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Department of Veterinary Pathology (NJF, MLJ, BAK) and Prairie Diagnostic Services (KHW), Western College of Veterinary Medicine, 52 Campus Drive, Saskatoon, SK, Canada
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Abstract |
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Key words: Dogs; formalin-fixed; histiocytoma; immunohistochemistry; mast cell tumor; paraffin-embedded tissue; skin.
Diagnosis of canine cutaneous round cell tumors based on histopathology alone is often challenging. Different round cell tumors may have a similar morphologic appearance, and even special stains may not be useful, particularly for poorly differentiated tumors. An accurate diagnosis is important in determining prognosis and treatment. Immunohistochemistry (IHC) has been used as an adjunct to light microscopy in the diagnosis of various neoplasms, including round cell tumors. In veterinary medicine, antibodies that have been validated for specific use in humans are often utilized for IHC; however, these antibodies have rarely been validated for animal species.7 Because IHC is a relatively expensive diagnostic procedure, pathologists tend to use one or two antibodies to confirm diagnostic suspicions, but these results may not be reliable if the specificity of these antibodies is in question. Although certain antibodies may work in well-differentiated tumors, they are often less effective in poorly differentiated tumors that are also more difficult to diagnose histologically.
Tryptase, chymase, and serotonin are found in mast cell granules, and tryptase and chymase are mast-cell specific.12 Serotonin is a preformed biogenic amine stored in mast cell granules,26 but it is also present in other tissues. In the dog, there are three subtypes of mast cells, based on content of tryptase and chymase: mast cells containing tryptase only, mast cells containing chymase only, and mast cells containing both tryp-tase and chymase, the latter being the most common subtype in canine skin.12,27 The subtype of mast cell involved in canine cutaneous mast cell tumors has not been elucidated, although one canine mast cell tumor examined by Welle et al.28 contained both tryptase and chymase. Immunohistochemical and enzyme-histochemical staining methods for tryptase and chymase have been described.12,28
Canine cutaneous histiocytoma is a tumor of epidermal Langerhans' cell origin.17 Langerhans' cells in canine cutaneous histiocytomas express leukocyte antigens characteristic of dendritic cell differentiation, including CD1a, CD1b, CD1c, and CD11c. Expression of CD1 molecules distinguishes Langerhans' cells from macrophages. A diagnostic problem arises because these antigens are only detectable in fresh or frozen tissue, which is not often available to the veterinary pathologist. Currently, the most useful immunohistochemical criteria for identifying histiocytic cells in formalin-fixed, paraffin-embedded (FFPE) tissues are CD18 positivity, coupled with negative results for CD3 and CD79a to rule out lymphocytic origin.1,17 CD18, a nonspecific antigen involved in leukocyte adhesion, is present on the surface of all leukocytes.4 Neoplastic histiocytes also consistently express MHC class II.17 MHC class II staining is more specific for dendritic cells than is CD18, but T and B lymphocytes express MHC class II and will stain positively.11,14 CD1a is the most reliable marker for Langerhans' cells in the epidermis;17 however, the only commercially available monoclonal antibodies for use in FFPE tissues were developed for use in humans and have not been investigated in the dog.
Lymphosarcoma and plasmacytoma are differential diagnoses for round cell tumors. T lymphocytes express CD3 and CD18 and, in the dog, both resting and activated T cells express MHC class II.11,14 B lymphocytes express CD79a, CD18, and MHC class II.14 Macrophages and granulocytes express 10-fold more CD18 than do lymphocytes, and in FFPE tissues, lymphocytes may have undetectable CD18.14 Plasmacytomas stain positively for CD79a approximately 80% of the time15 and may be positive for MHC class II.
We selected a panel of monoclonal antibodies recognizing the following antigens: tryptase, chymase, and serotonin for mast cells; MHC class II, CD18, and CD1a for histiocytes; CD3 for T lymphocytes; CD79a for B lymphocytes and plasma cells; and one histochemical stain (naphthol AS-D chloroacetate for chy-mase activity). These were applied to FFPE sections of canine cutaneous tumors histologically diagnosed as mast cell tumors, histiocytomas, lymphosarcomas, and plasmacytomas, as well as unidentified round cell tumors. In addition, toluidine blue was applied to those tumors diagnosed histologically as mast cell tumors. With the exception of CD1a, these antibodies were chosen based on their previous application to FFPE tissues, the samples most often available to diagnostic pathologists. The primary objective of this study was to evaluate a panel of immunohistochemical stains and one histochemical stain in assisting the diagnosis of 72 canine cutaneous round cell tumors. Mast cell tumors and histiocytomas were the main focus, but lymphoid tumors were included to properly evaluate the stains. Of particular interest was identifying reliable markers for poorly differentiated round cell tumors.
