Mammary gland tumor (MGT) is the most common tumor in intact female dogs (24). Canine MGT is highly heterogeneous in its histological types and biological behaviors (24). Around 50% of canine MGTs show malignant behavior, and the most frequent is simple carcinoma (15,28). Mammary comedocarcinoma is a histologic subtype of carcinoma characterized by a central necrotic area surrounded by neoplastic luminal epithelial cells within the ducts involved (7). Among canine MGTs, mammary comedocarcinoma is considered to be rare, accounting for 1.5% and up to 8.4% (7,21,28). Canine mammary comedocarcinoma is invariably associated with poor prognosis (3,7,10,20). Emerging studies have recognized the prognostic value of canine MGT-infiltrating lymphocytes, including CD4⁺ and Tregs (4,5,9). Mammary comedocarcinoma contains immune infiltrates in dogs (10,17,19). Herein, we report clinical, clinicopathological, histological, and immunophenotypic features of mammary comedocarcinoma in a dog, providing immunophenotypic evidence of an immune suppressive microenvironment.

A 15-year-old, intact female, Great Pyrenees dog was referred to the Veterinary Medical Teaching Hospital at Jeju National University for evaluation of mammary masses. During history evaluation, it was discovered that mammary masses had grown rapidly within a month. Physical examination found mammary masses affecting the right 3^rd to 5^th mammary glands. The largest mass size was 5.4 × 5.7 × 5 cm (Fig. 1). The tumor was solid, and the margins were indistinct with adhesion to the surrounding tissues. There was no peripheral lymphadenopathy. Complete blood count, serum chemistry, blood gas analysis, and coagulation parameters were within normal limits, except for mild hypophosphatemia (2.2 mg/dL; reference interval (RI), 2.5-6.8 mg/dL) and increased alkaline phosphatase activity (473 U/L; RI, 23-212 U/L). Thoracic and abdominal radiographs were within normal limits (Supplementary Data 1).

Figure 1. Gross morphology of the mammary masses in a dog. The mammary masses are solid, movable, and attached to the underlying tissue. The masses are palpated as a single lesion that affects the right 3^rd to 5^th mammary glands.

Fine needle aspiration (FNA) was performed on the 4^th and 5^th mammary masses. Microscopic evaluation of fine needle aspirates revealed a moderate cellularity of two epithelial cell types, which were often intermingled in clusters. One was characterized by a dense aggregate of epithelial cells with a scant rim of lightly stained cytoplasm that often appeared invisible (Fig. 2A). The other was characterized by large round to polygonal cells sporadically filled with faintly eosinophilic globules in the abundant basophilic cytoplasm and an eccentric meniscus-like nucleus (signet ring cells) (Fig. 2B, C, asterisk). The intracellular globules morphologically resembled Melamed-Wolinska (MW) bodies in that they appeared as one to several, round to oval, hyaline-like, red to pink cytoplasmic inclusions of varying sizes. The nucleus contained dense, granular to homogenous chromatin with no prominent nucleoli. In the background, there were numerous small lymphocytes, including plasma cells (Fig. 2D). Necrotic debris and cholesterol crystals were often found. Considering rapid growth, large mass size, and cellular atypia, the cytologic diagnosis was a malignant mammary tumor, favoring adenocarcinoma. Regional mastectomy was performed on the masses, which were subject to histologic examination. Representative mammary masses were fixed in 10% neutral-buffered formalin, processed, and embedded in paraffin wax. Three-micrometer sections were prepared and stained with hematoxylin and eosin (H&E). At low magnification, the tumor showed a multilayered to solid carcinomatous area intermingled with an intense desmoplastic stroma (Fig. 3A). The carcinoma appeared as distended ductal structures lined by a multilayered epithelium, often lacking lumina, and with surrounding necrotic debris in a comedo pattern (Fig. 3B). Approximately, 15 mitotic figures were observed per ten fields at 400× magnification. Invasion of neoplastic cells into lymphatic vessels and metastasis to regional lymph nodes were observed (Fig. 3C). Multifocal areas of immune infiltrates were also observed, exhibiting a concentric pattern and distributed closely around the vessels within the tumoral stroma (Fig. 3D). The histopathologic diagnosis was high-grade (grade III) mammary comedocarcinoma with nodal metastasis.

