Fine Needle Aspiration Biopsy

of Malignant Tumors in

Dogs and Cats:

A Report of 102 Cases

Michele Menara, Michel Fontaine and Michel Morin

Department of Veterinary Pathologyand Microbiology, Faculty ofVeterinary Medicine, University ofMontreal C.P. 5000, Saint-Hyacinthe,Quebec J2S 7C6

IntroductonFine needle aspiration biopsy

(FNAB) is not a new technique.The pioneers ofFNAB are Martin andEllis from Memorial Hospital forCancer and Allied Diseases in NewYork who described its use on 65 humanpatients with malignant tumors in 1930

(1). From the same hospital, Stewartreported 2,500 cases in 1933 in whichFNAB was performed with excellentdiagnostic results (2). During the nextthree decades, FNAB was replaced bythe rapidly growing field of histo-pathology in the United States (3, 4). Inthe 1950's, FNAB gained popularity inSweden because ofthe scarcity ofquali-fied pathologists (4, 5, 6). The successobtained with FNAB in Scandinaviawas reflected by the marked increase ofits use every year. In 1983, it wasestimated that more than $1.5 millionwas saved every year at the KarolinskaInstitute, since the majority of theFNAB were performed in out-patient

clinics. Since the beginning of the 70's,use ofFNAB has increased in the UnitedStates and Canada although the rate ofgrowth is slower than that in Europe(5, 6).The efficacy of FNAB reported in

the human literature for the diagnosis ofmalignancy (based on histologicalevaluation of the lesion or follow-up ofthe patient) ranges between 82% and91% with 0.1-2% false positives (7-12).Reports of the efficacy of FNAB andthe cytological description of thedifferent types of tumors are still scarcein the veterinary literature (13). Fineneedle aspiration biopsy is inexpensive,fast, simple and nontraumatic. Thetechnique is commonly used to samplesuperficial and radiographically discer-nible lesions. This article presents theresults obtained with FNAB andcytology of 97 malignant tumors andfive benign lesions (included in this studybecause they were suspected of beingmalignant on cytological evaluation)from 83 dogs and 19 cats. Histologicalevaluation of the lesion was done sub-sequent to the cytological evaluationand the results were compared. The goalof this study was to determine the effi-cacy of FNAB for the diagnosis ofmalignancy and determination of itscellular origin in dogs and cats.

Materials and MethodsThe standard technique of FNAB wasused (Figure 1) and is described else-where (6, 8, 14). A syringe holder(Aspiration Gun, The Everest Co., Inc.,Sherman Street, Linden, New Jersey)was used to facilitate the aspiration. Fordeep lesions (i.e. lung, bone), a 22gauge spinal needle was used for theaspiration following radiographic locali-zation. The technique oflung aspirationis well described (15). The aspiratedmaterial was rapidly expressed ontoglass slides and spread as for a standard

504Can Vet I Volume 27 No. 17 fleremh.r 1Q.

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onrgnwasinorgtedf al nttumorsandfivebeninl in 83 dosand 19 cats.Htoloclam onothe lesionswa p d in each case.

..Y ..:. O.. ..Malgnanywsetecteby cyogy69% ofthe malignanttumot The celtl-iaroriginof e det74% of thfe cases. C ir ofmlgacanddeterniainoclua

origin permitd an eady dia dprognsis. Sinc fin nede asiatobiopsy and:cytlog cannot'dente.yrule out ma , a s landhist shouldbedoghmalgnac is useced sdicayannot confirmed cytolog ly. Spe ensof good qultatdequtecaboratiobetween the and

ea nd andand sufcetknowledge~ ofand cyooyaebscrequireeti oobtainin pgod resuts with fine:neiedlaspiration biopsy an cytoloy.

Key Words: Fine needle, ,biopsy, cytolog, cancer, dogs Cts,nalignancy:criteria.L

Reprint requests to Dr. M. Fontaine

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blood smear (16). When the materialwas too thick, a "squash-prep" wasmade (14). The smears obtained wererapidly air-dried and stained withWright's stain.

