Immunohistochemical Detection ofIntermediate Filament Proteins in

Formalin Fixed Normal and Neoplastic Canine Tissues

Michel M. Desnoyers, Deborah M. Haines and Gene P. Searcy

ABSTRACT

Normal and well differentiatedneoplastic canine tissues were immu-nohistochemically stained for keratin,vimentin and desmin intermediatefilament proteins using commerciallyavailable monoclonal antibodies.Keratin was detected in 56 of 57carcinomas, vimentin in 59 of 62sarcomas and desmin in three of fourmuscle cell tumors. Most normal andneoplastic tissues expressed only onetype of intermediate filament; excep-tions were one hemangiosarcoma andone pulmonary carcinoma in whichthere was coexpression of vimentinand keratin proteins. Since immuno-histochemical detection of interme-diate filaments has tissue-specificdistribution in the majority of welldifferentiated canine neoplasms, thesestains may be useful in the differentialdiagnosis of anaplastic canine tumors.However, the monoclonal antibodiesto cytokeratin which were tested inthis study failed to detect intermediatefilaments in liver, pancreas andsalivary glands which suggests thatthese antibodies may also be unable todetect epithelial tumors derived fromthese tissues. In addition, in nineneoplasms, the normal tissues adja-cent to neoplastic cells failed to stainfor the intermediate filament normallyexpressed. When this occurs, evalua-tion of intermediate filament expres-sion is invalid for the determination oftissue of origin of the neoplastic cells.

RESUME

Des tissus canins normaux etneplasiques bien differencies ont etecolores histochimiquement pour lakeratine, la vimentine et la desmine,proteines filamenteuses interme-diaires, 'a l'aide d'anticorps monoclo-naux disponibles commercialement.La keratine, la vimentine et la desmineont respectivement ete decelees dans56 cas d'epithelioma sur 57, 59 cas desarcomes sur 62 et trois cas sur quatrede tumeur des cellules musculaires. Lamajorite des tissus normaux etneoplasiques presentaient seulementun type de filament intermediaire saufun cas d'hemangiosarcome et un casd'epithelioma pulmonaire ou de lavimentine et de la keratine ont etedecelees. Etant donne que la detectionhistochimique des filaments interme-diaires a une distribution tissulairespecifique dans la majorite desneoplasmes canins bien differencies,ces colorations peuvent etre utilespour le diagnostic differentiel destumeurs canines anaplasiques. Toute-fois, les anticorps monoclonauxcontre la cytokeratine, lesquels ont eteevalues dans la presente etude, n'ontpas permis de deceler des filamentsintermediaires dans le foie, le pancreaset les glandes salivaires, suggerant queces anticorps ne pourraient probable-ment pas deceler des tumeurs epithe-liales originant de ces tissus. De plus,pour neuf neoplasmes, les tissusnormaux adjacents aux cellules

neoplasiques ne se sont pas colorespour les filaments intermediairesnormalement presents. Dans de tellescirconstances, la detection des fila-ments intermediaires n'est pas valablepour la determination du tissud'origine des cellules neoplasiques.(Traduit par Dr MichelFontaine).

INTRODUCTION

Intermediate filaments are cyto-skeletal proteins found in mostmammalian cells (1). There are fivetypes of intermediate filaments.Keratin filaments are present solely inepithelia, vimentin in mesenchymalcells, desmin in striated, cardiac andsmooth muscle, neurofilaments innerve cells and glial fibrillary acidicprotein in glial cells (1). Tissue-specificdistribution of intermediate filamentproteins is retained in most neoplasticcells (2,3) and their detection is used inthe characterization of anaplastictumor cell populations in humans(1,4). Recently, intermediate filamentdetection has been evaluated in somecanine tumors and while only rela-tively small numbers of tissues havebeen examined, their expression seemsto be tissue specific (5-8). Intermediatefilament expression in tumor cells maybe demonstrated by immunohisto-chemical stains (2,9-12). lmmunohis-tochemical detection of intermediatefilaments may be accomplished witheither monoclonal (13) or polyclonal

Department of Veterinary Pathology (Desnoyers, Searcy) and Department of Veterinary Microbiology (Haines), Western College of VeterinaryMedicine, University of Saskatchewan, Saskatoon, Saskatchewan S7N OWO.Reprint requests to Dr. D.M. Haines.Supported by grants from the Companion Animal Health Fund, Western College of Veterinary Medicine, Saskatoon, Saskatchewan and the Universityof Saskatchewan President's NSERC Fund.

