s. . . . is ‘ , .I .' . . u . , , . . . . 53.5%.. as. _ , . r ... . a. . s i. :53; . ”$5: 3&3 x. . . . . .1 . . . . ‘ , . . , an... :93. . (1:11 2‘ a .. 2.3. . , i V . . .i . , ‘ ‘ , . . .l at! . , c . khan. n. .8! , ,5. iv. .3 33:33.1. ‘ . “Eve: 5?... . w... . ...y..h... . 1 u .. . : ‘ . .. .2. 95!. . 7.6.». s .r, .1 .M. I} 55. Mr»! .. . . angliuflthav... .I :Efivaflfz ‘ q (.1 V...) .Mvhtig. 5 iguana .32..-.» , V a . . x 3 in?! . 5 .. a i‘. . . afifiau. .. (r3 . s . . an... . cammbflotflmwm r V but. . t , .. Rh... 41.40“. . .Hliz i .FA .M..z.i :iila {that}... {‘3‘ . .1 5‘ . A. .3? lei... 0V1.“ . . . .. . a .. in.-. .55.. 3...” .1 . . . : I. V . .1 ‘ . ygfifimfiwmfi ‘ . ifiafi. a .1007 This is to certify that the dissertation entitled Prognostic Classification of Canine Cutaneous Mast Cell Tumors and the Characterization of the role of the c-KIT Proto- Oncogene in Canine Cutaneous Mast Cell Tumors presented by Joshua D. Webster has been accepted towards fulfillment of the requirements for the Doctoral degree in Pathobiology and Diagnostic Investigation and Comparative Medicine and Integrative Biology Major Profesfi‘s Signature June 13, 2006 MSU is an Affinnative Action/Equal Opportunity Institution LIBRARY Michigan State University PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. MAY BE RECALLED with earlier due date if requested. DATE DUE DATE DUE DATE DUE 2/05 p:/CIRC/DaleDue.indd-p.1 PROGNOSTIC CLASSIFICATION OF CANINE CUTANEOUS MAST CELL TUMORS AND THE CHARACTERIZATION OF THE ROLE OF THE c-KIT PROTO-ONCOGENE IN CANINE CUTANEOUS MAST CELL TUMORS BY Joshua D. Webster A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Comparative Medicine and Integrative Biology Pathobiology and Diagnostic Investigation 2006 Cu 1 .. . LC ABSTRACT PROGNOSTIC CLASSIFICATION OF CANINE CUTANEOUS MAST CELL TUMORS AND THE CHARACTERIZATION OF THE ROLE OF THE c-KIT PROTO-ONCOGENE IN CANINE CUTANEOUS MAST CELL TUMORS BY Joshua D. Webster Canine cutaneous mast cell tumors (MCTs) are one of the rmxn: common neoplastic diseases iii dogs, anmi have an extremely variable biologic behavior ranging from a benign, solitary inass ix) a. potentially' fatal :metastatic: disease. Due to the high prevalence of canine MCTs, their variable biologic: behavior, time physical, emotional, and. financial costs associated with various treatment protocols, accurate prognostication. of canine MCTs is critical in order to identify patients that will benefit most from adjunct radiation enui chemotherapy; Currently, histologic grading is the major prognostic and therapeutic determinant for canine iMCTs as several studies have shown. a .significant association between histologic grades and survival. However, the marked degree of inter-observer variation associated with histologic grading, and the predominance of intermediate grade MCTs, has led many to question the relevance of the current system. An additional concern with the treatment and prognostication of canine cutaneous MCTs re. .3 ]. J. r. L; x... U I a. 3 .FJ L. C. 3 Q? 's "g ...‘ is the current lack of knowledge in terms of the biology of these tumors. Therefore, in light of our current knowledge gap and the need for improved prognostication of canine MCTs, the goals of this dissertation were: 1. to identify novel markers and characterize previously described markers for the prognostication of canine MCTs; and 2. to characterize time role <1f the crICUI proto-oncogene ill the pathogenesis of canine cutaneous MCTs. The studies described in this dissertation elucidate the utility of KIT staining patterns, c—KIT mutations, and proliferation markers such ansiKi67 and AgNORs iii the prognostication of canine cutaneous MCTs, and demonstrate the inadequacy of tumor depth, tumor location, tryptase staining' patterns, and PCNA immunostaining for prognostication. Additionally, the studies described in this dissertation clarify the role of time c—KIT proto-oncogene iii the pathogenesis cxf canine MCTs, demonstrating“ c—KIT"s importance in time progression of this disease. In summary, the results of these studies strengthen the current body of knowledge of canine MCTs both in terms of cflagnostics and basic biology, and these results should serve as building blocks for further hypotheses and future studies. .3 \r . . . J .v. . . .. 14 v .n .l. .‘ L. .J . .. .A .. N; C. . . . a V. mfl» - w. WJ. t a ‘J u .. A. w. L A: .3. a: ACKNOWLEDGEMENTS I would. first .like tx> acknowledge nu; mentors, Drs. Matti Kiupel anui Vilma Yuzbasiyan—Gurkan, vflu> have guided me through.tmy doctoral studies balancing independence and guidance along the way. II would also like to acknowledge my Ph.D. committee members Drs. James Resau, Micheal Scott, and Barbara Kitchell who have furthered my scientific development during my Ph.D. Additionally, I would like to acknowledge Drs. Patrick Venta and Donna Housley, and Lee Alexander for technical expertise and experimental problem solving and support; RoseAnn Miller, Dr. John Kaneene, Dr. Robert Tempelman, and. Elizabeth. Hamilton, for assistance with experimental design and statistical analyses; Sue Sipkovsky and.Ihm Jeff Landgraf, fin: assistance vfliflt the microarray experiments; Elizabeth Kruszewski and Angela Sosin vflu> helped with tjma sequencing of time kinase domain of c—KIT; Patty‘ Schultz, Kelli Cicinelli, TONI Wood, and Scot Marsh (n? the histopathology laboratory an: DCPAH for technical assistance; eumi Dr. Daniel Zemke, vflu) began the initial characterization of c-KIT mutations in canine cutaneous IMCTs, which VNMS the foundation. for this dissertation. iv . v * \ k. v v -‘ ___‘ _n“- TABLE OF CONTENTS LIST OF TABLES .......................................... Vii LIST OF FIGURES .......................................... ix INTRODUCTION .............................................. 1 CHAPTER 1 IMPACT OF TUMOR DEPTH, TUMOR LOCATION, AND MULTIPLE SYNCHRONOUS MASSES ON THE PROGNOSIS OF CANINE CUTANEOUS MAST CELL TUMORS ......................................... 39 Introduction ........................................ 40 Material and Methods ................................ 42 Results ............................................. 45 Discussion .......................................... 49 CHAPTER 2 USE OF KIT AND TRYPTASE STAINING PATTERNS IN THE PROGNOSTICATION OF CANINE MAST CELL TUMORS ........... 63 Introduction ........................................ 64 Material and Methods ................................ 68 Results ............................................. 73 Discussion .......................................... 75 CHAPTER 3 THE ROLE OF c—KIT IN TUMORIGENESIS: EVALUATION IN CANINE CUTANEOUS MAST CELL TUMORS ............................... 86 Introduction ........................................ 87 Materials and Methods ............................... 91 Results ............................................. 99 Discussion ......................................... 102 CHAPTER 4 EVALUATION OF THE KINASE DOMAIN OF c—KIT IN CANINE CUTANEOUS MAST CELL TUMORS ............................. 116 Introduction ...................................... 117 Materials and Methods ............................. 121 Results ........................................... 124 Discussion ........................................ 126 A..- .—. H .4- v- a...»_ —— -. V 5. \~.. ""- ~"~_ ‘g‘ l'i l I CHAPTER 5 CELLULAR PROLIFERATION IN CANINE CUTANEOUS MAST CELL TUMORS: ASSOCIATION WITH c-KIT AND PROGNOSTIC MARKERS...135 Introduction ....................................... 135 Materials and Methods .............................. 141 Results ............................................ 150 Discussion ......................................... 154 CHAPTER 6 EVALUATION OF PROGNOSTIC MARKERS ASSOCIATED WITH THE PROGRESSION OF CANINE CUTANEOUS MAST CELL TUMORS IN DOGS TREATED WITH THE COMBINED CHEMOTHERAPY VINBLASTINE AND PREDNISONE .............................. 166 Introduction ....................................... 166 Materials and Methods .............................. 169 Results ............................................ 177 Discussion ......................................... 180 CHAPTER 7 IDENTIFICATION OF CANDIDATE GENES ASSOCIATED WITH THE PROGRESSION OF CANINE MAST CELL TUMORS: A PILOT STUDY...189 Introduction ....................................... 189 Materials and Methods .............................. 191 Results ............................................ 198 Discussion ......................................... 200 CONCLUSIONS ............................................. 207 REFERENCES .............................................. 213 vi :v 10. 11. 12. LIST OF TABLES Mast cell tumor outcomes by signalment .............. 57 Distribution of additional MCT development and mortality among dogs with MCTs of different tumor depths .............................................. 58 Distribution of additional MCT development and mortality among dogs with MCTs in different body locations ........................................... 58 Distribution of additional MCT development and mortality among dogs with multi-centric mast cell disease ............................................. 59 Univariate analysis of risk factors for MCT outcomes by body location and depth .......................... 6O Unviariate analysis of risk factors for MCT outcomes (significant @ p < 0.3) ............................. 61 Reduced* multivariate proportional hazard regression model for survival analysis for days to distant MCT develOpment after the original diagnosis ............ 62 Reduced* multivariate proportional hazards regression model for survival analysis for death ............... 62 Distribution of recurrent disease and deaths among KIT staining patterns of canine cutaneous MCTs .......... 83 Multivariate analysis results of KIT staining patterns II and III in canine cutaneous MCTs predictive ability for recurrence, death due to MCT and death due to any cause ............................................... 84 Distribution of recurrent diseases and deaths among tryptase staining patterns of canine cutaneous MCTs.85 Mutation and case description for cases with ITD c-KIT mutations .......................................... 114 vfi :H 13. 14. 15. 16. 17. Primers used for PCR amplification of c-KIT exons 16- 20 and size of expected PCR products for each primer pair ............................................... 134 Univariate and multivariate logistic regression analysis between Ki67, AgNOR, PCNA, and Ag67 and incidence of subsequent tumor development and MCT- related mortality .................................. 164 Univariate and multivariate proportional hazards analysis between Ki67, AgNOR, PCNA, and Ag67 and time until subsequent tumor and MCT—related mortality..”165 cDNA probe labeling of intermediate and mutant canine MCTs for microarray hybridization .................. 204 Differentially expressed genes in malignant vs. intermediate canine mast cell tumors ............... 205 vfii :4 10 11 12. 13. LIST OF FIGURES Tumor depths identified for prognostic evaluation...55 Tumor locations defined for prognostication ......... 56 Immunohistochemically stained sections of canine cutaneous mast cell tumors .......................... 80 Local recurrence survival curve for dogs with cutaneous mast cell tumors with different KIT staining patterns ................................... 82 Overall survival curve for dogs with cutaneous mast cell tumors with different KIT staining patterns....82 Laser capture microdissection of neoplastic canine cutaneous mast cell tumors ......................... 109 2% agarose gel of PCR amplified c-KIT exon 11 and intron 11 from LCM extracted DNA from canine MCTs..110 Sections of canine cutaneous mast cell tumors (skin) stained with anti—KIT antibodies and counterstained with hematoxylin (magnification: 100x oil) representing three patterns of KIT localization identified in neoplastic canine mast cells ......... 111 Kaplan—Meier Survival Curve: Relative frequency of survival vs. time in months for canine cutaneous mast cell tumor patients with and without identified c-KIT mutations .......................................... 112 .Correlation between ITD c—KIT mutations and KIT protein localization in canine MCTS ................ 113 .Schematic diagram of the receptor tyrosine kinase KIT ................................................ 131 Immunohistochemical staining patterns of canine cutaneous MCTs staining with anti-KIT antibodies...l32 Schematic Diagram of Primer Design for Polymerase Chain Reaction ..................................... 133 ix mfg 14 15. l6. 17 18 19. 20 21 22. 23. 24 25. .Amino acid alignment of the kinase domain (exons 16- 20) of canine and human KIT ........................ 133 Histochemical and immunohistochemical staining for proliferation markers in canine cutaneous MCTs ..... 158 Kaplan—Meier survival curve evaluating the time until local recurrence of canine MCT patients classified based on Ki67 protein expression ................... 159 .Kaplan—Meier survival curve evaluating the time until local recurrence of canine MCTs patients classified based on Ag67 values ............................... 160 .Relative frequency of MCT—related mortalities in canine cutaneous MCTs based on their Ki67 and Ag67 indices ............................................ 161 Kaplan—Meier survival curve evaluating the time until MCT-related mortality of canine MCT patients classified based on Ki67 protein expression ........ 162 .Association between cellular proliferation and the presence of c—KIT mutations or aberrant KIT protein localization ....................................... 163 .Kaplan—Meier survival curves of time to treatment failure of canine MCTs classified based on prognostic markers ............................................ 185 Kaplan—Meier survival curves of survival times of canine MCTs classified based on prognostic markers.186 Kaplan—Meier survival curves of time to treatment failure (A) and survival time (B) of grade III MCTs treated with either surgery alone or with vinblastine and prednisone ..................................... 187 .Kaplan-Meier survival curves showing percent survival of KIT pattern III MCTs(A), and MCTs with ITD c-KIT mutations (B) treated with vinblastine and prednisone compared to those treated with surgery alone ....... 188 Diagram of tumor necrosis factor (TNF) signaling...206 K J.\. .k, . ”l 3. . .. .1 E 2 .AJ A. v .‘ PN . ‘ vs. C .1 ... .1 .3. . . .3 .. . I C r. s. c. .5 1 . my . s. .3 I. .. .2 ..;H .v \u INTRODUCTION .MCT.Biology Canine cutanemns mast cell tumors (MCTs) accounting for 7-21% of all cutaneous neoplasms*4. Clinically, canine MCTs often present as solitary neoplastic masses in the skin or subcutaneous tissue in older dogs, however a small proportion of canine MCT patients may have multiple synchronous masses at the tjmma of diagnosis””. The mean age of onset is approximately 9 years of age, but MCTs have been reported in dogs as young as 2 weeks of age and in dogs as old as 19 years of agem7. All breeds are affected by MCTs, although several breeds such as the boxer, bulldog, Boston terrier, Weimaraner, and Labrador retriever have been reported to have an increased incidence of mast cell disease“‘”. Canine MCTs occur in males and females at an approximately equal frequencyh7. Human mast cell diseases are rare, primarily affect infants and juveniles, and commonly have a favorable ll prognosisa’ In contrast, canine MCTs are common and have M. Early studies based on a variable biologic behavior“- necropsy findings reported that up to 96% of MCTs metastasize; however, these reports were grossly biased towards animals that died as a direct result of their mast cell disease due to the inclusion of only necropsy ,_‘ "w ._,x....»~ r . Q» .3 .44. uv¢\ «VI .3 ..: bkt findingsmle. Several more recent studies have reported a much lower rate of metastasis and mast cell related 8,16,17 O mortality' In the event of metastases, MCTs most commonly metastasize txa the regional limufli nodes, followed by the spleen, the liver, and the bone marrow5'7. Solitary metastases in other viscera occur at much lower rates. In some cases, cutaneous MCTs may be associated with systemic mastocytosis, involving multi-organ metastases and mast cell infiltration. Systemic mastocytosis carries an extremely poor prognosis. In one retrospective evaluation of 16 dogs with systemic mastocytosis, 14 of the dogs evaluated luxi a. primary cutaneous IMCT and 88% CH? these animals died as a result of their MCTML Primary gastrointestinal MCTs appear to be clinically distinct from cutaneous MCTs, as they are most commonly seen in toy breed dogs, and are associated with an extremely poor prognosis with a 39.1% 30—day survival rate and 8.7% 180-day survival rate. Gastrointestinal. MCTs also occur at £1 much. lower incidence than their cutaneous counterpartsfih Ilka to the unique physiology of mast cells and their ability 11) release vasoactive anxi inflammatory mediators, such as histamine, serotonin, and heparin, both canine and human MCT patients may develop either a local or systemic para-neoplastic syndrome as a :result. of' mast cell degranulation. Most commonly this para—neoplastic syndrome will occur locally' and. consist. of jpain, swelling, ulceration, local bleeding, and wound dehiscence. In some cases, especially in time presence~ of systemic :mast cell disease, the para-neoplastic syndrome may be remarkably more severe, characterized by gastric ulcerations, coagulopathies, and systemic hypotension, which may be fatal”;”“”l. canine.MCT Prognostication Histologic Grading Due ix) the variable biologic behavior (n5 canine MCTS and the potentially fatal outcome associated with this disease, an accurate diagnosis and prognostication is critical in order to determine the most appropriate therapeutic strategy for a given tumor. Cytology and histopathology cmni be routinely ‘used tx> diagnose canine MCTs without much difficulty, but accurate prognostication of these tumors can be more challenging. Currently, histologic grading is the most commonly used prognostic and therapeutic determinant for canine MCTs, as several studies have found a significant association between histologic grade and survival“”“. The two most commonly used histologic classification systems for canine cutaneous MCTs, described In! Bostock iii 1973 aumi Patnaik Ill 1984, 1.. .. A: I .. ... I, .. E .3 v. j. . 