6 Matti Kiupel Michigan State University, USA Mast cell tumors (MCTs) are very common in dogs, less common in cats, and uncommon to rare in other species. The great majority of MCTs in animals occur as solitary nodules in the skin; however, in humans solitary MCTs are rare. Subcutaneous MCTs should be distinguished from cutaneous MCTs in dogs because they have contrasting biological behaviors. Less common sites of MCTs in animals include intestinal MCTs (see Chapter 13), MCTs in visceral organs, and rare MCTs at other locations such as the oral cavity or craniomediastinum. Whether MCTs in visceral organs represent the primary sites of origin or metastases from known or unknown cutaneous MCTs is not always clear. In particular in cats, synchronous occurrence of MCTs in spleen, liver, and other organs has been reported as disseminated mastocytosis and most likely represents a multicentric neoplastic disease process. Mast cell leukemia is rare in most species but approximately 40% of cats with MCTs have mastocytemia if buffy coat preparations are examined. MCTs are primarily seen in adult animals, but there are interesting and rare variants of proliferative diseases of mast cells seen in puppies, calves, and foals that are referred to as mastocytosis and which may spontaneously regress. These unusual entities have some similarities with a disease in humans called urticaria pigmentosa. Fortunately, the vast majority of cutaneous MCTs can be diagnosed easily from fine‐needle aspirates or H&E‐stained histologic sections. Although most cutaneous MCTs in dogs, cats, and horses are commonly cured by complete surgical excision, a portion of canine cutaneous MCTs and a few cases in cats and horses will spread to the local lymph nodes or cause widespread metastasis. A considerable effort has been made in dogs and to a degree in cats to characterize the histologic and molecular features of MCTs that behaved more aggressively. The behavior of cutaneous MCTs in other species is less defined due to their rare occurrence. The majority of this chapter will focus on these topics and primarily for dogs and cats. The cell of origin of MCTs is quite obviously a mast cell. However mast cells are a highly heterogeneous cell population based on their differences in biochemical, histochemical, ultrastructural, and functional characteristics. In rodents, mast cells are divided into formalin‐resistant mesenchymal (connective) and formalin‐sensitive mucosal mast cells. Formalin‐fixed mesenchymal mast cells have easily visualized metachromatic granules that stain with toluidine blue or Giemsa. Formalin‐sensitive mucosal mast cells require Carnoy’s fixation to visualize their granules with special stains (e.g., Alcian blue, Giemsa etc.). The differences in staining characteristics are most likely attributed to the greater concentrations of highly sulfated acid mucopolysaccharides in mesenchymal mast cells. Mucosal mast cells have lower levels of sulfated acid mucopolysaccharides and of catecholamines, 5‐hydroxytryptamine and histamine. Currently, human mast cells are divided based on their content of the mast cell–specific proteases tryptase and chymase. This leads to the recognition of three subtypes: mast cells that only contain tryptase, mast cells that contain chymase and carboxypeptidase, and mast cells that contain all three proteases and cathepsin. Formalin‐resistant and ‐sensitive mast cells have been detected in dogs and chymase and tryptase have been detected in canine mast cells. Knowledge of how fixatives can influence staining of mucosal mast cells is important if mucosal MCTs are to be recognized. Canine cutaneous mast cells are similar to the subtype of human mast cells that contain only tryptase. The highest density of mast cells in dogs can be found in the dermis, especially in the proximity of hair follicles and vessels and close to the stratum basale of the epidermis. In contrast, the density of mast cells in subcutaneous fat is very low. Cutaneous MCTs are derived from mesenchymal mast cells that contain only tryptase while comparable studies need to be done to further characterize subcutaneous and visceral MCTs. Mast cells are derived from pluripotent hematopoietic stem cells of the bone marrow. In contrast to other hematopoietic stem cells, mast cells leave the bone marrow as precursor cells and these undifferentiated cells circulate in the blood prior to differentiating within the connective tissue or mucosa into mature mast cells of the various types. These mature mast cells retain proliferative potential even in a fully differentiated stage. While differentiation of mucosal mast cells is regulated by various interleukins that are released from T cells, including IL‐3, IL‐4, IL‐9, and IL‐10, differentiation of mesenchymal mast cells is driven by the stem cell factor or c‐kit ligand that is secreted by fibroblast and bone marrow cells. This stem cell factor binds to the transmembrane tyrosine kinase receptor KIT that is encoded by c‐kit. The KIT receptor is composed of an extracellular domain that binds the ligand, a transmembrane domain, and the intracellular domain. Binding of the ligand activates the intracellular tyrosine kinase, leading to phosphorylation of intracellular molecules and thereby activation of signaling pathways that are essential for mast cell proliferation and differentiation. Mutations in c‐kit have been shown to play a central role in the oncogenesis of approximately 20–30% of canine and a much smaller percentage of feline MCTs. Cutaneous MCTs in dogs with mutations in exon 11 of c‐kit tend to be more aggressive tumors. Therapy with tyrosine kinase inhibitors in dogs has proven to be effective against MCTs with c‐kit mutations. The older literature does not distinguish between cutaneous and subcutaneous MCTs. Information published prior to 2011 that referred to cutaneous MCTs also included subcutaneous MCTs in their data and therefore some historical information in this chapter is a combination of cutaneous and subcutaneous MCTs. When authors distinguished the two types of MCT then that information is provided separately. The important differences are that subcutaneous MCTs tend to be less aggressive than cutaneous MCTs, and separate, but similar, means of evaluation should be