Submitted October 24, 1989.

Can J Vet Res 1990; 54: 360-365360

antibodies. Monoclonal antibodies tointermediate filaments are commer-cially available (1,4) and their limitlesssupply provides an advantage overpolyclonal antibodies which can beobtained only in finite amounts.Limitless quantities of antibodies ofdefined reactivity allow the standardi-zation of immunohistochemical tech-niques within and among differentlaboratories. Standardization of thesestains will improve the utility of thistechnique to assist in tumor diagnosis.

Intermediate filament detection hasbeen occasionally used for tumoridentification in dogs, but this is notyet a reliable means of tumor diagno-sis. For this to be reliable it isnecessary to examine a large numberof well differentiated tumors andnormal tissues to predict the accuracyof the test for evaluation of anaplastictumors. It is particularly important toexamine the staining of tissuessubjected to routine fixation andprocessing procedures as these are thespecimens most likely to be submittedfor diagnostic evaluation.The goal of this study was to

evaluate the immunohistochemicaldetection of the three most widelydistributed intermediate filaments,keratin, vimentin and desmin informalin-fixed paraffin-embeddednormal canine tissues and welldifferentiated neoplastic tissues usingcommercially available monoclonalantibodies.

MATERIALS AND METHODS

NORMAL TISSUES

Tissues from 18 organs from normaladult dogs were fixed in neutralbuffered 10% formalin for 48 h, thenembedded in paraffin. These tissueswere: skin, trachea, lung, small andlarge intestine, liver, pancreas, sali-vary gland, oesophagus, mammarygland, testicle, thyroid, kidney, heart,skeletal muscle, lymph node, spleenand brain.

NEOPLASMS

One hundred and nineteen welldifferentiated tumors were chosenfrom those submitted to the PathologyDepartment, Western College ofVeterinary Medicine, Saskatoon,Saskatchewan. These consisted of 57

carcinomas (20 mammary glandcarcinomas, 19 squamous cell carcino-mas and 18 pulmonary carcinomas)and 62 sarcomas (20 osteosarcomas,19 fibrosarcomas, 19 hemangiosarco-mas, two rhabdomyosarcomas andtwo leiomyosarcomas). Tumors wereclassified according to histopathologi-cal criteria (14,15).

ANTIBODIES

Commercially available monoclo-nal antibodies were selected based onreports of their effectiveness in thedetection of intermediate filaments informalin-fixed, paraffin-embeddedhuman (1,10,1 1) and canine (6) tissues.Antibodies tested were: anti-keratinA.E.1/A.E.3 (Boehringer MannheimBiochemicals, Indianapolis, Indiana),anti-vimentin clone D9 (BiogenexLaboratories, Dublin, California),anti-vimentin clone 9 (Sanbio bv,Uden, The Netherlands, distributed byDimension Laboratories, Missis-sauga, Ontario) and anti-desmin clone33 (Sanbio bv, Uden, The Nether-lands, distributed by DimensionLaboratories, Mississauga, Ontario).

