Anaplastic large-cell lymphoma or null-cell lymphoma is a clinical entity reported in people, classified according to the unique appearance of large pleomorphic cells that express CD30. Null-cell lymphoma has also been described in dogs when neither CD3 nor CD79α is expressed by the tumor. We describe a case of lymphoma in the dog in which neoplastic cells did not express routine B- or T-lymphocyte markers on flow cytometry or immunohistochemistry; however, cells immunohistochemically labeled for CD30. The dog in our case died 5 mo after initial presentation, confirming a poor prognosis. Identification of further similar cases in dogs would provide additional prognostic information for this subset of lymphomas. CD30 may also serve as a potential therapeutic target in anaplastic large-cell lymphomas

Garden, O A

Pittaway, R

Polton, G

Priestnall, S L

Scurrell, E J

Suárez-Bonnet, A

Szladovits, B

Wu, Y

English

 Anaplastic large-cell lymphoma or null-cell lymphoma is a clinical entity reported in people, classified according to the unique appearance of large pleomorphic cells that express CD30. Null-cell lymphoma has also been described in dogs when neither CD3 nor CD79α is expressed by the tumor. We describe a case of lymphoma in the dog in which neoplastic cells did not express routine B- or T-lymphocyte markers on flow cytometry or immunohistochemistry; however, cells immunohistochemically labeled for CD30. The dog in our case died 5 mo after initial presentation, confirming a poor prognosis. Identification of further similar cases in dogs would provide additional prognostic information for this subset of lymphomas. CD30 may also serve as a potential therapeutic target in anaplastic large-cell lymphomas. </jats:p

Rachel Pittaway

Ying Wu

Balazs Szladovits

Alejandro Suárez-Bonnet

Emma J. Scurrell

Oliver A. Garden

Gerry Polton

Simon L. Priestnall

Crossref

Journal of Veterinary Diagnostic Investigation

Diagnosis of anaplastic large-cell lymphoma in a dog using CD30 immunohistochemistry

Pittaway, Rachel

Wu, Ying

Szladovits, Balazs

Suárez-Bonnet, Alejandro

Scurrell, Emma J.

Garden, Oliver A.

Polton, Gerry

Priestnall, Simon L.

accedaCRIS (Universidad de Las Palmas de Gran Canaria)

RVC Repository (Royal Veterinary College)

