Prognostication of Canine and Feline Mast Cell Tumors and c-Kit PCR Testing

Canine cutaneous mast cell tumors:

Canine cutaneous mast cell tumors (MCTs) are one of the most common neoplasms in dogs, representing up to 21% of all canine cutaneous neoplasms. Their variable biologic behavior makes them one of the most frustrating neoplasms for veterinary practitioners in terms of advising clients regarding their prognosis and, therefore, recommended therapy. For decades, histologic grading has been used as the sole criterion to predict the biological behavior of canine cutaneous MCTs. The inconsistency of the traditional histologic grading of MCTs has made it difficult to determine prognosis and to select a treatment plan. A newly developed 2-tier histologic grading system has provided a highly reproducible approach to predict the behavior of canine cutaneous MCTs, as accurate grading remains the cornerstone of MCT prognostication. However, supplementation of histologic grading with modern molecular tests and interpretation of results with respect to clinical findings is essential to determine an accurate prognosis and the best therapeutic approach.

Why should we NOT utilize the historical 3-tier grading systems anymore?

The historical 3-tier grading systems, e.g. Bostock or Patnaik systems, suffer a number of limitations that essentially eliminate them from accurate daily use in surgical pathology. Most importantly, as has been shown by numerous studies, is an inter-observer variation that only results in approximately 60% consistency of applying the same grade among different pathologists. The reason for this poor reproducibility lies in the ambiguous and arbitrary features that determine the different histologic grades. As an example, grade 1 MCTs according to the Patnaik system are only confined to the superficial dermis and located in interfollicular spaces. Confirming confinement of neoplastic cells to the superficial dermis is easily affected by the plane through which histologic sections are taken and methodology of tumor sectioning. Furthermore, a recent study investigated tumor depth of canine cutaneous MCTs as an independent parameter and found no associated prognostic significance. Much more arbitrary is the role of the mitotic count in determining grade in the Patnaik classification. While grade 1 MCTs have no mitoses, grade 2 tumors can have a count of 0 to 20 mitoses in 10 high power fields (hpf) and grade 3 MCTs can have a count anywhere from 30 to 60 mitoses per 10 hpf. The huge range of possible mitotic counts within each grade has been one of the major reasons for poor reproducibility of this grading system among pathologists. In addition, the methods by which the mitotic count should be assessed were not detailed and neither grading system used evidenced based methods to determine the cutoffs for mitotic counts for each defined grade, such as receiver-operating characteristic analysis. The mitotic count values for grade 2 and grade 3 MCTs also conflict with more recently published cut-off values that vary from 5 to 7. Secondly, the historical 3-tier grading systems were not correlated with measure prognostic outcomes other than total survival time and didn’t analyze data using multivariate survival analysis. Neither grading system was evaluated based on disease free intervals, time to progression or cause of death, whether due to MCT-related disease or other reasons.

Why should we utilize the novel 2-tier grading system?

A new two-tier grading system for canine MCTs is now used by many pathologists to more accurately identify more aggressive tumors and to eliminate interobserver variation. According to this system, MCTs are graded as high-grade or low-grade based on the number of mitoses, presence of multinucleated cells (3 or more nuclei) or bizarre nuclei, and degree of karyomegaly. As has been shown by a number of independent studies around the globe, the clear definition of specific histologic features for each grade has resulted in a high inter-observer consistency and reproducibility of this grading system. In contrast to previous grading systems, a higher grade predicts the risk of metastasis and MCT associated mortality rather than only a shorter overall survival time. A low grade designation has been shown to be associated with significantly longer survival times and a decreased risk of metastasis.

Why should we NOT combine the novel 2-tier grading system with a historical 3-tier system?

A number of recent studies have proposed to combine the Patnaik system with the 2-tier system by documenting a more accurate differentiation of grade 2 MCTs into grade 2 high and grade 2 low for predicting survival. Not only are most of these studies based on survival data rather than predicting the risk of MCT associated mortality and/or metastasis, they all fall trap to a fundamental error. By combining the 2-tier system with any of the historical 3-tier systems, we automatically lose the consistency and reproducibility of the 2-tier system and end up with a much higher inter-observer variability due to the high inter-observer variability associated with the 3-tier systems. In all studies that propose a combination of these grades, grading using a 3-tier system was performed by either a single pathologist or represent a consensus grade of 2 to 3 pathologists; such methodology would not produce consistent results in daily practice. Furthermore, in the initial study that proposed the 2-tier system, the consensus grading of the Patnaik system was statistically compared to the new 2-tier system. An analysis of variance for the Cox proportional hazard model assessing the association of the proposed 2-tier grading system and the Patnaik grading system showed that when fitting the proposed 2-tier grading system first, the Patnaik grading system was not statistically significant, i.e. it did not contribute any additional information to predicting survival than what was provided by the 2-tier system. However, when the Patnaik grading system was fitted first, the proposed 2-tier grading system was still significant in the model, showing that the 2-tier grade denoted additional statistically significant information about survival. As such, the 2-tier grade is a better predictor of survival than grading using the traditional Patnaik 3-tier system.