IMMUNOHISTOCHEMICAL STAINING

Five micron sections of normal andneoplastic tissues were applied to glassslides coated with 0.1% poly-D-lysine(MW 157,400, Sigma Chemical Co.,St-Louis, Missouri). The sections wererehydrated by sequential immersion inxylene, graded concentrations ofethanol, and tap water.The tissue sections were immuno-

histochemically stained using anavidin-biotin peroxidase complex(ABC) method (16). The sections werecovered with 5% normal horse serumin phosphate-buffered saline (PBS)(0.1 M, pH 7.4) for 30 min at roomtemperature to saturate nonspecificprotein-binding sites. Normal serumwas gently removed and the sectionscovered with the anti-intermediatefilament monoclonal antibody. Afterincubation in a humid chamber,sections were washed twice in PBS,followed by 30 min incubation withbiotinylated horse antimouse IgG(Vector Laboratories, Burlingame,California) diluted 1:200 in PBS.Following washing in PBS, the slideswere incubated for 30 min in a solutionof0.5% hydrogen peroxide in methanolto inactivate endogenous peroxidase

activity. The ABC solution, preparedaccording to the manufacturer's direc-tions (Vectastain ABC kit, VectorLaboratories, Burlingame, California),was applied for 60 min. The sectionswere washed in PBS, then incubatedwith 1 mg/mL solution of 3, 3-diaminobenzidine tetrahydrochloride(DAB) (Electron Microscopy Sciences,Fort Washington, Pennsylvania) and0.1I% hydrogen peroxide in PBS for 5min. The sections were lightly counter-stained with hematoxylin.The optimal staining procedure for

each intermediate filament was deter-mined by staining normal caninetissues either untreated or followingtreatment with proteolytic enzymes.The tissues were stained followingdigestion at 37°C for either 15 or 30min with protease (Type XIV SigmaChemical Co, St Louis, Missouri)0.05% in PBS, trypsin (Gibco Labora-tories, Paisley, Scotland) 0.1% in PBSsupplemented with CaCl2 0.1% pH7.8, or pepsin (Fisher Scientific Co,Fair Lawn, New Jersey) 0.04% in PBS.The optimal staining, which was usedin all subsequent tests of tumors, wasobtained with monoclonal antibodiesto keratin on tissues digested withprotease for 15 min at 37°C whiletissues stained with monoclonalantibodies to vimentin and desminwere untreated.Optimal staining periods and

temperatures were chosen followingtrials in which normal tissue sectionswere incubated with the anti-intermediate filament monoclonalantibodies either for 2 h at roomtemperature or for 16 h at 4°C. Therewere no appreciable differences in theintensity or distribution of immuno-histochemical staining between thesetwo protocols with antibodies detect-ing keratin and vimentin. There wasenhanced staining intensity with theantibody detecting desmin when tissuesections were incubated at 40 C for 16h. In all subsequent tests, the monoc-lonal antibodies detecting keratin andvimentin were incubated on the tissuesections for 2 h at room temperatureand the antibody detecting desmin for16 h at 4°C.The optimal dilution for each of the

monoclonal antibodies was deter-mined by staining normal tissuesections. Antibodies were tested atserial doubling dilutions from 1 /25 to

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1/400. The optimal dilutions, chosento produce strong and diffuse specificstaining with minimal nonspecificbackground marking, were as follows:1) Anti-keratin A.E.1/A.E.3: 1/100,2) Anti-vimentin clone D9: 1/400, 3)Anti-vimentin clone 9: 1/ 50, 4) Anti-desmin clone 33: 1/25.

All tissues were stained with eachantibody in duplicate. Controls forimmunohistochemical stains weresections of normal canine tissuescontaining epithelium, mesenchymeand muscle, stained concurrently withthe tumor sections. Other controlswere sections in which the monoclonalantibody detecting intermediate fila-ments was replaced by PBS.Tumor sections were graded inde-

pendently by two of the authors(DMH and MMD) estimating thepercentage of staining neoplastic cellsas 0%, less than 50% or more than50%. Normal tissue constituents,when present in sections of neoplastictissue, were also evaluated for inter-mediate filament detection. Positivecells were those with cytoplasmicdeposits of brown DAB chromogen.

