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Keywords:

  • canine;
  • grade;
  • histologic margin;
  • local recurrence;
  • mast cell tumour

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

Completeness of mast cell tumour (MCT) excision is determined by assessment of histologically tumour-free margins (HTFM). The HTFM width necessary to prevent local recurrence (LR), recognized as histologic safety margin (HSM) in human oncology, has not been defined. We hypothesized that HTFM width would correlate with risk for LR and high-grade tumours would require wider HTFM than low-grade tumours. Records of dogs with completely excised MCTs were included. Signalment, two-tier tumour grade, tumour size, HTFM width, recurrence and therapy data was collected. High-grade (n = 39) tumours were more likely to recur than low-grade (n = 51) tumours (35.9% versus 3.9%), P < 0.0001, with no association between HTFM width and LR. Twenty-nine percent of low-grade tumours had HTFM less than 3 mm; none recurred. Narrow (≤3 mm) histologic margins are likely adequate to prevent LR of low-grade tumours. High-grade tumours have significant risk of LR regardless of HTFM width.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

Complete surgical excision is crucial for long-term control and a prerequisite for a cure for many solid tumours; this is also the case in most canine mast cell tumours (MCT).[1-4] The surgical guidelines for MCTs are well established in the literature and suggest that these tumours be resected with surgical margins of 2–3 cm laterally and one tissue plane in depth in order to achieve ‘complete’ margins and minimize the risk of local recurrence (LR).[1, 5] The association between surgical margins and histopathological margins, however, has not yet been well studied in canine MCTs making it difficult to interpret how the histologically tumour-free margin (HTFM) correlates with the risk of LR. In recent years, many pathologists routinely report the width of the HTFM surrounding the excised tumour on histopathology reports. However, there are currently no established guidelines on how to interpret and integrate this information into treatment decisions.

As a model and example of how the HTFM can be used to establish a histologic safety margin (HSM), we observe that an HSM has been assessed in basal cell carcinoma (BCC) in people. An HSM is defined as ‘the distance [from the edge of the neoplastic tissue] to the closest resection margin that could be determined microscopically’, and this value is associated with LR of BCC.[6] BCC is a common skin tumour in humans, and complete surgical excision is the standard of care. These tumours are most commonly developed in sun-exposed areas such as the head and neck where wide surgical excision is less desirable for cosmetic reasons. There are two histologic subtypes of BCC evaluated by Auw-Haedrich et al.: solid and fibrosing, and the risk of recurrence is higher for the fibrosing type.[6] The recommended clinical or surgical margin for solid BCC is 2–3 mm, whereas the clinical margin for fibrosing BCC is at least 5 mm. To reduce the incidence of LR, it is recommended that an HSM of ≥0.2 mm be achieved for solid BCCs, but 0.2 mm may not be adequate for fibrosing BCC.[6] We aimed to establish similar guidelines for canine MCTs by defining the HSM for both low- and high-grade tumours.

The purpose of this study was to evaluate associations among the width of the HTFM, tumour grade, tumour size and LR rate in canine MCTs. We aimed to identify the smallest width of the HTFM associated with long-term local tumour control in both low- and high-grade MCTs in order to provide practical treatment guidelines (an HSM) for clinical application. Our hypothesis was that a small (≤3 mm) HTFM width, large tumour size and high tumour grade would be associated with increased risk of LR. In addition, we hypothesized that high-grade tumours would require a wider histologic margin, and therefore a wider HSM, than low-grade tumours to achieve long-term local control.

Methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

Study population

Medical records from dogs presented to the Matthew J. Ryan Veterinary Hospital, University of Pennsylvania with either grade II or grade III MCTs diagnosed between 1996 and 2010 were reviewed. All MCTs were excised with complete surgical and histopathological margins. Dogs were excluded if they received additional local therapy including surgical revision or external beam radiation therapy. In addition, patients were excluded if blocks or pathology slides were not available for histopathologic review or if follow-up data could not be obtained. Patients were not excluded, however, if they received neo-adjuvant or adjuvant systemic therapy.

