NEW PERSPECTIVES WITH CANINE MAST CELL TUMORS Brian Husbands, DVM, Diplomate ACVIM (oncology)

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1 NEW PERSPECTIVES WITH CANINE MAST CELL TUMORS Brian Husbands, DVM, Diplomate ACVIM (oncology) Overview of Canine Mast Cell Tumors Mast cell tumors are the most common canine skin tumor in dogs accounting for approximately 20% of all skin tumors. The etiology of mast cell tumors in dogs has not been determined. While mast cell tumors are more commonly seen in mixed breed dogs, several breeds to be at higher risk for developing mast cell tumors than others. These include brachycephalic breeds (boxers, English Bulldogs, pugs, and Boston terriers), Labradors, golden retrievers, Rhodesian ridgebacks, Viszlas, schnauzers, beagles, and Shar peis. In these breeds, there may be an underlying genetic abnormality that leads to higher risk of developing a mast cell tumor. History and Clinical Signs: The majority of canine mast cell tumors arise in the skin or subcutaneous tissue. Primary mast cell tumors have been rarely reported to develop in other areas in the body (gastrointestinal tract, spleen, other sites). In many patients, the skin or subcutaneous mass is incidental. In some cases, it is known to have been present for many months prior to evaluation. Subcutaneous mast cell tumors are often soft and have been misdiagnosed as a lipoma based on palpation alone. While most patients present with a solitary skin/subcutaneous mass, up to 15% of patients will have more than one tumor simultaneously (Withrow, 2013). In some patients, clinical signs attributable to mast cell disease are present. These can include local inflammation and discomfort secondary to the tumor in/under the skin as well systemic signs as a result of the release of histamine, heparin, and other substances and their effect on the body. Diagnostics: Most mast cell tumors are easily diagnosed with needle sample/cytology. Baseline labs are recommended depending on the overall health status of the patient in considering anesthesia and surgery. Staging tests that are often considered in the assessment of a patient with mast cell neoplasia includes sample the regional lymph node (if accessible), thoracic imaging (to evaluate distant lymph nodes and screen for additional comorbidities), abdominal ultrasound with sampling the liver and/or spleen and any internal enlarged lymph nodes, and a bone marrow aspirate. There is newer data out suggesting that in most cases, sampling the regional node would be a reasonable initial approach in dogs with mast cell neoplasia (see below). If the regional lymph node sample is free of metastasis, it is less likely that there is remote/distant metastasis. In patients undergoing extensive surgery (reconstructive surgery or amputation) or radiation therapy, additional staging is often recommended to ensure that there is not distant metastasis before such an undertaking. Therapy:

2 Surgery is the treatment of choice for solitary mast cell tumors and potentially for dogs with more than mast cell tumor. The standard recommendation for mast cell tumor removal has been removal of the visible mass with 2-3cm gross margins lateral to the tumor and one facial plane deep to the tumor. With this approach, most mast cell tumors will be cleanly excised with adequate margins (more than 5mm on histopathologic evaluation). For small tumors, or those in locations (distal limbs or head/face) that make a larger resection challenging or impossible there is the potential to remove less tissue and achieve optimal outcomes (see below). In patients with solitary, low grade mast cell tumors that have been cleanly excised with adequate histopathologic margins do not require additional therapy. Most oncologists feel that completely excised mast cell tumors with 5mm margins lateral and deep to the tumor is considered adequate without the need for additional local therapy (additional surgery OR radiation therapy to the site). Typically monitoring the incision site and a thorough examination for any new skin masses, enlarged regional nodes, and abdominal organomegaly is recommended serially after the initial diagnosis. Patients diagnosed with a high grade mast cell tumor are considered at risk for the development of metastasis. Between 60 and 90% of patients with a high grade mast cell tumor will develop metastatic disease. If there is no evidence of metastasis, excision with adequate surgical margins is recommended for local control in addition to systemic therapy/chemotherapy. There are a variety of chemotherapy options available. Post-operative chemotherapy options that are most frequently used include vinblastine injectable chemotherapy, vinblastine injectable alternating with CCNU (lomustine) oral chemotherapy, or toceranib. Other treatment protocols have been reported as well. Prognosis: There have been a number of prognostic factors that have been reported including: a) Tumor grade: see more below b) Clinical stage of mast cell cancer: those that have metastasis at the time of diagnosis have shorter survival expectations than those that stage clean. c) Clinical behavior prior to removal: tumors that have been present for prolonged periods of time (months to years) tend to be low grade. d) Location: Subungual (nailbed), oral, and other mucous membrane sites are often more aggressive than other sites. Preputial and perineal mast cell tumors also tend to have a more aggressive behavior than other sites. e) Breed: Boxers and other brachycephalic breeds tend to develop lower grade mast cell tumors. Mast cell tumors that develop in Shar peis have the potential to behave more aggressively. The focus of this lecture will be new perspectives of mast cell cancer in dogs: I) I keep seeing reference to a new MCT grading system and I am not sure why there was a change and what the change means to me and my patient.