used. Extracutaneous MCTs or mast cell leukemia occur, but are uncommon (intestinal tract) or rare.1,2 Cutaneous MCTs are the most frequently diagnosed malignant skin neoplasm in dogs, representing up to 21% of skin neoplasms. There is no known sex or age predilection, but the risk of developing cutaneous MCTs increases with age and the mean age of dogs developing MCTs is 9 years.3 MCTs have been described in 2‐week‐old puppies, however, multiple cutaneous mastocytic proliferations have also been described in a pup. This puppy developed multiple skin nodules at 3 weeks of age and the lesions spontaneously regressed over 28 weeks and were not visible by 35 weeks of age and no lesions recurred by 2 years of age (Figure 6.1).4 Whether these lesions are neoplastic is unknown and they may represent a syndrome similar to urticarial pigmentosa in humans that has been referred to as mastocytosis in an attempt to differentiate this syndrome from true MCTs. Mastocytosis has also been reported in a foal.5 Figure 6.1 Mastocytosis, 8‐week‐old dog. This pup developed skin nodules at 3 weeks of age which increased in number and size over the next 10 weeks but then started to regress. All lesions spontaneously resolved by 35 weeks and at 2 years of age no lesions had recurred. The masses ranged from 1 to 5 cm in diameter and contained well‐differentiated mast cells on histology.4 (Source: Davis et al., 1992.4) There is a predisposition for MCTs in numerous breeds, the most commonly affected breeds being boxers, labrador and golden retrievers, shar‐peis, bulldogs, Boston terriers, pit bull terriers, fox terriers, weimaraners, cocker spaniels, Rhodesian ridgebacks, dachshunds, Australian cattle dogs, beagles, schnauzers, and pug dogs.3,6–8 Boxers and pug dogs tend to develop MCTs that are less biologically aggressive, whereas shar‐peis are predisposed to develop MCTs that often behave more aggressively at a younger age.6,7 The degree of cutaneous mucinosis is also significantly associated with more aggressive behavior of cutaneous MCTs in shar‐peis.7 MCTs can develop anywhere on the body, but cutaneous MCTs followed by subcutaneous MCTs are the most common sites in dogs. There is a higher incidence of MCTs on the hindlegs of boxers, pug dogs, Boston and Staffordshire terriers. Rhodesian ridgebacks more commonly develop MCTs on the tail and English setters on the head and hindlegs.3,6,8–10 Primary extracutaneous MCTs may develop in the gastrointestinal tract (see Chapter 13), oral cavity and tongue, conjunctiva, salivary gland, nasopharynx, larynx, spinal cord, urethra, liver, spleen, and lung (Figure 6.2A–C). When MCTs are present in multiple tissues they mostly likely represent metastases of primary cutaneous lesions.11,12 Disseminated MCTs are very rare in dogs, as is mast cell leukemia. Figure 6.2 Primary extracutaneous mast cell tumor, 9‐year‐old dog. (A) This is an enlarged lymph node that was located along the aorta that was expanded by infiltrating neoplastic mast cells. The location of the primary MCT was unknown, but spleen and liver were diffusely infiltrated by neoplastic mast cells. There was no history of a prior cutaneous MCT. (B,C) MCTs were also found in other unusual sites, such as periosteum of a rib (B) and spinal canal dorsal surface of the meninges (C). MCTs were widely disseminated in this dog: spleen, liver, lymph nodes. MCTs this widely disseminated are unusual in any species. It is not known if these are metastases or multicentric origin. (Images courtesy of Erica Noland.) Cutaneous MCTs present most commonly as a solitary lesion, but multiple simultaneous skin masses occur and have been reported in boxers, Boston terriers, golden retrievers and pug dogs (Figure 6.3A).9,10,13 In one study, 56% of pug dogs with MCTs had multiple simultaneous tumors, ranging from 2 to 7 that occurred at distinct anatomic locations.6 Figure 6.3 Cutaneous mast cell tumors, dog. (A) Multiple simultaneous cutaneous MCT in a boxer dog. (B) Large MCT with extensive cutaneous swelling and erythema. (C) Gastric hyperemia and hemorrhage in a dog with cutaneous MCT. Neoplastic mast cells can release histamine which may stimulate gastric H2 receptors, causing ulceration and bleeding through hypersecretion of hydrochloric acid. (D) Hairless, raised, well‐circumscribed, erythematous, and focally necrotic canine cutaneous MCT. (E) Ulcerated and erythematous MCTs arising on the nasal planum. (F) Multiple MCTs located periorally and along the mucocutaneous junction of the muzzle. MCTs located in the muzzle are often aggressive. (Images courtesy of Douglas Thamm.) Local and systemic paraneoplastic signs are frequently seen and are linked to the release of histamine, heparin, and proteases. Some dogs develop erythema and wheal formation (Darier’s sign) when an MCT is manipulated during examination, which may cause degranulation of neoplastic mast cells. Local swelling, erythema, and pruritus are primarily mediated through histamine H1 receptors (Figure 6.3B), while histamine‐induced gastrointestinal ulceration is primarily mediated through H2 receptors (Figure 6.3C). Stimulation of gastric H2 receptors by MCT causes hydrochloric acid oversecretion and gastric hypermotility, which infrequently manifests as ulceration, vomiting, gastrointestinal hemorrhages, anorexia, and abdominal pain. Some dogs have developed secondary anemia due to iron deficiency from gastrointestinal bleeding. Peritonitis has been reported due to perforation of the gastric ulcers. Rarely, an acute anaphylactic reaction or collapse of the dogs can be caused by sudden, massive release of histamine in dogs with large MCTs or widespread tumors. Cutaneous MCTs have a highly variable gross appearance, ranging from nodular rashes to diffuse swellings or, most commonly, hairless, raised, erythematous tumors (Figure 6.3D). The size ranges from a few millimeters to large‐sized masses. Well‐circumscribed, hairless, solitary lesions tend to be slow growing and are often present for months. Ulcerated and pruritic MCTs are usually poorly circumscribed (Figure 6.3E). They tend to grow rapidly and additional, smaller tumors are often in close proximity. MCTs that appear grossly as aggressive (large, invasive, severely ulcerated) are usually malignant; however, MCTs that