Figure 2. Fine needle aspiration of mammary comedocarcinoma in a dog. (A) Dense aggregates of neoplastic cells with minimal cytoplasm are present. Neoplastic cells have mild anisocytosis and anisokaryosis. (B) Large globoid cells with finely granular eosinophilic cytoplasm are intermingled with neoplastic cells observed in panel A. (C) An intracytoplasmic finely eosinophilic globule (asterisk) compresses the nucleus to one side of the cell. A lymphocyte (arrow) and an eosinophil (arrowhead) are present next to neoplastic cells. (D) Numerous small lymphocytes and plasma cells (arrow) are found. (E) Minimal cellular and nuclear atypia are present in mammary comedocarcinoma. Note that globoid cells contain intracytoplasmic structures filled with homogeneous or stippled eosinophilic material (arrow in insert). (F) Fine needle aspirates of the axillary lymph node. Note the presence of epithelial clusters but no lymphocytes in the background, suggesting lymph node parenchymal effacement. H&E stain, A, F: 200×, B: 200×, enlarged, C: 1,000×, D, E: 400×, insert: 1,000×, enlarged, Scale bars = 50 μm (A, B, F), 10 μm (C, insert), and 20 μm (D, E).

Figure 3. Histopathology of mammary comedocarcinoma in a dog. (A, B) Comedo pattern characterized by central necrosis surrounded by multilayered to solid carcinomatous is present. (C) Neoplastic cells are disseminated into lymphatic vessels, leading to structural destruction of the lymph node. Asterisks indicate a nest of neoplastic cells. (D) Note the perivascular immune cell infiltration. Asterisks indicate blood vessels. H&E stain, A: 40×, B, C: 100×, D: 400×, Scale bars = 500 μm (A), 200 μm (B, C), 50 μm (D).

In canine mammary carcinomas, the perivascular lymphocytic pattern was unusual (4) and has been associated with histologically aggressive subtypes, immunosuppressive environment, and poor prognosis (6,9). However, no clinical relevance is available in canine mammary comedocarcinoma. To characterize the immune infiltrates, we performed immunophenotyping using immune histochemistry (IHC) and flow cytometry. IHC was performed in the serial H&E-stained sections. After hydration and heat-induced antigen retrieval, endogenous peroxidase was depleted by incubating slides in 0.3% hydrogen peroxide for 1 hour. Sections were incubated at 4°C overnight with anti-CD3 and PAX5 antibodies (Table 1), and then incubated with horseradish peroxidase-conjugated secondary antibody for 1 hour at room temperature. IHC revealed a prominent perivascular infiltration of CD3⁺ lymphocytes (Fig. 4A, B) and to a lesser extent PAX5⁺ B lymphocytes (Fig. 4C, D). These lymphocytes were distributed uniformly within the immune infiltrates. Using antibodies and reagents (Table 1), peripheral blood mononuclear cells (PBMCs) and tumor-infiltrating leukocytes (TILs) were subject to paired flow cytometric analysis (Fig. 5). The TILs were prepared from the 4^th and 5^th mammary masses using the protocol, as previously described (12). The gating strategy using PBMCs is present in Fig. 5A, B. Flow cytometry revealed that a majority of tumor-infiltrating CD45⁺ leukocytes were of T cell origin with a predominance of CD4⁺ (34%) and CD8⁺ (19%) T cells (Fig. 5C). CD11b⁺ myeloid and CD21⁺ B cells were also recruited into the tumor. Of note, there was a preferential infiltration of tumor-infiltrating CD25⁺FOXP3⁺ Tregs (3.9-fold increase in the total number) and CD21⁺ B cells (14-fold increase in the total number) when compared to blood. Meanwhile, the proportion of the circulating immune subsets in healthy dogs is present in Supplementary Data 2.

Figure 4. Immunophenotyping of mammary comedocarcinoma in a dog. Immunohistochemistry. Inflammatory T and B cells display strong membranous to cytoplasmic immunostaining of CD3 (A, B) and PAX5 (C, D), respectively. (A) CD3⁺ T cells show diffuse infiltrative pattern within neoplasm. (B) Enlarged section from panel A. T lymphocytes are heavily infiltrated into the supporting stroma adjacent to neoplastic cells. (C) PAX5⁺ B cells are moderately scattered in the inflammatory infiltrates adjacent to the neoplasm. (D) Enlarged section from panel C. IHC stain, Mayer’s hematoxylin counterstain. A, C: 100×, B, D: 400×, Scale bars = 200 μm (A, C), 50 μm (B, D).