   RVC OPEN ACCESS REPOSITORY – COPYRIGHT NOTICE  This is the author’s accepted manuscript of an article published in the Journal of Veterinary Diagnostic Investigation.  The final publication is available at SAGE Journals via https://doi.org/10.1177/1040638718760965.  The full details of the published version of the article are as follows:  TITLE: Diagnosis of anaplastic large-cell lymphoma in a dog using CD30 immunohistochemistry  AUTHORS: Rachel Pittaway, Ying Wu, Balazs Szladovits, Alejandro Suárez-Bonnet, Emma J. Scurrell, Oliver A. Garden, Gerry Polton, Simon L. Priestnall JOURNAL TITLE: Journal of Veterinary Diagnostic Investigation PUBLICATION DATE: 19 February 2018 (online) PUBLISHER: SAGE Publications DOI: 10.1177/1040638718760965   Page 1 of 13 Diagnosis of anaplastic large-cell lymphoma in a dog using CD30 1 immunohistochemistry 2  3 Rachel Pittaway,1* Ying Wu,* Balazs Szladovits, Alejandro Suárez-Bonnet, Emma J. 4 Scurrell, Oliver A. Garden, Gerry Polton,† Simon L. Priestnall† 5  6 Departments of Pathobiology and Population Sciences (Pittaway, Szladovits, Suárez-Bonnet, 7 Priestnall) and Clinical Science and Services (Wu, Garden), The Royal Veterinary College, 8 University of London, North Mymms, United Kingdom; North Downs Specialist Referrals 9 Bletchingley, Surrey, United Kingdom (Polton); CytoPath Ltd, Ledbury, Herefordshire, 10 United Kingdom (Scurrell). Current address: Department of Clinical Sciences and Advanced 11 Medicine, University of Pennsylvania School of Veterinary Medicine, Matthew J Ryan 12 Veterinary Hospital, Philadelphia, PA (Garden). 13  14 1Corresponding author: Rachel Pittaway, Department of Pathobiology and Population 15 Sciences, The Royal Veterinary College, University of London, Hawkshead Lane, North 16 Mymms, UK. rpittaway@rvc.ac.uk 17 * Joint first authors. 18 † Joint last authors. 19  20 Short running title: Diagnosis of canine ALCL using CD30 immunohistochemistry 21 22 Page 2 of 13 Abstract. Anaplastic large-cell lymphoma or null-cell lymphoma is a clinical entity reported 23 in people, classified according to the unique appearance of large pleomorphic cells that 24 express CD30. Null-cell lymphoma has also been described in dogs when neither CD3 nor 25 CD79α is expressed by the tumor. We describe a case of lymphoma in the dog in which 26 neoplastic cells did not express routine B- or T-lymphocyte markers on flow cytometry or 27 immunohistochemistry; however, cells immunohistochemically labeled for CD30. The dog in 28 our case died 5 mo after initial presentation, confirming a poor prognosis. Identification of 29 further similar cases in dogs would provide additional prognostic information for this subset 30 of lymphomas. CD30 may also serve as a potential therapeutic target in anaplastic large-cell 31 lymphomas. 32  33 Key words: CD30; dogs; immunohistochemistry; lymphoma; null-cell lymphoma. 34 35 Page 3 of 13 A 10-y-old male neutered Beagle dog was presented to North Downs Specialist Referrals 36 (Bletchingley, Surrey, UK) with unilateral right-sided mandibular lymphadenopathy. Prior 37 medical history included dorsal laminectomy and disc fenestration of an intervertebral disc 38 extrusion (T13/L1) the preceding year. He had also been diagnosed with sciatic neuritis by 39 histologic examination of a sciatic nerve biopsy specimen 9 mo prior to presentation. The dog 40 had received intermittent treatment with prednisolone and cyclosporine. Three months before 41 presentation, the dog developed severe anemia as a result of gastrointestinal hemorrhage, and 42 prednisolone therapy was stopped. 43 Hematologic examination at the time of presentation indicated no evidence of 44 cytopenias, and cytologic evaluation of a fine-needle aspirate (FNA) of the enlarged 45 mandibular lymph node was carried out by a board-certified veterinary clinical pathologist (B 46 Szladovits). The nucleated cell population was dominated by medium-to-large lymphocytes 47 (Fig. 1). Cells had mostly round, occasionally indented, and rarely lobed, central-to-48 paracentral nuclei that were typically 1.5–2× and occasionally ≥3× the size of red blood cells. 