Journal of Veterinary Diagnostic Investigation

http://vdi.sagepub.com/ Gastric B-Cell Lymphoma with Mott Cell Differentiation in a Dog

Gabrita De Zan, Valentina Zappulli, Laura Cavicchioli, Linda Di Martino, Eriberta Ros, Giorgia Conforto and Massimo Castagnaro J VET Diagn Invest 2009 21: 715 DOI: 10.1177/104063870902100521 The online version of this article can be found at: http://vdi.sagepub.com/content/21/5/715

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J Vet Diagn Invest 21:715719 (2009)

Gastric B-cell lymphoma with Mott cell differentiation in a dog

Gabrita De Zan,1 Valentina Zappulli, Laura Cavicchioli, Linda Di Martino, Eriberta Ros, Giorgia Conforto, Massimo Castagnaro
Abstract. A gastric lymphoid tumor with involvement of regional lymph nodes and spleen was diagnosed in an 8-year-old crossbreed male dog with a 6-month history of gastrointestinal disease. Despite surgical excision and palliative therapy (prednisolone and cimetidine), the dog was euthanized due to worsening of clinical signs. At necropsy, multiple white, solid, nodular, infiltrative masses were observed in the stomach, duodenum, spleen, liver, and lungs in association with generalized lymph node enlargement. Cytology, histology, histochemistry, immunohistochemistry, and electron microscopy revealed that the neoplastic cell population was composed of B lymphocytes that contained variable amounts of round periodic acidSchiffpositive cytoplasmic globules consistent with Russell bodies. The tumor most likely represented a variant of Bcell neoplasia with extensive Mott cell differentiation. Key words: B-cell lymphoma; dogs; electron microscopy; immunohistochemistry; Mott cell. origin, in contrast to the primarily B-cell origin of intestinal lymphomas in humans.1 An 8-year-old crossbreed male dog was presented for clinical examination with a 6-month history of vomiting, ptyalism, anorexia, weight loss, hematemesis, and melena, which had worsened in the previous 2 months despite symptomatic treatment. On clinical examination, the dog was in poor general condition with deep-seated epigastric pain and abdominal distension. Routine hematology revealed mild leukocytosis (13,500/ml; reference [ref.] interval: 6,00012,000/ml), slight lymphopenia (1,350/ml; ref. interval: 1,4403,600/ml), and moderate monocytosis (1,485/ml; ref. interval: 0480/ml) consistent with a stress leukogram. All values in the biochemical profile were within the reference interval. Survey abdominal radiographs showed a focal perigastric opacity, and abdominal ultrasound revealed an infiltrative, hyperechogenic, 8 cm 3 4 cm transmural mass involving most of the gastric fundus and a 2-cm diameter perigastric mass that was consistent with an enlarged perigastric lymph node.

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Lymphomas are one of the most common tumors of dogs, and the 2 dominant anatomic forms are multicentric and intestinal lymphoma.5 In addition, lymphoma is the most frequently reported intestinal tumor of hematopoietic origin in the dog. According to a 2004 report, primary gastrointestinal lymphomas of dogs are usually of T-cell

From the Dipartimento di Sanita ` Pubblica, Patologia Comparata ed Igiene Veterinaria, Facolta ` di Medicina Veterinaria, Universita ` degli Studi di Padova, Agripolis, Legnaro, Padova, Italy (De Zan, Zappulli, Cavicchioli, Castagnaro); Clinica Veterinaria Privata San Marco, Laboratorio dAnalisi Veterinarie San Marco, Padova, Italy (Di Martino); and Ambulatorio veterinario dott.sse Ros e Conforto, Pordenone, Italy (Ros, Conforto). 1 Corresponding Author: De Zan Gabrita, Dipartimento di Sanita ` Pubblica, Patologia Comparata ed Igiene Veterinaria, Facolta ` di Medicina Veterinaria, Universita ` degli Studi di Padova, Agripolis, Viale dellUniversita ` , 16, 35020 Legnaro, Padova, Italy. gabrita.dezan@unipd.it

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Figure 1. A heterogeneous cell population is composed mainly of atypical lymphoid cells containing variable numbers of clear globules consistent with Russell bodies (arrow), which are also distributed in the background of the preparation. There are scattered immature lymphocytes (arrowhead) and rare plasma cells. Numerous free nuclei are also present. Fine-needle aspirate; B-cell lymphoma with Mott cell differentiation; gastric wall; dog. May-Gru nwald/Giemsa stain. Bar 5 40 mm.

