Alys A. Harshbarger
Dr. Harshbarger is a second-year clinical pathology resident at Oklahoma State University. She grew up in Virginia and obtained her DVM from Virginia–Maryland College of Veterinary Medicine. Before beginning her residency, she completed an internship in food animal medicine and surgery at Oklahoma State University. In her free time, she enjoys playing the fiddle and spending time with her husband and 3 dogs.Read Articles Written by Alys A. Harshbarger
James H. Meinkoth
DVM, MS, PhD, DACVP
Dr. Meinkoth is a professor in the department of veterinary pathology at Oklahoma State University, where he has worked for 30 years. He completed his veterinary degree at Oklahoma State University and, after a short time in private practice, went to Washington State University for a residency in clinical pathology. His main professional interest is in diagnostic cytology, and he routinely gives continuing education lectures and has contributed chapters to several textbooks on the topic. In his spare time, he spends way too much time and money going to Bruce Springsteen concerts across the country.Read Articles Written by James H. Meinkoth
Mast cell tumors (MCTs) are common in dogs, making up approximately 20% of canine skin tumors. Their biological behavior is variable, and grading provides significant prognostic information for use in making treatment decisions.1
Histopathologic Grading of Mast Cell Tumors
Currently, histopathologic evaluation is considered the gold standard for grading MCTs. These grading systems have evolved over the years. Historically, a 3-tier system (Patnaik system) has been used, defining MCTs as grade I, II, or III. Grade I tumors are well differentiated with a low metastatic rate (7% mortality from tumor-related disease) and grade III are poorly differentiated with a high metastatic rate (94% mortality from tumor-related disease).2 Patnaik grade II tumors tend to be overrepresented, generally accounting for 42% to 78% of MCTs, but sometimes cited as comprising 91% of MCTs.3-5 Reports have shown significant interobserver differences in grading MCTs with the Patnaik system.3,6 The biological behavior of grade II tumors is variable, with up to 20% being aggressive,7 and together, these factors lead to uncertainty in the appropriate management of many patients using the Patnaik system.
In 2011, a 2-tier classification system was proposed by Kiupel et al, defining MCTs as either low grade or high grade.6 In this system, criteria for a high-grade MCT include the presence of any of the following characteristics:
- ≥7 mitotic figures in 10 high-power fields (HPF)
- ≥3 multinucleated cells (at least 3 nuclei) in 10 HPF
- ≥3 bizarre nuclei in 10 HPF
- Karyomegaly (nuclear diameters vary by at least twofold in ≥10% of cells)
Classification is based on the histologic area with the most mitotic figures or nuclear variation. Kiupel et al found that there is good interobserver consistency in categorization with this system and a high correlation between grade and clinical outcome.
In the 2013 Oncology-Pathology Working Group (OPWG) consensus statement on grading of cutaneous MCTs, the application of both the Patnaik and Kiupel grading schemes was suggested. In 2021, the OPWG suggested incorporation of the 2 grading systems to better predict behavior of some tumors and study survival rate.7 MCTs are thus categorized as:
- Grade I/low-grade
- Grade II/low-grade
- Grade II/high-grade
- Grade III/high-grade
Cytologic Grading of Mast Cell Tumors
Cytology is a quick, inexpensive method routinely used to diagnose MCTs. Generally, these tumors exfoliate high numbers of cells that typically contain large numbers of small, round, purple granules, making diagnosis straightforward.
The cellular features that compose the Kiupel grading system can be evaluated on cytologic preparations. Several recent studies investigated how well cytologic features correlate to histologic grade. Two of these studies directly applied the Kiupel system to cytologic preparations.8,9 Histopathology was considered the gold standard for grading, and overall, both studies found good correlation between MCT grades determined cytologically versus histologically. Cytologic grading matched histologic grading in 94% of cases in both studies. Some cytologic limitations were identified, including fewer numbers of cells for evaluation and lower occurrence of mitotic figures on cytologic preparations compared with histologic preparations.
