0095-1137/88/020236-08$02.00/0
Copyright C 1988, American Society for Microbiology
Evaluation of Methods for Detecting
Human
Papillomavirus
Deoxyribonucleotide Sequences
in
Clinical
Specimens
DEORAJ CAUSSY,1 WILLIAM ORR,2 A. DEAN DAYA,2 PAULA ROTH,3 WILLIAM REEVES,4
AND WILLIAMRAWLSl 2*
Molecular Virology and Immunology Program,' Department of Pathology,2 andDepartmentof Obstetricsand Gynecology, McMaster University, Hamilton, OntarioL8N3Z5, Canada, and Divisionof Epidemiology, Gorgas
MemorialLaboratory, Panama City,Republic of Panama4
Received 23 July 1987/Accepted21October 1987
Specimens from 26 condylomatous lesions, 24 invasivecancercells,and 33 cervices, without evidence of the diseases,weretested forthepresenceofhumanpapillomavirus (HPV) types 6, 11, 16, and 18 by Southern blot hybridization, in situ filter hybridization, orin situtissuehybridizationmethods. Atotal of89% (23 of 26)of thecondylomatous lesionscontained HPVDNAs,asdetermined byone or moreof themethods. Thepositive rates for the detection ofHPV DNA in condylomas by the different methods were 82% for Southern blot hybridization, 62% for in situ filter hybridization, and 72% for in situ tissue hybridization. Among the specimens frompatients withcancer, HPV DNAwasfoundin83%(19of23) byone or moreof the methods. Positive rates of89 and 70%, respectively, wereobtained forcancer lesions tested bythefilter in situ and
Southern blothybridization methods; however, only30% of those lesionswere positive by the in situ tissue hybridization method.Thirteenpercentof the control cerviceswerepositiveforHPV DNAbyone ormoreof theassays. With respect toall disease categories,the methods hadcomparable sensitivities and specificities, exceptforthein situ tissuehybridization method,whichrevealedaspecificity of 72% for condylomatous lesions
and 30% for invasivecancer cells.
Papillomaviruses (PVs) belongto a subgroup of Papova-viridae whicharecharacterizedbyasmall, double-stranded,
circular DNAgenome(25).The PVsarespecies specificand exhibitahigh degree of tissue tropisms. Inhumans, over45 types of human PVs (HPVs) have been isolated from
pa-tients with such diverse clinical conditions as cutaneous warts, anogenital warts, laryngeal papillomas, and epider-modysplasia verruciformis (17). In addition, specific
geno-types of HPV have been implicated as the cause of histo-pathological changes in cervical intraepithelial neoplasia (CIN) lesions and in thegenesis of invasive cervicalcancer
(28). The prevalence of HPV types 11 and 6 has been reportedto be between 85 and 100% in benign condylomas andbetween 18 and 30% inadvanced CIN lesions(7, 9, 26). In contrast, HPV type 16 seems to prevail in more severe
forms of CIN and invasive cervical cancers, with the
re-ported prevalence ratesvarying from 17to100% in invasive cervical cancers(5, 6, 21, 28). HPV type 18 has also been associated withinvasivecervicalcancers,but itsprevalence islower than thatof HPVtype 16 (4, 28).
Progress inelucidating theepidemiology and pathogenesis of the HPVs has been hampered by the lack of suitable conventional laboratory techniquestoculture the viruses in vitro. Vegetative replication of PVs is confined to the specific host andtothe degree of differentiation of the host cells(16). Only the terminally differentiating keratinocytes of theparticular hostseemtobe permissivetoinfectionby PVs (17). Therefore, the diagnosis of PVs in clinicalsamplesrests uponthepresenceof complete virionsasshownby electron microscopy (1), thepresenceof virion structural antigensas shown by immunological methods (13), or detection of virus-specific DNA sequences by molecular hybridization methods.
The success of electron microscopy in the diagnosis of
*Corresponding author.
HPV depends on the demonstration of the HPV virions, while that of the immunological tests dependson the pres-enceofthecommonPVstructuralantigens.Virionassembly and expression of the late structural antigens takes place only interminallydifferentiatedcells. Thus,both virions and antigens are absent in grade 3 CIN lesions, which, by
definition, are undifferentiated(19). Consequently,the
sen-sitivity of these tests in the diagnosis of HPV from these lesions ranges between 40 and 70% (17). Furthermore, neitherassayiscapable ofdistinguishingthesubtypesof PV thatareresponsible for the infection. Theselimitationshave
beenlargelyovercomebycloning theHPVDNAinbacterial plasmids, since this permits the development of viral probes which can be used in molecular hybridization assays to detect specific viral DNA sequences. Three such methods have been developed: Southern blot hybridization, in situ filterhybridization, and in situ tissue section hybridization. The purpose of this study was to define the relative sensi-tivities andspecificities of these methods for detecting HPV DNA sequences in papillomatous lesions, cervical cancer cells, and cervices without evidence of the diseases.
