Vet Pathol 30:195-203 (1993)

Granulated Round Cell Tumor of Cats M .

F.

M cENTEE,

S.

H ORTON,

J.

BLUE , AN D

D. J.

M EUTEN

Department of Pathobi ology, Co llege o f Vet erin ary Medi cin e, Unive rsity of Te nnessee, Kn oxville, TN (MFM); College o f Veterinary Medi cin e, No rth Caro lina Sta te Uni versit y, Raleigh, NC (SH , DJM ); and New York State College of Veterinar y Medi cine, Co rne ll Unive rsity, Ithaca, NY (JB) Abstra ct. Morphologic and biologic features o f five felin e granulated ro und cell tumors were com pared with those previou sly rep orted to be of globule leu kocyte and lar ge granular lymphocyte origin. Th e five cats ra nged from 6 to 9 years of age and present ed with non specific gastroi ntestina l signs. Fo ur of th e five cats were tes ted for felin e leuk emi a virus and were negative by enzyme-linked im m unoso rbent assay. Th e neopl astic process in volved the a bdo m ina l cavity in all cases , with a pred ilection for the distal sma ll intestin e and mesent er y. Th e liver and peripheral and th or acic lym phoid tissues were also spo radic ally affected . Neoplastic ro und cells co ntai ned 0.5- 1.5-J,l m eosino philic cytoplasm ic granules that were difficult to di scern on cas ua l obse rva tio n with hem at oxylin an d eosin sta in but were dee p blu e and eas ily visua lized when stain ed with ph osph otungstic acid -hema tox ylin. In two cases, epit heliu m in the affected ileum and liver contained un usuall y large num bers of a ppa rently normal globule leuk ocytes. Ultras tructura lly, the tum or gra nules tended to cluster at one nu clear pole and were spind le to ro und in sha pe with variably dense co ntents. Some gra nules co ntained a den se "cap" at one end or int ern al crystalloid bars that d istort ed the gra nule membran e. Th e tum ors rep ort ed her ein are simil ar to all three o f th e previously reported felin e gra nulated round cell tum ors and prob abl y hav e a common cellular origin.

Key words: Ca ts; globule leukocyte; large granu lar lymphocyte; mas t cell; neo plasia .

Three granulated round cell tumors (GRCT) have been reported in cats. The globule leukocyte, in two cases, a nd th e large gra nular lymph ocyte (LGL), in one case, were pr oposed as cells o f origin based on morph ologi c a nd histo ch em ical co m parisons with normal cells and with LGL in vol ved in th e Iymphoproliferati ve diseases o f human be ings and F-344 rat s.8.9 •14 Harvey et al.!' recently suggested that two of th ese feline neoplasms may ha ve a co m mon origin but did not co m m it to what th at origin might be. The ontogen y and fun ct ion of globule leuk ocytes rem ains spec ulati ve,1alth ou gh so me reports indi cate th at th ey ma y be derived fro m mu cosal ma st cells;1 5.16.22 tum or s o f globule leuk ocytes have not been reported in other species. LGL have natural killer (NK) cell activity and are well cha racte rized in lab oratory rodents a nd human beings." NK cells and LGL ha ve al so been de scribed in

cats" -" G lobule leukocytes, granulated int raepitheli al lymph ocytes (IEL; so me IEL have morph ologic features o f LGL6.28 but ha ve, as a who le, minimal NK-cell activity"), mast cells, enterochromaffin cells, pla sma cells, eosin ophils, and other m yelogenous granul ocytes are pre sent withi n th e intestinal tract " and th erefore co uld produce granulated cell neoplasm s withi n th e a bdo me n. A lthough cytomorpho logy, histoch emist ry, a nd pattern of neopl asti c gro wth will d istingui sh tu-

m ors ca used by m an y of th ese cells, th e lack of specific markers for various feline cell types, as well as a poor under standing of th e origin a nd fun cti on of cells such as th e globule leu kocyte , mak es definiti ve di agno sis difficul t. The purpose o f th is study was to cha racterize a group o f feline G RC T a nd to co m pa re th em with previousl y reported cases.

