J Clin Pathol 1990;43:554-559

Adenoacanthoma of the endometrium:morphological changes induced by humanpapillomavirus

W F Kealy, P G Annis, J A Barry, J M Hogan

AbstractThe observation of koilocyte-likefeatures in the squamous epithelium ofsome endometrial adenoacanthomasprompted an investigation into a pos-sible viral aetiology. These changesclosely resemble those that occur in theectocervical mucosa which are acceptedas morphological evidence of humanpapillomavirus (HPV) infection. Sec-tions of 87 hysterectomy specimensremoved for endometrial carcinomaover 12 years, together with preoperativecurettings, were reviewed for thepresence of acanthomatous change andfor appearances suggestive ofHPV infec-tion. The ages of the women ranged from36 to 84 years, average age 62-6. Lightmicroscopical examination showedkoilocytosis, papillary formations, andintranuclear eosinophilic inclusionsof both squamous and glandular epi-thelium in some tumours. Immunocyto-chemistry and DNA in situ hybridisationindicated the presence ofHPV antigen insquamous and glandular cells, and peri-nuclear virus particles characteristic ofHPV were seen on electron microscopi-cal examination in those cells withnuclear inclusions.HPV probably infects endometrial

adenocarcinomas directly from thecervix but it is unlikely that it has anaetiological role. It is possible, however,that in addition to being a "passenger,"the virus may stimulate squamousmetaplasia in some adenocarcinomas ofthe endometrium and may also exertsome influence on their behaviour.

Department ofHistopathology,Regional Hospital,Wilton, Cork, IrelandW F KealyP G AnnisJ A BarryJ M HoganCorrespondence to:Dr W F KealyAccepted for publication5 April 1990

Adenoacanthoma of the endometrium is a

malignant epithelial tumour composed of bothglandular and squamous elements in whichthe squamous component is histologicallybenign."I The squamous parts of the tumoursare considered to arise as a result of meta-plastic glandular cells,"' but there is littleagreement about the amount of squamousepithelium which should be present before anadenocarcinoma may be called an adenoacan-thoma.'1'7 We have been impressed by thefact that the squamous epithelium present inuterine adenoacanthomas often displays cyto-plasmic vacuolation and koilocyte-likechanges, the appearances of which resemblethose produced by human papillomavirus(HPV) infection of ectocervical epithelium.

With this in mind, we reviewed the sections ofall uterine carcinomas received in thislaboratory over 12 years and attempted toestablish the presence of HPV in those with asquamous component.

MethodsSections of hysterectomy specimens removedfor endometrial carcinoma between 1977 and1988 inclusive were re-examined for thepresence of a squamous component. The oc-currence of squamous epithelium of benignhistological appearance intimately minglingwith the tumour, however small in amount,was accepted as evidence of acanthomatouschange. Note was also taken of epithelialchanges which might be ascribed to HPVinfection, such as cytoplasmic vacuolation andkoilocyte-like change. Sections of cervix,when present, were also reviewed for similarepithelial appearances and for the presence ofsquamous metaplasia. Preoperative endo-metrial curettings were also re-examined.

In two hysterectomy specimens showingadenoacanthoma of the endometrium multiplecircumferential blocks of the total cervix weretaken, which extended from the ectocervix tothe lower uterine cavity, to ascertain whetherthere was a direct connection between cervicalmetaplastic epithelium and the endometrium.Immunohistochemistry for HPV was perfor-med on sections of 23 adenoacanthomas andthe accompanying cervix, and six endometrialcurettings were also included. An immuno-peroxidase method using a polyclonal anti-papillomavirus antibody was used followed byvisualisation with an avidin-biotin complexdetection system (Dako). DNA in situ hy-bridisation for HPV was applied to sections ofadenoacanthoma in 11 cases using biotiny-lated probes specific for HPV types 6/1 1,16/18, and 30/50. (Enzo diagnostics).In two instances the hysterectomy specimen

was received fresh and unfixed and multiplerandom blocks of the endometrial carcinomawere taken into glutaraldehyde for electronmicroscopical examination. Selected areas offormalin fixed, paraffin wax embedded blocksof endometrial adenoacanthomas were alsoreprocessed for ultrastructural examination.

