Introduction

Mast cell tumor (MCT) is the most common cutaneous neoplasm in the dog, comprising ≤21% of all skin tumors.^1–3 These tumors exhibit a wide variety of clinical behavior, ranging from benign to highly malignant and rapidly lethal. Because of this disparate behavior of canine MCTs, there has been significant effort to identify prognostic factors to determine which tumors are likely to act aggressively. Some prognostic factors that have been previously explored include tumor characteristics (size, growth rate, recurrent tumor), histopathologic grade, stage, anatomic location, surgical margin status, mitotic count, microvessel density, and c-kit mutation status.^4–21

Tumor grade is generally considered to be the most reliable predictor of prognosis.^7,8,10,22 The three-tiered (Patnaik) grading scheme was used widely for many years to categorize canine MCTs.^7,22 This grading scheme was limited by frequent disagreement between pathologists, and the majority of MCTs are identified as intermediate grade.^10,21,23 For this reason, a two-tier (Kiupel) histopathologic grading scheme was introduced to improve agreement between pathologists and more accurately prognosticate the clinical course of disease.^10,24 Twenty-three to 35% of cutaneous MCTs are considered high grade with the Kiupel grading scale.^10,24–26 Dogs with Kiupel high-grade tumors are more likely to develop metastasis and experience tumor-related death than those with low-grade tumors.^10,21,24,26

Regional lymph node metastasis (stage 2) has variably been associated with a worse outcome in dogs with cutaneous MCT in prior studies.^9,20,26–33 Approximately 20% of dogs with cutaneous MCT are found to be stage 2 at the time of diagnosis, although this number increases to closer to 50% of cases when examining high-grade MCTs or tumors of specific anatomic locations (oral, muzzle, perioral).^{19,26,28–30,34,35} In dogs with stage 2 disease, local control of both the primary tumor and the metastatic lymph node have a positive impact on survival, although the role of chemotherapy is less well-defined in low-grade tumors.^30,36–39 Two retrospective studies showed dogs with Patnaik grade II, stage 2 MCTs treated aggressively with local control and systemic chemotherapy achieved median survival times (MSTs) over 3 yr.^32,38

There have been attempts to standardize histopathologic evaluation of metastatic lymph nodes. Weishaar et al. (2014) described a classification system (HN0–HN3) of MCT metastasis within regional lymph nodes, which predicted clinical outcome. Kiupel grading was not assigned to any of the primary tumors in that study. As such, it remains unknown if Kiupel high-grade tumors are more likely to have metastatic lymph node(s) assigned a higher classification than low-grade tumors.

Hayes et al. (2007) showed that dogs with grade III MCTs that were surgically excised and who received adjuvant vinblastine/prednisone chemotherapy did not reach an MST after 429 days. However, dogs with metastatic disease at the time of presentation had a significantly shorter MST of 322 days. Six of eight dogs with metastasis at presentation were considered to have stage 2 disease, but survival in these dogs was not evaluated separately. Additionally, the metastatic lymph node was not excised in every dog, so some had gross disease at the time they received chemotherapy.

Hume et al. (2011) also evaluated outcomes in dogs with grade III MCTs, including a small subset (n = 5) who were stage 2 at diagnosis. In those dogs with regional lymph node metastasis, the overall survival time was 176 days, but variable treatment protocols were used and not all underwent lymph node extirpation. In a retrospective review by Miller et al. (2016), dogs with stage 2 high-risk MCTs treated with cytotoxic chemotherapy or the tyrosine kinase inhibitor masitinib had an MST of 203 days, but these dogs received variable treatments, and some had gross disease at the time they underwent systemic therapy.

Data indicate that therapy aimed at addressing metastatic lymph nodes significantly influences the outcome in dogs with stage 2 MCT. Mendez et al. (2020) found that any treatment to locoregional lymph nodes (extirpation and/or radiation therapy) in dogs with MCT appears to prolong overall survival time in dogs with various stages of disease and treatment protocols.

