Cervical thymoma originating in ectopic thymic ... - Wiley Online Library

Veterinary Clinical Pathology ISSN 0275-6382

Cervical thymoma originating in ectopic thymic tissue in a cat Ana Lara-Garcia1, Maxey Wellman2, Mary Jo Burkhard2,3, Cecilia Machado-Parrula2, Victor E. Valli4, Paul C. Stromberg2, C. Guillermo Couto1,3 1

Department of Veterinary Clinical Sciences and Veterinary Teaching Hospital, 2Department of Veterinary Biosciences, College of Veterinary Medicine, and 3OSU Comprehensive Cancer Center, The Ohio State University, Columbus, OH; and 4Department of Pathobiology, College of Veterinary Medicine, University of Illinois, Urbana, IL

Key Words Cervical, ectopic thymus, feline, flow cytometry, PCR, thymoma Correspondence Ana Lara-Garcia, DVM, MSc, PhD, DACVIM, Department of Veterinary Clinical Sciences and Veterinary Teaching Hospital, College of Veterinary Medicine, The Ohio State University, Columbus, OH 43210 E-mail: [email protected] DOI:10.1111/j.1939-165X.2008.00061.x

Abstract: An 11-year-old female spayed domestic shorthair cat was referred to The Ohio State University Veterinary Teaching Hospital (OSUVTH) for evaluation of a 6 4 3.5 cm mass in the left midcervical region causing increased respiratory sounds and lateral deviation of the trachea. A fine needle aspirate of the mass was obtained before referral and the cytology results were compatible with a reactive lymph node. Immunocytochemistry showed increased numbers of CD31 T lymphocytes and small numbers of CD201 and CD79a1 medium to large lymphocytes. Differential diagnoses from the referral pathologist were T-cell-rich B-cell lymphoma and feline Hodgkin’s-like lymphoma. A subsequent fine needle aspirate performed at the OSU-VTH showed similar results. On flow cytometry the majority of cells were CD31 T lymphocytes that were double positive for CD4 and CD8 (73%), compatible with either a double-positive (CD41CD81) T-cell lymphoma or lymphocytes from ectopic thymic tissue. The mass was surgically removed. Histopathology and immunohistochemistry of the mass revealed a predominant population of CD31 small lymphocytes and small numbers of medium to large lymphocytes with moderate anisocytosis and anysokaryosis. A population of cytokeratin-positive epithelial cells surrounded small microcystic structures filled with eosinophilic material and structures interpreted as Hassall’s corpuscles. These findings were consistent with thymic tissue and a diagnosis of ectopic thymoma was made. PCR results for lymphocyte antigen receptor rearrangement (PARR) were negative. The cat had no evidence of disease 16 months after removal of the mass. To our knowledge this is the first report of an ectopic cervical thymoma in a cat. The clinical and diagnostic features of this unusual case will be useful in helping veterinarians and pathologists obtain a presurgical diagnosis and establish a prognosis for similar lesions.

Case Presentation An 11-year-old female spayed domestic shorthair cat (6.2 kg) was referred to The Ohio State University Veterinary Teaching Hospital (OSU-VTH) for evaluation of a mass located in the left midcervical region. The mass initially was noticed by the owners approximately 1 year before referral. On the initial visit to the local veterinarian, the mass measured 3 3 3 cm. A fine needle aspirate of the mass yielded 1 mL of hemorrhagic fluid, which was submitted to a diagnostic laboratory (IDEXX Laboratories, Worthington, OH, USA) and interpreted on cytologic examination as a hematoma by

the pathologist. The mass continued to increase in size gradually, and the cat developed increased respiratory sounds approximately 8 months later. On a second visit to the local veterinarian, another fine needle aspirate of the mass was obtained and submitted to the same laboratory, where it was interpreted by a clinical pathologist as a reactive lymph node. However, immunocytochemical stains of the cytology specimen done at the laboratory showed a predominant population of CD31 T lymphocytes and a few scattered medium to large lymphocytes positive for CD20 and CD79a. Differential diagnoses reported by the clinical pathologist included T-cell-rich B-cell lymphoma and

c 2008 American Society for Veterinary Clinical Pathology Vet Clin Pathol 37/4 (2008) 397–402