appear well differentiated should not be assumed to be benign. On cut surface cutaneous MCTs are white or pink, sometimes with foci of hemorrhage. Their margins are not distinct. Subcutaneous MCTs are located in the subcutis at any place on the body, but the legs, back, and thorax are the most common sites, accounting for 60% of these tumors.14,15 Multiple tumors are uncommon; 95% are single masses (16 multiple, 290 single).15 They cause the skin to bulge, but they do not enter the dermis and are rarely ulcerated. They often present as soft, fleshy masses that appear grossly similar to lipomas. Many subcutaneous tumors are not well circumscribed, making it difficult to accurately determine margins based on palpation or visualization during surgical excision. In cases of metastatic spread, there may be evidence of local lymphadenopathy or organomegaly via abdominal palpation or imaging. Subcutaneous MCTs are located in the subcutis and are surrounded by adipose tissue (Figure 6.4A). Although some tumor cells may extend dorsally into the deeper dermis, the majority of the tumor is subjacent to the dermis and epidermis. If the tumor is located in the epidermis or outer dermis it is a cutaneous MCT and if the majority of the tumor is below these anatomic locations it is subcutaneous. These anatomic locations also mean they are evaluated differently for prognoses. Until fairly recently some subcutaneous MCTs were probably diagnosed as grade 2 cutaneous MCTs because of their deeper location. The three‐tier grading scheme should be replaced by the two‐tier grading scheme for cutaneous MCTs, and subcutaneous MCTs should be categorized as a separate entity. Subcutaneous MCTs are not encapsulated, the majority are infiltrative (n = 163), some are well circumscribed (n = 50) and many are a combination of these two patterns (n = 90).15 Figure 6.4 (A) Subcutaneous MCTs are located in the subcutis and are surrounded by adipose tissue. (B) A characteristic pattern of well‐differentiated MCTs are rows or ribbons of neoplastic mast cells. (C) Most subcutaneous MCTs are composed of well‐differentiated mast cells that are round, monomorphic, and have abundant basophilic granules in their cytoplasm. Nuclei are uniform and round, nucleoli are not visible, and mitoses are rare or absent. (D) A low Ki67 index is characteristic of the majority of subcutaneous MCTs; only one nucleus is labeled red in this field. (E) One large AgNOR as depicted here is typical for most nuclei in subcutaneous MCTs. (F) Perimembranous labeling of KIT is observed in most subcutaneous MCTs. Many cutaneous and subcutaneous MCTs have distinctive histologic patterns that are recognized at low magnifications. In some tumors the neoplastic cells form rows or ribbons (Figure 6.4B). Some tumors will have a marked amount of edema and hemorrhage that cause the formation of distinct blue foci where the tumor is located. In others, eosinophils are so numerous that an MCT is suspected at first observation. The neoplastic cells in cutaneous and subcutaneous MCTs look identical; the differentiation of subcutaneous from cutaneous is based on gross and subgross examination. Tumor cells may be individualized and their cell borders distinct or they may be packed so closely that cell borders are not discernible. At higher magnifications the neoplastic mast cells are round to polygonal with round central to slightly eccentric nuclei. The cytoplasm is of moderate amount, pale pink and contains granules which stain light gray/blue with hematoxylin and eosin (H&E) or purple with metachromatic stains (Figure 6.4C). Eosinophils are almost always found in canine MCTs and can sometimes be the predominant cell type. Many tumors will have aggregates of eosinophils that are beyond the borders of the tumor and these should not be interpreted as part of the tumor when evaluating margins. Collagenolysis, sclerosis, edema, necrosis, and secondary lymphocytic inflammation are often seen in MCTs. When these secondary lesions are severe, they can mask neoplastic cells and make assessment of surgical margins difficult. MCTs are not encapsulated; however, the well‐differentiated tumors are well delineated and margins can easily be identified. Less‐differentiated tumors tend to be infiltrative and their margins are not easy to identify. More detailed descriptions of the microscopic features of neoplastic mast cells in various stages of differentiation are given in the section about histologic grading. Most neoplastic mast cells resemble their normal counterparts, making the diagnosis relatively easy in cytologic or histologic preparations. The real challenges are determining margins and grading, both of which are done with histology and/or molecular tools. Cutaneous MCTs are typically diagnosed with cytology and the cells are characterized by discrete, individualized, small to medium‐sized round cells with cytoplasmic metachromatic granules. In some examples the cytoplasmic granules are so numerous and densely stained they obscure the nuclei, while in others the granules are inconspicuous and must be searched for at high magnifications. Tumor cells that are heavily granulated will have free granules scattered in the background and the diagnosis is straightforward. However, visualizing nuclei and cytologic features for grading may be difficult in these tumors. Although cytoplasmic granules interfere with determination of mitotic count (MC), immunohistochemistry using a red chromogen for Ki67 will allow for easy enumeration of proliferating cells. Mast cell granules stain reliably with Wright’s stain (Figure 6.5A). Even in poorly differentiated MCTs some granules can be observed in cytologic preparations (Figure 6.5B) that may not be appreciated in histologic samples even after staining with Giemsa, toluidine blue, or other histochemical stains for mast cells (acid fast, Luna’s, etc.). Granules occasionally do not stain with “Diff‐Quik” (Figure 6.5C), especially in cats. Before ruling out MCT in a cytologic preparation of a round cell tumor that was stained with Diff‐Quik, consider staining the same slide or different slides with Wright–Giemsa. Using methanolic‐based stains as opposed to aqueous‐based will enhance visibility of mast cell granules and granules in large granular