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Int. J. Gynecol. Obstet., 1990,31: 145-152 International Federation of Gynecology and Obstetrics
Occurrence of human papillomavirus DNA types 16 and 18 (HPV-16/l@ in cervical smears as compared to cytological findings H.G. KiicheP, A. Teichmannb,
N. Eckardt”,
P. Arendtb, W. Kuhnb and R. Thomssena
OCenterof Hygiene and Human Genetics of the University, Department of Medical Microbiology, Kreuzbergring 57, D-3400 Giittingen and “Department of Obstetrics and Gynecology of the University, Robert-Koch-Strasse 40, D-3400 Gtittingen (FRG) (Received June 27th, 1988) (Revised and accepted January 30th. 1989)
Abstract
Cervical smears from 2336 women were examined for the presence of HPV-16118 by dot-blot hybridization using 32P-labeled HPV-X6/18 DNA under high stringency conditions. The hybridization data were compared with cytological findings classified according to Papanicolaou. The ages of the patients ranged from under 20 to over 70 years. Ninety-eight (4.40/o) of the 2237 cytologically normal cervical samples (Pap I and II) were HPV-16/18 positive. Thirteen out of 32 (40.6%) samples showing signs of mild and moderate dysplasia (Pap IUD) were found to be HPV-16118positive. In 5 out of 7 (71.4%) samples from women with severe dysplasia or carcinoma in situ (Pap IV) and in 9 out of 25 (32. I Yo) samples from patients with invasive cervical carcinoma (Pap V) HPV-16/18 DNA was detected. Thirty-two smears were from women with severe unspecific cervical inflammation (Pap III). Two (6.2Yo) out of them were HPV-Id/18 positive. Normal smears showed an apparent age-dependent pattern of HPV-16/18 positivity with a peak prevalence of 10.6% among women younger than 20 years old. The majority of premalignant lesions was detected among women younger 0020-7292/90/$03.50
0 1990 International Federation of Gynecology and Obstetrics Published and Printed in Ireland
than 40 years old; whereas all invasive lesions were from women older than 39 years. Compared to the HPV-16/18 prevalence rate in normal smears, abnormal smears harbored HPV-/-16/18 DNA approximately 9 times more frequently. This finding supports the hypothesis that HPV-16/18 may be involved in the development of cervical cancer. Keywords: Cervical smears; Papanicolaou cytology; Cervical dysplasia; Cervical carcinoma; HPV-16/18; Dot-blot hybridization. Introduction
Epidemiologic studies support the involvement of a sexually transmitted agent in cervical carcinogenesis. Several attempts have been made to identify the nature of this infectious agent. Recently, attention has been focused on human papillomaviruses (HPV) as possible candidates. Of more than 50 different HPV types [20], only a few have been identified in tissue originating from cervical and genital lesions. It has been shown by DNA hybridization techniques that HPV-6 and -11 are predominantly present in genital warts and low grade dysplasias Clinical and Clinical Research
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Kochel et al.
[6,12]; whereas HPV-16/18 are associated with the majority of cervical carcinomas and of high grade dysplastic lesions [2,5,8]. In rare cases DNA of HPV-3 1 [17], -33 [l] and -39 [ 181has also been detected. The genomes of HPV-16 and -18 have been usually found to be integrated into the cellular genomes of cancer cells [9] and of cell lines derived from cervical carcinomas [25]. The presence of HPV genomes has also been demonstrated for corresponding metastatic tissue [ 151. In cancer cells the viral DNA is not only transcriptionally active [25,27], but also specific early proteins have been identified [28]. This suggests that the presence of the viral genome and the permanent expression of certain viral functions may be necessary for the induction and maintenance of the malignant phenotype. Because of their association with either benign or malignant cervical lesions the HPV types mentioned above may be classified as low risk (HPVd/ll) and high risk types (HPV-16/18 and other related HPV-types). The early detection of the so-called high-risk types may help to identify women at risk for developing cervical carcinoma. However, up to now an etiological relationship between a HPV-16/18 infection and cervical cancer has not been established. For this purpose we have initiated a follow-up study on a large cohort of patients. In order to specify the number of patients necessary for this followup investigation, we have attempted to determine the prevalence rates of HPV-16/18 in normal and abnormal cervical epithelia. We examined 2336 cervical samples from women between under 20 and over 70 years of age living in and around Gottingen (Northern West Germany). In addition to exfoliative (Papanicolaou) cytology the analysis was performed by dot-blot and in some selected cases by Southern-blot hybridization. The DNA assay and the cytological classification were performed independently (double blind). This report will show that the presence of HPV-16/18 correlates well with cervical dysInt J Gynecol Obstet 31