MATERIALS ANDMETHODS
Study population and preparation of samples. Samples
were obtained from three categories of patients from two
different populations. The first category consisted of 26 Canadian patients with genital condylomas. These patients
were seenatuniversity-affiliated clinics inHamilton, Onta-rio. Weobtained specimens from6penile,6vulvar, 2 groin, and 12 cervico-vaginal lesions. The second category con-sisted of 23 Panamanian women with newly diagnosed
cervical cancers. The third category consisted of 21 Cana-dian and 12 Panamanian patients who had undergone
hys-terectomy for diseases other than neoplasia. Histologic examinationconfirmed the diagnoses of condylomaor inva-sivecervicalcancerinthefirst and second categories andthe
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HPV DETECTION METHOD EVALUATION 237 absence of these lesions in the third category. Although
patients in the thirdcategory wereconsiderednormalforthe purposes of this study, histologic examination revealed 9 patients with cervicitis, 11 patients with cervical metaplasia, and 13patients with unremarkable cervices.
We attempted to collect cells fromthe lesion from each patient forin situfilterhybridization and then to obtain two biopsies of the lesions. This was not achievedin all cases. For the in situ filter hybridization method, cells from the cervicalos orcervical lesionswerecollected witha cotton-tipped swab and suspended in 4 ml of phosphate-buffered saline. The number of cells was counted in a hemacytom-eter, and the cells were stored at -20°C until they were tested. Thecellcountsranged from
104
to107epithelialcells persample. Biopsies intended for the Southern blot method were snap-frozen in liquid nitrogen for transportationto the laboratory, where they were subsequently stored at -70°C until they were tested. Biopsies for the histopathological examination and in situ tissue hybridization methods were fixed in 10% buffered, neutral Formalin and processed by routine histologic methods.For the in situ filter hybridization method, the samples werethawed, and thecellswerepelletedat2,000 x gfor10 min and suspended in400,ulofabufferconsisting of5 ,ugof denatured salmonsperm DNA permlin6x SSC(lx SSC is 0.15 M sodium chloride plus 0.015 M sodium citrate [pH 7.0]).Theextraction ofDNAfrom
biopsies
forSouthernblot analysis was done as described previously (18) by using proteinase K digestion and phenol-chloroform extraction. Sections for use in the in situ tissuehybridization
method were cut to a 5-,um thickness from paraffin-embedded blocks. The sections were placed onpoly-D-lysine (Sigma
ChemicalCo., St. Louis, Mo.)-coated glass slides and fixed by baking the slides at 60°Covernight.
Probe DNA andradiolabeling. Recombinantplasmids con-taining HPV type 6C, 11, 16, and 18 DNAs were kindly provided byL.Gissman andE.deVilliers(Deutsches
Krebs-forschung
Zentrum,Heidelberg,
FederalRepublic
of Ger-many). For use as probes in the in situ filter hybridization and Southern blot hybridization methods, the HPV DNAs were excised from the plasmids to avoid possiblefalse-positive
reactions because of the presence of pBR322 se-quences in some clinical specimens (2). The HPV DNAs were not excised from plasmids when they were used as probes inthe in situ tissuehybridizationmethod to enhance the signal associated with the probe (22). The DNA was labeled with either[32P]dCTP
or[35S]dCTP
(NewEngland
NuclearCorp.,
Lachine, Quebec, Canada)
with a commer-cial nick-translation kit(Bethesda
ResearchLaboratories,
Gaithersburg,
Md.),
and the instructions of manufacturer werefollowed.Aspecific activity ofgreater than108cpm/,ug
ofDNAwas achieved for each
probe.
Forall
hybridization
methods, two
probe
types wereused;
one consisted of amixture of HPV types 16 and 18
DNA,
while the second consisted ofamixture ofHPV types6C and 11 DNA.Insitu filter
hybridization.
Insitufilterhybridization
wasperformed by
a modification ofthe methods describedby
Wagneretal. (26)andGrunstein and
Hogness
(10).
Samples
(100 ,ul)were
applied
undervacuum to anitrocellulosefilter with a 96-well manifold apparatus(Schleicher
&Schuell,
Inc.,Keene,
N.H.).