3 Grading System Features for Canine Mast Cell Tumors: Patnaik system (three tiered) Kiupel system (two tiered) Mitotic Index-reported as a # Mitotic Index-reported # Degree of Cellularity-subjective Multinucleate cells-reported # Cellular Morphology-subjective Presence of bizarre nuclei-reported # Degree of invasion-subjective Karyomegaly in at least 10% of cells Degree of Stromal Reaction-subjective The most commonly used reference for mast cell tumor grading is the Patnaik grading system, reported in This three tiered grading system (low grade, intermediate grade, and high grade) has been incorporated in most of the published studies evaluating outcomes for dogs with mast cell tumors in the 35 years since the initial publication. The five parameters assessed with this grading system include: number of mitotic figures seen (mitotic index), cellularity, cellular morphology, degree of invasion, and degree of stromal reaction. Of the factors assessed, all features except mitotic index are subjectively reported by pathologists (as mild, moderate, or severe). Each of these parameters is assigned a number based on the severity of the change seen and a cumulative number is totaled. The grade is assigned based on the cumulative total reported. As a result of the number of features and the subjectivity involved in their assessment, wide variability in assessing mast cell tumor grades exist with the Patnaik grading system. This means that one pathologist might report a certain tumor as a low grade mast cell tumor and a second pathologist might report the same tumor as an intermediate grade mast cell tumor. Within the Patnaik grading system, mitotic figures (number of dividing cells) are evaluated. Two recent reports have evaluated the mitotic index (the number of mitotic figures seen in 10 HPFs) as the sole feature and its impact on outcome post-operatively. Mast cell tumors with a mitotic index of 5 or more per 10 HPFs are associated with shorter survival than those with a lower mitotic index. Due to the inherent subjectivity in applying the three tiered Patnaik grading scheme to canine mast cell tumors (low, intermediate, and high grades), a newer grading system with less subjectivity was proposed. This two tier grading system (low and high grade) was described by Kiupel, et al in This system evaluates the number of mitotic figures seen within the neoplastic cell population (mitotic index), the number of multinucleate neoplastic cells, the number of neoplastic cells with bizarre nuclei, and the % of mast cells with nuclear diameter varying at least two fold. As a result of the numerical criteria and the elimination of three available options for grade using the previous system, the repeatability in pathologist interpretation appears to have improved. There are three publications that have applied the Kiupel grading system to patients undergoing surgery for mast cell tumors and it appears to more accurately predict outcomes. In most of the patients where both of the systems have been applied to the mast cell tumor, low grade tumors with the Patnaik system remain low grade with

4 the Kiupel grading system. Similarly, Patnaik high grade tumors remain high grade with the Kiupel grading system. The intermediate grade (grade II) with the Patnaik system is more diverse in their categorization using the Kiupel system, meaning some are identified as low grade and some are high grade. The median survival time reported in the initial publication was not met at greater than 2 years for low grade mast cell tumors and 3.6 months for high grade mast cell tumors. Since the initial description and evaluation in predicting outcome in canine mast cell tumors in 2011, several groups have assessed this grading system in predicting outcome supporting the benefit of a two tiered grading system. II) I have diagnosed a mast cell tumor in a dog! My client is willing to do anything for their dog. What staging tests should be performed? Should every mast cell tumor have complete staging prior to therapy? When canine mast cell tumors metastasize, they tend to spread to the lymph nodes as well as the spleen and the liver. Bone marrow metastasis can be seen, but this is uncommon when compared to other sites (especially if the other sites are clear of metastasis). Other sites of metastasis are possible, but are very rare. There is controversy regarding the extent of staging needed as a minimum prior to therapy for a newly diagnosed mast cell tumor. Staging is never wrong as it adds information up front about the nature of that patient s particular cancer and has the potential to detect unrelated comorbidities. Finding advanced stage mast cell cancer at diagnosis in concerning, but