lymphocytes. Nuclei are often central, surrounded by abundant cytoplasm and have the appearance of a “fried egg.” Large numbers of eosinophils are commonly observed. Any round cell tumor that also has eosinophils admixed in should alert the possibility of MCT. In highly cellular samples there are usually low numbers of spindle cells. These are supporting stromal cells. The histological grading of MCTs is important prognostically and is accomplished with histology as presently there are no published criteria for cytological grading. Figure 6.5 Cytology of canine cutaneous MCTs. (A) Mast cell granules fill the cytoplasm and are extracellular in this MCT, Wright’s stain. The cytoplasm of well differentiated MCTs stain intensely with Wright’s or Diff‐Quik. However, the granules in some MCTs will not stain reliably with Diff‐Quik or any aqueous stain. Most Romanowsky stains such as Wright’s or Wright–Giemsa are methanolic based and will stain granules in mast cells and basophils. Aqueous‐based stains such as the common “dip” stains may not stain cytoplasmic granules in mast cells, basophils, or large granular lymphocytes.96 (Source: Allison and Velguth, 2010.96) (B) Poorly differentiated MCT in which some granules can be observed with Wright’s stain and another MCT (C) stained with aqueous Diff‐Quik in which granules are not visible. If a suspected MCT does not have visible granules with Diff‐Quik stain then consider staining additional slides with a methanolic‐based Rowmanosky stain such as Wright’s. See the appendix on Cytologic grading of canine cutaneous mast cell tumors (p. 952). Many low‐grade cutaneous MCTs are not encapsulated, but the margins of the tumors are very well delineated and easy to identify. However, in more aggressive MCTs the neoplastic cells invade the tissue surrounding the primary tumor, and evaluation of tumor margins is an important while challenging part of the prognostic evaluation. One of the most difficult aspects of this is differentiating neoplastic from non‐neoplastic mast cells. Many cutaneous MCTs have a reactive halo that is composed of edema fluid, inflammatory cells, mast cells, and reactive stromal cells surrounding newly formed capillaries. This halo can be several centimeters thick in larger tumors and makes it difficult to determine tumor margins grossly. Single mast cells as well as clusters of five or more mast cells, so‐called satellites, can be found within this halo. While some of these mast cells might be neoplastic cells invading the halo, others are “inflammatory” mast cells that were attracted to the tumor site by chemokines (e.g., RANTES, MIP‐1, SCF, TNF‐α). We are currently unable to differentiate neoplastic from non‐neoplastic mast cells. CD25 has been used successfully to identify neoplastic mast cells in human mastocytosis, but this has not proven to be definitive in canine tumors. Grade 1 canine MCT dual marked with KIT and CD25, while non‐neoplastic mast cells in healthy skin labeled with KIT and not CD25.16 However, a low number of non‐neoplastic mast cells in allergic dermatitis dual marked, hindering the usefulness of CD25 to identify neoplastic mast cells.16A large percentage of the tumor cells in grade 1 MCT labeled positively for CD25, but the percentage of positive cells decreased as the MCT became less differentiated. Therefore, in a higher grade MCT the utility of CD25 to identify neoplastic mast cells may be compromised.16 Based on the authors experience, in dogs CD25 is also expressed in a large number of non‐neoplastic mast cells and a study to utilize CD25 as a marker for complete surgical removal was unsuccessful. Development of a reliable marker to distinguish neoplastic from non‐neoplastic mast cells would be a great aid in the assessment of margins and regional lymph nodes. Until reliable markers are available we rely on how numerous the mast cells are and if they are in groups. Currently, satellites (clusters) are assumed to be composed of neoplastic cells, while single, well‐differentiated mast cells within the halo are considered to be “inflammatory.” Although it is easy to write these words, the distinction in many specimens is problematic. Fortunately a significant percentage of MCTs do not recur even when tumor cells are at or close to the margin. Clean margins are predictive of nonrecurrence, but oncologists and pathologists may be placing too much importance on the presence of tumor cells at the margin to predict recurrence. Histologic margins of MCTs should be reported, but surgeons must also report if they trimmed off any surrounding tissues or our assessment is compromised. This is especially true if the surgeon tried for wide margins and now has a relatively large specimen. Ideally, surgical margins should be inked and the MCT should be submitted for complete margin evaluation. Unfortunately, histologic margin evaluation is not based on a uniformly accepted method and many studies do not describe how margins were evaluated (e.g., tangential sectioning versus parallel slicing). Regardless, clean surgical margins are predictive of nonrecurrence of low‐grade MCTs.17 The reported recurrence rate following complete surgical excision of grade 2 tumors has been reported as 5–11%, with time to recurrence of 2–24 months.18,19 The recurrence rate following incomplete excision or narrow margins (<2 mm) of low‐ and intermediate‐grade MCTs has been reported to be between 6 and 30%, but the majority (85–90%) of low‐grade MCTs with incomplete margins do not recur.20–23 Recent studies have shown that low‐grade MCTs with low proliferation as determined by Ki67 and AgNORs (agyrophilic nucleolar organizer regions) are highly unlikely to recur, despite incomplete surgical margins.23,24 The definition of “recurrence” is important when reporting follow‐up data. Recurrence should be used when there is regrowth of an MCT at the site of the original excision. The diagnosis should be confirmed with histopathology or cytology, but not simply palpation. This is especially important for MCTs since dogs may develop multiple tumors over different time periods. We do not know whether MCTs that occur at a distance from the site of a previously completely excised tumor represent distant recurrences, metastases or de novo tumors. Presently there are