Figure 5. Immunophenotyping of mammary comedocarcinoma in a dog. Flow cytometry. Forward and side scatter density plots are used to exclude potential doublets. A viability dye is used to remove debris and dead cells. Following the selection of live singlets, gating strategies of circulating CD45⁺CD3⁺CD4⁺CD25⁺FOXP3⁺ Tregs (A), CD8⁺ T, CD11b⁺myeloid, and CD21⁺ B cells (B) are present. (C) Tumor-infiltrating immune subsets are present. Figures refer to the percentage of immune cells present in the quadrant or circle. (D) Paired analysis of circulating and tumor-infiltrating immune subsets indicated. Note the preferential infiltration of tumor-infiltrating regulatory T and B cells compared to circulating ones.

Table 1 . Antibodies and reagents used in this study.

Antibody/reagent	Clone	Vendor	Dye	Dilution
CD45	YKIX716.13	Invitrogen	PE	1:100
CD45	YKIX716.13	Novus Biologicals	AF700	1:100
CD3	CA17.2A12	Bio-Rad Laboratories	FITC	1:100
CD4	YKIX302.9	Bio-Rad Laboratories	AF647	1:100
CD8	YCATE55.9	Invitrogen	PE	1:100
CD21	CA2.1D6	Bio-Rad Laboratories	AF488	1:100
CD25	P4A10	Invitrogen	PE-Cy7	1:100
FOXP3	FJK-16s	Invitrogen	PerCP-Cy5.5	1:50
CD11b	M1/70	BioLegend	PE-Dazzle 594	1:100
CD3ɛ	LN10	Biocare	Not applicable	1:200
PAX5	DAK-Pax5	BD Biosciences	Not applicable	1:50
Fc receptor binding inhibitor	Not applicable	Invitrogen	Not applicable	1:100
Fixable viability dye	Not applicable	eBioscience	EF780	1:8,000

The patient was discharged, but after 2 months, new masses developed affecting the right 1^st to 2^nd mammary glands. The right axillary lymph node was enlarged. FNA was performed on the mammary masses and enlarged lymph node. The cytological features of epithelial cells were highly consistent with the initial cytologic findings, including large signet ring cells with faintly eosinophilic globules or MW-like bodies (Fig. 2E, arrow). The diagnosis of metastatic carcinoma to the lymph node was cytologically made (Fig. 2F). Chemotherapy was strongly recommended, but it could not be considered because the dog could not be brought to the veterinary hospital due to gait difficulties and intolerance of travel. The patient died after 127 days of the first presentation due to a deteriorating condition. Necropsy was not performed at the request of the owner.

Mammary comedocarcinoma is a unique and rare entity of mammary carcinoma (7,21). The comedo type of mammary carcinoma is histologically characterized by necrotic debris surrounded by a few layers of highly atypical epithelial cells (4,7). The involvement of a high histologic grade and highly proliferative neoplastic cells are postulated to be involved in the pathogenesis of underlying comedonecrosis (1,2,25). In dogs, mammary comedocarcinoma is invariably associated with a poor prognosis due to aggressive biological phenotypes characterized by a high histologic grade, invasiveness, nodal metastasis, and chemoresistance (7,10,13). This is consistent with our case, which showed recurrence, rapid deterioration of the condition, and death.

In our cytological findings, intracytoplasmic inclusions were very similar to MW bodies. Interestingly, a similar feature was reported in a simian case of mammary comedocarcinoma (23). MW bodies have often been observed in canine urothelial cell carcinomas associated with degenerative changes (15). Because there was no bladder mass in our case, we postulate that the MW-like bodies might represent a degenerative neoplastic alteration related to mammary comedocarcinoma. Given that there are no known cases of MW-like bodies in canine MGT, our case may highlight the importance of clinical awareness of these inclusions in canine mammary carcinomas.