Seventy-eight specimens wereobtained from animals presented to theSmall Animal Clinic of the Universityof Montreal and 24 specimens weresubmitted by veterinarians in privatepractice. The cytologic evaluation wasdone by Menard and Fontaine. Selec-tion of these cases was based on asubsequent histological diagnosis ofmalignancy. Cases with histologicaldiagnosis of benign tumors or inflam-matory lesions were included only whensuspected or diagnosed as being malig-nant on cytological evaluation.The tissue sample for histological

evaluation (Morin) was obtained eitherby surgery or at necropsy. The speci-mens were formalin-fixed, stained withhematoxylin, phloxine and safran (HPS)and interpreted according to standardcriteria (17, 18, 19).

The cytological criteria ofmalignancyused during this study are summarizedin Table I. Nuclear malignancy criteriaare the most important for identificationof malignancy. Nondividing (inter-phase) nuclei of malignant cells usuallycontain an increased amount of DNAand are larger than the interphasenucleus of a diploid benign cell of thesame origin (20, 21, 22, 23). Thisphenomenon is reflected by an increasednuclear/cytoplasmic ratio (20). Markedanisokaryosis (variation in nuclear size)is common in some malignant tumorsand is caused by abnormal mitosis (20,21). The chromatin of malignant cellsoften displays a coarse, dense granularitywhich is usually not observed in benigncells (21, 24). Aberrations of nuclearshape (lobulation, indentation, nuclearprotrusion) are frequent in cancer cells(20, 21, 24).

Multinucleated cells are suspected ofbeing malignant when they have anuneven number of nuclei or havemoderate to marked anisokaryosis (21,25). Nucleoli are present in cells under-going active RNA and protein synthesis(26). Normal cells can show onemedium size nucleolus (3-4pm) or oneto three small nucleoli (1-2 pum).Nucleolar aberrations in size, shape andnumber are important malignancy cri-teria. Malignant tumors often containcells with multiple prominent nucleoliof variable size and shape (21, 25). Thepresence of mitotic figures is a malig-nancy criteria only when they are

Figure 1. Fine needle aspiration biopsy technique. A 22 gauge needle connected to a20 mL syringe is introduced into the mass (a). A negative pressure of 15 mL isapplied (b). The needle is moved in three to five directions without withdrawing theneedlefrom the mass to obtain a representative sample (c). The negatvepressure isreleased and the needle is withdrawn (d). Smears are made immediately.

abnormal with an excessive number ofmitotic spindles or with abnormal dis-tribution of chromosomes (27).

Malignant cells can vary greatly insize within the same tumor (aniso-cytosis). Abnormal cellular shapes canalso be observed (24). Loss ofadhesionand cohesion are important character-istics of malignant cells which enablethe fine needle aspiration of a largenumber ofcells often in clumps with cellpiling and loss of nuclear polarity orindividualization of epithelial cells ontothe smears (21, 28). Malignant cellsoften have a marked cytoplasmicbasophilia as may be seen in any rapidlydividing or metabolically active cell(24). The amount ofcytoplasm can alsovary considerably between malignantcells which appear to be at the samestage of differentiation. Cytoplasmicvacuolation is a nonspecific changewhich may reflect cellular degenerationor the formation of a secretory product(24). Histochemistry can be used to

identify the nature of these vacuoles(20, 25, 29). The cytoplasmic appear-ance is primarily used to evaluate thedegree ofdifferentiation and the type oftumor while the diagnosis ofmalignancyis based primarily on the nuclear criteria(20, 21, 24). A "positive" diagnosis ofmalignancy was made in this studywhen at least four nuclear malignancycriteria were observed in a specimencontaining a minimum of thirty wellpreserved cells (24). If these criteriawere partially met, malignancy was"suspected". Specimens werejudged tobe of poor quality with "no evaluationpossible" in extreme cases when therewas excessive blood or inflammatorycell contamination, the cellularity waspoor, most ofthe cells were degenerate,or the smears were thick with loss ofcellular detail.