Thespecificity
of thehybridization
wascontrolled
by
testing
serial dilutions of Caskicells,
which contain 500copies
ofHPVtype 16 percell
(27),
and similar dilutions ofhumanembryonic
fibroblastcells,
which donotcontain HPV DNA. Cell
lysis
and DNA denaturation wereeffected
by
overlaying
the filter on Whatman 3MM filterpaper that hadbeen
previously
soaked in 1 MNaCl-0.5 M NaOH for 5 min, and theprocedure
wasrepeated
once more. The filters werethen neutralizedby
overlaying
them twicefor5minonWhatman 3MMfilter paper that had beenpreviously
soakedin 1.5 MNaCl-0.5MTris buffer(pH7.4).
The filters were
air-dried, placed
into aplastic bag,
and treated with 2mgofproteinase
Kper mlin10 mMTris(pH
7.8)-0.5%
sodiumdodecyl
sulfate-0.5 mM EDTA. The fil-ters werewashed threetimesinchloroform(5mineach)and then weregiven
one finalwash in 2x SSC. The DNA was fixed on the filterby baking
at80°C
for 2 h. HPV DNAsequences
were detectedby
a modification of the method describedby
Thomas(24).
Filters wereprehybridized
for2 to3h at42°C
inaprehybridization
bufferconsisting
of50% formamide-5x SSC-50mMsodiumphosphate (pH 6.5)-250
,ug of
sonicated,
denatured salmon sperm DNA perml-5x Denhardtsolution, containing
0.02% each of bovine serumalbumin,
Ficoll(Pharmacia
FineChemicals, Piscataway,
N.J.),
andpolyvinylpyrrolidone.
Thehybridization
buffer consistedof4partsofprehybridization buffer,
1partof50%
(wt/vol)
dextransulfate,
and 5 x 105 cpm of 32P-labeledspecific
DNAprobes
perml. Priortouse, thehybridization
cocktailwasboiledfor5minat
100°C
and then cooledonice toachieve denaturation ofthe DNA.Following
themethods described inpreviously
published
reports(20, 26),
hybrid-ization was carried out under
stringent
conditions at42°C
(which corresponded
to17°C
below themelting point
[Tm
-17°C])
overnight,
and the filters were washed four times undernonstringent
conditions(Tm
-42°C)
at42°C
in2x SSCcontaining
0.1% sodiumdodecyl
sulfate,
with each washlasting
1 h. The filters were enclosed in SaranWrap
andexposed
for 1 to 3days
toX-ray
film at-70°C
by
using
anintensifying
screen(Eastman
KodakCo., Rochester, N.Y.).
The filters were
subsequently
washed understringent
con-ditions(Tm -17°C)
by
using nonstringent
washconditions,
except that the
procedure
was carried out at68°C.
A final wash in 0.1 xSSC-0.1%
sodiumdodecyl
sulfatewascarried out at roomtemperature
for 15min,
andautoradiography
was doneasdescribed above. All
autoradiograms
werereadindependently by
threeobservers,
and thosespecimens
that were recorded aspositive by
two or more observers were scored aspositive.
Southern blot
analysis.
Southern blotanalysis
was doneessentially
as describedpreviously
(18).
Briefly,
following
phenol-chloroform
extraction,
the DNAwasdigested
witharestrictionenzyme
(BamHI)
andfractionatedby
agarosegel
electrophoresis.
The DNAwasthen transferred to nitrocel-lulose filterby
thetechnique
describedby
Southern(23)
andprehybridized
andhybridized
asdescribed above forthe in situ filterhybridization
method. Inkeeping
with standardpractice
(4,
7, 9, 18),
however,
hybridization
wascarriedoutin
20%o
formamideat42°C
undernonstringent
conditions(Tm
-42°C).
Thefilters
werethenwashedasdescribed above for the in situ filterhybridization
method. For the purposes ofcomparison,
the results obtained for the Southern blot method understringent
washing
conditionswereregarded
asbeing
comparable
to those obtained for theremaining
two assays understringent
hybridization
andwashing
condi-tions. Theautoradiograms
werescoredby
threeobservers
asdescribed above for the in situ filter
hybridization
method. In situ tissuehybridization.
A modification of the method describedby
Haase et al.(12)
was used for in situ tissuehybridization.
Paraffinwasremovedfrom the tissue sections withxylene,
and the tissue sections weresequentially
hy-drated in
descending
grades
of alcohol. The DNA wasdenatured in situ
by
treating
thesectionin 0.2 NHCI
for10 VOL. 26,1988on April 11, 2020 by guest
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1 2 3 4 5 6 7 M
r *10.2
u.~~~~~~4.
