Introduction

Histopathological grading according to the Patnaik scheme has historically been the most commonly used prognostic factor for canine mast cell tumors (MCTs); however, significant interobserver variability has been documented.^1–6 In addition, a large percentage of MCTs are classified as intermediate grade, and, since these tumors have varied biological behavior, additional predictors of prognosis would help to guide staging and treatment recommendations.^1–3,6 Such prognostic factors include markers of cellular proliferation, such as argyrophilic nucleolar organizer regions (AgNORs), Ki-67, and mitotic, as well as expression patterns of kit and occurrence of internal tandem duplication (ITD) mutations in exon 11 of the c-KIT gene.^7–16 These factors may provide supplementary information in addition to tumor grade to further predict the clinical behavior of canine MCTs.

The average number of AgNORs per nucleus and the Ki-67 index are markers of cellular proliferation that have been associated with prognosis in canine MCTs.^10,11 AgNORs are involved in ribosomal RNA transcription, and their number increases with increased cell proliferation.¹⁷ Ki-67 is a protein that is expressed in all active phases of the cell cycle but not in resting cells; therefore, the presence of Ki-67 indicates that a population of cells is dividing.^18,19 The average number of AgNORs per nucleus and the Ki-67 index have been shown to be directly associated with the risk of MCT recurrence (both local and distant) and risk of metastasis at time of death as well as death due to MCT.^7,8,10,11 The product of multiplying these two scores resulted in a better prediction of biological behavior of MCTs compared to using each one as an individual parameter.¹⁰ The average number of AgNORs per nucleus is higher in recurrent versus nonrecurrent MCT.⁷ AgNOR numbers and Ki-67-index have also been reported to be inversely related to survival time for dogs with MCTs, and a higher Ki-67 index was associated with a higher risk of MCT-related death, specifically for dogs with grade II tumors¹¹. Ki-67 index was directly associated with the tumor grade, and it was higher for all dogs that died as well as the subpopulation of dogs with grade II MCT that died compared to those that survived.⁹

One study evaluated the value of proliferation indices for predicting local recurrence of incompletely excised MCT.²⁰ Although individual proliferation indices were not significantly associated with the risk of local recurrence, the combination of Ki-67 and proliferating cell nuclear antigen scores was significantly associated with that risk.²⁰ A similar study found that a higher Ki-67 index was associated with shorter survival, but no association between the Ki-67 index and local recurrence and/or metastasis in dogs with incompletely excised grade II MCT was reported.¹² In one study evaluating subcutaneous MCTs, no association was found between survival and KIT staining pattern, number of AgNORs, or Ki-67 index, while in another study, these variables were significantly associated with local recurrence and metastasis.^21,22

The c-kit gene encodes KIT, a receptor tyrosine kinase found on the surface of several cell types, including mast cells. Mutations in the juxtamembrane coding region of c-kit have been documented in canine MCTs, and the presence of these mutations is positively associated with tumor grade.^23–25 Canine MCTs with ITD mutations in exon 11 of c-kit were significantly associated with an increased incidence of recurrent disease and death.¹⁶ Another study found no statistically significant association between ITD mutations in exon 11 of c-kit and likelihood of recurrence and/or metastasis.²³ The presence of c-kit mutation and cytoplasmic KIT staining has also been associated with an increased Ki-67 index and higher numbers of AgNORs.¹⁰ The expression of KIT can be detected with immunohistochemistry, and the staining pattern and intensity has been associated with an increased risk of local recurrence and decreased survival time.¹⁵ KIT staining pattern and presence of ITD mutation in exon 11 of c-kit have also been significantly associated with shorter disease-free interval and overall survival in dogs with MCTs treated with surgery with or without radiation therapy to the primary tumor followed by systemic chemotherapy.²⁶

