What Is Your Diagnosis? Cutaneous Mass in a Dog

A 5-year-old female neutered miniature dachshund was presented to the University Veterinary Teaching Hospital of Camden (UVTHC) at Sydney University with a cutaneous mass on her dorsal neck. The patient had been diagnosed with atopic dermatitis and treated with cyclosporin for the past 7 months and appeared otherwise healthy with no significant clinical symptoms or abnormalities noted during the physical exam. Cytology smears from fine-needle aspirates were examined. Photomicrographs are shown below (Figure 1). Cytologic interpretation: Most likely panniculitis. Concern for underlying round cell neoplasia, particularly cutaneous lymphoma. The slides showed low nucleated cellularity with fair preservation and against a highly vacuolated and proteinaceous basophilic background that contained frequent pale basophilic cytoplasmic fragments [lymphoglandular bodies], many bare nuclei and degenerated cells, and rare ribbons of pale pink fibrillar material [collagen]. The cells consisted of pleomorphic round cells present individually and in small aggregates admixed with low numbers of non-degenerated neutrophils, and macrophages/histiocytes, fewer eosinophils and small lymphocytes, and rare mast cells. Moderate numbers of adipocytes were seen in poorly preserved aggregates frequently intermixed with inflammatory cells. The pleomorphic round cells varied in size (12–16 μm) and showed a high nucleus-to-cytoplasm ratio, moderately basophilic cytoplasm, irregular nuclear shapes, fine to coarse chromatin, distinct large nucleoli often bizarrely shaped, and often prominent anisonucleoliosis. There was moderate anisocytosis and anisokaryosis. These cells were likely of lymphoid origin, though some resembled histiocytic cells. The mass (2.0 × 1.5 cm) was surgically excised with minimal margins and submitted for histopathologic evaluation. Histologically, it was characterized by a dense infiltrate of atypical pleomorphic round cells extending into the subcutaneous tissue, fat lobules, and deep dermis, occasionally rimming adipocytes (Figure 2). The cells ranged from small to intermediate in size, with irregularly round to oval nuclei, and showed moderate anisokaryosis and mild anisocytosis. Thirty mitotic figures, including some atypical ones, were observed in a 2.37 mm2 area. Additionally, there were variable numbers of larger cells resembling histiocytes/macrophages, fewer mast cells, a few neutrophils, scattered plasma cells and eosinophils. Immunohistochemistry (Figure 3) showed that the atypical pleomorphic round cells were CD3 (1:500; polyclonal rabbit anti-human CD3; Dako) positive but CD79b (AT107-2; 1:20000; monoclonal antibody; BioRad) negative, with occasional CD79b immunopositive cells (presumably resident B lymphocytes). Additionally, the presumed histiocytes/macrophages present in both the subcutaneous infiltrates and dermis showed Iba1 (1:200; concentrated polyclonal antibody; Biocare Medical) cytoplasmic immunoreactivity. The Iba1 status of the CD3-positive atypical pleomorphic round cells, the population of interest for potential neoplastic transformation, was carefully evaluated. The Iba1 staining was interpreted as inconclusive in the atypical pleomorphic round cell population due to overlapping morphology with the presumed histiocytes/macrophages that consistently labeled Iba1 positive. Numerous CD3-positive lymphocytes and Iba1-positive histiocytes/macrophages were observed scattered throughout the dermis, along with fewer CD79b-positive lymphocytes. While the CD3-positive cells could potentially represent neoplastic cells, they did not form a distinct mass nor were they present in sufficient numbers to mimic the features seen in the subcutis. No immunopositive cells were detected in the epidermis. Controls were appropriately positive for all markers (CD3, Iba1, CD79b). Final diagnosis: Panniculitis with atypical lymphoid infiltrate. Findings raise suspicion for a non-epitheliotropic cutaneous T-cell lymphoma, most consistent with subcutaneous panniculitis-like T-cell lymphoma (SPTCL). For completeness, PARR (PCR for antigen receptor rearrangement) analysis was performed on DNA extracted from paraffin-embedded tissue samples that were sent to an external laboratory. The analysis was conducted using the manufacturer's recommended protocol (ReliaPrep FFPE gDNA Miniprep System; Promega Corporation, Madison, WI, USA). PCR reactions were performed on the MiniAmp Plus Thermal Cycler (Thermo Fisher Scientific, Waltham, MA, USA). The PCR reactions were size-separated by capillary electrophoresis. All PCR analyses were performed on the ABI Genetic Analyzer (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). The PARR assay targeted T-cell receptor (TCR) gamma and beta genes, as well as immunoglobulin heavy chain (IgH) genes; however, the specific primer sets used were not disclosed. The results were polyclonal, indicating no evidence of a clonal lymphoid population. Additionally, the external laboratory reported a detection rate of approximately 89% for clonal rearrangements in histologically confirmed