Chalfon et al. (2022) also evaluated the impact of lymphadenectomy in dogs with Kiupel high-grade stage 2 MCTs and showed that both time to progression and MST were increased in dogs who underwent lymphadenectomy. This study reported an MST time of 371 days in the dogs treated with lymphadenectomy and variable adjuvant therapy, although not all dogs had adequate local control, and adjuvant therapy was variable and consisted of cytotoxic chemotherapy, a tyrosine kinase inhibitor, or both.

The purpose of this study was to expand upon previous literature by reporting outcome and prognostic factors in a specific subset of dogs with high-grade, stage 2, cutaneous MCTs treated with adequate local control via surgery with or without radiation therapy and adjuvant cytotoxic chemotherapy.

Materials and Methods

Electronic medical records of dogs presenting to the University of California at Davis William R. Pritchard Veterinary Medical Teaching Hospital for treatment of MCT between 2005 and 2019 were retrospectively reviewed. Inclusion required a histopathologic diagnosis of Kiupel high-grade cutaneous MCT with metastasis to a locoregional lymph node (stage 2) confirmed by cytology and/or histopathology. Dogs were required to undergo tumor staging with complete blood count, biochemical panel, and abdominal ultrasound before surgery. Liver and spleen aspirates were performed at the clinician’s discretion. Adequate local control of the primary tumor and the metastatic lymph node was required and was defined as either complete surgical excision of the primary MCT (histopathologic margins free of tumor cells) and lymph node excision, or incomplete excision of the primary MCT followed by definitive radiation therapy to the excision site plus excision or radiation therapy of the metastatic lymph node. Dogs must also have received adjuvant cytotoxic chemotherapy with either vinblastine and prednisone or vinblastine, 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU), and prednisone.

Dogs were excluded if the primary MCT was mucocutaneous or subcutaneous or if visceral metastasis was discovered before treatment. Evidence of visceral metastasis was determined via cytologic evaluation. Dogs were excluded if cytology revealed clustering of well-defined mast cells or if mast cells with atypical morphology were observed. Dogs were also excluded if they had received cytotoxic chemotherapy before referral; however, neoadjuvant chemotherapy prescribed for the MCT in question at the referral institution was permitted. Treatment with corticosteroids before local control was allowed.

Data collected from the medical records included patient signalment, body weight, gross tumor characteristics (anatomic location, longest tumor diameter, presence of ulceration), and initial staging results (complete blood count [CBC], chemistry profile, thoracic radiographs, abdominal ultrasound, cytology of liver and spleen if collected, and cytology and/or histopathology of metastatic lymph node). Gross tumor characteristics, including the presence of ulceration, were determined based on review of the medical record. Treatment protocols, including date of surgery, surgical margins obtained, assigned Patnaik and Kiupel grades, mitotic count, chemotherapy and radiation therapy protocols, and outcome data were also obtained. When available, tumor samples were reviewed by a single anatomic pathologist (K.D.W). Mitotic counts were generated by counting mitotic figures across 2.37 mm² in accordance with recently standardized nomenclature.⁴¹ The stage of disease immediately before surgery was assigned based on the World Health Organization criteria. Lymph node (HN) classification was assigned based on criteria outlined in Weishaar et al. (2014) when possible.

Restaging, including abdominal ultrasound, was recommended at the midpoint and end of the assigned chemotherapy protocol and then every 3 mo after completion of chemotherapy for 2 yr. These diagnostics were performed outside of this timeline if indicated by clinical presentation.

Follow-up information was obtained primarily from medical record review and supplemented with phone calls to referring veterinarians and owners. Overall survival was defined as the number of days between initiation of treatment (either surgery or chemotherapy for those patients who received neoadjuvant chemotherapy) and death or censoring. Overall survival was censored for dogs alive at the time of data collection and for those dogs lost to follow-up. Survival was estimated using the Kaplan-Meier method, and the log-rank test was used to compare survival between groups. Progression-free interval (PFI) was defined as the number of days between initiation of treatment (either surgery or neoadjuvant chemotherapy administration) and documented or suspected disease progression (local recurrence, metastasis, and/or development of a new MCT). PFI was censored for dogs alive or lost to follow-up without evidence of local recurrence or metastasis. Factors evaluated for influence on survival included tumor size, location, ulceration, Patnaik grade, mitotic count, lymph node HN classification, chemotherapy protocol, use of radiation, and development of local recurrence. When there was disagreement in Patnaik grade or mitotic count between the original pathology report and the pathologist review, results of pathology review were used in statistical analyses. All statistics were performed using Graphpad Prism version 8.2.1.

