Biology and Etiology

Mast cells arise from bone marrow, are located in connective tissues throughout body, and are an important component of immune system. Mast cells play a central role in inflammatory and immediate allergic reactions. Mature mast cells store inflammatory mediators, including histamines, proteases, chemotactic factors, cytokines, and arachidonic acid metabolites. These may induce paraneoplastic syndromes in dogs with MCT.

MCT Epidemiology & Risk Factors

MCT are the most common cutaneous tumor in dogs, accounting for 16 to 21% of skin tumors. Risk factors include

• Age: Higher incidence in older dogs (mean 9 years old) but also reported in young dogs.
• Breed: Mixed breeds most common; Boxers, Boston Terriers, Labradors, Beagles, Schnauzers.
• Gender: Not correlated.

MCT etiology: The cause is largely unknown and they are rarely associated with chronic inflammation or application of skin irritants. There is no clear evidence of viral cause. The role of estrogen and progesterone are poorly defined. Genetic alterations are also not completely understood, but the following have been associated: p53 - tumor suppressor, p21 and p27 - cell cycle regulation, c-KIT - RTK for stem cell factor.

C-Kit is receptor tyrosine kinase (RTK) that binds to stem cell factor (SCF), the ligand. STF-kit interactions promote development of mast cells from hematopoietic progenitors. RTK are enzymes that transfer phosphate from ATP to cell protein and the phosphate is on the amino acid tyrosine. These are important in signal transduction and cellular activity, like cell division. Dysregulation in c-kit may promote mast cells’ uncontrolled growth or survival. Thirty to 50% of dogs with MCT have c-kit mutations, essentially causing the cells to be stuck in “on” position, leading to continuous c-kit activation and unregulated cell growth. The mutation is internal tandem duplications (ITD) in exons 11 & 12 (in the juxtamembrane domain). ITD mutations associated with increased recurrence and death. Mutations have also been identified in exons 8 and 9.

Clinical Presentation

MCT are most common in dermal and SQ tissues. The sites are: trunk 50–60%, limbs 25%, and head & neck least common. They have a varied appearance. They are typically solitary, but 11–22% has multiple lesions.

The tumor clinical appearance is associated with histologic differentiation. Well-differentiated MCT are typically single, 1–4 cm diameter, slow growing, rubbery, non-ulcerated, alopecic, and present 6+ months. Undifferentiated MCT are large, rapidly growing, ulcerated and irritated, and the surrounding tissue is edematous and inflamed. Small satellite nodules may be present.

Disease symptoms can be complicate by signs attributable to release of histamine, heparin and other vasoactive amines. Darier’s sign refers to the wheal & flare in surrounding tissues following manipulation of the MCT and is caused by mast cell degranulation. GI ulceration causes vomiting possibly with blood, melena, anorexia, and abdominal pain. GI ulceration is due to histamine stimulation of H receptors on parietal cells that increased HCI. GI ulceration is noted in 35–83% necropsy specimens.

Prognostic Factors

• Histologic grade
• Clinical stage
• Location
• Systemic signs
• Recurrence
• Tumor size
• Mitotic index (Ml)

Diagnostics

Preliminary diagnosis is typically made with fine needle aspirate. On cytology, the neoplastic cells are small to medium sized round cells, with abundant, small, uniform cytoplasmic granules that stain purplish-red (metachromatic).

To Stage or Not to Stage? (Or I’ve Done My Aspirate, Now What)

Should you do surgery first or diagnostics prior to surgery? Diagnostics may include incisional biopsy for grade, LN FNA, CBC, chemistry panel, UA, AUS ± liver/spleen FNA, bone marrow cytology, and buffy coat?

WHO Clinical Stage

There are problems with current staging, and the usefulness of WHO is being questioned. Stage III is vague, and combines multiple cutaneous nodules with large infiltrating tumor. There was a presumption that multiple tumors represent systemic metastasis vs. de novo MCT. In addition, the diagnosis of metastasis based on presence of mast cells in LN FNA, but mast cells are also present in healthy animals. There is also questionable value of buffy coat smears and bone marrow in staging.

Histologic Classification: 3-Tiered

Grade 1: clearly defined cytoplasmic boundaries with regular, spherical or ovoid nuclei, rare or absent mitosis, abundant large deep staining cytoplasmic granules.

Grade 2: closely packed cells with indistinct cytoplasmic boundaries, N:C ratio lower than anaplastic type, infrequent mitoses, and more granules that anaplastic type.

Grade 3: highly cellular, frequent mitoses, undifferentiated cytoplasmic boundaries, nuclei irregular in size and shape, sparse cytoplasmic granules.

