Materials and Methods

Records of all dogs evaluated at Sonora Veterinary Specialists for MCTs from January 1995 to June 2004 were reviewed. One portion of the data was compiled retrospectively (n = 24 dogs), and another portion was accumulated prospectively (27). The prospective study protocol was approved by the internal ethics review committee at Sonora Veterinary Specialists, and owners’ consent was obtained.

Retrospectively compiled data—Criteria for inclusion in the retrospective portion of the study were a diagnosis of primary cutaneous MCT; treatment with neoadjuvant prednisone; no treatment of the mass with prednisone or other corticosteroid prior to initial evaluation at Sonora Veterinary Specialists; no chemotherapy other than prednisone prior to follow-up evaluation; and complete record information, including the dog's age, sex, breed, weight, clinical signs at first evaluation, mean mass diameter and location, diagnosis confirmed by examination of a fine-needle aspirate (n = 2) or biopsy (47) specimen, and response to treatment.

At initial evaluation, masses were characterized as de novo (mass on a dog with no prior history of MCT), recurrent (mass at or adjacent to a previous MCT excision site), or subsequent (mass at a new location on a dog with a history of MCT). The term subsequent was used because it could not be determined definitively whether masses in the last group were metastatic or de novo in nature.

Baseline measurements and objective response data were recorded according to WHO criteria¹⁷ and summarized (Appendix 1). In most retrospective cases, cross-sectional area was only recorded at initial evaluation, and only the categoric response was recorded after neoadjuvant prednisone treatment.

Other data obtained for analysis included month of onset, mass description, results of diagnostic tests used to stage tumors (eg, CBC, abdominal ultrasonography, radiography, buffy coat analysis, and analysis of bone marrow and lymph node aspirates), and histologic reports. Histologic reports were reviewed for tumor grade (according to the Patnaik scheme)¹⁹ and margin quality. Surgical margins were categorized as complete (no tumor within 1 mm of the surgical margin), complete but close (mast cells within 1 mm of the surgical margin), or incomplete (mast cells at the surgical margin). Records were also reviewed for evidence of postoperative complications that might be attributable to prednisone administration (eg, delayed wound healing, wound infection, thromboembolic disease, gastric irritation, and colitis).

Tumor resection was performed according to the 3-cm lateral margin–1 intact fascial plane deep margin rule¹ when possible, and resection margins were determined on the basis of posttreatment mass size. Prior to treatment with neoadjuvant prednisone, the hair over the MCT was shaved and the border of the grossly apparent mass was traced with a permanent marker for identification after the treatment period. When a mass was no longer identifiable because of complete response, the resection margin was just outside the grossly evident border of the mass prior to prednisone administration.

Prospective data collection—Dogs evaluated for MCT from February 1999 to June 2004 were eligible for inclusion in the prospective portion of the study if they met the described criteria and had grossly measurable MCTs that could not be completely resected without amputation or use of reconstructive techniques because of tumor location, size, or both. Assignment of dogs to treatment group was not randomized but was performed on an alternating basis according to date of initial evaluation, with dogs assigned to the low-dose treatment group receiving neoadjuvant prednisone at a dosage of 1.0 mg/ kg (0.45 mg/lb), orally, every 24 hours and dogs assigned to the high-dose treatment group receiving neoadjuvant prednisone at 2.2 mg/kg (1.0 mg/lb), orally, every 24 hours. No criteria other than date of initial evaluation were used to determine group assignment. Tumor dimensions were recorded before and after neoadjuvant prednisone treatment and are reported according to RECIST¹⁸ criteria and tumor volume (Appendix 1). The RECIST criteria for categoric response to treatment were also applied to tumors on prospectively enrolled dogs. These criteria are equivalent to WHO criteria, except that the definition of progressive disease is more stringent (Appendix 2).

Twenty months after conclusion of the prospective portion of the study, follow-up information was retrospectively gathered from medical records and via telephone conversations with owners. Information requested included whether subsequent MCTs had developed, date on which disease progression was diagnosed, site of subsequent MCTs, treatment received for subsequent MCTs, whether the dog was alive or dead, date of death, and cause of death (MCT, other disease, or unknown).

Statistical analysis—Statistical analyses were performed with a commercial statistical software package.^a For descriptive purposes, continuous variables are expressed as mean ± SD or median and range. Categoric variables are expressed as a percentage of the total.