Results

Seventeen dogs met all inclusion criteria and were included in statistical analysis (see Table 1). The patient population was primarily composed of older, large, pure breed dogs. The median age was 7 yr (range, 4–11 yr), and the median weight was 34.2 kg (range, 4.5–55.6 kg). There were seven spayed females, eight castrated males, and two males. Represented breeds included Labrador retriever (n = 3), golden retriever (n = 2), Doberman pinscher (n=2), and single numbers of other pure breeds (n = 9). Only one mixed-breed dog was included.

TABLE 1 Clinical Characteristics and Outcomes in Dogs with High-Grade, Stage 2 MCTs Treated with Adequate Local Control and Cytotoxic Chemotherapy

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One dog (case 6) was reported to have systemic clinical signs at the time of presentation, consisting of hyporexia, lethargy, and pain associated with the primary tumor. The remaining 16 patients did not have any documented systemic clinical signs.

All dogs in this study had their primary tumor localized to either a limb (n = 13) or the head/muzzle (n = 4). The largest tumor diameter was available in 15 cases, and the median tumor size was 2 cm (range, 0.4–15 cm). This measurement was obtained before any neoadjuvant chemotherapy administration. Only one dog (case 6) was receiving corticosteroids at the time that the largest tumor diameter was recorded. This dog had received prednisone 0.5 mg/kg by mouth once daily for 10 days. Eight of 17 (47%) tumors were ulcerated.

CBC and chemistry results at the time of surgery were available for all patients. Three dogs (cases 6, 11, and 13) had mild liver enzyme elevations that were attributable to corticosteroid administration. These three dogs received corticosteroids between 6 days and 2 mo before blood work being performed. One dog (case 1) also had a Veterinary Cooperative Oncology Group-Common Terminology Criteria for Adverse Events version 2.0 grade 1 elevation of total bilirubin with normal liver enzyme values, and one dog (case 5) had a grade 1 creatinine elevation at the time of baseline blood work but a urine specific gravity of >1.050, so prerenal azotemia was suspected.⁴² At subsequent chemistry rechecks, the creatinine normalized. No other significant abnormalities were observed.

Eleven of 17 dogs had thoracic radiographs available for review before treatment, with no evidence of mast cell neoplasia found. All 17 dogs had abdominal ultrasound (AUS) performed before treatment. Aspirates of the spleen and liver were obtained based on clinician and owner discretion. Fourteen of 17 (82.4%) had a fine-needle aspirate and cytology of the spleen. Of these, 7 of 14 (50%) were reported to have splenomegaly, a mottled appearance, or splenic nodules observed on ultrasound. A total of 8 patients also had liver aspirates performed. Six of 8 (75%) dogs who had the liver aspirated were reported to have hepatomegaly or changes in echogenicity (4 hypoechoic, 1 hyperechoic, and 1 “coarse appearance”). In dogs who had aspirates of the spleen and/or liver, no cytologic evidence of MCT metastasis was found. The 3 patients who did not have fine-needle aspirate of the liver or spleen performed had no visible changes on AUS or signs of systemic illness. Of the 2 dogs who were receiving corticosteroid at the time of AUS, one (case 6) had a normal appearing liver, and one (case 11) was reported to have an enlarged, hypoehoic liver. Both dogs had spleen aspirates performed, and case 6 also had liver aspirates performed, none of which showed evidence of MCT metastasis. Case 11 did not have liver aspirates performed.

Fifteen of 17 dogs had aspirates of locoregional lymph nodes performed before surgery, all of which were suspicious of or consistent with metastatic MCT. Selection of the draining lymph node was determined based on anatomic location, physical examination findings, and/or appearance on ultrasound. No patients underwent sentinel lymph node mapping procedures. Fourteen of these 15 dogs had the lymph nodes removed, which then confirmed metastatic MCT via histopathology. One dog (case 1) did not have the metastatic lymph node removed surgically, but that lymph node was subsequently treated with definitive radiation therapy. The lymph node in this dog was normal in size on physical examination but cytologically metastatic. The remaining 2 dogs did not have aspirates of the lymph nodes performed before surgical removal of the lymph nodes, which then were confirmed to be metastatic via histopathology.