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Materials and Methods |
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A panel of monoclonal antibodies was applied to deparaffinized 4-µm sections of each skin tumor. The primary antibodies recognized tryptase (1 : 50, AA1, DAKO, Carpinteria, CA), chymase (1 : 500, CC1, NeoMarkers, Fremont, CA), MHC class II (1 : 150, TAL.1B5, DAKO), CD18 (1 : 4, CA16.3C10, P. Moore, UC-Davis, Davis, CA), and CD3 (1 : 25, CD3-12, Novocastra Laboratories Ltd., Newcastle upon Tyne, UK). Staining was done using a Benchmark staining platform (Ventana Medical Systems Inc., Tucson, AZ), with the BMK iVIEW DAB Paraffin detection kit (Ventana Medical Systems Inc.) using a streptavidin-biotin amplification system. Antigen retrieval was performed as follows for all antibodies except CD18. Heat retrieval consisted of applying cell conditioner #1 (this and other reagents are proprietary and included in the kit from Ventana Medical Systems Inc.) to skin sections and then heating to 95°C for 8 minutes, followed by 20 minutes at 100°C. I-VIEW inhibitor was added and incubated for 4 minutes at 42°C. For CD18 antigen retrieval, protease 2 was applied and incubated for 10 minutes. All slides were then incubated with primary antibody for 32 minutes, followed by I-VIEW biotin immunoglobulin for 8 minutes, I-VIEW SA-HRP for 8 minutes, I-VIEW DAB and H2O2 for 8 minutes, and I-VIEW copper for 4 minutes. Slides were counterstained with hematoxylin and bluing reagent.
Three different anti-human CD1a clones (ICA04, Neo-Markers; MTBI, Novocastra Laboratories Ltd.; JMP30, Novocastra Laboratories Ltd.) for use in FFPE tissues were tested on select canine tumors at different dilutions using both heat and protease antigen retrieval but were not applied to every tumor in the study.
Serotonin (1 : 100, YC5/45, Medicorp, Montreal, PQ, Canada) and CD79a (1 : 2, HM47/A9, NeoMarkers) staining was done with a Codon Histomatic slide stainer (Fisher Scientific Co., Edmonton, AB, Canada), using an avidin-biotin immunoperoxidase technique previously described by Haines and Chelack,7 with the exception that antigen retrieval for CD79a was performed by heating sections to 95°C for 20 minutes in a citrate buffer, pH 6.0, instead of by proteolytic digestion. Primary antibodies were incubated overnight at 4°C. Secondary antibodies were incubated for 30 minutes at 37°C. Stains were completed with a 30-minute incubation with avidin-biotin complex at 37°C. Diaminobenzidine (Cedarlane Laboratories Ltd. Canada, Hornby, ON, Canada) was used as the chromogen substrate. Positive control slides were included in each run and examined for appropriate staining.
Each tumor was evaluated for chymase activity with an enzyme histochemical stain using naphthol AS-D chloroacetate as substrate (Procedure No. 91, Sigma-Aldrich Canada Ltd., Toronto, ON, Canada). This chymase stain has been used for normal canine mast cells in FFPE tissues.12,13,27,28 It is a modification of the original staining procedure described by Benditt and Arase,3 who demonstrated the selectivity of chloroacyl naphthol esters for chymotryptic enzymes compared with tryptic enzymes. Tumors that were identified as mast cell tumors histologically were stained with toluidine blue (BDH Laboratory Supplies, Poole, UK) for comparison with tryptase and chymase IHC and chymase histochemistry.
A blinded examiner (NJF) reviewed all the slides for each stain consecutively to ensure consistency of evaluation. Sections were classified as positive or negative based on staining characteristics of tumor cells. Slides that were difficult to interpret due to faint staining of tumor cells were restained. Expected results for each tumor type are summarized in Table 1. Our panel was not designed to differentiate plasma-cytoma from B-cell lymphosarcoma; thus, these results are grouped as one diagnosis.
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Results |
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Of the 21 tumors that were diagnosed histologically as mast cell tumors (MCTs), 17 (80.9%) had staining patterns characteristic of MCTs. All (7/7; 100%) grade I MCTs, 6/7 (85.7%) grade II MCTs, and 3/7 (42.9%) grade III MCTs were positive for both tryptase antigen and chymase activity (i.e., chymase histochemical stain; Fig. 1a, b). One grade III MCT was positive for toluidine blue despite being negative for tryptase antigen and chymase activity. This tumor was also positive for CD18 (Fig. 2). The four remaining tumors included two that were positive for only CD18 and therefore designated as undetermined. These tumors were both histologically diagnosed as grade III MCT. All MCTs diagnosed by stain panel were negative for MHC class II; however, many MCTs contained numerous MCH class II–positive nontumor cells.
