Janse Van Rensburg, Estrelita

Raubenheimer, Erich J.

van Heerden, Willem F. P.

Venter, Estelle H.

English

AJOL - African Journals Online

Detection of humanpapillomavirus DNA with insitu hybridisation in oralsquamous carcinoma in arural black populationEstrelita Janse Van Rensburg, Willem F. P. vanHeerden, Estelle H. Venter, Erich J. RaubenheimerIntra-oral carcinoma is the third most common malignancyamong men in developing countries, and carries a highmortality rate, particularly in Africa, where patients oftenpresent initially with lesions at an advanced stage. Thepresent study was undertaken to determine the prevalenceof human papillomavirus (HPV) DNA in oral squamouscarcinoma in the west of the Northern Transvaal, an areawhere a large number of new cases has been diagnosedover the past few years. Paraffin blocks from 66 cases (51men, 15 women; mean age 58,7 years) of oral squamouscarcinoma were randomly selected. Blocks containedsamples of both tumour and adjacent normal epithelium.The presence of HPV antigen was established by means -of immunocytochemistry and HPV DNA by in situhybridisation with radiolabelled probes for HPV-6, 11, 16and 18. Immunocytochemistry for viral antigen wasnegative in all the specimens. HPV-18 was detected innormal epithelium adjacent to the tumour in one case only.It appears from our study that HPV is of limitedimportance in oral squamous cell carcinogenesis in thepopulation studied.S Afr Med J 1995; 85: 894-896.Human papillomaviruses (HPVs) have a predilection forsquamous epithelium where they induce benign proliferativelesions such as warts on the skin, and papillomas andcondylomas on mucosal surfaces.' Certain types, especiallyHPV-16, 18 and 33, have been implicated in squamous cellcarcinogenesis because of their ability to immortalise humanDepartment of Medical Virology, University of Stelienbosch,Tygerberg, W. CapeEstrelita Janse Van Rensburg, M.MED. (VIROL)Department of Oral Pathology and Oral Biology, Medical Universityof Southern Africa, PO Medunsa, 0204Willem F. P. van Heerden, M.CH.D.Erich J. Raubenheimer, M.CH.9.Department of Tropical Diseases, University of PretoriaEstelle H. Venter, PHD.epithelial cells after transfection.'·3 HPV-16 and 18 arestrongly associated with high-grade premalignant lesionsand anogenital carcinomas! Oral HPV infections have notbeen studied to the same degree as those of the genitaltract, but are known to be associated with a variety of orallesions in man, including papillomas, focal epithelialhyperplasia, hyperkeratotic lesions, lichen planus andleukoplakia.' In view of the'obvious oncogenic potential ofsome HPVs and the close similarity between the oral andgenital mucosa, the possibility that certain HPV types mayplay a causative role in oral cancer does not seem tooremote. Certain HPV types have been detected in benig~oral lesions, as well as in oral squamous carcinoma." . ~Studies originating from different geographical areas showa variation in HPV positivity ranging from 0% to 76,4%.7.11This difference in positivity is due to different populationsstudied and different methods used in HPV DNA detection.According to the Federation Dentaire Internationa~ intra-oral cancer, of which squamous cell carcinoma accounts forthe majority of cases, is the third most common malj§nantdisease among men in developing countries.'2 The g'iortalityrate remains unacceptably high, particularly in Africa, whereadvanced lesions are common on presentation. Althoughoral squamous carcinoma rarely occurs before the age of 50years, Fleming et al. '3 found a significant difference in agedistribution between black and white South African men:33,4% of the blacks were below the age of 50 years, asopposed to 15,6% of white men. The authors suggest thatthe black group may have been exposed to carcinogenicagents at an earlier age than the white group.Our study was undertaken to determine the prevalence ofHPV DNA in oral squamous carcinoma at Ga-RankuwaHospital, a referral centre for the western NorthernTransvaal, an area in which a high number of new cases hasbeen diagnosed over the past few years.Material and methodsFormalin-fixed paraffin embedded blocks from 66 patients(51 men, 15 women, mean age 58,7 years) with oralsquamous carcinoma were randomly selected from