Categoric response was further grouped into response (complete or partial response) or no response (stable or progressive disease). Proportions between groups for categoric variables that were dichotomous were compared with the Fisher exact test (2 × 2 tables). Associations among response to treatment and signalment, initial mass characterization, mass location, and mass duration were determined with the χ² test.

A Student t test was used to assess whether there was a difference in prednisone treatment duration between treatment groups. To determine whether there was a reduction in MCT size (as determined by MaxD and tumor volume) after treatment, regardless of treatment group, the permutation test was used. To compare the efficacy of treatment dose with regard to tumor response (on the basis of MaxD and tumor volume), analysis of covariance adjusting on initial tumor size (both absolute change and percentage reduction) was used. Values of P < 0.05 were considered significant. Follow-up data gathered retrospectively were insufficient for significance testing but are reported descriptively.

Results

Combined data—Criteria for inclusion in the retrospective portion of the study were met in 49 dogs (including all 27 dogs included in the prospective study group). Eleven dogs had multiple masses, 2 dogs were treated twice with an interval of several months, and 1 dog was treated 3 times; overall, 65 masses in 53 treated cases were included.

Median age was 9 years (range, 9 months to 17 years). Sex distribution was 24 males and 25 females, of which 3 males and 3 females were sexually intact. Median weight was 27 kg (59.5 lb; mean, 25.9 ± 14.2 kg [57.0 ± 31.2 lb]; range, 4.5 to 52 kg [12 to 115 lb]). Eleven (22%) dogs were small (0 to 11.4 kg [0 to 25 lbs]), 7 (14%) dogs were medium sized (> 11.4 to 22.7 kg [> 25 to 50 lb]), and 31 (63%) dogs were large (> 22.7 kg). Fifteen (31%) dogs were mixed-breed; 5 (10%) were Golden Retrievers; 5 (10%) were Labrador Retrievers; 3 (6%) were Pugs; 3 (6%) were Shar Peis; 2 (4%) were Shetland Sheep Dogs; 2 (4%) were German Shepherd Dogs; 2 (4%) were Schnauzers; and 1 each was Queensland Heeler, Flat-Coated Retriever, German Shorthair Pointer, Weimaraner, Rhodesian Ridgeback, Cocker Spaniel, American Eskimo Dog, English Bulldog, Shih Tzu, Jack Russell Terrier, Poodle, and Bernese Mountain Dog.

Among the 65 masses, 47 (72%) were de novo masses at initial evaluation, 13 (20%) were recurrent masses at or in the immediate vicinity of a previous excision site, and 5 (8%) were subsequent masses distant from a previous excision site. For 4 of 6 dogs with multiple masses, the diagnosis of MCT was made for the first time at study entry. The duration of 25 masses was not included in the record, 17 masses had been evident for < 1 month, 11 masses had been evident for 1 to 3 months, 8 masses had been evident for 3 to 6 months, 2 masses had been evident for 6 to 12 months, and 2 masses had been evident for > 1 year.

Twenty-seven (42%) MCTs were on the trunk of the body, 24 (37%) were on the extremities, 9 (14%) were on the head, and 5 (8%) were in the perineal or inguinal region. Tumor attributes were described as fluctuant and ill-defined (n = 11), raised (9), erythematous (7), ulcerated (6), infiltrative (6), alopecic (5), pruritic (2), and abscessed (1); the remaining tumors were described nonspecifically as cutaneous or subcutaneous tumors.

Staging diagnostic tests were performed at the discretion of the attending clinician, and the diagnostic series was not complete for any case in the study (thoracic radiographs were not performed in any dog). World Health Organization tumor-node-metastasis staging was not reported for that reason. More extensive testing was primarily conducted for high-grade, multiple, and large tumors. Buffy coat analysis was performed in 17 of 49 (35%) dogs, and all results were negative for mast cells. Bone marrow aspiration was performed in 1 dog, and cytologic characteristics were normal. Aspiration of regional lymph nodes was performed in 8 dogs (4 with regional lymphadenopathy); of those, the cytologic assessment was reactive in 7 dogs and revealed metastasis from a nearby grade III MCT in 1 dog. Results of abdominal radiography were normal in 9 dogs, and splenomegaly was reported in 1 dog (abdominal ultrasonography and needle biopsy were not performed). Abdominal ultrasonography was performed in 14 dogs; of those, 9 results were considered normal, 2 dogs had generalized hepatomegaly (1 had normal cytologic results, and 1 had findings consistent with cholangiohepatitis), 2 dogs had generalized splenomegaly (in 1 of those dogs, cytologic findings were consistent with hyperplasia, and in the other dog, cytologic analysis was not performed but the spleen appeared normal in subsequent ultrasonographic imaging), and 1 dog had lymphadenomegaly (no cytologic assessment, but metastasis was suspected). Forty-eight masses were preoperatively diagnosed as MCTs by fine-needle aspiration and cytologic analysis, and 16 masses were preoperatively diagnosed by biopsy and histologic examination (1 grade I tumor and 15 grade II tumors). Both cytologic and histologic analyses were performed in 1 (grade III) tumor. All cytologic slides were examined by the attending surgical clinician or a clinical pathologist at an outside laboratory.

