Veterinary Clinical Pathology ISSN 0275-6382
BRIEF COMMUNICATION
Immunocytochemical detection of the class A macrophage scavenger receptor CD204 using air-dried cytologic smears of canine histiocytic sarcoma Yuki Kato1, Risa Funato1, Akihiro Hirata2,3, Mami Murakami3,4, Takashi Mori3,4, Koji Maruo3,4, Tokuma Yanai1, Hiroki Sakai1,3 1
Laboratory of Veterinary Pathology, Faculty of Applied Biological Sciences, 2Division of Animal Experiment, Life Science Research Centre, Comparative Cancer Centre, and 4Laboratory of Clinical Oncology, Department of Veterinary Medicine, Gifu University, Gifu, Japan
3
Key Words Dog, fine-needle aspiration, immunocytochemistry, neoplasia, round cell tumor, SR-A Correspondence H. Sakai, Laboratory of Veterinary Pathology, Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan E-mail: [email protected] DOI:10.1111/vcp.12190
Background: Cytologic diagnosis of canine histiocytic sarcoma (CHS) can be challenging because neoplastic histiocytes commonly show marked nuclear and cellular atypia and may resemble other pleomorphic malignant round cell tumors. Therefore, even on histopathologic examination, immunostaining is often necessary for a definitive diagnosis. Objectives: The objective of this study was to validate an anti-human CD204 antibody for immunocytochemical staining of air-dried smears for a rapid definitive diagnosis of CHS. Methods: Cytologic specimens were obtained from 10 dogs with CHS and 45 dogs with other tumors. After the cytologic evaluation of modified Giemsa-stained smears, acetone-fixed specimens were immunostained using mouse anti-human CD204 antibodies. All immunocytochemical specimens were assessed blinded and at high-power magnification (9 40 objective) in 10 randomly selected fields per sample. Parameters evaluated were the subjective staining intensity and location, and the proportion of positive cells. Results: All 10 CHS samples showed intense positive staining for CD204 in ≥ 50% of the cells, whereas the 45 other tumors were negative for CD204 staining. Conclusions: Immunocytochemistry of air-dried cytologic smears of CHS for CD204 is useful for a rapid confirmation of a cytologic diagnosis of CHS.
Cytology is being widely used in veterinary medicine, providing valuable information in cases of neoplasia. Cytopathology of neoplastic diseases is especially helpful as a diagnostic tool for round-cell tumors and often confirms the diagnosis.1,2 However, in canine histiocytic sarcoma (CHS), cytomorphology alone does not conclusively establish the origin of cells, although their high number in peripheral blood and atypical features may suggest a neoplastic disease, most likely of histiocytic origin.3–5 Therefore, when CHS is suspected for cytologic diagnosis, histopathologic and immunohistochemical examinations are often required to confirm the diagnosis.4,5 Morphologic features and the CD18+ (or MHC II+), CD3, and CD79a phenotype of the neoplastic cells are the most useful routine diagnostic criteria for a likely histiocytic origin.5
Previously, we showed that the class A macrophage scavenger receptor CD204 was a useful diagnostic marker for CHS.6 CD204 is a major macrophage receptor and plays important roles in host defense and in diseases such as atherosclerosis.7 A monoclonal antibody specific for CD204, clone SRA-E5, has been reported to recognize canine tissue-resident macrophages, including alveolar macrophages, hepatic Kupffer cells, splenic red pulp macrophages, sinus macrophages in lymph nodes, and interstitial macrophages in various organs.6,7 However, dendritic cells (DCs), such as the interdigitating cells of lymphoid tissues, and epidermal Langerhans cells are invariably negative for CD204.7 In this study, we performed CD204 immunocytochemical staining of air-dried smears to confirm
Vet Clin Pathol 43/4 (2014) 589–593 ©2014 American Society for Veterinary Clinical Pathology
589
CD204 immunocytochemistry of histiocytic sarcoma
Kato et al
cytologic diagnoses of CHS. If properly validated and made available, this antibody could be used to diagnose CHS more quickly, less invasively, and potentially at a lower cost. In this study, cases of 10 dogs with CHS and 45 dogs with other neoplastic diseases were retrieved from the database of the Laboratory of Veterinary Pathology, Gifu University, Gifu, Japan, based on their histologic diagnoses. Both cytologic and histologic
