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Cytogenetic evaluation of four canine mast cell tumors
Cytogenetic Evaluation of Four Canine Mast Cell Tumors Diana M. Stone, Peter B. Jacky, and David J. Prieur
ABSTRACT: We evaluated fl)ur (:anine (,~utane(ms mast (:(~11 tumors (:ytogeneti(:ally. All lout tum(n's (:(retained bath hypodipl(~id and hyl)erdipl(~id (:ells, an in(:r(~as~: in the numb~!r ol me3a(:entri(: chromosomes, ex(;hange cotd]gurations, and (:ells showit~g loss o!an X chr(;mosome. All ttml()rs (:(retained nmtaphases with chromosome gaps (rod breaks at Jre(lueneies greater tha~ ot)serw~(t spol~taneous chromosome breaks in normal (:ultured (:(mine peripheral blo()d l~mph(J(:vt~s. Three (~j the four tumors had a normal modal (:hromostm~e numt)e!r (ff 7~. The !ourth lam(~r had a m()dal (:hromosonle number of 93. ~vhi(:h r(~preset~ted 15% ()f the (:ells evala(ded from this tHIllor.
INTRODUCTION T h e r e h a v e been m a n y (:ytogenetic studies of h u m a n t u m o r (:ells and an increasing n u m b e r of n o n r a n d o m c h r o m o s o m e aberrations h a v e been d e t e c t e d in bu(na(~ leukemias and l y m p h o m a s and in solid t u m o r s [1, 2]. Little information, h o w e v e r , is available on a n i m a l neoplasia, and, in particular, naturally ()(:curring soli(I tumors. S t u d i e s of solid t u m o r s in a n i m a l s shouh] greatly facilitate our u n d e r s t a n d i n g of the o n c o g e n i c process, c a n c e r geneti(:s, and c a n c e r cytogenetics: e.g., t u m o r s in a n i m a l s can be f o l l o w e d o v e r t i m e for c y t o g e o e t i c changes, w h e r e a s huinan m a l i g n a n c i e s are f r e q u e n t l y r e m o v e d or treated w h e n d i a g n o s e d . The d o m e s t i c dog is a p o t e n t i a l l y useful a n i m a l m o d e l for study of s p o n t a n e o u s tumors. M a n y dogs live an e x t e n d e d life an(I are p r o v i d e d w i t h veterinary m e d i c a l care, p e r m i t t i n g d e t e c t i o n an(t diagnosis of m a l i g n a n c i e s . Of all domesti(: animals, dogs most i n t i m a t e l y share the h u m a n e n v i r o n m e n t , are expose(t to m a n y of the s a m e e n v i r o n m e n t a l (:ar(:inogens and m u t a g e n s as h u m a n s , and d e v e l o p m a l i g n a n c i e s i d e n t i c a l to m a n y h u m a n m a l i g n a n c i e s . C a n i n e c u t a n e o u s mast cell t u m o r s w e r e selected for cytogeneti(: e v a l u a t i o n because t h e y are one of the most c o m m o n skin t u m o r s in clogs, corot)rising 7 - 2 0 % of all skin t u m o r s in this species [3, 4]. In a d d i t i o n , these t u m o r s l)rogress sh)wly, though all are c o n s i d e r e d p o t e n t i a l l y inalignant [5]. C o n s e q u e n t l y , changes in c y t o g e n e t i c characteristics (:an be f o l l o w e d with p r o g r e s s i o n from relatively low-grade m a l i g n a n c y to quite high-grade m a l i g n a n c y . A n o t h e r r e l e v a n t feature of mast (:ell t u m o r s is the b r e e d - a s s o c i a t e d p r e d i s p o s i t i o n for this t u mor in dogs. Boxers h a v e 9.9 16] to 16.7 [ 7] the risk of d e v e l o p i n g (:utaneous mast cell t u m o r s as c o m p a r e d with o t h e r dogs. Thus,
From tlu~ Department of Veterinary Mi(:r~)[fiolo~yand l'atholo,~v, Washin,~ton ~tat~ (Iniw~rsity (I). M. ,%, D. 1. ILL IJullm;m. Washington, and l)el)artlnellt of l~athology/(]ytogeneih:s. Kaiser lh~rmalmllh~ Northwesl (P. B. ].), Portland. Ores,on. Address reprint requests to: Diana M. St(me. I).V.M., Ph.D., Department of Veterinary Mi(:robiology and Pathology, Washington State University, Pulhnan. WA 99164 7040. He(:e,ived luly 26, 1990: ac(:e,pted ,~eptember 6, 1990.
105 r; 19(31 Elsevier S(:iell(:ePublishing Co., In(:. 655 Avlmul! of lhe Americas, New York, NY 1()()1(1
(]an(:er (;enet (~y~ogcnci53:11}5 112 ( 1991 ) 0165-4608/91/S03,50
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this canine tumor may prove a useful model for both heritible and cytogenetic cancer studies of solid tumors. Our study was designed to determine whether cytogenetic abnormalities exist in canine mast cell tumors and, if so, to define the range of such abnormalities and determine whether any consistent cytogenetic changes could be detected.