3 w? .3 7".- ..‘C . _ \ § . s _ ~x¥ r. “C .3 . . . r. . .- «C 2.. A. .3 1.. .NA classify MCTs jinx) three histologic grades an; being well- differentiated tumors (Patnaik grade I, Bostock grade III), moderately differentiated tumors (Patnaik grade II, Bostock grade II), and poorly differentiated tumors (Patnaik grade III, Bostock grade I)“”“. The Patnaik classification system is the most widely used system for histologically grading canine MCTs and defines grade I MCTs as being well— differentiated. tumors located :Ui the superficial dermis, grade II MCTs as intermediately differentiated tumors located in the superficial and/or the deep dermis, and grade III MCTs as being of poor differentiation”. In this study 93% of dogs with grade I MCTs, 47% of dogs with grade II MCTs, and % of dogs with grade III MCTs survived greater than 1,500 days, and this association was found to be statistically significant“. In his earlier study, Bostock used similar morphologic criteria to classify canine MCTs, also finding a significant correlation between histologic grade and patient survivalhi .A major point of contention between time Patnaik enmi Bostock classification systems, however, is the role of tumor depth in the classification of these tumors. 111 the Patnaik system, tumor“ depth .is 51 primary’ criterion. used tx) differentiate low grade and intermediate grade iMCTs”, however Bostock does not include tumor depth in the histologic . G {—- V‘s ,-. y, -5 A. «J C a. E [II-Ii .. . a 4 . r h. r . . . . j a . . . « .VJ /. r. r . . .1 .3. e a. .3 .3 . QC. .. i. rip. A: v... .r . .v:. .C 1 «C .r .. Q. nu. a . .3 WI... classification of these tumorshfl Variation in the inclusion of tumor depth in the histopathologic classification of canine MCTs has led to a marked degree of inter-observer variation when evaluating low to intermediate grade MCTs Qle. However, despite the controversy that surrounds the use of tumor depth in the histologic grading of canine MCTS, only one study has commented on the independent prognostic significance of tumor depth in canine cutaneous MCTs, and only as a manor side note in a larger studyifl Despite the statistical significance of histologic grading, the utility and relevance of the current histologic grading system has been called into question on multiple occasions due to the predominance and questionable biology of intermediate grade tumors, which account for as many’ as 72% (H5 the tumors in some studies”'“, and. the marked degree of inter-observer variationxkafl Specifically, the ijfinma of inter—observer variability has been clearly demonstrated in 2 sets of studies that have evaluated variation among pathologists in the histologic grading of canine MCTs. In the first set of studies, 60 canine cutaneous MCTs were evaluated by 10 pathologists at a single institution. When each pathologist was allowed to grade according to his/her own set of histologic criteria, v.~‘ .3 ,WJ l a a 5.41 there was only 50.3% agreement among the pathologists, and when a standardized set of histologic grading criteria was used, specifically the Ehinaik histologic grading system, ,21 the total agreement only increased to 62.1%:0 Similarly, ha a second study in vflfl1fl1 95 canine cutaneous MCTs were histologically graded km! 31 pathologists from 113 different institutions, there was only a 63.1% concordance for grade I MCTs, 63% concordance for grade II MCTs, and 74% concordance :fllr grade IEEI MCngfl Together tflmxxa studies clearly demonstrate the limitations of the current histologic grading system and the need for novel and improved prognostic indicators for canine MCTs. Clinical Progostic.Markers Over the last 30 years, several clinical, histologic, and. .molecular‘ markers have ibeen evaluated. for the prognostication of canine MCTs. These markers include, but are run: limited th tumor location3&22 stageéd6fl7, growth rate”) durationLfl proliferation InarkerSHFLC DNA.jploidyfifi intratumor vascular density”, plasma histamine concentrationifi, and morphometryyi However, the prognostic value and utility of many of these markers have been variable, and to date no marker has been established as a gold standard for prognosticating canine MCTs. . ._ . . . r? v. _. .... .. 4 2:. a_ E LN «4 _ rg. * _ Vi a . «4. w. . . .ch ”we ”3 Ar”. .3 j. .t ,t i A: .. _. w... ._f. .3 my. C. 3 v. C. .3 r. f. rc.‘ .‘n V“ Vs... Certain tumor locations, specifically the inguinal and perineal region, have been associated with aa worse prognosis, although these associations have been largely based on the clinical impression of oncologists and pathologists“”“fl with relatively few statistical studies 2,;5—27, l 41. In a recent supporting this association retrospective study in which 68 cases of inguinal and perineal. MCTs ‘were evaluatemi for‘ prognostic factors, the authors concluded that bfifls :hi the inguinal enui perineal regions may have similar tumor-free intervals and survival times as compared to MCTs in cmher locations when treated with appropriate chemotherapy and radiation therapyfifi It must. be noted, however, that the authors of this study based these conclusions on a cxmmarison of their survival data. with. that (of time current literature and IN) control population consisting of bKfls :hi other anatomic locations were included in this study. Interestingly, the results of another recent study suggest that MCTs of the muzzle may be more locally aggressive, have eni increased rate (If local metastases, and may be associated with an increased incidence of MCT-related mortality“fl This study, however, also lacked 51 control populatrmi and conclusions must be extrapolated from the current body of literature, thereby placing limitations CH1 the conclusions that (XHI be made. . .AP‘ 4 ”\k :w i, a. is .v. _ . .rl.‘ .‘ . I. . 4.. . . .2 hi. ;. .. ,. ., .. I; x» . m A w» Ag .7; _‘. V. V‘ s» . . 4‘ ‘ ‘ »v .3 .3 r. _. av H; «V .. ._. a. L. ., rd. a .2 r” A aa _ (u. ~ ~ h .‘ a» H .F‘ vi .‘XI .5 c . a \ .. T .3 :. 3 I i . . .... r” S r m: 3. Although many clinicians and pathologists still contend that inguinal, perineal, and muzzle-associated MCTs are associated with more aggressive mast cell disease, a true case-control study evaluating a large population of dogs undergoing a standardize treatment regimen is still needed to support or refute this hypothesis. Clinical staging has also been Luxxi to prognosticate MCTs. However, the significance of multiple synchronous tumors, which are considered to be stage III disease according to the World Health Organization’s staging system for canine MCTs, has been highly debated. Emeviously, no differences in survival were found between dogs with stage I and stage III MCTs when treated with prednisone and vinblastineJfi However, in a second study, stage III MCTs were associated with an increased rate of metastases, but not an increased rate of local recurrence or a decrease in survival time, compared to stage I MCTs when treated with radiation therapy aloneyfl .Additionally, in a recent study evaluating an eclectically treated group of multi- synchronous MCTs, Mullins et al. suggested that multi- synchronous MCTs do not offer a worse prognosis compared to solitary tumors“, however a control population of solitary tumors was not included in this study. One potential cause for the discrepancies between these studies may be the fact v. r». 1‘ _ . v4, .. . l P. .14 vi 6: ”A. r“ 3. C S .3 A s a _ 6L .3 .74. L .\c .r h a s .i .3 h . .Nu .../v .1 ~VIJ4 .14 at. L. V... that each cu? these studies evaluated populations cm? dogs treated with cflstinct therapeutic protocols. Animals with multi—synchronous :mast cxflj_ tumors are likely 11) respond better to systemic chemotherapy than to local therapy alone, such. as surgery' alone or .radiation therapy' since their disease is more widespread. Therefore, these studies should be interpreted in light of the therapeutic protocol used in each study, and future studies should standardize the sampling populations used with regards to the treatment given to each individual patient in order to produce interpretable results. Additional clinical parameters that have been used to prognosticate MCTs include tumor duration”, Ixnxa of tumor growthhfl plasma histamine levelsyfi and detecthmn of nest cells III buffy' coat smearsqL44. Previouslyy Bostock: has shown that both the duration that a tumor has been present and the rate of tumor growth per week are significantly associated with survival. Specifically, Bostock showed that dogs with tumors that grow less than 1 cm9/ week and dogs with tumors that have been. present longer than 28 weeks have significantly increased survival as compared to dogs with tumors that grow more than 1 cmW/week and those that have been present for less than 28 weeks, respectivelyhf Recently, Ishiguro et al. investigated the relationship) between DMEF progression enui plasma. histamine concentrations (PHC). Although initial PHC levels could not predict survival, tine results CM? this study cfixi show that seven of the seven dogs that died due to MCT-related disease developed a marked hyperhistaminemia, with a median value of l4ng/mL compared to 4 dogs that survived whose PHC levels remained less tflmni lng/mL. These results suggest that PHC cxn1 be used to evaluate disease progression, but not for initial prognostication’fi One prognostic measure for canine MCTs that has fallen by the wayside is the evaluation of buffy coat smears*””. At one time, buffy coat smears were commonly used to detect mastocythemia in MCT patients in order to evaluate patients for systemic mast cell disease. However, it has been subsequently shown that mastocythemia may be associated with several dermatologic and inflammatory diseases, and that tine mastocythemia associated vnif1 other diseases may in fact be greater than when it occurs secondary to MCTs, thereby extinguishing the promise of this prognostic «;_‘.,4(1’4€3 tool . 10 a» ~¢. PL. . 3 T T .f r. i .4 Q L L L r ._ a. : S Ma. C. Cu «4 :u .C ‘- . Tumor-free.Margins Due tx> the high degree of inter—observer variability in histopathologic grading, several additional pathologic- based diagnostic markers lunma been evaluated iii order to improve the prognostication of the current histologic grading system. Since mast cells tumors consist of round cells that invade enui distribute themselves throughout. a given. tissue, evaluation. of innmn: margins is 51 critical part of the histologic evaluation of a canine MCT, as marginal mast cells are thought to be responsible for local tumor recurrence. In order to evaluate the significance of tumor-free vs. nontumor—free margins, Michels et al. compared the rate of local and distant relapse, and survival in 20 dogs with tumor—free margins, and in 11 dogs with nontumor—free margins?) This study found that dogs with nontumor-free margins had three-times greater incidence (if local relapse (2/ll \HL 1/20, respectively), although this was not statistically significant, and a significantly increased rate of relapse at 12 anxi 24, but not 6, months post—surgery. Additionally, 2/11 of the dogs with nontumor-free margins died. due to MCT-related diseases, compared to 0/20 dogs with tumor-free margins. Based (Hi the Ixuv data, ii: appears tjmn: complete surgical excision is likely' to result jil a reduced incidence of 11 . _ I. l .. .. . , I as _. .. .. .. .2. .1; .. C. r w: 7. n“ r. . .... C r. .c . i T i; a. . i .I v. A s E f E E 2.. c. y... .3 .4 .1 E l K .1 E i t E .T. .2 .f. .3 C I .T. E C T. w.. .C C . . 3 : r. N.“ S nu r c .m a. T. . r . . s. .3 A. .3 relapsing disease, and a decreased incidence of MCT-related mortality. These CDnClUSiOHS coincide Math tjma presumed biologic principles that leaving behind a single neoplastic cell could result in re—growth of the tumor. The major limitation CM? this study, however, is tflufi: the extremely small sample size, vflflrfl1 is likely t1) be responsible for the jhnfl< of statistical significance. 111 this study a relatively lrwv number (If cases (2/11) vniji nontumor-free margins luxi local relapse. These .results seem tun be in striking contrast to the hypothesis that a single neoplastic cell left behind can reconstitute the tumor, and therefore place into question the significance of these marginal cells. Since mast cell tumors may produce chemokines that can attract additional normal mast cells to the tumor sitemfi C ea major question continues to arise as no whether marginal mast cells represent neoplastic cells or recruited normal nwmn: cells. ID: is critical 11) define the molecular phenotype of these marginal cells in order to define ‘their‘ biologic: significance, and an: this time few conclusions can be made based on the low number of cases and the relatively low rate of tumor recurrence and MCT- related mortality in this study. 12 C. ”,4 .c ~ ‘ » 2» CV I ‘ J. . A L” .r; r: we ». ~. A. he ‘ h k a. o C s ”at. _ v . ~‘ A ‘1. \r~ WI l‘ .—.. x. Ty he. . r. M». - Proliferation.Markers As uncontrolled cellular proliferation is considered a hallmark cflf cancen”, pmoliferation nerkers, such an; Ki67 indices, PCNA indices, and AgNOR counts, have been used to 52‘, 60-66 prognosticate several humant"3'59 and canine“' neoplastic diseases including canine cutaneous MCTs. Ki67 is a nuclear protein that is expressed during all phases of the cell cycle, ibut If; absent III non-cycling' cellsgbfifi. The function of Ki67 during the cell cycle is unknown, although it appears to kxa necessary for cxfld. cycle progressionfi&69. Since Ki67 irs expressed ii1 all phases of tjua cell cycle, the relative jproportion <3f cxfldrs expressing" this protein has been used as a measure of the proliferation index, or the relative proportion. of neoplastic cells involved in cellular proliferation in a given tumorm’m'mtmtn. PCNA, or proliferating cell nuclear antigen, is an auxiliary subunit of IEUX polymerase—delta, tjma major eukaryotic replicative 75. PCNA.jrs also involved iii other nuclear DNA polymerase”' processes, most notably DNA repair”. Due to its role in DNA replication, PCNA is primarily expressed during the S- phase, or DNA synthesis phase of the cell cycle-””77. As such, PCNA has been used to determine the S-phase index, or the relative proportion of cells in the S-phase of the cell 7 0 cycle“”' Although. PCNA. expression. peaks during' the S- 13 phase, PCNA has a 20-hour half life78 and therefore may be expressed at other phases of the cell cycle. Despite PCNA’s long half-life, PCNA is still considered by many to be an S-phase specific markerm'wfl AgNOR (agyrophilic nucleolar organizer region) histochemical staining is a silver-based staining technique that stains areas of ribosomal RNA transcription in the nucleus, called AgNORs, due tx> 2 RNA transcription-associated proteins' affinities for silverfi. The number of AgNORs per nucleus is correlated vnij1 the rate (n3 cell proliferation anui tumor growthwfflfl which is thought to be due to the need for increased protein synthesis during cell cycle progression“% Ki67, AgNOR, and PCNA counts have all been shown to be independent prognostic factors for canine IMCTs, although the utility of these markers has varied between studies”- ‘””” In a study evaluating PCNA and Ki67 immunostaining in 120 MCTs treated with surgery alone, Abadie 6%: al. found that the mean number of Ki67 positive cells/1,000 cells was significantly associated. with survival. In this study, using cut—offs of 55 and 135 Ki67 positive cells/1,000 cells, lflfhs were classified into 13 distinct classes that were significantly different from 6%Mfl1 other 111 terms of survival, and grade II MCTs with greater than 93 Ki67 positive cells/ 1,000 were found to have significantly 14 v. v . . .3 .3 J I. E .1. l . 4 x ‘v v _.,.. H .j .P‘ w. .4. .1. .q. < . ‘_ 2. e. A: i; W43 CW ‘ ‘ s._ C. r. k. .3 t. A; .3 a. ..v _... ‘ . . a . . i .. .2 AC «C l . an a , . T .4... ‘ “.9 ~\~ § ~ a» NW§J .3 «V reduced survival as compared to grade II MCTs with less than 93 positive cells/1,000cellsj”. Additionally, in this study Abadie et al. found that the PCNA—positive cells were significantly greater in dogs that died due to MCT-related disease an; compared tx> those that survived, inn: there was significant overlap between these groups thereby eliminating the prognostic utility of this techniqueflfi In an earlier study, PCNA immunohistochemical staining was evaluated in a series 120 canine MCTs. Dogs with MCTS with greater than ZMH. PCNA-positive cells/5 .high. powered fields had significantly reduced survival as compared those with less than 261 PCNA-positive cells/5 high powered fields”9. AgNORs were also evaluated in this study, and it was found that dogs with an AgNOR count greater than 2.25/cell, based on the evaluation of 100 cells, had significantly decreased survival durations an; compared to those with less than 2.25%. In the original study evaluating AgNOR histochemical staining in canine MCTs, Bostock found 51 significant difference 1J1 survival between cases with >4 AgNORs/cell and those with less than 4 AgNORs/cell based on counting 100 cells. Additionally, in this study IN) dogs with eni AgNOR count CM? less than 1.7 AgNORs/cell resulted.:h1 MCT-related nwrtalityfl. Recently all three markers were evaluated in a single study, 15 Perv- ‘3»..A .r»~« ‘n..A‘ a A p .3 Qt. allowing for a comparison of these markers. In this study, AgNORs and. Ki67 were both associated. with disease progression, but Ki67 was determined to be the most: useful as it could distinguish potentially benign and malignant histologic grade II tumors using distinct cut-off values”. Although these studies demonstrate the prognostic significance of these proliferation. markers, the .methods and results of these studies are highly ‘varied, thereby confounding the interpretation and application of each individual study. Additionally, at this time, only 1 study has evaluated all three of these markers in a single cohort of animals*%, and no studies have evaluated the utility of using these markers as 51 panel rather tjmni as individual indicators of malignancy. Since cellular proliferation is a result of IMNji the number (Hf cycling cells i11 a given tumor and the rate of cell cycle progression, it is necessary tx> evaluate both the pmoliferation index (K167) and the rate of cellular proliferation (AgNORs) in order to gain a full understanding of a tumor’s cellular proliferation””fifl“. By looking at all three markers in a single cohort of animals the combined significance of these markers and a comparison of the prognostic significance of these markers can be made. Therefore, a single study evaluating all three of these markers in a cohort of dogs 16 E f .t i. L; r. .3 C. ;. .3 5.. £.. . . . . . ... . AJ 2.. w . v . . s... v. v." ..A d . fl.