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feline Hodgkin’s-like lymphoma.1,2 No medical treatment was instituted before referral. On admission to the OSU-VTH, the cat’s body condition score was 5/5. Abnormalities on physical examination included an audible respiratory stertor on inspiration and expiration, and a left midcervical mass, which was firm, nonpainful, partially movable, and measured 6 4 3.5 cm. The only abnormality on the CBC was hyperproteinemia (8.2 g/dL; reference interval, 5.6–7.4 g/dL). Serum biochemical abnormalities included mild hypercalcemia (11.4 mg/dL, reference interval 8.4–10.1 mg/dL), hyperalbuminemia (4.5 g/dL, reference interval 2.5–3.5 g/dL), and hypercholesterolemia (267 mg/dL, reference interval 65–200 mg/dL). Serum-ionized calcium and urine specific gravity were not measured. Results of feline leukemia virus antigen and feline immunodeficiency virus antibody tests (IDEXX, Westbrook, ME, USA) were negative. Results of a PCR test on an EDTA blood sample for Bartonella sp. (Laboratory of Infectious Diseases, College of Veterinary Medicine, North Carolina State University) were also negative. Ultrasonographic evaluation of the neck revealed a mass measuring 4 2 3 cm, with mixed echogenicity and a central cavity. The mass was well demarcated and did not involve surrounding structures. The mass was compatible with a cervical lymph node, although the left thyroid lobe could not be visualized. Results of abdominal ultrasonography were unremarkable. Thoracic radiographic abnormalities included right displacement of the cardiac silhouette, likely secondary to the presence of mild pectus excavatum. The caudal cervical trachea was focally narrowed and displaced to the right by the mass, and a small amount of gas was seen within the thoracic esophagus. A fine needle aspirate of the cervical mass was performed, and smears were prepared, air-dried, and stained with Wright-Giemsa. The smears were highly cellular, and contained predominantly (93%) small lymphocytes (Figure 1). A mild increase in the number of medium-sized lymphocytes (10–11 mm, 7%) and rare large lymphocytes (12–14 mm) were also seen. The cytologic findings were compatible with lymphoid hyperplasia or small cell lymphoma. Flow cytometry was performed from a fine needle aspirate specimen of the cervical mass using monoclonal antibodies for the following antigens: CD18 (panleukocyte marker, clone MHM23; Dako North America Inc, Carpinteria, CA, USA), CD5 (pan T-cell marker, clone FE1.1B11; AbDSerotec, Raleigh, NC, USA), CD4 (T-helper lymphocytes, clone vpg34; AbDSerotec), CD8 (cytotoxic T lymphocytes, clone vpg9; AbDSerotec), CD21 (B-cell

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Figure 1. Cytologic specimen of a cervical mass from a cat, with predominantly small lymphocytes and small numbers of medium-sized lymphocytes. Wright-Giemsa, 100 objective.

marker, clone B-ly4; BD Biosciences, San Jose, CA, USA), and CD14 (monocyte/histiocyte marker, clone Tuk4; AbDSerotec) using a FACSCalibur (BD Biosciences) flow cytometer and appropriate isotype controls (clone W3/25; AbDSerotec). Briefly, monoclonal antibodies for cell surface staining were directly conjugated to either fluorescein isothiocyanate or phycoerythrin and incubated with isolated cells for 30 minutes at room temperature. The cells were washed by centrifugation with phosphate-buffered saline and analyzed immediately. Typically 10,000 events were collected from the gated region. Analysis of flow cytometric data was completed using appropriate software (BD Biosciences). The cells were positive for CD18 (94%) and CD5 (92%). The majority of the cells were double positive for CD4 and CD8 (73%) but a small percentage (9% each) expressed only CD41 or CD81 (Figure 2). The cells were negative for CD21 and CD14. The lack of staining with CD21 and concurrent staining with T cell markers was consistent with either a doublepositive (CD41CD81) T-cell lymphoma or with thymic T-lymphocytes. The mass was surgically removed under general anesthesia the following day. On gross examination there were 2 masses, one of approximately 1.5 cm diameter that was attached to a larger 4 2 3 cm encapsulated mass with a central cavitation. On histologic examination, the smaller mass was a cervical lymph node that microscopically was normal. The larger mass was well encapsulated and hypercellular,


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Figure 2. Flow cytometric analysis of cells from the cervical mass. (A) The scatter plot shows a dense, uniform population of cells. The circled area (R1) was gated for analysis. Controls included unstained cells and cells labeled with isotype antibodies (not shown). Cells stained positively for CD18 (B) and CD5 (C), indicating T-cell lineage. (D) The majority of cells are CD4/CD8 double-positive and additional cells stain positively for CD4 or CD8, consistent with the thymocyte maturation process.