Fourteen dogs achieved adequate local control with surgery alone. Three dogs had incomplete surgical margins and then received definitive radiation therapy to the surgical sites. Dogs who underwent radiation therapy (n = 3) received a total dose of 48 Gray administered in daily 3 Gray treatments for 16 consecutive days (Monday through Friday). Two dogs had this protocol administered to both the primary tumor excision site and the lymph node extirpation site. One dog (case 1) did not undergo lymph node extirpation but had the same radiation protocol administered to both the primary tumor excision site and gross metastatic lymph node. Median time to starting radiation therapy after surgery was 20 days (range 14–21 days). Radiation therapy was delivered using a 6-MV linear accelerator^a. Nongraphic (manual) planning or computer-based planning was used, depending on the tumor location and risk to surrounding tissues.

Only six tumors had tissues available for histologic review. Original grading and grading during review were in agreement for 5 tumors. One tumor was originally classified as Patnaik grade III/Kiupel low grade and, after review, was reclassified as Patnaik grade II/Kiupel high grade. The change in the Patnaik grade was because of a lack of cellular atypia and nuclear features, whereas the increase in the Kiupel grade was attributed to a higher mitotic count identified on the pathology review. After this revision, all tumors were classified as high grade in the Kiupel grading scheme. Using the Patnaik grading scheme, 9 tumors (53%) were classified as grade II, and 8 (47%) were classified as grade III. Three tumors were not assigned a Kiupel grade at the time the original histopathology and were not available for histopathologic review. However, all 3 of these tumors had a mitotic count ≥7 in 2.37 mm² recorded in the original histopathology report, so it would fall into the criteria for high-grade tumors in the Kiupel scheme. The median mitotic count for all tumors was 14 per 2.37 mm² (range, 3–62 per 2.37 mm²).

Sixteen dogs underwent lymphadenectomy, and HN classification could be determined for 13 dogs through review of the tissue and/or the microscopic description in the histopathology report. One dog (7.7%) was classified as HN1, 5 dogs (38.5%) were classified as HN2, and 7 (53.8%) dogs were classified as HN3.

After adequate local control was obtained by surgery and/or radiation therapy, dogs received one of two chemotherapy protocols. Eight dogs (47%) received a vinblastine and prednisone protocol, and 9 dogs (53%) received an alternating vinblastine and CCNU protocol. The vinblastine and prednisone protocol was followed as published by Thamm et al (2006). The alternating vinblastine and CCNU with prednisone protocol was followed as published by Lejeune et al (2015). Dose escalations or decreases for both protocols were performed at the discretion of the clinician.

Chemotherapy was initiated during or after radiation therapy or as soon as possible following surgery. Two dogs received 1–2 doses of vinblastine at 2–2.5 mg/m² before surgical excision of the MCT in the neoadjuvant setting, and both received the alternating vinblastine and CCNU protocol after surgery.

The median time to begin chemotherapy after adequate local control was obtained was 19 days (range, 0–87 days). Two dogs had delays in initiation of chemotherapy after surgery because of administration of radiation therapy. The dog with the longest time to starting chemotherapy after surgery (87 days) had the primary tumor and metastatic lymph node excised through the referring veterinarian, and the reason for prolonged time to referral for chemotherapy was not described in the medical record.

Only 7 of the 17 patients completed the entirety of their originally prescribed chemotherapy protocol (2 dogs in the vinblastine and CCNU group and 5 dogs in the vinblastine and prednisone group). Of the 10 who did not finish the original chemotherapy protocol, 8 dogs had progression of their disease while undergoing chemotherapy. One dog who did not finish the original chemotherapy protocol developed elevated liver enzymes suspected to be secondary to CCNU administration and was switched to single-agent vinblastine chemotherapy. The last dog was lost to follow-up before completing the prescribed protocol.

