J Clin Pathol 1990;43:913-917

Detection and typing of human papillomavirususing the Vira Type "in situ" kit: comparisonwith a conventional dot blot techniqueB E Faulkner-Jones, VM Bellomarino, A J Borg, K Orzeszko, S M Garland

AbstractA new commercial kit (Vira Type "insitu", Life Technologies, Inc., MolecularDiagnostics Division, Guithersburg,Maryland, USA) for the detection ofhuman papillomavirus (HPV) types 6,11, 16, 18, 31, 33 and 35 in routinelyprocessed human anogenital tissue wascompared with a conventional dot blotassay for HPV 6, 11, 16 and 18. Bothsystems use double-stranded genomicDNA probes for the detection of typespecific HPV DNA. The probes used onthe dot blots were labelled with 32P andvisualised autoradiographically. TheVira Type probes were labelled withbiotin and visualised using a strep-tavidin-alkaline phosphatase conjugatewith NBT-BCIP substrate. Biopsyspecimens from the cervix, vagina, andvulva of 46 women were processed byboth methods and compared. The his-tological diagnoses ranged from benignchanges, to dysplasia, and invasive car-cinoma. Overall, 50% of biopsy speci-mens were positive for HPV DNA by dotblot hybridisation; only 39% werepositive by Vira Type in situ hybridisa-tion. Three of the specimens positive bythe Vira Type "in situ" kit showed nocross hybridisation and were the sameHPV type as the dot blot. A further 13showed cross hybridisation, but thestrongest Vira Type result correspondedto the dot blot results. One biopsyspecimen was positive for different HPVtypes by the two tests and one waspositive by Vira Type and negative bydot blot. Six biopsy specimens werenegative by Vira Type but positive by dotblot.

It is concluded that the Vira Type "insitu" kit has a similar specificity butlower sensitivity than the dot blothybridisation method for the detectionofHPV DNA.

Papillomaviruses are small, species specificDNA viruses which cause epithelial orfibroepithelial proliferations known as con-dylomas. Human papillomaviruses (HPV)have an icosahedral capsid and a covalentlyclosed, circular, double-stranded DNA gen-ome of about 8000 base pairs. HPV cannot becultivated in vitro, and serological testsremain insensitive and relatively non-specific.The viral genome can be found throughoutthe thickness of an infected and con-dylomatous epithelium, but virion production

and cytopathic effect are limited to the upperand terminally differentiated layers. Histologyand immunohistochemistry for capsidantigens are, in practice, relatively insensitive.Moreover, HPV produce the same cytopathiceffect regardless of type. Nucleic acidhybridisation techniques remain the most sen-sitive way of detecting infection, and the onlyreliable way of typing the HPV involved.'2Over 60 types of HPV have now been

identified and they exhibit tropism for dif-ferent epithelia. The anogenital area is consis-tently infected by types 6, 11, 16, 18, 31, 33and 35, together with other less commontypes, such as HPV 42. It has become clearthat types 6 and 11 are most often found inclassic condylomata acuminata and subclinical"flat warts," or in low grade dysplasias. Incontrast, high grade dysplastic lesions andinvasive carcinomas are much more likely tobe infected with HPV 16, 18, 31, 33 and 35.These HPV types can also be found in lowgrade lesions.3 In condylomatous lesions HPVgenerally exist as free intranuclear episomes,but in genital carcinomas and some dysplasiasthey are integrated into the host cell genome.

Controversy still exists about the frequencywith which low grade dysplasias progress tohigh grade dysplasia and to invasive carcin-omas, but progression does occur in a propor-tion of patients.4 Strong epidemiological andin vitro evidence suggests that lesions infectedwith HPV 16, 18, 31, 33 and 35 have a higherrisk of progression to invasive carcinoma thantypes 6, 11.56 A direct aetiological role forthese HPV in malignant transformation,however, is yet to be proved. It has beensuggested that the detection and typing ofHPV in anogenital lesions may help to predictthe clinical course of lesions, thereby allowingthe allocation of screening and treatmentprogrammes to be planned effectively.