Procedures

Medical records for all dogs with grade II and grade III MCTs with HTFM were reviewed. Breed, sex, date of birth, diagnosis date, tumour size and location, surgery date, neo-adjuvant and adjuvant systemic therapy, date of LR, date of death and cause of death were recorded. Three dimensional tumour size was obtained from the physical exam form or from the biopsy submission form in the medical record when available, and the long axis was recorded. Of the 90 tumours, 10 had no size recorded and were not used in statistical analysis regarding tumour size. Follow-up information was obtained either via phone or e-mail to the client or primary care veterinarian if the outcome data was not available in the medical records.

All tumours were trimmed using guidelines similar to those published by the American College of Veterinary Pathologists.[7] Tissues were sectioned at 5 µm and stained with haematoxylin and eosin. For the purpose of this study, one pathologist (MG) re-evaluated and graded biopsy samples using both the Patnaik grading system as well as the newly proposed two-tier grading system.[8, 9]

Mitotic index, tumour width (in mm) and width of HTFM (narrowest left and right lateral margins and narrowest deep margin) were recorded. Margins were measured with a diaphanous ruler and recorded in millimeters. Single mast cells and aggregates of eosinophils in the tissue were not considered to be part of the neoplastic process, but aggregates of mast cells were considered to be part of the neoplastic process. Mitotic index was determined by identifying the most cellular and mitotically active area of the slide when scanning at 10× magnification, and the number of mitoses in 10 consecutive high-power (400×) fields was recorded as the mitotic index.

LR was defined as a mass associated with or under the surgical scar diagnosed as a MCT by fine needle aspiration.[4] Cytologic diagnosis of MCT recurrence was obtained from the medical records.

There are three grades of MCTs defined by the Patnaik system with grade I tumours being the most differentiated and grade III tumours being the least differentiated. The grade is defined by histopathologic features such as invasiveness of the tumour, cellularity and mitotic index.[8] The two-tier grading system described by Kiupel and colleagues categorizes tumours as high grade or low grade. In this system, a tumour is characterized as high grade if any one of the following criteria is met: mitotic index ≥7, at least three multinucleated cells in 10 hpf, at least three bizarre nuclei in 10 hpf or karyomegaly.[9] Owing to the accordance between the two grading systems, we elected to use only the two-tier grading scheme in our statistical analysis.

Statistics

Continuous data were expressed as mean ± SD (standard deviation), unless not normally distributed, in which case median values and ranges were reported. Categorical data were expressed as percentages and frequencies. Fisher's exact test was used to evaluate the association between categorical data such as sex, breed, tumour grade by the two-tier system (high and low), tumour location (head and neck, trunk, extremity), treatment with chemotherapy, treatment with prednisone and LR of the tumour. Fisher's exact test was also used to evaluate the association between tumour grade and the use of chemotherapy. Student's t-test was used to evaluate the association between normally distributed continuous data such as age and the narrowest lateral margin with recurrence of the tumour. Because of non-normality of the data, the Mann–Whitney test was used to evaluate the association between tumour size, mitotic index, narrowest deep margin, and narrowest margin overall and recurrence of tumour. The Mann–Whitney test was also used to evaluate the association between tumour grade and the size of the tumour. All tests were two-tailed and P < 0.05 was considered statistically significant.

Logistic regression analysis was used to determine the effects of multiple variables simultaneously on LR. Two-way interactions among the main effects were investigated (tumour grade, tumour size, tumour location, use of chemotherapy and margin size). If not effect modifiers, variables with P < 0.20 in univariate analysis were evaluated in the multivariate analysis. A given variable was retained in the multivariable model when the value of P for that variable was ≤0.05. All analyses were completed using STATA 11 (College Station, TX, USA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

Tumours and grading

Ninety MCTs from 73 dogs were examined. Nine dogs had two MCTs included in this study, and seven of those nine dogs had both tumours excised on the same day. Four dogs had three tumours included in this study. Of these four dogs, all had two tumours removed no less than 6 months apart from occurrence of the third MCT, and all these particular tumours were assigned grade II by Patnaik criteria and low grade by two-tier criteria. According to the Patnaik grading system, 55 were grade II and 35 were grade III. Using the new two-tier system, 51 were low grade and 39 were high grade. Of the 35 grade III tumours by Patnaik criteria, 100% were classified as high grade according to the two-tier grading system. Four of the 55 grade II tumours (7.3%) by Patnaik criteria from four separate dogs were graded as high grade by the two-tier criteria. Of these, one of the four dogs (25%) had LR.