Is the novel 2-tier grading system all we need to predict biological behavior?

Neither grading nor mitotic count will ever be sufficient to identify every single MCT with aggressive biological behavior. While a high grade MCT has a very high risk of resulting in systemic disease, and additional therapy should always be considered, some low-grade MCTs may still exhibit aggressive biological behavior. While histologic features alone may suggest a less aggressive biologic behavior, approximately 5% of dogs with histologically low grade MCTs will die due to MCT-associated disease and up to 15% of dogs with low grade cutaneous MCTs may have regional lymph node spread at the time of presentation.
We therefore recommend a mast cell tumor prognostic panel in addition to histologic grading. The panel dramatically increases prognostic certainty and predicts response to targeted therapy. The panel that we offer at the Michigan State University Veterinary Disease Laboratory (formerly the Diagnostic Center for Population and Animal Health) includes cell proliferation analysis (Ki-67, AgNORs), c-Kit PCR to detect internal tandem duplication (ITD) mutations in exon 11 and exon 8, and KIT immunohistochemistry (IHC) to analyze expression of this tyrosine kinase receptor.

What does proliferation analysis tell us?

Mast cell tumors that have a high proliferation activity have been associated with a significantly worse prognosis and decreased survival times. Expression of the nuclear protein Ki-67 is used to determine the number of proliferating cells in a tumor by distinguishing cells that are in the cell cycle (growth fraction) from resting cells. The number of AgNORs correlates with the speed of cell proliferation, i.e. the time it takes a cell to pass through the cell cycle and to divide into two cells. Research indicates that neoplastic cell proliferation activity cannot be predicted reliably by using a single measure, but this combination Ki-67 index and AgNOR score is highly correlated with survival and metastasis, which results in better prediction of prognosis. In studies comparing proliferation indices to survival time, the strongest statistical association with decreased survival time was seen in tumors with AgNOR x Ki67 indices >54, but there was also an association between decreased survival time and tumors with >23 Ki67 positive nuclei per grid. Sixty percent of dogs with MCTs with a combined AgNOR × Ki-67 score >54 died due to the MCT within 12 months of the initial diagnosis. Mast cell tumors with AgNOR x Ki67 indices of ≤54 and ≤23 Ki67 positive nuclei per grid were not associated with a poor prognosis. If chemotherapy is considered, cutaneous MCTs with a high combined AgNOR × Ki-67 score, but no internal tandem duplication (ITD) mutations in exon 11 of c-Kit or an aberrant KIT pattern, have a better response rate to a combination therapy of prednisolone-vinblastine than to treatment with tyrosine kinase inhibitors (TKIs).
Of equal importance, proliferation activity does not only predict survival, but also local recurrence. Low grade MCTs with a low proliferation index, as determined by a low Ki-67 index and a low AgNOR x 67 score, are highly unlikely to recur, even if surgical margins are incomplete.

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Figure 1. Proliferation activity of canine cutaneous mast cell tumors (MCTs) can be determined by evaluating the Ki-67 index and the average number of AgNORs per nucleus with the combination of both best reflecting proliferation activity. (A) While the growth fraction (all cells in G1, S, G2 and M phases) can be determined by the Ki-67 index (red), mitotic figures (blue) are only observed in the M phase and the mitotic count therefore represents a phase index. Proliferating cell nuclear antigen (PCNA; green) is another phase index parameter that labels cells in the S phase, but its long half-life and role in DNA repair can cause additional weaker expression in the G1, G2 and M phases. (B) There are strong differences in the percentage of MCT-related mortality between dogs with low or high proliferation indices as determined by Ki-67 alone, and even more accurately by the combined AgNOR × Ki-67 score. The average number of AgNORs per nucleus correlates with proliferation speed. MCTs with low numbers of AgNORs per nucleus have a less aggressive biological behavior (C), while large numbers of AgNORs per nucleus are associated with a worse prognosis (D). Low grade MCTs with a low Ki-67 index (E) rarely recur regardless of the cleanliness of the margins, while MCTs with a high Ki-67 index have a high risk to cause systemic disease (F). AgNOR silver staining method (C, D). Anti-Ki-67 antibody immunohistochemistry, Fast Red chromogen, hematoxylin counterstain (E, F).
Reprinted from “Sledge DG, Webster J, Kiupel M. Canine cutaneous mast cell tumors: A combined clinical and pathologic approach to diagnosis, prognosis, and treatment selection”, Vet J. 2016 Sep; 215: 43-54.” with permission from Elsevier.