thearchives of the Department of Oral Pathology at MEDUNSA.These blocks contained samples of both tumour and.adjacent normal epithelium. Sections 5 ~m thick were cutand used for light microscopy and in situ hybridisation. Allsections were evaluated for epithelial chan'ges consistentwith HPV infection. These include verrucous hyperplasiawith hyperparakeratosis and the presence of koilocytes.ImmunocytochemistryAll sections were examined for the presence of HPVstructural proteins (group-specific papillomavirus capsidantigens) with an ABC immunoperoxidase kit (LipshawCorporation, Detroit).HPV DNA in situ hybridisation (ISH)All sections were mounted on 3-aminopropyltriethoxysilane-coated slides,'4 deparaffinised and rehydrated by sequential, 894 Volume 85 No. 9 September 1995 SAMJ--~----I~immersion into xylene and ethanol and digested withproteinase K (0,05 mg/ml) to expose the fixed target DNA.After post-fixation in 4% paraformaldehyde, slides wereprehybridised for 30 minutes at 52°C, prior to the applicationof the denatured probe solution. DNA probes for HPV-6, 11,16 and 18, cloned in pBR322, were used. These were kindlyprovided by Or E.-M. de Villiers of the HumanPapillomavirus Reference Center, DKFZ, Heidelberg,Germany. The probes were labelled with 32p dCTP accordingto the multiprime DNA labelling system (Amersham, UK).Hybridisation was allowed to take place at 52°C for 16 hoursin a humidified chamber. There were two post-hybridisationwashes in a 2 x SSPEl50% formamide solution and one in50% formamide, 0,1 % SOS, 2 x SSC; each wash lasted1 hour at 37°C. Slides were dehydrated through gradedethanol containing 0,3M NH acetate and then dipped inLM-1 emulsion (Amersham, UK). After a 4-day exposuretime at 4°C, slides were developed (1Iford, Ciba Geigy),rinsed briefly in water and fixed for 5 minutes in Hypamfixative (lIford). Sections were counterstained withhaematoxylin and eosin before mounting.Assay sensitivity was confirmed with two known positivecontrol slides (one of cervical intra-epithelial neoplasia andthe second, a carcinoma of the vulva positive for HPV-6 and16 respectively). Assay specificity was confirmed byhybridisation of plasmid vector pBR322 on all sectionsinvestigated.Results. In none of the biopsies were HPV-associated morphologicalchanges found in the normal epithelium adjacent to thecarcinoma. HPV-antigen expression could also not bedemonstrated by means of the ABC immunoperoxidasetechnique in any of the tumour sections or in the adjacentnormal epithelium.HPV-18 DNA was detected by in situ hybridisation in the'normal epithelium adjacent to the carcinoma in a single case(Fig. 1).Fig. 1. Mic~ograph showing the presence of HPV-18 DNA ascondensations of black-silver grains superimposed on the nucleus(x 200), with inset of high-power detail of positive cell Ix 400).SAMJARTICLESDiscussionThe oral mucosa is continuously exposed to minor trauma,micro-organisms and chemical factors such as tobacco andalcohol, which may act synergistically with HPV, leading tothe development of carcinoma.'5.1.This study determined the prevalence of HPV infection intumours and adjacent normal epithelium in cases of oralsquamous carcinoma. The detection system used wasselected because in situ hybridisation is useful forretrospective biopsy screening, as it permits detection,typing and localisation of HPV DNA in paraffin-embeddedtissues that are readily available in a department ofpathology. Radiolabelled probes, instead of biotinylatedones, were used because of their superior sensitivity.9Viral antigens were not demonstrated in any of our 'casesby immunocytochemistry. The use of immunocytochemicalmethods is seriously limited by the fact that HPV antigensare only expressed in productive infections. Therefore, false-negative results may be obtained in infections where viralstructural proteins are not likely to be expressed, as incancers, because viral expression is inversely related to thedegree of neoplasia."HPV-18 DNA was found in the normal oral mucosaadjacent to the tumour in only 1 of the 66 casesinvestigated. Other studies of oral carcinoma found adifferent pattern with HPV positivity within the tumour but alower detection rate in the adjacent normal mucosa.,,·,8.19HPV detection in oral carcinoma in studies from