‘ . a s V . L .L 3 t. E . . .3. C . ~ I I h . . . 3 S .. i c I .1. law ~ .. .J. :3 A u \ treated with a standardized therapeutic protocol is needed in order to evaluate the true relevance of these markers, both alone and in combination. Image Analysis In addition ix) routine light macroscopic techniques, image analysis software has been used to develop additional markers for the classification and prognostication of canine MCTs”*fi. In a study evaluating the nuclear morphology of 24 canine MCTs using image analysis software, Strefezzi et al. found a significant difference in nuclear area, mean diameter, and nuclear perimeter between grade I and grade III and grade II and grade III MCTs. However, no differences were found between grade I and grade II tumors, which, an; mentioned previously, are time most difficult to differentiatefl The utility of these findings is limited since no survival analysis was performed, and a relatively low number (Hf cases were analyzed. Therefore, 51 larger study with complete survival analysis is needed in order to determine time biological significance (Hf these variations in morphology. Image analysis software has also been used to determine the prognostic significance of intratumor microvessel density in canine MCTsfi. Subsequent to immunohistochemically labeling endothelial cells with factor VIII—related antigen (von Willebrand’s factor), 17 Preziosi (a: al. used image analysis software tx> determine the number of microvessels/mm: in a series of MCTs in order to determine the association between intratumor vessel density and survival. Based on the cut-off value of 14.1 vessels/mm‘, dogs with MCTs with a high microvessel density were found to have a significantly decreased survival time and cancer-free interval, as compared.tx> dogs with tumors with lxwv microvessel density. In tint; study, intratumor micro-vessel density was found to be an independent prognostic indicator based on multivariate regression analysis. Although kxfifli of these studies anxa relatively small, they do demonstrate the potential power and utility of image analysis 1J1 cancer prognostication. There are, however, significant limitations to these techniques, such as the time constraints, cost, and the current availability of image analysis software an: veterinary diagnostic laboratories. In the future, image analysis-based techniques may be routinely used in the prognostication of neoplastic diseases, but these current limitations prevent the routine inxa of image analysis 111 a veterinary diagnostic setting. Despite the plethora of potential markers that have been evaluated for the pmognostication of canine MCTs, no single :marker‘ has Ibeen clearly' shown to 1x3 consistently 18 , .3 . .. 2.. .3 C l .. T .. . . 3 .1 r r i C e , E f .5 S . C r .. -3 I. z. .5 2. . . E C. .i. E l i C E aw S 3. .4 ... . s .. s a. . v. v. n. i. 2 z. 0 z, A: c. .l ,a. V». .5 :l ; .si .1, r r 4.. ., C. C. a: .3 C . . C M T: I _ _ 3. _ . i . 3 C S E .. C. C. .. Mei «~“W_ predictive of MCT behavior. In light of this, it is probably unrealistic to assume that a single marker will ever be able to accomplish such a daunting task. Instead, a prognostic panel for canine MCTs may be established that combines clinical, pathological, and molecular markers for the accurate prognostication and tjmmapeutic determination of canine MCTs. .Mblecular Pathology of canine.MCTs p53 Despite the attention canine cutaneous MCTs have received 111 terms (Hf prognostication, especially 1J1 terms of histopathologic evaluation, much less attention has been given to the molecular biology or molecular pathogenesis of these tumors. Using' immunohistochemistry, expression. of the p53 tumor suppressor gene in canine MCTs has been described III 2 independent studies“”w. The focus of each of these studies was to evaluate the prognostic significance (n5 p53 expression 111 canine PKHEL and_ each study concluded that despite p53 expression in a large number of canine cutaneous MCTs, detection or the relative proportion of p53-positive cells was not useful in terms of prognostication. The major linutation CH? these studies, however, its that there vmme 1M) attempts characterize the biologic significance (Hf p53 expressitni in. these tumors. 19 q—f, v--- . .«f a .. . . a ,. ,. . I .4 . H . ~ . . . 1 . r” . Q .7. v.“ Av“. .>4 .5» . A L. g L . fi . . 5; rv Flu :4 2‘ c; v; a.¢ .._ C. 3. .3 C. C. S C. t .L .7. t 3 C. t. c . For example, null mutations in p53 may provide a chemo— resistant or radiation resistant phenotype by allowing the neoplastic cells to ignore DNA damage and to continue through the cell cycle. Additionally, as p53 mutations may be responsible for chemo-resistance and resistance to radiation therapy, these studies could have also been strengthened. by assessing' the prognostic significance of p53 expression and p53 mutations in a population of animals given a standardized therapeutic protocol, in order to determine the role these play on chemo-resistance in canine MCTs. p21 and p27 Expression. of time cyclin-dependent kinase inhibitors p21 and p27 has also been evaluated in canine MCTs in a single study“%, In this study, 47 IMIMS were evaluated for p21 and p27 expression using immunohistochemistry in order to test the hypothesis that p21 and p27 expression is lost in canine MCTs with advanced disease, as characterized by having a higher histologic grade. Although there was a tendency for higher histologic grade tumors to have a loss of' p27 expression, there» was also an increased relative proportion of grade II and grade III MCTs that had moderate to marked expression. of jp27. Additionally, this study found an increased p21 expression in higher histologic 20 .: «xv grade MCTs, vflflxfli is contrary ix) the authors’ hypothesis that there should be loss of expression of these genes in canine MCTs. Overall, the significance of these results is very hard tx3 determine, as IN) clear associations between histologic grade and jp21 enui p27 expression. were found. Therefore, these results provide no evidence of a potential role of p21 and p27 in the progression of canine MCTs. DNA ploidy In order to determine the role that DNA ploidy and chromosomal aberrations play' in canine iMCTs, Ayl et al. conducted a retrospective study comparing DNA ploidy in 40 MCTs, using flow cytometry with propidium iodide staining, with patient survival, histopathologic grade, tumor-free survival, and clinical stage”. 72.5% of the MCTs evaluated in this study were diploid, suggesting a low incidence of chromosomal aberrations in canine MCTs. Although there was an overall low incidence of aneuploidy in the MCTs evaluated, the investigators did find that a relatively higher proportion of grade III MCTs were aneuploid, although this was not statistically significant. In this study, aneuploid tumors also tended to 1x3 associated with decreased survival times, although this was not statistically significant, and were found to be significantly associated with an1 increased clinical stage 21 I» i: . _ V a 2.. ru. A: .2 .2... n .. W. .3 2.. a . S when. stage 1. tumors vnnxa compared t1) all other' clinical stages combined. These results suggest that changes in DNA ploidy are rare events in canine MCTs, but they may be associated with more aggressive forms of mast cell disease when they do occur. Currently, the nature of aneuploidy in canine jMCTs in 'unknown, but knowledge of the associated genetic changes may shed light on additional genes that may be responsible for the progression of canine MCTs. ABC Transporters Malignant canine MCTs commonly have a poor response rate tx> chemotherapeutics”7, suggesting tjmu: these tumors may express ATP—binding cassette transporters that are potentially responsible for drug efflux from the neoplastic cell and therefore the development a multi-drug resistance phenotypeflfl. In a recent study, Miyoshi et al. evaluated a series of 44 canine MCTs for the expression of two of these transporters, P-glycoprotein and multidrug-resistance- associated protein”fl In this study the authors found expression of at least one of these two proteins in 26% of the MCTs evaluated, and the expression of these proteins suggested an inverse correlation with histologic grade. At first glance these results are optimistic, suggesting that the majority of canine MCTs, especially those of higher histologic grade should be sensitive to standard 22 chemotherapeutics. However, the INK: transporter' family' is an extremely large protein family of which several members have been associated with multi-drug resistancew. Therefore, although P-glycoprotein and. multi-drug- resistance-associated protein are not likely to play a significant role in the efficacy of chemotherapeutic treatment of canine MCTs, it is possible that other ABC transporters do play a role in the chemo-resistance of canine MCTs. The Role of the c-KIT.Proto-oncogene in canine MCTS c—KIT Of the genes that have been evaluated for a potential role 1J1 canine MCTs, time role of time c—KIT proto-oncogene has been most clearly defined“”m’WK The c-KII’ proto- oncogene encodes the type III receptor tyrosine kinase, KITV. The KIT protein consists of an extracellular ligand- binding domain consisting of 5-immunoglobulin-like loops, a transmembrane domain, a negative-regulatory juxtamembrane domain, and. a split cytoplasmic kinase domainwbwfi The receptor tyrosine kinase KIT is expressed in multiple cell types including hematopoietic progenitor cells, melanocytes, mast cells, interstitial cells (If Cajal, and gemn cells, where ICUF has been shown t1) be important for 100-105 cell survival, differentiation, and proliferation 23 .3 .I w» c... a; .3 .. : . .II t v o a r. .9 S k. c . .2 a. .3 C .1 A.» .l i N44 .A‘ .N Additionally, KIT runs been shown tx> be important for mast cell chemotaxis, fibronectin adhesion, and degranulation”%’ 31. The ligand for ICUF is stem cxfld. factor (SCF, also known. as Jmast Icell growth factor, KIT ligand, and. steel factor)"“""”;‘1”, which is also expressed by multiple cell types including fibroblasts, stromal cells and endothelial cells”“. Mice with either c—KIT or SCF null mutations are characterized kn/ hypopigmentation, sterility, anemia, and mast cell depletion, further demonstrating c—KIT and SCF’S essential role in the survival, differentiation, and proliferation of hematopoietic cells, germ cells, melanocytes, and mast cells“‘”1”l”. c—KIT in Human Cancer In recent years, the c—KII’ proto-oncogene has been implicated in several distinct neoplastic diseases, including gastrointestinal stromal tumors (GISTs)MEflZK mastocytosis: ”L”, germ. cell tumorslM‘Lfi, small cell lung cancerlfl’Lfl, prostate cancerlH'L”, and acute myeloblastic leukemiaH’i'l‘1 1J1 humans, enmi mast cmflJ. tumorsobqubgB and 1 3 .; - - . in canines. Potential gastrointestinal stromal tumorshq' activating mutations have been identified in several different exons of the c-KIT proto-oncogene in a variety of cancers. In human gastrointestinal stromal tumors patients, germline mutations have been identified in exon 8 24 I. x... .C r: . L. “a .3 .1 .a i. Y. 3 ~... C. 7. .: of the extracellular domainn3, exon 11 of the juxtamembrane domainlm'ng, and exons l3 and 17 of c-KIT’s kinase domainug; however, the majority of sporadic c-KIT mutations in GISTs have been identified in exon 11, and to a lesser extent in exon 9 of the extracellular ligand-binding domainl”. In human mastocytosis patients, point mutations occur most commonly in exon 17 at codon 816, usually resulting in the replacement (If valine ikn: aspartate. However, additional substitutions at codon 816 have been characterized in human mastocytosis; patients, resulting 1J1 valine, tyrosine, .and phenylalanine substitutionslfl. Additionally, 23 point mutation resulting in.ai lysine substitution.:fi1r glutamate at codon 839 has been characterized in some pediatric mast cell patientsuq, and a 3bp deletion at codon 419 in exon 8 has been identified in a family with a history of mastocytosis and GISTSL”. In addition to those found in GISTs and mastocytosis patients c-KIT mutations have also been identified. in exons 11 and it7 111 human germ. cell tumorslfl”hm. Although tjmz true biological significance (fl? each of these mutations has Inn: been thoroughly characterized, it has been previously shown that mutations in exons 11 and 17 of the c-KIT proto-oncogene result in constitutively activated KIT products in the absence of ligand“8“f&ly1 25 . . 1.. . f. z. :. :._ .. ;. r. .: .r. 2,. 2. 3 a. : a. L. E .. a” .3 .r” v. E v.“ C. .Q A: C .J r. u. .n... .«iq .vp. ry. «v wk. .3 4‘ ~\» ”N Y. .. bk ..A W.“ .3 Additionally, different c-KIT mutations have been associated with distinct clinical forms of human mast cell diseaseX3”3“1”, and with distinct tumor locations, phenotypes, and more aggressive disease in GIST patientstflJtVlfl, further suggesting a key role for c—KIT in the progression of these diseases. Interestingly, the presence and location of c-KIT’ mutations has also been associated with response to receptor tyrosine kinase inhibitors, such as imatinib mesylate (Gleevec). Specifically, it has been shown that tumors with c-KIT mutations in exon 11 are significantly more likely to respond tx> c—KIT inhibitors, snufli as Gleevec, an; compared to tumors with exon 17 mutations or tumors that express KIT but lack c—KIT’mutations altogetherLH’H7J‘8 With the discovery of Gleevec, there has been an increased interest in identifying tumors that contain c-KIT mutations, cm: aberrantly express KIT. In light CM? this, aberrant KIT expression has been characterized in a number of human neoplastic diseases, including uterine leiomyosarcomas ”, small cell carcinoma of the urinary bladdermg, follicular thyroid carcinomaSMH, endometrial carcinomaslm, acute lnyeloblastic leukemia’”, prostatic -1:9 carcinomas“”, and small cell lung cancerhfl The significance of aberrantly expressed KIT has been 26 H‘ U: A‘ .-. .,‘~ .4 -4, ’4‘ w 4.. 1 .V“ -.._ 7‘1.‘ ... A. "V’w J-‘i ‘ ‘n- -fo “r -. ~».,’ ’.. "—Ul ‘ ”a “‘4. r'?‘> ‘4‘ . #‘_ M ‘r.,.'_ “V . -. .‘ \‘fi\,_ L. V.‘ "‘ ( ;) characterized for sxnma cancers, Inn: for many, time role of KIT still needs to be elucidated. In some tumors, such as small cell lung cancer, concurrent SCF production by the neoplastic cells creates EH1 autocrine/paracrine signaling loop which results in the constitutive activation of KIT‘L4’W“. A. truncated iSOfOIHl of KIT (TR—KIT) has been identified i11 other" tumors, such an; prostatic: carcinomas 141,144 and colon cancerlw' In humans, TR—KIT originates in intron 15 cm? the C either bind tx> its ligand cm: autophosphorylate. However, instead of dimerizing and signaling through traditional KIT pathways, TR-KIT acts as a scaffolding protein, which binds and activates the Src—like kinase Fyn. Upon activation, Fyn is able to bind and phosphorylate the STAR family RNA binding protein Sam68, which then acts as a scaffolding protein, facilitating the interactions between Fyn and Phospholipase Cvl (PLCyl). This leads to the activation of PLCVIH‘. In prostate cancer patients, TR-KIT has been found in neoplastic cells, but is absent from the surrounding :normal tissue. Additionally, TR-KIT If; more 27 r — «\s. commonly found in advanced forms of prostate cancer, thereby implicating TR—KIT in the progression of this diseaset’. c-KIT in canine cancer Similar to human cancers, c—KIT expression has been described in multiple canine neoplastic diseases using immunohistochemistry, including'1mammary’ gland. adenoma and carcinoma, malignant melanoma, seminoma, interstitial cell tumor, granulosa cell tumor, ovarian papillary adenocarcinoma, gastrointestinal stromal tumors, and Imast 5‘41-{V‘i:',l.'4, l L, 1414 cell tumors“' In the majority of these cancers, KIT expression has been described as being weak, with variable expression in the few cases that were evaluated ”. 0f the canine neoplastic diseases that have been shown to express KIT, KIT expression has been most consistently' described jjl MCTs enmi GISTs, which, tend tx> have strong diffuse KIT expression throughout the majority of these tumorsq”m'“4”ifl In 2003, two laboratories independently described KIT expression ll) canine gastrointestinal stromal tumors. The relative frequency of KIT expression in GISTs varied between these two studies from 52% immuno-positivity (ll/21 GISTs)”3 to 100% immuno-positivity (5/5 GISTs)“4, but both studies described strong, diffuse immuno-reactivity in the 28 2. vWAv .na ”.10- ,1» Y‘ .. U . _ ‘3. v. «v :. .2 -w. ..