MethodsThe study group comprised 46 women referredbetween February 1987 and July 1988 to theDysplasia Clinic of the Royal Women's Hosp-it.al, Melbourne, because of an abnormalcervical smear. Colposcopically directed targetbiopsy specimens were taken and bisected;failing that, adjacent target biopsy specimenswere taken. One sample was sent for HPVDNA detection and the other for routinehistological analysis.

HPV DNA DETECTION BY DOT BLOTHYBRIDISATIONBriefly, DNA was extracted with phenol-

Microbiology andAnatomical PathologyDepartments, TheRoyal Women'sHospital, 132 GrattanStreet, Carlton,Victoria, Australia,3053B E Faulkner-JonesVM BellomarinoA J BorgK OrzeszkoS M GarlandCorrespondence to:Dr B E Faulkner-JonesAccepted for publication11 June 1990

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chloroform after initial proteinase K digestionand then precipitated with ethanol.7 Quantita-tion was performed by comparison withlambda phage (Hind III) DNA markers aftergel electrophoresis. Duplicate samples of 3 jgof DNA were dotted on to nylon membranes(Zeta Probe, Bio Rad) using a 96-well vacuummanifold (Biodot apparatus, Bio Rad) andbaked for 30 minutes at 80'C (instructionmanual for DNA dot blotting, Bio-Rad). Gen-omic HPV DNA probes cloned into plasmidpBR322 (HPV 6, 11, and 16 into the Bam Hlsite and HPV 18 into the Eco Rl site) werekindly supplied by Professor Harald zurHausen, Heidelberg, West Germany. Plasmidswere transformed into Escherichia coli HB 101and then isolated by caesium chloride ethidiumbromide gradient ultracentrifugation. HPVinsert DNA was recovered using the appro-priate restriction enzyme and purified byagarose gel electrophoresis. Inserts werelabelled using 32P dATP (Amersham UK)either by oligolabelling or by using the Multi-prime DNA labelling kit (Amersham) to aspecific activity of2-6 x 10' cpm/pg ofDNA.8One of the pair of filters produced for each

patient was hybridised with combined HPV16/18 probes and the other with pBR322 DNA.Hybridisation was performed using 12-14 ngof radiolabelled probe/ml at high stringency(Tm - 20'C) for 22 hours. Filters were washedonce in 2 x SSC at room temperature forseven minutes, once in 2 x SSC (0-15Msodium chloride, 0-15M sodium citrate), 0 1%sodium dodecyl sulphate (SDS) at room tem-perature for 15 minutes and three times at 65'Cfor one hour in 0-1 x SSC and 0-1% SDS.The filters were left to autoradiograph for one tofive days at - 70'C with KodakXAR-5 film andtwo intensifying screens. Both filters were thenstripped of probe and one rehybridised withcombined HPV 6/11 DNA and the other withpBR322 DNA, using the same conditions.

HPV DNA DETECTION USING VIRA TYPE KIT

Specimens sent for routine histological analysiswere used for in situ hybridisation. Controlsections, prepared from cell blocks of HeLa,CaSki, and SiHa cell lines, were also used.Tissue processing, pretreatments, hybridisa-tion and detection were carried out accordingto the manufacturer's instructions with someminor modifications. Briefly, the procedure wasas follows. Tissue was fixed in 10% bufferedformol-saline for a variable period, paraffin waxembedded, and then cut at 4-5 gm. Sevensections per case were floated on to silanatedslides, air dried and then baked for at least 30minutes at 58°C before dewaxing throughxylene and dehydrating in absolute ethanol. Aprotease/hydrochloric acid digestion step wasfollowed by a TRIS-buffered saline wash anddehydration through graded ethanols. Forty to80 microlitres of hybridisation buffer contain-ing biotinylated HPVDNA probes were addedto each of the sections as follows: the first wasprobed with combined HPV 6/11, the secondwith HPV 16/18, and the third with HPV 31/33/35. One positive and one negative controlprobe (human placental DNA and pBR322

DNA, respectively) were included each timeand the remaining two sections were stainedwith haematoxylin and eosin for histologicalassessement.

