Oral malignant melanoma with rhabdoid cell differentiation and myogenic features in a dog

Abstract

Malignant melanoma (MM) is difficult to diagnose because of its pleomorphic morphology and diverse protein profile. We describe a case of canine oral MM that exhibited rhabdoid cell differentiation. The tumor contained two cell types: typical MM cells and aggregated rhabdoid cells. Immunohistochemically, both types of neoplastic cells were positive for vimentin and desmin. Typical MM cells are positive for melanocytic markers; a subset of rhabdoid cells is negative for melanocytic markers and positive for myogenin. Consequently, the tumor was diagnosed as an MM with rhabdoid cell differentiation. This case highlights a potential diagnostic pitfall, as the morphological and immunohistochemical features may closely mimic those of collision tumors and myogenic tumors.

Malignant melanoma (MM) is a tumor derived from melanocytes distributed throughout the body. Oral MM is characterized by a high incidence and marked malignancy in dogs [14, 16]. Histopathologically, canine MM exhibits variable pigmentation and highly diverse cellular morphologies, including epithelioid, spindle, and oval-to-polygonal cells, and rarely contains signet-ring cells [8]. Despite its marked malignancy, oral MM in dogs is often difficult to diagnose because of its highly diverse cytological morphology.

Rhabdoid cells are generally characterized by an abundant eosinophilic cytoplasm with glassy inclusion bodies and eccentrically located nuclei. Reports of tumors with rhabdoid cells in dogs are limited [9, 17, 19], and only one has described MM with rhabdoid features [3]. We recently encountered a case of canine MM containing rhabdoid cells showing desmin expression; its pathological characteristics were described.

A 15-year-old castrated male Toy Poodle presented with a 5 cm × 5 cm × 3 cm nodule around the mandibular canine tooth (Fig. 1a). Part of the mass was ulcerated. Partial mandibulectomy was performed (Fig. 1b). The surgical margins were clean, with approximately 1 cm margin of normal tissue.

Fig. 1.

Macroscopic appearance of the tumor. a) The mass shows an irregular, reddish surface. b) Cut surface of the mass. The nodule measured 5 cm × 5 cm × 3 cm. Bar=1 cm.

No metastasis to lymph nodes or other organs was observed at surgery. All surgical specimens were fixed in 10% neutral-buffered formalin. Paraffin-embedded samples were sectioned at 3-µm thickness and stained with hematoxylin and eosin (HE). Special stains, including periodic acid–Schiff (PAS), Alcian blue (pH 2.5), phosphotungstic acid hematoxylin (PTAH), Masson’s trichrome, and Fontana–Masson, were used to characterize the cytoplasmic features of rhabdoid cells. Immunohistochemistry (IHC) was performed using primary antibodies listed in Table 1. A peroxidase-labeled secondary antibody (Histofine Simple Stain MAX-PO (Multi); Nichirei Biosciences Inc., Tokyo, Japan) was used. Color was developed with ImmPACT^® NovaRED and sections were counterstained with Mayer’s hematoxylin. The immunohistochemical staining results were semi-quantitatively graded as follows: −, 0% positive cells; ±, 1–5% positive cells; +, 6–25% positive cells; ++, 26–75% positive cells; and +++, 76–100% positive cells.

Table 1. Primary antibodies and immunostaining protocols.

Antibody	Clone	Dilution	Supplier	Antigen retrieval
Vimentin	V9	1:25	Dako (Glostrup, Denmark)	MW, pH 6.0, 90°C, 10 min
Desmin	D33	Prediluted	Dako	MW, pH 6.0, 90°C, 10 min
S-100	Polyclonal	1:100	Dako	AC, pH 6.0, 121°C, 20 min
α-SMA	1A4	1:100	Dako	No treatment
Myogenin	F5D	1:50	Santa Cruz (Dallas, TX, USA)	AC, pH 6.0, 121°C, 20 min
MelanA	A103	1:50	Dako	MW, pH 9.0, 95°C, 20 min
PNL2	PNL2	1:100	Abcam (Cambridge, UK)	AC, pH 6.0, 121°C, 20 min
SOX10	EPR4007-104	1:500	Abcam	AC, pH 9.0, 121°C, 20 min
Ki-67	MIB-1	1:100	Dako	AC, pH 9.0, 121°C, 20 min

MW=Microwave, AC=Autoclave, pH 6.0 =citrate buffer (pH 6.0), pH 9.0 =Tris-EDTA buffer (pH 9.0).