evaluation were available for all selected cases between April 2011 and July 2012 at the Gifu University Veterinary Teaching Hospital, Gifu, Japan. Per definition, all CHS cases in this study were CD18+, CD204+, CD3, and CD79a (or CD20), as previously described.5,6 The non-CHS group comprised 13 roundcell tumors (7 mast cell tumors, 4 lymphomas, and 2 extramedullary plasmacytomas), 18 epithelial tumors (6 squamous cell carcinomas, 4 mammary tumors, 3
A
B
C
D
E
F
Figure 1. Cytology and immunocytochemistry of canine histiocytic sarcomas (CHS) for CD204 expression. (A) CHS of inguinal subcutis dog no. 2. Tumor cells are round to polygonal, of variable size, and have variably abundant pale vacuolated cytoplasm. Nuclei were round to oval, and nucleoli were occasionally prominent. Bi- to multinucleated atypical giant cells and mitoses were seen occasionally. Romanowsky-based stain. (B) CD204 immunocytochemistry of DH82 cells derived from a canine CHS case. Note positive cytoplasmic and membranous staining for CD204. (C) CD204 immunocytochemistry of a CHS case in a subiliac lymph node, dog no. 8. Neoplastic histiocytes including multinucleated atypical giant cells show strong cytoplasmic and membranous positivity for CD204. (D–F) CD204 immunocytochemistry of representative non-CHS B-cell lymphoma (D), plasmacytoma (E), and hepatocellular carcinoma (F). CD204-positive macrophages (arrow) in a CD204-negative tumor. (B-F) 3,3’-Diaminobenzidine tetrahydrochloride chromogen. All bars = 50 lm.
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Vet Clin Pathol 43/4 (2014) 589–593 ©2014 American Society for Veterinary Clinical Pathology
Kato et al
CD204 immunocytochemistry of histiocytic sarcoma
hepatocellular carcinomas, 3 adenocarcinomas, and 2 anaplastic carcinomas), and 14 nonepithelial tumors (5 hemangiopericytomas, 3 osteosarcomas, 2 malignant melanomas, 1 seminoma, and 3 undifferentiated sarcomas). These tumors were diagnosed by histopathologic examination and immunohistochemistry (if needed). None of the dogs had received antitumor therapy (eg, chemotherapy and radiation) prior to diagnosis. Fine-needle aspiration specimens or impression smears were used for cytologic diagnosis of the tumors. In addition, the tumors were biopsied using special needles such as trephine or core biopsy needles. Cytologic smears were fixed in absolute methanol for 2 min and then stained with a Romanowsky-based stain (Hemacolor; Merck KGaA, Darmstadt, Germany). Smears were evaluated for cell preservation, cellularity, and features of malignancy. Tissue samples were fixed in buffered formalin and embedded in paraffin. Next, 3lm sections were stained with hematoxylin and eosin (H&E) and submitted for immunohistochemistry when necessary for confirming the diagnosis. Cytologic specimens were air-dried and stored either at 4°C for up to 1 week, or at 20°C for up to 6 months. Storage conditions and time were validated earlier with fine-needle aspiration specimens kept for 1 week at 4°C or 6 months at 20°C, the specimens were collected from cutaneous granuloma and normal liver in dogs. Smears were fixed in cold acetone (4°C) for 3 min immediately after storage. They were rinsed in phosphate-buffered saline (PBS, pH 7.6) for 15 minutes and placed in 0.3% hydrogen peroxide in absolute methanol for 10 min to inactivate endogenous peroxidase activity. After another 10 min-rinse in PBS, they were incubated with 5% normal goat serum (Dako, Glostrup, Denmark) in PBS for 30 min to prevent
nonspecific binding of the primary antibody. Specimens were then incubated with monoclonal mouse anti-human CD204 antibodies (1:1600 dilution, clone SRA-E5; TransGenic Inc., Kobe, Japan) in an incubation chamber overnight at 4°C. For visualization, the smears were developed with 3,30 -diaminobenzidine solution (DAB+, Dako) and counterstained with Mayer’s hematoxylin. Canine DH82 cells (European Collection of Cell Cultures, ECACC No. 94062922) originally derived from a dog diagnosed with disseminated histiocytic sarcoma and cultured on circular coverslips (at 37°C in an atmosphere containing 5% CO2 in Eagles MEM supplemented with 1% nonessential amino acid and 15% fetal bovine serum) were used to determine optimal antibody dilution and as a positive control, while incubation PBS alone was used as the negative control.8 All smears were examined by 2 pathologists unaware of the cytologic or histologic diagnosis. Each pathologist first read the immunocytochemistry slide and