J Clin Pathol 1988;41:280-283

Malignant progression of laryngeal papillomaassociated with human papilloma virus type 6 (HPV-6)DNA

A P ZAROD,* J D RUTHERFORD,t G CORBITTtFrom the *Department of Otolaryngology, Manchester Royal Infirmary, Christie Hospital and Holt RadiumInstitute, Manchester, the tPathology Department, The Stopford Building, University ofManchester, and thetNorth Manchester Regional Virus Laboratory, Manchester Royal Infirmary

SUMMARY A case of laryngeal squamous papilloma in the early stages of development showedhistological features suggestive of virus infection. Five years later positive evidence of HPV-6infection was obtained at a time when the lesion had developed into a squamous cell carcinoma. It isconcluded that this case represents a complete example of the virus to papilloma to carcinomasequence, and as far as is known, is the first reported case of its kind in the larynx.

For over 100 years papilloma has been recognised asbeing the most common benign tumour of the larynx.Because of the morphological similarity to papillomaswhich occur in other sites where a viral origin has beenconfirmed, laryngeal papilloma has long been con-sidered to be of viral origin. Only recently has thepresence of papilloma virus (HPV) been confirmedwithout doubt and established to be the same viruswhich infects the tissues of the genital tract.

In 1880 Mackenzie was the first to record thatlesions in children tend to be multiple and recurrentwhereas in adults they tend to be single and curablewith one excision.' By 1973 there were conflictingreports on the presence of viral particles in laryngealpapilloma when examined by electron microscopy.23In 1980 papilloma virus capsid antigen was detected byLack et al using immunoperoxidase techniques.4 In1982 Mounts et aP showed that viral genomes wereclosely related to papilloma virus HPV type 6, usingDNA hybridisation techniques. Human papillomavirus type 6 has been linked with oral, genital, andlaryngeal papillomas. In 1983 Gissman et al foundHPV type 11 in laryngeal papilloma and HPV type 6and HPV type 11 in genital papillomas.6 Carci-nomatous change in pre-existing papillomas fromother sites and species has also been observed.79

Accepted for publication 25 September 1987

Case report

A 72 year old female non-smoker had had repeatedmicrolaryngoscopic procedures for recurrent laryn-geal papillomas since the age of 65. In 1984 when thecarbon dioxide laser became available at the ChristieHospital and Holt Radium Institute she was referredfor further treatment by laser vapourisation. Previousprocedures for recurrent hoarseness due to clinicallyand histologically benign laryngeal papillomas consis-ted ofmicrolaryngoscopic excision oftumour from theleft vocal cord.At the time of laser treatment papilloma was noted

covering the anterior one third of the left vocal cord,the base of the epiglottis under the tubercle encroach-ing on the anterior comissure, and the floor of the leftventricle. Two biopsy specimens were taken from theanterior one third of the left vocal cord using cupforceps. One specimen was sent for conventionalhistological staining and the other for HPV typing.The residual papilloma was vapourised by the laser.Results showed the presence ofinvasive squamous cellcarcinoma and HPV type 6 infection.Although the staging was probably that ofa T,NOMO

laryngeal carcinoma, she was clinically classified asT2NAM0 because papillomatous infection was notlimited to one vocal cord. She received a radical courseof radiotherapy and remained disease free at the timeof writing.

280

Malignant progression oflaryngealpapilloma associated with HPV-6

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Pathology

Sections of the laryngeal biopsy specimens from 1979,1980, 1981 and 1984 were reviewed. All were substan-tial cup forceps biopsy specimens. The first two were ofbenign papillomata and both showed koilocytoticatypia; neither showed the key features of verrucouscarcinoma as outlined by Michaels'°-that is, keratin-isation, parakeratosis, mononuclear inflammatory cellinfiltrate of the connective tissue core, crowding ofbasal cells and wide fields of large intermediate cells(figs 1 and 2). The third specimen from 1981 showedkoilocytosis and obvious cellular atypia (fig 3). The1984 biopsy specimen showed unequivocal evidence ofwell differentiated squamous cell carcinoma showingkeratin pearls and microinvasion (fig 4). All fourspecimens were taken from the left vocal cord. UsingDNA hybridisation techniques, dot blotting, andSouthern transfer analysis" human papilloma virustype 6 (HPV-6) was isolated from the final biopsyspecimen taken in 1984.