‘ majority of the tumors that expressed KIT. Aside from the identification of KIT expression in canine GISTs, Frost et al. also identified potential activating mutations in exon 11 of c-KIT in two of the four tumors evaluated. These mutations consisted of aa six base EEUJT deletion, deleting Try556 and Ly3557, and a three base pair duplication of Gln555 in (Hue case, and a 1?‘U3 C transition, substituting ProS75 lint leu575 jJ1 a second casel”. Although IK) large scale studies have evaluated the prevalence of c-KIT mutations in canine GISTs, the consistent expression of c— KIT’ in the :majority' of canine GISTs tumors, the identification (Hf potential activating Hmtations, enui the known role c—KIT plays in human GISTs suggests a potential role of c-KIT in the progression of canine GISTs. However, in the future, further studies are needed to characterize the prevalence enmi the biologic and clinical significance of both KIT expression and c—KIT mutations in canine GISTs. Mutations in the c—KIT proto—oncogene and aberrant KIT expression, characterized by increased cytoplasmic protein localization, have IMKHI described iJ1 canine FREE» and are thought to play' a key role in the progression of this diseasegbgLQWfiG. C have c—KIT mutations vflufii compared tx> the entire spectrum cu? canine MCTs, as seen in the former studym”%k Additional discrepancies have also been reported in terms (n? the incidence (Hf deletions ij1 canine MCTs. As mentioned previously, in EH1 evaluation cm? 88 canine MCTs Zemke et al. found deletions in 4.54% of the MCTs evaluatedmfi However, in.ea subsequent study Jones et al. failed to find any deletions in exon 11 of the C-KIT proto- oncogene in 25 MCTSMG. Although this discrepancy may be a result of a small sample size, the forward primer used by Jones et al. for PCR amplification and sequencing was complementary to the sequence where Zemke et al. had previously reported deletions. Therefore, due to the design of this forward primer, these primers would not amplify the mutant allele in the majority of deletions and only the normal allele would amplify in heterozygotes, thereby inhibiting the detection of most, if not all deletions that were similar to those previously . Q ‘ 3 described”””(. 31 v ¢ h" by VJ. «C _ Cc ...... . a x ._ .r .2 v; . ~\— ~“ . ,lt . In light of these discrepancies, it is likely that c~ KIT mutations occur in 15-20% of all canine MCTs, but may occur in, as :many as 30-50% of high grade or malignant MCTshhmfi Despite the relatively low incidence of exon 11 c—KIT mutations, only two studies, in which only 6 and 11 MCTs were included, respectively, have screened additional c—KIT domains for pmfiential activating Hmtationsm’%. Therefore, future studies are needed to evaluate canine MCTs fin: additional c—KIT mutations, such as nmfiations in exon 17 of the kinase domain as seen in human mastocytosis patients14'mjfljs that HEW? also play 51 role III the progression in canine MCTs. Early studies suggest that c-KIT’ mutations play a significant role in time progression. of canine :mast cell disease. IPrevious studies .by CNN? laboratory' have shown that deletions and ITD c—KIT mutations are significantly associated. with higher histologic grade IMCTsmfi and. work from another group has shown that MCTs with ITD c—KIT mutations are twice as likely to locally recur and twice as likely to metastasize as compared to MCTs that lack ITD c- KII‘ mutations, although these differences were not statistically' significantl“. Together these data suggest that mutations in the c-KIT’ proto-oncogene may play a significant role in the progression of canine MCTs, and 32 Ly ~\» 24 .v . ‘~. .nq VJ: TC C. "w I ______ therefore may serve as 51 potential target for treating MCTs. Additionally, the presence of these mutations may be used fin: prognostication anxi identification (n5 MCTs that may respond to targeted therapies, such as receptor tyrosine kinase inhibitors. .MCT Treatment Currently, canine cutaneous MCTs are primarily treated with surgical excision alone or in combination with radiation and/or chemotherapy depending on the clinical stage anui histologic grade cu? the tumor. In time face of clean surgical margins, solitary low- and intermediate grade MCTs that do not involve the local lymph nodes are commonly treated with surgery alone, followed by careful observation. of time surgical site for 'tumor recurrence5fl. Three centimeter margins lateral and deep tx> the MCT have classically been considered to be ideal for complete tumor resection, but. a .recent study suggest that 2-cm lateral margins and one fascial plan deep may be adequate for complete excision of histologic grade I and grade II MCTSH7. However, no grade III MCTs were included in this study and since histologic grade is usually not determined by the time of surgical excision, the results of this study may not be applicable in a real world setting. If adequate surgical margins are INN: obtained from.an1 intermediate or 33 high—grade MCT, re-operation and/or local radiation therapy is commonly used to prevent tumor recurrence. If regional lymph rxxka or systemic metastases are pmesent, (n: if the tumor is run: amenable II) resection, chemotherapeutics may also Ix; added tx> the therapeutic regimen. Several single and Hmlti-agent chemotherapeutic pmotocols lmnma been used to treat canine MCTs. These include prednisone, vincristine or lomustine alone; prednisone and vinblastine in combination; prednisone, cyclophosphamide, and vinblastine in combination; or cyclophosphamide, vincristine, prednisone, and hydroxyurea in combination. Despite the wide array of chemotherapeutic protocols available, the overall response rate for canine MCTs extremely variablem7. Coinciding with the discovery of c- KIT mutations in canine MCTs, recent focus has been directed towards the use of receptor tyrosine kinase inhibitors in the treatment of canine MCTs, especially those MCTs with activating c—KIT mutations, which are likely tx> have aa better clinical response tx> these novel drugs. In ea recent phase II clinical trial, time receptor tyrosine kinase inhibitor SU11654 was shown to cause objective tumor shrinkage in 11/22 canine MCTs and a total response rate of 55% with minimal and tolerable toxicitiesmg. Interestingly, an increased response rate 34 was seen in canine MCTs with ITD c-KIT mutations (9/11) as compared to those that lacked c—KIT mutations (2/11). Furthermore, although run: statistically' significant, dogs with 11K) c—KIT mutations tended to lunme a longer survival duration than those that lacked ITD c-KIT mutations (36.9 weeks vs. 15.4 weeks, respectively)”8. This utility' of SU11654 has been further validated, as it has been shown to modulate KIT phosphorylation and down-stream signaling pathways, such. as ERKI 1/2 in canine iMCTs based. on the evaluation of serial pre- and. post-treatment biopsiesms. Although 8011654 is not currently available, these data demonstrate (flue potential for ‘targeting c—KIT 111 canine cutaneous MCTs in the future, which may provide the greatest lunxa for definitively treating aggressive canine MCTs. Purpose At the time of the inception of this dissertation project in late 2001, histologic grading and the overall clinical picture were the only criteria routinely used to prognosticate canine MCTs. At that time, the criteria used to evaluate a given patient's clinical picture was largely based (M1 the clinical experiences and .haterpretations <3f oncologists and pathologists, with few studies available to support or refute these clinical assessments. 35 Additionally, as mentioned previously, the discrepancy between the histologic grading of pathologists and the question of tjua role tumor depth plays ill the histologic grading of canine MCTs further confounded the prognostication of canine MCTs. Although studies had been published evaluating the IHKB of proliferation markers for prognosticating MCTs, the discrepancies between studies, the use of image analysis software, and the labor intensive methods used to evaluate these markers prohibited their use in a routine diagnostic setting. Similarly, aside from the identification of KIT expression in canine MCTs, the identification of activating juxtamembrane domain mutations in tjma c—KIT proto-oncogene, and time associations between these mutations and. histologic grade, minimal conclusive data were available with regards to the IKfl£3(Df c-KIT in canine MCTs. The primary goals of time work described in the subsequent chapters of this develop multiple MCTs and male CkXflS with MCTs have a decreased survival time as compared to females. Several previous studies have found that boxers have a predilection for the development of cutaneous MCTsE'8'12 and, although rmfl: well described ill the literature, boxers are commonly thought to be predisposed to the development of multiple MCTs ill a single patient. (Mn: study confirms the results of previous studies and supports the hypothesis that boxers are predisposed to multi—centric mast cell disease. The predisposition for multiple MCTs in older dogs may be explained by the fact that older dogs have accumulated more mutations in their genomic DNA and are therefore more likely to develop cancer in general. It would be logical that these dogs have an increased 52 likelihood of having multiple transformed mast cells and therefore multiple MCTs at the time of diagnosis. In this study male dogs were also found to have decreased survival duration as compared to females. Similarly, a previous study found that female dogs treated with a multi-agent chemotherapeutic protocol had a more favorable prognosis as compared to malesl‘”. Estrogen and progesterone receptors have been found in canine MCTs, and may explain this difference in survival between males and females, however, the exact role of hormone receptors in canine MCTs has not been elucidatedHQ'MU. Future studies are necessary to verify the sex-specific differences in survival for dogs with cutaneous MCTs and to determine the role of hormones and hormone receptors in the development of canine cutaneous MCTs. This study defines the prognostic significance of tumor depth, location and multiple synchronous tumors for surgically removed canine cutaneous MCTs. It should be emphasized that no single clinical parameter can be used to accurately define the prognosis for every given patient. Only by evaluating multiple prognostically significant parameters concurrently can the biological behavior of canine cutaneous MCTs be more accurately predicted. Additionally, new technologies based on the molecular 53 tflology of MCTs are needed to elucidate the relationship of Hmltiple cutaneous lflflks occurring ill a single (km; as well as to identify MCTs with metastatic potential. Figure 1 (A—D): Tumor depths identified for prognostic evaluation. A. superficial dermis. Neoplastic mast cells lie immediately below the epidermis and do not extend below the adnexal structures. B. Superficial and deep dermis. Neoplastic mast cells extend from the superficial dermis, immediately below the epidermis and extend into the deep dermis and subcutaneous adipose tissue. C. Deep dermis. Neoplastic mast cells are isolated in the deep dermis and the peri—adnexal region. D. do not extend into Neoplastic mast cells extend and thiscularature invasion. invade into the underlying muscularature. 55 Ventral Figure 2: Tumor location was defined for prognostic purposes as: 1. Head and neck (dark gray); 2. Trunk (black); 3. Extremities (white); 4. Inguinal/ perineal region (light gray). 56 Table 1: Mast cell tumor outcomes by signalment Additional MCT Development Death due to MCT Local Distant Risk Factor X2 p Odds 95% 0.1. X2 p Odds 95% X2 p Odds 95% Ratio Ratio C.l. Ratio C.l. Age < 3 yrs .9949 - - .9906 - - .9926 - - 3 - 5 yrs .1673 .23 .03 — 1.85 .2447 .56 .21 - .0520 .13 .02 - 1.50 1.02 6 - 9 yrs .3771 1.81 .49 - 6.75 .9023 1.05 .46 - .2260 1.84 .69 - 2.40 4.95 > 9 yrs .3883 1.85 .46 - 7.54 .1004 2.06 .87 - .1566 2.09 .75 - 4.90 5.78 Wt. < 10 lbs .9954 - - .9913 - - .9935 - (lbs) 10 - 30 lbs .0710 3.60 .90 - 14.45 .9895 - - .8064 1.21 .27 - 5.41 30 — 50 lbs .5105 .50 .06 - 4.01 .4230 1.47 .57 - .5034 1.49 .47 - 3.81 4.75 50 — 70 lbs .2869 .32 .04 - 2.60 .4119 .63 .21 - .1418 .22 .03 - 1.84 1.67 > 70 lbs .7477 1.24 .33 - 4.62 .1620 1.84 .78 - .4221 1.54 .54 — 4.35 4.38 Sex Male .2815 2.07 .55 - 7.74 .6561 1.21 .53 - .0206 2.23 1.13 - 2.75 4.37 Sterile .9644 1.05 .13 - 8.41 .2165 .51 .17 - .0138 .26 .09 - 1.49 .76 Breed Mixed .4530 1.70 .43 — 6.81 .2268 .47 .14 - .8661 1.10 .36 - 1.59 3.42 Labrador .3315 .36 .04 - 2.87 .4667 1.41 .58 - .9340 1.05 .34 - 3.43 3.26 Golden .7199 1.46 .18 - 11.77 .9384 1.06 .25 - .5211 1.63 .37 - Retriever 4.52 7.16 Boxer .9941 .99 .12 - 7.96 .2768 1.82 .62 - .8497 1.15 .26 - 5.36 5.09 Other .9518 1.04 .26 - 4.19 .7446 .86 .36 - .4716 .66 .21 - 2.10 2.05 57 :1 42k finfle 2: Distribution of additional MCT development and nwrtality among dogs with MCTs of different tumor depths. Additional MCT De th Development* Mortality P Local Distant MCT Total Deaths Deaths Superficial Dermis . a . 2 . rs . r:- .7$~ . (n=6) 1 (16 7 ) 0 (O 0 ) 1 (16 ) (33.3%) Superficial/ Deep ,. n ,, 16 7 13.09 15 27.8w 8 14.8: Dermis (n=54) ( ’) ( 3) ( ’3) (29.63:) Deep Dermis (n=10) 0 (0.0%) 2 (20.0%) 0 (0.0%) 1 (1.0%) Muscle Invasion . ,, n g 7 (n=28) 3 (10.76) 7 (25.0 ) 7 (25.0”) (25 On) * Six dogs had local and distant MCT development number of dogs in column is 29) (total Table 3: Distribution of additional MCT development and mortality among dogs with MCTs in different body locations. Additional MOT Mortalityfi . Development Location* . Total Local Distant MCT Deaths Deaths _ ' r) q. 4 Head/ Neck (n—ll) 3 (27.3 ) 6 (54.5 ) 3 (27.3m) (36.4%) - _ 2 g 1 10 Extremeties (n—36) 4 (11.1 ) 8 (22.2 ) 3 (833) (27.8%) i . . 12 : r5 , ‘1; . 5‘ Trunk (n 43) 5 (11.6 ) 11 (25 62) 7 (16 3 ) (27.9%) Inguinal/ Perineal } g a 4 (n=11) 0 (0.0 ) 0 (0.0 ) 3 (27.3 ) (36.4%) * Three dogs had multiple synchronous MCTs at 2 distinct locations (total number of locations in column is 101) 1 Six dogs had local and distant MCT development and 2 of the dogs with synchronous MCTs had also additional MCT development accounting for 2 additional locations number of dogs in column is 29) £ Three (total dogs had multiple synchronous MCTs at 2 distinct locations and died during follow—up accounting for the 3 additional events in the table column is 27) 58 (total number of dogs in Table 4: Distribution of mortality among dogs with multiple additional MCT development synchronous mast cel l tumors. Additional MCT . Development Mortality . MCT Total Local Distant Deaths Deaths Multiple Synchronous 3 (30%) 2 (20%) 2 (20%) 5 (50%) Tumors (n=10) 59 PMCT Table 5: Univariate analysis of risk factors for outcomes by body location and depth. Outcome Factor Level X3 p Odds 95% Ratio C.I. Locat. Head—neck .2956 2.32 .48 — 11.19 Local MCT Extremities .8650 .89 .22 - Develop. 3.55 Trunk .5227 1.54 .41 - 5.72 Inguinal .9946 — - Depth Dermis/ .5630 1.85 .23 - reticularis only 14.83 Dermis to .8161 1.07 .29 — subcutaneous 4.01 tissues Subcutaneous .9933 - - tissues only Skeletal muscle .7103 1.30 .33 — layers 5.21 Locat. Head—neck .0332 2.94 1.09 — 7.95 Distant Extremities .6264 .80 .33 - MCT 1.95 Develop. Trunk .8996 1.05 .46 - 2.40 Inguinal .4949 .50 .07 - 3.70 Depth Dermis/reticularis .9918 - — only Dermis to .4855 1.35 .58 — subcutaneous 3.12 tissues Subcutaneous .6075 .68 .16 - tissues only 2.92 Skeletal muscle .7574 1.16 .48 - layers 2.83 Death due Locat. Head-neck .2958 1.95 .56 - to MCT 6.86 6O Table 6: Unviariate analysis of risk factors for MCT OLICCXDHKBS (significant @ p < 0.3) \ (Jutx30me Risk Factor Chi-square Odds Ratio 95% C.I. P Age .1282 1.98 .82 — 4.76 ‘ch:a]_IMCT Golden .1286 1.46 .18 — 11.77 Development Retriever _ Male .2815 2.07 .55 - 7.74 Location: .2956 2.32 .48 — 11.19 head-neck Animal age .0503 1.70 1.00 — 2.91 Mixed Breed .2268 .47 .14 — 1.59 Distxnnt MCT Boxer .2768 1.82 .62 — 5.36 Dexmilopment Sterilized .2165 .51 .17 - 1.49 Location: .0332 2.94 1.09 — 7.95 head-neck Age .0200 2.28 1.13 — 4.37 Male .0086 4.56 1.47 - 14.16 Sterilized .0138 .26 .09 - .76 Depth: .1711 1.96 .74 — 5.37 Skeletal Death due muscle to MCT layér Location: .2958 1.95 .56 — 6.86 Head-neck Location: .1678 .41 .12 — 1.45 Extremities Location: .1312 2.64 .75 - 9.34 Inguinal 61 Table 7% Reduced* multivariate proportional hazard regression model for survival analysis for days to distant lflfl‘devehnmwnt after the original diagnosis. * hfltial model contained age, sex, sterilization, breed, Imfltiphelesions, tumor location, and tumor depth [ Risk Factor Wald x2 p Hazard Ratio 958 C.I. (Location: head-neck” 3.53 0.0601 3.33 0.95—11.69 [ Age 5.98 0.0145 2.16 1.17—4.01 [ Boxer 3.89 0.0487 2.92 1.01—8.49 = 18.98, 5 d.f., p = 0.0019 Model Likelihood Ratio X; a — Retained in model to control for confounding Reduced* multivariate proportional hazards Table 8: regression model for survival analysis for death. * — Initial model contained age, sex, sterilization, breed, multiple synchronous tumors, tumor location, and tumor depth Risk Factor Wald X2 p Hazard 95% C.I. [ Ratio f Age 12.31 .0005 3.34 1.70 - 6.55 Male 5.42 .0199 2.85 1.18 - 6.89 Multiple 6.51 .0107 4.60 1.42 - 14.83 synchronous [ tumors Location: 3.50 .0613 3.93 .94 - 16.43 [ head-necka Model Likelihood Ratio X2 = 39.94, 7 d.f., p < .0001 a — Retained in model to control for confounding 62 CHAPTER 2 iNerxsterr JD, Kiupel M, Kaneene JB, Miller RA, Yuzbasiyan- Gurkan V (2004). Use of KIT and tryptase expression 'panrterjis as prognostic tools for canine cutaneous mast Geld. tunmms. Veterinary Pathology. 