Mater ials and Met hods Five st udy cases and two feline intesti na l mast cell neoplasm s were received as forma lin-fixed surgical biopsies. The oppo rt unity to per form a necropsy was not provided in any o f the study cases. Norma l ileum from a matu re cat was placed in Carne y's, 10% neutral buffered formalin , or Trump's fixativ e. Normal ileum from a mature mou se was fixed in 10% neut ral buffered formalin for immun ohi stochemi cal exam inatio n. Paraffin-embedd ed tissue was sectioned at 5 J,lm and sta ined with hem at oxylin and eosin, Wolbach 's Modi fied G iems a," 0. 5% toluidi ne blue (p H 4.0), Luna's method for eo sinophil or mast cell gran ules," phosphotungstic acid- hemat oxylin," per iodi c acid-Schiff, alcian blue for acid mu cin s (p H 2.5),-' azu re-A, or chloracetate esterase act ivit y. G ra nulat ed ro und cell tum or cells in sme ars from lymph nod es o f cat No s. 3 and 4 were exa m ined for chloracetate este rase , pero xidase, acid ph osph at ase, and leu kocyte alka line phosphatas e ac tiv ity.' Immu nohistochem ical exa mination, using ro utine pero xid ase- ant iperoxidase tec h n ique s, was per forme d on deparaffinized tissue sections with a prim ary ra b195

196

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Table 1. Age (year s)

Summary o f signa lment, clinica l signs , and the location of lesions in five cats.

Sex*

Breed]

6

F/ s

DLH

neg

2

6.5

M/c

DSH

not don e

3

8.3

F/s

DSH

neg

4

9

F/ s

DSH

neg

5

8

M/c

DSH

neg

Ca t No .

Vet ra tho l 30:2, 1993

FeL V Status j

Presentat ion

vomiting, anorexia ; 3 month wt loss I week anorexia and wt loss 3 m onth a no rex ia , vomiting, wt loss ; diarrhea on presentation lum ps on neck ; sporadic but in . . creasmg anorexia and lethargy

2 month vomiting, 2 week marked wt loss, good a ppetite

Signs

Lesion s

palpable abdom inal ma ss icterus, palpabl e abdominal mass palpable ab dominal mass

distal ileum and mesen tery mesentery, liver

en larged peripheral nodes; palpable ab dominal mass ; thoracic and pr escapular aspi rat es = granulated ce lls thickened j eju num

cranial m ed ias tinum , me sentery, peripheral lymph nodes (especia lly pr escapular)

mesenteric nodes, di stal ileum , cra n ia l mediastinum

j ejunum (liv er histologicall y normal)

* F/ s = spaye d fem ale; M/ c = cas tra ted male. DLH = dom esti c longha ir ; DSH = dom est ic sho rtha ir. :j: neg = negat iv e by enzy me-linked immunosorbent assay .

t

bit polyclonal ant ihi stamine ant ib od y (Ch cmicon , El Sugundo , CA ). Tissues wer e fixed in formalin (study ca ses) o r Trump's fixa ti ve (n orm al ileum) and processed for ultrastructu ra l exa m ina tio n.

Results A summary of clinical and pathologic findings from study cases (cat No. 1-5) arc presented in Table I. All cats presented with a I-week to 3-month history of weight loss, and most demonstrated anorexia , vomiting, and lethargy. On physical exam ination, four cats (Nos. 1- 4) had palpable abdominal masses, and one (No.4) also had en largement of peripheral lymph nodes. Rad iographs rev ealed a cranial mediastinal mass in two (Nos. 3, 4) of the three cats eva luated and thoracic fluid aspirated from cat No.4 contained granulated round cells. Clinical chemistry and hematologic changes were not diagnostic. Four of the five cats (Nos. 1, 35) were tested by en zyme-linked im m unosorbent assay for antibodies to feline leukemia virus at least once and were negative. Abdom inal palpation , surgery, or radiography iden tified abdominal masses in all five cats. The terminal ileum was affected in two cats (Nos. 1, 3) and another (cat No .5) had m ultifocal thickenings (1 ern) of the jej una l wall. Cat No .2 (Table 1) had extensive mes-