Neoadjuvant prednisone was prescribed at doses of 0.5 mg/kg (0.23 mg/lb) to 2.2 mg/kg in the combined data group, and was administered orally, once daily. In that group, 1 dog (1 mass) was treated with 0.5 mg/kg daily, 24 dogs (30 masses) were treated with 1.0 mg/kg daily, and 26 dogs (34 masses) were treated with 2.2 mg/kg daily. One dog was treated with 1.0 mg/kg daily for the original mass and with 2.2 mg/kg daily for a subsequent mass. One dog was treated with 2.2 mg/kg daily for the original mass and with 1.0 mg/kg daily for a locally recurrent mass. A third dog was entered into the study 3 times (for the original mass, a subsequent distant mass, and a locally recurrent mass) and was treated with 2.2 mg/kg each time. Signalment and mass characterization at the time of initial evaluation were not significantly different between the 1.0 and 2.2 mg/kg treatment groups, and the 0.5 mg/kg treatment group was too small for statistical comparison. Neoadjuvant prednisone was administered for a median 10 days (range, 3 to 60 days). The most common duration of treatment was 7 days (n = 18), followed by 10 days (16). Only 6 dogs received neoadjuvant prednisone for > 16 days, and only 1 dog was treated for < 7 days.

When response to treatment was grouped categorically, 7 of 53 (13%) cases were classified as a complete response, 30 (57%) were classified as a partial response, 10 (19%) were classified as stable disease, and 6 (11%) were classified as progressive disease (Figure 1). Given this classification scheme, 70% of the cases were considered to be responsive to prednisone treatment. Categoric response to treatment was not significantly related to dose of neoadjuvant prednisone (75% and 64% response rate for prednisone dosages of 1.0 and 2.2 mg/kg, respectively; P = 1.0). The difference in response between retrospectively (WHO criteria) and prospectively (RECIST criteria) enrolled cases was not significant (P = 0.77). Additionally, no aspects of signalment or mass characteristics were significantly related to categoric response to treatment.

After prednisone treatment, 59 of 65 (91%) masses were excised from 44 of 49 (90%) dogs. Fifty-one of 59 (86%) masses were diagnosed as MCTs on the basis of postexcisional histologic evaluation; in 7 (12%) samples, there was no evidence of MCT. Eight of 51 (16%) MCTs were grade I tumors, 39 (76%) were grade II tumors, and 4 (8%) were grade III tumors. One mass (2%) was not submitted for histologic evaluation at the owners’ request. Timing and grade of histologic diagnosis for the tumors were summarized (Figure 2). The distribution of grades for all 65 MCTs was 8 (12%) grade I tumors, 51 (78%) grade II tumors, 4 (6%) grade III tumors, and 2 (3%) tumors of unknown grade. The last 2 tumors were diagnosed by cytologic analysis; 1 was diagnosed in a dog with previous histologically confirmed grade II MCT at a different site, and the other was a first development of MCT.

Of the 17 dogs for which a pretreatment diagnosis of MCT was made on the basis of histologic assessment, 13 had surgical excisions; 1 of those specimens was not submitted for histologic analysis. For the 12 dogs with postexcisional histologic evaluation, 8 were assigned the same grade that had been assigned before treatment, 1 was reassigned from grade III to high grade II, and 3 had no remaining evidence of disease.

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Bar graph depicting the categoric response to treatment with neoadjuvant prednisone in 53 dogs treated for MCT. Bars represent the overall study population and subset populations that received different doses of neoadjuvant prednisone. CR = Complete response. PR = Partial response. SD = Stable disease. PD = Progressive disease.

Citation: Journal of the American Veterinary Medical Association 232, 1; 10.2460/javma.232.1.53

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Confirmation of margin quality was unavailable for most retrospective cases in the study but was reported for tumors in prospectively enrolled dogs. No postoperative complications attributable to prednisone administration were reported in any dogs.