RESULTS

Anti-keratin monoclonal antibo-dies A.E. 1/ A.E.3 darkly and diffuselystained the epithelia of skin (exceptbasal cells), gastrointestinal andrespiratory tracts. Thyroid follicularcells and renal tubular cells stainedpalely but diffusely. In the testicle,only the basal portion of seminiferoustubular cells stained. Monoclonalantibodies A. E. I/ A. E.3 failed to stainhepatocytes, acinar cells of thepancreas and salivary gland epithelialcells. Normal tissues of mesenchymalorigin were unstained with monoclo-nal antibodies A.E. I/ A.E.3.Monoclonal antibody D9 to vimen-

tin darkly stained connective tissue,endothelial cells, cartilage and muscle.There was no staining of normalepithelial tissue with this antibody.Normal striated, smooth and car-

diac muscle stained with monoclonalantibody 33 to desmin. Normalepithelial tissue and tissues of mesen-chymal origin other than muscle wereunstained.

Fifty-seven carcinomas and 62sarcomas including four muscle cell

tumors were immunohistochemicallystained for intermediate filamentproteins (Table I). Keratin wasdetected in 56 of the 57 carcinomas.Keratin was not detected in the normalor neoplastic epithelium in sections ofone pulmonary carcinoma. In severalcutaneous squamous cell carcinomas,staining was limited to keratinizedcells while basal cells were unstained(Fig. 1). The staining intensity in themajority of epithelial tumors was darkand homogeneous. There was stainingof a few spindle-shaped neoplasticendothelial cells in one hemangiosar-coma. All other mesenchymal tumorswere unstained.

Immunohistochemical staining forvimentin was dark and homogeneousin 59 of 62 sarcomas, including thefour muscle cell tumors (Fig. 2). Onepulmonary carcinoma showed coex-pression of keratin and vimentin, thestaining for keratin was present in cellsthroughout the tumor section whilethe staining for vimentin was confinedto cells at the periphery of neoplasticcell clusters. Vimentin was notdetected in any other epithelial tumor.In addition to monoclonal antibodyD9 to vimentin (Biogenex Laborato-ries), all normal and neoplastic tissueswere immunohistochemically stainedwith a monoclonal antibody tovimentin designated clone 9 (Sanbiobv). Staining with this antibody waspaler and fewer cells were stainedcompared with monoclonal antibodyD9. Seventeen of 62 mesenchymal

tumors including two muscle tumorswere unstained with monoclonalantibodies from clone 9 while onlythree mesenchymal tumors failed tostain with monoclonal antibody fromclone D9.Desmin intermediate filaments were

demonstrated in the two rhabdomyo-sarcomas and in one of two leiomy-osarcomas (Fig. 3). The intensity anddistribution of immunohistochemicalstaining for desmin was variable innormal and neoplastic muscle. Onerhabdomyosarcoma and one leiomy-osarcoma showed dark diffuse stain-ing of the neoplastic cells, while in onerhabdomyosarcoma only a few cellswere palely stained. Desmin was alsodetected in the basal portion of severaltubular epithelial cells of normalkidney adjacent to the unstainedleiomyosarcoma and in severalspindle-shaped cells and in the capsuleof one splenic hemangiosarcoma.

Immunohistochemical staining forintermediate filament proteins failedto stain normal tissues within sectionsof nine of 119 neoplasms (Table I). Inone pulmonary carcinoma, normalepithelial tissue and normal mesen-chymal tissue failed to stain for keratinand vimentin respectively, althoughnormal muscle stained weakly fordesmin. In another pulmonary carci-noma and in one hemangiosarcoma,normal mesenchymal tissue failed tostain for vimentin. Normal musclefailed to stain for desmin in onehemangiosarcoma, three pulmonary

TABLE I. Immunohistochemical staining of 119 formalin-fixed paraffin-embedded canine tumorswith monoclonal antibodies to intermediate filaments

Keratin Vimentin Desmin(percentage of cells stained)

Tumor type 0 <50 >50 0 <50 >50 0 <50 >50

Fibrosarcoma 19 0 0 1 5 13 19 0 0Hemangiosarcoma 18 1 0 2C 3 14 19e 0 0Osteosarcoma 20 0 0 0 8 12 20 0 0Mammarycarcinoma 0 1 19 20 0 0 20' 0 0Squamouscarcinoma 0 7 12 19 0 0 19 0 0Pulmonarycarcinoma la 2 15 1 7b 1 0 1 8d 0 0Rhabdomyosarcoma 2 0 0 0 2 0 0 1 1Leiomyosarcoma 2 0 0 0 0 2 1 1 0

aNormal epithelium failed to stain in this sectionbNormal mesenchymal tissue failed to stain in sections of two tumorscNormal mesenchymal tissue failed to stain in one sectiondNormal muscle failed to stain in sections of three tumorseNormal muscle failed to stain in one sectionfNormal muscle failed to stain in sections of two tumors

