Adenosquamous inflammatory mammary carcinoma in bitch: Can CA 15-3, CRP and LDH improve prognosis?

Inflammatory mammary carcinoma (IMC) is characterized by the presence of any subtype of aggressive carcinoma in association with an intense inflammatory reaction, in addition to the presence of tumor emboli. Considering IMC as a type of cancer with chronic inflammation, the complex relationship between inflammation, and alterations in cellular metabolism have highlighted the rationale assessment of cancer antigen 15-3 (CA 15-3), c-reactive protein (CRP), and lactate dehydrogenase (LDH). The present report had the objective to describe a rare case of adenosquamous carcinoma in mammary gland of a 10-year-old bitch, 7 kg, sexually intact, mixed-breed and how CA 15-3, CRP and LHD helped to determinate the clinical presentation of inflammatory carcinoma and the prognosis. Complete clinical staging, cell blood count and renal, and hepatic biochemistry was performed and did not reveal any significant abnormalities. CA 15-3 (9.46 IU/mL), CRP (5.3 mg/L), and LDH (287 U/L) were all above the references values reported by the literature. Although adenosquamous carcinoma of the mammary gland with inflammatory presentation in dogs is rare, the association of serum biomarkers could improve the patient's prognosis evaluation.


Introduction
Canine mammary tumors (CMTs) are the most common neoplasms in female dogs and inflammatory mammary carcinoma (IMC) is the most aggressive form of mammary neoplasia (Ribeiro et al., 2020;Soremo, 2020;Vascellari et al., 2016). Clinically, patients with IMC present with the rapid appearance of inflammatory signs including erythema, edema, firmness, pain, and warmth of the mammary skin, with or without a defined mass. It is locally aggressive and highly metastatic that is poorly responsive to treatment, resulting in a poor prognosis, with invariably short survival times after diagnosis (Cassali et al., 2020;Souza et al., 2009).
In 2011, a new detailed histologic classification of subtypes of CMTs was published by Goldschmidt et al. (2011) and described the adenosquamous carcinoma, that is composed of areas of carcinoma of any type admixed with foci where the neoplastic cells exhibit squamous differentiation and features of malignancy. Islands and cords of epithelial cells are seen with the formation of keratin pearls. Cells and nuclei are large; the cells produce intracytoplasmic keratin tonofilaments; and intercellular bridges may be found. A marked secondary acute and chronic inflammatory cell infiltrate often occurs in these neoplasms. The neoplasm has its origin either from squamous cells of the teat duct or from ductal epithelial cells that had undergone squamous metaplasia and neoplastic transformation (Goldschmidt et al., 2011).
Histopathologically, IMC is characterized by the presence of any subtype of aggressive carcinoma in association with an intense inflammatory reaction, in addition to the presence of tumor emboli within dermal lymphatic vessels, resulting in profuse oedema, because of the obstruction of lymph drainage (Cassali et al., 2014;Goldschmidt et al., 2011).
Considering IMC as a type of cancer with chronic inflammation, expression of diverse inflammatory mediators has been explored in search for an explanation for the severe aggressiveness of the disease. Few biomarkers are specific for a single tumor, but their association can increase diagnostic accuracy (Kabel, 2017). The complex relationship recently described between mammary tumor, inflammation, and alterations in cellular metabolism have highlighted the rationale for combined assessment of cancer antigen 15-3 (CA 15-3), C-reactive protein (CRP), and lactate dehydrogenase (LDH) (Hanahan & Weinberg, 2011). In CMTs, CA 15-3 (Campos et al., 2012;Campos et al., 2015;Manuali et al., 2012;Valencakova-Agyagosova et al., 2014), CRP (Planellas et al., 2009;Tecles et al., 2009;Szczubiał et al., 2018), and LDH (Campos et al., 2012) have been suggested as good biomarkers.
The present report had the objective to describe a rare case of adenosquamous carcinoma in mammary gland of female dog, and how CA 15-3, CRP and LHD helped to determinate the clinical presentation of inflammatory carcinoma.