Figure 2. A dense infiltration of irregularly round lymphoid cells with variable numbers of round cytoplasmic globules (arrows) are present. Histologic section; B-cell lymphoma with Mott cell differentiation; gastric wall; dog. Hematoxylin and eosin. Bar 5 10 mm.

Multiple ultrasound-guided fine-needle aspirations were obtained from the masses and routinely stained with MayGru nwald/Giemsa. The gastric mass and the suspected enlarged perigastric lymph node were surgically excised and submitted for histology. Palliative therapy with prednisolone (1 mg/kg/ twice a day) and cimetidine (5 mg/kg/3 times a day) was administered. Abdominal ultrasonography was repeated after 2 months and revealed a focal perigastric hyperechogenic area, possibly consisting of a second enlarged perigastric lymph node, and a 1-cm diameter, splenic nodule. Ultrasound-guided fine-needle aspirates were collected from both lesions. After 7 months of clinical recovery, gastrointestinal clinical signs recurred and the owner requested euthanasia. Postmortem examination revealed approximately 200 ml of serous thoracic effusion and multiple white, solid, round, 0.25-cm diameter, infiltrative masses involving the gastric wall, duodenum, spleen, liver, and lungs. The right axillary, sternal, para-aortic, hepatic, gastric, and mesenteric lymph nodes were enlarged with fatty cut surfaces and obscured cortico-medullary junctions. Selected tissues samples obtained at surgery biopsy and necropsy were fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at 3-mm, and routinely stained with hematoxylin and eosin. Replicate tissue sections also were stained by the periodic acidSchiff reaction and with Giemsa stain for the gastric samples. Immunohistochemistry was performed on replicate sections of stomach, lymph nodes, spleen, and lung. The primary antibodies recognized the following antigens: cluster of differentiation (CD)3,a CD5,a CD20,c CD45,c CD45RA,c CD79acy,a myeloid/histiocytic antigen,a B-lymphocyte antigen 36b; immunoglobulin A (IgA),c IgG,c and IgM.c A portion of formalin-fixed, paraffin-embedded, perigastric

lymph node was submitted for transmission electron microscopy (kindly performed by Prof. M. L. Valente, School of Medical and Surgical Sciences, Padova, Italy). Despite preservation artifacts, cytologic specimens revealed a heterogeneous cellular population of 1020-mm diameter round cells with indistinct borders and moderate to abundant amounts of cytoplasm. The cytoplasm was densely packed with numerous well-demarcated 0.31-mm diameter clear globules that were morphologically consistent with Russell bodies and often obscured nuclear detail. Individual nuclei measured 715 mm in diameter and were irregularly round with a fine chromatin pattern. Numerous cells showed an immature lymphoid or plasmacytoid differentiation together with variable numbers of intracytoplasmic clear vacuoles or globules consistent with Russell bodies. Mitotic figures were rare, and numerous disseminated clear globules (Russell bodies) were present in the background. Immature lymphocytes and plasma cells were rare. Numerous free nuclei were also present (Fig. 1). Histologic examination of gastric wall, duodenum, spleen, liver, lungs, kidney, and lymph nodes tissue revealed a dense, infiltrative, round cell population that was diffusely distributed or separated into nodular aggregates by minimal to thick fibrovascular stroma. The architecture of the lymph nodes was severely and diffusely effaced. In the stomach, there was a transmural distribution with occasional mucosal erosions. In the duodenum, focally extensive nodular infiltrates were observed within the muscular layers. Neoplastic cells were arrayed in multifocal to coalescing, irregularly round nodules that invaded the parenchyma of the spleen, lung, and liver. In the kidney, multifocal infiltrates of neoplastic cells were observed beneath the capusule and within the interstitium. Histologically, the neoplastic cells were morphologically similar to those seen in cytologic preparations (Fig. 2). These were mainly round cells, ranging in size from 7 to 15 mm in diameter with occasional cells reaching 50 mm in diameter. They also had variable amounts of cytoplasm filled with intensely eosinophilic, periodic acidSchiff-

