Cutaneous Mast Cell Tumor (Mastocytoma): Cyto- Histopathological and Haematological Investigations

Hosseini et al. Diagnostic Pathology 2014, 9:9 http://www.diagnosticpathology.org/content/9/1/9

CASE REPORT Open Access Cutaneous mast cell tumor (Mastocytoma): Cyto- histopathological and haematological investigations Ehsan Hosseini1, Behnam Pedram2, Ali Mohammad Bahrami1*, Mohammad Hossein Jaberi Moghaddam3, Javad Javanbakht4, Fatemeh Emami Ghomi5, Najme Jaberi Moghaddam6, Mobin Koohestani3 and Radmehr Shafiee7

Abstract Cutaneous mast cell tumours (MCTs) are the most common skin tumours in dogs. Due to the prevalence of canine MCTs and the variable biologic behavior of this disease, accurate prognostication and a thorough understanding of MCT biology are critical for the treatment of this disease. A cytologic diagnosis of mast cell tumor with evidence of prior hemorrhage was made, and the masses were surgically removed. Cytological evaluation of fine-needle aspirates from the cutaneous mass from the axillary comprised many well-differentiated, highly granulated mast cells with moderate numbers of eosinophils. Nuclei were varied in size and shape with high nuclear’to’cytoplasmic ratio, prominent nucleoli, marked atypical and mitotic figures. Microscopically, mass consisted of sheets of neoplastic round cells that formed nonencapsulated nodules in the dermis and infiltrated into the adjacent dermal collagen, and also there was diffuse subcutis invasion of round to pleomorphic tumor cells. Tumor cells had moderate to abundant cytoplasm, round to ovoid nuclei with scattered chromatin, and mitotic figures. In this tumor, cytoplasmic granules showed atypical metachromasia. In addition, eosinophils were scattered among the mast cells at the periphery of the nodules. The presence of eosinophils and the observation, at high magnification, of cells with cytoplasmic metachromatic granules. Invasion of the deep subcutaneous fat or cutaneous muscles were a common feature of grade III tumour. Finally, a diagnosis of grade III cutaneous mast cell tumor was made. Virtual slides: The virtual slide(s) of this article can be found here: http://www.diagnosticpathology.diagnomx.eu/ vs/4755249151157024. Keywords: Cytology, Pathology, Dog, Mast cell tumor, Grading

Background malignant, but their true metastatic potential is not en- Mast cell tumors (MCTs) are highly invasive and meta- tirely known [6]. static and are the most frequent round cell tumors in Round cell tumors consist of discrete cells that are dogs, comprising 16–21% of all cutaneous tumors diag- round to oval rather than fusiform. Included in this nosed [1,2], and are the most common skin tumors in group are mast cell tumor, histiocytoma, lymphosarcoma dogs [3], representing about 7% to 21% of all dog skin (including reticulum cell sarcoma) and transmissible tumors and 11% to 27% of malignant skin tumors in this venereal tumor. The differential diagnosis of round cell species [4]. Canine MCTs have variable biologic behaviors, tumors by histologic examination without concomitant ranging from solitary benign masses that can be cured cytologic characterization may, in some instances, de- with surgery alone to systemic and potentially fatal meta- pend more on age of animal, growth rate, location of static disease [5], and are always considered potentially tumor, number of tumors and lymph node involvement rather than histologic criteria. Mastocytosis is a rare group of diseases characterized by abnormal prolifera- * Correspondence: [email protected] tion and infiltration of mast cells within the tissues. The 1Faculty of Para Veterinary Medicine, Ilam University, Ilam, Iran Full list of author information is available at the end of the article skin is most frequently involved but mast cells also