MATERIALS AND METHODS Specimens of canine mast cell tumors were obtained by biopsy from four dogs presented to the Washington State University Veterinary Medical Teaching Hospital between June 1987 and May, 1989. Case MCT-01 was a 12-year-old female Labrador retriever; case MCT-02 was a 5-year-old female Sbar Pei; case MCT-03 was an 11year-old female Basset hound; and case MCT-O4 was a 6-year-old male Boxer. None of these dogs received any chemi(:al or radiation tberapy before tumor excision. Mast cell tumors were initially diagnosed by tumor aspirate and later confirmed by histopathology. Clinically, two of the (:ases (MCT-02 and MCT-03) were characterized by metastatic lesions of previously diagnosed solitary cutaneous mast cell tumors. Case MCT-02 had metastatic lesions involving the skin, lympb nodes, both kidneys, and tbe pancreas. Case MCT-03 had multiple lesions involving the skin. A (:omplete necropsy and histologic evaluation were not permitted in this dog: thus, only the cutaneous masses were observed and verified as being mast (:ell tumors. The other two cases (MCT-01 and MCT-04) were dogs with solitary, (:utaneous mast cell tumors with no evidence of metastasis. Sections of tumor for culture were obtained using sterile technique and pla(:ed in a Petri dish containing warmed (37°C) RPMI 1640 medium; short-term cultures were initiated using the t)rotocol of Yunis [8] as modified by the Kaiser Permanente Hospital Cytogenetics Laboratory in Portland, Oregon, for preparation of chromosomes from lymph node biopsies for cytogenetic study [9]. Sections of tuinor tissue were rubbed with a fine mesh screen to dislodge tumor cells. Aliquots of tumor cells were stained with toluidine blue to ensure that they were mast cells. Cells were washed twice in m e d i u m and resuspended at l × 10" cells/ml in warmed (37°C) R P M I t 6 4 0 m e d i u m s u p p l e m e n t e d with 15% fetal calf serum, 2% L-glutamiile, and containing penicillin (50 U/ml), streptomycin (50/xg/lnl). Cultures were established in 25-cm ~ flasks, each containing 5 ml suspe, nded tumor cells incubated at 37°C in 5% CO z. Cells were harvested for cytogenetic analysis at 1 hour, and at 4. 12. 24, and~or 48 hours of culture time, d e p e n d i n g on the n u m b e r of metaphases obtained in previously harvested samples. Thirty lninutes before cell harvest, 25/~1 of 10/~g/ml Colcemid was added to each flask. Cells were harvested and processed for metapbase chromosome spreads according to conventional methods with hypotonic treatment in 0.075 M KCI (5 minutes) and fixation in 3:1 lnethanol :acetic acid [101. Some preparations were standard-stained with Giemsa and others were aged at room temperature for 3 days and G-banded 111].
RESULTS Table 1 summarizes the cytogenetic characteristics of the four canine mast cell tumors. All tumors showed variation in the n u m b e r of chromosomes per cell, but three of the four tumors showed a normal canine modal chromosome n u m b e r of 78 at varying frequencies (MCT-01, MCT-03, MCT-04). The fourth tuinor (MCT-02) had a modal chromosome n u m b e r of 93 in 15% of the cells evaluated. Tbus, all tumors contained
44 41 76 74
86 109 83 156
(2n]
(yr)
MCT-01/F/11 MCT-02/F/5 MCT-o3/F/11 MCT-04/M 6
IlklnlbeFs
Range of chromosome Diploid (2n = 78) (%) 35.0 0.0 69.0 77.8
ttypodiploid (%)
50.0 33.3 14.0 5.5
15,0 66.7 17.0 16.7
Hyperdiploid (%) 20 27 29 18
Metaphases evaluated
Cytogenetic characteristics of four canine mast cell tumors
Case/sex/age
Table 1
78 93 78 78
Modal no.
÷ + +
Extra metacentrics
4 + + 4-
Loss of X-chromosoinc
20 7 10 6
Metaphases with chromosome breaks (%)
0 i + (I
Metastatic
"-.1
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e
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0
e! F i g u r e 1 A (;-banded hypt)diploi(I metal)hase tr(ml (:;mitre mast (:ell t t m m r MCT-02 f r o m a l'emale Shar Pei dog. T h e mctal)hase ctmsish~(t t)f 51 a(:i'¢)cimli'il; ( h r o l n o s o l n e s in,'-;lea(I of lh(! m)rmal t:anine, diploid lltllllbOr of 7,bl, Neitlmr of lhe, y;tlhll/(?hlt;l!tlh'i(: X chrolllOSOllley; was [)reselH in the m e t a p h a s e .
a substantial n u m b e r of both hypodil)h~id (Fig. 1) and hyperdiploid (Fig. 2) (:ells. The tumor with a modal n m n b e r of 93 showe(t extreme variation in (:tm)mosome numbers, whi(:h ranged from 41 to 109. All four tumors containe, d metaphases with extra meta(:e, ntri(: chromosomes. The canine karyotype is normally composed of all acro(:cntri(: (:hromosomes except tim submeta(:entric X and Y (:hromosomes. An increase in tmnor cell metacentric forms was observed in both hyt)o- and hyt)erdit)loid metal)bases (Fig. 3). In one case [M('T02) the hyperdi ph)id metal)bases contai ned a higher n m n b e r of abnormal meta(:entric forms than did the hypodii)loid metaphases. The other three cases bad insufficient numbers of either hypo- or hyperdiploid metaphases h) make a valid (:omparison. In none of the hypodiploid nletapbases (lid the increase in tnetat:entric forths a(:(:ount for the hyt)odiploidy. All tumors contained (Jells showing loss of an X chromosome. Exchange configurations in the form of quadrara(lials and telomeri(: associations were also observed in some tumors. All tumors (:ontaine(t metaphases with spontaneous chromosomal gaps or breaks. The percentage of (:ells containing these chromosome gaps or breaks varied from 6 to 20% of the cells. This was significantly greater than the observed spontaneous chromosomal breakage rate (<2%) in normal cultured canine peripheral blood lymphocytes from 17 dogs (Stone, 1989: u n p u b l i s h e d observations). Definitive identification of the origin of some observed melacentric chromosomes or of some karyotypic abnormalities su(:h as apparent deletions or translocations was impossible owing to the variability or resistan(:e of the tumor (Jell chromosomes t() Gbanding, overly condensed (:hromosomes, comparatively small numbers of analyzable initotic (:ells under sonle (:ir(:umstances, and the morphologic similarity of the small (:anine a(:rocentri(: chromosomes. Cytogeneti(: ewduation of stinmlate(I t)erit)heral
109
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,,C Y F i g u r e 2 A (Vbandcd hyperdil)loid mctat)h~tsc from (:allille lllttS( t:cll tumor M(;'I'-02. 'l'tw metaphase consisted of 104 c:hromosomes instead of l]m normal c;anim~ dil)hfid number of 78. No X chromosomes could b¢~ idt;ntificd.