if there is no evidence of metastasis or if there is loco-regional metastasis present (ie, the primary mast cell tumor with metastasis only to the regional lymph node), local therapy is recommended and can result in long term survival. In addition, patients with distant metastasis would benefit minimally from the removal of a mast cell tumor on the skin. Staging can guide the recommendations for the patient, but taking a logical approach seems warranted most of the time. In the past, smaller scale studies seemed to have mixed results regarding the benefit of complete staging. As a result we have been attempting to delve further into the benefit or lack thereof for complete staging. Two larger scale studies have surfaced in the past few years (Warland, et al, Vet Comp Onc, 2012 and Stefanello, et al, JAVMA, 2015) looking at the benefit of staging in dogs with newly diagnosed dermal mast cell tumors. Warland, et al. retrospectively evaluated 220 newly diagnosed mast cell tumor patients over 12 year period. These patient were staged prior to therapy. Patients had regional lymph node assessment when possible/accessible, an abdominal ultrasound, and thoracic radiographs performed. Just over ½ of the lymph nodes were assessed cytologically. Of these cases, 68 (31% of the entire population of 220 patients) were found to contain metastatic mast cell cancer. In the initial evaluation of these patients, distant metastasis was detected in 15 patients. All of the 15 patients with distant metastasis also had involvement of the regional lymph node suggesting that node assessment be a reasonable first step in staging a patient with a newly diagnosed mast cell. The second study (Stefanello, 2015) correlating a battery of staging tests at the initial diagnosis of mast cell cancer compared the new two tiered histologic grading and the traditional three tiered scheme with the presence of metastasis at

5 diagnosis in 386 dogs. All dogs had assessment of the regional lymph node, thoracic radiographs, abdominal ultrasound, and bone marrow sampling. All patients with irregularities in the liver and/or spleen had sampling performed to screen for metastasis and some patients with normal appearing liver/spleen were sampled (clinician preference). Regional lymph node metastasis was detected in 18% (n = 70) of the patients. Distant metastasis was detected in 4% (n = 16) of the patients, most frequently to the spleen (n = 5), liver (n = 2), or both (n = 6). Notably, 2 of the 16 patients had distant metastasis without regional lymph node involvement. When correlating the Kiupel two tiered grading scheme to those with metastasis, 15% (44 of the 295 low grade tumors) were found to have metastatic disease. The lymph node was involved in all but one of these cases. In correlating the high grade tumors with metastasis, 31% (28 of the 91 low grade tumors) were found to have metastasis at diagnosis. In this subset of patients, the lymph node was involved in all but one of the patients. The degree of staging is largely oncologist dependent, but as a starting point, I always recommend sampling/aspirating the regional lymph node when possible, even when it is palpably normal. Additional staging is strongly recommended if the node is deemed positive OR there are there are additional features that are worrisome about the case. Cues that I use to consider additional staging up front/prior to surgery include rapidly growing mast cell tumors, those that are large or markedly infiltrative into the tissue beneath, or for patients that are feeling off. III) What are the current recommendations for narrowly or incompletely excised mast cell tumors? Narrowly excised mast cell tumors: The definition of completely excised mast cell tumors with adequate margins has not been consistently described in the literature. Most oncologists feel that tumors that have been removed with a rim of normal tissue surrounding the tissue that is 5mm or greater on histopathology have had an appropriate surgical prescription. There is data to suggest that for low grade tumors, 3mm or greater may be sufficient in long term survival (Donnelly, Vet Comp Onc, 2013). In patients with high grade mast cell tumors, 5mm or greater of cancer free tissue is recommended. Incompletely Excised Mast Cell Tumors: With incompletely excised mast cell tumors, some patients do not develop recurrence. In most patients, it is impossible to accurately predict those that will or those that will not develop recurrence. Recurrent tumors have anecdotally been reported to behave more aggressively. Incompletely excised low grade tumors with a low mitotic index may have a lesser recurrence rate than tumors with a high mitotic index or those