Camus Grading System
To overcome these limitations, Camus et al developed a unique cytologic grading system that did not directly apply the histologic grading criteria to cytologic samples.10 They statistically evaluated various combinations of cellular features to create a grading scheme that correlated with not only histologic grade but also—and more importantly—patient survival. The cytologic feature that best correlated with 2-year survival was degree of granulation of tumor cells, followed by anisokaryosis, multinucleation, binucleation, and mitotic figures. In this cytologic grading scheme, tumors are classified as high grade if they meet either or both of 2 criteria:
- Poor granulation of the entire cell population
- The presence of 2 of the following features of atypia:
- Mitotic figures
- Binucleated or multinucleated cells
- Nuclear pleomorphism (shapes not including round and oval)
- Anisokaryosis (twofold variation in nuclear size, termed karyomegaly in Kiupel system)
Granulation of mast cells is categorized as heavily granulated (FIGURE 1), mixed granulation (a mixture of heavily granulated and poorly granulated cells), and poorly granulated (FIGURE 2). Only tumors in which all cells show poor granulation are categorized as high grade based on this criterion.
In contrast to the quantitative metrics of the Kiupel system, the Camus cytologic grading system considers the presence of any mitotic figures (FIGURE 3), binucleated (FIGURE 4) or multinucleated (FIGURE 5) cells, atypically shaped nuclei (bizarre nuclei or nuclear pleomorphism; FIGURE 6), or cells with twofold variation in nuclear size (FIGURE 7) a positive finding for that feature. The presence of any 2 of these features classifies a tumor as high grade.
Cytologic and histologic grading matched in 94% of cases.10 Of the cytologic low-grade MCTs, 98.5% were also called low-grade by histology. However, of the tumors cytologically classified as high grade, approximately one-third (31.8%) were determined to be low grade by histology. Thus, there is a tendency to overestimate tumor grade compared with histology, likely resulting from less stringent criteria (e.g., any mitotic figures seen versus ≥7 per 10 HPF). The authors point out that as a screening test, this is preferable to underestimating tumor grade, which might result in aggressive tumors being undertreated.
Paes Grading System
Cytologic grading schemes are likely to continue to evolve. A 2022 study by Paes et al modified the Camus cytology scale by including evaluation of the number of fibroblasts (FIGURE 8) and collagen fibrils (FIGURE 9) and eliminating binucleated cells and nuclear pleomorphism.11 Fibroblasts and collagen fibrils are both common findings in MCTs and are more common in low-grade tumors. In this study, finding only low numbers of these elements correlated with a reduced 1-year survival.
In all studies, variable numbers of cases had discordant results between cytologic and histologic grades. The overall agreement varied from 77% to 94%. Both the Camus and Paes systems tended to classify more tumors as high grade than did histology. However, the main goal of tumor classification is to predict patient outcome, and both systems correlated with patient survival approximately as well as, or better than, histologic grading.10,11 In fact, the study by Paes et al showed that in 11 cases called high grade on cytology but low grade on histology, there were 5 tumor-related deaths (45%), so it is possible that cytologic grade may be predictive of outcome even in histologically low-grade tumors.
There are potential limitations of applying cytologic grading schemes to MCTs. One is that nuclear features are often obscured in heavily granulated tumors, making evaluation of nuclear morphology difficult and potentially masking atypical features. Despite this potential challenge, cytologic grading classified more tumors as high grade than histology in the studies by Camus and Paes and grading by both methods significantly correlated with survival data.
A second concern is that cytology cannot distinguish cutaneous from subcutaneous MCTs. The current histologic grading systems are applied only to cutaneous MCTs.1,7 One study did include both cutaneous and subcutaneous tumors and still showed 85% agreement with histologic grading.12
A third potential issue is the type of stain used. Aqueous Romanowsky stains (e.g., Diff-Quik) may not stain mast cell granules as well as methanolic Romanowsky stains such as May-Grünwald-Giemsa (MGG). As granularity of mast cells is used in cytologic grading, the lack of granularity with aqueous stains may falsely indicate a high-grade tumor. In a recent study, approximately 18% of cytologic samples from primary MCTs showed less granulation with aqueous stains than with a standard methanolic stain.12 Notably, hypogranularity was significantly correlated with other cytologic evidence of malignancy. In this study, cytologic samples stained with both aqueous stains and MGG stain were graded using the Camus scheme and compared with histologic grading. While there were some differences in grading of individual tumors with different stains, the diagnostic accuracy was 85% with both types of cytologic stain when compared with histologic classification. This study included both cutaneous and subcutaneous MCTs.
