopathological classification (ER status, PR status, HER2 status and Ki-67 index), malignancy 106 grade, presence of tumour necrosis, lymphatic invasion, lymphocytic infiltration, cutaneous 107 ulceration, regional lymph node involvement and clinical stage (TNM system) (

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To assess leptin and ObR immunoreactivity were used a scoring system previously reported [6] 154 [13] [36] and the H-Score published by the American Society of Clinical Oncology (ASCO). The final IHC score was obtained by multiplying the positive cells (0=absence of staining; 1=all cells stained), by the highest staining intensity (Table 2), varying from 0 to 3, with tissue samples scored as 0 considered negative, while samples scored as 3 designated highly reactive. All slides were subjected 158 to blind scoring, by two independent and experienced pathologists.

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In addition, results revealed that body weight did not influence serum leptin and ObR levels, 182 both in the controls (p=0.0760 and p=0.8432, respectively, data not shown) and in the cancer group positive mammary carcinomas had lower serum leptin levels than healthy animals (0.00 pg/mL vs.

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The results obtained in this study showed that cats with mammary carcinoma have a reduced