that are deemed high grade, but this has not been evaluated on any large scale. Given this, in most cases, additional local therapy (additional surgery or radiation therapy) is recommended to reduce the chance of recurrence. Until recently, there was minimal data available assessing the benefit of additional local therapy compared to monitoring without additional local therapy. Boston, et al (Vet Surg, 2015) evaluated 64 patients (total of 70 tumors) retrospectively over a 10 year period for the impact of additional therapy for narrowly or incompletely excised canine mast cell tumors. Of the treatment groups for the 70 tumors assessed (64 patients), 33% underwent re-excision/surgery, 30% received local radiation therapy to the incision/scar, and 37%

6 underwent monitoring without additional local therapy. Median follow up time for the entire population was approximately 15 months (range 1.5 months to over 8 years). Recurrence rate between the groups: Additional surgery = 13% recurrence (3/23 MCTs) Radiation therapy to the incision = 10% recurrence (2/21) No additional local therapy = 38% (10/26). For those that did develop recurrence, the median time to recurrence was found to be shorter (399 days) in the group of patients that did not undergo a second surgery or radiation therapy (over 5 years in both groups). The statistical evaluation between those that received additional therapy and those that did not was significant as was the difference in time to mast cell tumor recurrence. As a result of the potential for recurrent mast cell tumors to be more challenging to treat and the results of this publication, additional therapy is recommended if feasible. IV) My patient has a mast cell tumor in an area that prohibits a large surgery (2cm or greater lateral margins) without reconstruction. Are there any surgical recommendations short of reconstructive surgery (or amputation for those on the distal limbs)? Our general recommendation for surgical excision of a known mast cell tumor is to excise 2-3cm lateral to the mast cell tumor and one facial plane deep to the tumor when feasible. In most cases, this will yield a 5mm or greater rim of cancer free tissue between the tumor and the lateral surgical margins. In larger patients with a dermal or subcutaneous mast cell tumor on the trunk, the neck, or the proximal limbs, this is usually accomplished without complication. In smaller patients, or those with a mast cell tumor on the distal limb, this can be challenging. Recent data evaluating outcome using a modified surgical approach to the standard surgical recommendation has been published (Pratschke, JAVMA 2013). This modified approach evaluated surgical removal of a dermal mast cell tumors with lateral margins that were equal to the diameter of the tumor up to 4cm (meaning that a 1.2cm mast cell tumor was removed with 1.2cm margins laterally on all edges and one fascial plane deep). In patients with tumors that were 4cm or greater, 4cm margins were used as the maximum margin lateral to the tumor. Forty seven tumors in 40 dogs were included in the evaluation. A ruler or calipers were used to measure the tumor and the distance around the tumor. Eight separate ink markings were made and these were joined to form a circle around the tumor to guide the surgery. Of the 47 tumors, there was only one dog with a tumor larger than 4cm. On pathology evaluation, 87% (41 of the tumors) were considered to be low grade tumors and 13% (6 of the tumors) were considered to be high grade tumors. 85% (40 tumors) had tumor free margins of 1mm or greater and 15% (7 tumors) were found to have incomplete MCT removal. The range of time for follow up in this collection of patients was between 1 day to over 1 year (median: 8 months). Of the 40 patients, 29 of these were re-evaluated at least 6 months following surgery or longer. Of these patients, 1 developed local recurrence 45 days after the initial surgery and a second patient developed mast cell tumors at other sites distant to the initial surgery site. Both of these patients had high grade mast cell