ResultsEighty seven hysterectomy specimens ofendometrial carcinoma were received into thislaboratory over the study period. Endometrialcurettings before hysterectomy were sent for

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Adenoacanthoma of the endometrium

examination in 63 cases. Squamous epitheliumwas identified in sections of endometrialadenocarcinoma in 50 hysterectomy cases, 46ofwhich showed acanthomatous change (fig 1),four were examples of adenosquamous carci-noma. Koilocyte-like changes of the squamousepithelium were present in 28 adenoacan-thomas (fig 2). These cellular changes occurredboth superficially and deeply within thetumours. Keratinisation was not a feature ofthe squamous epithelium and multinucleationwas rarely seen. In places the squamous andglandular cells adopted a rather papillary pat-tern. Round, relatively large, faintly eosino-philic nuclear inclusions were seen focally inboth glandular and squamous epithelium (figs3 and 4). Metastases from some of the endo-metrial carcinomas also showed focal benignsquamous epithelium and occasionallykoilocyte-like changes.Four of the accompanying cervices showed a

deficiency of ectocervix. Of the remaining 42,20 showed squamous metaplasia of the endo-cervical epithelium. Microscopic examinationof sections of the multiple circumferentialblocks taken from the whole cervix in the twocases showed focal squamous metaplasia, butdirect extension of the squamous epithelium tothe uterine cavity was not seen. Koilocyticchange in the ectocervical epithelium waspresent in 13 cases and of these, 10 also showedsquamous metaplasia. Intranuclear inclusionbodies of the type seen in the endometriumwere not present in either the ectocervical orendocervical epithelium. Preoperative endo-metrial curettings were performed in 32 instan-ces in those patients with adenoacanthoma.Seventeen of these showed acanthomatouschange of which 12 had koilocytic featuressuggestive of HPV infection. Eosinophilic

intranuclear inclusions were seen in some ofthesquamous cells ofa small number of curettings.

IMMUNOHISTOCHEMISTRYThis technique for HPV showed positive stain-ing in sections from 13 hysterectomies and oneendometrial curetting. The staining was dis-tributed in a rather focal manner, of varyingintensity, and in places affected both thenucleus and cytoplasm of both squamous andglandular epithelium. No cytological featuresaccounted for the variability of staining but thesquamous epithelium tended to stain moreintensely than the glandular cells. The intra-nuclear inclusions, however, tended to stainmore darkly in the glandular epithelium (fig 5).Sections of cervix showed focal staining of bothglandular and squamous cells. This positivity,however, was not often accompanied by similarstaining of the endometrium and indeed, theconverse was more usual. Immunoperoxidasestaining of a single ovarian metastasis withsquamous metaplasia was negative.

DNA IN SITU HYBRIDISATIONIn sections of endometrial adenoacanthomafrom 11 cases five tumours were negative forHPV. Three showed strong focal positivity forall the viral subtypes. The remaining threewere negative for subtype 6/1 1; of these,however, one showed positive staining of somesquamous cells for subtype 16/18 and anotherfor 31/35/51. The third showed positivity ofoccasional squamous and glandular cells forsubtypes 16/18 and 31/35/51, respectively. Theprobe tended to stain the intranuclear in-clusions only weakly but outlined the peri-nuclear area rather intensely (fig 6). In com-mon with most endometrial adenoacanthomas,those tumours showing positive immunohisto-

Figure 1 Adenoacanthomaof endometrium showingbenign squamousepithelium.

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Kealy, Annis, Barry, Hogan

Figure 2 Adenoacanthomaof endometrium showingkoilocyte-like change ofsquamous epithelium(Haematoxylin andeosin.)

Figure 3 Glandularepithelium with nuclearinclusions (Haematoxylinand eosin.)

chemistry and DNA in situ hybridisation were

all well differentiated carcinomas.

ELECTRON MICROSCOPYUltrastructural examination of the glutaralde-hyde fixed samples showed few of the lightmicroscopic features suggestive of viral infec-tion, as described above. Appearances verysuggestive of HPV infection, however, were

identified in many of the selected areas taken

from paraffin wax blocks. Examination of thenuclear inclusions showed that they consistedof fibrillary and granular material, the fibrils ofwhich ranged from 5 to 10 nm in thickness andthere was peripheral displacement of nuclearchromatin. Intranuclear viral particles werenot identified. Many cells, however, containedperinuclear aggregates of spherical electrondense bodies, not membrane bound, each ofwhich measured about 55 nm in diameter (figs

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Adenoacanthoma of the endometrium

Figure 4 Squamousepithelium with nuclearinclusions (Haematoxylinand eosin.)

Figure 5 Adenoacanthomaof endometrium withpositive staining ofglandular and squamousnuclei. (HPVImmunoperoxidase.)

7 and 8). Tissue preservation was not ideal andprecise morphological analysis was thereforeunsatisfactory. Nevertheless, the size of thesestructures and their arrangement in "rosarybead" and "mosaic" patterns were highly sug-gestive of papilloma virions.