F i g u r e 3 A (]-banded h y p c r d i l , l o i d metaph~nsc fronl c a n i n e mast c:~,~lllul~lor N.1(71'-02, wilh ~lll increased number of subme, tac;entric and metac:cmlrit: t:hromosC,lneS. Ei~,ht st~l)m~h~ccnlric: or II](,qilC:(HltI'iC (:hl'oI//OSOlll{,~S W(31"(!ichmtil'i¢~d f a r r o w s ) instead of tim llOrllldl [l~ll]~ll(~ c;~tllilll~ IllllIiht~l" of two subme, tac;entri(: X (:hromosom~s.
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1
110
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blood lympho(:yte cultures fron/ all four cases sh()we(t that each dog ha(I a normal diploid number (2(1 78) in lyInpho(:~/tes.
DISCUSSION The (:ells of many Inmmn malignant ne(Jl)hlsms have abnornml cytogeneti(: features [12[. There have been few reports of cytogeimtic analysis of neoplasti(: (:anine tissue, other than the canine transmissible venereal tumor (CTVT), which is a stable coitally transmitted allograft. CTVT has generally been shown to (:ontain 57 59 chromosomes, with a modal c h r o m o s o m e number of 59 [13-15]. This reduction in chromoson~e number is consistent with extra meta(:entric fusion (:hrom(isomes. Variation in ¢:hrom o s o m e number and other karyotypic almormalities have als() been report(xl in (:ells from cases of caniim lyml)h(isarcoma ]16-231. Karyotypi(: almormalities in lymphosar(:oma cells include hypodiploi(ty, hyperdiploidy, (:hange in the modal chromosome number, extra metacentric forms, (touhh; mimlt(; ((Imin) chromosome, s, exchange configurations, an(I loss 1)t an X (:hromosome. No single sl)ecific karyotypic abnormality characteristic of these (:ells has been identifie, d. There has been one report of a (:anine fibrosarc()ma with a redu(:e(] number (if (:hr(m)(is/mles and an increased number of metacentric forms 124]. Cutaneous mast cell tumors ;ire (:lassified as roun(t (:ell tumors, along with histio(:ytomas, lymphosarcomas, and CTVT 1251. The only report in the literature on a (:ytogenetic study of canine mast (:ell tumors involve(t analysis of two such tumors. Karyotypic analysis disclosed a pattern of reduction in the total number of chromosomes with an increase in the proportion of metacentric h)rms [261, similar to the pattern characteristic of CTVT, s(ime canine, lymphosar(:omas, and (:anine fibrosarcomas. In one dog, the modal (:hromosome munher (if the neoplasti(: mast (:ells was 40: in the other, the m(idal numl)er was 50. This cytogeneti(: analysis (if f(iur (:anine mast cell tulnors is the first report of both h y p o d i p l o i d an(t hyperdil)loi(t (:ells in the same tum(ir, and the first report of a canine mast cell tumor with a hyperdiploid modal (:hromosome lmmber. In this tumor (MCT02), the modal c h r o m o s o m e number ()f 93 was present in 15% of the (:ells, suggesting that this karyotypic abnormality was derived from a single progenitor (:ell. This report is also the first (lescription of chromosome, breaks, exchmlge (:(mfigurations, (:entri(: ftlsiolls, and loss of all X (;]lromos(ime in canine mast cell tmnor cells. The consistent loss of one X (:hromosome was reported in one case (if canine lymphosarcoina in a female Boxer dog [17]: 48 of 50 tumor (:ells examined (:ontaine(l only one X chromosonle, and the remaining two (Jells la(:ked b/)th X chromosomes. In the present study, one X chromosonle was missing in at least one metaphase in ea(:h (;ase, but in l l o n e of the cases was loss of the X chromosolne observed in a majority of the (Jells. In one female (tog [M(TI'-01), one (;-bande(I metaphase (:learlv lacked both X chromosomes. Loss of the X chrolnosonle in the canine mast (:t;11 tumors analyzed in this study may have been ran(lore event, together with loss of iildividual autosomes. The (:anine X, however, is easily identified, and the indivi(hml autosomes are not. I,oss of an X chroin(isome has prognosti(: signifi(:an(:e in some [roman leukemias. A poor prognosis has been associated with loss of an X (:hromosome in acute myelogenous leukenfia (:ells with an 8;21 translocation [27]. The signifi(:ance of the loss of an X c]lrolnosome in (;alliO(; neoplastic (;ells has not been (]etermilled. A tetrat)loid metaphase with a 2n 1)t 156 was observed in one (if tim (:anine lllast cell tumors in this study [MCT-04). Tetraploidv was reported in (me other (:anine neot)lasm, the leukeini(: (:ells of a dog with mono(:yti(: leukemia 1191. Although a rare observation in human leukenfias, tetraploi(lv is not u n c o m m o n in bovine leukemia 1281. There was no clear-cut correlation between the metastati(: and the nonmetastatic
Cytogenetics of C a n i n e Mast Cell T u m o r s
111
mast cell t u m o r s and the degree and kind of k a r y o t y p i c a b n o r m a l i t i e s present. O n e of the metastatic t u m o r s (MCT-02} s h o w e d a m a r k e d variation in ( : h r o m o s o m e n u m ber, but the o t h e r metastatic mast (:ell tuinor {MCT-03) was not s u b s t a n t i a l l y different from the two nommetastati(: mast (:ell t u m o r s in terlns of variation in c h r o m o s o m e n u m b e r or the o t h e r k a r y o t v p i c a b n o r m a l i t i e s e v a l u a t e d . O n e (if the nonmetastati(: mast (:ell tunlors (MCT-01) e x p r e s s e d the highest percentage of cells w i t h (:hromos o m e s breaks. The k a r y o t y p i c a b n o r m a l i t i e s o b s e r v e d in c a n i n e mast cell t u m o r (:ells w e r e similar in several respects to those reported for callille l y m p h o s a r ( : o m a s and fibrosar(:omas. In mast (Jell tumors, the c h r o m o s o m a l (:hanges w e r e quite variable: generally, the most f r e q u e n t l y o b s e r v e d (:hallges w e r e hypo- or hyf)erdil)loidy, an alm()rmal m o d a l c h r o m o s o m e nunfl)er, and an increase in metacentri(: forms. As with cytogenetic: findings of (:anine lymphosar(:()mas, no single k a r y o t y p i c feature (:haracterized the c a n i n e mast (:ell t u m