44.2
FIG. 1. AutoradiogramofSouthern blothybridizationanalysis. DNAwasextractedfrombiopsysamples,cleavedwith the-restric-tion enzyme BamHI, and analyzed as described elsewhere (18). Lanes 1 to 4, DNA fromcondylomatouslesions;lanes 5to7,DNA
from invasive cancercells. Arrows indicate molecularweight mark-ers (M),inkilobases,asdeterminedby electrophoresisof HindIlI-digested lambdaphageDNA. Theblotwasprobedwith HPV type 16 DNA and washed under stringent condition (Tm -17°C). The typical episomal HPV moleculeisexemplified bythesignalin lane 1,while patterns consistent with viralintegrationinto cellularDNA areexemplified bythesignalsin lanes 5 and7.
min, and the sections were then treated with a 25 ,ug of proteinaseKin0.05MTris
hydrochloride (pH 7.4)
at37°C.
Thesections werethentreated witha4%
paraformaldehyde
solution in
phosphate-buffered
saline,
washed inphosphate-buffered saline
containing
0.2%(wt/vol) glycine,
followedby
awash
in phosphate-buffered
saline.The sectionswerethen dehydrated in ascendinggrades
of alcohol and air-dried.Hybridization
cocktail contained 2xSSC,
400 ,ug of dena-tured salmon sperm DNA per ml, and various concentra-tions of formamide. Formamide concentrations of 10%(Tm
-42°C) and
50%
(Tm -17°C)wereused fornonstringentand stringent wash conditions,respectively.Tothehybridization
solUtion was added 105 cpm of 35S-labeled specific
HPV
DNA per ml. The whole hybridization cocktail was dena-turedby boilingfor5 min at
1000C,
chilledonice, and made to 10 mM indithiothreitol
(12).Hybridization
was carried out at 37°C for 18 h, and the sections were washed as follows: 30 min at roomtemperature in2x SSCfollowed by 1hat420C in 2x SSC. The slideswerethenwashedfor2to 3 daysin three changesofasolution
containing 50% form-amide, 2xSSC,
0.01 M Tris hydrochloride (pH 7.4), and 0.001MEDTA. Theslideswerethenrinsed threetimes for 15 min in 2x SSC and dehydrated in ascending grades of alcohol. Autoradiography was carried out by dipping the slides in meltednuclear
track emulsion(NTB2;
Kodak Canada Ltd., Toronto, Ontario), which was diluted in dis-tilled water, as directed by the manufacturer. The slides were dried in the dark for0.5
h and exposed at 4°C for 4 weeks in a light-proofplastic box containing calcium sulfate as adessicant. Before the slides were developed, they were equilibrated to roomtemperature
andimmersed for 3 min in developer (D-19; Kodak), followed by 1 min in 1% aceticacid
and 3 min in fixer(Kodak). After 10 min of washing in distilled water, thesections were lightlystained with hema-toxylin and then eosin and were independently scored by three readers, asdescribed above for the two otherhybrid-ization methods.
Cytopreparation
ofCaski cells and Siha cells, which harbor 500 and 10 copies of HPV-16(27),
respectively, were included to determine the lowest copy numberofHPVgenomes detectablebythis method.
Statistical analysis. The
reliability
of thehybridization
methods inthe
diagnosis
ofHPV wasexaminedby
percent agreement andby using unweighted
Cohenkappa
statistics (8). The percent agreement is the number ofsamples
for which identicalresults were obtainedby
two methods rela-tive to the total number ofsamples examined. Use of the kappa statistics is done in anattempt tocorrectthepercent agreementforagreement thatoccursby
chancealone.RESULTS
Representative results obtained
by
each of the three hybridization methods are shown inFig.
1 to 3. The HPV DNAfromcondylomatous
lesions existedasepisomal
mol-ecules, asexemplified
by the appearance ofan 8-kilobase fragment afterdigestion
with thesingle-cut
enzymeBamHI and Southern blotanalysis (Fig.
1,lane1).Asimilaranalysis
ofHPV DNA extracted from invasive cervical cancercells sometimes
yielded
aseriesofbands(Fig. 1,
lanes 5and7),
afeaturewhich is consistentwith the fact that the HPV DNA isintegrated intothecellular DNA
(4).
The type of
hybridization signals
observedby
the in situ filterhybridization methodis shown inFig.
2. Itappears that by this method HPV sequences can be detected within aggregates of cells(26),
giving
rise to a discrete dot(as
exemplified by samples in
Fig.
2,rowsDandG,
column9,
or rows FandG, columns 11and12)
thatfillsthe whole well. However, no correlation between the number ofcells ini-tially present inthesamples
and theintensity
of thesignal
1
2 3
4
5
A
ar0
B *
c ~i
0
D 'E
Z. G H
o
6 7
8 9101112
*
e
0
*
..
