554 J Clin Pathol 1990;43:554-559 of the endometrium:

morphological changes induced by human J Clin Pathol: first published as 10.1136/jcp.43.7.554 on 1 July 1990. Downloaded from papillomavirus

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

Abstract With this in mind, we reviewed the sections of The observation of -like all uterine received in this features in the squamous of laboratory over 12 years and attempted to some endometrial adenoacanthomas establish the presence of HPV in those with a prompted an investigation into a pos- squamous component. sible viral aetiology. These changes closely resemble those that occur in the ectocervical mucosa which are accepted Methods as morphological evidence of human Sections of specimens removed papillomavirus (HPV) . Sec- for endometrial between 1977 and tions of 87 hysterectomy specimens 1988 inclusive were re-examined for the removed for endometrial carcinoma presence of a squamous component. The oc- over 12 years, together with preoperative currence of squamous epithelium of benign curettings, were reviewed for the histological appearance intimately mingling presence of acanthomatous change and with the tumour, however small in amount, for appearances suggestive ofHPV infec- was accepted as evidence of acanthomatous tion. The ages of the women ranged from change. Note was also taken of epithelial 36 to 84 years, average age 62-6. Light changes which might be ascribed to HPV microscopical examination showed infection, such as cytoplasmic vacuolation and koilocytosis, papillary formations, and koilocyte-like change. Sections of , intranuclear eosinophilic inclusions when present, were also reviewed for similar of both squamous and glandular epi- epithelial appearances and for the presence of thelium in some tumours. Immunocyto- squamous . Preoperative endo- chemistry and DNA in situ hybridisation metrial curettings were also re-examined. indicated the presence of HPV antigen in In two hysterectomy specimens showing http://jcp.bmj.com/ squamous and glandular cells, and peri- adenoacanthoma of the endometrium multiple nuclear virus particles characteristic of circumferential blocks of the total cervix were HPV were seen on electron microscopi- taken, which extended from the ectocervix to cal examination in those cells with the lower uterine cavity, to ascertain whether nuclear inclusions. there was a direct connection between cervical HPV probably infects endometrial metaplastic epithelium and the endometrium. directly from the for HPV was perfor- on September 25, 2021 by guest. Protected copyright. cervix but it is unlikely that it has an med on sections of 23 adenoacanthomas and aetiological role. It is possible, however, the accompanying cervix, and six endometrial that in addition to being a "passenger," curettings were also included. An immuno- the virus may stimulate squamous peroxidase method using a polyclonal anti- metaplasia in some adenocarcinomas of papillomavirus antibody was used followed by the endometrium and may also exert visualisation with an avidin-biotin complex some influence on their behaviour. detection system (Dako). DNA in situ hy- bridisation for HPV was applied to sections of adenoacanthoma in 11 cases using biotiny- Adenoacanthoma of the endometrium is a lated probes specific for HPV types 6/1 1, malignant epithelial tumour composed of both 16/18, and 30/50. (Enzo diagnostics). glandular and squamous elements in which In two instances the hysterectomy specimen the squamous component is histologically was received fresh and unfixed and multiple benign."I The squamous parts of the tumours random blocks of the endometrial carcinoma are considered to arise as a result of meta- were taken into glutaraldehyde for electron plastic glandular cells,"' but there is little microscopical examination. Selected areas of Department of agreement about the amount of squamous formalin fixed, paraffin wax embedded blocks , epithelium which should be present before an of endometrial adenoacanthomas were also Regional Hospital, may be called an adenoacan- Wilton, Cork, Ireland reprocessed for ultrastructural examination. W F Kealy thoma.'1'7 We have been impressed by the P G Annis fact that the squamous epithelium present in J A Barry uterine adenoacanthomas often displays cyto- Results J M Hogan plasmic vacuolation and koilocyte-like Eighty seven hysterectomy specimens of Correspondence to: Dr W F Kealy changes, the appearances of which resemble endometrial carcinoma were received into this Accepted for publication those produced by human papillomavirus laboratory over the study period. Endometrial 5 April 1990 (HPV) infection of ectocervical epithelium. curettings before hysterectomy were sent for Adenoacanthoma of the endometrium 555