differentgeographical regions show a great variation. An Americanstudy evaluating 10 squamous carcinomas detected HPV ina single case with ISH and polymerase chain reaction(PCR),' while two other American studies could not detectHPV in any of their specimens with ISH alone.8,9 Tsuchiya etal. 10 from Japan detected three positives from 30 samplesusing ISH while 76,4% of oral carcinomas in a Taiwan studywere positive for HPV-16 according to the Southern blottechnique." In the last study mentioned, a high incidence ofbetel quid chewing and smoking was found in the patientsstudied.The low prevalence of HPV in our collection can beexplained in several ways: (I) the lesions studied may nothave a viral aetiology; (it) the possibility in the lesions studiedof the occurrence of HPV genomic sequences other than theones used by us as probes, cannot be ruled out; (iit)transformed cells may contain altered viral DNA notdetectable by the probes used; and (iv) the method is notsensitive enough to detect low viral copy numbers. The ISHtechnique is highly sensitive in cases where individual nucleicontain a high copy number of the target DNA but often failsto detect cases in which subgenomic fragments of the viralDNA have been incorporated into the host genomeYo Theextremely sensitive PCR may demonstrate a higher positivityrate than ISH. In a study by Watts et al. ,21 ISH detected HPVin 60% of the oral squamous carcinoma cases examinedwhile PCR could detect it in 90%. The same degree ofincreased sensitivity was not found in two other studieswhere results show a high degree of correlation between thetwo methods used.T." Although we did not use PCR in thepresent study, we feel that the outcome would not havebeen significantly different, given that only 1 case was foundpositive by means of ISH.SAMJ Vo/umeS5 No.9 September /995~The present study, in which 66 cases of oral squamouscell carcinoma were reviewed retrospectively, is the largestseries to have been investigated so far. It appears that inthese cases of squamous cell carcinoma, HPV-associatedoral infection occurs only rarely in the black population ofthe western Northern Transvaal.The authors wish to thank Mrs H. van der Wait for secretarialservices and Or A. Gunders for help in preparing the manuscript.The study was supported by grants from thePoliomyelitisResearch Foundation, Sandringham, and the Medical ResearchCouncil of South Africa.REFERENCES1. Chang F. Role of papillomaviruses. J Clin Pathol 1990; 43: 269-276.2. Woodworth CD, Bowden PE, Doniger J, et aJ. Characterization of normal humanexocervical epithelial cells immortalized in vitro by papillomavirus types 16 and 18DNA. Cancer Res 1988; 48: 4620·4628.3. Woodworth CD, Doniger J, Di Poalo JA. Immortalization of human foreskinkeratinocytes by various human papillomaviru5 DNAs corresponds to theirassociation with cervical carcinoma. J Viral 1989; 63: 159-164.4. Zur Hausen H. Human papillomaviruses in the pathogenesis of anogenital cancer(Mini-review). Virology 1991; 184: 9-13.5. Scully C, Cox MF, Prime SS, Maitland NJ. Papillomaviruses: the current status inrelation to oral disease. Oral Med 1988; 65: 526-532.6. Chang F, Syrjanen S, Kellokoski J, Syrjanen K. Human papillomavirus (HPV) infectionsand their associations with oral disease. J Oral Pathol Med 1991; 20: 305-317.7. Shroyer KR, Greer RO. Detection of human papillomavirus DNA by in situhybridisation and polymerase chain reaction in premalignant and malignant orallesions. Oral Surg Oral Med Oral Patho/1991; 71: 708-713.8. Zeus MS, Miller CS, White OK. In situ hybridization analysis of humanpapillomavirus DNA in oral mucosal lesions. Oral Surg Oral Med Oral Pathol 1991;71: 714-720.9. Young SK, Min KW. In situ DNA hybridization analysis of oral papillomas,leuko"plakias, and carcinomas for human papillomavirus. Oral Surg Oral Med OralPa/hol 1991; 71: 726-729.10. Tsuchiya H, Tomita Y, Shirasawa H, Tanzawa H, Sato K, Simizu 8. Detection ofhuman papillomavirus in head and neck tumors with DNA hybridisation andimmunohistochemical analysis. Oral Surg Oral Med Oral Pathol 1991; 71: 708-713.11. Chang KW} Chang CS, Lai KS, Chou MJ, Choo KB. High prevalence of humanpapillomavirus infection and possible association with betel quid chewing andsmoking in oral epidermoid carcinomas in