Diagnostic challenge in veterinary pathology: Pruritus in a dog with a mixed cellular infiltrate

Clinical History

An 11-year-old, castrated male German Shepherd dog initially presented with a 9-month history of pruritus and multifocal erythematous skin lesions, alopecia, and mild weight loss. Skin lesions waxed and waned without complete resolution and newer lesions developed in other body regions. Erythematous macules and alopecia began on the ventral abdomen and progressed to the chest and dorsum. The patient received 0.27 mg/kg cetirizine (twice daily for 20 days), 22 mg/kg amoxicillin/clavulanic acid twice daily for 20 days, 0.5 mg/kg oclacitinib (once daily for 30 days), and 6 mg/kg ketoconazole (once daily for 21 days), in the prior 5 months without significant improvement. There had been no changes in the dog’s appetite or water intake. A complete blood cell count and a serum biochemistry profile performed 3 months prior to referral revealed no abnormalities and thyroid hormone T4 was within the normal reference range.

Gross Findings

The dog was bright, alert, and responsive on physical examination, with abnormalities limited to the integument. Dermatologic examination revealed multifocal to coalescing erythematous macules and patches, and plaques with scale, crust, and erosions to ulcers distributed along the neck, cranio-ventral and lateral thorax, abdomen, lower extremities, footpads, and preputial region (Fig. 1).

Figure 1.

Erythematous macules and patches, and plaques with erosion, crust, and alopecia, cervical and thoracic skin, German Shepherd dog: (a) multifocal to coalescing erythematous macules and patches, and plaques with scaling, crusting, and ulcerations distributed along the neck and (b) higher magnification of similar lesions along the lateral thorax.

Differential Diagnosis

A neoplastic process such as cutaneous lymphoma was the primary differential diagnosis due to the history, age of onset, and clinical presentation. Generalized superficial pyoderma in conjunction with atopic dermatitis and pemphigus foliaceus were considered differential diagnoses. Both superficial pyoderma and pemphigus foliaceus skin lesions can be pruritic and show signs of erythema and crusting. Reactive histiocytosis was considered less likely as a differential diagnosis due to the lack of nodules.

Laboratory Findings

Skin cytology using Diff Quick stain revealed an accumulation of neutrophils and few corneocytes. There were no observed acantholytic keratinocytes or microorganisms. Four, 8-mm punch biopsies were obtained from the inguinal, neck, and cranio-ventral and lateral thoracic skin lesions; fixed in formalin; and processed for histological examination. Limited clinical history was received with biopsy submission.

Microscopic Findings

Microscopic findings included multifocal, poorly delineated, infiltrates of a mixed population of neutrophils, small mature lymphocytes, plasma cells, histiocytes, eosinophils, and mast cells. Inflammation and edema expanded the superficial dermis and loosely surrounded adnexa superficially but did not extend into the subcutis (Fig. 2a). Depending on the section, the epidermis exhibited multifocal erosion, orthokeratotic hyperkeratosis, serocellular crusts, multifocal subcorneal pustules, and moderate hyperplasia (Fig. 2b, c). Apocrine glands were mildly ectatic (Fig. 2a, d). Special stains (Gram, Ziehl-Neelsen, Periodic-Acid-Schiff-Hematoxylin) did not reveal the presence of infectious agents.

Figure 2.

Inflamed, nonepitheliotropic T-cell lymphoma, skin, dog: (a) superficial infiltrates (arrows) expanding the dermis and apocrine gland ectasia (Hematoxylin and eosin [HE]). (b) Subcorneal pustules within the epidermis (arrow) and dermal expansion by edema, neoplastic round cells, and inflammation (HE). (c) Neoplastic round cells and mixed inflammation expand the dermis (HE). (d) Low numbers of inflammatory and neoplastic round cells surround, but do not invade, adnexal structures (HE). (e) Atypical round cells with mixed inflammation and occasional mitotic figures (arrows) (HE). (f) Membranous and cytoplasmic round cell immunolabeling of CD3.

An initial diagnosis of mixed, multifocal, superficial dermatitis was made based on the presence of a loosely organized, mixed inflammatory infiltrate multifocally present within the dermis. The primary differential diagnoses were allergic dermatitis with potential secondary bacterial infection, pemphigus foliaceus with secondary infection, though no acantholytic cells were identified, or another immune mediated process such as reactive histiocytosis, with an atypical histopathologic presentation.

Further Investigation and Diagnosis

Additional clinical history was discussed between the clinician and the pathologist after apparent discordance between clinical suspicion of a neoplastic process and histopathologic findings of mixed inflammation. Re-evaluation identified atypical, intermediate to large round cells initially interpreted as histiocytes scattered throughout the dermis and surrounding, but not infiltrating, adnexa (Fig. 2d). These round cells, amongst the dermal rarely infiltrated the overlying epidermis. The cells had distinct borders with low to moderate amounts of eosinophilic cytoplasm; round to ovoid, central nuclei with open chromatin; and 1 or 2 prominent nucleoli (Fig. 2e). Anisocytosis and anisokaryosis were mild to moderate with an occasionally high nuclear to cytoplasmic ratio and rare binucleated to multinucleated cells. The mitotic count for the atypical round cells was 4 per 2.37 mm² with occasional bizarre mitoses. On immunohistochemistry, the atypical round cells exhibited weak to strong, membranous to cytoplasmic immunoreactivity to CD3 (Fig. 2f). The round cells were separated by mixed inflammatory populations, as previously described. Fewer, small mature lymphocytes within the neoplastic population exhibited strong, membranous immunoreactivity to CD20 and CD79.