Histologically, the tumor consisted of the following two major components: a predominant area showing typical features of MM with epithelioid to spindle-shaped cells, and a minor rhabdoid cell area. Local invasiveness was not observed in any part of the lesion.

In a typical MM area, a large portion of the tumor was poorly demarcated and exhibited solid proliferation with sparse fibrous stroma, forming fascicular or sheet-like growth patterns (Fig. 2a). It comprised spindle-shaped and polygonal cells with eosinophilic cytoplasm, vesicular nuclei, and prominent nucleoli. Some neoplastic cells also contained brown pigments, which were stained black with Fontana–Masson. Mitotic activity was observed in 41 mitoses per 10 high-power fields (HPFs).

Fig. 2.

Histological features of canine oral malignant melanoma (MM) with rhabdoid cell differentiation. a) Typical MM area. The tumor exhibited a sheet-like growth pattern, and neoplastic cells comprised polygonal morphology with eosinophilic cytoplasm, vesicular nuclei, and prominent nucleoli. A part of neoplastic cells had melanin pigments (inset). b) Rhabdoid cell area. The area mainly comprised rhabdoid cells (inset) and occasionally contained pleomorphic neoplastic cells. Rhabdoid cells had an eosinophilic cytoplasm with a glassy inclusion body and an eccentric nucleus (arrowheads). Hematoxylin–eosin (HE). Bar=50 μm.

In contrast, the rhabdoid cell area occupied only a small portion of the tumor (Fig. 2b). This area primarily comprised rhabdoid neoplastic cells, characterized by abundant eosinophilic cytoplasm containing glassy inclusion bodies and eccentrically located nuclei. The rhabdoid cells were aggregated, and mitotic activity was 30 mitoses per 10 HPFs. These two areas were relatively well demarcated but showed intermixed components in portions of the interface (Fig. 3). The staining results showed that the cytoplasm of rhabdoid cells was negative for PAS, Alcian blue, PTAH, and Masson’s trichrome. In contrast to typical MM cells, the rhabdoid cells were negative for Fontana–Masson.

Fig. 3.

Histological features of an intermixed component composed of typical malignant melanoma (MM) cells and rhabdoid cells. Typical MM cells (white arrowheads) and rhabdoid cells (black arrowheads) were present in the same area. Hematoxylin–eosin (HE). Bar=50 μm.

The results of the immunohistochemical analysis are summarized in Table 2. Typical MM cells were positive for vimentin (score: +++), desmin (score: ++, Fig. 4a), Melan A (score: +, Fig. 4b), PNL2 (score: ++, Fig. 4c), and SOX10 (score: +++, Fig. 4d), but negative for cytokeratin AE1/AE3, S-100, α-smooth muscle actin (α-SMA), and myogenin. In contrast, rhabdoid cells were positive for vimentin (score: +++), desmin (score: +++, Fig. 5a), and myogenin with a small subset (score: ±, Fig. 5b), but negative for Melan A, PNL2, SOX10, S-100, and α-SMA. Notably, in rhabdoid cells, the positively stained cytoplasmic areas for the intermediate filaments vimentin and desmin corresponded to the inclusion bodies observed on HE staining. Furthermore, the immunohistochemical profile of each cell type in the intermixed components was similar to those described above. Both areas showed a high Ki-67 labeling index in the neoplastic cells, as summarized in Table 2.

Table 2. Results of immunostaining of typical melanoma area and rhabdoid cell area.

Tumor areas	Vimentin	Desmin	S-100	α-SMA	Myogenin	MelanA	PNL2	SOX10	Ki-67b)
Typical melanoma area	+++	++	-	-	-	+	++	+++	49.7%
Rhabdoid cell area	+++	+++	-	-	±	-	-	-	65.3%

a) Score: -, 0% immunopositive cells; ±, 1–5% immunopositive cells; +, 6–25% immunopositive cells; ++, 26–75% immunopositive cells; +++, 76–100% immunopositive cells. b) positive rate for Ki-67.

Fig. 4.

Immunohistochemical staining of a typical malignant melanoma (MM) area. a) Neoplastic cells were partly positive for desmin. b) Neoplastic cells were partly positive for Melan A. c) Neoplastic cells were partly positive for PNL2. d) Nuclei of neoplastic cells were positive for SOX10. Bar=20 μm.