Taiwan. J Med Virol 1989; 28: 57-61.12. Johnson NW. Oro-facial neoplasms: Global epidemiology, risk factors' andrecommendations for research. Report of the Commission on Oral Health, Researchand Epidemiology of the Federation Dentaire International, London, 1990.13. Fleming 0, Shear M, Altini M. Intraoral squamous cell carcinomas in South Africa. JDent Assoc South Atr 1982; 37: 541-544.14. Pringle JH, Primrose L, Kind CN, Talbot IC, Lauder L In situ hybridizationdemonstration of poly-adenylated RNA sequences in formalin-fixed paraffin sectionsusing a biotinylated oligonucleotide poly mprobe. J PathoJ 1989; 158: 279-286.15. Wynder EL, Muskinski MH, Spirak JC. Tobacco and alcohol consumption in relationto the development of multiple primary cancers. Cancer 1977; 40: 1872-1878.16. Pindborg JJ. Epidemiology of oral cancer and precancer. In: Pindborg JJ, eel. OralCancer and Precancer. Bristol: John Wright & Sons Ltd, 1980: 126-128.17. Syrjanen KJ. Human papillomavirus (HPV) infections of the female genital tract andtheir associations with intra-epithelial neoplasia and squamous cell carcinoma.Pa/hol Annu 1986; 21: 53-87.18. Howell RE, Gallant L. Human papillomavirus type 16 in an oral squamous carcinomaand its metastasis. OraJ Surg Oral Med Oral Patho/1992; 74: 620-626.19. Chang F, Syrjanen S, Nuutinen J, Karja J, Syrjanen K. Detection of humanpapillomavirus (HPV) DNA in oral squamous cell carcinomas by in situ hybridizationand polymerase chain reaction. Arch DermatoJ Res 1990; 282: 493-497.20. Higgs TW, Moore NJ, Badawi DY, Taub FE. Type-specific human papillomavirusdetection in formalin-fixed, paraffin-embedded tissue sections using nonradioactivedeoxyribonucleic acid probes. Lab lnvest 1990; 63: 557-567., 21. Watts SL, Brewer EE, Fry TL. Human papiJIomavirus DNA types in squamous cellcarcinomas of the head and neck_ Oral Surg Oral Med Oral Pathol 1991; 71: 701-707.Accepted 9 Nov 1993.Assessment of quality oflife by clinicians -experience of a practicalmethod in lung cancerpatientsRaymond Abratt, Gary ViljoenObjective. To evaluate a practical method (brief scale) ofassessing the quality of life in patients with lung cancer.Design. To compare the scores obtained by means ofthe brief scale with those obtained on formal tests.. Thebrief scale consists of an Outlook score (measuringpsychological status) and a Support score (measu.';ingpsychosocial support). The formal tests were the f10spitalAnxiety and Depression Scale (HADS) for psychologicalstatus, and the Rotterdam Symptom Checklist (RSCL) andSpitzer QL-Index for quality-of-Iife assessment.Setting. Lung cancer follow-up clinic, Groote SchuurHospital.Participants. A total of 40 patients selected by randomsample.Main outcome measures. The correlation between thebrief scale and standard formal tests.Results. The HADS indicated that psychologicalmorbidity was present in 30% of patients. Both the RSCLand the Spitzer QL-Index indicated a significantly poorquality of life in 25% of patients. The Outlook scorecorrelated with both psychological status and quality oflife. The Support score correlated with psychologicalstatus but not with the assessment of quality of life. It did,however, correlate with the independent evaluation ofsocial support in the Spitzer QL-Index.Conclusions. The brief scale is a cost-effective anduseful tool for quality of life assessment in the clinicalmanagement of patients with lung and other cancers.S Atr Med J 1995; 85: 896-898.The practice of medicine is concerned not only withprolongation of the survival of patients, but also with theirquality of life. However, although SUbjective assessment ofthe factors affecting quality of life may form part of theclinician's management of a patient with lung cancer, it mayDepartment of Radiation Oncology, Groote Schuur Hospital, CapeTownRaymond Abratt, M.MED. RAD. [T.)Cape Psychosocial Oncology Trust, Cape TownGary Viljoen. MAVolume 85 No.9 September 1995 SAMJ-

Detection of human papillomavirus DNA with in situ hybridisation in oral squamous carcinoma in a rural black population

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Detection of human papillomavirus DNA with in situ hybridisation in oral squamous carcinoma in a rural black population

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