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accumulate in the bone marrow, gastrointestinal tract, lymph reaction. Invasiveness was assessed as follows: non- nodes, spleen, and liver. Mast cell tumors in association with invasive (tumours confined to the superficial dermis and oncocytes thus should be considered supportive evidence of interfollicular spaces); moderately invasive (tumours an FNA diagnosis of adenolymphoma. Mastocytosis should with lower dermal and limited subcutaneous tissue inva- be considered in the differential diagnosis of urticaria and sion); highly invasive (tumours with massive infiltration solitary mastocytomas may remain active into adoles- of subcutaneous and deep tissue). Mitotic activity was cence, raising concern for systemic progression [7-13]. assessed on toluidine blue-stained sections, five high MCTs are most often graded histologically by the scheme power fields (hpfs) being evaluated in each case with a × described by Patnaik et al. [14]. Tumours of grade I are cir- 40 objective. The data were expressed as mean number cumscribed, mainly dermal in location, and consist of well- of mitoses per hpf (mitotic index) and tumours were differentiated mast cells with prominent metachromatic classified according to the following grading system: 0 cytoplasmic granules. Poor cellular differentiation, ag- (mitotic figures absent); 1 (mitotic index ranging from 0 gressive growth pattern, moderate to high mitotic activ- to 2 mitoses/hpf); 2 (mitotic index > 2/hpf). Invasiveness ity, mitotic atypia and sparse to absent cytoplasmic and mitotic activity assessment were used to define the granulation are the major features of grade III tumours. histological grade, and also as individually evaluated Grade II MCTs constitute an intermediary form be- parameters. tween the two other grades, but they tend to infiltrate more deeply than grade I neoplasms [14,15]. MCTs are Hematological findings routinely diagnosed by cytology and histopathology. A complete blood work was performed. No changes were noticed in the erythrogram, however, leukogram Case presentation changes included leukocytosis (55,245 leukocytes/μL), Animal ethics neutrophilia (27,644 neutrophils/μL) with mild regenera- The animal was placed in shade, in standard conditions, tive left shift (4,376 band cells/μL). water ad libitum, and without restriction of movement according to the guidelines of Institutional Animal Ethical Cytological findings Committee of the Tehran University of Animal Science, Cytological examination indicated mast cells with nu- Iran. Surgery was performed under aseptic conditions and merous metachromatic stained granules. Nuclei were sedation by injection of Xylazine hydrochloride (0.05 varied in size and shape with high nuclear’to’cytoplasmic mg/kg) followed by 2% Lignocaine hydrochloride. ratio, prominent nucleoli, marked atypical and mitotic figures. The background was filled with granules from Clinical case reports ruptured cells and a moderate eosinophils were also In October 2013, a 5-year-old, 7 kg, female black terrier present (Figure 2A and 2B). dog was presented to the clinic at the college of Veterinary Medicine, university of Tehran, with a growing mass Histopathological findings around axillary and mobility problems related to the mass, Histopathologically, MCT tumour cells were less well and euthanasia was applied upon the owner’s request and circumscribedbyconnectivetissueandneoplastic also taking into consideration the age of the animal and cells often exhibited an aggressive behavior, high cel- the size of the mass. The mass spreading into subcutis is lularity, cellular pleomorphism, and various morpho- of proximal right front leg, was of 2.8 × 2.5 cm size and logic patterns. 185 g weight (Figure 1). The mass was surgically resected However, in some regions of the tumor tissue shows and an impression smear of the tissue was prepared and neoplastic cells extending into the subcutis. Neoplastic stained with Giemsa. The rest of the tissue was subjected cells had distinct cell borders and widely variable amounts to histopathologic evaluation. of pale to brightly eosinophilic cytoplasm. Furthermore, One formalin-fixed, paraffin wax-embedded section of tumour had neoplastic cells containing numerous fine eo- each neoplasm was stained with haematoxylin and eosin sinophilic to basophilic cytoplasmic granules consistent (HE). Tumour was classified independently by two pa- with mast cell granules and also highly pleomorphic thologists in order to confirm the diagnosis according to tumour cells with a high mitotic index, anisokaryosis and the WHO criteria [16]. Also tumour was graded accord- infiltration of the surrounding dermis and subcutis were ing to the criteria proposed by Patnaik et al. [14] and observed (Figure 2C and 2D). Kiupel et al. [17], as well, moderately or poorly differentiated (grades I, II or III, respectively) [14,17]. These cri- Pathology grading of cutaneous mast cell tumor teria included the following histomorphological features: In this case is characterized by one or more of the fol- extent of tumour (invasiveness), cellular morphology, lowing criteria: at least 3 mitotic figures in 5 hpf, at least size of cytoplasmic granules, mitotic activity and stromal 2 multinucleated (2 or more nuclei) cells in 5 hpf, at Hosseini et al. Diagnostic Pathology 2014, 9:9 Page 3 of 5 http://www.diagnosticpathology.org/content/9/1/9

Figure 1 Cytohistophatology of dermal mass from the axillary of the dog. Representative photomicrographs of mass cell tumuor of Patnaik grade. (A and B) grade III. High-grade mast cell tumour was characterized by highly pleomorphic tumour cells with a high mitotic index, anisokaryosis and infiltration of the surrounding dermis and subcutis. Haematoxylin and eosin stain. Bar, 50 μm. the smears show n were prepared at the initial presentation and stained with Wrigth-Giemsa solution. Histopathologically, The presence of eosinophils and the observation, at high magnification, of cells with cytoplasmic metachromatic granules and also, neoplastic cells often exhibited an aggressive behavior, high cellularity, cellular pleomorphism, and various morphologic patterns. Eosinophils are present at the periphery of the mass (Figure 1C and D, H&E, 400x). least 2 bizarre nuclei (highly atypical with marked indenta- the majority of cases, but forming an accurate prognosis is tions, segmentation, and irregular shape) in 5 hpf; karyo- more challenging [5]. Prognostic factors of significance in- megaly (ie, nuclear diameters of at least 10% of neoplastic cluded grading (cytology and histopathology), staging (re- mast cells vary by at least 2-fold). The selected field was it gional and distant metastases), breed, tumour localisation that was most highly mitotically active or had the highest and treatment (surgery, radiation and chemotherapy). degree of anisokaryosis. Cytological examination after fine needle aspiration is Canine cutaneous MCTs were graded according to the useful in establishing the diagnosis but histopathology is Patnaik and the Kiupel systems [14,17]. Grade III MCT needed for grading [18]. Cytology often is helpful in the was poorly demarcated accumulations of poorly differen- diagnosis of MCTs because of the characteristic appear- tiated mast cells with severe nuclear pleomorphism. In- ance of mast cells with routine staining. As is the case vasion of the deep subcutaneous fat or cutaneous with findings from other species, Wright–Giemsa stain muscles were a common feature of grade III tumour. Fi- resulted in more intensely stained granules in the neo- nally, a diagnosis of grade III cutaneous mast cell tumor plastic mast cells. The mechanism of the differences in was made. staining is unclear. Several subtypes of mast cells have been identified in humans and dog based primarily on Discussion granule contents and biological function. Dogs are at risk for cutaneous MCT, which accounts for This study clearly showed a cellular infiltrate of mast up to 21% of all skin tumours [3]. The diagnosis of cells, mononuclear cells and eosinophils in the thick- MCT by cytology or histopathology is straight forward in ened, hyperplastic, and hyperkeratinized epidermis. Mast Hosseini et al. Diagnostic Pathology 2014, 9:9 Page 4 of 5 http://www.diagnosticpathology.org/content/9/1/9

