Abstract
Cutaneous lymphomas are uncommon in people and companion animals. The tumors can be broadly categorized into epitheliotropic and nonepitheliotropic forms, which appear to have different biological behaviors. The present case describes a feline cutaneous epitheliotropic lymphoma. Masses in a 9-year-old cat were first identified on the tail. The cat was treated with chemotherapy, but additional skin masses developed on the flank, face, and ears. Local radiation induced transient tumor regression, but eventual dissemination prompted euthanasia 13 months after initial tumor appearance. Granular lymphocytes were consistently detected on blood smears, and histologically, the tumor involved the skin and superficial subcutis. Tumor lymphocytes expressed cluster of differentiation 3 (CD3) and perforin molecules, suggestive of a cytotoxic phenotype. Location, histopathological features, and perforin expression were similar to a distinct entity in human medicine designated primary cutaneous, CD8-positive, epidermotropic, cytotoxic, T-cell lymphoma.
Feline lymphoma is a diverse entity that can be categorized according to anatomical location,
24
cytomorphology,
25
biological behavior, immunological phenotype,
25
and association with retroviruses such as
A 9-year-old, male, neutered Domestic Shorthair cat was referred to the University of Guelph Veterinary Teaching Hospital (Guelph, Ontario, Canada) for staging and treatment of lymphoma. Approximately 10 weeks before referral, the owner first noted a mass on the proximal portion of the tail. A few days later, a second mass appeared on the distal portion of the tail, and the tail was amputated. Histopathological examination of both tail masses indicated lymphoma. Approximately 6 weeks after the first mass was noted, an additional mass was identified over the remaining base of the tail. This mass appeared to grow rapidly. It was excised as well and again confirmed to be lymphoma. Histopathologically, the tumors consisted of aggregates of large lymphocytes that infiltrated the dermis and the superficial subcutis and destroyed the normal skin architecture (Fig. 1). In addition, lymphocytes were present as small intraepithelial nests, compatible with Pautrier microabscesses. The neoplastic lymphocytes had scant cytoplasm and central oval nuclei with prominent single nucleoli. Approximately 2-fold variation in size was noted among cells and nuclei. Small lymphocytes and large numbers of macrophages were scattered throughout the lesion, but mitotic activity was minimal.
Expression of clusters of differentiation 3 (CD3) and 79a (CD79a) molecules was assessed in tissue sections from the tail mass. Slides were deparaffinized by immersion in xylene and hydrated in a series of ethanol solutions. For antigen retrieval, the slides were immersed in buffer (ethylene-diamine tetra-acetic acid buffer, pH 8, for CD3; citrate buffer, pH 6, for CD79a), heated to 95°C in a microwave, and rapidly chilled to room temperature in an ice bath. Slides were exposed to hydrogen peroxide and universal blocker, a incubated for 30 min at room temperature with the primary antibodies (CD3 = A0452; CD79a = HM57 a ), washed, and then secondary antibodies a (goat antirabbit/antimouse, conjugated to horseradish peroxidase) were applied for 30 min. Positive staining was visualized with Nova Red horseradish peroxidase substrate, and the slides were counterstained with hematoxylin. Negative control sections consisted of substitution of the primary antibody with preimmune rabbit serum (CD3) and mouse immunoglobulins or buffer lacking primary antibody (CD79a). Positive control sections included in each run consisted of normal feline lymph node. Immunohistochemistry results revealed strong positive staining for CD3 in the lymphocytes within the epidermis and in those infiltrating the superficial subcutis (Fig. 2). A few clusters of CD79a-expressing lymphocytes were present in the deep dermis. Based on the findings, the tumor was classified as cutaneous epitheliotropic T-cell lymphoma.