In addition, mitotic index can be assessed using routinely stained histopathology slides and has some prognostic significance for canine MCTs. One report including 32 MCTs of three different grades found that mitotic index was prognostic for survival, but no cutoff value was established.¹³ These findings were supported by more recent studies, which found that mitotic index inversely correlated with survival for grade II and grade III MCTs and with survival, disease-free interval, and risk of metastasis and local recurrence, with cutoff values of five and four, respectively.^14,27 In addition, a higher mitotic index was associated with an increased risk of metastatic disease, but the location of metastatic disease and how it was diagnosed were not defined. There is currently no agreed upon cutoff value for mitotic index to distinguish between MCTs with a low and high risk of local recurrence, metastasis, and shorter survival, as different studies have reported different values; however, a specific mitotic index value of 7 has been proposed as part of the new grading system for canine cutaneous MCTs.^6,13,14,27

In many of these studies, however, it is unclear if the MCT-related deaths were due to the recurrence of the primary tumor, growth of de novo cutaneous tumors, or regional or distant metastatic disease. The association between these molecular markers and the presence of regional lymph node metastasis has not yet been investigated. In addition, given the sometimes conflicting reports in the literature regarding the prognostic significance of the individual parameters described above, evaluating all of the parameters simultaneously in a set of tumors may provide a more accurate prediction of the behavior of canine cutaneous MCTs. The regional lymph node is typically the first site of metastasis in most aggressive MCTs; therefore, the ability to predict the risk of regional lymph node metastasis using various prognostic parameters may provide more accurate information and also help to guide the treatment of dogs with cutaneous MCTs.⁵ For example, if a dog were found to be at increased risk of lymph node metastasis, then specific treatment aimed at the regional lymph node, such as surgery or radiation therapy, may be recommended.

The purpose of this study was to compare the average number of AgNORs per nucleus, the Ki-67 index, the mitotic index, the KIT staining pattern, and the presence of ITD mutations in exon 11 of c-kit between dogs with grade II MCTs with and without regional lymph node metastasis at the time of diagnosis.

Materials and Methods

A medical records search was conducted to identify dogs with a histopathological diagnosis of MCT. Dogs with grade II tumors that underwent diagnostic fine-needle aspiration or biopsy of at least one regional lymph node within 1 mo of the diagnosis of MCT were included. Only grade II tumors were included because of their varied biological behavior, in the hopes of being able to use assessments of average number of AgNORs per nucleus, the Ki-67 index, the mitotic index, the KIT staining pattern, and the presence of ITD mutations in exon 11 of c-kit to predict the risk of lymph node metastasis at diagnosis. Dogs that received treatment prior to MCT excision or lymph node evaluation (other than surgical excision of the primary tumor) were excluded. Dogs with metastatic disease beyond regional lymph nodes were excluded, but dogs with multiple cutaneous MCTs were included as long as at least one regional lymph node was evaluated from at least one of the tumors. Dogs were excluded if insufficient primary tumor tissue was available for molecular analysis. Data recorded from medical records included signalment, diagnosis date, location and duration of each MCT, tumor stage based on lymph node evaluation, and whether the MCT was a de novo or recurrent tumor. Recurrent tumors were defined as MCT that occurred in the same location from which a MCT had previously been removed according to the dog’s medical history. Duration of MCT was defined as the time from when the owner or veterinarian first noted the tumor until it was treated with surgery, radiation therapy, or chemotherapy. Cytological lymph node assessment for metastatic disease was based on previously published methods.²⁸ Specifically, lymph node metastasis was defined as a cytological classification of “certain metastasis” based on the prior study, and all samples were evaluated by the pathologist who was involved in developing that classification scheme.²⁸ Histopathological lymph node metastasis was assessed by one pathologist and diagnosed based on large sheets of clustered neoplastic mast cells within subcapsular and medullary sinuses and within corticomedullary tissue. Tumors with no evidence of lymph node metastasis were classified as stage I, while tumors that were associated with lymph node metastasis on cytology and/or histopathology were classified as stage II.⁵