and consisted of a predominant population of small lymphocytes and small numbers of medium-sized lymphocytes and also some large lymphocytes, which had moderate anisocytosis and anisokaryosis. Epithelial cells surrounded small microcystic structures, filled with light eosinophilic, homogenous material that was interpreted as Hassall’s corpuscles (Figure 3). Multifocal, small areas of coagulation necrosis were observed as well as several foci of acute hemorrhage. Bacterial culture of both masses yielded no growth. Immunohistochemical staining of paraffin-embedded tissue was performed with primary monoclonal antibodies (Dako) to CD3 (human pan-T-cell marker), BLA36 (human B-cell antigen 36), CD79a (B-cell antigen receptor complex), and cytokeratin (epithelial marker) using an avidin–biotin–peroxidase method as previously described.1 Positive cytoplasmic cytokeratin staining was observed only in the structures that were histologically consistent with Hassall’s corpuscles (Figure 4). The majority (80%) of lymphoid cells were positive for CD3, with scattered B-cell follicles that stained positively with BLA36 and CD79a. The staining pattern was most consistent with a thymoma.

To rule out the possibility of small cell lymphoma involving ectopic thymic tissue, a stained smear of the fine needle aspirate of the cervical mass was submitted to Dr. Anne Avery at the College of Veterinary Medicine at Colorado State University for PCR for

Figure 3. Histologic section of the cervical mass showing mature small lymphocytes and small microcystic structures filled with light eosinophilic homogeneous material interpreted as Hassall’s corpuscles. H&E, 100 objective.


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Figure 4. Positive cytoplasmic staining for cytokeratin AE1/AE3 of the structures consistent with Hassall’s corpuscles in histologic sections. Avidin–biotin–peroxidase, 40 objective.

antigen-receptor rearrangement (PARR) assay.3 Clonal rearrangements of B-cell or T-cell receptor genes were not detected. The cat recovered uneventfully from surgery, and remained free of disease 16 months after surgery.

Discussion Although lymphoid neoplasms rarely present as solitary cervical masses in cats, initial differential diagnoses in this cat included T-cell-rich B-cell lymphoma and Hodgkin’s-like lymphoma as well as lymphoid hyperplasia. In a report of 8 cats diagnosed with T-cellrich B-cell lymphoma, all had enlargement of a single submandibular or cervical lymph node. Lymph node enlargement occurred rapidly in 3 of the cats and there was no evidence of disease 6 months after surgical removal in 3 cats. The histopathologic features of the abnormal lymph nodes from those cats were identical to those of nodular lymphocyte predominant (lymphocytic and histiocytic) Hodgkin’s lymphoma in people.1 In another review of 20 cats with what the authors defined as feline Hodgkin’s-like lymphoma based on histopathologic similarities to Hodgkin’s lymphoma in humans, the most frequent clinical presentation was unilateral or bilateral mandibular or cervical lymphadenopathy. Follow-up was available for 15 cats, 11 of which had no treatment and lived from 7 months to 4 years or were euthanized for a disease other than lymphoma. In these cats, histopathology revealed phenotypic heterogeneity of the cells, which included T and B lymphocytes, Reed-Sternberg cells, lacunar cells, and lymphohistiocytic variants. The majority of cases resembled lymphocyte predominant