recorded the results. Next, the H&E and immunohistochemistry slides were read and the results were recorded. In case of discrepancies in the diagnosis or scores, the final diagnosis was established after deliberations between the 2 pathologists. However, there were no discrepancies in the CD204 immunocytochemistry assessments. All immunocytochemical preparations were assessed under high-power magnification (9 400) in 10 randomly selected fields per sample and evaluated according to the intensity of staining (, negative; +, faint; 2+, moderate; 3+, marked), and the median percentage of positive tumor cells was calculated. Only intact cells were evaluated. The Romanowsky-stained cytologic preparations of CHS were highly cellular, and all cases showed similar neoplastic cell morphology. The tumor cells
Table 1. Location of tumors, and intensity and proportion of histiocyte staining positive for CD204-immunocytochemistry in fine-needle aspirates (FNA) or impression smears (IS) from 10 dogs with histiocytic sarcoma.
No.
Breed
Site
Sampling
Intensity*
Percentage of Positive Cells (Median from 10 Fields at 4009 Magnification)
1 2 3 4 5 6 7 8 9 10
Pembroke Welsh Corgi Pembroke Welsh Corgi Pembroke Welsh Corgi Pembroke Welsh Corgi Flat-Coated Retriever Flat-Coated Retriever Shetland Sheepdog Miniature Schnauzer Doberman Golden Retriever
Lung Inguinal subcutaneous mass Lung Lung Elbow joint mass Intra-abdominal mass Intra-abdominal mass Subiliac lymph node Lung Axillar subcutaneous mass
FNA FNA FNA IS FNA IS FNA FNA FNA IS
2+ 3+ 3+ 2+ 3+ 2+ 2+ 3+ 3+ 2+
70 100 100 80 100 50 70 100 90 80
*, negative; +, faint; 2+, moderate; 3+, marked.
Vet Clin Pathol 43/4 (2014) 589–593 ©2014 American Society for Veterinary Clinical Pathology
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CD204 immunocytochemistry of histiocytic sarcoma
Kato et al
were round to polygonal with anisocytosis, had variably abundant pale vacuolated cytoplasm, and round to oval indented nuclei with occasional prominent nucleoli. Bi- to multinucleated atypical giant cells and mitotic figures were occasionally seen (Figure 1A). The optimal positive cytoplasmic and membrane staining with the CD204 antibody was seen at a 1:1600 dilution, tested on DH82 cells from a caninedisseminated histiocytic sarcoma (Figure 1B). All 10 CHS cases expressed CD204, and positive cells showed distinct, brown cytoplasmic staining (Table 1, Figure 1C), the percentage of CD204positive tumor cells was ≥ 50%. No CD204-positive cells were observed in the negative control or in the neoplastic cells of all other 45 tumors, although CD204 was expressed by resident and infiltrating macrophages (Figure 1D–F). Canine histiocytic sarcoma is an aggressive canine neoplastic disease, presenting with solitary or multiple lesions that spread rapidly.9 CHS is classified into localized and disseminated subtypes.2,4 Disseminated histiocytic sarcoma (HS), previously referred to as malignant histiocytosis, is characterized by disseminated lesions that arise simultaneously in multiple organs such as the skin, spleen, liver, lymph nodes, and bone marrow.2,4 A less common variant of HS, displaying prominent hemophagocytosis and anemia as a dominant clinical feature, is termed “hemophagocytic HS.”10 While localized and disseminated HS originate from dendritic cells or tissue macrophages, hemophagocytic HS appears to arise from resident macrophages in the red pulp of the spleen.5,9–12 In this study, immunocytochemical staining of CD204 showed good specificity and sensitivity. Although CD204 is expressed not only by neoplastic histiocytes but also by resident and infiltrating macrophages, these cells can be easily distinguished based on cellular and nuclear atypia. Interestingly, non-CHS canine cutaneous histiocytomas are negative for CD204.6 We evaluated immunocytochemistry for CD204 using air-dried smears to establish a CHS diagnosis. In veterinary medicine, air-dried smears are increasingly used for immunocytochemical staining, particularly for tumor classification and related prognosis and progression assessment.13,14 Immunocytochemistry represents a very valuable diagnostic method in high-risk patients who cannot undergo general anesthesia for invasive procedures such as a surgical biopsy. This includes cases with severe anemia and coagulopathies. Moreover, immunocytochemistry for CD204 could be used to diagnose CHS in the lung, a difficult site for tissue biopsy.