Why do we analyze KIT protein expression and occurrence of c-Kit mutations?

The c-Kit gene encodes the receptor tyrosine kinase KIT, which plays a central role in mast cell survival, proliferation, differentiation and migration. ITD mutations in exon 11 of c-Kit have been detected in about 20 to 30 percent of canine cutaneous MCTs. MCTs with such mutations are highly aggressive, but are twice as likely to respond to treatment (60% response rate) with tyrosine kinase-inhibiting (TKI) therapies, such as Kinavet or Palladia, than dogs with MCTs without mutations (31.3% response rate). ITD mutations in exon 8 of c-Kit are less common and have been detected in 2 to 5 percent of canine cutaneous MCTs. Recent studies have shown that dogs with MCTs with a mutation in exon 8 have a better prognosis than dogs with MCTs with a mutation in exon 11, and even dogs with MCTs with no mutation in exon 8 or 11 (M. Kiupel, unpublished data). Regardless, dogs with MCTs that have any activating mutations in c-Kit will most likely respond to therapy with TKIs.
Aberrant KIT expression patterns have been associated with higher histologic grade of canine MCTs and decreased survival, as well as an increased incidence of local recurrence. MCTs with KIT pattern 1 (perimembrane labeling) have not been associated with a poor prognosis. Dogs with KIT patterns 2 (focal perinuclear or stippled cytoplasmic with decreased perimembrane labeling) and 3 (diffuse cytoplasmic labeling) have a lower disease free survival time and overall survival time compared to those with expression pattern 1. In one study, 30% of dogs with KIT pattern 3 were dead within 6 months and 20% of dogs with pattern 2 were dead within 10 months. Activating mutations in c-Kit are commonly associated with aberrant KIT protein localization; however, not all MCTs with aberrant KIT protein localization have activating mutations in c-Kit. It has been speculated that MCTs with aberrant KIT expression (patterns 2 and 3) will most likely respond to TKI therapy.

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Figure 2. Internal tandem duplication (ITD) mutations in exon 11 of c-Kit and KIT expression patterns are important prognostic indicators in canine mast cell tumors (MCTs). (A) Dogs with MCTs that have mutations in exon 11 of c-Kit have significantly shorter survival times than dogs with MCTs that lack such mutations. (B) KIT pattern 1 is defined by perimembranous labeling in >90% of neoplastic cells. (C) KIT pattern 2 is defined by focal perinuclear or stippled cytoplasmic labeling, and loss of perimembranous labeling, in >10% of neoplastic cells. (D) KIT pattern 3 is defined by diffuse cytoplasmic labeling in >10% of neoplastic cells. Anti-KIT antibody immunohistochemistry, 3,3′-diaminobenzidine (DAB) chromogen, hematoxylin counterstain (B–D).

Reprinted from “Sledge DG, Webster J, Kiupel M. Canine cutaneous mast cell tumors: A combined clinical and pathologic approach to diagnosis, prognosis, and treatment selection”, Vet J. 2016 Sep; 215: 43-54.” with permission from Elsevier.

Recommendations for prognostication of canine cutaneous MCTs

MCT grading, staging (not discussed here), cell proliferation analysis, c-Kit PCR and KIT IHC results, as well as margin assessment (see discussion in previous newsletter), are all linked to MCT-associated survival and metastasis. While each test can be run independently, prognoses developed from interpretation of all tests offer the highest level of certainty, especially for low grade MCTs. The following flow charts provide an overview of a prognostic approach based on clinical and pathological considerations.

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Figure 3. Decisions regarding pursuit of additional local therapy at the site of excision of a cutaneous mast cell tumor (MCT) should be made with respect to completeness of excision (determined by complete margin study), histologic grading using the two-tier system and evaluation of proliferation indices, including Ki-67 index, AgNOR counts and the AgNOR × Ki-67 score. If margins of the excised MCT are free of neoplastic cells (are clean), there is no indication for additional local therapy, regardless of histologic grade and results of evaluation of proliferation indices. There is also no indication for further local therapy if neoplastic cells extend to the surgical margins (are ‘dirty’), and the MCT is histologically of low grade and has low proliferation indices, as there is only minimal difference in local recurrence rate between such tumors with clean or ‘dirty’ margins. In one study, only 6/86 dogs with such MCTs had local recurrence. Of these, recurrence occurred in only 1/40 dogs that had complete excision and in only 5/46 with incomplete margins. There were no statistically significant differences in recurrence between dogs with complete and incomplete excision. There was no significant difference in overall survival between dogs that had initial complete or incomplete surgical excision of a cutaneous low grade MCT with low proliferation activity. However, local therapy, such as secondary surgery or radiation therapy, should be considered if margins are ‘dirty’ and the MCT is of high grade, or is of low grade but has a high proliferation index.