Prospective data—Records of the 36 dogs entered in the prospective portion of the study were analyzed separately. Twenty-seven (75%) dogs completed the study and met the criteria for inclusion. Nine dogs were excluded from the prospective study group: 4 dogs did not return for follow-up measurements or surgery, 3 dogs had only a categoric response to treatment (WHO criteria) recorded (data for these dogs were included in the retrospective data and reported under combined data results), 1 dog had no posttreatment measurements or response recorded, and the record for 1 dog was lost. Of the 27 dogs included in the prospective study group, 8 had multiple masses, and 2 were evaluated twice for solitary masses, yielding 38 masses evaluated overall. With assignment to the low- and high-dose treatment groups alternating by date of evaluation, 12 (44%) dogs with 17 (45%) masses were assigned to the 1.0 mg/kg dose prednisone treatment group (low-dose group), and 16 (59%) dogs with 21 (55%) masses were assigned to the 2.2 mg/kg dose prednisone treatment group (high-dose group). One dog that was evaluated twice was treated for the original mass at 1.0 mg/kg of prednisone and, 14 months later, with 2.2 mg/kg for the locally recurrent mass. Another dog that was evaluated twice was included in the high-dose group both times, 6 months apart.

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Flow chart summarizing tumor grading and diagnostic testing for 65 cutaneous MCTs. *Tumor grade unknown. See Figure 1 for key.

Citation: Journal of the American Veterinary Medical Association 232, 1; 10.2460/javma.232.1.53

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Overall median age of the prospective study group was 9 years (mean, 8.8 ± 3.3 years; range, 6 to 16 years). Sex distribution was 15 males and 12 females. Median weight was 31.8 kg (70 lb; mean, 28.1 ± 15.4 kg [61.9 ± 34.0 lb]; range, 4.5 to 52.3 kg [10 to 115 lb]). Six dogs were small, 4 were medium sized, and 17 were large-breed dogs. Signalment characteristics of the 2 treatment groups were not significantly different.

In the low-dose group, 12 masses were de novo tumors, 2 masses were recurrent at or adjacent to a previous excision site, and 3 masses were a subsequent mass distant from a previous excision site. Five masses were on extremities, 10 masses were on the trunk, 1 mass was in the perineal or inguinal region, and 1 mass was on the head or neck. In the high-dose group, 17 masses were de novo tumors, 4 masses were recurrent at or adjacent to a previous excision site, and no masses were considered to be a subsequent mass. Twelve masses were on extremities, 5 were on the trunk, 2 were in the perineal or inguinal region, and 2 were on the head or neck. Tumor characterization at initial evaluation and mass location were not significantly different between treatment groups.

Treatment duration, pretreatment tumor size, and tumor response for the prospective cases were summarized (Table 1). Neoadjuvant prednisone was administered for a median 9.0 days (range, 7 to 35 days; mode, 7 days [n = 13]; only 2 dogs received treatment for more than 16 days). Mean reduction in MaxD was significant in both treatment groups (35.4% [P = 0.03] and 48.8% [P < 0.001] in the low-and high-dose groups, respectively; overall P < 0.001). Median MaxD reduction was 45.2% for all cases, and 21 of 29 (72%) cases had > 25% reduction in MaxD. In both treatment groups, the larger masses underwent greater absolute change in MaxD; however, there was no relationship between initial tumor burden and percentage reduction in MaxD. After adjusting for initial tumor volume, the difference in mean percentage reduction of MaxD between treatment groups was not statistically significant (32.8% and 49.1% for the low-and high-dose groups, respectively; P = 0.27). Percentage reduction in MaxD and categoric response to neoadjuvant prednisone between treatment groups were compared (Figure 3).

Mean reduction in tumor volume was significant in both treatment groups (52.5% and 78% in the low- and high-dose treatment groups, respectively; P < 0.001 for each group and overall). Median reduction in tumor volume was 80.6% for all masses, and 33 of 38 (87%) masses underwent > 25% reduction in tumor volume. In both treatment groups, the larger masses had greater absolute change in tumor volume; however, there was no relationship between initial tumor volume and percentage reduction in tumor volume. After adjusting for initial tumor volume, the difference in mean percentage reduction of tumor volume between treatment groups was not significant (50% and 80% for the low-dose and high-dose groups, respectively; P = 0.09).