o r s in tills s t u d y or in the one p r e v i o u s report. A m o n g (]omesti(: a n i m a l s , e x c e p t for CTVT, no single or (:onsistent (:ytogeneti(: a b l m r m a l i t y that chara(:terizes a specific n e o p l a s m has [)een (les(:ribed. An in(:rease in the n u m b e r of m e t a c e n t r i c ( : h r o m o s o m e s (:haract0rizes all types of c a n i n e n e o p l a s m s that h a v e been cytogeneti(:ally e v a l u a t e d 113 17, 19, 20, 2 2 - 2 4 , 261. T h e s e n o v e l metacentri(: c h r o i n o s o m e s reflect Robertsoifiall-type transh)cations or centric fusions. In this study, (:entric fusions a l o n e coul(t not a(:count for the h y p o d i p l o i d y o b s e r v e d in any (if the tumors. Cells from at least one of these tumors, M C T - - 0 2 , w e r e (:haracterized by the lwper(tipl()i(t m e t a p h a s e s h a v i n g a h i g h e r n u m ber of inetacentric forms than the h y p o d i p l o i d m e t a p h a s e s . Centric fusion of (:hromos o m e s in this t u m o r may have [)een a p r i m a r y e v e n t f o l l o w e d by stfl)sequent in(:rease in the n u m b e r of c h r o m o s o m e s . R o b e r t s o n i a n t r a n s l o c a t i o n s are not a c o m m o n feature of h u n l a n solid tuinors. That they c h a r a c t e r i z e d all types of c a n i n e solid t u m o r s so far e v a l u a t e d may partly reflect the large n u m b e r of acrocentri(:s available for R o b e r t s o n i a n fusion in this species. W h e t h e r the s a m e c a n i n e a(:rocentric c h r o m o s o m e s are i n v o l v e d in the t e n ( r i o fusions in the different (:anine t u m o r s has not yet been d e t e r m i n e d , h n p r o v e d c h r o m o s o m e b a n d i n g u l t i m a t e l y will p e r m i t identifi~:ation of the specific c h r o m o s o m e s i n v o l v e d , as well as other possibly c o n s i s t e n t cytogenetic a b n o r m a l t i e s . T h e role of gene and c h r o m o s o m a l r e a r r a n g e m e n t s in neoplasti(: (Jells is of major interest in s t u d y i n g h u m a n cancer. S i m i l a r o n c o g e n i c processes probably operate in (:anine m a l i g n a n c i e s , and identification of c a n i n e m a l i g n a n c i e s with a b n o r m a l (:yfogenetic features is an i m p o r t a n t step in d e v e l o p i n g a n i m a l mode, Is for neol)lasia. Supported in part by Grant No. RR0(/515 from the Nalional Institutes of Health and by grants from the Morris Animal Foundation and lhe American Cancer Society.
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7. Peters JA (1969]: Canine masto(:ytoma. Excessive risk as related to ancestry. I Natl Can(:er lnst 42:435-443.
8. Yunis IJ (1981): New c h r o m o s o m e techniques in tile study of h u m a n neoplasia. Hum Palhol 12:540-54(3. 9. A n o n y m o u s (1986): Laboratory Procedure, Cytogenetics. Protocol for Preparation of (]hrom o s o m e s From Cultured Bone Marrow, Peripheral Blood and Lymt)h Node as Relates to (]hromosome Studies in Malignan(:y. Kaiser Perlnanente Hospital Cytogeneti(:s Laboratory. Portland, Oregon. 10. Priest ]H (19771: Medical Cytt)genetit:s anti Cell Culture, 2n(t Ed. Lea & Febiger, Philadelphia. 11. Seabright M (19711: A rapi(l b a n d i n g l e c h n i ( t u e for h u m a n (:hromosomes. Lancet 2:(`)71-972. 12. Sandberg AA (1980): The C h r o m o s o m e s in [tumat~ Cancer and Leukenfia. Elsevier S(:ien(:e Publishing (]o., New York. 13. Barski G, Corneferl-Jensen FR (1966): (]ytogeneti(: study of Sticker venereal sarcoma in European (togs. J Nat] Cancer Inst 37:7{~,7-7(,17. 14. Murray M, lanes ZH, Martin WB {196(`)1: A study of tim cytology and karw~type of the, canine transmissiblewmere, al lum()r. Res Vet S(:i lt):565 568. 15. Oshimura M, Sasaki M, Makino S (1973): (]hronmsonud banding patterns in primary and lranst)lante(l venereal lmiiors of lilt*, dog. I Natl (]allcer lnsl 51:11{)7 1203. 16. Basrur PK, (;ilman PW (1966]: (]]Iromosonm studies in (:anine lymphosar(:olna. (]ornell Vet 56:451-46(.). 17. Benjamin SA, Noronha F (1(.)67): (]ytogenelic studies in (:anine lyml)hosar(:oma. Cornell Vet 57:526 542. 18. Hare WCI), Weber WT, M(:Feely RA, Yang T ( 1966): (]ytogetlelit:s in the (tog an(l (:at. I Small Anita Pra(:t 7:575-592. 19. Grindem CB (19831: Morl)hologic, (:yto(:hemi(:al an(t (:ytogencti(: t:haracterization of dog an(t cat leukemic (:ells. Vet S(:i 44:200. 20. Grindem CB, Buoen LC {1986): (]ylogenetic analysis of leukaemic t:ells in the (log. A reporl of 10(:ases and a review of the literature.] Comp Pathol 96:623 635. 21. Swayne DE, Michalski K, Mc(]au D (1987): Cutalmous lylnphosar(:oma with a|mormal (:hro ll]OSOIlleS ill (t (tog. I Comp Palhol (`)7:(}0,9-614. 22. ldowu L (1976): Observations (m the chromoson]es of a l y m p h o s a r c o m a in a dog. Vet Re(: 99:103. 23. Mile, s (]P, Moldavannu (;, Miller ])G, Moore A (1970]: C h r o m o s o m e analysis of (:anine lymphosarcoma: Two cHses involving I)robable centric [usioll. Am J Vet Res 31:783-790. 24. Sonoda M, Nhyama M, Mori M (197(I): A (:ase of t:anille, fibrosar(:oma with abnormal c h r o m o s o m e s , lpn I Vet Re,s 18:145 151. 25. Duncan J, Prasse K (1979): Cytology of canhle C/llaneotls round (:ell tumors. Mast (:ell tumor, histiocytoma, lyml)hosar(:oma an(t transmissible, venereal tumor. Vet Pathol 16:673-679. 26. Post IE, NoroIlhtl F, Richard (;(; (1970): (]anine lllaS| (:ell leukemia. In: (]omparatiw,~ Leukemia Research. Pro(:c~edings of tim International Symt)osimn on (]omparative Leukemia Research, RM l)utcher, ed. S. Karger, New York. 27. Golomb HM (1982): (]hromosome abnormalities in adult acute leukemia: Biologic and the, rapeutic significance. In: Adult l,eukemias I, CD Bloomfield, ed. Marlinus Nijhoff, The Hague, pp. 127-153. 28. Hare W(]D, Yang TI, McFeely RA (1967): A survey of (:hromosome findings in 47 cases of bovine lymphosar(:oma (leukemia). J Natl (ranter lnst 38:383-392.