0
FIG. 2. Autoradiogram obtained by the in situ filter hybridization method. Exfoliated cervical cells were trapped on nitrocellulose
paper, lysed, digested with proteinase K, and then hybridized as
described in thetext.Rows A to H,columns1to 9,Clinical samples frompatients. Theinset (rows A to D, columns 11 and 12) shows
that there was no hybridization signal when a 10-fold dilution of
humanfibroblast cells, ranging from 104 to 10Ù cells per well, was
included as a negative control. A positive control consisted of
similar dilutions of Caski cells, which harbor HPV type 16 se-quences (rows E to H, columns 11 and 12). Any samples that showed signals comparable to or weaker than those in row H, columns 11 and 12,were scored negative; and any signal stronger
than thesewerescoredpositive.Aclear positive signal is
exempli-fiedby the signal inrowA,column3; and a clearnegative signal is
illustrated in row A, column 1. Some borderline signals (row A,
column6;rowB,column6; rowC,column 2; and row F, columns 6and7)arealsoapparent.Thesewere considerednegative for the purposesoftheanalysis.
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HPV DETECTION METHOD EVALUATION 239
was observed. By using various dilutions of HPV type 16 containing Caskicells (Fig. 2,rows EtoH, columns 11 and 12), hybridization signals were clearly identifiable in wells
that receivedasfewas 100cells (Fig. 2, rowG, columns 11 and 12). Similar experiments with anHPV-negative human
fibroblastcell lineyieldednodetectable hybridizationsignals
at the highest concentration tested (Fig. 2, rows A to D, columns 11 and 12).
When the in situtissue hybridization methodwasusedfor
the detection of HPV sequences in condylomatous lesions,
the positive silver grains were observed to be unevenly distributed in the different layers of the epithelium. The signals wereweakest in the basal layers and progressively
increased in intensity toward the terminally differentiated epithelialcells and the koilocytes, which showed the strong-est signals (Fig. 3a and b). Positive sections from patients with cancer appeared to show strong reactivity in and aroundcells, at'theperiphery of the invasivesquamous cell
carcinoma islands; but most of the invasive cells were
negative (Fig. 3d). Essentially nohybridization signals were seen in the majority of tissues from histologically normal
patients, including the morphologically normal epithelium of the columnar cells or the underlying stroma from patients with condyloma and invasive cervicalcancer.Occasionally,
focal-positive signals were seen in the underlying stroma (Fig. 3f), in areas of inflammatory infiltrate, or along the
basement membrane. However, these specimens reacted with all HPV and vectorprobes under stringent hybridiza-tion conditions. Similar observations have been made by Gupta and co-workers (11), and we concur with their
con-clusions andregard these stromal reactions asnonspecific.
TheoccurrenceofHPV DNAswasfoundtovary
accord-ing to clinical condition. Of 26 histologically confirmed condylomas, 23(89%)werefoundtocontain HPV DNAs by
one ormoreof the methods. A total of19 specimens (73%)
had HPVtypes6C- and 11-related DNAsequences; 5(20%)
had HPV types 16 and 18 DNA, and 3 (12%) reacted with HPVtypes6C and11 DNA only under nonstringent hybrid-ization conditions. Among patients withcancer,HPVDNA wasfound in 19 of 23 (83%) patients by one or more of the
methods. These19 specimens contained HPVtypes 16- and 18-relatedDNA, and 13 (57%) also reacted with HPVtypes 6C and 11 DNA probes. Cervical specimens from women
with no histologie evidence of genital neoplasia or
condy-lomas hybridizedtoHPV sequencesto alesserextentthan did those frompatients in the othertwocategories. Among
such samples, 4 of 31 (13%) contained such sequences.
Interestingly,histologicevidence ofmetaplasiawasfound in
all the cervices from which positive specimens were
ob-tained.
The occurrence of specific types of HPV according to clinical condition, asdetermined by each of the
hybridiza-tion methods, issummarized in Table 1. A total of 62% of the condylomatous lesions werepositive forany HPV DNAby
the in situ filterhybridization method, 82%werepositive for
HPV DNA by the Southern blot method, and 72% were
positive for HPV DNA by the in situ tissue hybridization method.Amongpatients withcancer,89% of the specimens werepositive by the in situ filter hybridization method, 70% were positive by the Southern blot hybridization method,
and 30% were positive by the in situ tissue hybridization
method. Only 9.5% of the specimens from patients withno
histologic evidence of neoplasiaorcondylomawerepositive
for HPV DNA by the in situ filter hybridization method, while 13% of thespecimens were positive by the Southern
blothybridization method and 6% were positive by the in
situ tissuehybridization method.