Case report
A 10-year-old, 7kg, sexually intact, female, mixed-breed dog was presented to the veterinary hospital reporting 30 days of erythematous skin on the last two mammary glands (M4 and M5) of the right chain, sized 8.0x3.0x0.5cm, with pain on palpation, and limb edema, with lameness, and vulvar edema in association (Figure 1).
The superficial inguinal lymph nodes were enlarged by palpation. The patient's heart rate, respiratory rate, and rectal temperature measured within normal limits. The patient's food and water intake were reportedly normal.   (Carney et al., 2011;Kaneko et al., 2008;Valencakova-Agyagosova et al., 2014).
Based on clinical and laboratory findings, palliative treatment with piroxicam (0.3 mg/kg PO, SID) and tramadol (3mg/kg PO, TID) was recommended (Greghi et al., 2021;Souza et al., 2009). 15 days after, the owner came back reported partial resolution of the left hind limb edema, lameness, and pain and erythema. However, the patient developed evidence of systemic disease (anorexia and lethargy) coupled with progressive deterioration of the clinical status. Given the clinical conditions of the patient, the owners opted for euthanasia.
A necropsy was performed and reveal thickening of the bilateral mammary gland, enlargement of bilateral inguinal lymph nodes, severe infiltration of fat and muscle tissue, and subcutaneous edema. Samples were collected and then fixed in 10% buffered formalin and cross-sections of the fragments were taken and routinely processed for histopathology.
Histopathologic evaluation form mammary gland and regional lymph node indicated the presence of neoplastic proliferation consisting of tubular structures coated by malignant glandular epithelium with areas of malignant squamous differentiation, as well marked fibrous stroma that support an intense infiltrate of lymphocytes, plasmocytes and hemosiderophages, and foci of necrosis. Neoplastic emboli were observed in several dermal lymphatic vessels. Neoplastic cells were present in lymph nodes.
The histopathologic results were consistent with a high grade adenosquamous carcinoma of the mammary gland with clinicopathologic presentation of IMC (Figure 3).

Discussion
Although the clinical signs in this patient were consistent with IMC, a definitive diagnosis was required. Cytologic examination performed on abnormal tissue confirmed the epithelial origin of the neoplasm, and histopathology showed the presence of tumor emboli in the dermal lymphatic vessels (Goldschmidt et al., 2011).
Based in the values of serum biomarkers, it was possible to infer that the case was a CMT with a poor prognosis. Few biomarkers are specific for a single tumor, but their association can increase diagnostic accuracy (Kabel, 2017). Ryuet al.
(2019) suggest that the combined dosage of CRP with other serum biomarkers can increased the diagnostic accuracy for detecting cancer. Despite that work indicate the association of more than one biomarker in cases of cancer in dogs, there are no works evaluating CA 15-3, CRP and LDH simultaneously in cases of CMT. Valencakova-Agyagosova et al. (2014) proposed a cut-off value of 5.0 to 7.0 IU/mL, with 100% sensitivity and 95.0% specificity, and suggested the application of CA 15-3 as a biomarker of first choice with high sensitivity and specificity for CMT. Until now, there have been non consensus for reference intervals indicated for CA 15-3. Largest tumors with lymph node metastasis (Campos et al., 2012;Campos et al., 2015) and higher grades based on histopathological analysis (Manuali et al., 2015) exhibit significantly higher concentration of CA 15-3.
Since CRP and LDH exhibit non-specific increase in various situations (Kaneko et al., 2008;Carney et al., 2011), they could not be applied individually as the biomarkers for CMT. Although elevated CRP and LDH are not the biomarkers of cancer, they may serve as the cofactors that increase the diagnostic accuracy to detect CMT. Female dogs that exhibited larger nodules (Szczubiał et al., 2018), ulcerated skin (Planellas et al., 2009;Szczubiał et al., 2018), distant metastasis (Szczubiał et al., 2018), and high clinical stages (Tecles et al., 2009) exhibited higher concentrations of CRP. Campos et al. (2012) demonstrated a positive correlation between LDH concentration and clinical staging.
The patient in the present case received piroxicam as a palliative treatment. The use of a COX inhibitor as palliative therapy in cases of canine IMC has shown encouraging results and should be considered as a single agent for the treatment of this tumor type, this therapy significantly increased survival time in dogs than in those treated with traditional chemotherapy protocols (Souza et al., 2009). In the present case, a brief clinical improvement was reported by the owner after treatment with piroxicam was begun. Rasotto et al. (2017) reported that adenosquamous carcinoma present a slower disease progression (18 months), distant metastases were common, and that was the CMT that most commonly recurred. This tendency for local relapse might be attributable to the squamous component of the tumor, and the risks of dying from tumor-related causes was high.
Adenosquamous carcinoma were usually grade III, the histologic grade was significantly associated with tumor-specific overall survival, local recurrence, and distant metastases (Goldschmidt et al., 2011;Peña et al., 2014;Rasotto et al., 2017). In the present report most of these facts were found except for the time of progression, that was much shorter than reported, probably because of high grade and IMC association.

Conclusion
The association of serum biomarkers CA 15-3, PCR and LDH can improve the prognosis of the dog affected by adenosquamous carcinoma of the mammary gland with inflammatory presentation.