plasia and carcinoma. It will also demonstrate that HPV-16/18 are widespread in women with normal cytology irrespective of the age of the patients. Materials and methods Sample collection Cervical samples were obtained from women being treated by the outpatient clinic of the University Women’s Hospital of Gottingen for their routine Pap-smear. A combined ecto- and endocervical sample was taken with cotton swabs. After preparation of the Pap-smear the cotton swabs were transferred to 4 ml of phosphate buffered saline (PBS, pH 7.4) in 10 ml conical plastic tubes Nuembrecht, Germany). This (Sarstedt, procedure ensured that the same sample was used for the cytological examination as for hybridization analysis. The samples were either immediately processed or stored overnight at 4 OCprior to processing. Extraction of DNA The plastic tubes containing the swabs were vigorously shaken on a Vortex mixer. The swab was removed and the suspended cervical cells were collected by centrifugation at 3000 x g for 5 min. The supernatant was discarded, the cells resuspended in the remaining PBS and transferred to 1.5 ml Eppendorf tubes. After a second centrifugation step, the PBS was removed and the cells were lysed in 300 ~1 of 50 mM Tris-Cl (pH 7.4), 10 mM EDTA, 50 mM NaCl, 2% SDS in the presence of 200 pg/ml proteinase K. Incubation was at 50°C for at least 2 h. Lysates were extracted with an equal volume of phenol/chloroform After centrifugation the (50: 50, w/v). aqueous supernatants were transferred to new Eppendorf tubes and the DNA was ethanol precipitated. The DNA was pelleted by centrifugation and dissolved in 300 ~1 of TE (pH 7.4). One hundred ~1 of the DNA solutions were stored at - 20°C for further analysis, such as Southern-blot hybridization. The remaining 200 ~1 DNA solution were processed as described below.
HPV-16/18
Probe preparation Human papillomavirus types 16 and 18 DNAs cloned in pBR 322 were kindly provided by Dr. zur Hausen, DKFZ, Heidelberg, Germany. Viral DNA from HPV-16 and HPV-18 was isolated from the plasmids by restriction digestion with BamHI and EcoRI, respectively. HPV DNA was separated from the vector by agarose gel electrophoresis and subsequent electroelution in dialyse bags. DNA was radiolabeled by nicktranslation [23] in the presence of [a32P]dCTP (Amersham, Braunschweig, Germany). Specific activity of the radiolabeled DNA was between 1 and 3 x lo8 cpm/ c(g. For the determination of the amount of cellular DNA a human repetitive DNA sequence was prepared in the following manner. Human DNA extracted from the liver was digested with BamHI and cloned into the BamHI site of pBR 322. E. coli cells were transformed [ 141 and resulting ampicillin resistant bacterial colonies were hybridized in situ with 32P-labeled total human DNA. One positive clone was selected and its plasmid DNA was isolated by routine methods [19]. This plasmid DNA was 32P-labeled and characterized by Southern-blot hybridization against BamHI restricted DNA from humans, guinea pigs and the fungus Candida albicans. The plasmid called pNANB 2/63 reacted specifically with human DNA producing a continuous smear and some minor bands on the autoradiogram. This repetitive DNA was not further characterized. Prior to use, it was 32P-labeled as described above. Dot blot hybridization To the remaining 200 ~1 of the DNA solution l/10 volume of 3 M NaOH was added and the DNA denatured at 60°C for 1 h. After being neutralized by the addition of an equal volume of 2 M ammonium acetate, the DNA was bound to a nitrocellulose membrane (BA 85, Schleicher and Schuell, Dassel, Germany) by means of a manifold filtration device (Minifold SRC 96, Schleicher and
in cervicalsmears
147
Schuell, Dassel, Germany). As a control a standard series of HPV-16/18 DNA ranging from 0.5 to 100 pg was additionally applied to the membranes. In order to determine the relative amount of cellular DNA, l/10 aliquots of the DNA solutions were filtered through a separate membrane in the same order. One to 5 pg of DNA from human placenta served as a control. Prior to filtration the membranes were soaked in 1 M ammonium acetate for 10 min. After filtration the membranes were baked for 1 h at 80°C in a vacuum oven and then washed in prewash solution 1191at 42OC for 1 h. Before hybridization, the membranes were prehybridized in 100~l/cm20f 50% formamide, 5 x SSC, 5 x Denhardt’s solution, 0.25% lithium dodecylsulfate and 100 pg/ml of sheared and denatured salmon sperm DNA at 42OC overnight. Hybridization was conducted at 42OC for 36 h in 100 Ccl/cm2of the same mixture described above except that 1 x lo6 cpm/ml of 32Plabeled HPV-16/18 DNA was added. To the filters used for the determination of the amount of human DNA lo5 cpm/ml of the 32P-labeled human repetitive element was added. Southern-blot hybridization Selected samples were also tested for the presence of HPV-16118 DNA by Southernblot hybridization. For this purpose the DNA extracted from cervical cells was digested with Hind111 and subjected to agarose gel electrophoresis. After alkali denaturation and subsequent neutralization, the DNA was transferred onto nitrocellulose membranes and hybridized as described above. After hybridization all filters were subjected to three consecutive washings under stringent conditions (0.5 X SSC, 0.1% SDS, 65OC). Filters were exposed to Kodak XAR X-ray films for l-7 days. Autoradiograms were evaluated by visual inspection and comparison with the standard series. DNA samples which did not hybridize with the repetitive element were excluded from this study. Clinical and Clinical Research
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Kochel et al.