The sensitivities and specificities of each hybridization method were first computed with respect to disease type. The sensitivity of a test is defined as the proportion of
subjects with the disease andapositivetestresult,while the specificity ofatest is defined as the proportion of subjects without the disease and a negative test results (8). The results ofan analysis in which the- patients with condyloma orinvasivecancer werepooledare summarized in Table 2. The Southern blothybridization method exhibited a sensi-tivity of 76% and a specificity of 87%. The in situ filter
hybridization method had a sensitivity of 72% and a
speci-ficity of 90%, while the in situ tissue hybridization method had an overall sensitivity of 50% and a specificity of 93%. However, the sensitivity of the in situ tissue hybridization method differed between diseasecategories: itwas72%for
condylomas and 30% for invasive cancers. The relative sensitivities, specificities, and reliabilities of the hybridiza-tion methods indetecting HPVsequences aresummarized in
Table3.By using the Southern blothybridization methodas
the standard, the in situ filter hybridization method had a
sensitivity of 66% and a specificity of 88% and showed
significant agreement beyond chance (kappa = 0.54; P <
0.0001).With respecttothe Southernblot method,the in situ tissue hybridization method had a sensitivity of 61% and a
specificity of86%and showed significantagreement(kappa TABLE 1. Occurrence ofHPVtypebyclinicalconditions
No.positive/no.tested(%)for thefollowingHPVtypes:
Hybridization Histologic
assaya diagnosis 6 and11b 16 and 18b 6,11,16, Otherf Total
and18"
Insitufilter Condyloma 9/26(35) 3/26(12) 1/26(4) 3/26(12) 16/26(62)
Invasivecancer 4/18(22) 8/18(44) 4/18(22) 0/18 (0) 16/18 (89)
Normal 2/21(9.5) 0/21 (0) 0/21(0) 0/21 (0) 2/21 (9.5)
Southernblot Condyloma 8/17(47) 1/17(6) 2/17 (12) 3/17 (18) 14/17 (82)
Invasivecancer 0/20(0) 13/20(65) 0/20(0) 1/20 (5) 14/20 (70)
Normal 1/31 (3) 1/31(3) 0/31 (0) 2/31 (6) 4/31 (13)
Insitu tissue Condyloma 13/25(52) 3/25 (12) 2/25(8) (0) 18/25 (72)
Invasivecancer 2/23(7) 3/23(13) 3/23(13) (0) 7/23 (30)
Normal 2/33(6) 0/33 (0) 0/33 (0) (0) 2/33 (6)
aThe same numberwasnotavailablefortesting byall three assays.
bSpecimensweredetected understringentconditions ofhybridizationfor thespecificHPV.
cSpecimensweredetectedonlyundernonstringentconditions ofhybridizationwithanyofthe HPV DNAprobes. VOL.26, 1988
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240 CAUSSY ET AL.
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é* r îFIG. 3. Autoradiographic signals seenbythein situtissuehybridization method.Paraffin-embedded biopsy safnpleswereprocessedby
standardhistologicmethodsandhybridizedwith3S-labeledHPVprobesunderstringent hybridization conditions(T,,, -17°C),asdescribed in thetext.HPV type6C and11probeswereused forcondylomatous tissue, and HPVtypes 16and18probeswereusedfor invasivecancer
tissue.(a)Typical condyloma. (b) Higher magnification of positive koilocytes inacondyloma. (c)Hematoxylinstaining ofaninvasivetumor
that showedapositive signalin theinvasive cells (d).(e)Hematoxylin staining of invasivetumorsthatshowedapositive signalin thestromal
cells of thetumor(f). Barforpanela, 0.2mm; bars for panelsb tof,0.04mm.
J.CLIN. MICROBIOL.
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HPV DETECTION METHOD EVALUATION 241
TABLE 2. Sensitivities and specificities of different hybridization methodsin detecting HPV withreferencetodisease
Hybridization No. tested Sensitivity (%) Specificity (%) % Agreement Cohenkappa Pvalue
method statistic
Southern blot 63 76 87 80 0.62 0.0019
Insitu filter 65 72 90 78 0.51 <0.0001
In situ tissue 83 50 93 68 0.39 <0.0001
a Results for specimens of condyloma and invasive cervical cancer were pooled.
=0.50; P<0.0001). Pairwisecomparison of the in situ filter and in situ tissue hybridization methods similarly showed significantagreement(kappa = 0.55;P < 0.0001).
DISCUSSION
Our aim in this study was to compare the sensitivities,
specificities, and reliabilities of molecular hybridization methods in detecting HPVsequencesunder various clinical
conditions. To this end,weapplied the Southern blot, in situ
filter, and in situ tissue hybridization methods tospecimens ofcervical swabs and biopsies from histologically normal cervices, condylomatous lesions, and invasive cervical can-cercells.