examination in 63 cases. Squamous epithelium intranuclear inclusions were seen in some ofthe was identified in sections of endometrial squamous cells ofa small number ofcurettings. adenocarcinoma in 50 hysterectomy cases, 46 ofwhich showed acanthomatous change (fig 1), IMMUNOHISTOCHEMISTRY J Clin Pathol: first published as 10.1136/jcp.43.7.554 on 1 July 1990. Downloaded from four were examples of adenosquamous carci- This technique for HPV showed positive stain- noma. Koilocyte-like changes of the squamous ing in sections from 13 and one epithelium were present in 28 adenoacan- endometrial curetting. The staining was dis- thomas (fig 2). These cellular changes occurred tributed in a rather focal manner, of varying both superficially and deeply within the intensity, and in places affected both the tumours. Keratinisation was not a feature of nucleus and cytoplasm of both squamous and the squamous epithelium and multinucleation glandular epithelium. No cytological features was rarely seen. In places the squamous and accounted for the variability of staining but the glandular cells adopted a rather papillary pat- squamous epithelium tended to stain more tern. Round, relatively large, faintly eosino- intensely than the glandular cells. The intra- philic nuclear inclusions were seen focally in nuclear inclusions, however, tended to stain both glandular and squamous epithelium (figs more darkly in the glandular epithelium (fig 5). 3 and 4). Metastases from some of the endo- Sections ofcervix showed focal staining of both metrial carcinomas also showed focal benign glandular and squamous cells. This positivity, squamous epithelium and occasionally however, was not often accompanied by similar koilocyte-like changes. staining of the endometrium and indeed, the Four of the accompanying cervices showed a converse was more usual. Immunoperoxidase deficiency of ectocervix. Of the remaining 42, staining of a single ovarian with 20 showed squamous metaplasia of the endo- squamous metaplasia was negative. cervical epithelium. Microscopic examination of sections of the multiple circumferential DNA IN SITU HYBRIDISATION blocks taken from the whole cervix in the two In sections of endometrial adenoacanthoma cases showed focal squamous metaplasia, but from 11 cases five tumours were negative for direct extension of the squamous epithelium to HPV. Three showed strong focal positivity for the uterine cavity was not seen. Koilocytic all the viral subtypes. The remaining three change in the ectocervical epithelium was were negative for subtype 6/1 1; of these, present in 13 cases and of these, 10 also showed however, one showed positive staining of some squamous metaplasia. Intranuclear inclusion squamous cells for subtype 16/18 and another bodies of the type seen in the endometrium for 31/35/51. The third showed positivity of were not present in either the ectocervical or occasional squamous and glandular cells for endocervical epithelium. Preoperative endo- subtypes 16/18 and 31/35/51, respectively. The metrial curettings were performed in 32 instan- probe tended to stain the intranuclear in- ces in those patients with adenoacanthoma. clusions only weakly but outlined the peri- http://jcp.bmj.com/ Seventeen of these showed acanthomatous nuclear area rather intensely (fig 6). In com- change of which 12 had koilocytic features mon with most endometrial adenoacanthomas, suggestive of HPV infection. Eosinophilic those tumours showing positive immunohisto-

Figure 1 Adenoacanthoma of endometrium showing on September 25, 2021 by guest. Protected copyright. benign squamous epithelium. 556 Kealy, Annis, Barry, Hogan

Figure 2 Adenoacanthoma of endometrium showing koilocyte-like change of

squamous epithelium J Clin Pathol: first published as 10.1136/jcp.43.7.554 on 1 July 1990. Downloaded from (Haematoxylin and eosin.)

Figure 3 Glandular epithelium with nuclear inclusions (Haematoxylin and eosin.) http://jcp.bmj.com/ on September 25, 2021 by guest. Protected copyright.

chemistry and DNA in situ hybridisation were from paraffin wax blocks. Examination of the all well differentiated carcinomas. nuclear inclusions showed that they consisted of fibrillary and granular material, the fibrils of ELECTRON MICROSCOPY which ranged from 5 to 10 nm in thickness and Ultrastructural examination of the glutaralde- there was peripheral displacement of nuclear hyde fixed samples showed few of the light chromatin. Intranuclear viral particles were microscopic features suggestive of viral infec- not identified. Many cells, however, contained tion, as described above. Appearances very perinuclear aggregates of spherical electron suggestive of HPV infection, however, were dense bodies, not membrane bound, each of identified in many of the selected areas taken which measured about 55 nm in diameter (figs Adenoacanthoma of the endometrium 557

Figure 4 Squamous epithelium with nuclear inclusions (Haematoxylin and eosin.) J Clin Pathol: first published as 10.1136/jcp.43.7.554 on 1 July 1990. Downloaded from

Figure 5 Adenoacanthoma of endometrium with positive staining of glandular and squamous nuclei. (HPV Immunoperoxidase.) http://jcp.bmj.com/ on September 25, 2021 by guest. Protected copyright.