enteric involvement, which precluded evaluation of the in testinal tract. Enlarged mesenteric lymph nodes were specifically ide ntified in cat NO.3. Cat No . I died of complications 2 days aft er surgery, and cat Nos. 2 and 5 were euthanatized with a poor prognosis after continued clin ical illness. Cat No.3 was euthanatized 7 months after surgery because of seizures and paraplegia of undetermi ned cause. Cat NO.4 (animal with the least signs of systemic illness) was tr eated with vincristine, predn isolone, and cytoxan and has been in remission for approximately 20 months , except for a persistently enlarged prescapular lymph node that regressed to norm al size approximately 16 months following initiation of therapy. Aspirates of the enlarged node consistently yielded neoplastic round cells with morphologic features similar to those in previo us reports.r-! ' Nuclei were ind ented to round and contained coarse ly granular to co rded chromatin . A perinuclear region of pale staining cytoplasm contained ro und to oblong granules that were purple in air-dried WrightGeimsa-stained preparations. Diagnoses were made on the basis of biopsies, whi ch incl uded samples from an abdominal mass (cat No.3) , left prescapular lym ph node (No. 4), liver and mesenteric lymph node (No. 2),jej unum (No .5), and ileum (No.1). Histologically, these samples contained a d if-

Table 2.

197

Felin e G ra nulated Neo plasms

Vet Peth e l 30:2. 1993

Re sults* of h ist o ch emical sta ini ng of cyt opla smic gra n u les in formal in-fi xed, paraffi n -embedded feline ti ssu e s.

G ran u lated ro u nd ce ll tum o r (st ud y ca se s) G lob u le leu ko cyte t u mor N o . I x G lob u le leu ko cyt e tum o r N o . 2' 4 Large gran u la r lymph o cyt e tum or (feli ne)" Intest in al m a st ce ll

neoplasm s'[

HEt

PT AH t

T B§

Gie msa

re d

+

a

a / re d

re d

+

a

a

NO

a

re d

+

a

a

NO

+

NY

+

a

a

NO

a

blue

a

+

+

a

+

rcd

+

a

a /red

red blue

a a

a

a

+

+

Lun a ll

ato

+

PAS#

+

N ormal ce lls of the felin e inte stine G lo b u le leukocyte s Intraepithelial lym p h o cytes M a st ce lls

a to a a

+

a a a

* a = negati ve (no metach rom asia with T B and Gicmsa); NV = not visible at less tha n 1,000 x magnificati on ; ND = not don e. t HE = hem at oxylin and eos in. t PTAH = phosph otungstic acid-hema tox ylin. § T B = tolu idin e blue. Lun a for mast cells and Azu re-A dem on strate metach rom asia simi lar to th at of T B and G iemsa sta ins (indicated as + ). II Luna stai n for eosino phils. Differences in staining intensity, attr ibutab le to histotechni cal subjectivity in the differenti ati on step, wou ld be elimi nated if all sections were stained sim ultaneo usly, in- batch. # PAS = per iodic acid -Schiff. ~ Previou sly repo rted inte stinal mast cell tum ors.' fuse or locall y extens ive infiltrati on of round cells that effaced lymph nod e ar chitectu re and extended fro m th e small int estinal lamina propri a transmurall y to th e serosal sur face and adjacent mesentery. Liver from th e icteric cat (No. 2) contained aggregat es of granulated cells in po rtal areas, adjacent hepatic plates, and scattered throughout the parench yma ; bile canaliculi were di stended with secretion . Neoplastic cells were similar in all granulated round cell tumors (GRCT) and ranged from 10 to 17 11m in diameter with mostly indistinct cytoplasm ic borders and pale eosinophilic to clear cytoplasm of variable abundance. Nucl eus: cytoplasm rati os ranged from I : I to I : 2. Th ere was individual cell necrosi s and, in larger ma sses, central ar eas of necrosis. Most cells conta ined 5-1 2 (range of 4-30) ro und to elongate eosinophilic granules of various sizes that were often clustered in a perinuclear location (Fig. I). Elongated granules typically radi at ed from a central focus point. Mo st nucl ei were ecce ntrically located , co ntai ned clumped hetero chromatin and were ind ented , irregular, or round . Nucl eoli were prominent in one tumor where chro m atin was finely granular and di spersed. Eight to 40 m itoti c figures were present per ten high-power (400 x ) field s. Mo st neoplastic infiltrates contained ma crophages with accumulations of int ern ali zed eosinophilic granular debris. Ileal mucosa from cat No. I conta ined tumor cells and a remarkable number of cells in the adjacent villou s epithelium with morphologic features of normal