HPV DNAsequences werefound in 70% of patients with
invasive cancer by using the Southern blot hybridization method, and most of the positive samples contained HPV types16- and 18-related DNA. Thispercentageis consistent with that for HPVtype16reported previously byus(18) and
others(7). By the Southern blot hybridization method, HPV DNA was detected in 82% of the condylomatous lesions
examined. This percentage agrees with that reported by
Crum and colleagues (5), but is somewhat less than that reported by Schneider and colleagues (20). Only 13% of histologically normal ectocervical biopsies were found to contain HPV DNAsequences by the Southern blot
hybrid-izationmethod. This finding is inagreement with the obser-vation of MacNab and co-workers (14) but is lower than that observedby Meanwell and co-workers (15), who found that 35% of histologically normal ectocervices contained HPV type 16sequences.
Wagner and colleagues (26) adapted the in situ filter hybridization method for detecting HPV DNA in exfoliated cervicalcells. Basedontheresults of the study by Grunstein and Hogness (10), wemodified this method toincrease the
recovery of HPV DNA and digested cellular proteins and
mucus to reduce the nonspecific background. Using this modified in situ filter hybridization method, we found that cells from 62% of the condylomatous lesions and 89% ofthe cervical cancers contained HPV DNA sequences. Our
re-sults for invasive cervicalcancer casesarein agreement with those of Schneider and colleagues (20), who found HPV DNA in four of four cases. They also detected viral se-quencesin sixofsix womenwithcondylomatous lesions, a
frequency thatwasgreaterthanthatobserved inourstudy.
They also foundfewerpatients withnormalepitheliumthat
waspositive for HPV DNA.
Application of the in situ hybridization method to the detection of vegetative replication of PV in tissue sections
was first reported by Orth and colleagues (16). Using
35S-labeled probes, we found that 60% of condylomas were
positive for HPVtypes6C and 11. Thisrateis in agreement with that observed by Gupta and colleagues(11),who found HPVDNA in58% of the condylomatous tissues tested, but lower than the 100% positive rate observed by Beckmann and colleagues (3), who used a biotin-labeled probe. Our
sample size was significantly larger than those used in
previous studies andmayrepresenta morereliable estimate.
Whenwe applied the in situ tissuehybridization methodto invasivecancerlesions, wefound only 30% of the sampleto bepositive for HPVtypes16and 18 DNA. Wealso observed that6%of histologically normal cervices reacted with HPV probes by this method.
The evaluation of theaccuracyofatest restswith knowl-edge of whether the disease is trulypresent.Inourstudywe
evaluated the respective sensitivities and specificities of each of the assays with respect to histologic evidence of disease.Our estimates of sensitivity andspecificity could be distortedby selection bias sincewe definednormalpatients as those whose cervices showed no histologic evidence of
genital neoplasticorcondylomatous disease after
hysterec-tomy. However, about one-third of these cervices were
foundtohaveinflammatoryormetaplastic changes andwere
nottruly normal. A furthersourceofpotential selection bias couldresult from the fact thatwepooled the prevalence data
fromtwodifferentpopulations toestimate the test
parame-ters. When all the disease categories are considered to-gether, the Southern blot hybridization method gave the
highest sensitivity (76%), followed by the in situ filter hybridization method (72%) and finally by the in situ tissue hybridization method (50%). A converse pattern was seen
for the specificity; the most specific assay was the in situ tissue hybridization method (93%), followed by the in situ filter hybridization method (90%) and the Southern blot hybridization method (87%). The sensitivity ofthe in situ tissue hybridization method, however, differed between disease categories, being similar to the other methods in detecting HPV DNA in condylomatous lesions but less sensitivewhenappliedto invasivecancercells.
Theaccuracy ofone method versus anothercanalso be expressed in terms of relative sensitivity and specificity. Whenweused the Southern blothybridizationmethodas a
referencetoevaluate the other methods for the detectionof
TABLE 3. Relative sensitivities andspecificitiesofdifferenthybridizationmethods indetectingHPV
Hybridizationmethod No. tested sensitivityRelative(%) specificity
Relative
(%) e%AgrnementArent statisticCohn PvalueSouthern blota versusinsitu filter 56 66 86 80 0.54 <0.0001
Southern blota versusin situ tissue 72 61 86 77 0.50 <0.0001
Insitua tissueversusinsitufilter 59 66 86 79 0.55 <0.0001
aTechnique usedas astandard in thecomparison.
VOL.26, 1988
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HPVDNA, therelative sensitivitiesandspecificities of the in situ filter and tissue hybridization methods were not different from each other. The percent agreement beyond
chance,
ascomputed by
theunweighted
Cohenkappasta-tistic,
showedgood
agreement(kappa
=0.50;
P<0.0001).