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positive, 110-mm diameter Russell bodies that frequently obscured the nucleus. Nuclei were irregularly round, 57-mm diameter, rarely indented, and vesicular or with clumped chromatin and contained multiple eosinophilic nucleoli. Mitotic figures were rare (02 per 403 field of view). Intermixed with this population were occasional immature lymphoid or plasmacytoid cells. The former were characterized by a high nuclear-to-cytoplasmic ratio, scant basophilic cytoplasm, and round or indented nucleus that was 714 mm in diameter with a fine chromatin pattern. Plasmacytoid cells contained scant, homogenous, basophilic cytoplasm that at times exhibited a clear perinuclear halo. Individual nuclei were centrally located, round, and approximately 10 mm in diameter and contained hyperchromatic, clumped chromatin. Both the lymphoid and plasmacytoid cells contained variable numbers of Russell bodies. Similar to the cytologic findings, rare immature lymphocytes and plasma cells also were observed. Multifocal necrosis and occasional vascular invasion were noted. Giemsa staining for the detection of Helicobacter spp. organisms in the gastric samples was negative. After immunohistochemical staining, the neoplastic lymphocytes had diffuse, specific but variable, cytoplasmic reactivity with anti-CD79acy, antiB-lymphocyte antigen 36, anti-CD45, anti-CD45RA, and anti-CD20 antibodies. Scattered lymphocytes and lymphoid aggregates that were intermingled with the neoplastic cell population had moderate cytoplasmic staining with anti-CD3 antibodies and intense cytoplasmic staining with anti-CD5 antibodies. Scattered histiocytic cells stained with the anti-myeloid/ histiocyte antigen antibody. Anti-IgM, anti-IgA, and antiIgG antibodies produced inconsistent, weak cytoplasmic staining and, therefore, were considered negative. Despite some cellular disruption due to formalin-fixation and paraffin embedding, ultrastructural examination of 1 perigastric lymph node allowed the identification of variable numbers of osmiophilic, cytoplasmic structures within ectatic cisternae of the rough endoplasmic reticulum. These cells were identified as Mott cells (Fig. 3). Rare plasma cells and immature lymphocytes were also observed. Based on the cytologic, histologic, immunohistochemical, and ultrastructural findings, the present case was diagnosed as a lymphoid neoplasm with diffuse Mott cell differentiation. This tumor most likely originated from the gastric wall and resulted in multiple visceral and lymph node metastases. Specific cytoplasmic staining for CD79acy, B-lymphocyte antigen 36, CD45, CD45RA, and CD20 polypeptides indicated that the neoplastic lymphocytes were of B-cell origin. A similar lymphoid neoplasm has been infrequently described in humans and diagnosed by some authors as a Mott cell tumor.3,13 Only 2 cases have been diagnosed recently: 1 neoplasm originated in the stomach in association with Helicobacter pylori infection,3 and 1 neoplasm originated in the rectum.13 An additional gastric Mott cell neoplasm also has been reported by Russian investigators.7 In human medicine, an unusual gastric lesion called Russell body gastritis occurs in association with H. pylori infection. This form of gastritis is characterized by localized

Figure 3. Electron micrograph with 5 irregularly round cells that contain variable amounts of cytoplasmic osmiophilic material within ectatic cisternae of the rough endoplasmic reticulum (arrows) consistent with Russell bodies. These cells were identified as Mott cells. Electron micrograph; B-cell lymphoma with Mott cell differentiation; perigastric lymph node; dog. Bar 5 2 mm.