globule leu kocytes (Fig. 2). Cells with eos inophilic cytopl asmi c gran ules that were slightly sma ller and paler than th ose within th e epithelium di stended superficial lact eal s in some villi. Cells di stinctly similar to globul e leukocytes were also prominent within th e biliary epith elium and hepatic plat es of cat No.2 (Figs. 3, 4). Nuclei of the globule leukocyte-lik e cells in both cases resembled those of th e tumor cells, which had an extraepithel ial location and sm aller, paler granules. Results of histochemical studies are presented in Ta ble 2. Granules of the tumors described in thi s repo rt, tho se of the three previously reported cases , and those of normal globule leukocytes a ll stained dark blue with ph osph otungstic acid-hematoxylin (PT AH) (Figs. 2, 3), were eosinophilic (granules in hematoxylin and eo sin [H E]-stained sections were d ifficult to disc ern on cas ual ob servation), and lacked m eta chromasia with toluidine blu e and Gi em sa sta ins. GRCT granules in th e study cases stained with periodi c acid-S chiff(PA S), but previou sly reported cases had variably positi ve granules. Neoplastic cells in sme ars and ti ssue sections were uniforml y negati ve for chlorace tate esterase , perox idas e, acid phosph ata se, and leukocyte alkalin e phosph at ase acti vit y. Ultrastructurally, nucl ei were ind ent ed (Fig. 5) or, less oft en , round with peripheral heterochromatin and a single nucl eolus of variable size. Cytoplasmi c membranes had smooth contours with occasional clathrin pits. In one case (cat No .4), short filopoid pr ojections

198

McEntee et a1.

Vet Pathol 30:2. 1993

Fig. 1. Abdo mi na l mass ; ca t No .3 . T umo r sectio n I ~ m thic k shows cells co nta ining ro und to spindle-shaped gra nules of vario us sizes th at radia te aro und a central perinu clear focal point. T olu id ine blu e. Bar = 27 ~ m . Fig. 2. Ileum ; cat No. I. Tum or infiltrat ion of th e lamina propri a. Th e tumor cells (arrows) and the globule leu kocytelike cells in the epitheli um both conta in gra n ules th at stai n dark blue. PT AH . Bar = 30 ~m . Fig. 3. Liver ; cat No .2. Perip ort al infiltrate of gra nulated neoplast ic round cells (arrow) co ntaining PT AH-stained blue gra n ules. T he biliar y epithelium and hepati c cords co ntai n a large number of globul e leu kocyte-l ike cells. G ran ules in these cells are larger than th ose in th e tum or cells but also stained int en sely blu e. PT AH . Bar = 30 ~m . Fig. 4. Electro n m icr ograph . Liver ; ca t No.2. Bile du ctul e co ntains a histologically normal globule leu kocyte (arrow) . G ra nulated tumo r cells are in the adjace nt peripor tal a rea (arro whead). Bar = 1.8 ~m .

were scatte red over the surfac e of the cells, but there was no ev ide nce of int erdigitation with adja cent processes. Mo st cells were organelle poor , containing few round to elongate mitochondria, profiles of rough endoplasmi c reticulum (occasionally in parall el arrays),

smoo th and coated vesicles, multivesicular bodies, myelin figures, and polyribo som es. On e or two centriol es, Golgi structures, and membran e-bound granules were typically adjacent to the indentation in a perinuclear location (Fig. 5).