7 tumors with >1mm surgical margins. Overall, this approach has some limitations and the data in the section above regarding the long term outcome without additional therapy is concerning regarding the potential for recurrence, but it may be a feasible approach for select patients. V) The pathologist is suggesting the MCT prognostic panel and I am not sure what this panel is and if it will benefit my client/patient. The MCT panel has become popular and is often recommended by pathologists after a canine mast cell tumor has been diagnosed. It is offered by Michigan State University and Colorado State University and includes evaluation of cell proliferation markers (Ki-67, PCNA, and AgNOR), c-kit PCR to detect internal tandem duplication (ITD) mutations in exon 11 and exon 8, and KIT immunohistochemistry to evaluate expression of this tyrosine kinase receptor on/in the neoplastic mast cells. The panel requires tissue (either tissue in formalin or processed slides from the original pathology service forwarded to MSU). a) Proliferation Indices: Ki-67, PCNA, and AgNOR immunohistochemistry: Ki-67 evaluates the number of proliferating mast cells in the tissue and has been found to be predictive of survival in dogs with mast cell tumors in a number of studies. It appears to correlate with histologic grade relatively well (low Ki-67 are more common in low grade MCTs and high Ki-67 is more frequently detected in high grade MCT). PCNA (proliferating cell nuclear antigen) is seen in highest concentration in the DNA synthesis phase of the cell cycle. When evaluated in canine mast cell tumors, higher numbers of cells with PCNA present has been associated with overall survival in dogs with mast cell tumors. AgNOR (agyrophilic nucleolar organizing region) is also associated with cellular proliferation in neoplastic canine mast cells. The number of AgNORs correlates with the speed of cell proliferation and can help to predict mast cell tumors that might have a more aggressive nature. Data assessing these proliferation indices indicates that neoplastic cell proliferation cannot be reliably predicted using a single measure, but when used in combination have the potential to predict metastasis and overall survival in canine mast cell cancer. b) Tyrosine kinase c-kit and KIT evaluation: c-kit gene mutation (exon 11 and exon 8) and KIT expression: c-kit gene mutations in exon 11 of c-kit have been detected in 20-30% of canine cutaneous MCTs. MCTs with such mutations tend to be highly aggressive. Mutations in exon 8 of c-kit are less common and have been detected in two to five percent of canine cutaneous MCTs. Tumors with either of these mutations are expected to respond to tyrosine kinase inhibitors (TKIs), such as toceranib (Palladia), although inherent resistance to these drugs is possible as with any cancer/chemotherapy interaction. KIT expression in canine MCTs: Immunohistochemistry to assess for the KIT protein in mast cells has shown that the presence and location of expression has also corresponded with survival. KIT is the tyrosine kinase protein receptor that exists on normal mast cells and contributes to normal mast cell function. Aberrant KIT expression patterns characterized by increased

8 intracytoplasmic expression and loss of membrane-associated/diffuse expression have been linked with decreased survival in dogs with MCTs. MCT grading, cell proliferation analysis, c-kit mutations, and KIT IHC results have correlated with aggressive behavior in canine mast cell tumors and overall survival. When all of the features assessed in the panel are concerning or alternatively, all are more benign, the combination is easy to interpret and helpful in guiding the most appropriate therapy for the patient (ie, a more intense, multimodality approach in therapy is indicated for those with a c-kit mutation, intracytoplasmic expression of the KIT protein, and high Ki-67 and AgNOR proliferation indices). The biggest limitation with this panel is when two or three of the tests suggest a more aggressive behavior and two or three of the tests suggest a more benign behavior. If this occurs, I typically will scrutinize the results and look at that test individually regarding the possibility of aggressive mast cell cancer behavior. To date, we have not been able to compare each of the individual tests against one another to determine if one or a specific combination of two weigh heavier than the others in interpretation of the panel with predicting outcome. I tend to perform/recommend the Mast Cell Tumor Prognostic Panel in patients that have a mast cell tumor has been deemed a low grade tumor, but is not behaving as a benign tumor (ie, rapid growth or invasive) OR in cases where the pathologist feels less confident in their ability to comfortably grade the mast cell tumor as either a high grade tumor or a low grade tumor based on some specific feature of concern. With the two tiered grading system, the second scenario is uncommon. As a note, c-kit mutation testing is available as a single test from Michigan State University as well (ie, ordering the entire panel is not a requirement). For cases where systemic therapy is indicated, this test can help in selecting therapy (ie, a TKI like toceranib OR traditional chemotherapy with vinblastine and/or CCNU). Additional references available by request.