Thirty-six of 38 (95%) masses were excised. Overall, 30 of 36 (83%) masses were grade II MCTs and 2 of 36 (6%) were grade I MCTs. Three of 36 (8%) masses had no MCT remaining (complete response) on histologic examination. The owner declined to submit the mass for histologic examination on 1 dog (high-dose group); in that dog, the mass was biopsied (grade II MCT) before surgery. Response to neoadjuvant prednisone was mediocre (16.7% reduction in MaxD and 27.1% reduction in tumor volume), and the limb was amputated to yield a wide tumor-free margin. Two dogs did not have surgery. Adequate numbers of grade I or grade III MCTs were not available to evaluate the relationship between tumor grade and response to treatment.

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Box-and-whisker plot depicting MCT reduction in MaxD and categoric response to treatment with neoadjuvant prednisone by treatment group in dogs enrolled in the prospective portion of the study (n = 27). Boxes represent the middle quartiles for each treatment group, and the median for each group is indicated by the color change. See Figure 1 for key.

Citation: Journal of the American Veterinary Medical Association 232, 1; 10.2460/javma.232.1.53

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Summary of initial tumor size and response to neoadjuvant prednisone by treatment group for 27 dogs prospectively enrolled in a study in which response to neoadjuvant prednisone treatment and surgical excision in dogs with cutaneous MCT was evaluated.

Citation: Journal of the American Veterinary Medical Association 232, 1; 10.2460/javma.232.1.53

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Thirty-one of 35 (89%) masses evaluated histogically had complete resection margins. Three masses from 3 dogs in the low-dose group had incomplete margins, 1 at each of the carpus, antebrachium, and axilla. One mass from the high-dose group had an incomplete margin; that mass was a recurrence at an incision site on the trunk that developed 11 months after the first definitive surgery (ie, surgery at which complete gross resection was performed). Three of the dogs with incomplete margins were postoperatively treated with adjunctive chemotherapy (eg, with cyclophosphamide, vincristine, and prednisone). The other dog received no additional treatment. Margin quality was not related to response to neoadjuvant prednisone (decrease in MaxD ranged from –3.0% to 85.7% for masses with incomplete margins, and decrease in tumor volume ranged from 0% to 99.2%; P = 0.22). Histologic diagnosis, grade, and margin quality were not significantly different between treatment groups. The dog in the low-dose group that did not undergo definitive surgery was treated with intralesionally injected methylprednisolone acetate for an MCT near the lip. The dog in the high-dose group that did not have surgical treatment received radiation therapy for a locally recurrent MCT at the carpus.

Long-term follow-up—Tumor recurrence was not specifically investigated in this study; however, follow-up information was obtained after the study concluded for 21 of 27 (78%) dogs in the prospective portion of the study. Five dogs were alive with fol-low-up times of 6 to 70 months (median, 18 months), and 16 dogs died or were euthanatized 23 days to 111 months after first diagnosis of MCT. Nine of 21 (43%) dogs had no further development of MCT (4 were alive, and 5 were deceased) with follow-up times of 6 to 70 months. Of the 12 (57%) dogs with subsequent MCT development (disease-free interval of 1 to 44 months), 9 were euthanatized or died for reasons related to MCT at 2 to 52 months after initial diagnosis and treatment. Two dogs with subsequent MCTs died of unrelated causes 18 and 87 months after initial diagnosis. The remaining dog developed a subsequent MCT 6 months after treatment (by excision) and was still alive at 24 months.

Seventeen dogs had complete resection of margins at surgery; 9 had subsequent development of MCT (2 local masses, 1 local mass with systemic spread, 4 regional masses, 1 distant mass, and 1 mass for which no location was identified). Three of 17 dogs with complete resection of margins had no evidence of disease on a postexcisional biopsy specimen. Two of those dogs had no subsequent MCT development (follow-up times, 21 and 33 months). The third dog developed subsequent disease at a new site 4 months after excision of the first MCT. Three of the 4 dogs with incomplete margins had subsequent development of MCT at 3, 14, and 44 months after surgery (2 local masses and 1 regional mass with systemic spread). One dog with incomplete resection of margins had no recurrence but was euthanatized 10 months after diagnosis of MCT because of hemoabdomen associated with splenic hemangiosarcoma and liver metastasis. The overall local recurrence rate was 23.8%, whereas the local recurrence rate for dogs with complete resection margins was 17.6%.