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Histiocytoma
Of the 19 tumors that were diagnosed histologically as histiocytoma (HCT), 7 (36.8%) tumors were clearly identified as HCT using the stain panel (CD18 and MHC class II positive; Fig. 3a, b), 1 (5.7%) was diagnosed as B-cell lymphosarcoma (BLSA) or plasmacytomas (PCTs), and the remaining 11 (57.9%) could not be definitively diagnosed based on the stain panel alone and were designated as undetermined. While these tumors were positive for CD18 and MHC II, they also contained CD3- and/or CD79a-positive cells. It was impossible to determine whether the cells staining positively for CD3 and CD79a were infiltrating lymphocytes associated with HCT regression or whether they were neoplastic cells (Fig. 4). All of the three tumors histologically diagnosed as atypical HCT (HCTA) were designated as undetermined by the stain panel.
There were four tumors not histologically diagnosed as HCT that were clearly diagnosed as HCT by the stain panel: two PCTs, one MCT grade II, and one unidentified tumor.
The kappa statistic comparing agreement between the histologic diagnosis of HCT and the stain panel diagnosis was poor, at 0.29 (0.07–0.51). The agreement between MHC class II and CD18 was fair to good, at 0.50 (0.27–0.72), when the stains were compared across all tumors.
Lymphosarcoma and plasmacytoma
T-cell lymphosarcoma was the most common (10/13; 76.9%) stain-panel diagnosis for histologically diagnosed lymphosarcoma, based on positive CD3 staining (Fig. 5). Tumors that were positive for CD79a were diagnosed as BLSA or PCT because the stain-panel results are the same for these two tumors (our panel was not designed to differentiate these tumors; Fig. 6).
Unidentified round cell tumors
A diagnosis was obtained for 4/5 (80%) histologically unidentified round cell tumors. The tumor that was negative for all stains was designated as undifferentiated, but could also be a PCT because only about 80% of PCTs express CD79a.15
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Discussion |
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We found a strong agreement between tryptase IHC and chymase HC (kappa = 1.00). This suggests that either stain could be used to aid the diagnosis of a MCT. We did not evaluate toluidine blue staining with a kappa statistic; however, toluidine blue staining detected one more MCT than did either tryptase IHC or chymase HC, suggesting that it is as good or better for detecting MCTs. Also, toluidine blue staining is likely to be readily available in a diagnostic histopathology laboratory and is a good first choice for confirmation of a diagnosis of MCT in the dog.
The finding of equal detection of tryptase and chy-mase in MCTs in this study may reflect the predominant mast cell subtype in normal canine skin.12,27 A negative result with one or both of these stains does not definitively rule out MCT as a diagnosis, and stains that detect other mast cell–specific substances (e.g., to-luidine blue for sulfated glycosaminoglycans) may be useful for diagnosis. This may explain one case, which was negative for both tryptase antigen and chymase activity but positive for toluidine blue.
Of MCTs positive for tryptase antigen and chymase activity, 6/16 (37.5%) were also positive for CD18. Higher grade, less differentiated MCTs expressed CD18 more often than low-grade, well-differentiated ones. This result was unexpected; however, high levels of CD18 expression have been described in immature cultured human mast cells24 and in a low percentage of normal human skin mast cells.25 The tumors in our study that expressed only CD18 may have been poorly differentiated, immature MCTs that had not yet developed the mast cell–specific proteases tryptase and chy-mase within their granules.
None of the MCTs was positive for MHC class II expression. However, many of the MCTs contained nontumor, MHC class II–positive cells resembling normal dendritic cells, which has not been previously described.
One grade I MCT that was positive for tryptase antigen and chymase activity was also positive for CD3 expression. CD3 is closely associated with the T-cell receptor and is reported to be expressed only on the surface of mature T cells and thymocytes.15 The staining pattern of CD3 in this tumor was granular and cytoplasmic rather than membranous, as would be expected in T cells. This suggests that these neoplastic cells were producing a molecule within their granules that was antigenically similar to CD3, and thus recognized by the CD3 monoclonal antibody. Ozaki et al.18 examined 39 gastrointestinal MCTs using various monoclonal antibodies, including CD3 and CD79a. None of the tumors was positive for CD3, but 7/39 (17.9%) tumors were positive for CD79a.
Several authors have described the use of chymase IHC to detect mast cells in both canine MCTs28 and human skin;9 however, the chymase antibody that we used consistently stained both tumor cells and adjacent nontumor cells and was not useful. In our study, all tumors were negative for serotonin, including those that were positive for tryptase and chymase. The reason for the absence of serotonin staining in our MCTs is unclear. Mast cells in canine fundic mucosa do not contain detectable serotonin,2 and this may be true of mast cells in canine skin as well.