49 There was moderate anisokaryosis and a finely granular to mildly clumped chromatin pattern. 50 Nuclei had moderately distinct, small-to-medium–sized nucleoli, and occasional cells had a 51 single large nucleolus. Cells had unusually large amounts of mid-blue cytoplasm with low-to-52 moderate numbers of small vacuoles, and prominent perinuclear clearing (Golgi zone), 53 commonly around the entire nucleus. The mitotic rate was high (≥3 per five 40× fields). 54 Small lymphocytes were rare; occasional neutrophils, rare eosinophils, and rare plasma cells 55 were present. A diagnosis of lymphoma was considered most likely; however, the cellular 56 morphology was considered unusual given the large amount of cytoplasm within the cells. 57 Thoracic radiography and abdominal ultrasonography were unremarkable. Bone marrow 58 evaluation was not performed, and the patient was diagnosed as having stage 1 lymphoma 59 Page 4 of 13 according to the World Health Organization’s clinical staging system for lymphoma in 60 domestic animals.4 61 Flow cytometric analysis was carried out on the FNA samples of the enlarged lymph 62 node in order to immunophenotype the lymphoma. Samples were stained with canine-specific 63 or cross-reactive fluorochrome-conjugated monoclonal antibodies (mAbs) against both extra- 64 and intracellular antigens (Table 1). Seven-color staining was performed. Cells were stained 65 with mAbs for cell surface antigens for 20 min. Following incubation in buffer (Intracellular 66 fixation and permeabilization buffer, Thermo Fisher Scientific, Paisley, UK) overnight, the 67 cells were stained with mAbs against intracellular antigens for 30 min. All incubations were 68 performed at 4°C in the dark, and cells were washed twice following each of the incubation 69 periods. Flow cytometric data were acquired (FACS Canto II flow cytometer, BD 70 Biosciences, Oxford, UK) and analyzed (Flowjo software, Tree Star, Ashland, OR). All gates 71 were defined by using isotype and “fluorescence minus one” controls. Flow cytometric 72 analysis of viable cells of the FNAs yielded the impression of a predominantly medium-sized 73 population, consistent with the cytologic appearance. The population was negative for all 74 lymphoid antigens, with a high proportional expression of the proliferation marker Ki-67. 75 The enlarged lymph node was surgically excised and, following fixation in 10% 76 neutral-buffered formalin, was processed routinely and embedded in paraffin wax. Sections 77 (4 µm) were stained with hematoxylin and eosin. Histologically, ~90% of the node was 78 effaced by a dense, highly infiltrative, nonencapsulated, poorly differentiated round-cell 79 neoplasm. The neoplastic cells were round-to-oval with distinct cell borders and variable 80 amounts of eosinophilic cytoplasm that usually contained a single, hyperchromatic nucleus 81 that was 2–3× the size of a red blood cell. There was marked anisocytosis and anisokaryosis, 82 including frequent macrokaryosis (Fig. 2). Frequently, some cells exhibited large vesicular 83 nuclei with distinct indentations or binucleation with multiple distinct, magenta, ovoid 84 Page 5 of 13 nucleoli. Fifteen mitotic figures were observed within 10 high-power (400×, 2.37 mm2) 85 fields, including occasional bizarre mitoses. Numerous individual neoplastic cells were either 86 necrotic or apoptotic. There were many fewer small (mature) lymphocytes within the 87 remaining nodal architecture and large central areas of coagulative necrosis (tumor necrosis 88 and/or the result of prior FNA sampling). The capsule was thickened by fibrous connective 89 tissue that was infiltrated by moderate numbers of small (reactive) lymphocytes and small 90 foci of neutrophils. Subcapsular and medullary sinuses were congested or contained low 91 numbers of hemosiderophages. The neoplastic tissue appeared contained by the thickened 92 capsule within the sections examined. A diagnosis of high-grade large-cell lymphoma was 93 given. 94 Immunohistochemistry (IHC) was performed on serial sections (Bond-Max autostainer, 95 Bond polymer refine detection system, Leica Biosystems, Newcastle-upon-Tyne, UK). 