Tumor grade was confirmed by one pathologist using the Patnaik grading system.¹ Mitotic index, Ki-67 index, average number of AgNORs per nucleus, KIT staining pattern, and ITD mutations in exon 11 of c-kit were evaluated by two authors (M.K., T.T.) who were blinded to the clinical case information (including tumor stage). Mitotic index was determined by counting the number of mitotic figures in 10 high-power (400x) microscopic fields that represented the most mitotically active areas according to previous methods.^6,14 Detection and analysis of average number of AgNORs per nucleus, Ki-67 index, KIT staining pattern, and ITD mutations in exon 11 of c-kit were performed according to previously published methods.^10,15,16 For three tumors, only two or three (rather than five) grid areas could be evaluated to determine the Ki-67 index due to limited tumor sample size.

Results

Sixty-six cutaneous MCTs from 64 dogs were included (Table 1). Two dogs had two MCTs, while the remaining dogs had one MCT. Fifteen tumors were stage II, while 51 tumors were stage I. Of the 15 dogs with lymph node metastasis, four were diagnosed on histopathology only, five were diagnosed on cytology only, and six were diagnosed on both histopathology and cytology. Neither of the dogs with two MCTs had lymph node metastasis, so they were placed into the stage I category. Lymph node cytology only was performed in 45 dogs, lymph node biopsy only was performed in 5 dogs, and 14 dogs had both lymph node cytology and lymph node biopsy. Of the dogs that had both lymph node cytology and biopsy, the results of both tests were in agreement in six dogs with stage II tumors and 5 dogs with stage I tumors. Two dogs were diagnosed with lymph node metastasis on biopsy, but not cytology; one dog was diagnosed with lymph node metastasis on cytology, but not biopsy. These tumors were classified as stage II, according to our study methods. Five tumors were recurrent in the area of a previous MCT removal (Table 2). Biopsy tissue and/or cytological and/or histopathological evaluation of the regional lymph node was not available from the original MCT for these dogs, so only the recurrent tumor (for which biopsy tissue and cytological and/or histopathological regional lymph node evaluation was available according to our inclusion criteria) was used for the analyses. The time from when the original MCT was removed until the recurrent MCT was diagnosed ranged from 3 mo to 2.5 yr. Mean values for the cellular proliferation indices, mitotic index, KIT staining pattern, and frequency of ITD mutations in exon 11 of c-kit in dogs with stage I and stage II MCTs are presented in Table 3. In both of the dogs with two MCTs, one tumor had a KIT staining pattern of two, and the other tumor had a KIT staining pattern of one. None of those four tumors had an ITD mutation in exon 11 of c-kit. Three dogs had a mitotic index of greater than 7 (actual values 8, 9, 13), and one of these three dogs had lymph node metastasis.

TABLE 1 Demographics of Study Population

View Fullscreen

TABLE 2 Tumor Stage, Method of Lymph Node Evaluation, and Number of AgNORs/Nucleus for Each of the Five Recurrent Mast Cell Tumors in the Study

View Fullscreen

TABLE 3 Ki-67 Index, Average Number of AgNORs/Nucleus, Product of Ki-67 Index and Average Number of AgNORs/Nucleus, Mitotic Index, KIT Staining Pattern, and Occurrence of ITD Mutations in Exon 11 of C-kit According to Tumor Stage

View Fullscreen

On logistic regression analysis comparing the effect of multiple variables on the presence of lymph node metastasis, there were two variables with P < .20: average number of AgNORs/nucleus (P < .08) and recurrent MCT (P < .07) (Table 4). None of the other variables examined were associated with lymph node metastasis. On multivariable analysis, both variables remained in the model with P < .05. Controlling for recurrent MCTs, for every 1 unit increase in the average number of AgNORs/nucleus, the odds of lymph node metastasis increased by 2.8 (95% confidence interval [CI] 1.0–8.0; P = .049). Controlling for the average number of AgNORs/nucleus, dogs with recurrent MCTs had 8.8 greater odds of lymph node metastasis compared to dogs without recurrent MCT (95% CI 1.1 to 69.5; P = .039).