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Discussion

Benign and malignant tumours associated with virusesare a well established phenomenon in animals.'2Bovine papilloma virus BPV-4 has been detected onlyin benign lesions. BPV- 1 and BPV-2, however, havebeen shown to transform cells and produce malignanttumours in a wide variety of species.'3 The Shopepapilloma virus has been identified in both benign andmalignant tumours.'4 In man epidermodysplasiaverucciformis HPV 5 or 8 has been detected in 82% ofinvasive lesions in which it was possible to detect HPVgenome.'5

Benign to malignant transformation as a result ofviral infection is gradually being accepted as a processwhich may occur in man. Current interest is alsocentered on HPV virus in cervical carcinoma. Crum etal concluded that the presence of HPV-16 correlatedwith the presence of mitotic figures in flat warts of thecervix and that these warts were a precursor ofinvasivecancer of the cervix.'6 In some veruccous carcinomas

281

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Fig 3 Biopsy specimenfrom 1981 showing koilocytosis andcellular atypia.

ofthe genital tract HPV-6 and HPV-1 1 sequences havebeen shown'7 and some vulval carcinomas containsequences related to HPV- 16.8 Our case seems torepresent an example of virus to papilloma to carci-noma sequence in the human larynx.

Although verrucous carcinoma may be mistaken forsquamous papilloma, careful review of the earlyspecimens and comparison with known squamouspapillomas confirmed our belief that the originalbiopsy specimens were of genuine squamouspapilloma.

Koilocytosis as a marker of viral infection inmucosal surfaces is well recognised.'0 An aetiologicalviral role in our case was implicated by the fact thatkoilocytosis was seen in the biopsy specimen takenwhen the patient first presented in 1979 and insubsequent specimens taken in 1980 and 1981.Although it is possible to show the presence of viralparticles by both electron microscopy2 and immuno-peroxidase methods,4 these tend to be time consuming,

Fig 4 Biopsy specimen from 1984 showing squamous cellcarcinoma characterised by nuclear pleomorphism, abnormalmitotic.figures, and microinvasion.

lack sensitivity, and do not give a high yield of positiveresults. In tumour tissue viral genome expression isusually limited, and production of virus specificprotein is either very small or non-existent. Recentadvances in DNA hybridisation techniques give asensitive technique for viral DNA detection and HPVtyping. In our patient the presence of HPV-6 DNAwas doubly confirmed by using dot blotting andSouthern transfer analysis.

It is interesting to note that the type of papillomavirus isolated here was of a different type (HPV-6) tothose commonly associated with carcinoma of thecervix (HPV-16),"6 although it is of the same type asone of those associated with benign condylomata ofthe uterine cervix (HPV-6 and HPV- I 1).6

In this case, malignant change occurred without anyincrease in symptoms. It is therefore recommendedthat all specimens be sent for histological examinationand that pathologists should specifically look forfeatures of viral infection and early atypia and

282

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Malignant progression oflaryngeal papilloma associated with HPV-6 283emphasise the presence of these in biopsy reports sothat clinicians may be alerted to the risk of cancerouschange.We would point out that the adult onset papilloma

is histologically indistinguishable from the juveniletype'9 but behaves in a different way clinically. Thejuvenile form is associated with florid papillomatosisbut no malignant change unless the lesion isirradiated.20 The papilloma viruses implicated (types 6and 11) are the same as those seen in benign con-dylomata of the cervix, implying that transmissionmay occur during passage down the birth canal. Theadult type, by contrast, is less florid clinically but morelikely to undergo spontaneous malignant change.2'Using cytological hybridisation techniques, Brichaceket al associated Epstein-Barr virus with supraglotticlaryngeal carcinoma in three of five patients withEpstein-Barr virus antibodies.22 In our case of glotticcarcinoma associated with a previous history oflaryngeal papilloma and HPV-6 infection the malig-nant change may have induced by the virus.We confirm the association of human papilloma

virus with laryngeal papilloma and that carcin-omatous change may take place in pre-existing benignpapillomas. We believe that our case illustrates the linkbetween these phenomena and postulate that the virusmay be the initiating event in the papilloma tocarcinoma sequence. This is analogous to the situationin the uterine cervix and adds further weight to theaccumulating evidence implicating viruses asaetiological agents in cancers in man.