41:371-377. 63 CHAPTER 2 USE OF KIT AND TRYPTASE EXPRESSION PATTERNS AS PROGNOSTIC TOOLS FOR CANINE CUTANEOUS MAST CELL TUMORS Introduction Canine cutaneous mast cell tumors (MCT) are one of the most (xxmmni neoplasms 111 dogs, accounting 1km? 7—21% of all cutaneous tumors 1J1 dogsh4. The biological behavior evaluate the prognostic significance of KIT and tryptase immunohistochemical staining patterns in canine cutaneous mast cell tumors. The KIT protein is a tyrosine kinase receptor that is ea product of time c—KIT proto-oncogeneqh which is expressed in numerous tissues including glioblastoma cells, term pdacenta, brain, erythrobd precursors, melanocytes, basophils and mast cellsngmLhfl. The ligand for the KIT receptor, stem cell factor (SCF), also called mast. cell growth. factor, has ]flUltipl€* effects (n1 mast cells including proliferation, maturation, migration, degranulation, suppression. constitutively activate the KIT tyrosine kinase despite time absence of time SCF 65 F ligandgl'gg’. It has been proposed that mutations in the jinctamembrane domain. may' play' a (critical role in the neoplastic transformation of mast cells tumors in 31f. dogsghgms Sui one study, mutations III the juxtamembrane domain were more prevalent in histologic grade II and III MCTs compared to grade I MCTsC’B. In another study, MCTs with tandem duplication mutations were twice as likely to recur and twice as likely to metastasize as those without the mutation, although the association between recurrence and metastasis, and the presence of the mutation was not statistically significant”. Expression of the KIT receptor in MCTs, and the detection.lM3T and death due to any cause. I_ Local 1 Death due to MCT Death due to l KIT recurrence ; any cause I Staining 0‘ Hazard. “V Hazard Hazard 1 Pattern p—value ratio! p~value ratio p-value ratio Staining i Pattern 0.0079 35.82 0.0056 20.10 0.004 7.52 Staining Pattern 0.0131 51.14 0.0004 76.54 0.0192 6.03 III 84 Table 1J3 Distribution (If recurrent between ICUF protein. localization and the presence of c-KIT mutations exists in canine MCTs. These data clearly implicate an important role of c—KIT in 90 the pror high prr (11 “U 'U (I) (‘1) '1 (I) 1:"? ‘1va m:| {13:05.1 {-1 Vase 3.: ‘ g\_,_‘ A Cl): allcq'r‘ltr; \v the progression of canine cutaneous MCTs. Considering the high prevalence of MCTs in dogs and the central role c-KIT appears 11) play iii the tumorigenesis of rmnnv canine MCTs, canine cutaneous MCTs pmovide 6N1 excellent spontaneous in vivo model for studying the molecular biology of ceKIT in human anmi animal neoplastic diseases. Enrthermore, canine cutaneous MCTs are an excellent model for the treatment of cancers that are driven by c—KIT and can be used in clinical trials for testing chemotherapeutics aimed at targeting the c-KIT proto-oncogene. Materials and Methods Case selection, tissue samples, and survival data Sixty canine cutaneous MCTs from. 60 different dogs submitted to Michigan State University’s Diagnostic Center for Population and Animal Health between 1998 and 2001 were included iii this study. Cases vmnme included iii this study solely based on the meeting of all inclusion criteria. Inclusion criteria for this study were as follows: 1. all cases were previously diagnosed as canine cutaneous MCT. The diagnosis of canine cutaneous mast cell tumor and the histologic grade of each tumor was confirmed by a veterinary pathologist. 2. all cases were treated with surgical excision as time only primary treatment modality; i.e., IN) chemotherapy (n: radiation therapy imms used. 3. 91 . i z . C e S e .5 1 mt. S . f F . i . d - i L a i I e r .C u Q I O mot r L t O. Q m .. S .1 I S Q. 3 a ,. .4 .3 r S a no r e. a e W .1. S a e S r T. a n r m. . S Dr S Moi acre 1.. t .. i v c, a u u e c u C i e a s i 3 r t S r o 3 C C Vi. h r L .. i s .1. mo. complete follow—up data from the referring veterinarian was available. 4. adequate formalin—fixed paraffin embedded tissue for DNA extraction and immunohistochemistry was available. Complete follow—up data for each case included age, sex, breed, weight, number of masses, location of mass, time before excision, medication at the time of surgery, diagnostic tests timfl: were performed, recurrence, tumor margins, metastasis, survival time and cause of death. Histologic grading of canine MCTs was performed in conjunction with a Hmlti-institutional review of the current histologic grading system for canine cutaneous MCTs, iii which Z11 pathologists participated iii the histologic grading CM? 95 canine MCTSLQ Histologic grades represent a consensus of those results. Laser capture microdissection and DNA extraction Laser capture microdissection (LCM) was used to isolate neoplastic mast cells for DNA extraction and subsequent PCR amplification of c-KIT exon 11 and intron 11 in order ti) identify IlT) CaKIT mutations. Five to '7 um sections of each fOrmalin-fixed, paraffin-embedded MCT was dehydrated eumi stained vniii hematoxylin 1mm? laser capture microdissection. Two-thousand to four—thousand neoplastic mast cells were extracted from each tumor sample using the Pixcell laser capture microdissection system with Macro LCM 92 X *n m U) 33 H FJ. Fl) luv (I) -nClLJ protc— caps (Arcturus, Mountain View, CA) (Figure 6). Extracted cells adhered to the Macro LCM caps were incubated overnight in 50 ul of DNA extraction buffer (10 mM Tris pH 8.0, 1 mM EDTA, 1% Tween) and 1.5 ul of 15 mg/ml Proteinase K (Roche, Indianapolis, IN) at 37°C. Samples were centrifuged at 14306 )< g fOr 55 minutes, and Pioteinase K was inactivated by heating at 95°C for 8 minutes. PCR amplification of c-KIT exon 11 and intron 11 Polymerase chain reaction (PCR) amplification was performed using 51 previously described. primer“ pair that flanks exon 11 and the 5’ end of intron 11”°, which includes the pmeviously described ITD region of time c—KIT proto—oncogene in canine MCTslTlm9b9&gmlm”H°. Polymerase chain. reactions vmnme prepared :hi a. 25 in. total reaction volume, with 5 ul LCM extracted DNA, 5 pmol of each primer, 0.5 units of Taq polymerase (Invitrogen, Carlsbad, CA), and final concentrations of 80 uM dNTPs, 2 mM MgC13, 20 mM Tris- HCl, and 50 ul KCl. Cycling conditions were as follows: 94°C for 4 minutes; 35-45 cycles at 94°C for 1 minute, 55°C for 21 minute, and 72°C tin: 1 minute; 72°C for 53 minutes. Amplified. products and ZEN) Hmtations were 'visualized. by agarose gel electrophoresis on a 2% agarose gel after ethidium bromide staining (Figure 7). 93 \e . 4:. erl‘L‘ 4 f: .1: Top-"I DNA p u r . i d . la a IFL m a c a x M. a c r . a a t l p. U3 n o C .1 Hi we r C r a .. C e e M .1 u o e a w C +1 .3 e . C .q m. i i u c a m in a a. a. a e .e r e i F S C D p S I... we 8 W a DNA sequencing Mutant c-KIT alleles were identified by agarose gel electrophoresis and DNA fragments were excised for DNA purification. DNA was purified using the Qiaex II gel purification. kit (Qiagen, ‘Valencia, CA) according ti) the manufacturer’s protocol. DNA fragments were subcloned into Topo vectors using the Topo cloning kit (Invitrogen, Carlsbad, CA) and subsequently chemically transformed into competent E. (miLi cells according' to time manufacturer's protocol. c—KIT’ clones were sequenced either using an automated sequencing technique using fluorescently labeled dideoxy—nucleotides, with capillary electrophoresis and detection using an ABI sequence analyzer (Foster City, CA) at Michigan State University's Genomics Technology Support Facility, or by manually sequencing with the Thermo Sequenase Radiolabeled Terminator Cycle Sequencing kit (USB Corporation, Cleveland, OH) and 33P-labelled dideoxy- nucleotide triphosphates according to the manufacturer's protocol, followed by 48—72 hr exposure to Biomax MR Scientific Imaging Film (Kodak, Rochester, NY). Immunohistochemistry Tissue sections of canine cutaneous MCTs were used for immunohistochemical evaluation (n5 KIT protein localization as previously' described“”. In. brief, 5 run sections of 94 formalin—fixed paraffin—embedded tissue were deparaffinized in xylene, rehydrated in graded ethanol and rinsed in distilled water. Endogenous peroxidase was neutralized with 3% hydrogen peroxide for 5 minutes. Antigen retrieval was achieved by incubating slides in a citric buffer antigen retrieval solution Ukflqh Carpinteria, CA) in 51 steamer (Black 6; Decker, Towson, Inn for 20 nUin and non-specific immunoglobulin binding was blocked by incubation of slides for 10 min with a protein—blocking agent (Dako, Carpinteria, CA). Using an autostainer, slides were incubated. for 1M) minutes ‘with 51 rabbit anti-human c—KIT antibody (Dako, Carpinteria, CA) at airiilution of 1:100. A. streptavidin-immuno peroxidase staining procedure (Dako, Carpinteria, CA) was used for immunolabeling. The immunoreaction was visualized with 3,3’-diaminobenzidine substrate (Dako, Carpinteria, CA). Sections were counterstained vaii Mayer’s hematoxylin. Positive and negative immunohistochemical controls were included in each run. Known canine MCTs were used as positive controls. Negative controls were canine MCTs that were treated identically as routine sections, except the 30 minute incubation with primary antibodies was replaced with a 30 minute incubation with buffer. IKLT staining patterns and protein localization for each MCT was characterized as 95 ing nvest ‘— ‘ D e s E . e A. c .1 . r... . a .. E C E f e. . e S E cit d a _ nu . . l e L . H A J. . AL C. L A; i» r . l 1 +1.. 5 9 d m“ t at t a e r a: . i a . . AC G . . r H S D .3 I e a C n f e D. .3 5 m- i S Dr A c . . C I 3 S O being peri-membrane (KIT staining pattern I), focal or stippled cytoplasmic (KIT staining pattern II), <1r diffuse cytoplasmic protein localization (KIT staining pattern III) as previously describedmg (Figure 8). The evaluation of KIT protein localization was performed by' 51 single investigator (JDW) in order to eliminate inter—observer variability. Tissue microarray and immunofluorescence One millimeter cores that were microscopically selected to be representative of each tumor were taken from paraffin—embedded MCT tissue blocks and were placed in a common recipient paraffin block. Mast cell tumors included in the tissue array were chosen based on the availability of tissue for transferring ti) the recipient block. This resulted in 42 MCT samples from 42 cases being represented on the tissue microarray. The recipient block was subsequently' heated at 37°C for approximately 1-hour in order to create a cohesive block. Five micrometer sections were cut and deparaffinized in xylene, and subsequently dehydrated in graded alcohol with a final rinse in distilled water. Twenty minute steam retrieval in a citric buffer solution (Dako, Carpinteria, CA) was used for antigen retrieval. Non-specific antibody binding was performed with 5% donkey serum with blocking buffer. 96 Slides were incubated with primary rabbit anti-human c-KIT (Dako, Carpinteria, CA) antibodies at ea dilution of 1:100 overnight in a humidity chamber at 4°C. Sections were then incubated with Cy-3 labeled secondary antibodies and nuclei were counter stained with 4',6-Diamidino-2-phenylindole (DAPI). Mean immunofluorescence was quantified for each tumor sample using a Perkin Elmer Scan Array (Perkin Elmer, Wellesley, MA). Statistics Univariable Analyses: Before developing multivariable models, each risk factor was evaluated for its association with MCT outcomes. Univariable proportional hazards model were developed for each risk factor for each outcome, and the level of association was assessed through the risk factor’s p—value iii the model. Risk factors with p) less than or equal to 0.20 were considered for inclusion in the multivariable model, which included the tum) variables, 0— KIT mutation status and KIT staining patterns. MUltivariable Logistic Regression .Models: Logistic regression 'models were developed for the occurrence of outcomes associated with MCTs, including recurrence of local MCTs, occurrence of kxe between 9—15% iii all MCTs. However these mutations may occur in as many as 50% of high grade canine MCTs““”. Previously our laboratory has shown that increased cytoplasmic KIT protein localization in neoplastic mast cells is associated with.kxflii a decreased disease—free and overall survival of dogs with cutaneous MCTsl'E’g. In this 104 H I e d S u e e o r S D. -‘—‘ ..‘. 1- en: "‘ curren subset .r. E a c r c C a mi Dr C v..* H..." Aiv 7.... .5 .. i a. C C .C S S a r c a r .l u h C U, Q.» 1‘ r Cu study, we identified a significant association between the presence of ITD c—KIT mutations and changes in KIT localization iii canine cutaneous MCTs. Seven CH? 9 MCTs with c—KIT mutations had aberrant KIT protein localization. Although the significance of this relationship is not currently clear, this may suggests that ITD c-KIT mutations may be responsible for aberrant KIT localization in a subset of canine MCTs. Two cases with c-KIT mutations did not have aberrant KIT localization, and remain as outliers to this hypothesis. However, the mutation in one of these MCTs was located within intron 11 only, and therefore could be spliced out during mRNA processing and may not be biologically significant (case 9). It is also important to note that the dog with the intronic c-KIT mutation (case 9) was still alive with no report of local or distant recurrence at 2N) months post—surgery. Furthermore, significant statistical relationships between ITD c-KIT mutations and both the incidence of (p=-0.0052) and time until MCT-related deaths (p=0.0267) are preserved when this mutation. is rmm: considered..as E1 biologically' significant mutation. A potential explanation for the absence of cytoplasmic KIT localization ii1 the other DKHT that had EH1 ITD c—KIT 105 TD T .L I a C E E: E 1'1 .0... . ‘ 1A ilk; 1V1. Y a e t . h r T t a a a s t C 1 l .h. to E C I o i t 3 t A- r C E m o . .... S E f .H S O . - i .Q a .. t r. . e r e r t I o. .t. e r . .1. h . Via a a an a.» . a a h A: QV e Ti m... .0 pl“ 4 a C. 4N“ .G C L ...W P. re .4 a 15 Rd “F“ mutation may be that this tumor only recently acquired the mutation, and the changes in KIT localization may not have occurred yet EH: the time CM? surgical excision. However, ITD c—KIT mutations and changes in KIT localization may represent separate events that occur independent of one another ii1 the progression of canine cutaneous MCTs. This hypothesis is supported by the fact that 26 MCTs included in this study' had. aberrant KIT localization. without the presence of ITD c-KIT mutations. This data (inflii also indicate that 111 addition ti) a direct causal relationship> between. the ITD Imitations and aberrant KIT localization, other factors may be responsible for aberrant KIT localization in canine cutaneous MCTs without ITD c—KIT mutations. The primers that were used in this study do not allow for the detection of the previously reported deletions in canine MCTs, since the forward primer is located in the region of c-KIT that has been reported to 3 .C) , 9 (‘3 be deleted in a small subset of canine MCTs“ Therefore, although rare, (HimH: c-KIT mutations enmi1 as deletions in the juxtamembrane domain may be responsible for the aberrant. protein. localization iii those «cases 111 which vme did not identify ITD c-KIT’ mutations. In summary, the correlation. between. ITD c-KII’Imitations EHmi aberrant iKIT localization leads to many interesting questions regarding 106 overa- I .c l n E I .n u H O 9 ff 5 e t b K'. -I‘» T. -. i. e we. . i n o a I c C C Cu .1 1i .d m blgnali Pd 0 i... .c a c. . 1.1. . .1.o . 11* e by .. r k: b ‘ 1C n 1‘ hr... )4 Q .3 cc .1 11 .Q . W S We h ha 0“ in. o G a U c 1 . l. C .C C C A O S the functional significance (if this relationship EHmi the overall functional significance of aberrantly localized KIT when ITD c—KIT mutations are not present. Current work in our laboratory is focused (H1 the further characterization of aberrantly localized KIT and (H1 functional studies to better elucidate the :relationshit> between. ITD c-KIT mutations and the aberrant localization of KIT. No significant relationship vmms found :ni this study between the presence of ITD c-KIT mutations or the aberrant localization of KIT and the level of KIT protein expression as measured by moan ihmmnofluorescence ii1 a tissue microarray. These results suggest that constitutive activation of KIT due to ITD mutations or changes in signaling pathways through aberrant KIT localization may be more important in the pathogenesis of canine MCTs than over—expression of KIT and subsequent increases in receptor sensitivity to its ligand. In order to clarify these observations, these results need to be verified using additional techniques to quantify' KIT protein levels in canine IMCTs. Additionally, further studies need ti) be conducted in order to elucidate the functional significance of aberrantly localized KIT and the effects it has on signaling in neoplastic mast cells. 107 Spontaneous neoplastb: diseases EHme commonly seen iii dogsL3, and in many cases share similar morphologic, clinical, and molecular characteristics to human neoplastic diseases. Therefore, these tumors are an excellent in vivo model of spontaneous neoplasia that may' be utilized to better understand the roles of various genes and proteins in the progression of neoplastic diseases, and to serve as model systems for testing the safety and efficacy of novel - 1 :3": , l 1') therapeutic agentsH 1. Canine cutaneous MCTs are one of the most common neoplasms in dogs and, unlike human mastocytomas, often have an aggressive behavior that can result in death. Due to the high incidence of canine MCTs, and the central role that c-KIT plays in MCT tumorigenesis, canine MCTs can serve as an excellent in vivo model for studying its role in time progression. of this and. other human and animal neoplastic diseases. We propose canine MCTs as ea spontaneous .hi vivo model :fin: clinical trials aimed at determining the safety and efficacy of novel targeted chemotherapeutic agents involving c—KIT signaling pathways. 