ABSTRACT: We evaluated fl)ur (:anine (,~utane(ms mast (:(~11 tumors (:ytogeneti(:ally. All lout tum(n's (:(retained bath hypodipl(~id and hyl)erdipl(~id (:ells, an in(:r(~as~: in the numb~!r ol me3a(:entri(: chromosomes, ex(;hange cotd]gurations, and (:ells showit~g loss o!an X chr(;mosome. All ttml()rs (:(retained nmtaphases with chromosome gaps (rod breaks at Jre(lueneies greater tha~ ot)serw~(t spol~taneous chromosome breaks in normal (:ultured (:(mine peripheral blo()d l~mph(J(:vt~s. Three (~j the four tumors had a normal modal (:hromostm~e numt)e!r (ff 7~. The !ourth lam(~r had a m()dal (:hromosonle number of 93. ~vhi(:h r(~preset~ted 15% ()f the (:ells evala(ded from this tHIllor.
INTRODUCTION T h e r e h a v e been m a n y (:ytogenetic studies of h u m a n t u m o r (:ells and an increasing n u m b e r of n o n r a n d o m c h r o m o s o m e aberrations h a v e been d e t e c t e d in bu(na(~ leukemias and l y m p h o m a s and in solid t u m o r s [1, 2]. Little information, h o w e v e r , is available on a n i m a l neoplasia, and, in particular, naturally ()(:curring soli(I tumors. S t u d i e s of solid t u m o r s in a n i m a l s shouh] greatly facilitate our u n d e r s t a n d i n g of the o n c o g e n i c process, c a n c e r geneti(:s, and c a n c e r cytogenetics: e.g., t u m o r s in a n i m a l s can be f o l l o w e d o v e r t i m e for c y t o g e o e t i c changes, w h e r e a s huinan m a l i g n a n c i e s are f r e q u e n t l y r e m o v e d or treated w h e n d i a g n o s e d . The d o m e s t i c dog is a p o t e n t i a l l y useful a n i m a l m o d e l for study of s p o n t a n e o u s tumors. M a n y dogs live an e x t e n d e d life an(I are p r o v i d e d w i t h veterinary m e d i c a l care, p e r m i t t i n g d e t e c t i o n an(t diagnosis of m a l i g n a n c i e s . Of all domesti(: animals, dogs most i n t i m a t e l y share the h u m a n e n v i r o n m e n t , are expose(t to m a n y of the s a m e e n v i r o n m e n t a l (:ar(:inogens and m u t a g e n s as h u m a n s , and d e v e l o p m a l i g n a n c i e s i d e n t i c a l to m a n y h u m a n m a l i g n a n c i e s . C a n i n e c u t a n e o u s mast cell t u m o r s w e r e selected for cytogeneti(: e v a l u a t i o n because t h e y are one of the most c o m m o n skin t u m o r s in clogs, corot)rising 7 - 2 0 % of all skin t u m o r s in this species [3, 4]. In a d d i t i o n , these t u m o r s l)rogress sh)wly, though all are c o n s i d e r e d p o t e n t i a l l y inalignant [5]. C o n s e q u e n t l y , changes in c y t o g e n e t i c characteristics (:an be f o l l o w e d with p r o g r e s s i o n from relatively low-grade m a l i g n a n c y to quite high-grade m a l i g n a n c y . A n o t h e r r e l e v a n t feature of mast (:ell t u m o r s is the b r e e d - a s s o c i a t e d p r e d i s p o s i t i o n for this t u mor in dogs. Boxers h a v e 9.9 16] to 16.7 [ 7] the risk of d e v e l o p i n g (:utaneous mast cell t u m o r s as c o m p a r e d with o t h e r dogs. Thus,
From tlu~ Department of Veterinary Mi(:r~)[fiolo~yand l'atholo,~v, Washin,~ton ~tat~ (Iniw~rsity (I). M. ,%, D. 1. ILL IJullm;m. Washington, and l)el)artlnellt of l~athology/(]ytogeneih:s. Kaiser lh~rmalmllh~ Northwesl (P. B. ].), Portland. Ores,on. Address reprint requests to: Diana M. St(me. I).V.M., Ph.D., Department of Veterinary Mi(:robiology and Pathology, Washington State University, Pulhnan. WA 99164 7040. He(:e,ived luly 26, 1990: ac(:e,pted ,~eptember 6, 1990.