Vet !'ath ol 30:2. 1993

Feline G ra n ulated Neoplas ms

199

Fig. 6. Electron micrograph. Abdominal mass; cat No. 3. Perinuclear granules are oriented around centrioles. Bar =

Fig. S. Eleetron micrograph. Jejunum, tumorous infil trate; cat No.5. Tumor cells contain indented nuclei and perinuclear granules with intragranular crystaloid bars. Bar =

1.3 Jlm .

G ra n ules differed in size, sha pe, co nte nt, and number from cell to cell and from tum or to tum or. Most were round and 0.5- 1.4 urn in di am et er , but so me were as large as 3.7 Jlm . Spind le-sha ped gra n ules were up to 3.3 urn long and 0.8 Jlm wide, co ntai ned faint electr on-lucent longitud inal lin es, and were arranged in a spo kelike manner aro und a centriole(s) (Fig. 6). T he maj orit y o f granules were filled with a co mpact am orph ou s to fibrillar or granular mat eri al. Occasional granules had a rim that was either relati vely electr on lucent o r den se, some granules conta ined smaller electronden se cores and/ or m embrano us profi les, and others had a d iscrete round to ovoid ca p of electro n-de nse material at on e pole. Two tum ors co ntai ned granules with electro n-dense bars (up to 30%) th at often d istorted th e granule m embran e (Fig. 5). Bars were 0.0230.22 Jlm wide by 0.5-2.0 Jlm lon g, with lon gitudinall y oriented fibril s. No rma l felin e intestinal mu cosa co ntained gran ulat ed intrae pithe lial lym phocy tes (l EL), ma st cells, and intraepithelial globule leukocy tes , all of which were identi fiabl e in forma lin-fixed , HE-stained sections. Very fine, loosely aggrega ted eos ino ph ilic gra n ules were ev ident at 400 x in IEL stained with HE , but th ese granules were not visible with any of th e other stai ns used in thi s study, including PTAH (Table 2). G lobule leukocyte granules in form alin-fixed sections were stro ngly positi ve with PTAH and negati ve to moderately positi ve with Luna's stai n for eos ino phils. These granules were negative with PAS and lacked metachro-

0.54 Jlm.

masia with Giemsa and tol uidine blu e stai ns. Cytoplasm ic gran ules of globule leukocytes in sectio ns fixed with Ca rnoy's so lutio n were not visi ble with any of the a bove stai ns, inc lud ing H E; wisps of eos ino philic ma terial in th e cytoplas m may have been gra n ules th at were d isru pted by thi s fixat ive. Subm ucosal and proprial m ast cells were metach romatic with tolu idine blue a nd Giemsa in both form alin- and Ca rnoy's solutio nfixed sectio ns . They were, however, rare in formal in fixed sections and co m mon (approxi ma tely tenfold m or e) in sections fixed with Ca rnoy's solutio n. Mast cell gra n ules were not vis ible in formalin- or Ca rnoy's so lutio n-fixed sectio ns using either PTAH or PAS stai ns . U lt rastruc tura l features o f normal globule leukocytes (Fig. 7) and other cell typ es were as pr evi ou sly described ." In G RC T exam ined immunoh istochemicall y, less th an 5% of th e cells were posit ive for histamine in two of th e tumors (th ese cells may ha ve been inte rspersed nonneopl asti c ma st cells), and th e othe r three tum or s were co m pletely negati ve. T wo felin e intestinal mast cell tum ors co ntained zs10% histam ine-positi ve cells. Both o f th ese mast cell neoplasm s co ntai ned man y cells with me tac hro matic gra n ules, non e o f whic h sta ined with PT AH. No rma l felin e cuta neo us m ast cells in for malin-fixed sect ion s were m od erate to strongly positive for histam ine as were normal subm ucosal intes tinal mast cells. Weakl y positive cells were scattered in th e intestinal lamina pr opria. G lobule leukocytes in th e ileum of normal cats and ca ts with GR CT were negat ive. Murine lam ina pr op ria mast cells in a normal sect ion o f ileum were faintl y positi ve for histamine, wh ereas ma st cells in th e mesentery and seros a were stro ngly pos itive; th is pattern o f stai ni ng is co nsis ten t

200

McEnt cc ct al.