Coverslips were carefully applied to excludeall air bubbles and the slides were then heatedat 95'C for 15 minutes. Hybridisation wascarried out for two hours at 37'C in a humidifiedchamber. Coverslips were removed by gentleagitation in TRIS-buffered saline with bovineserum albumin and three post-hybridisationwashes performed using the same buffer at37'C. Forty to 80 microlitres of streptavidin-alkaline phosphatase conjugate were added tothe sections which were then incubated for 20minutes at 37'C in a humidified chamber. Theslides were washed with TRIS-buffered salineand then immersed in nitroblue tetrazoliumchloride (NBT)-5-bromo-4-chloro 3-indolylphosphate p-toluidine salt (BCIP) sub-strate in TRIS-saline buffer for one hour at37'C. The reaction was terminated by washingin distilled water and sections were then coun-terstained with nuclear Fast Red for 15 min-utes. Sections were washed again with water,dehydrated through ethanols, cleared in xylenethen mounted in DEPX (Gurr, BDHchemicals). The whole procedure, from sec-tioning to histological assessment, can be per-formed during a normal working day.

ResultsOverall, 50% (23/46) ofbiopsy specimens werepositive for HPV DNA by dot blot, with sevenpositive for HPV types 6 and 11 and 16 positivefor HPV types 16 and 18. The detection limitfor the dot blot assay was taken as 12-5 pg ofHPV DNA. The correlation ofHPV type withhistology is given in table 1.

Vira Type "in situ" detected HPV DNA in39% (18/46) of specimens, with six positive fortypes 6 and 11 and 12 positive forHPV types 16and 18 (figs 1 and 2). The correlation withhistology is shown in table 2. Twenty twobiopsy specimens were negative by both assays.In three cases positive for HPV DNA the ViraType and dot blot result agreed and there wasno cross hybridisation with Vira Type results.A further 13 cases showed cross hybridisationwith the Vira Type test (fig 2D). As suggestedby the manufacturers, if a signal was evident inmore than one probe group, the strongestsignal was taken to represent the viral type, andin these 13 cases this agreed with the dot blotresult. One biopsy specimen was positive forHPV DNA by both assays but for differenttypes and one biopsy specimen was positive forHPV DNA by Vira Type "in situ" hybridisa-tion but negative by dot blot. Six biopsyspecimens were positive by dot blot butnegative by Vira Type "in situ" hybridisation.The correlation between dot blot and ViraType results is given in table 3.

Positive signals were obtained using ViraType "in situ" for the CaSki and HeLa celllines (500-600 copies of HPV 16 and 10-50copies ofHPV 18, respectively), but the signalin HeLa cells was very weak. No signal wasobtained for the SiHa cell line (one copy ofHPV 16).

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Table 1 Correlation ofHPV types by dot blot hybridisation with histological assessment of cervical biopsy specimens

Histology

InvasiveHPV squamous

HPV DNA type Normal infection CIN I CIN II CIN III carcinoma Total

HPV 6/11 1 4 1 - - 1-vulva 7HPV 16/18 - 4 - 2 9 1-cervix 1 6Negative 14 5 2 1 1 - 23Totals 15 13 3 3 10 2 46

Figure 1Photomicrograph of atypical vulval condylomaacuminatum. (A) Sectionshowing a koilocytosis(haematoxylin and eosin).(B) Lower magnificationshowing a positive signalwith a combined HPV 6/11 DNA probe: the signalis strongest in the upperlayers of the epithelium(alkaline phosphatase-NBT-BCIP and nuclearFast Red counterstain).