Nine weeks after the initial mass was noted, the owner identified a new nodular mass on the inner aspect of the left pinna. The cat was again referred to the University of Guelph Veterinary Teaching Hospital for tumor staging and additional treatment. During examination, the cat was bright, alert, and responsive. The mass on the pinna was raised, hairless, and ulcerated, and approximately 10 × 6 mm in diameter. Two other areas of skin thickening and crusting were identified on the caudal dorsum. Both submandibular lymph nodes were mildly enlarged. Thoracic radiographs revealed a focal area of consolidation in the left caudal lung lobe and a severe, diffuse bronchial lung pattern. Abdominal ultrasound detected mild enlargement of hepatic lymph nodes. No abnormalities were found in the biochemistry profile. Results of serum enzyme-linked immunosorbent assay testing for FeLV antigen and FIV antibodies were negative. Fine needle aspirates were obtained from the mass on the pinna and from the enlarged submandibular lymph nodes. The ear mass aspirates consisted of highly pleomorphic round cells with variable nuclear to cytoplasmic ratio and dark, irregularly stained cytoplasm, and rare cytophaga (Fig. 3). Nuclei were large, with finely reticulated chromatin pattern and occasional gigantic nucleoli, often exceeding the size of erythrocytes. Five- to 6-fold anisocytosis and anisokaryosis, occasional multinucleation, and mitotic figures were noted. Because of the profound cytological atypia, the sample was interpreted as “malignant round cell tumor.” The lymph node aspirates contained a more monomorphic population of medium-sized lymphocytes comprising approximately 80% of all nucleated cells. In light of the cat's previous diagnoses, anaplastic cutaneous lymphoma with involvement of lymph nodes was considered most likely. A complete blood cell count (CBC) indicated no numerical abnormalities, but on slide evaluation, the majority of lymphocytes had convoluted nuclei and cytoplasmic eosinophilic granules, suggestive of a cytotoxic or natural killer (NK) phenotype (Fig. 4). The cat was started on the Madison-Wisconsin (M-W) chemotherapy protocol, 18 and the ear mass decreased in size. However, during the next 6 weeks, 3 new cutaneous masses (on the bridge of the nose, the flank, and the right pinna) developed and increased in size despite therapy. The owners declined further aspirates or biopsies of the masses. Due to the limited response, 2 months after start of M-W therapy, treatment was switched to lomustine (50 mg/m 2 PO) b with continuation of prednisone (5 mg/kg PO q 12 hr) c therapy. During the subsequent 10 days the masses continued to increase in size, additional cutaneous masses arose, and neutropenia developed; therefore, lomustine was discontinued in favor of radiation therapy. The bridge of the nose and the right and left pinnae were irradiated with 3 fractions, each of 8 grays (Gy), delivered weekly. The size of the irradiated masses decreased by half after 2 treatments, and the masses became unpalpable at the time of the third treatment.
Skin section of a mass from the base of the tail. A lymphoid infiltrate in the dermis and superficial subcutis has altered normal skin architecture. Note intraepithelial lymphoid aggregates (arrows). Hematoxylin and eosin. Bar = 20 μm.
Lymphocytes in the skin mass uniformly express the cluster of differentiation 3 (CD3) molecule. Immunohistochemical stain with hematoxylin counterstain. Bar = 20 μm.
Cytological appearance of a fine needle aspirate of the ear mass. Note medium to large lymphocytes, multinucleated cells with cytophaga (arrow), and gigantic nucleoli (insert). Wright stain. Bar = 10 μm.
Blood smear from cat with epitheliotropic lymphoma. Note lymphocyte with irregular-shaped nucleus and eosinophilic granules in the cytoplasm. Wright stain. Bar = 10 μm.
Three months after the third radiation treatment, new masses arose on the left pinna. Another 8 Gy was added for a total of 32 Gy in 4 treatments. At the same time, the consolidated left caudal lung lobe, assumed to be affected by lymphoma, was irradiated with 8 Gy in a single treatment. Weekly CBCs during chemotherapy and radiation therapy revealed no numerical abnormalities other than intermittent neutropenia. Granular lymphocytes with atypical nuclei comprising 70–90% of all lymphocytes were noted on blood smears during the entire time. Their presence was suggestive of a cytotoxic phenotype resulting from circulating tumor cells. Therefore, immunohistochemical staining for perforin was performed on sections of formalin-fixed, paraffin-embedded tissues from the amputated tail mass. The antibody was a polyclonal antirat perforin antibody d that bound a 66-kD protein expressed in feline lymphocytes on Western blotting (unpublished observations), consistent with the expected size of perforin. 14 The same protocol as for CD79a staining was applied, except primary antibody incubation was 1 hr. Control tumor sections were incubated with preimmune rabbit serum instead of primary antibody (negative control), and a feline large granular lymphoma section was used as a positive control (Fig. 5). Prominent granular cytoplasmic staining was present in all lymphocytes in the positive control section (but not hepatocytes) and in approximately 80% of lymphocytes comprising the tail mass, suggesting a cytotoxic phenotype (Fig. 6).