Fig. 7. Electron micrograph . Ileum ; contro l cat. G lobule leuk ocyte is within the mu cosa o f the ileum. Th e granules of these cells o ften co ntai ned electro n den se "caps" at one pole (arrowhea d). Bar = 1.4 JIm .

with th e lower levels of hi stamine pr esent in mucosal m a st cells.' ? D isc uss ion The five neoplasms pr esented and th e three previ o us ly reported tumors o f putati ve globule leukocyte a nd large granular lymphocyte (LG L) origin are different from other felin e round cell tumors. Features that are common to thi s group o f gran ulated round ce ll tumors (GRCT) and help di stinguish them from other felin e neoplasms include I) cyto plasm ic gran ules that arc eosinophilic in hematox ylin and eosin -stained sections, stain deep blue with phosphotungstic acid-hematoxylin (PTAH), lack m etachromasia, and are ultrastructurall y heterogenous, 2) a pr edilection for abd ominal in vol vem ent (primarily di stal small intes tine and m esenteric lym ph nod es), and 3) a felin e leu kem ia virus (Fe LV) enzyme-linked immunosorbent assay -negative sta t us . We agree with Harve y et al. 13 that th e tw o previously re porte d neoplasm sv'" proba bly o riginated from th e sa me ce ll lin e and sugges t th at th ese two cases, our five st udy cases, and th e ca se reported by Finn and Schwartz" represent th e sa me entity. Subsequent di scussion is ba sed on this as sumpti on , gro uping th e eight ca ses together and noting unique indi vidual features. GRCT behave in a malignant m anner following a progressive and, in most in st an ces, fatal course. M ost cats present wit h advanced di sea se and nonspecific signs usuall y referable to th e gastrointestinal tract. Many ha ve palpable abdominal masses and , occasionally, pe-

Vet Pat hol 30:2. 1993

ripherallymph node enla rgement. Definiti ve di agn osis requ ires hi stopathologic or cyto logic demonstration of ro und ce lls co nta ini ng di st in ct blue PTAH-stained cytoplasmic granules. PTAH see ms to be a specific sta in for th ese granules, but th e gra n ular co m po ne nt to whic h th e sta in is binding is unkn own . PTA H has a high a ffi nity for ba sic a m ino ac ids and sta ins proteins blue whe n th ese clu st er in o ne segment of th e m olecul e." Protein s in Lew y bodies'? and M allory bodies!" sta in blue with PTAH , as do so me normal cyto plas m ic structure s (i.e., mitochondria). Therapy wa s attempted in o nly two of th e eight reported cases, which pr ecludes ade q uate evaluation of tumor responses to treatment. In o ne ca t, th erapy was not ben eficial." In th e other cat (No.4) , remission oc curred within several week s and ha s lasted for 2 yea rs, suggest ing that some GR CT may respond to th erapy. It is not clear what cell lin e is responsible for th ese neoplasms. Po ssibilities include Band T lymphoc ytes, LGL (i.e. , granulated intraepitheliallymphocytes [IELl) , globule leukocyt es, mucosal o r cuta neo us mast cells, and eosino phils or other m yelogen ou s cell typ es. R esults of hi stoch emical a nd ultrastructural ex a m ina tio n a rc not co m pa tible with a n eosino philic, neutrophil ic, B lymphocyti c, o r m on ocyti c o rig in. Felin e eosi no phil gra n ules co nta in a unique den se cylindrica l struc ture with regul arl y spaced circ um ferentia l lamellae," a nd th e cyto plas mic granules (Mott bodies) of B ce ll tum ors (myelo m as) are di stingui sh ed by ribosomes a lo ng th e gran ule membranes (rou gh endo plas mic reti culum). Neutrophil granules, although lacking truly di stinct ultr astructural m orphology, co nta in chlo racetate es te rase and/ or alkaline phosphatase activities.' The infi ltrati ve visce ra l m asses and progres sive clinical co urse are typical of feline lym pho m a, but cyto plasmic granulation ha s not been reported in this di sea se. The distrib ution of infiltrates is also different from that see n in feline lym pho m a. '? Intestinal and m esente ric lymph node involvement is consistent with th e a limentary form of lymphoma, but th e cra n ia l m edi as tin um (thy m us) is not usually a ffected in thi s form o f lymph osarcoma, no r a re th e intes tines involved in th e th ymic form of lymphoma. At lea st two of th e tum ors descr ibed in thi s rep ort a nd one in a previous re po rt 14 in vol ved th e intes ti ne as well as th e cra nial m ediastinum. In addition, all cats with GRCT th at were tested for FeLV (6/8) were negat ive, whe re at least 1/2 to 1/ 6 cats over 7 years o f age with felin e lymph oma a re FeLV positive ;'- wh ich suggests that FeLV is not in vol ved in th e form ation (tra ns formatio n) of G RCT . Sim ila rities between th e G RCT and lymphoproliferati ve di seases of human bein gs> and F344 rats 27•35 in vo lv ing LGL are lim ited to m orphologic features of th e ce lls. Although human ca ses have been described in whi ch neoplastic LGL in filtr ates have been pred omina ntly, or solely, lim ited to th e small intestine with