presence of multiple large nodules, the extension into the dermis, and the poorly differentiated mast cells could be comparable to grade III described for dogs. Re- cently, a new 2-tier histologic classification for canine cutaneous tumors has been proposed [22]. This grading system is based on the presence of mitotic figures, multinucleated cells, bizarre nuclei, and karyomegaly to predict biological behavior, likely time to metastasis, new tumor development, and survival time. In the present case, although the neoplasia did not show any of these histological parameters, the presence of multiple cutaneous tumors suggests a malignant behavior. Grade III tumours have a very poor prognosis even with treatment, which might discourage owners from extensive surgery. This may explain the discrepancy in surgery performed in MCT cases of different grades. This result agrees with the findings by Murphy et al. [23] that showed dogs with Figure 2 Macroscopical examination of a cutaneous cell tumor in a black terrier. The mass spreading into subcutis is of proximal poorly differentiated tumours had a significantly shorter right front leg. survival time. These results suggest that all multiple cutaneous MCTs should be treated as individual neoplasms and prognosis should be dependent on individual vari- cells and eosinophils, as opposed to the mononuclear ables such as grade and not related to the number of tu- cells of delayed hypersensitivity, predominated, suggest- mours present, which was shown not to affect survival. ing an immediate hypersensitivity reaction. Once acti- Mitotic index and size were not found to be independent vated, mast cells at the edge of a wound are known to predictors of survival. release inflammatory mediators within injured tissue by degranulation [19]. Thus, it would seem that immediate Conclusions hypersensitivity reactions may be responsible for the In conclusion, this study described cytohistopathology fea- development of skin lesions due to tumor in dogs. How- tures of canine cutaneous MCTs of the skin region. The ever, the present results suggest that the intense prolifera- histological features of these tumours would suggest that tion of mast cells following tumor occurs predominantly most should be classified as high-grade MCTs. A cutane- in the skin. ous MCT may be diagnosed on the basis of observing the The results of this study suggest that cellular prolifera- histopathological pattern described in present study. tion plays a significant role in the progression of canine Moreover, the use of grading system for a tumor type may MCTs. Although the results of this study confirm the re- be helpful in distinguishing this type of neoplasm from sults of previous studies that have shown the prognostic other malignancies with similar morphology. Further- significance of cellular proliferation in canine MCTs more, this study demonstrated that in canine cutaneous [14,15,18,19] cellular proliferation should not be evalu- MCTs, may be used as a sensitive prognostic indicator of ated as a single prognostic factor for canine MCTs but post-surgical tumour behaviour; in addition, it is related to should be evaluated in tandem with additional prognos- other histopathological variables that are widely accepted tic indicators. Furthermore, the histologic characteristics as having prognostic value. of the MCT cells in this affected dog was moderate to abundant cytoplasm, round nuclei with scattered chro- Competing interests matin, fibrous stroma, and eosinophil infiltration. Little The authors declare that they have no competing interests. necrosis was seen. Authors’ contributions Several grading systems have been proposed to classify EH, BP and AMB participated in the histopathological evaluation, performed canine mast cell tumors. The system most commonly the literature review, acquired photomicrographs and drafted the manuscript used classifies the tumor from grades I to III, depending and gave the final histopathological diagnosis. MHJM, FEGH, JJ, NJM, MK and RSH designed and carried out all the experiments and the principal on how well the mast cells are differentiated, mitotic ac- investigators of the laboratory in which the research were performed and tivity, location within the skin, invasiveness, and the contributed to the interpretation of the data and writing of the manuscript. presence of inflammation or necrosis, with grade III be- All authors read and approved the final manuscript. ing the most aggressive (characterized by the presence of Acknowledgements undifferentiated, immature mast cells with a high poten- The authors thank staff of the Department of pathology, Faculty of tial for metastasis) [20,21]. In the current case, the Veterinary Medicine, Tehran University for their valuable technical assistance. Hosseini et al. Diagnostic Pathology 2014, 9:9 Page 5 of 5 http://www.diagnosticpathology.org/content/9/1/9

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