Results Sensitivity and specificity of the method In order to evaluate the method employed different amounts (0.1-5 pg) of DNA extracted as described above from HeLa cells harboring integrated HPV-18 DNA [25], and from a HPV negative human cell line were applied to four separate nitrocellulose filters. A standard series of each of the HPV types 6, 11, 16 and 18 served as a control. Filters were separately hybridized with purified and 32P-labeled HPV-6, 11, 16 or 18 DNA. Under the described conditions HeLa cell DNA hybridized to HPV-18 only. No crosshybridization was visible between the different HPV types and human cellular DNA. In addition, the different HPV types did not normally hybridize with each other. An exeption was HPV-6 and -11 which did cross-hybridize; this is due to their striking sequence homology. When HPV-16/18 DNAs were hybridized with their homologeous counterparts the detection limit was 0.5 pg. However, when DNA from cervical smears was examined it was often impossible to decide whether those weak hybridization signals were due to small amounts of HPV16118 DNA or to large amounts of cellular DNA. This was especially the case after prolonged exposure of the autoradiograms. Therefore, exposure times were limited to a maximum of 3 days and the samples causing signals equivalent to less than 1 pg of HPV DNA were considered HPV- 16/ 18 negative. HP V-l 6/l 8 prevalence in cervical smears
Of the 2336 cervical smears which were examined for the presence of HPV-16/18, 2237 appeared cytologically normal (Pap I/ II). Ninety-eight (4.4%) of these were HPV16118 positive (Table I). The distribution of HPV- 16/l 8 positive cases within the normal group seemed to be age-dependent. The highest prevalence rates were found among women of less than 20 (10.6%) and between 20 and 29 years of age (7.7%) (Table II). The Int J Gynecol Obstet 31
Table 1.
Prevalence of HPV-16/18 in relation to cervical cytology (N,, number of investigated smears; N+ , number of HPV-16/18 positive smears; %total, percentage of N, in relation to the total number of investigated smears; %N,, percentage of smears positive for HPV-16118). Cytological diagnosis
HPV-16/18 prevalence
Pap
N
%total
I/II
III IUD IV V
2237 32 32 I 28
95.1 1.4 1.4 0.3 1.2
Total
2336
N+
%N,
98 2 13 5 9
4.4 6.2 40.6 71.4 32.1
127
5.4
other age groups showed a relatively uniform distribution of HPV-16118 positivity ranging from 2.5 to 4.2%. Thirty-two women had signs of mild or moderate dysplasia (Pap IIID). Thirteen (40.6%) were HPV-16/18 positive. Seven women had severe dysplasia or carcinoma in situ (Pap IV); HPV-16/18 being detected in 5 women (71.4%). Twentyeight smears came from cancer patients (Pap V); 9 of them (32.1%) were associated with HPV-16/18. As a control we extracted DNA as described above from 17 cervical carcinoma biopsies and found 10 (59%) positive for HPV-16/18 (not shown in tables). The age distribution of cervical carcinoma patients differed from that of women with premalignant lesions. Pap IIID and IV were most common among women between under 20 and 39 years of age; whereas all Pap V smears were from women over 39 years of age. The category Pap III included smears from women suffering from severe unspecific cervical inflammation or a cervical carcinoma which could not definitively be identified from Pap smears. One hundred four samples from women between 20 and 29 years of age (same cohort) were also tested for the presence of HPVd/ 11. Ninety women had normal smears; eleven were HPV-16/18 and 1 HPV-6/11 positive.