108 Figure 6: Laser capture microdissection of neoplastic canine cutaneous mast cell tumors (magnification: 10X). Laser capture microdissection (LCM) was performed using archival formalin—fixed paraffin—embedded tissue sections. DNA was extracted from captured cells and PCR amplification was performed in order to identify c-KIT mutations. A: Hematoxylin stained section of MCT prior to microdissection. B: Section of MCT following microdissection. C: Laser capture microdissected cells adhered to cap. 109 Figure 7: % agarose gel of PCR amplified C-KIT exon 11 and intron 11 from LCM extracted DNA from canine MCTs. L: 100 bp ladder; M: heterozygous for normal allele (19lbp) and mutant allele (250bp) with a upper band representing heterodimerization of normal and mutant alleles; N: 191 bp homozygous normal allele; NC: negative control (no template). 110 Figure 8: Sections of canine cutaneous mast cell tumors (skin) stained with anti~KIT antibodies and counterstained with hematoxylin (magnification: lOOX oil) representing three patterns of KIT localization identified in neoplastic canine mast cells. A: KIT staining pattern I, consisting of peri—membrane protein localization with little to no cytoplasmic protein localization; B: KIT staining pattern II, consisting of focal to stippled cytoplasmic staining; C: KIT staining pattern III, consisting of diffuse cytoplasmic staining. 111 __L _. 1.00 H .2 E 3, 0.75 3 8 g 0.50 3 U .5 o 0.25 > in 2 . . .. E 0.00 . .............. .t .................. ‘. ................ , ..... ‘ _ ‘ ' 0 10 20 ' 30 ' .40 50 . Time (months) ‘ ;.;.;.;.;. c-K’T Mutation '- n0 Mutaflon Figure 9: KaplaneMeier Survival Curve: Relative frequency of survival vs. time in months for canine cutaneous mast cell tumor patients with and without identified c—KIT mutations. The presence of duplication mutation in the c- KIT proto-oncogene was significantly associated with a decreased survival duration (p= 0.0068, HR: 6.23 (1.66— 23.40)). 112 Relative Frequency of Mutations 40 35~ 30- 25- 205 15* 10 L ‘ 2 3 a--- KIT Staining Pattern Figure 10: Correlation between ITD c-KIT mutations and KIT protein localization in canine.MCTs. A significant association was found between the presence of c—KIT mutations and the cellular localization of KIT in canine MCTs (p= 0.046). 7/9 (77.8%) of MCTs with ITD c-KIT mutations had aberrant KIT localization in neoplastic MCTs. 113 Table 12: Mutation and case description for cases with ITD c-KIT mutations. Case Size Location KIT Local Distant MCT— No. (bp) Grade‘ Staining Recur. Recur. related Patternt (months (months Death ) ) (months) 1 45 Exon 11 3 3 None None 0.5 2 45 Exon 11 2 2 None None None at 29.1 3 45 Exon 11 3 3 None 0.5 0.5 4 45 Exon 11 2 2 0.5 0.5 0.5 5 60 Exon 11/ 3 2 1 1 1 Intron 11 6 54 Exon 11/ 3 3 2 2 3 Intron 11 7 60 Exon 11/ 3 3 None 0.6 0.6 Intron ll 8 57 Exon 11/ 2 1 None None None Intron 11 at 7.3# 9 15 Intron 2 1 None None None 11“ at 20.4 114 Histologic grading was performed based on the Patnaik histologic grading system for canine cutaneous MCTs (45). KIT staining patterns were classified as described by Webster et al., 2004 (36). i Dog no.8 died at 7.3 months due to causes unrelated to mast cell disease. Mutation in dog no. 9 consisted of a 24bp poly-T insert with a 15 bp duplication, which was located entirely in intron 11. An additional A to G transition was also identified in the duplicated sequence preceding the poly—T insert. 115 CHAPTER 4 Webster JD, Kiupel M, Yuzbasiyan-Gurkan V (2006). Evaluation of the kinase domain of c—KIT in canine cutaneous mast cell tumors. BMC Cancer 6:85. 116 CHAPTER 4 EVALUATION OF THE KINASE DOMAIN OF c-KIT IN CANINE CUTANEOUS MAST CELL TUMORS Introduction The c—KIT proto-oncogene encodes the type III receptor tyrosine kinase KIT, which consists of an extracellular ligand binding domain, a transmembrane domain, a negative regulatory juxtamembrane domain and a split kinase domain”— (Figure 11). In healthy humans as well as in dogs, c—KIT is expressed by multiple cell types including mast cells, germ cells, melanocytes, and hematopoietic precursor cells 1“”lw'””“l”1”fl Notably, iii:mast cells ICUF and iii; ligand stem cell factor (SCF, also known as mast cell growth factor)””“ I 11"» l"—.1.) have been shown to be involved in cell survival, proliferation, differentiation, chemotaxis, ””411 In human degranulation, and fibronectin adhesion patients, mutations in time c—KIT proto-oncogene have been implicated in the pathogenesis of multiple neoplastic diseases, including mastocytosis, germ cell tumors, and gastrointestinal stromal tumors (GISTs) 1M'MZJZL1“““°”°°. The locations of these c—KJU‘ mutations vary' between the different. neoplastic: diseases. The ‘vast imajority <5f mutations characterized in human patients with mastocytosis 117 occur at codon 816 in exon 17, which encodes a portion of the kinase domain of KITLB'lM'UF’. Mutations in germ cell tumors have been found in both the juxtamembrane domain and the kinase domain (Hf c-KIT”°JEL while mutations iii GISTs tend to occur in exon 11 of the juxtamembrane domain of c- KITK°”“”“”. Despite their variation in location, both juxtamembrane domain and kinase domains c—KII’ mutations result in a constitutively activated KIT protein that is phosphorylated in the absence of the ligand”°”2%13“u°. In recent years, c—KII’ has been implicated in the pathogenesis (Hf canine cutaneous mast cell tumors (MCTs), which are one of the most common neoplasms in dogs“°. Internal tandem duplications (ITDs), deletions, and point mutations have been identified in the juxtamembrane domain of c-KII' in canine cutaneous MCTsM'%’%. The reported incidence (Hf c-KIT mutations has varied between different studies. In two studies that screened randomly selected cases submitted, mutations were identified in 15% of canine ‘l 4‘ l MCTsT’“;. However, another study reported c—KIT mutations in 50% of canine MCTs that were seen in a referral oncology practicel°. This discrepancy in the incidence of c—KIT mutations in canine MCTs is most likely due to the variations in case selection between these studies with the higher percentage reflecting ascertainment bias at the 118 referral practice. Internal tandem duplication c—KIT mutations EHme the most commonm””°, and therefore the most extensively studied c-KIT mutation in canine MCTs. All of the ITD mutations that have been studied thus far have been found ti) produce E1 constitutively activated product, thereby implicating c—KIT in the progression of canine MCTsM'W””°. Previous work by our laboratory has shown that c—Kli’ mutations are significantly associated. with. higher histologic grade MCTsQ‘If', and that MCT patients with ITDs have a significantly worse prognosis as compared to patients without ITD mutations, thereby further implicating c-KIT in the progression of canine MCTSNH. In addition to c—KII' mutations, aberrant KIT expression and more specifically, the aberrant localization of KIT has been described in canine cutaneous MCTszmTlm. Recently (nu: laboratory 1mm; identified 13 patterns (If KIT protein localization iii canine MCTs: 21. peri-membrane KIT localization UCUP pattern ll; 2. cytoplasmic stippling to focal ICUF localization UCUF pattern 2); 3. diffuse cytoplasmic KIT localization (KIT pattern 3), as shown in Figure 12. Recent studies have shown that canine MCTs that have 53 primarily cytoplasmic pattern (Hf KIT protein localization UCUF patterns 2 EHmi 3) have E1 significantly worse prognosis, in terms of both their disease—free 119 interval EHmi survival duration, EH3 compared ti) MCTs that have Ea primarily peri-membrane pattern (if KIT localizationfia. Furthermore, we tmnme found that ZEN) c—KIT mutations in canine MCTs are significantly associated with aberrant ICUF localization iii neoplastic IMHH: cells. However, a substantial number of canine MCTs have aberrant KIT localization, but do rmH: have ITD imutationslafl This suggests that additional factors, aside from ITD c—KIT mutations may be responsible for aberrant KIT localization in neoplastic mast cells. Despite the high incidence of c-KIT kinase domain mutations in human patients with mastocytosis, only a total of 18 canine MCTs and 3 canine MCT cell lines have been QLQ° For a evaluated.:flir kinase (fiflMIhl mutations iii c—KIT more detailed evaluation of possible c—KIT kinase domain mutations ii1 canine MCTs, vme screened exons 16—20 (Hf the phospho-transferase portion of time kinase domain CHE c—KIT for" mutations that. may' contribute ti) the progression. of these tumors. Additionally, in order to determine if mutations in exon 17 of c—KIT, where the majority of mutations occur in human mastocytosis patients, were responsible for aberrant KIT localization, exon 17 was screened :U1 18 MCTs with. aberrant KIT localization. that lack ITD c-KIT mutations. However, no mutations or 120 polymorphisms were identified in exons 16-20 of any of the canine MCTs that were examined. Materials and Methods Study Population Samples fromi 33 canine cutaneous IMCTs fromi 33 dogs were obtained. from. archival formalin-fixed. paraffin- embedded tissues that had been submitted to the Diagnostic Center for Population and Animal Health at Michigan State University. Single paraffin tiocks vnii1 neoplastic tissue were selected. for each tumor EHmi each tumor ‘was histologically graded based on the Patnaik histologic grading system” for canine cutaneous MCTs. DNA Isolation from formalin-fixed paraffin embedded tissues Tissue samples for DNA isolation were selected within the tumor boundaries, as identified by histologic evaluation. Approximately 2 mm5 tissue samples were obtained from each block for DNA extraction. DNA was isolated as previously described“”“”. In brief, 400 in. of digestion buffer (50 mM Tris, pH 8.5, 1 mM EDTA, 0.5% Tween) was added ti) each sample. Samples were heated ti) 95%: for 10 minutes followed tux heating iii a microwave EH: full power twice for 30 seconds. Samples were thoroughly vortexed between each heating step. Five ul of 15 mg/ml proteinase K was added to each sample, and the samples were subsequently 121 incubated overnight EH: 42°C. Proteinase I< was inactivated EH: 95%: for 10 mdnutes. Samples were then centrifuged and 200 pl of the reaction was aliquoted for use as template in PCR. Amplification of c—KIT exons 16—20 PCR amplification was carried out using primer pairs that flanked exons 16, 17, 18, JEL EHmi 20 in order to sequence each exon iii its entirety and time 10—20 nucleotides that flank each exon (Table 13, Figure 13). Twenty-five microliter PCR reactions were prepared with Eiiil DNA at a 1:25 dilution, 5 pmol of each primer, 0.5 units of Taq polymerase (Invitrogen, Carlsbad, CA), and final concentrations of 80 uM dNTPs, 2 mM MgC12, 20 mM Tris—HCl, and 50 ul KCl. Cycling conditions for amplifying exons 16, 17, 18, and 20 were as follows: 94°C for 4 minutes; 40 cycles EH: 94%: for 1 mdnute, 60°C tin: 1 minute, and 72°C for 1 mdnute; 72°C tin: 5 minutes. Cycling conditions were similar for EHKH1 19, except E1 54°C annealing temperature was used instead of a 60°C annealing temperature. Amplified products were Visualized by agarose gel electrophoresis on a 2% agarose gel stained with ethidium bromide. Sequencing of c—KIT exons 16—20 Amplified products were gimflimi in groups (Hf 7-10 for DNA sequencing whenever possible, Ems previously described 122 1“}. This pool EHmi sequence method has rmmHi shown ti) allow for the detection of minor alleles at frequencies as low as 5%. If clean sequences were not obtained from pooled samples, then samples were sequenced individually. DNA to be sequenced was separated on a 2% agarose gel, and DNA fragments were excised for DNA purification. DNA was purified using time Qiaex 11: gel purification idi: (Qiagen, Valencia, CA) according to manufacturer’s protocol. DNA sequencing was carried out using the Thermo Sequenase radiolabeled terminator cycle sequencing kit (Amersham, Piscataway, NJ), fOIlowing manufacturer’s instruction. Sequence reactions were separated on a 6% denaturing polyacrylamide gel, vflmii1 was dried EHmi exposed ti) BioMax MR scientific imaging tfiimi (Kodak, Rochester, DUN for 72 hours for visualization. DNA isolation from laser capture microdissected tumor samples Eighteen Pain; with aberrant KIT localization, but no ITD c—KIT mutations were identified as rmni:< 10 mm grid area was counted using a 1 cm: 10 x 10 grid reticle at 400x magnification. The number of immuno-positive cells per grid area was evaluated over 5 high powered fields and subsequently averaged in order to obtain an average S-phase index III the case of PCNA immunostaining, and the proliferation index in the case of Ki67 immunostaining. Evaluation of AgNOR histochemical staining In order to determine the average AgNOR count/cell in each tumor, AgNORs were counted in 100 randomly selected neoplastic mast cells throughout the tumor at 1000x magnification. Individual AgNORs were resolved by focusing up and down while counting within individual nuclei. Average AgNOR counts/cell was then determined based on averaging the counts within these 100 random neoplastic cells. Laser capture nficrodissection euui analysis CM? c-KIT mutations Laser capture microdissection (LCM) vuus used to isolate neoplastic mast cells for DNA extraction and subsequent PCR amplification of c-KIT exon 11 and intron 11 in order to identify ITD c—Kll’ mutations as previously 146 describedmg. Five to 7 um sections of each formalin-fixed, paraffin—embedded bflflk; were stained vnlji hematoxylin. and dehydrated in graded alcohol for laser capture microdissection. Two-thousand to four-thousand neoplastic mast cells were extracted from each tumor sample using the Pixcell laser capture microdissection system with Macro LCM caps (Arcturus, Mountain View, CA). Extracted cells adhered to the Macro LCM caps were incubated overnight in 50 ul of DNA extraction buffer (10 rml Tris gfli 8.0, 1 IM4 EDTA, 1% Tween) and 1.5 ul of 15 mg/ml Proteinase K (Roche, Indianapolis, IN) at 37°C. Samples were centrifuged at 1,306 >< g for 53 minutes, anui Proteinase I<‘was inactivated by heating at 95°C for 8 minutes. PCR amplification of c—KIT exon 11 and intron ll Polymerase chain reaction (PCR) amplification was performed using a previously described primer pair that flanks exon 11 and the 5’ end of intron 11M6, which includes the previously described ITD region (H? the c—KIT . . so 0:0"15, *, proto-oncogene in canine MCTle‘L‘”‘m 4 ”ble. Polymerase chain. reactions vmnxa prepared :hi a. 25 in. total reaction volume, with 5 ul LCM extracted DNA, 5 pmol of each primer, 0.5 units of Taq polymerase (Invitrogen, Carlsbad, CA), and final concentrations of 80 uM dNTPs, 2 mM MgC13, 20 mM Tris- HCl, and .50 in. KCl. Cycling' conditions ‘were an; follows: 147 94°C for 4 minutes; 35-45 cycles at 94°C for 1 minute, 55°C for 21 minute, and 72°C fin: 1 minute; 72°C fer 55 minutes. Amplified. products enui III) mutations were visualized. by agarose gel electrophoresis on a 2% agarose gel after ethidium bromide staining. Statistical Analysis Univariable Analyses: Before developing multivariable models, each risk factor was evaluated for its association with MCT outcomes. Univariable proportional hazards model were developed for each risk factor for each outcome, and the level of association was assessed through the risk factor’s p—value III the model. Risk factors with p) less than or equal to 0.20 were considered for inclusion in the multivariable model. MUltivariable Logistic Regression .MOdels: Logistic regression models were developed for the occurrence of outcomes associated with MCTs, including recurrence of local. MCTs, recurrence of «distant IMCTs, and. death associated with. MCTs. In addition to risk factors of interest, animal signalment. (age, sex, ‘weight) were included in the multivariable model to account for their effects (n1 model outcome. Results were reported an; odds ratios: an odds ratio less than one means the likelihood of the occurrence of an event is reduced, while an odds ratio 148 greater than (MK? indicates the likelihood of en1 event is increased” Odds ratios equal tx> 1 indicate that tjma risk factor' neither increases run: decreases the .likelihood account for tjmdr effects (n1 model outcome. Tflma effects cu? risk factors on days to events was reported as hazard ratios. Comparable to odds ratios, hazard ratios less than one indicate that the risk factor increases time to outcome, while hazard ratios greater than one indicate that the risk factor decreases time to outcome. 