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Fig. 1. Squamous cell carcinoma immunohistochemically stained with monoclonal antibodies tocytokeratin. The keratinized epithelial cells are intensely stained, the basal cells are unstained.Formalin-fixed parafrin-embedded tissue, avidin-biotin complex immunoperoxidase andhematoxylin counterstain. (Bar = 25 ,um).

carcinomas and two mammarycarcinomas.

DISCUSSION

Fifty-six of 57 canine carcinomasand 59 of 62 canine sarcomas stainedwith monoclonal antibodies to keratin

and vimentin respectively. This isconsistent with previous studies whichshowed that a majority of caninetumors stain in a tissue specific fashionfor intermediate filaments (6-8) andsuggests that these stains may beuseful in the classification of anaplas-tic neoplasms.

Fig. 2. Osteosarcoma immunohistochemically stained with monoclonal antibodies to vimentin.The majority of the neoplastic cell population is stained. Formalin-fixed paraffin-embedded tissue,avidin-biotin complex immunoperoxidase stain and hematoxylin counterstain. (Bar = 50 Mm).

Vimentin and keratin coexpressionwas demonstrated in one pulmonarycarcinoma and in one hemangiosar-coma. Coexpression of intermediatefilaments has been reported in humanpulmonary, renal, endometrial, thy-roid, ovarian and gastric adenocarci-nomas (10,17-19) but, to our knowl-edge, coexpression of theseintermediate filaments has not beenreported in hemangiosarcomas. Neo-plastic cells may manifest abnormal oraberrent synthesis of proteins whichmay result in coexpression of interme-diate filaments (20). There are con-flicting reports concerning the degreeof differentiation of tumors whichcoexpress vimentin and keratin. Inone study (10) more differentiatedpulmonary tumors coexpressed inter-mediate filaments. However, anotherstudy (17) found that a significantlyhigher percentage of moderately andpoorly differentiated pulmonaryneoplasms showed coexpression oftwo intermediate filaments. Ourstudy, confined to well differentiatedtumors, demonstrated keratin andvimentin coexpression in only twotumors. Whether coexpression of twoor more intermediate filaments willoccur more frequently in well differen-tiated or in poorly differentiatedcanine neoplasms will require furtherstudies. There was evidence of desminand keratin coexpression in renaltubular epithelial cells adjacent to anunstained leiomyosarcoma. Keratinand vimentin coexpression has beenreported in normal renal tubules (1)but there are no previous reports ofdesmin and keratin coexpression inthese cells. The significance of thisfinding is uncertain.Desmin intermediate filaments were

demonstrated by monoclonal anti-body immunohistochemical stainingin three of four muscle tumors. This isconsistent with a previous report inwhich a polyclonal antibody was usedto demonstrate desmin in 14 of 22canine smooth muscle tumors (5). Thedetection of desmin in formalin-fixedparaffin-embedded normal tissues wasless consistent than that of otherintermediate filaments. Formalinfixation has a deleterious effect ondesmin antigenicity and this interme-diate filament may be best demon-strated in frozen sections (4).Moreover, desmin is usually confined

363

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Ji.... .........

......................~~~~~~~~~~~~~~~~~~

''7, 4 4 -k~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~....

AX~ ~~~~~~~~~~~~~~~~~~~1*~~~~~~~~~~~~~~iFM4<~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.....