Histologic grade is a prognostic for biologic behavior and clinical outcome, and an accurate predictor for metastatic behavior. Low grade: <10%, intermediate grade: low to moderate, high grade: 55–96%. Metastasis is typically to local LN, liver and spleen, bone marrow.

Mitotic Index (MI)

Mitotic index is an indirect measure of cell proliferation based on number of mitotic figures, and is a strong prognostic factor. It can be performed during routine histology and is easier than other proliferation markers that require additional immunohistochemical staining (AgNOR, Ki-67).

Romansik 2007, Vet Path, 148 dogs: MI was significantly increased with grade, but there was no association MI and tumor recurrence.

MI was associated with metastasis and survival:

• Overall, <=5/10 HPF, MST 70 months; >5/10 HPF, MST 2 months.
• For grade II, <=5/10 HPF, MST 70 months;, >5/10 HPF, MST 5 months.
• For grade III, <=5/10 HPF, MST not reached, >5/10 HPF, MST <2 months.

Note MI not consistent from study to study & expect that the cutoff will continue to be refined as more studies evaluate mitotic index.

Molecular Prognostic Factors: Key Findings

Increased Ki67 and AgNORS both significantly associated with decreased survival. MCT with aberrant KIT localization or ITD c-KIT mutations are associated with increased cell proliferation. Multivariable analysis showed Ki67 were significantly associated with incidence & rate of subsequent tumor occurrence at original tumor site, rate of MCT at distant site.

The New 2-Tiered Grading System

Unfortunately there is inter-observer variation among pathologists, and pathologists tend to opt for grade 2 when it is borderline between grade 1 & 2. If more pathologists are calling tumors grade 2, the prognostic value is weakened. Based on the original work by Patnaik, there is ∼50/50 chance of 5 year survival for grade 2 tumors.

A 2-tiered system had been developed and is based on the number of mitoses (< or >7), presence of multinucleated cells or bizarre nuclei, and karyomegaly (increased nuclear size). High grade tumors significantly associated with shorter time to metastasis mast cell tumor associated mortality, shorter overall survival time. MST for high-grade MCT <4 months vs. >2 years for low-grade MCT.

This system is still relatively new and should continue to be validated in future studies.

Other Diagnostics

Buffy coats have been used historically but are unreliable. There can be high number of false positives with inflammatory skin disease, parvovirus, regenerative anemia, traumas, other cancers (McManus 1999).

Spleen and liver cytology: According to Finora 2006, routine FNA of normal liver and spleen on AUS is not useful but then another study concluded cytology was indicated whether AUS is normal or abnormal (Stefanello 2009).

Treatment

Surgery: Surgery All That You Need?

Surgery is the ideal treatment in areas amenable to wide resection. The recommendations for margins have historically 3 cm, but 2 cm lateral margins may be adequate for most (Simpson 2004). For small and lower grade, extensive deep margins are just as crucial: 1 fascial plane deep, 4 mm deep margins. The majority of naive dermal MCT are intermediate or low grade will be cured with surgery alone, provided site is amenable.

Location: In some locations, wide margins are often not possible (i.e., distal limb). In my opinion, amputation is probably too aggressive. But further therapy will likely be needed after surgery. Post-operative options include external beam RT, scar revision, and chemotherapy

Surgery and margins: Histologic assessment of margins may be unreliable. Thirty-seven point five percent (37.5%) recur with clean margins, and 36% have no recurrence with incomplete margins. In Seguin 2001: grade 2 MCT with clean excision, only 5% completely removed grade 2 recur, 5% metastasized, and 11% developed 2nd MCT. Note that microscopic formalin-fixed parameters do not reflect margin size at surgery. Tissue shrinkage of up to 30% for cutaneous tissues occurs.

Radiation therapy (RT) is recommended when wide surgical excision not feasible. Monotherapy has varying control rates 40 to 45 Gy 1 year control rates of 50, but surgery to achieve clinical stage O (microscopic disease) followed by full course RT, (typically 15 treatments over 3 weeks) has high control rates of 85–95% 2-year control rates for grade 1 and 2. Should do prophylactic nodal irradiation (PNI)? It is probably unwarranted in low to intermediate grade MCT.

Chemotherapy: Single-Agent

• Vincristine McCaw 1997: Not effective for measurable disease
• Vinorelbine: Grant 2008: Overall response rate (ORR) 8%
• Single-agent Vinblastine
• Henry 2007: ORR 12%
• Rassnick 2008L Higher dose higher RR (27 vs. 12%)
• Greater GI toxicities and grade 3 or 4 neutropenias
• Single-agent lomustine: Rassnick 1999: ORR 42%, Neutropenia was dose limiting toxicity
• Single-agent hyroxyurea: Rassnick 2010: ORR 28%, overall remission duration 60 d, Neutropenia was dose limiting toxicity

Steroids As a single agent in grade 2–3 (McCaw 1994), ORR was 20% (5 of 25). But prior to RT for non­resectable grade 1–3 (Dobson 2004), RR was higher at 75% (18/24). When given prior to surgery (neoadjuvant) for grade 1–3 pre-op (Stanclift 2008), ORR was 70%.