accumulations of Mott cells and plasma cells. Despite its monomorphic, plasmacytic appearance, this lesion is not considered neoplastic because of its polyclonal immunoreactivity.8 In the neoplasm of the present report, the possibility of a similar inflammatory lesion was considered but excluded based on the presence of metastases, the morphologic appearance, and the absence of Helicobacter spp. microorganisms in gastric samples. In 2008 and 2009, analogous cases of Mott cell tumors were reported in dogs,6,12 and all were characterized by primary involvement of the gastrointestinal tract, as in the present report. However, the tumors in the analogous cases were less advanced than those of the present case. The atypical lymphoid population described in human cases was mixed and consisted of centrocyte-like cells, plasmacytic cells, and Mott cells. In addition, these lesions were associated with peripheral secondary or scattered atrophic lymphoid follicles, leading the authors to postulate a possible origin from the mucosa-associated lymphoid tissue. Thus, mucosa-associated lymphoid tissue lymphoma with marked Mott cell differentiation was suggested as the most likely diagnosis.3,13 Based on plasmacytic differentiation of mucosa-associated lymphoid tissue lymphomas, a differential diagnosis of extramedullary plasmacytoma has also been suggested.4 In the present canine neoplasm, there was no evidence of residual mucosa-associated lymphoid tissue, and an origin from this lymphoid follicular tissue appeared less likely. In addition, the typical morphology of an extramedullary plasmacytoma was not observed9,15 in cytologic samples or

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in histologic sections, excluding the diagnosis of a classical extramedullary plasmacytoma. Moreover, the CD20-positive staining of the neoplastic cells of the current case suggested a B-cell lineage without differentiation to mature plasma cells. Cluster of differentiation 20 is usually expressed in B-cell precursors and in mature B lymphocytes but is normally lost during differentiation towards plasma cells. A 2007 report found CD20 to be the least likely representative marker in canine plasmacytomas.10 No data are available regarding CD20 expression in the few cases of Mott cell tumors reported in humans.3,7,13 Cluster of differentiation 20 detection was described only in the first veterinary report.6 In the same report, immunohistochemical staining for Pax-5 confirmed that the cellular population was lymphoid, leading to speculation of a direct transformation into Mott cells.6 The same conclusion, based on the absence of morphologic and ultrastructural aspects of plasma cell differentiation, was also reported in the second veterinary publication.12 In contrast, the 2 cellular populations in the present case had either immature lymphoid or plasmacytoid features in association with variable amounts of Russell bodies, suggesting different developmental stages toward Mott cells in both cellular populations. The diffuse, intense period acidSchiff staining of the cytoplasmic globules and the ultrastructural localization within ectatic cisternae of the rough endoplasmic reticulum were compatible with Russell body formation. Russell bodies are composed of condensed immunoglobulins within dilated cisternae of the rough endoplasmic reticulum in B-cell populations. They are associated with a high proliferative rate of mature B cells and aberrant formation of mutant and nonsecretable immunoglobulin molecules.14 Russell bodylike structures or intracisternal granules have also been identified in secretory cells that are unable to fully degrade certain insoluble molecules (i.e., zymogen granules of the exocrine pancreas).14 Segregation of these aggregates within the cisternae of the endoplasmic reticulum promotes cellular survival by maintaining the secretory pathway and the cellular traffic.14 The neoplasm of the present report had an unexpected lack of immunohistochemical staining for immunoglobulins. Usually, extramedullary plasmacytomas in dogs stain positively for immunoglobulins, especially monoclonal light-chain reactivity.9 In humans, sporadic Mott cell tumors revealed a diffuse, monotypic IgG kappa chain profile.3,13 Both previous veterinary reports of similar neoplasms had positive staining results for immunoglobulins. The first report described an IgM lambda chain profile of the Mott cells. In the second report, reactivity for IgM was present in the Mott cells of dog 1 while reactivity to both IgG and IgM was observed in the Mott cells of dog 2.6,12 Although immunoglobulin reactivity is a standard and sensitive test for plasma cell identification, some conditions may prevent immunoglobulin detection. Some evidence of low specificity of immunoglobulin detection has been attributed to nonspecific surface immunoglobulin adsorption, internalization of Ig-opsonins complexes within macrophages, and binding of immunoglobulins to a variety of cell types via Fc receptors.11 Too few cases of lymphoid neoplasia

with Mott cell differentiation have been investigated to make definitive conclusions regarding the validity of immunohistochemical detection of immunoglobulins. In the present neoplasm, the possibility of low specificity in immunoglobulin detection cannot be completely excluded. To further classify this unusual neoplasm, the presence or absence of a monoclonal gammopathy should have been assessed, as well as the clonality of the tumor cell population. Unfortunately, these analyses were not performed because serum and urine were not submitted by the clinician for protein electrophoresis and frozen tissue was not collected for molecular analysis. Acknowledgements. The authors thank Prof. P. F. Moore and Dr. V. K. Affolter for performing the immunohistochemical staining and Prof. Maria Luisa Valente and Dr. Mila Della Barbera for performing transmission electron microscopy.