Signalment and tumour location

Represented breeds were mixed breed (n = 22, 24.4%), Labrador retriever (n = 11, 12.2%), golden retriever (n = 10, 11.11%), boxer (n = 6, 6.6%) and Shar-Pei (n = 5, 5.5%). Several other breeds were represented in low numbers. The mean age of dogs included in the study was 8.85 years (SD ± 2.85). The tumour location was categorized into areas of the body as follows: trunk (n = 49, 54.4%), extremity (n = 26, 28.89%) and head and neck (n = 15, 16.67%).

Chemotherapy

Of the 90 tumours included in the study, 32 dogs with 38 tumours were treated with cytotoxic chemotherapy, of which 11 tumours recurred locally. One dog received chemotherapy on two separate occasions for two separate tumours. Twenty seven of 39 high-grade tumours (69%) were treated with chemotherapy while 11 of 51 low-grade tumours (21.5%) were treated with chemotherapy. Of the 14 high-grade tumours that recurred locally, 10 were treated with chemotherapy. Of the two low-grade tumours that recurred locally, one received chemotherapy. Cytotoxic chemotherapeutics used were vinblastine, vinblastine/cyclophosphamide or lomustine. All but four of the dogs that received chemotherapy were administered concurrent prednisone.

Recurrence and predictive factors

The primary endpoint in this study was LR. The association between LR and several of the studied clinical and histopathological factors according to both univariate and multivariate analysis are presented in Table 1.

Table 1. Association between local recurrence and clinical and histologic variables in 90 completely resected mast cell tumours from 73 dogs
VariableMedian (range)Univariate P-valueMultivariate P-value
  1. Mitotic index was determined by identifying the most cellular and mitotically active area of the slide when scanning at 10× magnification, and the number of mitoses in10 consecutive high-power (400×) fields was recorded as the mitotic index. Tumour size was obtained from the medical record when available, and the longest axis was used in statistical analysis. Tumour grade was determined by one pathologist using the criteria of the two-tier grading system.[9] NS, not significant.

Age P < 0.2NS
Recurred10.9 years (2.6–13.6)  
Did not recur8.9 years (2.2–14.3)  
Mitotic index P < 0.001NS
Recurred6.5 MI (0–50)  
Did not recur0 MI (0–40)  
Size P < 0.03NS
Recurred3.0 cm (0.2–10)  
Did not recur2.0 cm (0.2–10)  
Chemotherapy P < 0.03NS
Recurred11/16 treated (69%)  
Did not recur27/74 treated (36%)  
Recurrence by grade P < 0.001P = 0.001
High grade14/39 recurred (36%)  
Low grade2/51 recurred (4%)  

No association was found between recurrence and the narrowest lateral, narrowest deep or narrowest overall HTFM width in this study, and therefore no HSM could be established. High-grade tumours were found to be significantly more likely to recur than low-grade tumours (36% and 4%, respectively, P < 0.001). Only two low-grade tumours recurred; the narrowest HTFM in these two cases were 4 and 20 mm. Thirty percent of low-grade tumours had histologic margins smaller than or equal to 3 mm, and none of these particular tumours recurred. Figure 1 depicts the margin distribution of the narrowest margin (either left or right lateral or deep) for all low- and high-grade tumours. The median narrowest HTFMs in low-grade versus high-grade tumours was 6 mm (range 1–30 mm) and 4 mm (range 1–30 mm), respectively. This difference was not statistically significant, P = 0.29. Figure 2 depicts the narrowest margin distribution of high-grade tumours, comparing those that did not recur versus those that did. The median narrowest HTFMs in high-grade tumours that recurred and that did not recur were 4.0 mm (range 1–20 mm) and 4.0 mm (range 1–30 mm), respectively. This difference was not statistically significant, P = 0.53.

image

Figure 1. Most narrow histologic margin (lateral or deep) in high-grade versus low-grade completely resected mast cell tumours. Histologic margin distribution for all low- and high-grade tumours. The median narrowest HTFMs (lateral or deep) in low-grade versus high-grade tumours was 6 mm (range 1–30 mm) and 4 mm (range 1–30 mm), respectively. HTFM, histologically tumour-free margin.*indicates outliers.

