Canine Mast Cell Tumor

This slideshow gives all the basic information about Canine Mast Cell Tumor such as introduction to mast cells, mast cell tumor, diagnosis, grading of tumors, the immunohistochemistry of the tumors, treatment etc.

1. CANINE MAST CELL TUMOR MADE BY: Snehal K. Salunkhe PRINCIPAL INVESTIGATOR: Dr. Pradip R. Chaudhari DEPARTMENT/LAB: Small Animal Imaging Facility (Advanced Centre for Training, Education and Research in Cancer, Navi Mumbai)

2. INTRODUCTION TO MAST CELLS Mast cells are immune cells of the myeloid lineage and are present in connective tissues throughout the body. Mast cells are originated from pluripotent progenitor cells of the bone marrow, and mature under the influence of the c-kit ligand and stem cell factor in the presence of other distinct growth factors provided by the microenvironment of the tissue where they are destined to reside.  Mast cells are found in all vascularized tissues except for the central nervous system and the retina . Mast cells are located at the junction point of the host and external environment at places of entry of antigen (gastrointestinal tract, skin, respiratory epithelium ). Mast cells are located in areas below the epithelium in connective tissue surrounding blood cells, smooth muscle, mucous, and hair follicles .

3. FIGURE 1: FACTS ABOUT MAST CELL SOURCE: https://www.naughtylittlemastcells.com/get-started/

4. FIGURE 2 : ( A) Mast cells (arrows) are seen aligned along the wall of a blood vessel (V) and in the mesentery window. Toluidine blue. Bar = 25µm. ( B) Mature peritoneal mast cell is replete with electron dense secretory granules. N, nucleus; SG, secretory granule. Transmission electron microscopy. Bar = 1 µm.

5. FIGURE 3: MAST CELL STRUCTURE SOURCE: https://www.dreamstime.com/stock-image-allergy-image24417861 FIGURE 4: MAST CELL STRUCTURE SOURCE: https://www.dreamstime.com/stock-illustration-mast-cell-allergen-bound-to-specific-ige-resulting-degranulation-vasoactive-amines-created-adobe-image72564939

6. INTRODUCTION TO CANINE MAST CELL TUMOR (MCT) Mast cell tumor (MCT) represents a cancer of a type of blood cell normally involved in the body’s response to allergens and inflammation. MCT is the most common skin tumor in dogs; it can also affect other areas of the body, including the spleen, liver, gastrointestinal tract, and bone marrow . Certain dogs are predisposed to MCT, including brachycephalic (flat-faced) breeds such as Boston Terriers, Boxers, Pugs , and Bulldogs, as well as retriever breeds , though any breed of dog can develop MCT . When they occur on the skin, MCT vary widely in appearance. They can be a raised lump or bump on or just under the skin, and may be red, ulcerated, or swollen. In addition, many owners will report a waxing and waning size of the tumor, which can occur spontaneously, or can be produced by agitation of the tumor , causing degranulation. Several anatomical sites have been associated with more aggressive tumors and a more guarded prognosis; these sites include mucocutaneous junctions, oral mucosal, muzzle , subungual locations, and perineal and scrotal MCT

7. DIAGNOSIS Diagnosis before treatment is always advantageous, and given the variability of MCT appearance and how common they are, fine needle aspirates should be taken of all skin masses before excision. Cytology is considered a highly sensitive method for the diagnosis of MCTs . It is generally believed that cytology cannot accurately and reliably predict tumor grade, particularly using the Patnaik system in which tissue invasiveness is assessed, so it should not be used to provide a prognosis . However, some cytological features, such as cells being highly pleomorphic, can suggest a higher grade and a more aggressive behavior. Blood work (complete blood count and chemistry panel) and urine sampling are typically performed at the initial visit and provide important information regarding a patient’s overall health and ability of the patient to handle treatment. Abdominal ultrasound – This will assess abdominal organs for evidence of MCT spread CT Scan – This imaging identifies the precise location and size of MCTs for detailed surgery or radiation planning Tissue Biopsy – This biopsy allows microscopic evaluation of the primary MCT, providing confirmation of diagnosis and a tumor grade (low or high)

8. FIGURE 5: How to perform a fine needle aspiration for the cytology of a mast cell tumor (MCT ) MCTs usually exfoliate well, so syringe suction is rarely required. A 23 g 5/8” or 1” needle should be inserted into the mass and, once through the skin, moved quickly, backward and forward, while moving the direction of the needle. This allows a small core of cells to form within the needle. The contents of the needle are deposited onto a microscope slide using a syringe prefilled with air. The sample is gently smeared using a second slide. No pressure should be applied between the two microscope slides, as this will damage the cells. The slide should be allowed to air dry before staining or submission to a clinical pathologist. Cytology of a canine MCT, showing typical granular round cells (mast cells) along with a high number of eosinophils (Picture: Tim Williams)