96 Primary antisera were specific for CD3 (polyclonal rabbit anti-human, Dako, Ely, UK; 1 in 97 500 dilution; antigen retrieval in buffer pH 9.0 buffer [ER2, Dako] for 30 min); CD79α 98 (monoclonal mouse anti-human, Dako; 1 in 100 dilution; antigen retrieval in pH 9.0 buffer 99 [ER2] for 10 min); CD18 (mouse anti-canine, clone CA1.4E9, University of California 100 Davis, CA; 1 in 20 dilution; with enzymatic antigen retrieval [Enzyme 1, Dako] for 10 min); 101 and CD30 (monoclonal mouse anti-human, clone Ber-H2, Dako; 1 in 30 dilution; antigen 102 retrieval in pH 9.0 buffer for 20 min). Immunohistochemically, the neoplastic cells diffusely 103 exhibited specific membranous labeling for CD30 (Fig. 3) and did not express CD3, CD79α, 104 or CD18. Negative (Fig. 4) and positive controls were processed with the evaluated slides and 105 were labeled appropriately. A diagnosis of high-grade, poorly differentiated null-cell 106 lymphoma or anaplastic large-cell lymphoma (ALCL) was given. 107 Two months after lymph node removal, lymphadenopathy was noted in the same 108 anatomic site. Cytologic evaluation confirmed a diagnosis of lymphoma. Blood evaluations, 109 Page 6 of 13 thoracic radiography, and abdominal ultrasonography were repeated. Bone marrow aspirate 110 and biopsy of inflamed gingivae were also performed with no evidence of lymphoma at other 111 sites. 112 Treatment was implemented with a modified L-CHOP protocol. No measurable 113 improvement was noted in response to single L-asparaginase (400 IU/kg IM; Medac, Wedel, 114 Germany), vincristine (0.7 mg/m2 IV; Hospira UK, Warwickshire, UK), or 115 cyclophosphamide (250 mg/m2 PO; Star Pharmaceuticals, Harrow, UK) treatments, so the 116 protocol was abandoned. Further treatments using doxorubicin (30 mg/m2 IV; Pharmachemie, 117 Haarlem, The Netherlands), lomustine (70 mg/m2 PO; Medac), and chlorambucil (5 mg/m2 118 q48h PO; Aspen Pharma, Dublin, Ireland) also failed to achieve a beneficial effect. The 119 patient underwent a course of palliative radiation therapy (5× once wk 7 Gy fractions) by 120 which time lymphoma was also noted in the left mandibular and the right prescapular lymph 121 nodes. All detectable lymphoid tissues responded favorably to radiotherapy; however, 122 complete remission was not achieved. 123 While receiving treatment, a mass arose on the left upper eyelid and, 2 d after 124 completion of the radiotherapy course, the patient showed markedly reduced alertness. The 125 patient was anesthetized for MRI of the head and neck and cerebrospinal fluid analysis, 126 which gave no indication of lymphoma in the central nervous system; however, there were 127 multiple enlarged lymph nodes of the head and neck. The eyelid mass was removed and was 128 composed of neoplastic cells similar to those described within the mandibular lymph node. A 129 plasma cell origin to this neoplasm was also considered at this stage; however, cells did not 130 exhibit immunohistochemical labeling for MUM-1 (performed at an external commercial 131 laboratory; MUM-1/IRF-4 monoclonal mouse anti-human, Dako; 1 in 100 dilution; antigen 132 retrieval in pH 9.0 buffer [Envision FLEX target retrieval solution, Dako] for 30 min). 133 Neoplastic cells exhibited similar expression of CD30 and did not express CD3 or CD79a. 134 Page 7 of 13 The patient failed to make a satisfactory recovery from anesthesia and died 3 d later. Autopsy 135 was not performed. 136 ALCL was first recognized as a clinical entity in people in 1985 and was classified 137 according to the unique appearance of large pleomorphic cells that express CD30, originally 138 termed Ki-1.3 ALCL has previously been described in dogs, with 0.8% of 608 cases of canine 139 lymphoma being classified as null-cell type given the lack of expression of either CD3 or 140 CD79α.6 Further reports of null-cell lymphomas in dogs are rare. The use of CD30 in dogs 141 has been described in a study characterizing testicular neoplasms, in which CD30 was not 142 expressed,8 and in a case of canine pulmonary lymphomatoid granulomatosis in which a 