592
Disclosure: The authors have indicated that they have no affiliations or financial involvement with any organization or entity with a financial interest in, or in financial competition with, the subject matter or materials discussed in this article.
References 1. Duncan JR, Prasse KW. Cytology of canine cutaneous round cell tumors. Mast cell tumor, histiocytoma, lymphosarcoma and transmissible venereal tumor. Vet Pathol. 1979;16:673–679. 2. Valli VE. Veterinary Comparative Hematopathology. 1st ed. Iowa, USA: Blackwell Publishing; 2007:505–521. 3. Rossi S, Gelain ME, Comazzi S. Disseminated histiocytic sarcoma with peripheral blood involvement in a Bernese Mountain dog. Vet Clin Pathol. 2009;38:126–130. 4. Fulmer AK, Mauldin GE. Canine histiocytic neoplasia: an overview. Can Vet J. 2007;48:1041–1043; 1046– 1050. 5. Affolter VK, Moore PF. Localized and disseminated histiocytic sarcoma of dendritic cell origin in dogs. Vet Pathol. 2002;39:74–83. 6. Kato Y, Murakami M, Hoshino Y, et al. The class A macrophage scavenger receptor CD204 is a useful immunohistochemical marker of canine histiocytic sarcoma. J Comp Pathol. 2013;148:188–196. 7. Tomokiyo R, Jinnouchi K, Honda M, et al. Production, characterization, and interspecies reactivities of monoclonal antibodies against human class A macrophage scavenger receptors. Atherosclerosis. 2002;161:123–132. 8. Wellman ML, Krakowka S, Jacobs RM, Kociba GJ. A macrophage-monocyte cell line from a dog with malignant histiocytosis. In Vitro Cell Dev Biol. 1988;24:223– 229. 9. Valli VE, Jacobs RM, Parodi AL, Vernau W, Moore PF. Histological Classification of Hematopoietic Tumors of Domestic Animals. Washington: Armed Forces Institute of Pathology; 2002:60–61. 10. Soare T, Noble PJ, Hetzel U, Fonfara S, Kipar A. Paraneoplastic syndrome in haemophagocytic histiocytic sarcoma in a dog. J Comp Pathol. 2012;146:168–174. 11. Moore PF, Affolter VK, Vernau W. Canine hemophagocytic histiocytic sarcoma: a proliferative disorder of CD11d+ macrophages. Vet Pathol. 2006;43:632–645. 12. Hayden DW, Waters DJ, Burke BA, Manivel JC. Disseminated malignant histiocytosis in a Golden Retriever: clinicopathologic, ultrastructural, and immunohistochemical findings. Vet Pathol. 1993;30:256–264.
Vet Clin Pathol 43/4 (2014) 589–593 ©2014 American Society for Veterinary Clinical Pathology
Kato et al
13. Giancarlo A, Nazare C, Chiara P, et al. Subcutaneous embryonal rhabdomyosarcoma in a dog: cytologic, immunocytochemical, histologic, and ultrastructural features. Vet Clin Pathol. 2010;39:499–504.
CD204 immunocytochemistry of histiocytic sarcoma
14. Hoinghaus R, Hewicker-Trautwein M, Mischke R. Immunocytochemical differentiation of canine mesenchymal tumors in cytologic imprint preparations. Vet Clin Pathol. 2008;37:104–111.