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Hodgkin’s lymphoma or mixed cellularity Hodgkin’s lymphoma.2 Although the slow progression of the mass in the cat in this report could fit with the clinical course reported for feline Hodgkin’s-like lymphoma, its cystic nature, the negative PARR results, and the absence of Reed-Sternberg cells, lacunar cells, or lymphohistiocytic cells in cytology or histopathology specimens made a diagnosis or T-cell rich B-cell lymphoma unlikely.4–5 Lymphoid hyperplasia also was considered unlikely based on the size of the mass and the immunohistochemistry results. The Hassall’s corpuscles and cytokeratin-positive cells in the histologic sections were consistent with a mass of thymic origin. Unfortunately, in the original fine needle aspirate specimens of the mass, no evidence of epithelial tissue was found so immunocytochemical staining for cytokeratin was not performed. Cytokeratin-positive cells in cytology specimens in conjunction with the CD4CD8 double-positive lymphocyte population by flow cytometry would have provided a preoperative diagnosis of thymoma. The presence of thymic tissue in the cervical region could represent metastasis from a thymoma, local extension of a mediastinal thymoma derived from the orthotopic thymus, or neoplastic transformation of ectopic thymic tissue. Lack of involvement of the associated lymph node by gross and histologic evaluation, and the absence of a cranial mediastinal mass on ultrasound and thoracic radiographs, made metastasis and local extension less likely, and supported the diagnosis of a thymoma originating in ectopic thymic tissue. Ectopic thymic tissue may be found anywhere from the proximal neck, where it develops anatomically during fetal life, through its caudal pathway of migration to the anterior mediastinum, its final position at birth. Cervical ectopic thymic hyperplasia has been reported in children and less frequently in adults, with a higher prevalence on the left side and in males.6–7 Eighty to 90% of the lesions in people are asymptomatic; 6% cause clinical signs such as stridor, dyspnea, and dysphagia due to compression of the trachea or esophagus. Recommended treatment is complete surgical resection, because the lesions can undergo neoplastic transformation. The prognosis after removal is excellent, with no recurrences reported.7–8 Cervical ectopic thymic tissue has been described recently in laboratory mice, in which it may explain the failure of some strains to develop peripheral lymphopenia and the absence of tolerance following neonatal thymectomy (ectopic thymic tissue anatomically and functionally resembles the thoracic thymus). The prevalence of ectopic thymus varied from 33% to 90%, depending on the strain.9,10


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Thymomas are tumors of thymic epithelial origin with variable, sometimes marked infiltration of nonneoplastic lymphocytes. In human patients, 30%–87% of lymphocytes are CD4CD8 double positive, which except for a stage in normal thymocyte maturation is an otherwise unusual pattern of surface antigen expression. Flow cytometry has been shown to be highly accurate in distinguishing mediastinal or ectopic thymoma from lymphoma in people.11,12 In a study in which flow cytometry and PARR was performed in samples from dogs with mediastinal thymoma or lymphoma, all but one of the dogs with thymoma had 4 40% (range 10%–65%) CD4CD8 double-positive lymphocytes and clonal lymphocyte populations were not observed. Of the dogs with lymphoma, only 1 had lymphocytes that co-expressed CD4 and CD8; that dog also had a clonal rearrangement of the T-cell receptor gene.13 In our case, 73% of the lymphocytes in the mass were CD4CD8 doublepositive and there was no clonal rearrangement of antigen receptor genes, which demonstrates that flow cytometry and PARR can also be a useful diagnostic tool to diagnose ectopic thymoma and rule out lymphoma preoperatively in cats. Thymomas are rare in cats. The most common presentation is a mediastinal mass, usually in old cats (median age, 10 years). Most thymomas are benign, but this space occupying disease can cause severe clinical signs due to impairment of respiratory capacity or compression of surrounding vital structures. According to prior studies, thymomas in cats have a low metastatic rate of around 20%, with metastases to hilar lymph nodes and lung most commonly reported. This makes surgical excision the treatment of choice with the potential of cure for cats with this neoplasm.14–16 Only 2 cases of thymomas in cats have been reported arising from ectopic thymic tissue. In one cat, the neoplasm was located in the pericardial sac without evidence of a mediastinal mass. In the other cat, the thymoma involved the pericardial sac as well as the anterior mediastinum, making it difficult to determine the primary site.17 Ectopic thymoma in people can be found in the cervical region, thyroid gland, lungs, and pleura. In humans, o 30 cases of ectopic cervical thymoma have been described.17 Interestingly, although ectopic thymic tissue is more commonly found in males, there is a female predisposition for cervical thymoma, with a female to male ratio of 9:1. The age of presentation is 50 years or older. Patients present with a cervical mass, which often is diagnosed as a thyroid nodule. The prognosis after removal is good.18,19 To our knowledge, ectopic cervical thymoma has not been described previously in cats. Flow cytometry


and PARR, both of which can be performed on cells from a lymph node aspirate,20,21 can be useful noninvasive diagnostic tools to rule out lymphoma and establish a diagnosis of thymoma based on a high proportion of CD4CD8 double-positive lymphocytes and lack of clonal rearrangements of lymphocyte antigen receptor genes. This is important because in addition to the more favorable prognosis for thymic neoplasms, the treatment of choice for thymomas is surgery, whereas lymphomas are usually treated with chemotherapy.

Acknowledgments The authors would like to thank Sarah Leavell for her excellent FACS technical assistance.

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