It isapparent,however,
thatagreementwas notachieved inasubstantial
number ofcases. This residual lack ofagreement could beexplained by sampling
variation.Two main types oferrors are
commonly
encountered in assayssuchasthose described here. These includesampling
errors, in which material
representative
of the lesion is not included in thespecimen,
andinterpretive
errorsinreporting
theresultsof the assay. Thereisa
degree
ofsubjectivity
ininterpreting
theresults of thehybridization techniques
used inthisstudy,
andweattempted
tominimize thisby accepting
theconsensusof three
independent
observers.However,
nospecific
measures were undertakento control forsampling
error. The in situ
hybridization
method on tissue sections showed that theHPVDNAispredominantly
confinedtothe uppercell
layers
of theepithelium.
For the in situ filterhybridization method,
unlike in thebiopsy,
thesurface of the lesionwassampled; thus,
anendocervical swab should havecaptured
thosepositive
cells.
Useofanysampling
method in whichalesion is missedyields
alowpositive
rate,irrespec-tive of the limit of detection of the
hybridization
method used to test suchsamples.
In thisregard,
sampling
in ourstudy
was notcolposcopically
directed. The in situhybrid-ization method for tissue section is most vulnerableto this
sampling variation,
because notonly
can the lesions be missedby biopsy
butthey
canalsobe missedby
sectioning.
Indeed,
in somespecimens
that were testedby
the in situ tissuehybridization method,
thelesionswerepoorly
repre-sented.Thus,
sampling
errorprobably
accounted for asubstantialamountof the
disagreement
thatwas observed. The relative copy number of the HPV genomes in the varioussamples
mayalsoseriously
affect the limit of detec-tionby
thehybridization
methods. In ourhands,
a recon-structionexperiment
indicated that the Southern blot method could detect 0.2 pg of HPVDNA,
whichcorre-sponds
to0.2to0.3copies
ofHPV DNA percell(18). By
the in situ filterhybridization method,
ontheotherhand,
0.1 pg of HPV DNA could bedetected,
an amount that is notidentifiable
by
the Southern blothybridization
method(26).
This couldaccountfor the
slightly higher positivity
ratefor HPVDNAforcancer casesby
theinsitu filterhybridization
methodthan
by
theSouthern blothybridization
method. Itis difficulttoestimatedirectly
the limitofdetection
of theHPV genomesby
in situhybridization
in tissue sections. Eventhough
asingle
positive
cellcontaining multiple copies
ofthe HPV genomecantheoretically
bedetectedby
thismethod, it ispossible
thatfewer than10copies
ofthe HPV genome per cell cannot be detected. In support of thisargument is our observation that Sihacells,
which contain 10 copies ofthe HPV type 16 genome, as determined by the Southern blot method(27),
werenegative bytheinsitu tissuehybridization method(data
not shown). With our length of exposure,however,
wehavebeenable to detect 500 copies of the HPV genome,aswasobservedby usingCaski cells (27). This limit ofdetection isslightly
better than that observed by Crum andcolleagues
(5), who detected 800 copies of the HPV genomein theirsystem. Specimens fromanumber of cancer cases werepositive by
the Southern blot hybridization method butwerenegative
by the in situ tissuehybridization method. This suggeststhat the majority of cancers in some lesionsmight
harbor less than 500 copies ofthe HPV genome percell.
In conclusion, we found each of the methods to have advantages and disadvantages. While the Southern blot hybridization method permitted the unambiguous identifica-tion of thephysicalstateof the HPVgenomesand exhibited thehighest specificity, itsuffered fromarelianceonunfixed biopsies and a tedious DNA manipulation that required a specialapparatus.The in situ filterhybridization method isa noninvasivetechniquethatisquickandsimpleandthuswell suited forepidemiological studies; however, it suffers from the inability toinherently exclude reactionsas nonspecific. Thesimplicity of the in situ tissue hybridization method and its ability to detect HPV DNA sequences in paraffin-em-bedded sections lends itselftoretrospective studies. Its chief drawback is thatitmaybe of limited sensitivity indetecting HPVsequences in a substantialportion of invasive cervical cancers.
ACKNOWLEDGMENTS
We thank Sharryn Putns, Debra Browning, Carol Lavery, and Gudrun Goettsche for technical assistance; Maria Majela Brenes, Maria Elenade laGozia,and Don ManardQuerero,who conducted field work inPanama;and Ian Stewart and Rosa de Britton for aid in collecting clinical specimens.
Financial supportwasprovided bytheNational Cancer Institute (Canada), byPublic Health Service grant NCI CP EB 41026 from the National Cancer Institute (U.S.), and Public Health Service grant IROICA 42042 from the National Cancer Institute(U.S.). D.C. is a National CancerInstitute (Canada) predoctoral fellow.
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