These malignancy criteria were notapplicable to all tissues. Since lympho-cytes ofdifferent stages ofdifferentiationnormally have high nucleocytoplasmic

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ratio with moderate anisocytosis, abetter indication oflymphosarcoma wasa monotonous population of lympho-cytes at the same stages of differen-tiation in which case anisocytosis oranisokaryosis was often mild. Severalclassifications are used to describe thedifferent types of lymphosarcoma (6,18, 30-35). The lymphosarcomasobserved during this study were classi-fied as stem cell, lymphoblastic,prolymphocytic, lymphocytic and his-tiocytic lymphosarcomas according toMoulton's criteria (18).

Mastocytomas were diagnosed basedon the presence of intracytoplasmicmetachromatic granules. The presenceof cytological malignancy criteria wasreported but these tumors were alwaysconsidered at least potentially malignantregardless of their degree of cellulardifferentiation (18). In each case, his-tological examination ofthe tumor wasrecommended to evaluate the degree ofinvasiveness (36).

ResultsThe correlation between the results fromthe histological and cytological exami-nations is presented in Table II.

Malignancy was detected by cytology in71% and 60% ofthe malignant tumors ofdogs and cats respectively, and in 69%of the cases when dog and cat resultswere combined. The cellular origin ofthe lesion was determined in 72% and87% of the dog and cat tumors respec-tively, and in 74% ofthe cases when theresults for both species were combined.Of the 24 cases submitted by veteri-narians in private practice, malignancy

was diagnosed by FNAB in 11 cases(46%) and was suspected in eight othercases (33%). In 67% ofthese specimens,the quality was moderate to poor.Seventy-eight specimens were obtainedfrom animals presented at the SmallAnimal Clinic of the Faculty ofVeterinary Medicine of the UniversityofMontreal. Seventy-seven were malig-nant tumors and five were benignlesions based on histological evaluation.Malignancy was diagnosed in 56 of the73 malignant tumors (77%) by FNABand suspected in 15 others (20%). Thespecimens were of moderate to poorquality in 34% of the cases.One false positive diagnosis of

malignancy was made for a subcuta-neous chronic inflammatory lesion.The FNAB specimen was moderatelycellular with a marked degree of aniso-cytosis and anisokaryosis, coarse granu-lar chromatin, prominent large nucleoli,and high nuclear-cytoplasmic ratio.These cells appeared to be regenerativemyocytes on histological examination(Figure 2).

Malignancy was detected cytologic-ally in sixteen out of eighteen bonetumors. The aspiration ofosteosarcomasyielded specimens with good cellularity.The cells were round or oval, oftenindividual with a variable amount ofbasophilic cytoplasm which containedvacuoles and eosinophilic granules ofvariable size and number. Nuclei wereround or oval, eccentrically placed withmarked anisokaryosis and prominentmultiple nucleoli of variable size. Fineneedle aspiration biopsy from most ofthe cases (8 of 11) contained clusters ofcells embedded in a dense, homo-geneous, eosinophilic matrix mostcompatible with osteoid tissae (Figure3). Giant, multinucleated cells wereobserved in all cases of osteosarcomaand anaplastic sarcoma and in a fewcases of fibrosarcoma. The well differ-entiated bone fibrosarcomas yieldedoval or spindle shaped cells withmarked anisocytosis and anisokaryosisand small amounts of basophilic cyto-plasm often containing small eosino-philic granules. The nuclei were oval,centrally located and contained multiplenucleoli of variable size.

Fine needle aspiration biopsy fromthyroid carcinoma yielded specimenscontaining a large number of round oroval cells, individual or in clusters, witha small amount of pale and vacuolatedcytoplasm. In two cases the cells hadproduced an homogeneous pale eosino-philic material consistent with colloid.