HPV-16/18
Cytological findings and HPV-16/18 prevalence in relation to age of the women (N, number of smears; N + positive smears; % + , percentage of smears positive for HPV-16118).
Table II. HPV-16/18 Age
in cervicalsmears
Pap I/II
Pap IV
149
, number of
Total
Pap V
Pap III
Pap IIID
N
N+
N
N+
%+
N
N+
N
N+
%+
N
N-k
% +
-
4 11
2 6
50.0 54.5
4
3
-
-
-
71 343
9 34
12.6 9.9
-
N
N+
%+
70
229
f
3.1
8
2
-
-
1
-
10
4
40.0
248
13
5.2
-
Four samples contained cells consistent with mild or moderate dysplasia; three harbored HPV-16/18 and one HPVd/l 1. One patient with severe dysplasia was neither HPV-16/18 nor HPVd/l 1 positive. In total 13% of women of this patient group were HPV-16118 and 2% HPV-6/11 positive (not shown in tables). Southern-blot analysis Because dot-blot hybridization cannot provide information on the physical state of the viral DNA, selected samples from different Pap-groups were additionally examined by Southern-blot hybridization. However, a detailed analysis was precluded by limited amounts of material. Extracted cellular DNA was digested only with the restriction enzyme Hind111 which does not cleave either HPV-16 or -18. If the HPV-DNA has become integrated into the cellular genome, the digestion with Hind111 will result in autoradiographic bands appearing above the position of the monomeric episomal form II (open circular). Otherwise, the three isomeric forms of episomal HPV DNA should be visible. However, if only Hind111 is used, integrated DNA and oligomerized forms of DNA episomal cannot be reliably distinguished. In all 12 samples which were examined, episomal forms of HPV-16/18 DNA were
present (Fig. 1, FI, covalently closed circular; FII, open circular; FIII, linearized form). Additional high molecular weight bands which may be indicative of integrated viral DNA were almost exclusively detected in premalignant and malignant lesions (Pap IIID, IV, V, Fig 1, lanes 5, 6, 8, 10, 11, 12). Viral DNA of high molecular weight was also found in a Pap III smear from a women 70 years of age (Fig. 1, lane 3). Discussion
The presence of HPV-16/18 DNAs in cervical cancer cells cannot provide evidence of an etiological role for these viruses in the development of cervical carcinoma. The possibility that they are passengers rather than causative agents cannot be excluded. We have therefore initiated a follow up study which will attempt to answer the question whether papillomaviruses are able to induce and maintain transformation of the normal cervical epithelium. First, the prevalence rate of HPV-16/18 in cervical smears from women of different age groups was examined in relation to their cytological classification. Using dot-blot hybridization for this purpose, we found an overall HPV-16/18 positivity of 5.4% irrespective of the cytological diagnosis. When correlated to different age groups, the HPV- 16/l 8 prevalence rate Clinical and Clinical Research
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Kochel et al.
-FII -FIII
--FI
Fig. 1. Southern-blot analysis of HPV-16/18 DNA sequences in cervical smears of different Pap groups. DNA samples were digested with Hind111 and separated on a 0.7% agarose gel, transferred to nitrocellulose, and hybridized under stringent conditions as described in Materials and methods. Lanes 1 and 2, Pap II; lanes 3 and 4, Pap III; lanes S-10, Pap IIID; lane 11, Pap IV; lane 12, Pap V; lane 13, positive control, i.e. 20 pg of covalently closed circular plasmid DNA consisting of HPV-16 DNA and pBR 322. HPV-16/18 specific signals were visualized after exposure to X-ray film for 7 days. Positions of the monomeric episomal forms (FI, FII, FIII) of HPV-16/18 genomes are indicated by arrows.