We thank Mr R T Ramsden for permission toreport his patient who was subsequently treated underthe care of Mr W T Farrington, the generosity ofDr R Dodge for loaning histological material, and theResearch Grants Committee of the CentralManchester Health Authority for funding the DNAhybridisation studies.

References

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3 Cook TA, Cohn AM, Brunschwig JP, Goepfert H, Butel JS, RawlsWE. Laryngeal papilloma: etiologic and therapeutic considera-tions. Ann Otol Rhinol Laryngol 1973;82:649-55.

4 Lack EE, Jenson AB, Smith HG, Healy GB, Pass F, Vawter GF.

Immunoperoxidase localisation of human papillomavirus inlaryngeal papillomas. Intervirology 1980;14:148-54.

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7 Grussendorf E, Gahlen W. Metaplasia ofa verucca vulgaris into aspinocellular carcinoma. Dermatologica 1975;150:295-9.

8 Syverton JT. The pathogenesis of the rabbit papilloma to carcin-oma sequence. Ann NY Acad Sci 1952;54:1 126-40.

9 Gisser SD. Papovavirus and squamous cell carcinoma. HumPathol 1981;12:190-3.

10 Michaels L. Pathology ofthe Larynx. Heidelberg: Springer-Verlag,1984, 159-73, 258-65.

11 Southern EM. Detection of specific sequences among DNAfragments separated by gel electrophoresis. J Mol Biol1975;98:503-17.

12 McNeill PE, Laird HM, Jarrett WFH, et al. In: Essem M, TodaroG, zur Hausen H, eds. Viruses in naturally occurring cancers.New York: Old Spring Harbour Laboratory, 1981:215.

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14 Stevens JG, Wettstein FO. Multiple copies of Shope virus DNAare present in cells ofbenign and malignant non-virus-producingneoplasms. J Virol 1979;30:891-8.

15 Orth G, Jablonska S, Jarzabek-Chorzelska M, et al. Characteris-tics of the lesions and risk of malignant conversion associatedwith the type ofhuman papillomavirus involved in epidermodys-plasia verucciformis. Cancer Res 1979;39:1074-82.

16 Crum CP, Ikenberg H, Richart RM, Gissman L. Humanpapillomavirus type 16 and early cervical neoplasia. N Engl JMed 1984;310:880-3.

17 Durst M, Gissman L, Ikenberg H, zur Hausen H. A Papillomavirus DNA from a cervical carcinoma and its prevalence incancer biopsy samples from different geographic regions. ProcNatl Acad Sci USA 1983;80:3812-15.

18 Green M, Brackmann K, Sanders P, et al. Isolation of humanpapillomavirus from a patient with epidermodysplasiaverruciformis: presence of related viral DNA genomes in humanurogenital tumours. Proc Natl Acad Sci USA 1982;79:4437-41.

19 Capper JWR, Bailey CM, Michaels L. Squamous papillomas ofthe larynx in adults: a review of 63 cases. Clin Otolaryngol1983;8:109-18.

20 Majoros M, Devine KD, Parkhill EM. Malignatit transformationof benign laryngeal papillomas in children after radiationtherapy. Surg Clin North Am 1963;43:1049-61.

21 Mounts P,Kashima H. Association of human papilloma virussubtype and clinical course in respiratory papillomatosis. Laryn-goscope 1984;94:28-33.

22 Brichacek B, Hirsch I, Sibl 0, Vilikusova E, Vonka V. Associationof some supraglottic laryngeal carcinomas with EB virus. Int JCancer 1983;32:193-7.

Requests for reprints to: Dr G Corbitt, PrincipalMicrobiologist, North Manchester Regional VirusLaboratory, Manchester Royal Infirmary, Oxford Road,Manchester M 13 9WL, England.