Vet Clin Pathol 43/4 (2014) 589–593 ©2014 American Society for Veterinary Clinical Pathology
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BRIEF COMMUNICATION
Immunocytochemical detection of the class A macrophage scavenger receptor CD204 using air-dried cytologic smears of canine histiocytic sarcoma Yuki Kato1, Risa Funato1, Akihiro Hirata2,3, Mami Murakami3,4, Takashi Mori3,4, Koji Maruo3,4, Tokuma Yanai1, Hiroki Sakai1,3 1
Laboratory of Veterinary Pathology, Faculty of Applied Biological Sciences, 2Division of Animal Experiment, Life Science Research Centre, Comparative Cancer Centre, and 4Laboratory of Clinical Oncology, Department of Veterinary Medicine, Gifu University, Gifu, Japan
3
Key Words Dog, fine-needle aspiration, immunocytochemistry, neoplasia, round cell tumor, SR-A Correspondence H. Sakai, Laboratory of Veterinary Pathology, Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan E-mail: [email protected] DOI:10.1111/vcp.12190
Background: Cytologic diagnosis of canine histiocytic sarcoma (CHS) can be challenging because neoplastic histiocytes commonly show marked nuclear and cellular atypia and may resemble other pleomorphic malignant round cell tumors. Therefore, even on histopathologic examination, immunostaining is often necessary for a definitive diagnosis. Objectives: The objective of this study was to validate an anti-human CD204 antibody for immunocytochemical staining of air-dried smears for a rapid definitive diagnosis of CHS. Methods: Cytologic specimens were obtained from 10 dogs with CHS and 45 dogs with other tumors. After the cytologic evaluation of modified Giemsa-stained smears, acetone-fixed specimens were immunostained using mouse anti-human CD204 antibodies. All immunocytochemical specimens were assessed blinded and at high-power magnification (9 40 objective) in 10 randomly selected fields per sample. Parameters evaluated were the subjective staining intensity and location, and the proportion of positive cells. Results: All 10 CHS samples showed intense positive staining for CD204 in ≥ 50% of the cells, whereas the 45 other tumors were negative for CD204 staining. Conclusions: Immunocytochemistry of air-dried cytologic smears of CHS for CD204 is useful for a rapid confirmation of a cytologic diagnosis of CHS.
Cytology is being widely used in veterinary medicine, providing valuable information in cases of neoplasia. Cytopathology of neoplastic diseases is especially helpful as a diagnostic tool for round-cell tumors and often confirms the diagnosis.1,2 However, in canine histiocytic sarcoma (CHS), cytomorphology alone does not conclusively establish the origin of cells, although their high number in peripheral blood and atypical features may suggest a neoplastic disease, most likely of histiocytic origin.3–5 Therefore, when CHS is suspected for cytologic diagnosis, histopathologic and immunohistochemical examinations are often required to confirm the diagnosis.4,5 Morphologic features and the CD18+ (or MHC II+), CD3, and CD79a phenotype of the neoplastic cells are the most useful routine diagnostic criteria for a likely histiocytic origin.5
Previously, we showed that the class A macrophage scavenger receptor CD204 was a useful diagnostic marker for CHS.6 CD204 is a major macrophage receptor and plays important roles in host defense and in diseases such as atherosclerosis.7 A monoclonal antibody specific for CD204, clone SRA-E5, has been reported to recognize canine tissue-resident macrophages, including alveolar macrophages, hepatic Kupffer cells, splenic red pulp macrophages, sinus macrophages in lymph nodes, and interstitial macrophages in various organs.6,7 However, dendritic cells (DCs), such as the interdigitating cells of lymphoid tissues, and epidermal Langerhans cells are invariably negative for CD204.7 In this study, we performed CD204 immunocytochemical staining of air-dried smears to confirm
Vet Clin Pathol 43/4 (2014) 589–593 ©2014 American Society for Veterinary Clinical Pathology
589
CD204 immunocytochemistry of histiocytic sarcoma
Kato et al
cytologic diagnoses of CHS. If properly validated and made available, this antibody could be used to diagnose CHS more quickly, less invasively, and potentially at a lower cost. In this study, cases of 10 dogs with CHS and 45 dogs with other neoplastic diseases were retrieved from the database of the Laboratory of Veterinary Pathology, Gifu University, Gifu, Japan, based on their histologic diagnoses. Both cytologic and histologic