Discussion

The decision to investigate use of prednisone as a neoadjuvant treatment was made on the basis of previous reports of glucocorticoid administration for treatment of MCT and the frustrations and challenges of one of the authors (SDG) with regard to surgical management of MCTs that were difficult to resect because of large size or anatomic site. The effects of glucocorticoid administration on mast cells were recognized in the early 1950s,⁸ and not long after those initial studies, cortisone was first used for clinical treatment of dogs with MCTs.^9,10

In a 1994 clinical report¹¹ describing use of glucocorticoids for treatment for MCT, dogs with recurrent MCT were treated orally with prednisone at a dosage of 1 mg/kg daily for 28 days. In that report, only 20% of dogs had a 50% or greater reduction in tumor size. Those dogs were not examined before 28 days, and it is possible that a transient, more substantial reduction in tumor size was overlooked. It is also possible that because masses in the study population were recurrent, they had more aggressive biological behavior than the masses evaluated in the present study (72% of which were de novo tumors) and therefore a lower response rate. Dobson et al²⁰ briefly described administration of neoadjuvant prednisolone (40 mg/m²) for dogs with MCT for 10 to 14 days prior to radiotherapy. Response to prednisolone was recorded in 24 of 35 dogs, and 75% of tumors had mild to dramatic decreases in size, similar to findings in the present study. In the earlier study,²⁰ no complete responses were reported, 2 dogs had stable disease, and 2 tumors progressed in size. Whether response to prednisolone administered prior to radiotherapy was related to final outcome was not investigated.

The mechanism by which glucocorticoids affect MCTs in dogs is partially understood. Studies in which the effect of treatment with glucocorticoids on nonneoplastic mast cells in vitro (human and murine cell lines) and in vivo (murine models), undertaken to understand human mast cell–related inflammatory conditions such as asthma,^21,22,23 have revealed that glucocorticoids primarily affect fibroblasts and epithelial cells to reduce production of stem cell factor.^21,22 Fibroblasts and epithelial cells synthesize stem cell factor in the presence of inflammatory cytokines (ie, IL-1β).^23,24 Stem cell factor binds to the KIT receptor (a protein-tyrosine-kinase receptor on the surface of mast cells that is encoded for by the protooncogene, c-kit) and induces growth, differentiation, and chemotaxis; enhances release of histamine, leukotrienes, chemokines, and cytokines (eg, tumor necrosis factor-α, IL-3, IL-4, IL-5, and granulocyte macrophage colony–stimulating factor); and increases expression of adhesion molecules such as intercellular adhesion molecule-1 and lymphocyte function–asso-ciated antigen-1.^23,24 Glucocorticoids inhibit inflammation-induced synthesis of stem cell factor in vitro, and the effect is cancelled by concurrent administration of the glucocorticoid antagonist, RU486.²³ In vivo administration of stem cell factor reverses glucocorti-coid-induced apoptosis of mast cells, yielding further evidence for the relationship between glucocorticoids, stem cell factor production, and growth regulation of mast cells.²² Interestingly, increased synthesis of KIT protein in canine MCTs is significantly associated with histologic grade, cell differentiation, nuclear grade, and biological behavior,^25,26,27 findings that support the premise that suppression of stem cell factor production may be a mechanism for the effects of glucocorticoids on neoplastic mast cells in dogs.

In an earlier in vitro study,¹² glucocorticoid administration decreased MCT cell numbers, induced cell death as indicated by trypan blue staining, and had a progressively increasing effect throughout the 6-day incubation period.¹² In vivo, MCTs transplanted to mice continued to grow in the presence of prednisolone, but at a significantly slower rate, and growth rates were reduced in a dose-dependent manner.¹² In leukemic cells, glucocorticoid-induced apoptosis may result from antimitogenic signals (eg, downregulation of autocrine growth factors) that prevent autonomous proliferation and cause cell cycle deregulation.²⁸ Takahashi et al¹² suggested that the effects on MCTs may be similar to that on lymphoid cells because of shared hematopoetic origin. Support for this premise was revealed in an in vitro study involving a nonneoplastic murine cell line in which exposure to glucocorticoids reduced production of IL-4 and expression of intercellular adhesion molecule-1 via direct mast cell interaction; as a result, mast cell apoptosis ensued.²¹

In the present study, population signalment and clinical signs were similar to those in other stud-ies.^{6,7,11,14,15,29-33} Mass location was identified as head-neck, trunk, extremity, or perineal-inguinal on the basis of prior reports^34,35,36 that masses in the perineal-inguinal region or on the head or neck may be associated with a poorer prognosis. Since commencement of the present study, further reports^33,36-39 have refuted these earlier findings regarding the perineal-inguinal location in general. However, one³⁹ of those studies revealed the subset of preputial and scrotal MCTs to have a significantly shorter disease-free interval; survival time was not significantly different from dogs with MCT at other cutaneous locations. Similar to the recent reports, the present study revealed that mass location did not significantly impact response to treatment, although case numbers were insufficient to examine the impact of mass location on long-term outcome.