9. FIGURE 6: CRITERIA FOR GRADING OF CANINE MCT WITH HISTOPATHOLOGY SOURCE: https://www.slideshare.net/claire_mcg88/grand-rounds-47681713 GRADING OF CANINE MCTs

10. FIGURE 7: Mast cell tumors, skin, dog. Modified Wright’s stain . 1. Cytologic low grade. Highly granulated mast cells with minimal anisokaryosis . 2. Cytologic low grade. Mast cells of mixed granularity with minimal anisokaryosis ; most are poorly granulated with fewer highly granulated forms (arrows ). 3. Cytologic high grade. Mast cells are poorly granulated, display binucleation and multinucleation (arrowheads), and a few mitotic figures are observed (inset ). 4. Cytologic high grade. Mast cells are poorly granular and display anisokaryosis with some nuclei (black bar) >50% larger than others (red bar) and others showing nuclear pleomorphism , characterized by nonround nuclei (arrow).

11. GENETICS BEHIND CANINE MCTs Mutations in the c-KIT proto-oncogene have been implicated in the progression of several neoplastic diseases, including gastrointestinal stromal tumors and mastocytosis in humans, and cutaneous mast cell tumors (MCTs) in canines . Internal tandem duplications (ITDs), deletions, and point mutations have been identified in the juxtamembrane domain of c-KIT in canine cutaneous MCTs. The c-kit receptor (CD117) is a transmembrane protein with tyrosine kinase activity encoded by the oncogene c-kit. It is an important member of type III receptor tyrosine kinase family. The ligand for c-kit is stem cell factor (SCF) , a hematopoietic cytokine, which plays an important role in maintaining the survival of hematopoietic cells, promoting hematopoietic cell proliferation and differentiation, and regulating growth and development of hematopoietic cells. The canine proto-oncogene c-kit is located on chromosome 13 . C-kit is highly conserved in the evolution. And the c-kit receptor is composed of 976 amino acids ( aa ) divided into an extra-cellular domain with 519 aa , a trans-membrane domain with 23 aa , an intracellular tail of 433 aa consisting of a juxta -membrane domain and a tyrosine kinase domain inserted by about 80 amino acid residues.

12. FIGURE 8: Schematic representation of the structure of c-kit . The extra-cellular domain contains five immunoglobulin-like domains (D1~D5 ), D1~D3 of which as the key component of c-kit binding to SCF, and D4~D5 of which are the essential region of dimerization. The membrane region near the dimerization domain (exon 8 and exon 9 ), the intra-cellular domain close to the membrane and the kinase domain (exon 17) are often prone to mutations. MUTATIONS: EXON 11- 1663-1671 del, point mutation: 1676 T>A ITD of residues Pro580-Phe594 and Thr577-Pro588 are novel findings. c.1523A>T mutation in exon 9 ( Kobayashi et al., 2015 ).

13. TREATMENT SURGERY Despite the advent of new, licensed medications for the treatment of MCTs, surgery should be considered the treatment of choice for MCTs. Historically, a 3 cm margin of normal skin around the tumor and a deep margin of one fascial plane not invaded by the tumor has been recommended for MCTs. However, recent studies have shown that a 2 cm margin and one fascial plane beneath the tumour is adequate for complete excision of low-grade and most intermediategrade MCTs ( Fulcher and others 2006, Simpson and others 2004). 2. RADIOTHERAPY External beam radiotherapy has been described for therapy in some cases of MCT. The primary indication is in the treatment of low-risk MCT, in combination with a planned narrow surgical excision, where wide surgical excision is not possible. It is important to note that radiotherapy should not be seen as a means to salvage poor surgical planning: it should be part of the treatment plan from the outset. Detailed measurements, as well as preoperative, intraoperative and postoperative photographs of the tumour , tumour site, and any drain sites are required for accurate radiotherapy planning. Areas particularly sensitive to radiotherapy include the brain, eyes, mucous membranes, nasal planum , footpads and visceral organs , and additional consideration needs to be given if the tumor is close to one of these.