Thirteen of 17 dogs (76%) ultimately developed progressive MCT disease (local recurrence [n = 6], distant metastasis [n = 2], local recurrence and distant metastasis [n = 3] and/or a new MCT [n = 2]). When progressive disease did develop, 2 dogs underwent an additional surgical resection and additional chemotherapy treatment. One dog underwent an additional surgical resection and corticosteroid therapy. One dog underwent palliative radiation therapy and additional chemotherapy treatment. Four dogs pursued additional chemotherapy treatment in the gross disease setting. Four dogs were treated palliatively with corticosteroids alone. In one dog, it is unknown if additional therapy was pursued.

The MST for all dogs in this study was 259 days (range, 55–1722 days) (Figure 1). The median PFI for all dogs was 151 days (range, 55–1722 days). Thirteen dogs were dead or euthanized at the time of data collection, and 4 dogs were censored from the study. Only one dog who was dead at the time of data collection had a necropsy performed, which showed extensive metastatic disease. Another 11 dogs either had gross disease at the time they died or were euthanized for decreased quality of life suspected to be secondary to MCT disease. The remaining dog died at home with multiple comorbidities, and cause of death was not determined. Of the four dogs who were censored, two were alive and disease-free at the time of data collection 1115 days and 977 days after tumor excision, and two were lost to follow-up at 188 and 126 days.

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Factors found to be significantly associated with survival in this population of dogs included development of local recurrence, anatomic location, and presence of ulceration (Table 2). Nine of the 17 dogs ultimately developed local recurrence at a median of 78 days (range, 41–242 days), and their MST was 171 days (range, 55–496 days). One of these 9 dogs had undergone surgery (which resulted in incomplete margins) and followed with radiation therapy. The remaining 8 dogs had surgery alone but obtained complete margins. In the 8 dogs who did not develop local recurrence, their MST was 1354 days (P = .0008; range, 126–1722 days). Dogs with tumors of the head/muzzle had a worse outcome, with an MST of just 139 days (range, 55–496 days), compared with tumors at other locations, in which the MST was 387 days (P = .04; range, 113–1722 days). Dogs with ulcerated MCT had a shorter MST of 188 days (range, 55–496 days) compared with 1354 days (range, 126–1722 days) for dogs without ulceration (P = .01). Factors not found to be associated with survival in this population included tumor size, mitotic count, Patnaik grade, HN lymph node classification, use of radiation therapy, or chemotherapy protocol prescribed.

TABLE 2 Clinical Factors Evaluated Using the Log-Rank Test for Influence on Survival in 17 Dogs with Kiupel High-Grade, Stage 2 Mast Cell Tumors Treated with Local Control and Systemic Chemotherapy

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Discussion

This is the first study to report outcomes in a specific group of dogs with Kiupel high-grade, stage 2 MCTs treated with adequate local control and adjuvant cytotoxic chemotherapy. The results in this study show that these dogs can have a fair clinical outcome, with a MST of 259 days. The MST in this population of dogs was similar to that seen in a previous retrospective study evaluating the outcome in dogs with Patnaik grade III MCTs, in which the median overall survival was 257 days.²⁹ However, that study included dogs with varying stages of disease and treatment protocols, so results are not directly comparable. An MST of 176 days was reported in that study when Patnaik grade III stage 2 patients were assessed separately. In our current study, the dogs with Patnaik grade III, stage 2 MCTs comparatively had a more favorable outcome, with an MST of 259 days. Although numbers in both subgroups are very small, all dogs in the present study did receive cytotoxic chemotherapy, and this may have influenced the outcome.

All MCTs included in the current study were classified as Kiupel high grade, and there is limited data assessing outcomes in dogs whose tumors were graded using this scheme. In one retrospective study of 15 dogs with Kiupel high-grade MCTs treated with surgery and adjuvant CCNU and prednisone, the MST was 904 days.⁴³ Stage of disease was not clearly stated in that publication, but none of the dogs were specified to have lymph node metastasis at the time of diagnosis. As such, the difference in survival time between that study and the current study is presumed to be because of a higher stage of disease. Further research is warranted to determine whether a survival advantage exists between dogs with Kiupel high-grade, stage 1 versus Kiupel high-grade, stage 2 tumors when therapy with adequate local control and adjuvant chemotherapy is provided.