The primary difference between the present study population and those reported earlier is that cases were limited to those that had tumors difficult to resect (because of size, location, or both) as part of the selection criteria. The histologically complete margin rate of 89% obtained in this study compares well with values from previous studies,^6,7,29,30,32 especially considering the anticipated difficult nature of the present cases given that the selection criteria required evidence of grossly measurable MCT that could not be completely resected without amputation or use of advanced surgical techniques. When reviewed individually, each mass with incomplete resection margins had characteristics that made attaining an ideal margin difficult. Two of the masses arose on the distal part of an extremity, and despite a partial response to treatment with neoadjuvant prednisone, a full-circumference skin resection would still have been required to achieve a 3-cm lateral margin. One axillary mass likely would have necessitated limb amputation to achieve an adequate resection margin at the deep surface. The other mass lay within the incision field of a previous large resection on the thorax and had an apparently complete response to neoadjuvant prednisone. However, despite a 3-cm margin around the previous scar, the lateral margin of the resection was still incomplete.

Because many tumors in this study were large, the impact of initial tumor burden on response to treatment was investigated. Although larger masses had greater absolute changes in MaxD and tumor volume, the relationship between initial tumor burden and percentage change in MaxD or tumor volume was not significant. An additional interesting finding was that all tumors evaluated in the prospective study group that did not change in size or grew had an initial tumor volume < 5 cm³.

In the present investigation, dogs in the retrospective study group were treated with neoadjuvant prednisone at doses of 0.5, 1.0, and 2.2 mg/kg for 10 days, with the latter 2 doses being prescribed in all but 1 instance. These 2 doses are commonly used because of ease of computation and represent the low and high ends of typical clinical dose range for prednisone.⁴⁰ Because the initial impression was that both dosages were effective, they were subsequently investigated in the prospective portion of the study. It was found that treatment with prednisone reduced mass size, similar to earlier findings.²⁰ Both doses resulted in significant reduction in tumor, and 87% of prospectively treated MCTs had reduction in tumor volume of 25% or more, with a median 81% reduction. Seventy-two percent of prospectively treated MCTs had a reduction in MaxD of 25% or greater, with a median 45% reduction. There was no significant difference between the treatment groups, although many percentage reductions in the high-dose group were larger. The power of this study was lower than what was anticipated because of variable response to treatment among the study population and even within individuals (1 dog with 3 masses had reductions of –100%, 0%, and 50% in MaxD and –73%, 60%, and 99.8% in tumor volume). It is therefore possible that a significant difference between the treatment groups was not detected because of type 2 error. Via post hoc power calculation, it was estimated that at least 312 dogs (156 in each treatment group) would be necessary to reveal equivalence of tumor response induced by the 2 neoadjuvant prednisone doses investigated (on the basis of RECIST criteria). Interestingly, when tumor volume was used (also via post hoc power calculation), it was estimated that at least 70 dogs (35 in each treatment group) would be necessary to reveal a significant difference in tumor response between the 2 doses investigated.

For reporting purposes, WHO criteria were used for retrospective cases, and RECIST criteria were used for prospective cases. World Health Organization criteria were released in 1981 to standardize response reporting in neoplasia, but deficits were soon revealed in the system. In particular, progressive disease was suspected to be overreported, leading to early discontinuation of treatment in some patients.¹⁷ The RECIST criteria, first published in 2000, included a simplified reporting process (eg, on the basis of maximal tumor diameter for RECIST criteria vs determination of bidimensional product for WHO criteria) and a larger tumor burden increase cutoff (raised from 40% tumor volume increase to 73% tumor volume increase) for qualification as progressive disease.¹⁸ In addition, the unidimensional measurements used for RECIST criteria have a more linear relationship with tumor volume and cell number than the bidimensional product.⁴¹ Assuming tumors are spherical, the RECIST response categories were set to be otherwise equivalent to WHO criteria response categories for ease of comparison between new and old studies.^18,41 Multiple studies have revealed RECIST and WHO criteria to be comparable when RECIST criteria are retrospectively applied to existing studies.^18,41-43 The retrospective cases evaluated in the present investigation could not be recategorized with RECIST criteria because posttreatment measurements, other than objective response according to WHO criteria, were not recorded. However, the authors believe that combining the retrospective cases on the basis of WHO criteria and the prospective cases on the basis of RECIST criteria to assess the overall study population is validated by results of these prior studies. Additionally, there was no significant difference in response to treatment between retrospective and prospective cases in this study.