DiscussionThe light microscopic appearances of

koilocyte-like changes of the squamous epithe-lium of some adenoacanthomas and thepresence of large intranuclear inclusions inboth glandular and squamous cells suggest verystrongly that the changes are due to viralinfection. Positive staining for HPV antigen bythe immunoperoxidase technique, togetherwith its demonstration by DNA in situ hy-bridisation, confirms that it is present. Ultra-structural examination by electron microscopy,

557

Kealy, Anms, Barry, Hogan:!. z j .; . .; A * ......... . 0 .. i .*4? .* ,} :

ls.,, _ " §. ' s.;s. 6,

s^|v :a a

s .'.'.': .:_!11# k .... .. ...._,.' V *'.'::.. ..@.§

.:* m.

Figure 6 Glandular epithelium showing nuclear and perinuclear staiDNA in situ hybridisation.)

although unable to show intrannevertheless shown particles ofcompatible with HPV in theadjacent to the nuclear membrawith intranuclear inclusions. 1ing reinforces the appearancefhybridisation in which the nu,stained faintly but where theperinuclear positivity (fig 6). N

were demonstrable inside the nuclear in-clusions, but the peripheral condensation of

~'<'* '*rav nuclear chromatin and the presence ofgranularand fibrillary material were suggestive of viralinfection. The intranuclear fibrils had an

*.,,,;" ,e appearance similar to the type II fibrils des-cribed with early adenovirus infection'8 and in

-* these cases were possibly coupled with HPVM. A.4* antigen. The source of the squamous epithe-g hlium in adenoacanthomas of the endometrium

is unclear but the cells most likely arise as as '-X4;s result of metaplasia either through squamous

differentiation of a common stem cell, or of a''^.;2A glandular cell, and possibly instigated by oes-*2htt- $ X trogenS 34 11 19-22trogens.

We could not show direct continuity be-tween foci of squamous metaplasia of theendocervix and the endometrial cavity and thisfact would tend to support the metaplasticorigin of the benign squamous epithelium inadenoacanthomas. Equally, the finding thatacanthomatous change was present in a large

( ;@ t t percentage of uterine curettings of the tumourobtained shortly before hysterectomy wouldmilitate against the possibility of implantationof squamous epithelium from the cervix andthe stimulation of squamous metaplasia by thetrauma of curettage.

ining. (HPV The origin ofHPV in endometrial carcinomais a matter of conjecture and two routes arepossible: direct spread from the cervix, orinfection via the bloodstream. Both routes

Lclear virus, has would seem to be possible, but spread from thea size and shape cervix is probably more likely2324 and is suppor-cell cytoplasm ted by the presence of koilocytosis of the

ine of those cells ectocervical epithelium and positive immuno-['his latter find- peroxidase staining for HPV of the endo-s of the in situ cervical cells in a considerable number of cases.iclear inclusions The converse situation-that is, colonisation ofere was intense the cervix by spread from the endometrium[o virus particles originally infected through the bloodstream-

Figure 7 Fibrillarylgranular nucleus withperipheral displacement ofchromatin. Perinuclearnucleocapsids showing apalisading "rosary bead"formation.

Z.. .... li > \ ) 9

4~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1

y Aft~~~~~~~

4e

558

Adenoacanthoma of the endometrium

Figure 8 Fibrillarynucleus (bottom left) andperinuclear cytoplasmicvirions about 50 nm indiameter.

is unlikely, given the much more common HPVinfection of the cervix.

Viruses have regularly been implicated in thegenesis of tumours in both man and animals,and it is well known that HPV often infects theectocervical epithelium and is closelyassociated with carcinoma of the cervix.22 25 It isunlikely, however, that the virus has any part inthe pathogenesis of endometrial adenoacanth-oma. It is mqre likely that it exists in thetumour as a passenger rather than an aetio-logical agent, although it may cause squamousmetaplasia of the glandular epithelium in somecases. Whether the virus in any way influencesthe ultimate behaviour of the tumour is aconsideration for investigation. What seems tobe certain, however, is that the HPV does, fromtime to time, infect the epithelium of adeno-acanthomas of the endometrium causing lightmicroscopic and other changes which advertiseits presence.

In this study we confined our attention tothose endometrial carcinomas showing acanth-omatous change as it was the koilocyte-likechange in the squamous epithelium which firstprompted the investigation. The finding ofHPV infection of some of the glandular epith-elium was unexpected and may form the basisof further study.We thank Ms P Harte, who typed the script, Ms L O'Donovanfor technical assistance, and the secretarial staff of the SouthemTumour Registry, Cork.

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