limited molecular markers (e.g., detection of c‐kit mutations) to confirm a newly recognized MCT as different from the primary tumor. How these subsequent MCTs are labeled is important for the correlation of prognostic markers with biologic behavior. We believe the best way to categorize such additional MCTs is: “MCT at a different cutaneous site.” The development of another MCT at a different cutaneous site should be correlated with survival time and disease‐free interval (DFI). Metastases to the skin is an uncommon location for any tumor and until these subsequent MCTs are proven to be metastases they should not be reported as such. Two studies demonstrated that 2 cm skin margins and deep margins of one facial plain are sufficient for complete excision of low‐grade MCTs that are smaller than 4 cm.25,26 Another recent study demonstrated that excision of both grade 1 and grade 2 MCTs with a lateral margin of at least 1 cm did not result in recurrence.27 A deep margin of at least 4 mm was sufficient as long as the panniculus muscle or the underlying fascia or, in their absence, the superficial layer of the musculature were included.27 A modified margin approach of resecting MCTs with lateral margins equivalent to the widest measured diameter of the tumor and a minimum depth of one well‐defined fascial plane deep to the tumor also resulted in satisfactory local disease control.28 While lateral margins of at least 3 cm and deep margins of one fascial plane have been recommended for high‐grade MCTs, a histologic free margin distance that would prevent recurrence could not be determined.29 Up to 40% of high‐grade or grade 3 MCTs recurred despite “wide” margins.29 Also the grade of the neoplasm is usually not known while performing surgical excision. The risk of local recurrence (20%), metastasis (>80%), and death related to the MCT remains high for high‐grade MCTs. The 2‐year survival time for grade 3 MCTs is approximately 35%, versus 100% and 89% for grades 1 and 2, respectively.29 It is imperative that we use consistent methods to evaluate and report margins. This author’s recommendation is that margins should be evaluated using a combination of complete tangential margins for “cleanliness” and assessing distance of neoplastic cells to these margins based on radial sectioning of the MCT (Figure 6.6A,B). Numeric margins on radial sections should be reported as described in Chapter 2: M1 = margin infiltrated; M2 = margin is close, within 1–2 mm; M3 = margin is clean 2–5 mm; M4 = margin is clean >5 mm. This method will ensure consistency and provide the most accurate information for further therapeutic intervention and also allow for comparison of data between future studies. Figure 6.6 Margin evaluation of canine cutaneous MCT. (A) A combination of complete tangential margins for “cleanliness” and assessing distance of neoplastic cells to these margins based on radial sectioning is recommended for canine cutaneous MCTs. Areas of lateral skin margins are identified with red and deep tangential sections in white. Positions of radial cuts are identified by yellow lines. The sutures lines help orient the position of the mass in the animal. (B) Neoplastic mast cells extend to the inked margin, blue at base of image, H&E. Reports should state how many margins had tumor and a description of the neoplastic cells closest to the margin (e.g., neoplastic mass extending to margin; nests or individualized neoplastic cells extending to margin). Staging is considered the most important prognostic tool for canine cutaneous MCTs and is done by the oncologist using clinical criteria, histopathology, and cytology. Stage 1 is characterized by solitary tumors that are confined to the dermis without lymph node involvement. Stage 2 tumors are confined to the dermis, but the regional lymph nodes are affected. Stage 3 is characterized by multiple dermal tumors or large infiltrating tumors with or without regional lymph node involvement, and stage 4 is characterized by distant metastases. Unfortunately, a number of studies have reported various problems with the current staging system. In particular, the designation of stage 3 to dogs with multiple cutaneous MCTs is controversial. One study found no difference in outcome between stage 1 and stage 3 dogs, while stage 2 dogs had a worse prognosis than stage 3 dogs.30 Another study reported a DFI of more than 5 years in 54 dogs with stage 3 MCTs.13 Another study found that there was a worse prognosis if one of multiple skin tumors was high grade, whereas small tumor size (<3 cm) and location on the limb was associated with a better prognosis.10 A modified post‐operative WHO staging system has been proposed by Hayes et al., but stages of this system were not significantly associated with outcome in two studies that employed this system.31 A consensus panel of the European Society of Veterinary Oncologists and the Veterinary Cancer Society recommended, therefore, that the criteria between stages 2 and 3 should be switched, since lymph node spread carried a worse prognosis than simple detection of multiple cutaneous MCTs. Presence or absence of MCTs in the draining lymph node is one of the key components of staging cutaneous MCTs. In one study, the difference in survival time between stage 1 and stage 2 dogs was 6.2 versus 0.8 years.32 Furthermore, high‐grade MCTs had a much higher likelihood of metastasis to the regional lymph nodes.32 To determine regional lymph node spread in the current clinical setting, the anatomically closest lymph node is typically aspirated and evaluated cytologically prior to surgically removing the cutaneous MCT. If a lymph node is determined to be free of detectable metastases via cytology, then the recommendation is not to remove the node surgically. If lymph node metastasis is suspected, by either palpation or fine‐needle aspirate, the node is removed together with the primary cutaneous mass and both are examined histologically.33 Special stains (e.g., Giemsa or even immunohistochemistry for KIT) will aid in searching for mast cells. KIT immunohistochemistry will identify neoplastic and non‐neoplastic mast cells and may also detect other KIT‐positive cells (e.g., neoplastic melanocytes, neoplastic lymphoid cells, etc.). However, differentiating neoplastic