7 and 8). Tissue preservation was not ideal and koilocyte-like changes of the squamous epithe- precise morphological analysis was therefore lium of some adenoacanthomas and the unsatisfactory. Nevertheless, the size of these presence of large intranuclear inclusions in structures and their arrangement in "rosary both glandular and squamous cells suggest very bead" and "mosaic" patterns were highly sug- strongly that the changes are due to viral gestive of virions. infection. Positive staining for HPV antigen by the immunoperoxidase technique, together with its demonstration by DNA in situ hy- Discussion bridisation, confirms that it is present. Ultra- The light microscopic appearances of structural examination by electron microscopy, 558 Kealy, Anms, Barry, Hogan were demonstrable inside the nuclear in- clusions, but the peripheral condensation of ~'<'* '*rav nuclear chromatin and the presence ofgranular of viral and fibrillary material were suggestive J Clin Pathol: first published as 10.1136/jcp.43.7.554 on 1 July 1990. Downloaded from infection. The intranuclear fibrils had an 4~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1 *.,,,;" ,e appearance similar to the type II fibrils des- cribed with early adenovirus infection'8 and in Z...... li > \ ) 9 y Aft~~~~~~~ -* these cases were possibly coupled with HPV * antigen. The source of the squamous epithe- M. A.4 g hlium in adenoacanthomas of the endometrium is unclear but the cells most likely arise as a s-X4;' s result of metaplasia either through squamous differentiation of a common stem cell, or of a

*4? .* ,} : ''^.;2A glandular cell, and possibly instigated by oes- :!. z j.; . . 3 4 11 19-22 ;A*...... ls . 0 ..i . *2htt- $ X trogenStrogens. We could not show direct continuity be- .;s. 6, tween foci of squamous metaplasia of the s^|v :a a endocervix and the endometrial cavity and this 4e s .'.'.': .: _,.' V *'.':: fact would tend to support the metaplastic ' s of the benign squamous epithelium in .,, _" §. origin adenoacanthomas. Equally, the finding that .:* @.§m. acanthomatous change was present in a large ( ;@ t t percentage of uterine curettings of the tumour _!11# k...... obtained shortly before hysterectomy would militate against the possibility of implantation .... of squamous epithelium from the cervix and the stimulation of squamous metaplasia by the trauma of curettage. Figure 6 Glandular epithelium showing nuclear andperinuclear staiining. (HPV The origin ofHPV in endometrial carcinoma DNA in situ hybridisation.) is a matter of conjecture and two routes are possible: direct spread from the cervix, or infection via the bloodstream. Both routes although unable to show intran Lclear virus, has would seem to be possible, but spread from the nevertheless shown particles ofa size and shape cervix is probably more likely2324 and is suppor- compatible with HPV in the cell cytoplasm ted by the presence of koilocytosis of the adjacent to the nuclear membraine ofthose cells ectocervical epithelium and positive immuno- with intranuclear inclusions. 1['his latter find- peroxidase staining for HPV of the endo- ing reinforces the appearancefs of the in situ cervical cells in a considerable number ofcases. http://jcp.bmj.com/ hybridisation in which the nu,iclear inclusions The converse situation-that is, colonisation of stained faintly but where theere was intense the cervix by spread from the endometrium perinuclear positivity (fig 6). N[o virus particles originally infected through the bloodstream-

Figure 7 Fibrillaryl granular nucleus with peripheral displacement of on September 25, 2021 by guest. Protected copyright. chromatin. Perinuclear nucleocapsids showing a palisading "rosary bead" formation. Adenoacanthoma of the endometrium 559

Figure 8 Fibrillary nucleus (bottom left) and perinuclear cytoplasmic virions about 50 nm in diameter. J Clin Pathol: first published as 10.1136/jcp.43.7.554 on 1 July 1990. Downloaded from