lymphoid malignancies in dogs when testing formalin-fixed, paraffin-embedded tissue containing histologically suspicious cells. Therefore, the negative result reduced, but did not eliminate, the suspicion of lymphoma. The dog was started on lomustine orally every 3 weeks for a total of five treatments post-surgery in an effort to minimize the chances of recurrence and dissemination of the suspected lymphoma. To the author's knowledge, the patient has remained disease-free up to the present day. Primary cutaneous T-cell lymphomas (CTCL) can be broadly categorized into epitheliotropic and nonepitheliotropic forms, of which the former is most common. Epitheliotropic forms are identified histologically based on infiltration of the epidermis and/or adnexal structures by neoplastic lymphocytes [1-3]. Conversely, nonepitheliotropic forms of CTCL (NE-CTCL) have been less well characterized in dogs, and diagnosis is challenging in some cases, due to heavy infiltration of inflammatory cells [1, 2]. Most NE-CTCLs are classified as peripheral T-cell lymphomas, not otherwise specified (PTCL-NOS), according to the World Health Organization (WHO) classification. This category represents a highly heterogeneous group, although some specific lymphoma entities have been recognized. An indolent form in dogs named cutaneous lymphocytosis has been recently added to this list [1, 4]. A rare subtype recognized in humans (and more recently in dogs) is the subcutaneous panniculitis-like T-cell lymphoma (SPTCL), which is typically indolent in humans [1, 2]. This subtype is predominantly located in the subcutaneous adipose tissue and may resemble panniculitis in its early stages. In dogs, the biologic behavior of SPTCL appears to be more aggressive than typically reported in humans; however, some cases may exhibit a more indolent progression [1, 2]. In dogs, diagnosing NE-CTCLs is particularly challenging when inflammatory infiltrates, such as those present in this case, are a major component, leading to a heterogeneous dermal infiltrate rather than the more uniform infiltrate typically expected with lymphoma [1]. The polyclonal PARR result in this case highlights the diagnostic challenge posed by inflamed NE-CTCLs. The significant inflammation present in this case possibly contributed to this result, as inflammation in canine NE-CTCLs is known to interfere with PARR analysis due to the polyclonal T-cell infiltrate, and potentially low neoplastic clone levels [1]. Additionally, it is important to acknowledge that the diagnostic performance of PARR can be influenced by primer selection and interpretation criteria, and discordant results between laboratories have been reported in canine SPTCL cases [2]. It is interesting to note that previous reports of this condition consistently observed prominent histiocytic infiltrates, which led to the misdiagnosis of some cases as cutaneous reactive histiocytosis or histiocytic neoplasia in the absence of immunohistochemistry and T-cell antigen receptor rearrangement analysis [1, 2]. Similarly, in the cytologic examination of this case, presumed macrophages/histiocytes were mixed with the atypical pleomorphic round cell population, with overlapping morphologies. Cytologists should exercise caution when interpreting cases aspirated from cutaneous nodules or masses that feature a combination of a histiocytic-rich inflammatory infiltrate, a lipidic background, and an atypical, suspicious round cell population. Given the conflicting polyclonal PARR result and the ambiguity regarding the Iba1 status of the atypical pleomorphic round cells, a definitive diagnosis of cutaneous T-cell lymphoma, specifically SPTCL, remains challenging based on the current findings. While the histomorphology and CD3 positivity strongly suggest a lymphoid proliferation of T-cell origin, the possibility of atypical lymphoid hyperplasia or even a B-cell lymphoma or plasma cell neoplasm with aberrant CD3 expression, as reported previously [5], cannot be entirely excluded. Further investigations, such as flow cytometry on fresh tissue (if available in future cases) or a more comprehensive immunohistochemical panel, could provide additional diagnostic clarity; however, these were not available to the author for the current case. This case highlights the value of cytology as a screening tool in the diagnostic evaluation of cutaneous masses. Although cytology did not yield a definitive diagnosis, it effectively guided the next diagnostic steps, ultimately leading to a presumptive diagnosis based on histopathology and immunohistochemistry. Following surgical excision of the mass, the dog received chemotherapy, and, to the author's knowledge, no additional masses have been detected 11 months post-surgery. The authors acknowledge Veterinary Pathology Diagnostic Services' histologists and technicians for their provision of excellent technical assistance. Open access publishing facilitated by The University of Sydney, as part of the Wiley - The University of Sydney agreement via the Council of Australian University Librarians. The authors declare no conflicts of interest.