from non‐neoplastic mast cells is problematic. Numerous solitary mast cells are commonly detected within lymph nodes, especially when special stains are applied, but only aggregates or large numbers of mast cells are considered as evidence of metastatic spread by most pathologists. A classification scheme for mast cell infiltrates has been proposed for fine‐needle lymph node aspirates and has been shown to correlate with survival.32 This scheme identifies 2–3 aggregates of 2–3 mast cells as possible metastasis.32 More than 3 aggregates of 2–3 mast cells and/or 2–5 aggregates of more than 3 mast cells represent probable metastases.32 Certain metastasis is classified as large numbers of mast cells, and/or the presence of aggregates of poorly differentiated mast cells (pleomorphism, anisocytosis, anisokaryosis, decreased or variable granulation), and/or over 5 aggregates of more than 3 mast cells.32 One study, however, found a discrepancy between cytologic and histologic evaluation in 20% of examined cases, with histology detecting a higher percentage of affected nodes.34 In a recent study, lymph nodes were histologically classified based on the number infiltrating mast cells, their distribution, and the architectural disruption of the node.35 Nodes with overt metastases (HN3) were identified by disruption or effacement of normal nodal architecture by discrete foci, nodules, sheets, or masses of neoplastic mast cells.35 Nodes with early metastasis (HN2) were characterized by aggregates of more than 3 mast cells in sinuses (subcapsular, paracortical or medullary) and/or the parenchyma. Pre‐metastatic nodes (HN1) had >3 individualized mast cells in sinuses (subcapsular, paracortical, or medullary) and/or the parenchyma in a minimum of 4 HPF.35 Non‐metastatic nodes had <3, scattered, individualized mast cells in sinuses (subcapsular, paracortical, or medullary) and/or the parenchyma per HPF. The DFI for dogs with either HN3 or HN2 (21/41) nodes was significantly shorter (median survival time (MST) 804 days) than that for dogs with HN1 or HN0 (20/41) nodes (MST 1824 days).35 Adjuvant chemotherapy did not show an effect on either DFI or survival time for any group. In addition, some studies suggest that mapping of sentinel nodes using regional lympho‐scintigraphy combined with intra‐operative lympho‐scintigraphy and blue dye may be required to accurately select the lymph node with the highest risk of metastatic disease. In one study, 8 of 19 dogs (42%) had sentinel nodes with MCT metastases that would have been missed when sampling only the anatomically closest node.36 Furthermore, 7/12 MCTs with nodal metastases had a mitotic count (MC) ≤5.36 PCR to detect activating mutations in c‐kit should be considered on both the primary cutaneous MCT and the lymph node metastases for dogs where therapy with tyrosine kinase inhibitors is considered, since it will predict therapeutic response. Future studies need to be directed towards determining the molecular phenotype of mast cells in the tumor periphery or in lymph nodes in order to define their biologic significance. This should establish more objective criteria of how to assess tumor‐free margins and lymph node spread. Not every dog requires full staging, and examination of the tributary lymph node is sufficient for most low‐grade MCTs. However, if additional therapy is considered or a high‐grade MCT has been identified, full staging is recommended. As a minimum, fine‐needle aspirates of the tributary lymph node and abdominal ultrasound are recommended. For dogs with evidence of nodal metastasis, full staging is required to determine the best therapeutic approach and includes thoracic radiographs, abdominal ultrasound, and aspirates of spleen, liver, and bone marrow. While one study found that routine aspiration of an ultrasonographically normal‐appearing liver and spleen of dogs with cutaneous MCTs did not appear to be a clinically useful staging tool, another study reported shorter survival times for dogs with mast cell infiltrates in the liver and spleen that could be detected in ultrasonographically normal organs.37,38 Buffy coat smears are not useful for the diagnosis or staging of canine MCTs and higher numbers of mast cells were seen on the buffy coat smears of dogs with non‐MCT diseases compared to dogs with MCT.39 Assigning stage 4 is also controversial since clinical detection of distant metastasis is difficult. There are limited data on the true incidence of internal organ metastases of cutaneous MCTs due to a lack of autopsy follow‐up on most cases that die or are euthanized due to MCT‐associated disease. In one study, 4 of 17 dogs developed abdominal metastasis, with 3 dogs having grade 2 and 1 dogs a grade 3 MCT.40 The use of thoracic radiographs has been questioned by multiple oncologists and using abdominal ultrasound for staging is also controversial.41,42 Although one study found no changes on abdominal ultrasound in 50% of dogs with metastatic disease, other studies found abdominal ultrasound highly predictive, and in one study imaging correlated with fine‐needle aspirates of liver and spleen.41,42 Detection of neoplastic mast cells in bone marrow aspirates has been shown in multiple studies to carry a poor prognosis and in one study affected dogs had an MST of 43 days.1,2 Data such as this is influenced by the expertise of the individuals, technology selected, and decisions made by owners. The following will detail how to provide a prognosis for various types of MCT. Readers should also review the Appendix. Due to the prevalence and variable biologic behavior of canine MCTs, the cost of therapeutics, and the potential emotional stress to owners, it is important to accurately prognosticate cutaneous MCTs and to correctly select the most appropriate therapeutic approach. The majority of cutaneous and subcutaneous MCTs do not cause widespread metastatic disease. Although we can identify subcutaneous and less aggressive cutaneous MCTs with histological examination, approximately 10% of dogs with subcutaneous MCTs and 5% of dogs with low‐grade cutaneous MCTs will die due to MCT‐associated disease.15,43 Trying to identify these small subsets of MCT is a focus of considerable effort, but if successful we can provide an even more accurate