These findings indicated that both inflammatory and neoplastic processes might be involved. Therefore, a polymerase chain reaction for antigen receptor rearrangement (PARR) test^1,3,5,9 (Veterinary Diagnostics, Davis, CA, USA) was pursued on paraffin-embedded sections to confirm the disease process and diagnosis. This test was performed in triplicate using primer sequences to canine T-cell receptor gamma TRG(A), TRG(B), and TRG(C) and analyzed using electrophoresis as previously reported.^3,5,9 Sharp clonal spikes were identified in duplicate analysis for TRG(A), with no clonal spikes seen for TRG(B) or TRG(C).

Discussion

The heavily inflamed, mixed cellular infiltrate and significantly limited clinical history provided with the biopsy submission lead to an initial diagnosis of an inflammatory process. Communication of additional clinical history and suspicion of a neoplastic process between the clinicians and pathologists was necessary to reach the final diagnosis of canine inflamed, nonepitheliotrophic cutaneous T-cell lymphoma (NE-CTCL). ⁵ The diagnosis of inflamed NE-CTCL was supported by a combination of clinically distinctive, progressive, erythematous skin lesions in an aged dog, histopathologic findings of atypical CD3+ neoplastic round cells within a mixed inflammatory cell population, and a clonal PARR test revealing a sharp spike indicating monoclonal cell population.

Pruritus was a unique feature of the clinical presentation not previously associated with cases of inflamed NE-CTCL. ⁵ Pruritus potentially augmented inflammation due to self-trauma. Pruritus is nonspecific and is commonly associated with allergic or immune-mediate dermatitis, which were suspected from initial interpretation of microscopic findings. Therefore, this case illustrates how skin lesions of inflamed NE-CTCL can be accompanied by pruritus and mimic other inflammatory dermatoses.

The neoplastic cells were initially considered histiocytes within a mixed inflammatory cell infiltrate. The overall pattern of chronic, superficial pustular dermatitis was suggestive of allergic or immune-mediated dermatitis (pemphigus foliaceus) with secondary pyoderma or chronic surface trauma obscuring the primary process. Although amastigotes were not identified microscopically, the superficial distribution and heterogeneous inflammation were consistent with cutaneous leishmaniasis. These differentials were ruled out based on a lack of various key gross and microscopic findings and identification of the atypical round cells as CD3+, intermediate to large lymphocytes.

Histologically, typical cases of inflamed NE-CTCL are characterized by a homogenous dermal infiltrate of large, atypical and variably CD3+ lymphocyte population.^5,6 However, the diagnosis of NE-CTCL can be challenging when a heterogeneous cell population is present, which most commonly involves histiocytes, lymphocytes, and eosinophils. ⁵ In this case, additional inflammatory cells including large numbers of neutrophils, mast cells, and plasma cells, were present and obscured the neoplastic population. Histological differentiation between reactive histiocytosis and inflamed NE-CTCL can be challenging as both canine-reactive histiocytosis and inflamed NE-CTCL are characterized by heterogeneous inflammatory populations. ⁵ Inflammatory and neoplastic conditions may contain low numbers of mitotic figures, though the presence of bizarre mitoses could indicate a neoplastic process. Using an immunohistochemical panel to differentiate histiocytes from poorly differentiated, intermediate to large lymphocytes and clonality testing may improve chances for detecting the neoplastic population in these cases. ⁸

Lymphocyte clonality assessment using PARR testing has been utilized to discern inflammation and inflamed NE-CTCL. ⁵ Polymerase chain reaction for antigen receptor rearrangement testing detects clonal expansion of lymphocytes and is a highly sensitive and specific diagnostic tool for canine lymphoma, with around 90% sensitivity and specificity for most T-cell lymphomas. ³ However, lymphocyte clonal proliferation in histopathological skin samples is not an exclusive hallmark of neoplasia ¹ and can be observed with other lymphocytic inflammatory skin diseases. ⁷ Therefore, PARR testing as a single tool is not a standalone diagnostic test to diagnose malignancy, despite its high sensitivity and specificity, and PARR results should always be interpreted in the context of the clinical, histopathological, and immunophenotypic findings.

The prognosis for inflamed NE-CTCL is generally guarded to poor, with survival time ranging from 1 to 36 months after diagnosis.^4,6 Dogs with NE-CTCL have longer overall survival times than dogs with epitheliotropic CTCL. ² At the time of this writing and 4 months after diagnosis, the owners of this dog elected not to pursue any therapy as the dog remained systemically healthy. However, the skin lesions are still present and new skin lesions are still developing.

Inflamed NE-CTCL represents a diagnostic challenge due to its histopathologic resemblance to inflammatory processes. Providing an adequate clinical history with descriptions and distributions of lesions, and suspected differential diagnoses with the biopsy submission is essential to making an accurate diagnosis. Even so, multiple methodologies may be required for diagnostic confirmation of inflamed NE-CTCL, including immunohistochemistry and PARR testing.