evaluation were available for all selected cases between April 2011 and July 2012 at the Gifu University Veterinary Teaching Hospital, Gifu, Japan. Per definition, all CHS cases in this study were CD18+, CD204+, CD3, and CD79a (or CD20), as previously described.5,6 The non-CHS group comprised 13 roundcell tumors (7 mast cell tumors, 4 lymphomas, and 2 extramedullary plasmacytomas), 18 epithelial tumors (6 squamous cell carcinomas, 4 mammary tumors, 3
A
B
C
D
E
F
Figure 1. Cytology and immunocytochemistry of canine histiocytic sarcomas (CHS) for CD204 expression. (A) CHS of inguinal subcutis dog no. 2. Tumor cells are round to polygonal, of variable size, and have variably abundant pale vacuolated cytoplasm. Nuclei were round to oval, and nucleoli were occasionally prominent. Bi- to multinucleated atypical giant cells and mitoses were seen occasionally. Romanowsky-based stain. (B) CD204 immunocytochemistry of DH82 cells derived from a canine CHS case. Note positive cytoplasmic and membranous staining for CD204. (C) CD204 immunocytochemistry of a CHS case in a subiliac lymph node, dog no. 8. Neoplastic histiocytes including multinucleated atypical giant cells show strong cytoplasmic and membranous positivity for CD204. (D–F) CD204 immunocytochemistry of representative non-CHS B-cell lymphoma (D), plasmacytoma (E), and hepatocellular carcinoma (F). CD204-positive macrophages (arrow) in a CD204-negative tumor. (B-F) 3,3’-Diaminobenzidine tetrahydrochloride chromogen. All bars = 50 lm.
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Vet Clin Pathol 43/4 (2014) 589–593 ©2014 American Society for Veterinary Clinical Pathology
Kato et al
CD204 immunocytochemistry of histiocytic sarcoma
hepatocellular carcinomas, 3 adenocarcinomas, and 2 anaplastic carcinomas), and 14 nonepithelial tumors (5 hemangiopericytomas, 3 osteosarcomas, 2 malignant melanomas, 1 seminoma, and 3 undifferentiated sarcomas). These tumors were diagnosed by histopathologic examination and immunohistochemistry (if needed). None of the dogs had received antitumor therapy (eg, chemotherapy and radiation) prior to diagnosis. Fine-needle aspiration specimens or impression smears were used for cytologic diagnosis of the tumors. In addition, the tumors were biopsied using special needles such as trephine or core biopsy needles. Cytologic smears were fixed in absolute methanol for 2 min and then stained with a Romanowsky-based stain (Hemacolor; Merck KGaA, Darmstadt, Germany). Smears were evaluated for cell preservation, cellularity, and features of malignancy. Tissue samples were fixed in buffered formalin and embedded in paraffin. Next, 3lm sections were stained with hematoxylin and eosin (H&E) and submitted for immunohistochemistry when necessary for confirming the diagnosis. Cytologic specimens were air-dried and stored either at 4°C for up to 1 week, or at 20°C for up to 6 months. Storage conditions and time were validated earlier with fine-needle aspiration specimens kept for 1 week at 4°C or 6 months at 20°C, the specimens were collected from cutaneous granuloma and normal liver in dogs. Smears were fixed in cold acetone (4°C) for 3 min immediately after storage. They were rinsed in phosphate-buffered saline (PBS, pH 7.6) for 15 minutes and placed in 0.3% hydrogen peroxide in absolute methanol for 10 min to inactivate endogenous peroxidase activity. After another 10 min-rinse in PBS, they were incubated with 5% normal goat serum (Dako, Glostrup, Denmark) in PBS for 30 min to prevent
nonspecific binding of the primary antibody. Specimens were then incubated with monoclonal mouse anti-human CD204 antibodies (1:1600 dilution, clone SRA-E5; TransGenic Inc., Kobe, Japan) in an incubation chamber overnight at 4°C. For visualization, the smears were developed with 3,30 -diaminobenzidine solution (DAB+, Dako) and counterstained with Mayer’s hematoxylin. Canine DH82 cells (European Collection of Cell Cultures, ECACC No. 94062922) originally derived from a dog diagnosed with disseminated histiocytic sarcoma and cultured on circular coverslips (at 37°C in an atmosphere containing 5% CO2 in Eagles MEM supplemented with 1% nonessential amino acid and 15% fetal bovine serum) were used to determine optimal antibody dilution and as a positive control, while incubation PBS alone was used as the negative control.8 All smears were examined by 2 pathologists unaware of the cytologic or histologic diagnosis. Each pathologist first read the