is unlikely, given the much more common HPV 4 Alberhasky RC, Connelly PJ, Christopherson WM. Carci- noma of the endometrium. IV. Mixed adenosquamous infection of the cervix. carcinoma. Am J Clin Pathol 1982;776:655-64. Viruses have regularly been implicated in the 5 Novak ER, Nalley WB. Uterine adenoacanthoma. Obstet Gynecol 1957;9:396-402. genesis of tumours in both man and animals, 6 Marcus SL. Adenoacanthoma of the endometrium. Am J and it is well known that HPV often infects the Obstet Gynecol 1961;81:259-67. 7 Tweeddale DM, Early LS, Goodsitt ES. Endometrial http://jcp.bmj.com/ ectocervical epithelium and is closely adenoacanthoma. Obstet Gynecol 1964;23:611-9. associated with carcinoma ofthe cervix.2225 It is 8 Morrison DL. Adenoacanthoma of the uterine body. Obstet Gynecol Br Commonwealth 1966;73:605-10. unlikely, however, that the virus has any part in 9 Piver MS. Distant metastases of adenoacanthoma of the the of endometrial adenoacanth- endometrium. Am J Obstet Gynecol 1966;96:101 1-6. 10 Hopkin ID, Harlow RA, Stephens PJ. Squamous carcinoma oma. It is mqre likely that it exists in the of the body of the . Br J 1970;24:71-6. tumour as a passenger rather than an aetio- 11 Liu CT. A study of endometrial adenocarcinoma with emphasis on morphologically variant types. Am J Clin logical agent, although it may cause squamous Pathol 1972;57:562-73. metaplasia of the glandular epithelium in some 12 Silverberg SG, Bolin HG, DeGiorgi LS. Adenoacanthoma on September 25, 2021 by guest. Protected copyright. and mixed adenosquamous carcinoma of the endo- cases. Whether the virus in any way influences metrium. Cancer 1972;30:1307-14. the ultimate behaviour of the tumour is a 13 Aikawa M, Ng ABP. Mixed (adenosquamous), carcinoma of the endometrium; electron microscopic observations. consideration for investigation. What seems to Cancer 1973;31:385-97. be certain, however, is that the HPV does, from 14 Haqqani MT, Fox H. Adenosquamous carcinoma of the endometrium. J Clin Pathol 1976;29:959-66. time to time, infect the epithelium of adeno- 15 Blaustein A, Bigelow B, Demopoulos RI. Association of acanthomas of the endometrium causing light carcinoma ofthe breast with adenosquamous carcinoma of endometrium. Cancer 1978;42:326-9. microscopic and other changes which advertise 16 Christopherson WM. The significance of the pathologic its presence. findings in . Clinics in obstetrics and gynaecology. Philadelphia: WB Saunders, 1986:679-82. In this study we confined our attention to 17 Badib AO, Kurohara SS, Vongtama VY, Selimn MA, those endometrial carcinomas showing acanth- Webster JH. Biologic behaviour ofadenoacanthoma ofthe endometrium. Am J Obstet Gynecol 1970;106:205-9. omatous change as it was the koilocyte-like 18 Martinex-Palomo A, Le Buis J, Bernard W. Electron change in the squamous epithelium which first microscopy or adenovirus 12 replication. J Virol 1967; 1:817. prompted the investigation. The finding of 19 Seltzer V, Klein M, Beckman M. The occurrence of HPV infection of some of the glandular epith- squamous metaplasia as a precursor of of the endometrium. Obstet Gynecol 1977;49: elium was unexpected and may form the basis 34S-7S. of further study. 20 Kanbour AI, Stock RJ. Squamous cell carcinoma in-situ of the endometrium and fallopian tube as superficial exten- We thank Ms P Harte, who typed the script, Ms L O'Donovan sion of invasive cervical carcinoma. Cancer 1978;42:570-9. for technical assistance, and the secretarial staff of the Southem 21 Lauchlan SC. Metaplasias and neoplasias of Mullerian Tumour Registry, Cork. epithelium. Histopathol 1984;8:543-57. 22 Hausen HZ. Papilloma viruses in human cancer. Cancer 1987;59:1692-6. 1 Ng ABP, Reagan JW, Storaasli JP, Wentz WB. Mixed 23 Gupta JW, Saito K, Saito A, Fu YS, Shah KV. Human adenosquamous carcinoma of the endometrium. Am J papillomavirus and the pathogenesis ofcervical neoplasia. Clin Pathol 1972;59:765-81. Cancer 1989;64:2104-10. 2 Salazar DM, DePapp EW, Bonfiglio TA, Feldstein ML, 24 Roberts PF, Carron Brown J. Condylomatous atypia of the Rubin P, Rudolph JH. Adenosquamous carcinoma of the endometrial cavity. Case report to Br J Obstet Gynaecol endometrium. Cancer 1977;40:119-30. 1985;92:535-8. 3 Barrowclough H, Jaarsula KW. Adenoacanthoma of the 25 Drake M, Medley G, Mitchell H. Cytologic detection of endometrium: a separate entity or a histological curiosity? HPV infection. Obstet Gynecol Clin North Am J Clin Pathol 1980;33:1064-7. 1987;14:431.