Reprinted from “Sledge DG, Webster J, Kiupel M. Canine cutaneous mast cell tumors: A combined clinical and pathologic approach to diagnosis, prognosis, and treatment selection”, Vet J. 2016 Sep; 215: 43-54.” with permission from Elsevier.

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Figure 4. Decisions regarding pursuit of systemic therapy in dogs diagnosed with a cutaneous mast cell tumor (MCT) should be made with respect to clinical staging, histologic grading using the two-tier system, evaluation for abnormalities in KIT expression by immunohistochemistry (IHC) and PCR activating c-Kit mutations, and evaluation of proliferation indices, including Ki-67 index, AgNOR counts and the AgNOR × Ki-67 score. Systemic therapy should be considered for dogs that have evidence of metastasis on staging, or histologically high grade MCTs, or histologically low grade MCTs that have either high proliferation indices or activating mutations in exon 11 of the c-Kit gene or a KIT pattern 2 or 3 by IHC. There is no indication for further treatment in dogs that have no evidence of metastasis on staging and histologically low grade MCTs with low proliferation indices, no activating c-Kit mutations in exon 11 and a KIT pattern 1.

Reprinted from “Sledge DG, Webster J, Kiupel M. Canine cutaneous mast cell tumors: A combined clinical and pathologic approach to diagnosis, prognosis, and treatment selection”, Vet J. 2016 Sep; 215: 43-54.” with permission from Elsevier.

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Figure 5. Decisions regarding inclusion of tyrosine kinase inhibitors (TKI) in the treatment plan when systemic therapy is elected for dogs with mast cell tumors (MCT) should be made with respect to the results of PCR for activating mutations in exons 8 and 11 of c-Kit and the pattern of KIT expression, as evaluated by immunohistochemistry (IHC). If neoplastic cells of a given primary cutaneous or subcutaneous MCT, or a metastatic focus, are positive for an activating c-Kit mutation in exon 8 or 11, or have a KIT expression pattern of 2 or 3 by IHC, inclusion of a TKI in the treatment plan may be beneficial. If neoplastic cells are negative for c-Kit mutations by PCR and have a KIT expression pattern of 1, there is no indication for inclusion of TKIs in the treatment plan.

Reprinted from “Sledge DG, Webster J, Kiupel M. Canine cutaneous mast cell tumors: A combined clinical and pathologic approach to diagnosis, prognosis, and treatment selection”, Vet J. 2016 Sep; 215: 43-54.” with permission from Elsevier.

Feline mast cell tumors:

Unlike dogs, most cutaneous MCTs in cats are benign and cured by complete surgical excision. Occasionally, however, more aggressive feline cutaneous MCTs and systemic mastocytosis (generally affecting the spleen and/or liver) are seen. For these more aggressive forms, PCR to detect mutations in exon 8 of c-Kit can be performed. Similar to canine MCTs, feline MCTs with a c-Kit mutation are expected to respond to TKI therapy.

Specific prognostic cutoffs have not been established with regards to pattern of KIT expression or Ki67 and AgNOR related proliferation indices for feline cutaneous MCTs. Given the limited published data, no specific prognostic information can be given with reference to any of the evaluated criteria in the canine MCT prognostic panel. One study (Sabattini 2010) evaluated KIT expression and Ki67 expression in feline cutaneous MCTs with respect to prognosis. This study did suggest that clinical outcome in terms of mortality within the 2-year study period was statistically associated with Ki67 index, which was determined using image analysis to calculate the percent of cells positive for Ki67, and a KIT immunoreactivity score, which was determined by multiplying a score derived from the percentage of positive cells by a score determined by the intensity of labeling. In contrast to dogs, KIT pattern was not significantly associated with outcome and no discrete prognostic cutoffs for KIT or Ki67 labeling were described.

Submitted by:

Matti Kiupel, Dr. vet. med. Dr. habil., PhD, Dipl ACVP

Rebecca C. Smedley, DVM, MS, Dipl ACVP

Dodd G. Sledge, DVM, PhD, Dipl ACVP

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