immunocytochemistry slide and recorded the results. Next, the H&E and immunohistochemistry slides were read and the results were recorded. In case of discrepancies in the diagnosis or scores, the final diagnosis was established after deliberations between the 2 pathologists. However, there were no discrepancies in the CD204 immunocytochemistry assessments. All immunocytochemical preparations were assessed under high-power magnification (9 400) in 10 randomly selected fields per sample and evaluated according to the intensity of staining (, negative; +, faint; 2+, moderate; 3+, marked), and the median percentage of positive tumor cells was calculated. Only intact cells were evaluated. The Romanowsky-stained cytologic preparations of CHS were highly cellular, and all cases showed similar neoplastic cell morphology. The tumor cells
Table 1. Location of tumors, and intensity and proportion of histiocyte staining positive for CD204-immunocytochemistry in fine-needle aspirates (FNA) or impression smears (IS) from 10 dogs with histiocytic sarcoma.
No.
Breed
Site
Sampling
Intensity*
Percentage of Positive Cells (Median from 10 Fields at 4009 Magnification)
1 2 3 4 5 6 7 8 9 10
Pembroke Welsh Corgi Pembroke Welsh Corgi Pembroke Welsh Corgi Pembroke Welsh Corgi Flat-Coated Retriever Flat-Coated Retriever Shetland Sheepdog Miniature Schnauzer Doberman Golden Retriever
Lung Inguinal subcutaneous mass Lung Lung Elbow joint mass Intra-abdominal mass Intra-abdominal mass Subiliac lymph node Lung Axillar subcutaneous mass
FNA FNA FNA IS FNA IS FNA FNA FNA IS
2+ 3+ 3+ 2+ 3+ 2+ 2+ 3+ 3+ 2+
70 100 100 80 100 50 70 100 90 80
*, negative; +, faint; 2+, moderate; 3+, marked.
Vet Clin Pathol 43/4 (2014) 589–593 ©2014 American Society for Veterinary Clinical Pathology
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CD204 immunocytochemistry of histiocytic sarcoma
Kato et al
were round to polygonal with anisocytosis, had variably abundant pale vacuolated cytoplasm, and round to oval indented nuclei with occasional prominent nucleoli. Bi- to multinucleated atypical giant cells and mitotic figures were occasionally seen (Figure 1A). The optimal positive cytoplasmic and membrane staining with the CD204 antibody was seen at a 1:1600 dilution, tested on DH82 cells from a caninedisseminated histiocytic sarcoma (Figure 1B). All 10 CHS cases expressed CD204, and positive cells showed distinct, brown cytoplasmic staining (Table 1, Figure 1C), the percentage of CD204positive tumor cells was ≥ 50%. No CD204-positive cells were observed in the negative control or in the neoplastic cells of all other 45 tumors, although CD204 was expressed by resident and infiltrating macrophages (Figure 1D–F). Canine histiocytic sarcoma is an aggressive canine neoplastic disease, presenting with solitary or multiple lesions that spread rapidly.9 CHS is classified into localized and disseminated subtypes.2,4 Disseminated histiocytic sarcoma (HS), previously referred to as malignant histiocytosis, is characterized by disseminated lesions that arise simultaneously in multiple organs such as the skin, spleen, liver, lymph nodes, and bone marrow.2,4 A less common variant of HS, displaying prominent hemophagocytosis and anemia as a dominant clinical feature, is termed “hemophagocytic HS.”10 While localized and disseminated HS originate from dendritic cells or tissue macrophages, hemophagocytic HS appears to arise from resident macrophages in the red pulp of the spleen.5,9–12 In this study, immunocytochemical staining of CD204 showed good specificity and sensitivity. Although CD204 is expressed not only by neoplastic histiocytes but also by resident and infiltrating macrophages, these cells can be easily distinguished based on cellular and nuclear atypia. Interestingly, non-CHS canine cutaneous histiocytomas are negative for CD204.6 We evaluated immunocytochemistry for CD204 using air-dried smears to establish a CHS diagnosis. In veterinary medicine, air-dried smears are increasingly used for immunocytochemical staining, particularly for tumor classification and related prognosis and progression assessment.13,14 Immunocytochemistry represents a very valuable diagnostic method in high-risk patients who cannot undergo general anesthesia for invasive procedures such as a surgical biopsy. This includes cases with severe anemia and coagulopathies. Moreover, immunocytochemistry for CD204 could be used to diagnose CHS in the lung, a difficult site for tissue biopsy.