149 Results Eighteen dog breeds were represented in this study including 12 maxed tweed.ckxfiL 12 Labrador retrievers, 10 boxers, 6 golden retrievers, 2 fflflfihr 2 basset hounds, and 12 additional breeds that were represented by single dogs. The median age of the dogs included in this study was 7.93 years and ranged from 2 tx>lb4 years of age. Thirty-three female and 23 male dogs were included in this study. Eight of the fifty—six MCTs included in the study were histologic grade 14 41 MCTs were histologic grade 2 anui 7 MCTs were histologic grade 3. The average PCNA counts of the MCTs included in this study ranged from 6.00 to 243.20 positive cells per grid area vnlfli average count CM? 64.94 positive cells/grid area and a median of 44.50 positive cells. AgNOR counts ranged from 1.25 AgNORs/cell to 4.05 AgNORs/cell, with an average count of 2.29 AgNORs/cell and a median count of 2.21 AgNORs/cell. Ki67 counts ranged from 13 IX) 97 positive cells/grid area, vniji an average (n5 24.66 positive cells/ grid area and a median Ki67 count of 17.10 cells. The prognostic significance of Ki67, PCNA, and AgNOR counts were first evaluated ij1 a univariable statistical analysis in order to identify potentially significant variables for inclusion in a multivariable 150 I . statistical model controlling for age, gender, and breed (Tables Ih4 and 15). Additionally, since innmnr growth is determined by both the number of proliferating cells within a given tumor and tjma rams of cellular proliferation%7m, derived. variables were created. by' multiplying" the .AgNOR counts by either" the Ki67 counts (Ag67) or PCNA. counts (AgPCNA) 111 order tx> determine time utility (If evaluating the proportion of cycling cells, as measured by either the proliferation index (Ki67 count) or S—phase index (PCNA count), and the rate of cellular proliferation as measured by AgNORs in concert. According to multivariable analysis increased. Ki67 counts were significantly associated. with both EH1 increased incidence and Ixnxa of subsequent tumor occurrence an: the original surgical site (p=0.0111; p=0.0017, respectively), an increased rate of MCT occurrence an: sites distant from time original tumor site (p=0.0081), and an increased incidence of MCT-related mortality (p=0.0022). Increased. .AgNOR counts were significantly associated. with. both. an increased rate of subsequent tumor occurrence at 13K; original surgical site and at sites distant from the original surgical site (p=0.0435; p=0.0121, respectively), and vniji an increased incidence and rate of MCT-related mortality (p=0.0028; p=0.0017, respectively). Additionally, increased Ag67 151 counts were significantly associated with an increased incidence and rate of MCT occurrence at the original surgical site(p=0.0230; p=0.0021, respectively), an increased rate cu? MCT occurrence at (distant sites (p=0.0026), and the incidence and rate of MCT-related mortality (p=0.0053; p=0.0318, respectively), whereas AgPCNA counts were only associated with an increased incidence and rate of MCT-related mortality (p=0.048; p=0.0325, respectively). PCNA counts were not found to be of any prognostic significance following multi-variable analysis. In order to utilize these proliferation markers distinct cut points for each marker that differentiate between tumors with a favorable prognosis and those with a poor prognosis are needed. Cut-off values that discriminated MCTs associated with patient mortality from those that were not associated with mortality were determined for each proliferation marker. These cut-off values were based (m1 time mean value and 95% confidence interval of exufii marker‘ for lflfl? associated vnjji patient mortality and those that were not. Only the Ki67 and the Ag67 indices had. distinct cut off jpoints, of 23.08 and 53.95, respectively, that allowed for ea clear differentiation between these two populations of tumors 152 with. non-overlapping confidence intervals. According’ to multivariable analysis, MCTs with a Ki67 index greater than 23.08 cells/grid area (m: an Ag67 index greater than 53.95 were significantly associated vflxji an increased incidence (p=0.0262; p=0.0130, respectively) and rate (p= 0.0074; p=0.0109, respectively) of 1M1? occurrence an: the original surgical site (Figures 16 and 17, respectively) and with an increased incidence of MCT-related mortality (p=0.0016; p=0.0012, respectively) (Figure 18). Additionally; IMCTs with a Ki67 index greater than 23.08 were also significantly associated with an increased rate of MCT occurrence at distant sites (pr-0.0138), and an increased rate of MCT-related mortality (pz0.0171; Figure 19). In order to determine the associations between ITD c- KIT mutations enmi aberrant PU}? protein localization, and cellular proliferation in canine MCTs, each MCT included in this study was evaluated for ITD c-KIT mutations using PCR and aberrant KIT protein localization using immunohistochemistry. Internal tandenl duplication mutations were identified in the juxtamembrane domain of the c—KIT proto-oncogene of 9 of the 56 MCTs included in this study. According tx> multivariable analysis, canine MCTs with.IFND c—KIT mutations had 51 significantly greater proliferation index (p=0.0015), as measured by Ki67 153 immunostaining, and ea significantly' greater“ rate cflf cell cycle progression (p=0.0025), as measured. by' .AgNOR histochemical staining, than MCTs that lack such mutations. Additionally, we found that increased cytoplasmic KIT protein localization, as compared to peri-membrane protein localization. that its seen 1J1 normal, :non-neoplastic: mast cells, vwns also significantly associated.vnjji an increased proliferation index (p=0.0027), as measured by Ki67 immunostaining, and vnjjl an increased rate CH? cell cycle progression (p=0.0038), as measured by AgNOR staining (Figure 20). Discussion The results of this study demonstrate the significant role cellular proliferation. plays in. the progression of canine MCTs, as we have shown that both the rate of cellular proliferation, as measured by AgNORs, and the proportion of cycling cells, as measured by Ki67, are both significantly associated with the progression of canine MCTs. ihi light of these results, we recommend that AgNOR anxi Ki67 indices should 1x3 routinely evaluated 1J1 canine MCTs patients in conjunction with other prognostic markers, such as histologic grading, c-KIT mutations, and KIT staining patterns. 154 PI. _ In order to utilize proliferation indices in a routine diagnostic setting distinct break points are needed to differentiate between tumors that are likely to have a favorable prognosis and those that are likely to have a poor prognosis. Such distinct break points, with non- overlapping, cm: minimally over-lapping Sflfi% confidence intervals could only 1x3 defined for time Ki67 index, which had. a Ibreak point of 23.08 immuno-positive cells/ grid area, and the Ag67 index, which had a break point of 53.95. Based on these breakpoints Ki67 was better in terms of identifying MCTs that were associated with a decreased survival duration, whereas .Ag67 imas ea better 'marker for identifying' MCTs with. a (decreased disease-free interval. Therefore, based on these results MCTs should be evaluated for both their Ki67 and Ag67 indices, and should be evaluated in light of these break—points, in order to best identify patients that are likely to have subsequent local and distant MCT occurrences, and in order to identify patients that are likely' to succumb to their‘ mast cell disease. Although ITD c-KIT mutations and aberrant KIT protein localization have been well—characterized in canine cutaneous iMCTslmghqmgmlwmlw””fl, little 1J3 known. about downstream consequences of these mutations on the cellular 155 level. All of the ITD c—KIT mutations that have been characterized to date have been shown to produce a constitutively activated KIT protein in the absence of ”"3 and have also been associated with downstream ligandahqm ERK phosphorylationbm. Previous studies try our laboratory have shown that both ITD c-KIT mutations and aberrant KIT protein localizathmu in canine MCTs are associated with a worse prognosis, as compared to MCTs that lack, such mutations or have normal peri-membrane protein localization, respectively”””65. The results of this study suggest that 51 downstreant consequence (of crdifif mutations and aberrant KIT protein localization in canine MCTs may be an increase 1J1 cellular proliferation, kn/Iboth increasing the rate at which the cells enter the cell cycle and by increasing the rate at which the neoplastic cells progress through the cell cycle. Although these results are extremely intriguing enui provide preliminary 1J1 vivo data that support the hypothesis that c-KIT mutations and aberrant KIT protein localization cause an increase in cellular proliferation in canine cutaneous MCTs, further in vitro studies are needed to support these in vivo results. The results of this study demonstrate the utility of evaluating cellular proliferation, specifically Ki67 immunostaining and AgNOR histochemical staining, in the 156 1+ routine prognostication of results of this study studies that have shown the cellular proliferation in proliferation. should. inot prognostic factor in tandem with additional histologic grade, localization. 1K) date rm) distinguish between benign and each. prognostic: marker should additional makers 1J1 order to the disease and in order to canine confirm the canine be for canine MCTs, prognostic c—KIT mutations, MCTs. Although the results of previous prognostic significance of MCTsZflabaflr cellular evaluated. as 51 single but should be evaluated such markers as and aberrant KIT protein single marker will definitively malignant MCTs. Therefore, be considered in light of evaluate time full scope of make the best therapeutic determinations for a given patient. 157 Figure 15: Histochemical and immunohistochemical staining for proliferation markers in canine cutaneous MCTs. A: AgNOR. histochemical staining: AgNORs are identified as discrete, black nuclear foci (orginal magnification: 1,000X); B: PCNA immunohistochemical staining. Cells expressing PCNA are identified by dark brown nuclear staining (original magnification: 4OOX); C: Ki67 immunohistochemical staining. Cells expressing Ki67 are identified by magenta nuclear staining (original magnification: 400x). 158 .—‘L i: l Ki67 < .08 . . . . . J.- '. 5 - ' ................................. 'u-fi-i~A-on .n-u- -- -.. Inuln... :1”. 2, 2:55: 2 I? 32 2):: I ; o Ki67 a 23.03 ____*____ Relative Frequency Without Local Recurrence tn db- 1i- =h uh- #- 0 10 2b 30 40 50 Time (months) Figure 16: Kaplan-Meier survival curve evaluating the time until. local recurrence CH? canine DRIP patients classified based (n1 Ki67 Inxfimflrl expression. Patients vflifl1 greater than or equal to 23.08 Ki67 positive cells per grid area had a significantly shorter time to local recurrence compared to those with less than 23.08 Ki67 positive cells (p=0.0074). 159 I A967 < 53.95 ;.-o,'>-;.3‘:2, w:- ‘V-"'..-.',v.;'.:. - A C) . ooooooooooooooo A967 2. 53.95 Relative Frequency Without Local Recurrence '03 0 10 20 30 40 50 Figure 17: Kaplan-Meier survival curve evaluating the time until local recurrence of canine MCTs patients classified based on Ag67 values. Patients with Ag67 counts greater than or equal to 53.95 had a significantly shorter time to local recurrence compared to those with less than 53.95 (p=0.0109). 160 so- .................................................. Relative Frequency of MCT-related Mortality (%) <23.08 323.08 historical survival data. In order 11) better determine the efficacy of vinblastine and prednisone in the treatment of canine MCTs, an additional goal of this study is to compare the survival of dogs treated with vinblastine 168 and prednisone to those treated with surgery alone, when stratified based on known prognostic markers. Materials and Methods Case and tissue selection Twenty-eight canine cutaneous mast cell tumors from 28 dogs included in this study were identified as part of a larger retrospective study:6 with time goals of: l. evaluating the combination of vinblastine and prednisone as an adjunct chemotherapy for canine MCTs following surgical excision; and. 2. identifying clinical prognostic factors associated with this treatment. All cases were treated at the University of Wisconsin—Madison Veterinary Medical Teaching Hospital and were included in this study based on the following inclusion criteria: 1. absence of known severe concurrent disease; 2. complete staging; 3. no concurrent systemic anti-neoplastic therapy other then prednisone and vinblastine; 4. absence of measurable disease following surgery; 5. confirmed histologic diagnosis of canine cutaneous MCT; 6. adequate tissues available for c—KIT’ polymerase chain reaction and immunohistochemistry. Histologic grade of each tumor was confirmed according to the Patnaik histologic grading system for canine cutaneous MCTs by a single pathologist”. Complete clinical staging vmms performed :U1 all patients 169 including a complete physical exam, a complete blood count and a serum biochemistry profile, abdominal ultrasound, and regional lymph node palpation with or vdthout fine needle aspirate cytology. Lymph nodes were only considered positive for lymph node metastasis if they contained clusters or sheets of mast cells. Scattered individual mast cells were not sufficient in order to diagnose lymph node metastasis. c-KIT, tryptase and PCNA immunohistochemical staining For c—KIT, tryptase, and PCNA immuno-staining 5-um sections of formalin—fixed paraffin-embedded tissue were cut, deparaffinized iii xylene, rehydrated iii graded ethanol, and rinsed in distilled water. Endogenous o\0 peroxidases were blocked by incubating section in 3 hydrogen peroxide for 5 minutes and subsequently rinsed in distilled water. For c-KIT’ and tryptase immuostaining antigen retrieval was performed by incubating tissue sections in 21 citrate buffer antigen retrieval solution (Dako, Carpenteria, CA) in 51 steamer~ for 20 Ininutes and cooled for 20 minutes. No antigen retrieval was used for PCNA immuno-staining. Using a commercial autostainer (Dako, Carpenteria, CA) Non-specific antibody binding was blocked by incubating sections with a protein blocking agent (Dako, Carpenteria, (EU for‘ 10 Ininutes. Sections 170 were either incubated with mouse monoclonal anti-PCNA antibodies (PC10 cione; Ixflme Cytomation, Carpenteria, CA) at a 1:100 dilution, rabbit polyclonal anti-c—KIT antibodies (Dako 2 . e e i c >2 285. --------- HistoGradez 2.13.5. ......... . ................. . ................ 0 '0‘ d) o-o ' 3 g 3 5 KIT Pattemz g EA‘ ........ g '4‘ p .. ‘é Mi 3:? §=2< *5 ' *5 gKlT Pattern: E 'HlstoGrade3 E CE 0.0 . m 0.0 omlmedoadowintzbnum d2c'104005008t'101000121'111400 Time (days) Time (days) 1" 104 - "" 10« -—- ; a 3 ‘1 c 3 Tryptase1 D '5 5 . -- o 3. d) .8. >— >— 2 3 6 NoITDc-KITMutatlon 2 5 6 ,1 g *3: ' g E TryptaseZ g E 4.. I g. E ‘< ”1‘”: 11. T6 1.1. § 111 m 0 E. 5 2n '3 x: ‘2‘ - ‘ % 'TD C-K’T Mutation % """" .‘r ryptase3 a: 00 . m 0.0. 0 200 400 6(1) Bti] 1000 1200 1400 200 ‘00 5m 3m 1000 12m “00 Time (days) Time (days) Figure 21: Kaplan—Meier survival curves of time to treatment failure of canine MCTs classified based on prognostic markers. A: Histologic Grade (p=0.0062); B: KIT staining patterns (p=0. 009); C: ITD c—KIT mutations (p=0.0576); D: Tryptase (p=0.0286). 185 1.0 4 . _ « - .+———g- . . > _ Histo Grade 2 A > 1 U 3 , KIT Pattern 1 B 0 || 1 1 H . . l o 0 —~ _. < _ 3 g g a . 8' - 8' > KIT Pattern 2 u. 3 IL 3 0 m a in Z “6.41 3 “.4 - a 35 ° 4’ 0 s m .2. Histo Grade 3 a: .2 . ‘ . . KIT Pattern 3 0 0 0.0 - 0 1:00 21110 3000 0 1000 2000 3000 Time (days) Time (days) . 10* 'ér——+-—rTryptasei F ‘0 c > ‘n ...... . g D o a. ' ‘ 5 _ a. g _-B ' .Tryptasez §- g No ITD c-KIT mutations i g 9; ' I- g (’5’ .8. E $51 .2 - g 2 T3. 0 ‘4 ‘ ..................... '- o .4 1 a ————n 5 Q .......... a: '2‘ i a: '2 4 . Tryptase3 ITD c-KIT mutations j 0 ," . 0.0 . . ' r 0 mm mm mm 0 mm an) :nm Time (days) Time (days) Figure 22: Kaplan—Meier survival curves of survival times of canine MCTs classified based on prognostic markers. A: Histologic Grade (p=0.0004); B: KIT staining patterns (p=0.0502); C: ITD C~KIT mutations (p=0.0179); D: Tryptase (p=0.0924). 186 l 1.0: r 10 ; .. . C .8~ a _ i3 = e g E 5 hemotherapy E E S s ' t a U’ 0 > o . . r E 4% ' 3 5 g 2 g E 21 ESurgeryAlone ‘21 SurgeryAlone . o ............ . ............... ., . .i m ? 1 ' 0.0 . . 00 . :. . . . . 0 10 20 30 0 10 20 30 40 50 60 70 Time (months) Time (months) Figure 23: Kaplan-Meier survival curves of time to treatment failure (A) and survival time (B) of grade III MCTs treated with either surgery alone. Canine mast cell tumors treated with vinblastine and prednisone had a significantly increased time to treatment failure (p=0.002) and survival time (p=0.009) as compared to MCTs treated with surgery alone. 187 10* 1.0 . Chemotherapy A B > . >~ g .83 g .8-g Chemotherapy 0 - a — a ' ‘3? , :3: 2 gas ‘6‘ ............ Surgery‘uone g 2-5‘3 l-L :3 i , ll- : g m ............................ g m 2: 5 4. i 54‘ .................. , g I?! SurgeryAlone .b .2- 00 . . . . . , 00 . . . 0 1O 20 30 40 50 60 70 0 10 20 30 40 Time (months) Time (months) Figure 24: Kaplan—Meier survival curves showing percent survival of KIT pattern III MCTs (A), and MCTs with ITD c— KIT mutations (B) treated with vinblastine and prednisone compared to those treated with surgery alone. 