TABLE 4 Results of Logistic Regression Analysis Showing the Odds of Lymph Node Metastasis Associated with Each Variable

View Fullscreen

Receiver-operator characteristic analysis revealed that a higher average number of AgNORs/nucleus was directly associated with specificity and inversely related to sensitivity (Figure 1). For the seven AgNORs/nucleus values above 3.25, the specificity was at least 92.1%, but the sensitivity was 20% or lower. Average number of AgNORs/nucleus values ≥1.87 were associated with higher sensitivity (93.3%) but lower specificity (27.4%). The area under the curve was 0.65. No cutoff values were significantly associated with lymph node metastasis.

View Figure

• Download as Powerpoint
• Download Figure

Discussion

Our results suggest that the average number of AgNORs/nucleus and the presence of a recurrent MCT are associated with lymph node metastasis at the time of diagnosis in canine MCT. Only three dogs in this study had a mitotic index >7, consistent with a low-grade classification for the majority of MCTs included in this study.⁶

Since the average number of AgNORs per nucleus was found to be associated with lymph node metastasis, receiver-operator characteristic analysis was performed in an attempt to identify specific cutoff values that could be used to predict this occurrence. The area under the curve value (measure of the overall performance of the diagnostic test) was 0.65, which indicates that the average number of AgNORs/nucleus is not a clinically useful test to predict lymph node metastasis using surgical biopsies of canine MCT.²⁹ Furthermore, it was not possible to select a cutoff value that had both high sensitivity and specificity; therefore, we selected a cutoff value with high sensitivity because the average number of AgNORs per nucleus could be considered a screening test for risk of lymph node metastasis at diagnosis. However, this resulted in a specificity of only 27.4%, reflecting a high number of false positives at this particular cutoff. Therefore, using this particular test and cutoff to make clinical decisions regarding the use of chemotherapy might result in over-treating, i.e., using chemotherapy in patients that do not need it. Alternatively, high values for the average number of AgNORs/nucleus have high specificity and thus less chance of a false prediction of lymph node metastasis, but the low sensitivity might result in lower detection and thus possibly inadequate treatment of dogs that truly are at higher risk of lymph node metastasis.

The results of this paper expand the information of previous studies that have shown that cellular proliferation indices, including mitotic index and Ki-67 index, occurrence of c-kit mutations, and KIT staining pattern, are predictive of aggressive biological behavior (increased risk of local recurrence and tumor-associated death) of MCTs.^{7,8,10,11–15,23} However, none of these previous studies evaluate the association between these prognostic factors and the presence of regional lymph node metastasis as an individual parameter.

Local recurrence of MCTs has been associated with shorter survival times and higher risk of metastasis.^9,20 These results indicate that the biological behavior of recurrent MCTs may be different from new tumors, which is consistent with our findings that recurrent MCTs were significantly associated with lymph node metastasis.

There are conflicting reports regarding the prognostic significance of lymph node metastasis in canine MCTs.^28,30–35 These differences may, in part, be due to the impact of treatment of metastatic lymph nodes and inconsistencies in how lymph node metastasis is defined. Results from some studies suggest that in addition to chemotherapy, aggressive treatment of metastatic lymph nodes with surgery with or without radiation therapy can significantly improve outcome in both grade II and grade III MCTs.^36,37 These results demonstrate the importance of evaluating and treating regional lymph nodes in dogs with MCTs.