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image

Figure 2. Most narrow histologic margin (lateral or deep) in completely resected high-grade mast cell tumours with and without local recurrence. Margin distribution of high-grade tumours, comparing those that did not recur versus those that did. The median narrowest HTFM (lateral or deep) in high-grade tumours that recurred and that did not recur were 4.0 mm (range 1–20 mm) and 4.0 mm (range 1–30 mm), respectively. HTFM, histologically tumour-free margin.*indicates outliers.

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Treatment with chemotherapy was also significantly associated with tumour recurrence: 27/74 (36%) of the tumours that did not recur were treated with chemotherapy, compared to 11/16 (69%) of the tumours that did recur, P < 0.03. However, there was a significant association between the grade of the tumour and treatment with chemotherapy; only 21.5% (11/51) of the low-grade tumours were treated with chemotherapy while 69% (27/39) of the high-grade tumours received systemic treatment, P < 0.001. Additionally, low-grade tumours were significantly smaller than high-grade tumours with a median diameter of 1.5 cm (range 0.2–6 cm), versus 2.0 cm (range 1–12 cm), respectively, P < 0.008. Lastly, tumours that recurred were significantly larger than those that did not (P < 0.03) and had a significantly higher mitotic index than those that did not (P < 0.001), see Table 1.

On logistic regression analysis, there were no significant interactions. On univariate analysis, there were two variables, in addition to those noted above, with P < 0.2: treatment with prednisone (P < 0.19) and patient age (P < 0.20). On multivariable analysis, only tumour grade remained significant in the model with P < 0.05. High-grade tumours had 13.7 greater odds of recurrence compared to low-grade tumours (95% CI 2.9–65.2; P = 0.001). None of the other variables with P < 0.05 on univariate analysis (treatment with chemotherapy, tumour size and mitotic index) remained significant on multivariable analysis due to their association with tumour grade.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References

The results from this study confirm our hypothesis that high tumour grade is a significant risk factor for LR despite HTFM. The LR rate for completely resected high-grade tumours in this study is high (36%), while the recurrence rate in low-grade tumours appears similar to what has been reported by others evaluating outcome in dogs with grade II MCT (4–21%).[2, 3, 10, 11] However, contrary to our hypothesis, there was no association between LR and the width of the smallest HTFM, and an HSM could not be established.

On the basis of our results, dogs with low-grade tumours have a good outcome, characterized by a very low rate of LR (4%), regardless of the width of the HTFM. However, despite complete excision, high-grade tumours pose a clinical dilemma; 36% of these tumours recurred locally. LR of a high-grade tumour may result in an increased risk of metastasis, and metastasis commonly results in a fatal outcome.[12, 13] In recent literature, LR and lymph node metastasis were reported to be the most common causes of treatment failure in high-grade canine MCT.[12] Other investigators have reported a prolonged disease-free interval and survival in dogs with incompletely resected high-grade MCT treated with radiation therapy both at the primary site and draining lymph node.[13, 14] The substantial recurrence rate of high-grade tumours in this study supports the aforementioned finding that improved remission and survival can potentially be achieved in these patients through the addition of adjuvant loco-regional therapy.

On the basis of the recognized aggressive biological behaviour of high-grade MCTs, adjuvant systemic therapy has become the standard of care in order to delay or prevent metastasis. Although chemotherapy may theoretically have some efficacy in the microscopic or residual local disease setting, its performance as a sole treatment in the macroscopic disease setting has been disappointing.[15-17] Therefore, surgical revision or radiation therapy are typically the recommended modalities if loco-regional recurrence is a concern.[18]

The tissue taken for margin determination is selected based on gross evaluation of the sample, and vertical or ‘breadloafing’ cuts are made where the gross margins appear most narrow. However, the degree and specific extension/location of the microscopic disease into the surrounding tissues may be unpredictable, and gross examination of the tissue may not be adequate to select the most representative sections in all cases. It is possible that had every surgical margin been evaluated, more narrow or even incomplete margins may have been identified.