Vet Path ol 30:2, 1993

Feline Gr anulated Neopla sms

no ev ide nce of Ieukern ia. P- " splenomegaly and increased numbers of LGL in th e peripheral blood are key features of th e typi cal rodent and human diseases. One ca t had a lymphocytosis of gra nulated ce lls," but none of th e ca ts had splenic enla rgeme nt. GRCT cells were negati ve, or only weakl y positi ve," for acid ph osphatase, whe reas human LGL and F344 cell s contain thi s enzyme.e'-" Only scattered LGL granules from a case of F344 leukemia, sta ine d in o ur laboratory, were fa intl y blue with PTAH (data not shown). U lt ras tructural nuclear morphology a nd o rga ne lle distribution a re sim ilar in cells of felin e GRCT and the LGL proliferati ve diseases of human beings and rats. Granules tend to be larger, howev er, in th e cat GRCT, and th e morphologic diversity in granules among and within individual tumors is greater than in either the human or rod ent diseases. Intragranular parallel tubular arrays are apparently unique to human LGL and ar e not pre sent in rat LGU 4 •26 or feline GRCT ce ll granules. Granules o f normal felin e intestinal IEL did not stain blue with PTAH and were .:::; 0 .6 /lm in diam eter," as o pposed to GRCT granules, whi ch were up to 3 /lm in diamet er . Che ney et a l.' d eri ved a nd charac terized a LGL cell line from a felin e abdom ina l neoplasm that had features in common with GRCT, including intes ti nal a nd m esenteric lym ph node infiltration , cyto plas m ic gra nulatio n of th e tumor cells, and a FeL V-n egati ve sta tus. However, th e animal a lso had hepatospleno me ga ly and leukemia , and th e tumor cell granules d id not sta in blue with PTAH , were po sitive for chlora cet at e este rase and acid phosphatase, and were sm a ller th an GRCT granules. Therefore, this case more closely resembles human and rat LGL tumors than GRCT. The di stinction between felin e GRCT and an eq uine abdominal neoplasm of granulated round cell s " is also somewhat blurred. This equine tumor was beli eved to be of LGL origin, but, lik e GRCT of cats, the granules sta ined blue with PTAH. A mast cell or igin see ms improbable because th e G RCT granules in fixed secti ons were not m etachromatic with toluidine blue and th e granules of mast ce lls within ei the r intestinal mast cell tumors, exa m ine d in thi s study , or no rmal int estine did not stai n with PTAH . In additi on , mast cell granules in ei the r normal o r neoplasti c ce lls are more numer ou s a nd ultrastructurall y hom ogen eou s than th ose in GRCT, and th eir cytoplasmic m embranes typ icall y form villo us proj ections th at in terdigitate with th ose on adjacent cells.s-" One form of felin e mast cell neoplasia invol ves th e intestine, with spread to m esenteric lymph nodes, liver , a nd splee n.' Some of th ese tum o rs lacked m etachrom asia with toluidine blue and o nly sta ined faintly with PAS. It was not determined if granules in th ese cells would sta in with PTAH. Two di stinct populations of mast cells ha ve been well characterized in laboratory rodents. 10 Co nnective