A study evaluating dogs with Kiupel high-grade, stage 2 MCTs showed an MST of 371 days in those who underwent lymph node extirpation, compared with 250 days in dogs who did not have the metastatic lymph node removed.⁴⁰ Despite the fact that 16/17 dogs in the current study had lymphadenectomy performed, an MST of just 259 days was observed. The cause of this difference in survival between canine patients in these studies is not immediately apparent, and improved outcome would be expected in the study described here because of a more stringent local control inclusion criteria. There were differences in adjuvant therapy used in these studies, which may have contributed to the differing outcome, and further study is needed. Additionally, low case numbers in both studies may be a contributing factor.

In this study, the majority of the MCTs included exhibited a very high mitotic count, with a median of 14 mitotic figures seen per 2.37 mm² (10,400× high-power fields). Only 3 (18%) tumors exhibited a mitotic count ≤5 per 2.37 mm². Historically, mitotic counts of >5 or ≥ 7 mitotic figures in 2.37mm² have been correlated with a more aggressive course of disease and significantly shorter survival times.^6,10 A significant difference in survival time based on mitotic count was not found, but this comparison was limited by the small number of dogs with a low mitotic count. Further research on high-grade, low-mitotic count, stage 2 MCTs is warranted to determine if outcome is affected by this difference in mitotic count.

Tumor location on the head, ulceration of the tumor, and development of recurrence after local control were all associated with decreased survival time in this cohort of dogs. Only four dogs in the current study had an MCT located on the head or muzzle; however, the MST for these dogs was only 139 days. This poor prognosis is consistent with previous studies showing head/muzzle MCTs tend to display a more aggressive biologic behavior.¹⁹ Tumor ulceration was present in 47% of the dogs included in this study and was associated with a decreased overall survival time, consistent with prior studies.^30,44 This increased proportion of ulcerated MCTs is likely a reflection of this study including only high-grade MCTs.

Recurrence of MCTs after local control is more likely to occur in high-grade tumors and has been associated with decreased survival in prior studies.^27,30,44,45 Even though all MCTs in this study had aggressive local therapy, 53% of these tumors still recurred despite this treatment. Dogs who developed recurrence tended to do so soon after local control, at a median of 78 days, and the MST for these dogs was only 171 days. This finding is similar to previous studies⁴⁶ and an important point to discuss with owners of dogs diagnosed with high-grade MCTs because local control with complete excision appears to still be inadequate to control local disease in a significant portion of dogs.

This study did not show a survival advantage between two commonly prescribed cytotoxic chemotherapy protocols in this population; however, further evaluation in a prospective setting is warranted to better compare chemotherapy protocols for dogs with high-grade, stage 2 MCTs. This study focused on dogs who received cytotoxic chemotherapy protocols because these have been employed most frequently in the adjuvant setting for canine cutaneous MCT at our institution.^30,47,48 However, since the introduction of tyrosine kinase inhibitors (TKIs) such as masitinib and toceranib for the treatment of MCTs, more clinicians and owners may opt for adjuvant treatment with TKIs.^49,50 In a recent study of 28 dogs with high-grade or metastatic MCTs treated with a combination of vinblastine and toceranib, dogs in both groups had longer survival times (563 days for high-grade tumors, 434 days for stage 2 tumors) than what is observed in the current study.⁵¹ However, these results are not directly comparable because of differing patient populations. Prospective studies comparing systemic cytotoxic chemotherapy versus cytotoxic chemotherapy in combination with TKIs in dogs with high-grade stage 2 MCT are warranted. Data from the present study may serve as a useful basis for comparison to those results.