Fig. 5.

Immunohistochemical staining of the rhabdoid cell area. a) Neoplastic cells were positive for desmin. b) Nuclei of the neoplastic cells were focally positive for myogenin (arrowheads). The cells contained one or two centrally located nuclei. Bar=20 μm.

The patient was diagnosed with MM with rhabdoid cell differentiation and myogenic features. Most neoplastic cells exhibited characteristic features of MM, including melanin pigments, a nesting growth pattern, and pleomorphic cellular morphology. Typical MM cells were diffusely positive for melanocyte markers. This tumor was diagnosed as a malignant neoplasm because both typical MM cells and rhabdoid cells showed high mitotic activity. Differentiation from a collision tumor was challenging because a subset of these two cell types existed as independent components within the tumor. However, as shown in Fig. 3, part of the tumor exhibited intermixed components of the two cell populations and shared immunohistochemical characteristics of typical MM and rhabdoid cells, indicating a common origin rather than a collision tumor.

This case showed exceptionally rare features: rhabdoid cells characterized by abundant eosinophilic cytoplasm with glassy inclusion bodies on HE staining. In humans, rhabdoid cells are often observed in tumors, such as atypical teratoid/rhabdoid tumors [11], malignant rhabdoid tumors of the kidney [10], and soft tissue tumors [12]. In contrast to those in humans, reports on canine tumors containing rhabdoid cells are limited to different tumor types, including meningiomas [19], soft tissue sarcomas [17], and cutaneous extrarenal rhabdoid tumors [9]. In our case, IHC results revealed that the inclusion bodies comprised intermediate filaments, specifically vimentin and desmin. This immunohistochemical feature is consistent with previous reports of MM with rhabdoid cells in humans [2, 4,5,6,7, 18] and MM with signet ring cells in dogs [3, 8]. In a previous report of MM in dogs [3], neoplastic cells with rhabdoid features were described as “signet-ring cells.” However, we used the term “rhabdoid cells” because the cells in this case fulfilled the established morphologic criteria for rhabdoid morphology [9, 17, 19].

In our case, the rhabdoid cells and a subset of typical MM cells were positive for desmin, a myogenic marker, which is rare in canine MM. Only one report [15] has described a case of cutaneous melanoma exhibiting muscle differentiation. In humans, five cases of melanoma with rhabdoid cell differentiation and desmin expression have been reported [2, 6, 7, 18], and one case was negative for all melanocytic markers [2], similar to the rhabdoid cells in our case. These findings suggest that neoplastic cells exhibit marked dedifferentiation and acquire myocyte-like features. This phenomenon may be related to the neural crest origin of melanocytes, which retains its capacity for mesenchymal differentiation, including myogenic differentiation [1]. These unconventional features resembling myogenic differentiation complicate the differential diagnosis of MM from myogenic tumors and collision tumors.

Regarding the prognosis, Prieto-Torres et al. [13] reported that MM with rhabdoid cell differentiation may be associated with a poor prognosis in humans. However, only a few cases of MM with signet ring cells have been reported in dogs [3], so its prognosis remains unclear. Notably, the patient remained in a clinically good condition for at least 8 months after surgery, without evidence of tumor recurrence or metastasis. These findings suggested that rhabdoid cell differentiation does not necessarily predict an aggressive clinical course in dogs.

This case report has some limitations. Electron microscopy was performed using formalin-fixed tissue; however, ultrastructural details could not be evaluated because the tissues were formalin-fixed and showed a marked structural collapse. Molecular analysis was not conducted, and as this was a single case; conclusions about the prognosis or biological behavior remain limited.

This is the first reported case of canine malignant melanoma with rhabdoid cell differentiation and desmin expression, in which a large proportion of both typical cell and rhabdoid cell components were clearly demarcated, making distinction from a collision tumor difficult. We strongly recommend performing a panel of melanocytic marker staining when encountering oral tumors containing desmin-positive rhabdoid cells. These tumors may represent variants of MM and should be distinguished from muscle tumors. To clarify whether these findings are associated with a poor prognosis, as reported in humans, further case accumulation and analysis are required.

Conflicts of Interest

The authors declare no potential conflicts of interest with respect to the research, authorship, or publication of this article.