Approximately 10.5 months after the first mass was identified, and 1 month after radiation therapy was completed, the cat became lethargic and anorexic. Serum chemistry indicated azotemia (urea = 19.3 mmol/l, reference interval: 3.5–9 μmol/l; creatinine = 270 μmol/l, reference interval: 20–150 μmol/l), and urine specific gravity was 1.016. Radiographically, a large mass was now obvious in the left caudal lung lobe, and ultrasonographically, both kidneys were markedly enlarged and appeared irregular in shape. A focal segment of the small intestine had abnormally thickened walls, and the liver appeared hyperechoic and was enlarged. These findings were interpreted as likely dissemination of the cutaneous lymphoma, and the cat was subsequently euthanized. Necropsy was declined.
Cutaneous lymphoma is uncommon in people and companion animals 8,17,19,25,26 and can be classified into several subtypes: dermal nonepitheliotropic lymphoma and the main epitheliotropic variants (mycosis fungoids [MF], pagetoid reticulosis, and Sézary syndrome).5–8 In people, MF is the most common type of cutaneous T-cell lymphoma and accounts for more than 50% of all primary cutaneous lymphoma. 26 It is a proliferative disorder of CD4+ memory lymphocytes, or rarely CD4−/CD8+ lymphocytes, characterized by skin patches, plaques, or masses, which are often ulcerated. The infiltrate is epitheliotropic and may show intraepithelial clustering of lymphocytes, known as Pautrier microabscess. 11 Sézary syndrome is a leukemic variant of MF and associated with a less favorable prognosis. 11,26 Canine epitheliotropic lymphoma is a similar disease of older dogs that typically affects skin or mucus membranes and may have relatively slow (1–4 years) clinical progression. 16,20 The neoplastic cells are typically composed of CD8+/CD3+ lymphocytes with marked tropism for skin adnexal structures. 16,20 Cutaneous lymphoma with leukemia has occasionally been reported in dogs. 7,22
Feline liver section with large granule lymphoma infiltrate stained immunohistochemically with an antibody against perforin. Note prominent staining of cytoplasmic granules. Hematoxylin counterstain. Bar = 20 μm.
Skin mass stained immunohistochemically with an antibody against perforin. Note lack of staining in sebaceous gland. Granular cytoplasmic staining in lymphocytes is suggestive of a cytotoxic lymphocyte phenotype. Hematoxylin counterstain. Bar = 20 μm.
In cats, cutaneous lymphoproliferative diseases exhibit variable manifestations. There may be a benign 9 or malignant 4 behavior, lesions may be epitheliotropic 21,23 or restricted to the dermis, 4,13 and rarely there is association with leukemia. 21 Cats with cutaneous lymphoma or lymphocytosis were generally FeLV seronegative, 3,5,9,13,19,21,23 but in some cases, FeLV antigen or proviral DNA was demonstrated in tumor tissue sections by immunohistochemistry or polymerase chain reaction (PCR) amplification, respectively. 5,23
Cutaneous lymphocytosis is an uncommon disease of older cats. 9 It is characterized by slowly progressing, single or multiple lesions, which typically manifest with alopecia, erythema, and scaling (with or without crusting), and intense puritus. 9 Histologically, the lesions were composed of small, well-differentiated T-lymphocytes that exhibited some epitheliotropism but lacked obvious cytological features of neoplasia. 9 Feline cutaneous lymphocytosis is described as a benign disease that rarely progresses to systemic involvement, but which might be difficult to distinguish from early, small cell cutaneous lymphoma. 9
Feline cutaneous lymphomas are also diseases of older cats (mean age: 11 years) 17 and are rare malignant proliferations of T-lymphocytes. 4,8,25 Variants that are nonepitheliotropic appear to be more common than those that are epitheliotropic. 3,4,19 Lesions of nonepitheliotropic cutaneous lymphoma are characterized by diffuse or nodular growths and are infrequently associated with scaling and pruritus. 3,4,13 These tumors consist of expansile proliferation of small to medium T-lymphocytes between the superficial epidermis and the subcutis, with an admixture of plasma cells. 3,4,13,19 Surgical excisions of solitary tumors are typically followed by rapid recurrence. 3 Nonepitheliotropic lymphomas affecting the skin eventually progress to involve regional lymph nodes or viscera and are poorly responsive to chemotherapy or radiation therapy. 3,13