The advantage of using RECIST or WHO criteria instead of tumor volume is ease of use. Both WHO and RECIST criteria were developed for easy application to encourage more diligent measurement of lesions at initial evaluation and during treatment.^17,18 Reporting of tumor volume necessitates not only measurement but also more complex calculations. However, tumor volume is considered to more accurately reflect changes in tumor cell number than unidimensional (as with RECIST criteria) or bi-dimensional (as with WHO criteria) measurements.⁴¹ For this reason, we chose to report both RECIST and tumor volume for prospective study cases: RECIST to facilitate comparison of results with those from other studies, and tumor volume to approximate the actual change in tumor cell number. From a surgical perspective, it is the authors’ opinion that 3-dimensional change in tumor size is more applicable to assessing improved resectability than is computation of a single dimension (as in RECIST).

Because most MCTs were grade II masses (80% overall), it was not possible to evaluate the effect of tumor grade on response to treatment. The high rate of diagnosis of grade II tumors was consistent with results of other reports.^{6,7,13,29,30,32} Biological behavior of grade II MCTs can vary, as has been reported in other studies^25,33,44,45 and was evident in the dog in this study in which 3 different responses were seen. Multiple markers for MCTs are now used in combination as panels to facilitate prognostication for grade II MCTs.^{25,26,33,44-47} Further study of response to neoadjuvant prednisone treatment and the influence of tumor grade and marker status could shed more light on the biological behavior of MCTs.

Although long-term follow-up was not the primary aim of this study, follow-up data were obtained for 21 dogs. The overall rate of 57% for development of subsequent MCT is higher than percentages reported previously for grade II MCTs (16% to 53.6%),^29,30,31,33 but the local recurrence rate of 24% (17.6% for complete resections) is not exceptionally high (5% to 26% reported elsewhere).^{29,30,31,32,33} The higher rate for development of subsequent MCT may be a result of sampling bias secondary to the difficult nature (with regard to surgical resectability) of the tumors in dogs enrolled in this study. The recurrence rate may also be inflated because dogs enrolled in a prospective study are likely to have more diligent owners and, as a result, more follow-up information available. However, it must be considered that use of neoadjuvant prednisone only gave the appearance of a clean surgical margin via reduction of peri- or intratumoral inflammation when, in fact, disease still existed. Interestingly, one of the dogs with an incomplete surgical margin had regional, rather than local, recurrence of tumor, and recurrence took 44 months. This raises the question of whether mast cells seen at the margin represented residual tumor or nonneoplastic resident tissue mast cells. Further investigation of the influence of neoadjuvant prednisone use on disease-free interval and overall survival is needed to help answer these questions.

Another question that arises with the use of neoadjuvant prednisone is whether surgical resection of the mass on the basis of posttreatment tumor size will still yield complete margins. Our data suggest that surgical planning based on posttreatment size reliably yields tu-mor-free margins when the dog's anatomy enables adherence to the rule of leaving a 3-cm lateral margin and 1 fascial plane at the deep margin. Whether this would hold true for the more recently advocated 2-cm lateral margin remains to be investigated, although the authors suspect that it would. Studies investigating use of a 2-cm lateral margin for high-grade (high grade II and grade III), locally recurrent, and large (> 3-cm diameter) MCTs are needed before it can be universally recommended.

In the present study, only dogs with difficult-to-re-sect MCTs were enrolled because there was no foreseeable benefit of including dogs with MCTs that could be easily resected with an adequate surgical margin on the basis of mass size and anatomic site at the time of diagnosis. Additionally, there were initial concerns that use of preoperative glucocorticoids might increase the rate of postoperative complications and that surgical planning based on posttreatment mass size might yield incomplete surgical margins. The findings of no dog having postoperative complications attributable to prednisone administration and a tumor-free margin rate of 89% in the difficult resections investigated should allay these fears. It is the authors’ belief that the results support administration of neoadjuvant prednisone in dogs with MCTs to induce consolidation in masses in which adequate surgical margins cannot be confidently attained because of mass location, mass size, or a combination thereof. However, a larger study is still needed to determine the equivalence of or difference between the 2 prospectively investigated doses.