This study has significant limitations that warrant consideration, including the retrospective study design and small patient numbers, contributing to a possibility for type II error. Although this study aimed to assess a specific population of dogs with high-grade, stage 2 MCTs treated with uniform local and systemic therapy, the retrospective nature of this study produced inconsistencies in chemotherapy treatments (for example, inconsistent dose escalations of vinblastine). Medical record review may have underestimated certain tumor characteristics, such as the presence of tumor ulceration. Additionally, one dog was receiving corticosteroids at the time that the largest tumor diameter was recorded, so size of the tumor may have been underestimated in this case. Small case numbers also prohibited multivariate analysis. As such, it is not possible to determine how the prognostic factors we identified in univariable analysis (presence of ulceration, recurrence, or location of the tumor) might have interacted.

One major limitation of this study is that few cases had tissues available for histopathologic review. There can be differing opinions between pathologists when assigning MCT grade, and it is possible that this may have resulted in inconsistencies in the reported Kiupel and Patnaik grades. Of the six reviewed tumors, five had matching grades in pathology reports and pathology review, but it is unknown whether differences in assigned grade may have occurred in additional cases if more tissues had been accessible for review. This limitation also applies to pathologic classification of metastatic lymph nodes. In the current study, all cases were classified as stage 2 based on review of cytology and/or histopathology reports that concluded the lymph node contained metastatic MCT. Because of the time-frame of when these lymph nodes were excised, the HN0–HN3 lymph node classification system had to be retrospectively assigned to 13 of the excised lymph nodes, and 3 were unavailable for review. Although HN classification was not significantly associated with outcome in the current study (see Table 2), the MST for dogs with H3 metastasis was shorter at 259 days compared with an MST of 360 days for dogs with H1 or H2 metastatic lymph nodes. It is possible that dogs with different classifications of lymph node metastasis may benefit from different treatment recommendations, and there may be value in including this information in future prospective studies for this specific population of dogs.

All dogs included in the present study had metastasis identified in at least one peripheral lymph node. Determination of which lymph node(s) to aspirate was made based on data available to the clinician at the time. No dogs underwent sentinel lymph node mapping to identify metastatic node(s), which is a limitation of this study because MCTs have been shown to metastasize to lymph nodes outside of the nearest draining node.⁵² As such, it is possible that some dogs in the study population could have had additional lymph node metastasis that was not identified.

Although all patients were staged with a CBC, chemistry, and AUS before surgery, there were variable numbers of dogs who had thoracic radiographs and aspirates of the spleen and liver. Because of the retrospective nature of this study, it was not always clear in the medical record as to why clinicians elected to not aspirate the liver and/or spleen in some cases. Given the findings of Book et al. (2011)⁵³ and Pecceu et al. (2020)⁵⁴, it is known that the sensitivity of ultrasound for detecting mast cell infiltration of these organs is low (only 43–67% for the spleen and 0–29% for the liver). Although three dogs did have mild liver enzyme elevations, only one of these dogs had liver aspirates performed, so although corticosteroid administration was the suspected cause of these blood work changes, infiltrative mast cell disease cannot be excluded. Additionally, when aspirates of the spleen and/or liver were performed, medical records did not reliably indicate whether aspirates were obtained of normal or abnormal parenchyma. As such, reclassification of stage may have occurred with further diagnostics in some dogs.

Other limitations of this study include variable follow-up and restaging diagnostics of patients, which may have led to inconsistencies in identifying progressive disease and difficulty in accurately assessing PFI in these dogs. Therefore, survival was selected as the primary statistical endpoint because reliable survival data were available for most dogs in this study. As such, the reported survival times are affected by variables such as the choice of owners to pursue further treatment after progressive disease was identified or to pursue humane euthanasia.

Conclusion

Despite these limitations and the small number of patients included, the results of this study provide important data on the outcome for a specific population of dogs with high-grade, stage 2 MCT who received aggressive therapy. Dogs in this population can experience an MST of about 8.5 mo, and this can serve as a baseline for comparison with future studies in the same population. Dogs with Kiupel high-grade, stage 2 MCTs that are ulcerated, recur, or are on the head may have poorer outcome despite this aggressive therapy. This information may help owners and clinicians to have a more specific conversation about expected outcome for their dog if they elect to pursue aggressive treatment. Further studies in the prospective setting would be ideal to further evaluate the role of chemotherapy, TKIs, and radiation therapy in this patient population.