Epitheliotropic cutaneous lymphomas in cats appear to be rare, with only a few individual cases reported. 3,10,21,23 In affected cats, there are chronic, ulcerative, pruritic lesions and generalized lymphadenopathy. The lesions consist of proliferations of large, pleomorphic CD3+ lymphocytes in the superficial dermis, often accompanied by low numbers of plasma cells and histiocytes. The tumors typically destroy the dermal-epidermal distinction and are accompanied by discrete intraepithelial nests of lymphoid cells, compatible with Pautrier microabscesses. 3,10,21,23 Progression of the disease is variable and appears dependent on the nature of lesions at diagnosis. When the lesions are characterized by patches of erythema and alopecia, the tumor progresses slowly with minimal systemic involvement, 10 whereas tumor-stage lesions progress rapidly and disseminate to regional lymph nodes and occasionally viscera. 10,23 There is one report of Sézary-like syndrome in a cat with abnormal, cerebriform lymphocytes in peripheral blood and bone marrow. 21
The uncommon occurrence (or reporting) of epitheliotropic lymphoma in cats makes it difficult to accurately characterize this disease. The main features of the tumors in the present report were acute onset of nonpuritic nodular lesions, rapid recurrence after surgical excision, steady progression, relative resistance to chemotherapy, and moderate responsiveness to radiation. Hence, these tumors exhibited a greater behavioral similarity with nonepitheliotropic cutaneous lymphoma (solid masses, nonpruritic, and rapid growth) but were histologically of an epitheliotropic nature. The unique feature of this case was perforin expression, implying a cytotoxic tumor cell phenotype. The tumor appeared to have limited radiation sensitivity, but the radiation therapy was instituted at a relatively advanced stage of the disease.
Perforin is a cytotoxic pore-forming protein stored with granzyme in cytoplasmic granules of cytotoxic T-and NK-lymphocytes. 14 Extrusion of perforin by these lymphocytes results in pore formation in the target cell membrane causing osmotic lysis and facilitating entry of granzymes into the cytosol where they induce apoptosis. 14 In people, clonal diseases of cytotoxic lymphocytes are rare lymphoproliferative malignancies that manifest with distinct biological behavior ranging from indolent to very aggressive. 1 In veterinary medicine, it is likely that such tumors are more frequent than commonly appreciated, as antibodies reactive with the CD8 molecule in formalin-fixed tissue are lacking, and granules are difficult to appreciate in histological sections. Hence, appreciation of cytoplasmic granules and the potential for a tumor with a cytotoxic lymphocyte phenotype may arise mostly from cytological examination. In the present case, the tumor was assessed for perforin expression by immunohistochemistry in an effort to phenotypically link the neoplastic lymphocytes in the skin to the atypical lymphocytes in circulation. Because perforin is an intracellular protein, and fluorescently conjugated polyclonal antibodies for perforin are currently unavailable, a flow cytometric assay has not yet been established. However, prominent eosinophilic granules (lysosomes) in lymphocyte cytoplasm may be a consistent morphological feature associated with a cytotoxic pheno-type. Several features of the cutaneous lymphoma in the cat in the current case were reminiscent of a discrete entity in human medicine—-ulcerative masses of the ears and skin, pleomorphic tumor cells, rapid progression, epitheliotropism, expression of perforin, aggressive behavior, and chemotherapy insensitivity. In humans, this type of neoplasm consists of CD8- and perforin-expressing lymphocytes, 2,6,12 and it is referred to as cutaneous, CD8-positive, epidermotropic, cytotoxic, T-cell lymphoma. Although CD8 expression could not be assessed in the current case, the feline neoplasm had many other striking similarities to the human disease.
Footnotes
a.
Dako Canada Inc., Mississauga, Ontario, Canada.
b.
Lomustine, Bristol-Meyers Squibb Co., New York, NY.
c.
Prednisone, Apotex Inc., Weston, Ontario, Canada.
d.
Torrey Pines Biolabs, East Orange, NJ.