Mohs micrographic surgery (MMS) technique is the gold standard for removal of many human dermatologic malignancies.[19] In using this technique, all surgical margins are histopathologically evaluated intraopertively, and minimal unaffected tissue is removed. Tissue is frozen, and instead of the standard ‘breadloafing’ technique where vertical cuts are made through the submitted specimen, thin horizontal cuts of tissue are prepared. Each tissue slice is evaluated by the pathologist as the patient waits. If wider surgical margins are required to completely excise the tumour, the surgeon removes another thin tissue section in the area deemed incomplete by the pathologist.[20] This specialized technique is ideal for small tumours but would be technically difficult in veterinary medicine due to the large size of tumours in many cases and the time required for sample processing. Prolonged anesthesia time may be unacceptable to many clinicians. However, this technique has been reported in recent veterinary literature and may have utility in future investigations to determine HSMs for multiple companion animal malignancies.[21, 22]

A completely resected MCT is a common diagnosis in veterinary medicine, and our results suggest that a complete histologic margin, regardless of width, is adequate for long-term local control of low-grade MCTs. Twenty-nine (15/51) percent of the low-grade tumours had a histologic margin of 3 mm or less, and only 4% (2/51) recurred locally. Interestingly, both of these two dogs had margins wider than 3 mm. In fact, the recurrence rate for low-grade tumours was so low that further detailed evaluation of risk factors was not pursued for this sub-group. For these cases, the results from our study show that clean is clean. Thus, routine scar revisions in cases with narrow HTFM may not be necessary.

According to this study and others, grade is the most important predictor of LR in dogs, with 36% of high-grade MCTs recurring locally despite complete HTFM.[2, 3, 11] The question regarding the clinical management of completely resected high-grade tumours and how to identify the cases that would benefit from additional local therapy, could not be definitively answered in this study. Nevertheless, our findings are consistent with previous reports describing loco-regional recurrence to be a common cause of treatment failure in high-grade MCTs.[12] Therefore, it may be reasonable to offer either surgical revision or adjuvant radiation therapy (including the local lymph nodes) for dogs whose owners are interested in aggressive therapy, even when adequate histologic margins have been obtained.

As a secondary observation, use of the two-tier grading system in this study demonstrates the capacity of this system to identify biologically high-grade MCTs. Vascellari and colleagues recently noted that both the Patnaik and two-tier grading systems were significantly associated with patient prognosis.[23] However, the results from this study illustrate the need for additional studies to identify more reliable predictors or markers for LR in dogs with high-grade MCTs. Future studies may include the use of molecular markers, special stains, novel presurgical imaging and more extended evaluation of the surgical margin, perhaps, similar to MMS, in order to devise a patient-specific therapeutic plan for high-grade MCTs.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. References
  • 1
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    Chaffin K and Thrall DE. Results of radiation therapy in 19 dogs with cutaneous mast cell tumor and regional lymph node metastasis. Veterinary Radiology & Ultrasound 2002; 43: 392395.
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    Rassnick KM, Bailey DB, Flory AB, Balkman CE, Kiselow MA, Intile JL and Autio K. Efficacy of vinblastine for treatment of canine mast cell tumors. Journal of Veterinary Internal Medicine 2008; 22: 13901396.
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    Thamm DH, Turek MM and Vail DM. Outcomes and prognostic factors following adjuvant prednisone/vinblastine chemotherapy for high-risk canine mast cell tumour: 61 cases. Journal of Veterinary Medical Science 2006; 68: 581587.
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  • 23
    Vascellari M, Giantin M, Capello K, Carminato A, Morello EM, Vercelli A, Granato A, Buracco P, Dacasto M and Mutinelli F. Expression of Ki67, BCL-2, and COX-2 in canine cutaneous mast cell tumors: association with grading and prognosis. Veterinary Pathology 2013; 50: 110121.