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DiscussionTyping ofpapillomaviruses on routine anogen-ital biopsy specimens can be performed withinone working day using the Vira Type "in situ"kit. While generally well designed and user-

friendly, there was some scope for minorimprovements to the kit. The manufacturersrecommend that tissue be left in fixative for nomore than 24 hours. Unfortunately this doesnot always occur in practice and may con-

tribute to the lowered sensitivity of the kitrelative to the dot blot method. Additionally, as

emphasised by the manufacturers, sections cuton a dirty water bath increased the number ofbackground hybridisation signals, especiallyfrom bacteria in the water. Heating sections onto silanated slides overnight at 60'C, instead of30 minutes at 58'C, produced fewer lost sec-

tions and no appreciable reduction in signalstrength.

It was suggested to mark around the sectionwith a diamond pen to aid visualisation duringthe procedure, but this sometimes resulted inbroken slides after denaturation at 100'C. Analcohol/xylene insoluble felt pen was found tobe preferable. Although denaturing the sectionand probe DNA at 100°C on a heating block,several coverslips were forcibly dislodged bythe hybridisation buffer boiling beneath themwith obvious danger to the operator. Reducingthe temperature from 100'C to 95°C in a

hybridisation chamber and extending the timefrom five to 15 minutes avoided this danger andproduced an adequate signal with improvedmorphological detail and fewer lost sections.The other small modification which was foundto be useful was to increase the nuclear FastRed counterstaining time from five to 15minutes.

Signals were strongest in the upper layers ofthe epithelium and a strong signal provided no

problems with interpretation. Some tissue sec-

tions, however, showed non-specific peripheralstaining which could easily mask a weak signal.If more than one probe group produced a

positive signal, the strongest signal was taken torepresent the viral type and the others cross

hybridisation. A true mixed infection, how-ever, would be hard to interpret with thissystem. The hybridisation conditions used bythe kit are effectively of medium stringency,and cross hybridisation between HPV groups isinevitable. An alternative strategy would be toscreen at low stringency for all genital HPV toincrease the sensitivity of the test, and then totype at high stringency to improve the speci-ficity. This is already used by Life Tech-nologies Inc. in their Vira-Pap Vira-Type kitsfor the detection and typing ofHPV in cervicalscrapes by dot blot.The finding of two discrepant results could

be a consequence of analysing different, albeitadjacent, specimens which harbour differentHPV types. Additional HPV probes in the Vira

Table 2 Correlation ofHPV type by Vira Type "in situ" hybridisation with histological assessment of cervical biopsyspecimens

Histology

InvasiveHPV squamous

HPV DNA type Normal infection CIN I CIN II CIN III carcinoma Total

HPV61l1 - 4 ' - - 1-vulva 6HPV 16/18 - 2 - 3 7 - 12(*HPV3 /33/55)Negative 15 7 2 - 3 1 -cervix 28Total 15 13 3 3 10 2 26

*AIl biopsy specimens showing a positive signal with the HPV 31/33/35 group were strongly positive for HPV 16/18 and theresult read as HPV 16/18 positive with cross hybridisation.

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Figure 2Photomicrograph of vulvalintraepithelial neoplasia(VIN) grade III. Thispatient previously had asuperficially invasivesquamous carcinoma of thevulva. (A) VIN III(haematoxylin and eosin).(B) Same area asfigure2A showing a strongpositive sgnal with acombinedHPV 16118DNA probe. The signal isstrongest in the upperlayers of the epithelium,but positive nuclei can beseen towards the basallayer (alkalinephosphatase-NBT-BCIP). (C) High powerview offigure 2B to showintranuclear localisation ofHPV 16/18 DNA. (D)VIN III showing a weakpositive signal withcombinedHPV 31133135DNA probe. This weaksignal was taken torepresent crosshybridisation betweenHPV, most likely betweenHPV 16 and HPV 31 inthis case. (This biopsyspecimen was typed asHPV 16/18 positive).