143 population of cells expressed CD30.5 CD30 is a transmembrane protein receptor, of the tumor 144 necrosis factor receptor superfamily, which is normally expressed by activated B- or T-145 lymphocytes.2,7 Upon activation, CD30 influences cell growth and survival.1 146 We suggest that in cases in which lymphoma is strongly suspected histologically, 147 despite neoplastic cells failing to express routine T- or B-lymphocyte markers, CD30 may be 148 used to provide a diagnosis of null-cell lymphoma or ALCL. Given positive CD30 expression 149 and the unique appearance of large, pleomorphic neoplastic cells, a diagnosis of ALCL was 150 made. Identification of further cases of ALCL in dogs would provide additional prognostic 151 information for this subset of lymphomas. Therapeutics that target CD30 in people with 152 Hodgkin lymphoma and ALCL have been developed.1 The dog in our case had a survival 153 time of 5 mo following initial presentation, despite both chemotherapy and radiotherapy, 154 confirming a poor prognosis. 155 Declaration of conflicting interests 156 The authors declare no potential conflicts of interest with respect to research, authorship, and 157 publication of this article. 158 Funding 159 Page 8 of 13 Funding for this project was provided, in part, by the Petplan Charitable Trust (Wu). 160 References 161 1. Ansell SM. Brentuximab vedotin. Blood 2014;124:3197–3200. 162 2. Chiarle R, et al. CD30 in normal and neoplastic cells. J Clin Immunol 1999;90:157–164. 163 3. Jacobsen E. Anaplastic large-cell lymphoma, T-/null-cell type. Oncologist 2006;11:831–164 840. 165 4. Owen LN, ed. TNM Classification of Tumors in Domestic Animals. 1st ed. Geneva, 166 Switzerland: World Health Organization, 1980 167 5. Park HM, et al. Pulmonary lymphomatoid granulomatosis in a dog: evidence of 168 immunophenotypic diversity and relationship to human pulmonary lymphomatoid 169 granulomatosis and pulmonary Hodgkin’s disease. Vet Pathol 2007;44:921–923. 170 6. Ponce F, et al. A morphological study of 608 cases of canine malignant lymphoma in 171 France with a focus on comparative similarities between canine and human 172 lymphoma morphology. Vet Pathol 2010;47:414–433. 173 7. Sabattini E, et al. CD30 expression in peripheral T-cell lymphomas. Haematologica 174 2013;98:e81–82. 175 8. Yu CH, et al. Comparative immunohistochemical characterization of canine seminomas 176 and Sertoli cell tumors. J Vet Sci 2009;10:1–7. 177 178 Page 9 of 13 Table 1. Flow cytometry: staining antibodies, isotype controls, and fluorochromes. 179 Antigen Source Clone Isotype Fluorochrome CD3 Bio-Rad CA17.2A12 Mouse IgG1 FITC CD4 Thermo Fisher YKIX302.9 Rat IgG2a PE-Cyanine7 CD5 Bio-Rad YKIX322.3 Rat IgG2a PE CD8 Thermo Fisher YCATE55.9 Rat IgG1 PerCP-eFluor 710 CD21 Bio-Rad CA2.1D6 Mouse IgG1 Alexa Fluor 647 CD34 Bio-Rad 1H6 Mouse IgG1 PE CD45 Thermo Fisher YKIX716.13 Rat IgG2b eFluor 450 CD79b Bio-Rad AT107-2 Rat IgG1 FITC Ki-67 Thermo Fisher SolA15 Rat IgG2a eFluor 450 FITC = fluorescein isothiocyanate; PE = phycoerythrin. Sources: Bio-Rad, Watford, 180 Hertfordshire, UK; Thermo Fisher Scientific, Paisley, Renfrewshire, UK. 181 182 Page 10 of 13 Figure 1. A population of medium-to-large lymphocytes on cytologic evaluation of 183 mandibular lymph node aspirates from a dog. Modified Wright stain. Bar = 20 μm. 184  185   186 Page 11 of 13 Figure 2. Neoplastic cells within the mandibular lymph node of a dog exhibit marked 187 anisocytosis and anisokaryosis. Nucleoli are prominent, and mitotic figures are present. 188 H&E. Bar = 20 μm. 189  190   191 Page 12 of 13 Figure 3. Neoplastic cells within the mandibular lymph node of a dog exhibit specific 192 membranous labeling following CD30 immunohistochemistry. Bar = 20 μm. 193  194   195 Page 13 of 13 Figure 4. Neoplastic cells within the mandibular lymph node of a dog do not label for CD30 196 immunohistochemistry in the negative control. Bar = 20 μm. 197  198 

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Diagnosis of anaplastic large-cell lymphoma in a dog using CD30 immunohistochemistry

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