Single Agent Summary

Response rates (CR &PR) are variable.

• Vincristine 7%
• Lomustine 42%
• Steroids 20–75%

Combination Therapies

Studies have shown that combination therapies often improved efficacy.

1.  Vinblastine and prednisone, Thamm 1999: Pred @ 2 mg/kg/d, tapered and discontinued over 12–26 weeks; VBL @ 2 mg/m² every 1 to 2 weeks. Efficacy (gross disease) ORR 47%. Adverse effects (AE): 8/41 (20%), typically after 1st VBL, 15% mild, 5% severe and treatment discontinued.

2.  VBL, CTX and prednisone, Camps-Palau 2007: Pred @ 1 mg/kg/d, tapered and discontinued over 24–32 weeks; VBL @ 2–2.2 mg/m² every 3 weeks; CTX @ 200–250 mg/m² IV or PO every 3 weeks. Efficacy (gross disease, n=11), ORR 64% AE: Well-tolerated, Grade 1 neutropenia & grade 2 GI toxicity.

3.  Vinblastine, CCNU and prednisone, Rassnick 2010: Pred @ 2 mg/kg/d, tapered and discontinued over 24–28 weeks; CCNU @ 70 mg/m every 4 weeks, wk 1, 5, 9, then q 6 wk; VBL @ 3.5 mg/m² every 4 weeks, wk 3, 7, 11, then q 6 weeks. Prophylactic TMS for 1st treatments. Efficacy (gross disease) ORR 65%. Overall PFS for adjuvant 489 d (16 months), AE: febrile neutropenia. Persistent ALT 9%.

4.  Leukeran and prednisone, Taylor 2009: ORR 38%. Median response duration 533 d.

Tyrosine Kinase Inhibitors

Tyrosine kinase inhibitors (TKI) are drugs that inhibit RTK, and most block kinase binding to ATP competitive inhibitor of ATP binding which prevents phosphorylation of intracellular kinase domain of associated RTK and subsequent signal transduction. The drugs may also inhibit other RTK in split kinase family including VEGF and PDGFR.

Palladia

Palladia (toceranib phosphate) is an oral TKI that blocks activity of multiple receptors and selectively target split kinase family of RTKs. It exerts antiangiogenic and antiproliferative effects. The oral bioavailability is 77%. Palladia is labeled for dogs with grade 2 or 3, recurrent cutaneous MCT, + regional LN involvement, and was FDA approved in June 2009. Palladia is 1st anti-cancer drug FDA approved for dogs.

In the clinical field study of single agent Palladia (London 2009, Clin Cancer Research), this was a multi-center placebo-controlled, double-blinded study looking at the objective response (PR or CR) to single-agent Palladia @ 3.25 mg/kg EOD. The biologic response rate was 59.5% for all dogs (blinded + open): CR 16%, PR 31.3%, and SD 12.2%. The odds ratio of OR was 6.5x higher in Palladia-treated patients vs. placebo. Grade 2 MCT had better outcomes than grade 3, and had a significantly longer time to progression (TTP) and significantly longer response duration. Palladia was effective with or without c-Kit mutation (positive: 69% response; negative: 37% response).

Adverse events (AE): GI side effects were most common AES, >10% Palladia dogs (during blinded phase). Neutropenia was the most common lab abnormality. From the study the recommended dose for MCT is 3.25 mg/kg EOD. It is recommend to give with or without food (I recommend with food), give in evening, and adjust dose based on biweekly assessments for 1st 6 weeks, then every 4 to 6 weeks. Dose reductions of 0.5 m/kg are recommended with a minimum dose of 2.2 mg/kg EOD. If AE occur, drug holidays for up to 2 weeks. It is important to not be complacent in monitoring. Clients must be educated if any side effects are noted, stop medication, start-just-in case medications, and contact a veterinarian. Early recognition if side effects are critical when using Palladia!

TKI combination protocols are starting to be evaluated. Palladia given with vinblastine caused myelosuppression, and the dose of vinblastine was reduced significantly. The ORR of 71% is encouraging.

In conclusion, chemotherapy should be considered for: high grade/grade 3, dogs with distant metastasis, lymph node metastasis, C-kit positive dogs, dogs with high proliferation scores, and for non-resectable MCT in the neo-adjuvant setting. Dogs with multiple MCT in a short time period may also be considered for chemotherapy. Dogs with low grade/grade 1 or grade that have low proliferation scores and are c-kit negative can typically be managed with local therapy (surgery ± radiation).