20 1

tissu e mast cells, as found in th e skin, serosa l surfaces, a nd intes tina l muscul ari s a nd subm ucos a, sta in metachro m a tically aft er form alin fixati on. Mucosal mast ce lls, as found in th e lamina propria of th e intestin e, on ly ex hib it m etachromasia o f gra nules after fixati on in so lutio ns such as Ca rnoy's and are th erefore co nsidered formalin sensi tive.' There is ev ide nce that both typ es o f m ast ce ll originate from a co m m o n hem a topoi eti c ste m cell, with ac q uisitio n of unique ph en otyp es d et ermined by th e mi cro en vironment in which th ey a re located. 10,25 In this study, m etachromatic m ast ce lls were present in formalin -fixed sections of normal feline intes tinal lam in a pr opria but wer e markedl y increase d in number aft er Ca rn oy's fixation , suggesti ng that, as reported in human beings , two distinct mast ce ll populations exist in th e feline intestine. This form alin-sen sitive population of mast cell s cannot be co m plete ly ruled out as an origin of feline GRCT by m etachromatic staining becau se all tissu e samples were formal in fixed . However , th e fa ilure of normal mucosal m ast cell gra nules to sta in with PTAH , rega rdl ess of fixation, suggests th at this origin is unlikely. T wo of th e previ ously rep orted GRCT and those pr esented in thi s study ha ve m orphologi c and hist oche m ica l features sim ilar to th ose of normal globule leukocytes." U ltrastructura lly, th e gra nules in normal feline globule leukocytes co nta ined amorphou s to gra nula r materi al of va ria ble electro n den sit y and occasio na l electro n-de nse "caps" (Fig. 6) or crysta lline structures sim ila r to th ose in th e GRCT study cases. Elect ron-den se caps and crysta ls have been rep orted previously in a tumor believed to be of globule leu kocyte origin" and in normal felin e globule leukocytes .> In two cases, th e intestinal and bil iary epithelium adj ace nt to the neoplastic infiltrates contained excessive numbers of cells that would otherwise have be en id entified as typical globule leukocyte s. Although globule leukocytes can be found in th e inte sti na l and biliary epitheli um of cats, th ey are very rare in th e biliary tract and not nearly as numerous in th e intestine as was see n in ca t N o. I , whi ch suggest s a d irect or indi rect relation sh ip bet ween G RCTand th e intraepithe lial globule leukocyt e-like ce lls in th ese tissu es. It is unknown whe the r th e globule leukocyte-like cells represent d ifferentiated tumor ce lls or no rm al globular leukocytes induced by th e tum o r to prol iferate and/or mi grate. The currently a vail abl e in form ation suggests that G RCT are unique am on g felin e neopl asm s. The five tum ors de scribed in this re po rt a re m orphologica lly si m ilar to those previ ou sly described as felin e LG L/ glo bule leukocyte neoplasms, and th e morphologic, histoch emical , a nd ult rastructural features ar e most co ns istent with globule leukocytes or po ssibly cells in transition between mucosal mast cells and globule leu ko cytes. 15 • 16 ,2 2 A better understanding of th e normal

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ontogeny of these cells and identifi cati on with histochemica l or im m uno logic mark ers is necessary before the specifi c cell of or igin can be identified. T he progression and multiorgan pattern of G RCT infiltratio n resembl es both lymphoma and the visceral form of mast cell tum or in the cat, hence, all have a sim ilar clinical presentation . Altho ugh G RCT apparently carry a poor prognosis, too few cases have been treated to assess how they respond to therap y.

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Request repri nts fro m Dr. D. J . Me uten, College of Veter ina ry Med icine, No rth Ca ro lina Sta te U nive rsity, 4700 H illsboro ugh Street, Raleigh, NC 276 06 (USA).