Sources and manufacturers

a. Dako North America Inc., Carpinteria, CA. b. Novocastra Laboratories Ltd., Newcastle upon Tyne, UK. c. Prof. P. F. Moore, University of CaliforniaDavis, Davis, CA.

References
1. Chan JKC, Ng CS, Hui PK, Wong KF: 1995, Tumors of the lymphoreticular system (including thymus). In: Diagnostic histopathology of tumors, ed. Fletcher CDM, vol. II, pp. 805 927. Churchill Livingstone, New York, NY. 2. Coyle KA, Steinberg H: 2004, Characterization of lymphocytes in canine gastrointestinal lymphoma. Vet Pathol 41:141146. 3. Fujiyoshi Y, Inagaki H, Tateyama H, et al.: 2001, Mott cell tumor of the stomach with Helicobacter pylori infection. Pathol Int 51:4346. 4. Hussong JW, Perkins SL, Schnitzer B, et al.: 1999, Extramedullary plasmacytoma: a form of marginal zone cell lymphoma? Am J Clin Pathol 111:111116. 5. Jacobs RM, Messick LB, Valli VE: 2002, Tumors of the hemolymphatic system. In: Tumors in domestic animals, ed. Meuten DJ, 4th ed., pp. 119198. Iowa State Press, Ames, IA. 6. Kodama A, Sakai H, Kobayashi K, et al.: 2008, Bcell intestinal lymphoma with Mott cell differentiation in a 1-year-old miniature Dachshund. Vet Clin Pathol 37:409415. 7. Kokosadze NV, Kovrigina AM, Probatova NA: 2004, [MALT-lymphoma of the stomach with marked plasmocytic differentiation: a variant of Motts cell tumor]. Arkh Pathol 66(5):4042. In Russian. Abstract in English. 8. Paik S, Kim SH, Kim JH, et al.: 2006, Russell body gastritis associated with Helicobacter pylori infection: a case report. J Clin Pathol 59:13161319. 9. Platz SJ, Breuer W, Pfleghaar S, et al.: 1999, Prognostic value of histopathological grading in canine extramedullary plasmacytomas. Vet Pathol 36:2327. 10. Ramos-Vara JA, Miller MA, Valli VEO: 2007, Immunohistochemical detection of multiple myeloma 1/interferon regulatory factor 4 (MUM1/IRF-4) in canine plasmacytoma: comparison with CD79a and CD20. Vet Pathol 44:875884.

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Case Reports 11. Schrenzel MD, Naydan DK, Moore PF: 1998, Leukocyte differentiation antigens in canine cutaneous and oral plasmacytomas. Vet Dermatol 9:3341. 12. Stacy NI, Nabity MB, Hackendahl N, et al.: 2009, B-cell lymphoma with Mott cell differentiation in two young adult dogs. Vet Clin Pathol 38:113120. 13. Tanimoto A, Hamada T, Yamamoto T, et al.: 2002, MALT lymphoma with extreme plasma cell differentiation of the rectum. Am J Gastroenterol 97:18601862.

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14. Valetti C, Grossi CE, Milstein C, Sitia R: 1991, Russell bodies: a general response of secretory cells to synthesis of a mutant immunoglobulin which can neither exit from, nor be degraded in, the endoplasmic reticulum. J Cell Biol 115:983994. 15. Valli VE, Jacobs RM, Parodi AL, et al.: 2002, Plasmacytic tumors. In: Histological classification of hematopoietic tumors of domestic animals, ed. Schulman FY, second series, vol. VIII, pp. 3435. Armed Forces Institute of Pathology, Washington, DC.

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