prognosis and help guide therapeutic decisions. Furthermore, in one study, up to 15% of dogs with low‐grade cutaneous mast cell tumors were reported to have regional lymph node spread at the time of presentation.44 Additionally, almost 20% of dogs with low‐grade MCTs will develop another cutaneous MCT some distance from the first.43 It is not known if these subsequent MCTs are metastases or de novo masses, and some have classified these as recurrences. These additional MCTs are more likely new tumors, or tumors that were developing concurrently rather than metastases. Although the majority of MCTs are solitary, approximately 10–20% of dogs with MCTs have multiple tumors.10,45 The prognostic significance of multiple synchronous or multiple subsequently developing MCTs is currently uncertain. Although some studies found multiple synchronous MCTs to be associated with a worse prognosis, others found no association with prognosis or a better prognosis.10,45 A study of 280 dogs reported that 59 (21%) developed multiple MCT, and this event was not associated with a worse prognosis. There was no difference in survival at 12 and 24 months for 145 dogs with a single MCT versus 50 with multiple MCTs. Another study identified 10 dogs (10/95) with multiple synchronous MCTs that presented with a higher risk of systemic mast cell disease (5/10). Regardless, each tumor should be graded and evaluated separately and multiple MCT should not be the sole criterion for stage 3. Regardless of their pathogenesis, the development of additional MCTs is an issue for the owner and oncologist. If we could predict the likelihood that additional MCTs will occur it may help guide therapeutic decisions. Although inguinal and perineal cutaneous MCTs were thought to have a more aggressive biological behavior, this has not been confirmed in multivariate survival studies.45–47 In contrast, MCTs that originate from mucous membranes, especially from the muzzle, have a high metastatic rate (55–72%) to regional lymph nodes at time of diagnosis (see Figure 6.3F).48–50 In one study, MCTs that were located orally at the mucocutaneous junction or periorally in the haired muzzle had similar survival times.50 Metastasis to regional lymph node was shown to be a predictor of survival time in those dogs: dogs with MCTs in regional lymph nodes (n = 26) had an MST of 14 months, whereas MSTs were not reached during the study for dogs that did not have metastases detected (n = 10).50 For accurate prognostication, the ipsilateral lymph node(s) should be aspirated for cytology even if the node is not enlarged. Furthermore, histologic grading has not been established for these MCTs, but an MC greater than 5 has been shown to be a predictor of prognosis.50 However, the reported survival times could be fairly long (4 years) with a variety of treatments, even if metastasis had been detected in a regional node. Overweight dogs have also been shown to have a higher risk of developing MCTs, indicating an influence of obesity on the development of MCTs.8 Clinical signs suggestive of aggressive behavior include rapid growth, local irritation/inflammation, and invasion into adjacent tissues, epidermal ulceration, additional surrounding nodules, enlarged regional lymph nodes, and paraneoplastic signs.8,13,51 A clinical parameter first reported by Bostock was that if the MCT was present for more than 28 weeks prior to excision these dogs have a favorable prognosis.9 For owners who wish to treat beyond primary surgical excision there are now multiple microscopic and molecular parameters that can be assessed to provide a more accurate prognosis and help guide treatment options (Figure 6.7). The following paragraphs will review histologic grading and other prognostic factors based on our current knowledge of prognostically significant and therapeutically predictive parameters. It is important to recognize that the prognostic value of an individual parameter is based on a statistical evaluation of a population, but there are always exceptions in which the clinical, histologic, and molecular features favored a benign course, but the opposite was true for the clinical outcome, and vice versa. Figure 6.7 Prognostic flowchart for canine cutaneous MCTs. For detailed explanations please review the section on Prognostication. Currently, histologic grading including MC is the most frequently used prognostic and therapeutic determinant for canine MCTs. Historically, the most commonly used histologic grading systems described by Bostock in 1973 and Patnaik in 1984, classified MCTs into three grades: well‐differentiated tumors, intermediately differentiated tumors, and poorly differentiated tumors.9,52 Both schemes correlated grade with clinical outcomes, however, it is likely they will be replaced by a two‐tier scheme that classifies MCTs as low grade (Figure 6.8A) or high grade (Figure 6.8B).43 This two‐tier classification provides 97% inter‐observer consistency, and it predicts overall survival, MCT‐associated mortality, and time to new tumor development.43 The diagnosis of high‐grade MCTs is based on the presence of any one of the following criteria: (1) at least 7 mitotic figures in 10 HPF (Figure 6.8C); (2) at least 3 multinucleated (3 or more nuclei) cells in 10 HPF (Figure 6.8E); (3) at least 3 bizarre nuclei in 10 HPF (Figure 6.8F); (4) karyomegaly (nuclear diameters of at least 10% of neoplastic cells vary by at least two times) (Figure 6.8D).43 Figure 6.8 Grading of canine cutaneous MCTs using the two‐tier system. (A) Low‐grade MCTs are composed of monomorphic, well‐differentiated mast cells with round nuclei and medium‐sized visible cytoplasmic granules. They do not contain any of the four features used to classify high‐grade MCTs. (B,C) High‐grade MCTs are composed of less‐differentiated mast cells, which often have a mitotic count of at least 7 mitotic figures in 10 HPF. High power field is 40× objective and a 10× ocular that equates to 2.37 mm2.59 The number of eosinophils infiltrating MCT is highly variable. They are numerous in these two examples of high‐grade MCTs, and eosinophils are not assessed as a criterion to grade MCTs. (Source: Meuten et al., 2016.59) (D) Karyomegaly is a feature of high‐grade MCTs and is identified by 10% of neoplastic cells having a