592
Disclosure: The authors have indicated that they have no affiliations or financial involvement with any organization or entity with a financial interest in, or in financial competition with, the subject matter or materials discussed in this article.
References 1. Duncan JR, Prasse KW. Cytology of canine cutaneous round cell tumors. Mast cell tumor, histiocytoma, lymphosarcoma and transmissible venereal tumor. Vet Pathol. 1979;16:673–679. 2. Valli VE. Veterinary Comparative Hematopathology. 1st ed. Iowa, USA: Blackwell Publishing; 2007:505–521. 3. Rossi S, Gelain ME, Comazzi S. Disseminated histiocytic sarcoma with peripheral blood involvement in a Bernese Mountain dog. Vet Clin Pathol. 2009;38:126–130. 4. Fulmer AK, Mauldin GE. Canine histiocytic neoplasia: an overview. Can Vet J. 2007;48:1041–1043; 1046– 1050. 5. Affolter VK, Moore PF. Localized and disseminated histiocytic sarcoma of dendritic cell origin in dogs. Vet Pathol. 2002;39:74–83. 6. Kato Y, Murakami M, Hoshino Y, et al. The class A macrophage scavenger receptor CD204 is a useful immunohistochemical marker of canine histiocytic sarcoma. J Comp Pathol. 2013;148:188–196. 7. Tomokiyo R, Jinnouchi K, Honda M, et al. Production, characterization, and interspecies reactivities of monoclonal antibodies against human class A macrophage scavenger receptors. Atherosclerosis. 2002;161:123–132. 8. Wellman ML, Krakowka S, Jacobs RM, Kociba GJ. A macrophage-monocyte cell line from a dog with malignant histiocytosis. In Vitro Cell Dev Biol. 1988;24:223– 229. 9. Valli VE, Jacobs RM, Parodi AL, Vernau W, Moore PF. Histological Classification of Hematopoietic Tumors of Domestic Animals. Washington: Armed Forces Institute of Pathology; 2002:60–61. 10. Soare T, Noble PJ, Hetzel U, Fonfara S, Kipar A. Paraneoplastic syndrome in haemophagocytic histiocytic sarcoma in a dog. J Comp Pathol. 2012;146:168–174. 11. Moore PF, Affolter VK, Vernau W. Canine hemophagocytic histiocytic sarcoma: a proliferative disorder of CD11d+ macrophages. Vet Pathol. 2006;43:632–645. 12. Hayden DW, Waters DJ, Burke BA, Manivel JC. Disseminated malignant histiocytosis in a Golden Retriever: clinicopathologic, ultrastructural, and immunohistochemical findings. Vet Pathol. 1993;30:256–264.
Vet Clin Pathol 43/4 (2014) 589–593 ©2014 American Society for Veterinary Clinical Pathology
Kato et al
13. Giancarlo A, Nazare C, Chiara P, et al. Subcutaneous embryonal rhabdomyosarcoma in a dog: cytologic, immunocytochemical, histologic, and ultrastructural features. Vet Clin Pathol. 2010;39:499–504.
CD204 immunocytochemistry of histiocytic sarcoma
14. Hoinghaus R, Hewicker-Trautwein M, Mischke R. Immunocytochemical differentiation of canine mesenchymal tumors in cytologic imprint preparations. Vet Clin Pathol. 2008;37:104–111.
Vet Clin Pathol 43/4 (2014) 589–593 ©2014 American Society for Veterinary Clinical Pathology
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