188 CHAPTER 7 IDENTIFICATION OF CANDIDATE GENES ASSOCIATED WITH THE PROGRESSION OF CANINE MAST CELL TUMORS: A PILOT STUDY Introduction Canine cutaneous mast cell tumors accounting for 7—21% of all cutaneous neoplasmsb4. Canine MCTs have an extremely variable biologic behavior ranging from a solitary benign mass to a potentially fatal metastatic .;,l4,‘4t’) diseasemj' Currently, histologic grading is the primary prognostic and therapeutic determinant for canine cutaneous MCTsh”H'“H However, despite the significant associations that have been found between histologic grade and patient survival, the predominance of intermediate grade MCTs, the variable behavior associated with intermediate tumors, and the marked degree of inter- observer variation has led many to questions the relevance and utility of these classification systemsrmszmgq. Complete surgical excision is time primary treatment modality for canine cutaneous MCTs. In the event of incomplete surgical excision or a tumor that is unresectable, radiation is commonly employed as an adjunct local therapy, offering some benefit for local disease. In addition to surgery and radiation therapy, several 189 chemotherapeutic protocols have been employed for the treatment of systemic or non-resectable MCTs, with variable degrees (If successi7. ihi the absence (Hf accurate prognostic tools and a thorough understanding of the underlying biology of MCTs, these treatment modalities are of variable use. Previous work by our laboratory and by others has implicated time c-KIT proto—oncogene III the pathogenesis of canine MCTs“”M'W“%”5&1W””t. The c-KIT proto-oncogene encodes the receptor tyrosine kinase KIT“, which, in conjunction. with. its ligandi stem. cell factor (SCF) also ) 106,114,115 known as mast cell growth factor or steel factor , plays an important role in mast cell proliferation, 100,107—111 differentiation, survival, and chemotaxis Internal tandem duplications and deletions have been described in the juxtamembrane domain of c-KIT in canine MCTslmohqmqfllm, and those mutations that have been evaluated have been shown to lead to a constitutively activated form KIT in the absence of ligandfl'fl””5. Additionally, aberrant cytoplasmic localization of the KIT protein has also been described in 04,159 0 canine MCTs Previous work by our laboratory has shown that canine MCTs with ITD c-KIT mutations or aberrant KIT protein localization are significantly' associated. with a worse prognosis, as compared to those MCTs that lack ITD c— 190 KIT mutations or aberrant KIT localizationHQJGE respectively, and are also significantly associated with an increased rate of cellular proliferation in canine MCTs (Webster et al, unpublished data). Cancer is the result of a series of several genetic and epigenetic changesah While the significance of c~KIT in the pathogenesis of canine MCTs is clear based on the results of time above mentioned studies, i1: is likely that many more genes play a role in the progression this disease. In order tx> identify genes associated with the progression. of canine IMCTs, gene expression. profiles of high grade and intermediate grade MCTs were compared using ea custtmI designed CEHUJMB oligonucleotide Inicroarray. The results of this study identify several candidate genes that may play a role in the progression of canine cutaneous MCTs. Materials and Methods NUcroarray Design and production A targeted canine oligonucleotide microarray was designed to include genes associated with cancer, inflammation, embryonic and adult stem. cells, and. bone. Unique 60bp sequences with approximately equal melting temperatures were identified :UI 85d canine genes coding regions associated with the above biologic systems by the 191 Bioinformatics Core Facility of Michigan State University. Additionally, 60mers unique tx> 5 housekeeping genes, beta actin, beta-two microglobulin, glyceraldehyde 3-phosphate dehydrogenase, cyclophilin 2L and. ribosomal protein. L13a were also identified to be used as positive controls in all microarray experiments. Oligonucleotides (60mer) were selected to give unique and specific hybridization signals using the program OligoArray 2.0 developed specifically for design of oligonucleotide probes for microarrays using a thermodynamic approachrn. Oligonucleotides were then synthesized an: Michigan. State ‘University’s lMacromolecular Structural Facility for Harmoarray printing. Microarrays were printed on UltraGaps, Gamma Amino Propyl Silane coated slides (Corning, Corning, NY), at Michigan State University's Genomics Core Facility. Arrays were printed in duplicate on each slide, and each array consisted of 2 rows of 4 blocks, containing 120 features per block with a total of 960 features per array, and 1920 features per slide. Negative controls consisting of either empty features or random Oligonucleotides were included in each block, with a total of 73 empty features and 16 random Oligonucleotides included III each. array (146 anxi 32 jper slide, respectively). Positive controls consisting of the 5 housekeeping genes were included in alternating blocks in 192 the last. 5 positions of these blocks, with each housekeeping gene being printed a total of 4 times per array. Cases The tissue collection protocol used in this study was reviewed by the All University Animal Care and Use Committee (AUACUC) at Michigan State University, and this protocol was declared exempt as all tissues were obtained as part of the standard treatment of canine patients at the Veterinary Teaching Hospital at Michigan State University (MSU-VTH). All canine cutaneous MCTs presented to MSU-VTH for surgery or necropsy between July 2004 and December 2006 that were at least 2 cm in diameter were collected for this study. No criteria other than sample quantity and quality were used for inclusion into this study. All tissues obtained for this project were either obtained post-mortem or during surgical removal as part of the standard care for the treatment of each animal. Surgical samples of MCTs were obtained from time center' of 6%Mfl1 tumor ‘with. scalpel and thumb forceps or with a punch biopsy, avoiding tumor margins in order to facilitate histopathologic evaluations. Tissue samples collected for this study ranged from 40—500 mg. Each sample was snap frozen in liquid nitrogen and stored at -80°C until the time of RNA isolation. 193 RNA isolation For RNA isolation tissues were ground to a fine powder in liquid nitrogen using a mortar and pestal and cell lysis and RNA extraction was performed using the Versagene Fibrous Tissue RNA. extraction KIT (Gentra Systems, Mineapolis, MN) with on column DNase treatment according to the manufacturer's protocoli iRMA was initially quantified using tflma Nanodrop spectrophotometer (Wilmington, IND and RNA quality was assessed using the Agilent Bioanalyzer (Palo Alto, CA). Histopathology Sections of Efiflfll tumor' were routinely fixed :hi 10% neutral buffered formalin and paraffin embedded. The diagnosis of each canine cutaneous MCT was confirmed histologically CH1 5 inn sections stained vnifli hematoxylin and eosin and each tumor was histologically graded by a single investigator (JDW) using the Patnaik histologic grading system”. Identification of c—KIT mutations In order to identify ITDs and deletions in the juxtamembrane domain of the c—KIT proto-oncogene of canine cutaneous MCTs, laser capture microdissection was preformed and genomic DNA. was extracted from 5—7 um sections of formalin-fixed paraffin-embedded of canine MCTs as 194 1'1) 0.“. previously described1 When paraffin-embedded tissues were not available, DNA was extracted from tissue and RNA lysis buffer left tuna: from.IUU\ extractions. Specifically, 100 pl of tissue and lysis solution was combined with 100 pl of 4 M ammonium acetate, 1 ul of glycogen, and 500 pl of 100% ethanol and incubated for 10 Hdnutes an: -20°C and centrifuged at 1,306 x g for 10 minutes. Following centrifugation, the supernatant was decanted, and the pellet yum; rinsed vnlji 70% ethanol anxi centrifuged again for 1%) seconds an: 14,000rpms. The supernatant vwns again decanted and the pellet was dried at room temperature. When dry, DNA was eluted in 30 ul of distilled, de-ionized water and 2 ul of eluted DNA was used in each 25 ul PCR reaction. Polymerase chain reaction amplification of the juxtamembrane domain of time c—KIT proto-oncogene was preformed using fOrward (CATTTGTTCTCTACCCTAAGTGCT) and reverse (GTTCCCTAAAGTCATTGTTACACG) primers flanking exon 11, including the regions of where ITDs and deletions have been previously characterized in canine MCTsm'%”%J&3 Polymerase chain reactions were prepared in.a1 25 ul total reaction volume, with 5 ul LCM extracted DNA, 5 pmol of each primer, 0.5 units of Taq polymerase (Invitrogen, Carlsbad, CA), enui final concentrations of EMD'mM dNTPs, 2 195 mM MgCl;, 20 mM Tris-HCl, and 50ul KCl. Cycling conditions were as follows: 94°C for 4 ndnutes; 35-45 cycles at 94°C for Zl minute, 55°C fOr‘ZL minute, and 72°C lint 1 minute; 72°C for 5 minutes. Amplified products and ITD and deletion mutations were visualized by agarose gel electrophoresis on a 2% agarose gel after ethidium bromide staining. Reverse Transcription, labeling, and hybridization Five to fifteen micrograms of total RNA were concentrated to a final concentration of 312.5—937 ng/ul in 16 ul of DECP-treated water using vacuum centrifugation. cDNA was synthesized with the Superscript III Reverse Transcriptase (Invitrogen, Carlsbad, CA) using anchored oligo-dT primers and. amino :modified. deoxynucleotides and subsequently labeled. with either“ Cy3 or Cy5 fluorescent dyes as part of the Superscript Indirect cDNA. Labeling System (Invitrogen, Carlsbad, CA). Cy3 anui Cy5 labeling efficiencies were subsequently quantified using the Nanodrop Spectrophotometer (Nanodrop Technologies, Wilmington, DE), and equal picomoles of Cy3 and Cy5 labeled probes were hybridized to each slide in order to Hdnimize dye bias. Twenty to forty-five picomoles (Hf each labeled probe (Cy3 and Cy5) were concentrated using a Microcon tube (Millipore, Billerica, MA), and subsequently brought up to a final volume of JJI)1LL of Ambion Hybridization buffer 3 196 (Ambion, .Austin, TX) for ihYbridization. Prior to hybridization labeled. probes were heated to 70°C for 5 minutes in order to denature cDNA. All hybridization reactions were performed on the GeneTac Hbetation (Genomic Solutions, IUUI Arbor, DTI) using 51 step ckwni hybridization protocol consisting of 6 hr at 42°C, 6 hr at 35°C, and 6 hr at 30°C. Following the 18 fmnn: hybridization. step down, microarrays were washed in 2X SSC and 0.1% SDS at 37°C, followed by a tugh.snxingency wash consisting of 0.2x SSC and 0.1% SDS at 25°C, and were finally rinsed with a post wash buffer consisting of 0.2x SSC at 25°C. Microarray analysis Slides were scanned using the GeneTAC LS IV microarray scanner (Genomic Solutions, Ann Arbor, MI), and quantification tn? raw fluorescence aumi initial spot analyses were using' GenePix :microarray analysis software (Molecular Devices, Downington, PA). Statistical analyses of the microarray data was preformed using the Limma package for =oaémmo=§ mam Sod mnmd mam—.3294. .238. .585 £265 _m__o£ouco .238; 338.0 nomao Sam—Eon. 9.8 new 3.22.6.0 o 5205 3:30:82: ocom 838... m8... 8838. 205 :8 5.3.2.0 . owcmco o:_a> a. Son. 5.26:3". oEmz .muoadu Haoo umma ocflcmo oumHUoEuoucH .m> ucmceflama CH mocom commoumxo >HamflucouowMHm ”ha wHQmB 205 ( TNF) TRADD Apoptosis NF-KB ‘ ‘ Figure 25: Diagram of tumor necrosis factor (TNF) signaling. Upon ligand binding, tumor necrosis factor receptors aggregate allowing for downstream signaling through both apoptotic and NFKB pathways. TNF-receptor associated death domain (TRADD) and RIP are important for both signaling pathways. NFKB signaling is activated through their interactions with TNF-receptor associated factor 2 (TRAF2). The death receptor apoptotic pathway is activated through interactions with Fas associated death domain (FADD), which is involved in caspase 8 activation. The inhibitors of apoptosis proteins c—IAP2 has been shown to inhibit TNF induced caspase 8 activation. Arrows indicated differential expression seen in high grade MCTs. 206 CONCLUSIONS Due ti) the variable and potentially aggressive behavior of canine MCTs; the undesirable side effects associated with current therapeutics; and the emotional and financial stresses faced by owners, accurate prognostication of canine MCTs is of Ciitical importance°'7'l*”. Although the current Patnaik histologic grading system has been shown to be correlated with survival when evaluated. within. a large populationbfi the marked degree of inter-observer variation and the predominance of intermediate grade MCTs has led many to question the relevance of this systemi“2m¢b24. In light of these limitations, novel markers are needed for canine MCT prognostication. The studies described in this dissertation further identify and characterize multiple prognostic markers for canine cutaneous MCTs. Specifically, the results of this CUssertation demonstrate the utility of KIT staining patternsmg, c-KIT mutationsmz, Ki67 immunostaining, and..AgNOR. histochemical staining in the routine prognostication of canine cutaneous MCTs treated with surgery alone, cu: in combination with vinblastirme and. prednisone. Furthermore, the results of these studies demonstrate the inadequacies of tumor 207 depth”°, tryptase immunostainingwg, and S-phase evaluations in canine MCT prognostication. In these studies several markers have been shown to be significantly associated with disease progression. However, no single marker can clearly predict the biologic behavior of canine MCTs. Based on the results of these studies, we propose that a panel of markers including histologic grade, time evaluation of ICUF staining patterns, the assessment of cellular proliferation using Ki67 immunostaining' and..AgNOR. histochemical staining, and. the evaluation of c-KIT mutations, should be routinely used in the prognostication of canine MCTs. In the future, prospective studies should evaluate the use of these markers in combination, in tummy: to maximize their utility in a diagnostic setting. A prospective evaluation of these markers will allow for a more accurate assessment of each marker individually and a better understanding of how these markers can best be used in combination in a routine diagnostic setting. Additionally, as novel therapeutic protocols are developed the prognostic value of these markers should be re-evaluated, as these markers may vary in their association. ‘with. the outcome of specific treatments. 208 The studies described in this dissertation further define the role of the c-KIT proto-oncogene in the progressitmi of canine iflTTs. Specifically, these studies have shown timn: increased cytoplasmic localization (Hf KIT and 111) c—KIT mutations are significantly associated with decreased disease-free survival and survival durationumfl°3 These results strongly support the hypothesis that changes in the c-KIT proto-oncogene and the KIT protein play important roles in the progression of canine cutaneous MCTs. In our studies, ITD c—KIT mutations and aberrant KIT protein localization were associated with an increased rate of cellular proliferation as measured by AgNOR counts, and an increased proliferation index as measured by Ki67 immunostaining. These results suggest that one way KIT may promote the progression of canine MCTs is by increasing cellular' proliferation. Together, the results (n3 these studies suggest timn: changes in ECU? protein. localization and the presence of ITD c-KIT mutations may not only be useful as prognostic markers, but KIT may also be the best therapeutic target for canine MCTs. Despite the significant association. between. aberrant KIT localization and the progression of canine MCTs, little is known in terms of the true biologic significance of this 209 change. .Although vme have found 51 significant association between aberrant KIT protein localization and the presence of ITD c-KIT mutations, we have also found a substantial number of canine MCTs with aberrant KIT localization which lack ITD c-KIT mutationSHQ. Ihi these cases, aberrant KIT localization may result from additional mutations in c—KIT, although no mutations were found in the phospho-transferase region (Hf the kinase domain iii 32 MCTSMI. Other potential explanations for tinis change 1J1 KIT‘ protein. localization include time possible presence cxf aberrant truncated isoforms of KIT, as seen in human prostate canceer; increased recycling from the cytoplasmic membrane as a result of autocrine or paracrine signaling loops; or simply, defective post-translation modifications or intracellular transportation. In the future, additional in vitro and in vivo studies are needed not only to identify the cause of this aberrant KIT protein localization, but also to determine the downstream consequences of these changes. Recently, our laboratory has cloned the canine c- KIT transcript iii a GFP—tagged expression vector :nu order to begin these investigations. Since cancer is the result of the accumulation of genetic and epigenetic changes several additional genes aside from c-KIT must be involved in the progression of 210 canine cutaneous PMTTS. In. order ti) identify' additional genes involved in the progression of canine MCTs, the final project of this dissertation was to conduct a pilot microarray experiment comparing the gene expression profiles of a series of high grade MCTs to the expression profiles of £1 series of intermediate grade MCTs. In this study, we identified 17 candidate genes that were differentially expressed between these time populations of MCTs. Although the potential role of these candidate genes in the progression of canine MCTs is intriguing, these results are (Hug! the starting rmfljm: for' future studies. Differential expression cflf these genes needs ti) be confirmed first using quantitative reverse transcriptase PCR. Subsequently, time significance (if this (differential expression needs to be evaluated on the protein level using immunohistochemistry enmi western hdotting. These studies need to be followed by functional studies in order to clearly delineate the biologic significance of these changes. The results of the studies presented. in this dissertation significantly add to our current body of knowledge of canine cutaneous MCTs, both in the diagnostic setting and 1J1 our understanding (Hf the biology (if these tumors. Specifically, we have identified novel prognostic 211 factors, such as KIT staining patterns and c—KIT mutations, and have clarified the ‘utility' of previously identified prognostic factors, :mmii as tumor depth anmi proliferation markers in the routine prognostication of canine MCTs. These studies have also added to our understanding of the molecular biology of canine cutaneous MCTs, as the data provided in these studies further characterize the role of c-KIT in the progression of canine MCTs. Additionally, our pilot microarray study has identified several genes that may also be involved in the progression of canine MCTs. 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