105 r; 19(31 Elsevier S(:iell(:ePublishing Co., In(:. 655 Avlmul! of lhe Americas, New York, NY 1()()1(1
(]an(:er (;enet (~y~ogcnci53:11}5 112 ( 1991 ) 0165-4608/91/S03,50
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this canine tumor may prove a useful model for both heritible and cytogenetic cancer studies of solid tumors. Our study was designed to determine whether cytogenetic abnormalities exist in canine mast cell tumors and, if so, to define the range of such abnormalities and determine whether any consistent cytogenetic changes could be detected.
MATERIALS AND METHODS Specimens of canine mast cell tumors were obtained by biopsy from four dogs presented to the Washington State University Veterinary Medical Teaching Hospital between June 1987 and May, 1989. Case MCT-01 was a 12-year-old female Labrador retriever; case MCT-02 was a 5-year-old female Sbar Pei; case MCT-03 was an 11year-old female Basset hound; and case MCT-O4 was a 6-year-old male Boxer. None of these dogs received any chemi(:al or radiation tberapy before tumor excision. Mast cell tumors were initially diagnosed by tumor aspirate and later confirmed by histopathology. Clinically, two of the (:ases (MCT-02 and MCT-03) were characterized by metastatic lesions of previously diagnosed solitary cutaneous mast cell tumors. Case MCT-02 had metastatic lesions involving the skin, lympb nodes, both kidneys, and tbe pancreas. Case MCT-03 had multiple lesions involving the skin. A (:omplete necropsy and histologic evaluation were not permitted in this dog: thus, only the cutaneous masses were observed and verified as being mast (:ell tumors. The other two cases (MCT-01 and MCT-04) were dogs with solitary, (:utaneous mast cell tumors with no evidence of metastasis. Sections of tumor for culture were obtained using sterile technique and pla(:ed in a Petri dish containing warmed (37°C) RPMI 1640 medium; short-term cultures were initiated using the t)rotocol of Yunis [8] as modified by the Kaiser Permanente Hospital Cytogenetics Laboratory in Portland, Oregon, for preparation of chromosomes from lymph node biopsies for cytogenetic study [9]. Sections of tuinor tissue were rubbed with a fine mesh screen to dislodge tumor cells. Aliquots of tumor cells were stained with toluidine blue to ensure that they were mast cells. Cells were washed twice in m e d i u m and resuspended at l × 10" cells/ml in warmed (37°C) R P M I t 6 4 0 m e d i u m s u p p l e m e n t e d with 15% fetal calf serum, 2% L-glutamiile, and containing penicillin (50 U/ml), streptomycin (50/xg/lnl). Cultures were established in 25-cm ~ flasks, each containing 5 ml suspe, nded tumor cells incubated at 37°C in 5% CO z. Cells were harvested for cytogenetic analysis at 1 hour, and at 4. 12. 24, and~or 48 hours of culture time, d e p e n d i n g on the n u m b e r of metaphases obtained in previously harvested samples. Thirty lninutes before cell harvest, 25/~1 of 10/~g/ml Colcemid was added to each flask. Cells were harvested and processed for metapbase chromosome spreads according to conventional methods with hypotonic treatment in 0.075 M KCI (5 minutes) and fixation in 3:1 lnethanol :acetic acid [101. Some preparations were standard-stained with Giemsa and others were aged at room temperature for 3 days and G-banded 111].
RESULTS Table 1 summarizes the cytogenetic characteristics of the four canine mast cell tumors. All tumors showed variation in the n u m b e r of chromosomes per cell, but three of the four tumors showed a normal canine modal chromosome n u m b e r of 78 at varying frequencies (MCT-01, MCT-03, MCT-04). The fourth tuinor (MCT-02) had a modal chromosome n u m b e r of 93 in 15% of the cells evaluated. Tbus, all tumors contained
44 41 76 74
86 109 83 156
(2n]
(yr)
MCT-01/F/11 MCT-02/F/5 MCT-o3/F/11 MCT-04/M 6
IlklnlbeFs
Range of chromosome Diploid (2n = 78) (%) 35.0 0.0 69.0 77.8
ttypodiploid (%)
50.0 33.3 14.0 5.5
15,0 66.7 17.0 16.7
Hyperdiploid (%) 20 27 29 18
Metaphases evaluated
Cytogenetic characteristics of four canine mast cell tumors
Case/sex/age
Table 1
78 93 78 78
Modal no.
÷ + +
Extra metacentrics
4 + + 4-
Loss of X-chromosoinc
20 7 10 6
Metaphases with chromosome breaks (%)
0 i + (I
Metastatic
"-.1
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P
e
D
0
e! F i g u r e 1 A (;-banded hypt)diploi(I metal)hase tr(ml (:;mitre mast (:ell t t m m r MCT-02 f r o m a l'emale Shar Pei dog. T h e mctal)hase ctmsish~(t t)f 51 a(:i'¢)cimli'il; ( h r o l n o s o l n e s in,'-;lea(I of lh(! m)rmal t:anine, diploid lltllllbOr of 7,bl, Neitlmr of lhe, y;tlhll/(?hlt;l!tlh'i(: X chrolllOSOllley; was [)reselH in the m e t a p h a s e .