Of 19 tumors histologically diagnosed as HCTs, 11 (57.9%) could not be definitively diagnosed based on the stain-panel results. The most significant difficulty in making a diagnosis of HCT using the stain panel was differentiating regressing histiocytoma from T-cell lymphosarcoma. Others have reported difficulty in distinguishing lymphocytes and neoplastic cells.11 Tumors that were diagnosed as HCTs by the stain panel frequently contained CD3-positive nontumor cells with typical small mature lymphocyte morphology (i.e., round cells with small, round hyperchromatic nuclei and scant cytoplasm). In the undetermined cases, both small mature lymphocytes and larger cells with pleomorphic nuclei and abundant cytoplasm stained positively for CD3. It was impossible to determine, based on morphology, whether these pleomorphic CD3-positive cells were neoplastic or normal cells (i.e., lymphocytes). In these questionable cases, the histologic appearance of the tumor may be useful in reaching a diagnosis. The histologic appearance was not characteristic of T-cell lymphosarcoma, and in a diagnostic setting, these tumors likely would have been diagnosed as regressing HCTs without the aid of IHC. IHC may be more useful in aiding the diagnosis of HCTs that do not have a typical histologic appearance. The use of IHC for HCTs is probably best restricted to this situation.
There were also three tumors that contained CD79a-positive cells in addition to the CD3-positive cells, and these were similarly difficult to diagnose based only on the stain panel. Although regressing HCTs are generally infiltrated with CD3-positive T cells, B cells can occasionally be found within these tumors.11,17 A diagnosis of T cell–rich BLSA is unlikely in these cases because both normal-appearing small lymphocytes and pleomorphic cells were positive for CD3. In T cell–rich BLSA, IHC indicates T-cell origin of the small normal lymphocytes and B-cell origin of the large pleomorphic cells.23
In summary, the most reliable criteria for immunohistochemical diagnosis of canine cutaneous HCT were CD18 and MHC class II positivity coupled with negative staining for CD3 and CD79a. However, as discussed above, IHC is not always useful or recommended. In this study, by definition, an HCT had to be positive for both CD18 and MHC class II, which was necessary because of the nonspecific nature of CD18 and MHC class II. If both of these antibodies were not used, the tumor that was positive for CD18 only could have been incorrectly classified as an HCT, when in fact it was most likely an MCT based on positive staining with toluidine blue. Novel diagnostic methods may be useful in such cases, for example, the development of a canine CD1a monoclonal antibody that is effective in FFPE tissues. CD1a is the most reliable marker for Langerhans' cell differentiation in the epidermis, the cell of origin of canine cutaneous HCT.17 Currently, there is a canine-specific CD1a antibody available that is applicable only to fresh or frozen tissues and which detects an allotype of CD1a that is present in about 80% of dogs.17 Also, the use of only toluidine blue and methyl green pyronine to rule out MCT and PCT, respectively, does not allow diagnosis of HCT by exclusion because lymphosarcoma remains a differential diagnosis. IHC is a necessary next step.
Although it was not the primary focus of our study, we found that one of the epitheliotropic lymphosarcomas was of B-cell rather than T-cell origin. This was unexpected, as epitheliotropic lymphosarcoma in the dog is reported to be of CD8+ T cell origin.5,16 Nonepitheliotropic lymphosarcoma is reported to be mostly of T-cell origin in the dog, although it can be of B-cell origin.5 We found an equal distribution of T-cell and B-cell lymphosarcomas within our nonepithe-liotropic group; however, our sample size was small (n = 4). Diagnosis of non–T cell, non–B cell lymphosarcoma (of presumed natural killer cell origin) is rare in dogs;21 however, its existence presents a potential problem in the use of solely CD3 and CD79a to diagnose lymphosarcoma or to rule out lymphosarcoma in the diagnosis of HCT. Morphology would likely be useful in establishing a diagnosis given the granularity of these cells, perhaps combined with additional immunohistochemical stains.10
The stain-panel results were the same for both BLSA and PCT; however, the two could likely be distinguished based on morphology. Methyl green pyronine staining for RNA6 or immunohistochemical staining for immunoglobulin and kappa and lambda light chains8 could also be used to confirm the diagnosis. One histologically diagnosed PCT was designated as undetermined because it was positive for CD3 as well as CD79a.
Four of the five histologically unidentified tumors were given a final diagnosis based on the stain panel. The one tumor without a final diagnosis was negative for all stains. This may be because the tumor was poorly differentiated, or it may be one of the 20% of PCTs that are negative for CD79a.15 Methyl green pyronine staining may have been useful in ruling out PCT.
In conclusion, a minimum of five adjunct stains is most useful for the diagnosis of cutaneous round cell tumors in dogs: tryptase IHC, chymase HC, or toluidine blue for mast cells, MHC class II and CD18 IHC for histiocytes, and CD3 and CD79a IHC for lymphocytes (Fig. 7). Additional stains may be required depending on the outcome of the initial panel, especially for suspected HCTs.
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Acknowledgments |
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