HISTOLOGICAL CLASSIFICATION AND GRADING OF MALIGNANT MAMMARY NEOPLASMS

Sneha R. Thorat1, A.P.S. Brar2 N.K. Sood3 and Kuldip Gupta2
1M.V.Sc. Student, 2 Professor; Department of Veterinary Pathology, 3 Senior Veterinary Pathologist Teaching
Veterinary Clinical Complex, College of Veterinary & Animal Science, GADVASU, Ludhiana -141004 (Punjab.).
[Received: 18.6.2018; Accepted: 25.3.2019]
{DOI 10.29005/IJCP.2019.11.1.084-089}

Mammary tumors are the most common neoplasms that affect female dogs. The present study was conducted
on 40 cases of malignant canine mammary tumors. Among 40 malignant CMT, 30 (75%) were carcinomas, five
(12.5%) carcinosarcomas, four (10%) sarcomas and one other i.e. malignant mast cell tumor (2.5%). The carcinomas
further comprised of 13 simple carcinomas (32.5%), 10 complex carcinomas (25%), six solid carcinomas (15%) and
one special type i.e. squamous cell carcinoma (2.5%). The study revealed that majority of canine mammary tumors was
of simple carcinoma sub-type and of grade II category.
Keywords: Canine, grading, Histological classification, Malignant mammary tumor.

Canine mammary tumors (CMT) are among the most common neoplasms that affect female dogs with very high incidence. Majority of these tumors are malignant (Baba et al., 2016). CMT is of interest to both veterinary and human oncologists because of their similarity in many aspects. During the last 3-4 decades, these tumors have been
proposed as model for human breast cancer research (Lavalle et al., 2009, Gupta et al., 2012). Most commonly observed malignant tumor in women is the simple carcinoma, with more than one million cases and nearly
600,000 deaths occurring worldwide annually (Stewart and Kleihues, 2003). Metastasis remains the most threatening aspect of mammary carcinoma in both bitches and women, as most of deaths are recited because
of metastatic disease (Yan et al., 2008). Incidence of cancer is three times higher in bitches when compared to dogs (Merlo et al., 2008). Intact bitches are more affected than neutered. Occurrence of CMTs is higher in posterior mammary gland (Gupta et al., 2012) due to higher gland volume and abundant secretion during the lactation period that is approximately 40% of all mammary tumors (Baba and Catoi, 2007). Breeds such as Poodles, English Spaniels, English setters and Terriers exhibit a higher risk for mammary gland tumors, while in Boxers and
Chihuahua, the risk is minimal (Gupta et al., 2012). Prognosis of CMTs is directly related to factors such as tumor size, lymph node involvement, presence of distant metastasis, histologic type, histologic grade, nuclear
differentiation (Lavalle et al., 2009). The present study deals with 40 malignant cases of CMT with a view to find their histological classification and grading for determining their prognosis.

Materials and Methods
The present study was conducted on tissue samples from 40 cases of canine mammary tumors, out of which 25 were
retrospective cases and 15 prospective cases, collected from Department of Veterinary Pathology and Small Animal Clinics of the Department of Teaching Veterinary Clinical Complex, College of Veterinary and Animal Science, GADVASU, Ludhiana, respectively.

The mammary tumors were excised by mastectomy with or without the superficial inguinal lymph nodes. In cases of multiple tumors, the whole chain was resected. Representative tissue pieces were collected from multiple sites from excised tumors and fixed in 10% neutral buffered formalin, given overnight washings under running tap water
and dehydrated using ascending grades of ethyl alcohol followed by clearing with acetone and benzene. Thereafter, the tissues were embedded in paraffin wax (56oC). The sections of 4-5μm were cut and stained with routine H&E and Toludine blue.

The tumors were classified on the basis of latest WHO classification of mammary tumors of dogs as shown in tables 1 and 2.

Results and Discussion
All the tumors were classified into various histological sub-types and the results have been depicted in Table-3.

Carcinomas:
In the present study, 30 cases of carcinoma were recorded, which were further classified as simple carcinoma (n=13), complex carcinoma (n=10), solid carcinoma (n=6), and one case of squamous cell carcinoma.