a substantial n u m b e r of both hypodil)h~id (Fig. 1) and hyperdiploid (Fig. 2) (:ells. The tumor with a modal n m n b e r of 93 showe(t extreme variation in (:tm)mosome numbers, whi(:h ranged from 41 to 109. All four tumors containe, d metaphases with extra meta(:e, ntri(: chromosomes. The canine karyotype is normally composed of all acro(:cntri(: (:hromosomes except tim submeta(:entric X and Y (:hromosomes. An increase in tmnor cell metacentric forms was observed in both hyt)o- and hyt)erdit)loid metal)bases (Fig. 3). In one case [M('T02) the hyperdi ph)id metal)bases contai ned a higher n m n b e r of abnormal meta(:entric forms than did the hypodii)loid metaphases. The other three cases bad insufficient numbers of either hypo- or hyperdiploid metaphases h) make a valid (:omparison. In none of the hypodiploid nletapbases (lid the increase in tnetat:entric forths a(:(:ount for the hyt)odiploidy. All tumors contained (Jells showing loss of an X chromosome. Exchange configurations in the form of quadrara(lials and telomeri(: associations were also observed in some tumors. All tumors (:ontaine(t metaphases with spontaneous chromosomal gaps or breaks. The percentage of (:ells containing these chromosome gaps or breaks varied from 6 to 20% of the cells. This was significantly greater than the observed spontaneous chromosomal breakage rate (<2%) in normal cultured canine peripheral blood lymphocytes from 17 dogs (Stone, 1989: u n p u b l i s h e d observations). Definitive identification of the origin of some observed melacentric chromosomes or of some karyotypic abnormalities su(:h as apparent deletions or translocations was impossible owing to the variability or resistan(:e of the tumor (Jell chromosomes t() Gbanding, overly condensed (:hromosomes, comparatively small numbers of analyzable initotic (:ells under sonle (:ir(:umstances, and the morphologic similarity of the small (:anine a(:rocentri(: chromosomes. Cytogeneti(: ewduation of stinmlate(I t)erit)heral
109
~
It'
U
N
I.. ~' ~
,,C Y F i g u r e 2 A (Vbandcd hyperdil)loid mctat)h~tsc from (:allille lllttS( t:cll tumor M(;'I'-02. 'l'tw metaphase consisted of 104 c:hromosomes instead of l]m normal c;anim~ dil)hfid number of 78. No X chromosomes could b¢~ idt;ntificd.
F i g u r e 3 A (]-banded h y p c r d i l , l o i d metaph~nsc fronl c a n i n e mast c:~,~lllul~lor N.1(71'-02, wilh ~lll increased number of subme, tac;entric and metac:cmlrit: t:hromosC,lneS. Ei~,ht st~l)m~h~ccnlric: or II](,qilC:(HltI'iC (:hl'oI//OSOlll{,~S W(31"(!ichmtil'i¢~d f a r r o w s ) instead of tim llOrllldl [l~ll]~ll(~ c;~tllilll~ IllllIiht~l" of two subme, tac;entri(: X (:hromosom~s.
J
J
l
1
110
I). M. S t / , , , (, el.
blood lympho(:yte cultures fron/ all four cases sh()we(t that each dog ha(I a normal diploid number (2(1 78) in lyInpho(:~/tes.
DISCUSSION The (:ells of many Inmmn malignant ne(Jl)hlsms have abnornml cytogeneti(: features [12[. There have been few reports of cytogeimtic analysis of neoplasti(: (:anine tissue, other than the canine transmissible venereal tumor (CTVT), which is a stable coitally transmitted allograft. CTVT has generally been shown to (:ontain 57 59 chromosomes, with a modal c h r o m o s o m e number of 59 [13-15]. This reduction in chromoson~e number is consistent with extra meta(:entric fusion (:hrom(isomes. Variation in ¢:hrom o s o m e number and other karyotypic almormalities have als() been report(xl in (:ells from cases of caniim lyml)h(isarcoma ]16-231. Karyotypi(: almormalities in lymphosar(:oma cells include hypodiploi(ty, hyperdiploidy, (:hange in the modal chromosome number, extra metacentric forms, (touhh; mimlt(; ((Imin) chromosome, s, exchange configurations, an(I loss 1)t an X (:hromosome. No single sl)ecific karyotypic abnormality characteristic of these (:ells has been identifie, d. There has been one report of a (:anine fibrosarc()ma with a redu(:e(] number (if (:hr(m)(is/mles and an increased number of metacentric forms 124]. Cutaneous mast cell tumors ;ire (:lassified as roun(t (:ell tumors, along with histio(:ytomas, lymphosarcomas, and CTVT 1251. The only report in the literature on a (:ytogenetic study of canine mast (:ell tumors involve(t analysis of two such tumors. Karyotypic analysis disclosed a pattern of reduction in the total number of chromosomes with an increase in the proportion of metacentric h)rms [261, similar to the pattern characteristic of CTVT, s(ime canine, lymphosar(:omas, and (:anine fibrosarcomas. In one dog, the modal (:hromosome munher (if the neoplasti(: mast (:ells was 40: in the other, the m(idal numl)er was 50. This cytogeneti(: analysis (if f(iur (:anine mast cell tulnors is the first report of both h y p o d i p l o i d an(t hyperdil)loi(t (:ells in the same tum(ir, and the first report of a canine mast cell tumor with a hyperdiploid modal (:hromosome lmmber. In this tumor (MCT02), the modal c h r o m o s o m e number ()f 93 was present in 15% of the (:ells, suggesting that this karyotypic abnormality was derived from a single progenitor (:ell. This report is also the first (lescription of chromosome, breaks, exchmlge (:(mfigurations, (:entri(: ftlsiolls, and loss of all X (;]lromos(ime in canine mast cell tmnor cells. The consistent loss of one X (:hromosome was reported in one case (if canine lymphosarcoina in a female Boxer dog [17]: 48 of 50 tumor (:ells examined (:ontaine(l only one X chromosonle, and the remaining two (Jells la(:ked b/)th X chromosomes. In the present study, one X chromosonle was missing in at least one metaphase in ea(:h (;ase, but in l l o n e of the cases was loss of the X chromosolne observed in a majority of the (Jells. In one female (tog [M(TI'-01), one (;-bande(I metaphase (:learlv lacked both X chromosomes. Loss of the X chrolnosonle in the canine mast (:t;11 tumors