Limitations of this study are primarily due to the small sample size and retrospective nature. Only the Patnaik grading system was used, and since the sample size was limited to grade II MCTs, the majority of tumors in this study were low-grade according to the two-tier system; therefore, statistical comparisons were not made based on two-tier grading system classification. Only cutaneous MCTs were included in this study. Given recent literature that describes different behavior and prognostic factors in cutaneous versus subcutaneous MCTs, it is possible that different associations could have been found if we had included subcutaneous MCTs.^21,22,27 Size is another tumor variable that was intended to be analyzed, but that information was missing from many of the medical records. When sizes were recorded, they were often estimated, as opposed to actual measurements, making that variable difficult to include in this study. It is generally agreed that cutaneous MCTs that occur in distant locations from other cutaneous MCTs are de novo tumors, but it is possible that some tumors that were characterized as de novo were actually cutaneous metastases. The association between completeness of excision of the primary tumor and lymph node status was not evaluated in this study. Since lymph nodes that were evaluated within 1 mo of the diagnosis of MCT were included in the study, it is possible that complete or incomplete excision of the primary tumor could have affected the development of lymph node metastasis in that time frame.

Since only dogs with lymph node evaluation with cytology or histopathology were included, the study population could have been biased towards dogs with tumors on limbs, as it may have been more likely to obtain a diagnostic aspirate in those patients given the location and ease of sampling of regional lymph nodes. Furthermore, recent data suggest that marking of sentinel nodes may be required to accurately select the lymph node with the highest risk of metastatic disease.³⁸ In this study, the closest lymph node was typically aspirated or biopsied as part of the staging procedure, and it is therefore possible that the stage of disease was underestimated in some patients. However, metastasis to the anatomically closest regional lymph nodes has been shown to negatively impact survival in dogs with Grade III MCT.³⁷ Both histopathological and cytological samples of the regional lymph node were available for only 14 tumors in the study, and there was a discrepancy regarding whether or not metastasis was present in three of those samples. Such discrepancies have previously been reported, though that study found that cytology was more sensitive than histopathology.³⁹

Since cytological lymph node evaluation is more commonly performed in routine practice for MCT staging, and cytological evaluation of lymph node status has been shown to be prognostic, we felt that it was appropriate to include dogs that did not have both lymph node cytology and histopathology results available for this study.²⁸ Given the small number of dogs in this study that had both lymph node cytology and biopsy performed, it is difficult to know if the rate of discrepancy that we found between the two techniques is applicable to the population of dogs with MCTs as a whole. A larger, detailed study comparing surgical biopsies to cytological lymph node samples in a larger study population would be required to more objectively assess the diagnostic accuracy of either method.

The diagnosis of regional lymph node metastases of canine MCT is challenging, and there are currently no markers to differentiate neoplastic from nonneoplastic mast cells. It is also important to recognize that this study evaluated lymph node status at one single point in time, as opposed to determining the risk of later lymph node metastasis according to the same variables. The type of treatment administered to the dogs could affect the risk of future lymph node metastasis and would have presented a significant confounding variable in assessing the association between AgNORs/nucleus, the Ki-67 index, the mitotic index, KIT staining pattern, and the presence of ITD mutations in exon 11 of c-kit and development of later lymph node metastasis.

Conclusion

We conclude that the average number of AgNORs/nucleus and MCT recurrence are associated with regional lymph node stage in canine MCTs; however, no cutoff value for the average number of AgNORs/nucleus that could be associated with lymph node stage was determined. A larger study, particularly one that includes both cutaneous and subcutaneous MCTs, may allow for the establishment of a useful cutoff value for the number of AgNORs/nucleus. Future studies would include a prospective evaluation of the utility of using the average number of AgNORs/nucleus, the Ki-67 index, the mitotic index, the KIT staining pattern, and the presence of ITD mutations in exon 11 of c-kit for predicting the risk of lymph node metastasis at diagnosis and after surgery in canine cutaneous and subcutaneous MCTs. Because of the clinical implications of lymph node metastasis for dogs with MCTs, evaluation of regional lymph nodes for metastatic disease is an important part of the staging procedure, particularly in dogs with locally recurrent MCTs.