Simple carcinoma:
Thirty cases of simple carcinoma were reported, which were microscopically characterized by proliferation of only one cell type (Fig.1), resembling either luminal or basal epithelial cells with minimal stromal component. Based on the cellular pattern and the degree of differentiation. Simple carcinomas were further sub-classified as tubulopapillary (n=6) (Fig.2), tubuloacinar (n=2) with or without papillary projections at times giving rise to cystic appearance (n=1) (Fig.3). In addition, two cases of early simple carcinoma were also noted. In the present
study, among 13 carcinoma cases, mild to moderate inflammation was noticed in nine cases (69.24%), connective tissue invasion in 10 cases (76.93%), tumor infiltrating lymphocytes (TIL) in five cases (38.47%), epithelial mesenchymal transition (EMT) in eight cases (61.54%), necrosis in six cases (46.16%) and tumor thrombo-embolism in seven cases (53.85%) were observed as addition histological features.

Ten cases of complex carcinoma were recorded, which were microscopically characterised by proliferation of both
epithelial and myoepithelial components. The myoepithelial cells were arranged either in stellate or reticulate pattern (Fig.4). In the present study, EMT occurred in all cases of complex carcinoma, while necrosis in four
cases only.

Solid Carcinoma:
In the present study, 6 solid carcinoma cases were observed. On microscopic observation, the neoplastic cells were
predominantly arranged in solid sheets, cords, or masses, without lumina to form dense, irregularly sized lobules supported by a fine fibrovascular stroma (Fig.5). Mild to moderate inflammation was found in all the cases. Tumor embolism was recorded in two cases.

Special type of Carcinoma (Squamous cell carcinoma): In the present study, one case of squamous cell carcinoma was found. In which the neoplastic cells were arranged in this tumor in cords, sheets and trabaculae
with varying degree of squamous differentiation along with extensive thromboembolism.

Sarcomas:
Microscopically, all the four sarcomas were fibrosarcomas. It was observed that the neoplastic fibroblasts were arranged haphazardly and in concentric arrangement around the proliferating blood vessels and resembled somewhat the histological appearance of perivascular fibrosarcoma (Fig.6), as described by earlier workers
Misdorp (2002) and Gupta (2011). In addition, there was necrosis, tumor embolism and infiltration of neutrophils with abundant mitotic figures.

Carcinosarcoma:
In the present study five cases of carcinosarcoma were diagnosed. On microscopic observation it was observed that
the tumor contained cells resembling both malignant epithelial and connective tissue components in varying proportions. Appearance of carcinoma varied from early lesions to tubulo-acinar, cystic papillary and
solid to adeno-squamous types. The sarcomatous pattern comprised of varying degree of malignant connective tissue, mucin, chondroid and osteoid components (Fig.7). In the present study, among 5 carcinosarcoma
cases, inflammation and connective tissue invasion were recorded, EMT was observed in all cases and tumor embolism in 4 cases only.

Other tumor: Malignant Mast cell tumor:
One case of other type tumor included  the malignant mast cell tumor. The mast cells were scattered randomly below the epidermis and dermis. There were also significantly large number of eosinophils intermingled
with tumor cells, particularly in perivascular location. Mast cell tumor was confirmed by observing meta-chromatic granules using toluidine blue stain (Fig.8).

In the present study, the highest incidence of carcinomas was observed, followed by carcinosarcoma and sarcoma,
which is in accordance with previous findings of Ezerskyte et al. (2011) and Baba et al, (2016). On the contrary, Gupta et al. (2012) reported a higher incidence of carcinosarcoma as compared to carcinoma in dogs. In the present study, all malignant tumors had greater tumor angiogenesis, which, in turn, facilitates hematogenous
dissemination of neoplastic cells as reported earlier by Luong et al. (2006).

Grading of carcinomas:
All the 40 malignant CMT were graded into grade I, grade II and grade III as per Goldschmidt et al. 2011. Among 13
simple carcinoma cases, grade II was observed in nine cases, whereas, the remaining four cases were of grade I. Among six solid carcinomas, grade I was observed in one case, grade II was observed in three cases, while grade III was observed in two cases, while in complex carcinoma, grade I, II and grade III were observed in five, four and
one case respectively. All the four cases of fibrosarcoma belonged to Grade II category. Whereas, both squamous cell carcinoma and malignant mast cell tumor were of grade III type. The results of histological grading are depicted in table 4. Most of the CMT cases in the present study fell in grade I and II categories. Grade II and III mammary tumors are usually associated with poor prognosis.

Acknowledgments
The authors are thankful to University Grand Commission (UGC) New Delhi for providing financial support to carry out this research work under the UGC project “C-VPUGC- 12”.

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