analyzed in this study may have been ran(lore event, together with loss of iildividual autosomes. The (:anine X, however, is easily identified, and the indivi(hml autosomes are not. I,oss of an X chroin(isome has prognosti(: signifi(:an(:e in some [roman leukemias. A poor prognosis has been associated with loss of an X (:hromosome in acute myelogenous leukenfia (:ells with an 8;21 translocation [27]. The signifi(:ance of the loss of an X c]lrolnosome in (;alliO(; neoplastic (;ells has not been (]etermilled. A tetrat)loid metaphase with a 2n 1)t 156 was observed in one (if tim (:anine lllast cell tumors in this study [MCT-04). Tetraploidv was reported in (me other (:anine neot)lasm, the leukeini(: (:ells of a dog with mono(:yti(: leukemia 1191. Although a rare observation in human leukenfias, tetraploi(lv is not u n c o m m o n in bovine leukemia 1281. There was no clear-cut correlation between the metastati(: and the nonmetastatic
Cytogenetics of C a n i n e Mast Cell T u m o r s
111
mast cell t u m o r s and the degree and kind of k a r y o t y p i c a b n o r m a l i t i e s present. O n e of the metastatic t u m o r s (MCT-02} s h o w e d a m a r k e d variation in ( : h r o m o s o m e n u m ber, but the o t h e r metastatic mast (:ell tuinor {MCT-03) was not s u b s t a n t i a l l y different from the two nommetastati(: mast (:ell t u m o r s in terlns of variation in c h r o m o s o m e n u m b e r or the o t h e r k a r y o t v p i c a b n o r m a l i t i e s e v a l u a t e d . O n e (if the nonmetastati(: mast (:ell tunlors (MCT-01) e x p r e s s e d the highest percentage of cells w i t h (:hromos o m e s breaks. The k a r y o t y p i c a b n o r m a l i t i e s o b s e r v e d in c a n i n e mast cell t u m o r (:ells w e r e similar in several respects to those reported for callille l y m p h o s a r ( : o m a s and fibrosar(:omas. In mast (Jell tumors, the c h r o m o s o m a l (:hanges w e r e quite variable: generally, the most f r e q u e n t l y o b s e r v e d (:hallges w e r e hypo- or hyf)erdil)loidy, an alm()rmal m o d a l c h r o m o s o m e nunfl)er, and an increase in metacentri(: forms. As with cytogenetic: findings of (:anine lymphosar(:()mas, no single k a r y o t y p i c feature (:haracterized the c a n i n e mast (:ell t u m o r s in tills s t u d y or in the one p r e v i o u s report. A m o n g (]omesti(: a n i m a l s , e x c e p t for CTVT, no single or (:onsistent (:ytogeneti(: a b l m r m a l i t y that chara(:terizes a specific n e o p l a s m has [)een (les(:ribed. An in(:rease in the n u m b e r of m e t a c e n t r i c ( : h r o m o s o m e s (:haract0rizes all types of c a n i n e n e o p l a s m s that h a v e been cytogeneti(:ally e v a l u a t e d 113 17, 19, 20, 2 2 - 2 4 , 261. T h e s e n o v e l metacentri(: c h r o i n o s o m e s reflect Robertsoifiall-type transh)cations or centric fusions. In this study, (:entric fusions a l o n e coul(t not a(:count for the h y p o d i p l o i d y o b s e r v e d in any (if the tumors. Cells from at least one of these tumors, M C T - - 0 2 , w e r e (:haracterized by the lwper(tipl()i(t m e t a p h a s e s h a v i n g a h i g h e r n u m ber of inetacentric forms than the h y p o d i p l o i d m e t a p h a s e s . Centric fusion of (:hromos o m e s in this t u m o r may have [)een a p r i m a r y e v e n t f o l l o w e d by stfl)sequent in(:rease in the n u m b e r of c h r o m o s o m e s . R o b e r t s o n i a n t r a n s l o c a t i o n s are not a c o m m o n feature of h u n l a n solid tuinors. That they c h a r a c t e r i z e d all types of c a n i n e solid t u m o r s so far e v a l u a t e d may partly reflect the large n u m b e r of acrocentri(:s available for R o b e r t s o n i a n fusion in this species. W h e t h e r the s a m e c a n i n e a(:rocentric c h r o m o s o m e s are i n v o l v e d in the t e n ( r i o fusions in the different (:anine t u m o r s has not yet been d e t e r m i n e d , h n p r o v e d c h r o m o s o m e b a n d i n g u l t i m a t e l y will p e r m i t identifi~:ation of the specific c h r o m o s o m e s i n v o l v e d , as well as other possibly c o n s i s t e n t cytogenetic a b n o r m a l t i e s . T h e role of gene and c h r o m o s o m a l r e a r r a n g e m e n t s in neoplasti(: (Jells is of major interest in s t u d y i n g h u m a n cancer. S i m i l a r o n c o g e n i c processes probably operate in (:anine m a l i g n a n c i e s , and identification of c a n i n e m a l i g n a n c i e s with a b n o r m a l (:yfogenetic features is an i m p o r t a n t step in d e v e l o p i n g a n i m a l mode, Is for neol)lasia. Supported in part by Grant No. RR0(/515 from the Nalional Institutes of Health and by grants from the Morris Animal Foundation and lhe American Cancer Society.
REFERENCES 1. Yunis fl (1983): The chromosomal basis of Jmman neopJasia. Science 221:227 236. 2. Yunis ]J (1986): Chromosomal rearrangeulents, genes, and fragile sites in cancer: clinical and biological implications. [n: hnporiant Advances in Om:ology, VT De Vita, S Helhnan, SA Rosenberg, eds. I. B. Lippim:ott, Philadolphia, pp. 93 128. 3. Hottendorf GH, Nielsen SW (19671: Pathologic survey of 300 e,xtirpatc~d canine inash)cytomas. Zentrabl Veterinarmed 14:272 281. 4. Bostock I) (19731: The prognosis following surgical removal of illaSlO¢;Vtolnils ill dogs. J Small Anim Pratt 14:27-40. 5. Moulton JE (1978): Tumors in Domestic Animals. Uniw,'rsity of California Press, Berkeley, pp. 26 31. 6. Howard EB, Nielsen SW (1965): Neoplasia of the, Boxer dog. Am J Ve,t Res 26:112l 1131.
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7. Peters JA (1969]: Canine masto(:ytoma. Excessive risk as related to ancestry. I Natl Can(:er lnst 42:435-443.
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