......4

Fig. 3. Rhabdomyosarcoma immunohistochemically stained with monoclonal antibodies todesmin. There is diffuse staining of the tumor cells. Formalin-fixed paraffin-embedded tissue,avidin-biotin complex immunoperoxidase stain and hematoxylin counterstain. (Bar = 50 Am).

to one edge of the muscle cells and mayappear as a condensation (1) whichmay render its visualization moredifficult than keratin and vimentinwhich are present diffusely in thecytoplasm. Other markers, includingfast and slow myosin, actin andmyoglobin, can also be used foridentification of muscle tumors butdesmin has been shown to be the mostreliable immunohistochemical markerfor the diagnosis of humanrhabdomyosarcomas (12,21-23).

Several plump spindle-shaped cellsin one splenic hemangiosarcoma werestained for desmin. These cells couldeither be neoplastic endothelial cells orsmooth muscle myofibroblasts. Thelatter are normal constituents of thesplenic capsule and trabeculae. Sincethe splenic capsule stained for desminwhile neoplastic endothelial cellsforming blood channels did not, thestained cells were likely myofibroblasts.An important finding in the present

study was that in nine neoplasmsnormal tissues adjacent to tumor cellsfailed to stain for one or more of theexpected intermediate filament pro-teins. This was most likely due to theeffects of formalin fixation. Antigencross-linking by formalin is influencedby time and temperature. Formalinfixation exceeding 24 h has a delete-rious effect on tissue antigenicity (2).

Many tumors stained in this studywere fixed in formalin for an unknownperiod of time, possibly exceeding 48h. It is impossible to know if failure ofintermediate filament detection inneoplastic cells, in the same section, isdue to absence of the intermediatefilament or is due to fixation andprocessing alterations. When thishappens, evaluation of intermediatefilament expression is invalid for thedetermination of tissue origin of theneoplastic cells.The failure of monoclonal antibodies

A.E.1/A.E.3 to stain some normalepithelial tissues (including liver,pancreas and salivary glands) suggeststhat these antibodies are ineffective forkeratin detection in epithelial tumorsarising from those organs. Keratins area heterogeneous group of at least 19different proteins (24) and keratincomposition varies depending on theparticular epithelium (11). Normalhepatocytes, acinar cells of the salivaryglands and pancreatic epitheliumexpress mostly keratins 8 and 18(11,24). Keratin 8 is detected by theantibody A.E.3 but keratin 18 is notdetected by antibody A.E.3 or byantibody A.E. 1 (11). In the commer-cially available solution used in thisstudy, the ratio of antibody A.E. 1 toA.E.3 is 20:1. It is possible that keratin 8in these formalin-fixed tissues is of

insufficient quantity or antigenicity tobe detected by the amounts of antibodyA.E.3 present in this solution. Othercommercially available monoclonalantibodies such as antibody CAM 5.2are capable of recognizing keratin 18 (1)and may be useful for detection ofkeratin in these epithelial tissues andtumors.

This study demonstrated thatcommercially available monoclonalantibodies to intermediate filamentsdeveloped for use in the identificationof human tumors recognize similarantigens in normal and well differen-tiated neoplastic canine tissues. Inmost cases the intermediate filamentswere tissue specific, however, two of119 tumors expressed intermediatefilaments which differed from those inthe corresponding normal tissues.Since intermediate filament coexpres-sion can occur in neoplasms, it isimportant to stain tumors with a seriesof antibodies to assist in obtaining anaccurate diagnosis. It is also importantto guard against false negative stainingwhich may result from the effects offixation and processing. This isapparent when normal tissues presentwithin a neoplastic section fail to stainfor the expected intermediate fila-ment. In addition, since keratinsconsist of 19 different proteins, someanti-keratin monoclonal antibodiesmay not detect all keratins and,therefore, may fail to detect someepithelial neoplasms.The neoplasms chosen for this study

were well differentiated. The stainingof the vast majority of these tumorsconfirms the tissue-specificity ofintermediate filament expression incanine neoplasms and suggests thatthis may be a useful technique for theidentification of anaplastic tumors indogs. However the usefulness ofintermediate filament detection incanine tumor identification will onlybe shown when these stains have beenstandardized among many laborato-ries and a large body of informationconcerning these staining patterns hasbecome available.

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