Grading of MCTs provides significant prognostic information and can be useful in determining a treatment plan. Histologic evaluation is currently the gold standard for grading; histology also allows for evaluation of tumor margins. However, several studies show that cytologic grading schemes may be useful for predicting the histologic grade and independently correlate well with clinical outcome (BOX 1). Cytologic findings associated with poor outcome include poor granularity of the tumor cells or the presence of 2 or more atypical features, including anisokaryosis, mitotic figures, binucleation/multinucleation, nuclear pleomorphism, and reduced numbers of fibroblasts/collagen fibrils.
- Kiupel M. Mast cell tumors. In: Meuten DJ, ed. Tumors in Domestic Animals. Wiley Blackwell; 2020:176-201.
- Patnaik AK, Ehler WJ, MacEwan EG. Canine cutaneous mast cell tumor: morphologic grading and survival time in 83 dogs. Vet Pathol. 1984;21(5):469-474. doi:10.1177/030098588402100503
- Northrup NC, Howerth BG, Harmon CA, et al. Variation among pathologists in histologic grading of canine cutaneous mast cell tumors with uniform use of a single grading reference. J Vet Diagn Invest. 2005;17(6):561-564. doi:10.1177/104063870501700606
- Shaw T, Kudnig ST, Firestone SM. Diagnostic accuracy of pre-treatment biopsy for grading cutaneous mast cell tumors in dogs. Vet Comp Oncol. 2018;16(2):214-219. doi:10.1111/vco.12346
- Preziosi R, Sarli G, Paltrinieri M. Multivariate survival analysis of histological parameters and clinical presentation in canine cutaneous mast cell tumors. Vet Res Commun. 2007;31(3):287-296. doi:10.1007/s11259-006-3427-9
- Kiupel M, Webster JD, Bailey S, et al. Proposal of a 2-tier histologic grading system for canine cutaneous mast cell tumors to more accurately predict biological behavior. Vet Pathol. 2011;48(1):147-155. doi:10.1177/0300985810386469
- Berlato D, Bulman-Fleming J, Clifford CA, et al. Value, limitations, and recommendations for grading of canine cutaneous mast cell tumors: a consensus of the Oncology-Pathology Working Group. Vet Pathol. 2021;58(5):858-863. doi:10.1177/03009858211009785
- Hergt F, von Bomhard W, Kent MS, Hirschberger J. Use of a 2-tier histologic grading system for canine cutaneous mast cell tumors on cytology specimens. Vet Clin Pathol. 2016;45(3):477-483. doi:10.1111/vcp.12387
- Scarpa F, Sabattini S, Bettini G. Cytologic grading of canine cutaneous mast cell tumors. Vet Comp Oncol. 2014;14(3):245-251. doi:10.1111/vco.12090
- Camus MS, Priest HL, Koehler JW, Pierezan F, Costa MP, Lavalle GE. Cytologic criteria for mast cell tumor grading in dogs with evaluation of clinical outcome. Vet Pathol. 2016;53(6):1117-1123. doi:10.1177/0300985816638721
- Paes PRO, Horta RS, Luza LC, et al. Inclusion of fibroblasts and collagen fibrils in the cytologic grading of canine cutaneous mast cell tumors. Vet Clin Pathol. 2022;51(3):339-348. doi:10.1111/vcp.13098
- Sabattini S, Renzi A, Marconato L, et al. Comparison between May-Grünwald-Giemsa and rapid cytological stains in fine-needle aspirates of canine mast cell tumor: diagnostic and prognostic implications. Vet Comp Oncol. 2018;16(4):511-517. doi:10.1111/vco.12409