14. 3. ADJUVANT THERAPY Chemotherapy should be considered after surgical excision to reduce the risk of metastasis from a highgrade tumour , or an intermediate-grade tumour with features indicating a high risk of malignancy ( eg , high MI or ki67). Vinblastine, in combination with prednisolone, has been shown to be beneficial in increasing survival time over historical controls in the management of highgrade MCTs (two year disease-free interval 70 per cent) (Hayes and others 2007), and grade 2 to 3 MCTs with a high risk of metastasis (MST of 1305 days) ( Thamm and others 2006). The addition of lomustine ( CCNU ) to the combination of vinblastine and prednisolone has also shown favourable outcomes in grade 3 or metastatic MCTs , with a median progression-free survival ( PFS ) of 489 days ( Rassnick and others 2010). 4. TYROSINE KINASE INHIBITOR THERAPY AND PALLADIA PALLADIA is the first FDA-approved drug for canine cancer and is licensed to treat canine mast cell tumor. PALLADIA is a tyrosine kin ase inhibitor ( TKI ). TKIs have both antitumor and antiangiogenic properties, which mean s they can work against both the tumor itself and the blood vessels that supply it. Tyrosine kinase is an enzme that binds to a receptor on the surface of a cell and causes sign als to be conducted to the nucleus (the “brain” of the cell) that serve as growth factors f or the cell. The primary TK PALLADIA was designed to inhibit is c-kit. However, it also can inhibit platelet-derived growth factor receptor-beta ( PDGFR -b), vascular endothelial growt h factor receptor-2 ( VEGFR -2) as well. While these are new drugs, not all patients need tr eatment with them. Most mast cell tumors are cured with adequate surgical removal. H owever, there are some dogs that grow multiple masses, and some masses recur, spread, or are classified as high grade. These are patients that might need this treatment.

15. FIGURE 9: Suggested algorithm for clinical decision-making using histopathology and proliferation indices results

16. REFERENCES TITLE SLIDE IMAGE https:// jewishnews . timesofisrael .com/doctors-warn-against-home-testing-kits-for-cancer-causing- brca -mutations / INTRODUCTION TO MAST CELLS Krystel- Whittemore , M., Dileepan , K. N., & Wood, J. G. (2016). Mast Cell: A Multi-Functional Master Cell.  Frontiers in immunology ,  6 , 620. doi :10.3389/ fimmu .2015.00620 2. da Silva, E. Z., Jamur , M. C., & Oliver, C. (2014). Mast cell function: a new vision of an old cell.  The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society ,  62 (10), 698–738. doi :10.1369/0022155414545334 INTRODUCTION TO CANINE MAST CELL TUMOR ( MCT ), DIAGNOSIS, TREATMENT, GRADING OF CANINE MCTs 1. https :// www.vet. upenn . edu /docs/default-source/ ryan /oncology-handouts/final-canine- mct .pdf? sfvrsn =4 2. Warland  J,  Brioschi  V, Owen L , et al Canine mast cell tumours : decision-making and treatment In Practice  2015; 37: 315-332 . 3. https :// petcureoncology .com/mast-cell-tumors-in-dogs / 4. M. S. Camus , H. L. Priest , J. W. Koehler , E. A. Driskell , P. M. Rakich , M. R. Ilha , and P. M. Krimer . Cytologic Criteria for Mast Cell Tumor Grading in Dogs With Evaluation of Clinical Outcome , Veterinary Pathology 2016, Vol. 53(6) 1117-1123 The Author(s) 2016 5. http:// vetcares .com/ wp -content/uploads/2012/12/palladia.pdf

17. GENETICS BEHIND CANINE MCTs Webster , J.D., Kiupel , M. & Yuzbasiyan - Gurkan , V. Evaluation of the kinase domain of  c-KIT  in canine cutaneous mast cell tumors.  BMC Cancer   6,  85 (2006) doi :10.1186/1471-2407-6-85 Liang, J., Wu, Y.-L., Chen, B.-J., Zhang, W., Tanaka, Y., & Sugiyama , H. (2013). The C-Kit Receptor-Mediated Signal Transduction and Tumor-Related Diseases.  International Journal of Biological Sciences ,  9 (5), 435–443. doi : 10.7150/ ijbs .6087 Liang, J., Wu, Y.-L., Chen, B.-J., Zhang, W., Tanaka, Y., & Sugiyama , H. (2013). The C-Kit Receptor-Mediated Signal Transduction and Tumor-Related Diseases.  International Journal of Biological Sciences ,  9 (5), 435–443. doi : 10.7150/ ijbs .6087 https:// www. ncbi . nlm . nih .gov/gene/403811#genomic-regions-transcripts-products

18. THANK YOU