Anisocytosis and anisokaryosis wereprominent and nucleoli were multipleand large in two cases (Figure 4).The FNAB specimens from two lung

hemangiosarcomas consisted only ofblood with many immature red bloodcells probably indicating previoushemorrhage. Malignancy was detectedin two ofthe four lung adenocarcinomaswhich were primary in the lung in threecases and metastatic from an adrenalgland adenocarcinoma in one case. Thecells were often in clumps and had avery basophilic cytoplasm which wascommonly distended by vacuoles ofvariable size (Figure 5). These four caseswere all diagnosed as being ofglandularorigin and the differentiation between aprimary lung adenocarcinoma and ametastatic adenocarcinoma to the lungwas not possible cytologically.The cells from FNAB of squamous

cell carcinoma were often in clumpsand the nuclear details were obscuredbecause ofthe marked basophilia ofthecytoplasm. Malignancy was detected insix out of ten cases but the precisecellular origin was only determined infour cases. Cornified cells were observedin two cases (Figure 6).The adenocarcinomas were charac-

terized cytologically by clusters ofround cells, usually containing a largeamount of acidophilic or basophilicvacuolar cytoplasm and prominentnuclear malignancy criteria in the mostanaplastic tumors. Malignancy of thewell differentiated tumors had to beconfirmed histologically.

All the mast cell tumors were diag-nosed correctly based on the presenceofa variable amount ofintracytoplasmicmetachromatic granules with Wright'sstain (Figure 7). The aspirate ofone caseofliposarcoma was composed ofroundindividual cells with prominent malig-nancy criteria and the cytoplasm filledwith clear vacuoles of variable sizes(Figure 8). Both melanosarcomas con-tained cells with a variable amount ofmelanin granules (Figure 9). Malig-nancy was not diagnosed cytologicallyin one case because ofthe low cellularityof the specimen. The cells from bothcases contained several features ofmalignancy.

DiscussionThe degree of chronic inflammationfrom the FNAB specimen of the onlyfalse positive diagnosis of malignancyobserved during this study was under-estimated. Regenerative cells, fibro-blasts, and epithelial cells in chronic

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inflammatory lesions share severalcharacteristics with malignant cells (37).The degree of inflammation in anaspirate should never be overlooked inorder to avoid false positive diagnosesof malignancy (37).The lymphoblastic and prolympho-

cytic forms of lymphosarcoma wereeasily diagnosed cytologically, asreported by others (6, 28, 37). Thehistiocytic and lymphocytic (welldifferentiated) types were the most dif-ficult to diagnose cytologically asobserved previously (6, 37).The overall results obtained with

FNAB of bone tumors were very goodand compared to those reported pre-viously (14, 38, 39, 40). The moreanaplastic forms of fibrosarcoma weredifficult to differentiate from osteo-sarcomas cytologically.Mammary tumors were not aspirated

often as most of the time surgicalablation was performed with histo-logical examination of the lesion. Theresults obtained by FNAB from eightcases were poor. The specimens wereoften necrotic, with marked blood and

inflammatory cell contamination, orthe cells were in dense clumps with littlecellular detail. These results are consis-tent with those observed previously inveterinary medicine (13).The cytological appearance ofthyroid

tumors is well described in the literaturewith excellent results reported (6, 28,41, 42, 43). Only four cases wereaspirated during this study and theywere all correctly diagnosed.The squamous cell carcinomas were

difficult to diagnose cytologically. Thespecimens were often necrotic or hadpoor cellularity even if the aspirate wasrepeated two or three times. Sincethe tumors were often ulcerated, carehad to be taken to differentiate neo-plasia from hyperplasia secondary to a

chronic inflammatory lesion. Papani-colaou staining has been recommendedfor the diagnosis of squamous cellcarcinoma because keratin stains brightorange and nuclear detail is generallybetter than with Wright's staining (24).

Poor results were obtained withsubcutaneous fibrosarcomas. The cellu-larity was inadequate or the cells were

in clusters with poor cellular detail.Sufficient malignancy criteria to permitdifferentiation between a fibrosarcoma,a fibroma and granulation tissue wereoften not observed. The cells of thefibrosarcomas, hemangiopericytomasand hemangiosarcomas were often dif-ficult to differentiate and a diagnosis ofsarcoma was given with histopatho-logical examination of the lesionrecommended.