nuclear diameter that varies by at least two times. This is the most subjective criterion. (E) Another feature of high‐grade MCTs are at least 3 multinucleated (3 or more nuclei) cells in 10 HPF. (F) High‐grade MCTs can also be identified by having at least 3 bizarre nuclei in 10 HPF. High‐grade MCTs have one or more of the features listed in B, D, E, or F and low‐grade MCT have none of these. Fields with the highest mitotic activity or with the highest degree of anisokaryosis are selected to assess parameters. Low grade is based on the absence of each of these. Almost all low‐grade MCTs are also well circumscribed and the margins easy to identify.9,43,52 Approximately 90% of cutaneous MCTs are low grade.43 The MST was less than 4 months for high‐grade MCTs, and approximately 2 years for low‐grade MCTs.43 Although only 10 dogs had a high‐grade MCT, 9 of them died due to MCT‐associated disease.43 Five of these cases had metastases to internal organs, none of the dogs had cachexia. Only 4 (5%) of 85 low‐grade MCTs died due to MCT‐associated disease, but 14 (17%) of 85 dogs with low‐grade MCTs developed additional MCTs.43 Seven of 10 high‐grade tumors had additional MCTs in approximately 6 weeks.43 Other studies have confirmed the high inter‐observer consistency as well as the prognostic usefulness of the two‐tier system.44,53–55 In a study using 53 dogs, all pathologists identified 46 MCTs as low grade and 7 as high grade, and the mortality rate was 6% for low‐grade MCTs and 71% for high‐grade MCTs (n = 5) within a 12‐month follow‐up period.55 In the same study the concordance for identifying 22 grade 2 MCTs was 70%, with 17 dogs surviving more than 12 months.55 Similar results were reported in another study that confirmed a significantly higher inter‐observer consistency for the two‐tier compared to the Patnaik grading system. While 7 dogs with grade 3 MCTs had significantly reduced survival time and DFI compared to 40 dogs with either grade 1 or 2 MCTs, there was no significant difference in survival times between grade 1 and 2 MCTs.54 When using the two‐tier system, 19 dogs with high‐grade MCTs had significantly shorter survival time and DFI than 28 dogs with low‐grade MCTs.54 In a third study, 18 MCTs (13.1%) were identified as grade 1 according to Patnaik, 83 (61%) as grade 2, and 36 (26%) as grade 3.53 Only grade 3 MCTs were associated with a poor prognosis, but no significant difference between grades 1 and 2 was detected.53 The survival probability at 12 months was 100% for grade 1 MCTs, 87% for grade 2 MCTs, and 16% for grade 3 MCTs.53 All grade 1 MCTs were low grade in the two‐tier system, and all grade 3 were high grade. Among grade 2 MCTs, 71 (86%) were low grade, and 12 (14%) were high grade, with a 1‐year survival probabilities of 94% and 46%, respectively.53 In the most recent study of 386 cutaneous MCTs, all Patnaik grade 1 MCTs (n = 52) were classified as low‐grade MCTs, and all Patnaik grade 3 MCTs (n = 43) were classified as high‐grade MCTs according to the two‐tier system.44 Of the 291 Patnaik grade 2 MCTs, 243 (84%) were classified as low‐grade MCTs, and 48 (16%) were classified as high‐grade MCTs according to the two‐tier system.44 Dogs with Patnaik grade 3 MCTs were significantly more likely to have metastases at the time of initial examination than were dogs with grade 1 or 2 MCTs, and dogs with high‐grade MCTs according to the two‐tier system were significantly more likely to have metastases than were dogs with low‐grade MCTs.44 Fifty dogs had metastases to the local lymph nodes as determined by cytology and/or histology and 16 dogs had distant metastases. However, 3 of 52 (6%) dogs with Patnaik grade 1 MCTs, 48 of 291 (16%) dogs with Patnaik grade 2 tumors, and 44 of 295 (15%) dogs with low‐grade tumors according to the two‐tier system had metastases at the time of diagnosis, with 38 MCTs having only local lymph node spread.44 The authors concluded that although grading schemes were helpful for prognosis no scheme is completely accurate and staging remains the best overall predictor for cutaneous MCTs.44 Historically, the Patnaik classification system has been widely used and there is considerable follow‐up data and outcome assessments. It classified well‐differentiated MCTs as grade 1, intermediately differentiated MCTs as grade 2, and poorly differentiated MCTs as grade 3.52 Grade 1 MCTs are composed of distinct, round, monomorphic neoplastic mast cells, separated by collagen bundles that have a round nucleus, no nucleolus, and no or rare mitoses (<2 in 10 HPF).52 They are confined to the superficial dermis and are located in interfollicular spaces. Edema and/or necrosis are absent or minimal. Grade 2 MCTs are more cellular than grade 1 MCTs and the neoplastic cells are often pleomorphic, less basophilic than their normal counterpart, have indented nuclei with a single nucleolus and infrequent mitotic figures (0–2 per HPF).52 They are located in the superficial and/or deep dermis and may infiltrate the subcutis and subjacent skeletal muscle. Diffuse edema and necrosis are common. Grade 3 MCTs are more cellular and pleomorphic than grade 2 MCTs, and are composed of closely packed cells with irregularly shaped nuclei and multiple nucleoli. Multinucleate and bizarre cells are common, and mitotic figures are frequent (3–6 per HPF).52 Bostock’s classification also used three grades based on differentiation; however, they were organized differently.9 He used similar criteria, but added nuclei‐to‐cytoplasmic ratio, a criterion not used by Patnaik. A major difference between the Patnaik and Bostock grading systems is the role of tumor depth. In the Patnaik system, tumor depth is a primary criterion used to differentiate grade 1 and 2 MCTs, whereas Bostock does not utilize tumor depth. Inconsistent inclusion of tumor depth in the histopathologic classification of canine MCTs has led to a marked degree of inter‐observer variation when evaluating grade 1 and 2 MCTs.9,52
Mast Cell Tumors
General considerations
Canine mast cell tumors
Incidence, age, breed, and sex
Clinical characteristics
Sites and gross morphology
Histological and cytological features
Margin evaluation
Staging
Prognostication
Histologic grading of cutaneous MCTs
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