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,.r4 sjiv # _>e;Type kit (types 31, 33, and 35) and the use ofmedium stringency conditions could also per-mit cross hybridisation with other relatedHPV. These should not cross hybridise underthe high stringency conditions of the dot blot.Dot blot hybridisation, although less specificand less sensitive than Southern Blot hybridis-ation, is much less labour intensive and permitsrelatively rapid screening of large numbers ofclinical specimens. It still requires expensiveequipment and specialised technical skills.

The Vira Type kit, although less sensitivethan the dot blot assay, does have the advantageofnot requiring special expertise or equipment.In addition, archival tissue can be used forretrospective studies and the lesion associatedwith the HPV infection can be assessed.

Currently, management of CIN depends onclinical and histological assessment of indi-vidual women and HPV typing is thereforelargely of academic interest. If, however, theassociation between certain HPV types and

Table 3 Correlation of Vira Type "in situ" hybridisation result with dot blot result and histological assessment of cervical biopsy specimens

Vira type Histology"in situ"hybridisation Invasiveand dot blot squamousresults HPVDNA Normal HPV CIN I CIN II CIN III carcinoma Total

Negative by both assays Negative 14 5 2 - 1 - 22Positive by both assays, no cross hybridisation 6/11 - 2 1 - - - 3Positive by both assays, cross hybridisation 6/11 - 1 - - - 1-vulva 2

with Vira Type 16/18 - 2 - 2 7 - 11Positive by both assays- HPV 6/11 by Vira Type - 1 - - - - 1different HPV types HPV 16/18 by dot blotNegative by dot blot, positive by Vira Type HPV - - - 1 - - 116/18/31/33/35Positivebydotblot, HPV6/11 1 1 - - - - 2

negative by Vira Type HPV 16/18 - 1 - - 2 1-cervix 415 13 3 3 10 2 46

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carcinogenesis becomes more clearly estab-lished, HPV typing could have a role in themanagement of CIN. In order to assess thisassociation more data from prospective followup trials (with normal control groups) needs tobe obtained, with analysis of HPV types, thelesions involved, and progression or regressionrates. The Vira Type "in situ" kit would allowroutine pathology laboratories to type HPV inbiopsy material and provide some ofthese data.

We thank John Howlett, marketing manager, Gibco-BRL,Australia, and Dr Rosemary Versteegen of Life TechnologiesInc., Molecular Diagnostics Division, USA, for kindly provid-ing us with the Vira 'l'ype "in situ" kits; MrW Chanen and thestaff of the Dysplasia Clinic, Royal Women's Hospital, forproviding the clinical specimens; and Mrs Judy Jackson andMiss Voula Polites for typing the manuscript.

1 Pfister H. Biology & biochemistry of papillomaviruses. RevPhysiol Biochem Pharmacol 1984;99:1 11-81.

2 Lorincz AT. Detection of human papillomavirus infectionby nucleic acid hybridisation. In: Reid R, ed. Humanpapillomavirus. Philadelphia: WB Saunders, 1987:451-69.

3 Broker TR. Structure and genetic expression of papilloma-viruses. In: Reid R, ed. Human Papillomavirus. Obstetric& Gynecology Clinics ofNorth America. Philadelphia: WBSaunders, 1987:329-48.

4 McIndoe WA, McLean MR, Jones RW, Mullins PR. Theinvasive potential of carcinoma in situ of the cervix. ObstetGynaecol 1984;64:451-8.

5 McCance DJ. News on papillomaviruses. Nature1988;335:765-6.

6 Syrjanen K, Mantyjarvi R, Varynen M, Syrianen S, et al.Evolution of human papillomavirus infections in theuterine cervix during a long-term prospective follow-up.Appl Pathol 1987;5:121-35.

7 Appendix A: Biochemical Techniques. In: Maniatis T,Fritsch EF, Sambrook J. Molecular cloning: A laboratorymanual. New York: Cold Spring Harbour Publications,1982:436-78.

8 Feinberg AP, Vogelstein B. A technique for RadiolabellingDNA restriction endonuclease fragments to high specificactivity. Anal Biochem 1983;132:6-1 13.

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