Lipids are solubilized with Wright'sstain which is an alcohol based stain.Oil red 0 or Sudan stain would bebetter stains when a liposarcoma issuspected (29).The results obtained from the speci-

mens submitted by veterinarians inprivate practice were inferior to thoseobtained at the Veterinary SchoolHospital where the FNAB was done byclinicians more experienced with FNABand often repeated up to three timesuntil an adequate specimen wasobtained, which was often not the casefor external samples. The best resultswith FNAB reported in the literature are

(continued on page 510)

Can Vet J Volume 27, No. 12 December 1986 !Sfl7Can Vet J Volume 27, No. 12 December 1986 507

Figure 2. Subcutaneous chronic inflammatory esion falselydiagnosed as a malignant tumor on cytological evaluatiomAnisocytosisandanisokaryosis arepronounced with a coarse,granular chromatin, voluminous nucleol and high nuclear-cytoplasmic ratio. These cells appeared to be regenerativemyocytes on histological evaluation (Wright's stain).

Figure 4. Thyroid carcinoma containing round to oval cellswith prominent anisocytosis and anisokaryosis and largenucleoli. The cytoplasm is pale and vacuolated (Wright'sstain).

Figure3. Fine needleaspiratefrom an osteosarcoma with cellsembeddedin4 andapparently secreting, a dense, homogeneous,eosinophilic matrix most compatibl with osteoid tissue(arrows) (Wright's stain).

.'ii I

FigureS. Bronchoalveolarcarcinoma ofthe lung with two cells

containing large vacuoles displacing the nucleus eccentrically(Wright's stain)

508 Can Vet J Volume27.No.12December iPRA~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~---.. - - - . - ..-. - I- "W.-,

508 Can Vet J Volume 27, No. 12 December 1986

Figure 6. Squamous cellcarcionma containing a cornfied cel

(arrow) with angular shape, large amount ofpale, pink cyto-

plasm andadegeneratng nucleus. The othercelsalso haveanangular shape and seem to be in the process of cornufieation(Wright's stain).

Figure 8. Subcutaneous iposarcoma. The cells are mainlyseparateandfilled withcdear vacuolesofvariablesize(Wright'sstain).

Figure 7. Well differendated mast cell tumor. Cells arefilledwith small, roun4 purple granules which obscure nucleardetail (Wright's stain).

Figure 9. Subcutaneous melanosarcoma. The cells are very

anaplastic and contain black granules of variable sizes andshapes compatible with melanin granules (arrows) (Wright'sstain).

Can Vet J Volume 27, No. 12 December 1986509

Can Vet J Volume 27, No. 12 December 1986 509

(continuedfrom page 507)obtained in hospitals where the aspiratesare repeated several times to obtain arepresentative specimen (44, 45).The results were slightly inferior for

the diagnosis of malignancy of cattumors compared to dog tumors andthe opposite was true for the deter-mination of the cellular origin of thelesions. Dogs were much more numer-ous than cats for this study whichmakes comparison ofthe results betweenthese two species difficult. Half of thecat tumors were mammary glandcarcinoma, squamous cell carcinoma,or fibrosarcoma. The cytological detec-tion of malignancy was also difficult forthese tumors in dogs.As cytological evaluation is being

performed on lesions from an everincreasing number of organs, thecytologist needs an adequate knowledgeof the pathology and cytologicalappearance of these tissues. The greatestdanger of cytology lies in erroneousinterpretation of the samples byinexperienced observers without suffi-cient background in tissue pathologyand cytology (4).One of the major limits of FNAB is

the inability to definitively rule out thepossibility of malignant tumor. Whenmalignancy is still suspected clinicallyand has not been confirmed cyto-logically (after at least three aspirationsof different regions of the lesion), asurgical biopsy should be performed.The technique ofFNAB has multiple

advantages: it is fast, inexpensive, welltolerated by the animals and does notrequire anesthesia. No complicationswere observed during this study. Thebasic requirements for obtaining goodresults with FNAB cytology are a speci-men of good quality, adequate colla-boration between the cytologist and theclinician who should provide a completehistory of the case, and a strong know-ledge of pathology and cytology.Extensive literature describing thecytological appearance of tumors,inflammatory lesions, and commoncauses of false positives or false nega-tives of malignancy is available inhuman medicine. Until such infor-mation exists in veterinary medicine, avery conservative approach should betaken to avoid as much as possible falsepositive and false negative diagnosesof malignancy.References

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