Canine mammary comedocarcinoma, compatible with mammary carcinomas (6), induces immune infiltrates that mainly consist of lymphocytes and plasma cells (10,19). Our cytomorphologic and immunophenotypic investigation supports these findings. Of note, the infiltrative pattern was perivascular, an unusual pattern in mammary carcinomas (4), associated more with high histologic grades than other subtypes (6,9). The perivascular lymphocytic infiltration has been suggested to favor an immunosuppressive environment and negative prognosis in canine mammary carcinomas (6). By leveraging flow cytometric analysis of paired PBMC and TIL samples (12,26), we further demonstrated key immune subsets that could represent the functional immunogenicity of mammary comedocarcinoma, providing clinical relevance to immune infiltrates. For instance, compared to the periphery, we found a preferential infiltration of CD21⁺ B and CD4⁺CD25⁺FOXP3⁺ Tregs in the perivascular tumor niche. It should be noted that normal mammary tissues have absent or minimal Treg infiltration in non-tumor-bearing dogs (14,18). At the blood level, we also confirmed no changes in the proportion of circulating Tregs between our case and the healthy control group, consolidating our notion – the preferential Treg infiltration into the comedocarcinoma. In dogs, mature B cells are shown to preferentially infiltrate into invading areas or tertiary lymphoid tissue of canine mammary carcinomas (6,22). Tumor-infiltrating CD20⁺ B cells are associated with high-risk features of ductal carcinomas, including comedonecrosis (27). The expansion of CD4⁺ T cells is significantly associated with a poor prognosis in canine MGT (5), which could be further elaborated by tumor-infiltrating Tregs (TI-Tregs) due to their strong inhibition of anti-tumor immunity (8). Intriguingly, a study suggested that comedonecrosis in breast ductal comedocarcinoma correlates with stromal infiltration of immune-suppressive Tregs and exhausted PD1⁺ T cells during cancer progression (27). Likewise, TI-Tregs are significantly associated with necrosis in canine mammary carcinomas (8). While the clinical relevance of comedonecrosis among carcinomas needs further investigation, it may be related to the de novo generation of neoantigens derived from tumor necrosis (27). Therefore, our case strongly suggests that canine comedocarcinoma is highly immunogenic and shapes an immune-suppressive tumor microenvironment. Currently, no studies have investigated and characterized TILs with a specific focus on comedocarcinoma compared to other subtypes of mammary carcinomas in dogs. Thus, future studies will be of interest to investigate not only whether mammary comedocarcinoma has greater Treg infiltration than other types of mammary carcinomas in dogs, as demonstrated in humans (11,27), but also how comedonecrosis-induced Treg infiltration and ensuing immune suppression are associated with the biologically aggressive phenotype. Meanwhile, in our case, T cells were not distributed predominantly in the outermost areas in the immune infiltrates, nor did lymphocytes exhibit antigen-dependent polarity in histopathologic examination. Thus, it is unlikely that immune infiltrates are tertiary lymphoid structures, although the immune cell infiltrative pattern is highly perivascular and reflects an adaptive immune response (6). The migration of tumoral stroma-infiltrating CD8⁺ T cells to the neoplastic epithelial component correlates with the spontaneous remission of breast comedocarcinoma (16). Prospective studies are necessary to examine spatial and functional changes in tumor-infiltrating immune subsets in canine carcinomas, particularly the comedo type.

We report a case of a dog with mammary comedocarcinoma, providing valuable clinical, clinicopathological, histological, and immunophenotypic characteristics that can define this rare type of canine MGT. Our case provides a rationale for basic research that could map the immune landscape of mammary comedocarcinoma and identify key immune subsets for cancer progression.

The authors express sincere appreciation to all the pathologist, including Ha-Young Lim, from Green vet Laboratories involved with the pathology and immunohistochemistry reports used in this study. We thank Jin-Hyeon Kim (JNBIO Co., Ltd.), Jaewook Shin (BMIKOREA Co., Ltd.), and Ju-Hun Kim (BioApplications Inc.) for their academic and technical consultation on this study. This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (MOE) (#RS-2023-00241779). This research was also supported by Regional Innovation Strategy (RIS) through the NRF funded by MOE (#2023RIS-009).

The authors have no conflicting interests.