appeared to be age dependent. Women younger than between 20 and 29 years of age showed seemingly higher HPV- 16/ 18 infection rates than older women. This higher rate of HPV-16/18 positivity among young women correlates well with the prevalence rates of HPV in penile swabs from healthy men [ 131. Highest positivity rates (7.5%) were found among men between 16 and 35 years old; whereas among older men the HPV positivity declined to 1% . In cases of dysplasia the HPV-16/18 prevalence rates seemed to increase with the severity of the lesions (Pap IIID, 40.6%; Pap IV, 7 1.4%) but the difference was not statistically significant. However, other reports also describe a higher prevalence of HPV-16/18 in more severe dysplastic lesions [4,30]. This appears to support the hypothesis of an increase in HPV-16/18 prevalence with increasing lesion severity. Int J Gynecol Obstet 31
It has been demonstrated by molecular hybridization studies that about 65% of cervical carcinoma biopsies contained HPV- 16/ 18 DNA with some geographical fluctuations [ 111. In this study only 32.1% of smears from cancer patients were HPV- 16/l 8 positive, because smears from cervical carcinomas may be inappropriate for the detection of HPV due to the heterogeneous cell composition of the tumor and/or low copy numbers of HPV DNA in cancer cells [22]. This inadequacy is supported by the finding that approximately 60% of carcinoma biopsies which we examined were HPV- 16/ 18 positive. Previous studies dealing with the HPV-16/ 18 prevalence rates were mostly based on small numbers of patients and different hybridization techniques. However, de Villiers et al. [7] recently published a corresponding report on more than 9000 patients. Although they used the filter in-situ hybridi-
HPV-16/18 in cervicalsmears
zation method and pooled their HPV-6/11 and HPV-16/ 18 data, there are remarkable concordances between their results and ours. In particular, they described the same agedependent pattern of HPV positivity among women with normal smears. In agreement with our results premalignant and malignant lesions showed no consistent age-dependent pattern of HPV positivity. Without further documentation they also mentioned that HPV-16/18 were more prevalent in cervical smears than HPV-6/11. The infection rates for HPV-16/18 and HPV-6111 which we found in a small group of women aged between 20 and 29 years were 13% and 2%) respectively. However, these data have to be substantiated by screening larger groups of patients. De Villiers et al. [7] emphasized that their results probably underestimate the real HPV16/ 18 infection rate by a factor of 2 to 3. This could also be true for our examination. The problems regarding the sensitivity and applicability of the different hybridization methods have been widely discussed by Schneider 1241 and Gissmann et al. [lo]. The 9 times higher HPV- 16/ 18 positivity of abnormal smears in comparison to normal smears implies, but does not necessitate, that HPV-16/18 may be involved in the development of cervical carcinoma. Namely, the possibility that HPV DNA is more readily detected in abnormal than in normal tissue or that abnormal tissue is more prone to HPV infections cannot be disregarded. Therefore, a general screening of cervical scrapes for HPV infections as suggested by some authors [3,31] is not required at present. However, other authors observed a difference in the progression rate of mild dysplastic lesions to severe lesions depending on the HPV type [4,29,30]. Progression rates have been higher among women infected with HPV-16/18 than among those in whom HPV6111 was present. Whether the presence of HPV-16/18 DNA in cervical cells especially of its integrated form in premalignant lesions, which was described in this and in other
151
reports [16,26], represents a valuable diagnostic parameter remains to be defined. To establish the etiological relationship between HPV infections and cervical cancer, large prospective cytologic, colposcopic and virologic studies are necessary. However, the assessment of this relation will be made more difficult by the high HPV-16/18 prevalence rates among young women with normal cytology, particularly if an underestimation of the actual infection rate by a factor of 2 to 3 [7] is assumed. Acknowlegdment This study was supported by the BtittnerStiftung of the University of Gottingen. References Beaudenon S, Kremsdorf D, Croissant 0, Jablonska S, Wain-Hobson S, Orth G: A novel type of papillomavirus associated with genital neoplasias. Nature 321: 246, 1986. Boshardt M, Gissmann L, Ikenberg H, zur Hausen H: A new type of papillomavirus DNA, its presence in genital cancer biopsies and in cell lines derived from cervical cancer. EMBO 53: 1151, 1984. Burk RD, Kadish AS, Calderin S, Romney SL: Human papillomavirus infection of the cervix detected by cervicovaginal lavage and molecular hybridization: correlation with biopsy results and Papanicolaou smear. Am J Obstet Gynecoll54: 982, 1986. Campion MJ, Cuzich J, McCarice DJ, Singer A: Progressive potential of mild cervical atypia: Prospective cytological, colposcopic and virological study. Lancet ii: 937, 1986. Crum CP, Ikenberg H, Richart RM, Gissmann L: Human papillomavirus type 16 and early cervical neoplasia. N Engl J Med 310: 550, 1984. De Villiers EM, Gissmann L, zur Hausen H: Molecular cloning of viral DNA from genital warts. J Viral 40: 932, 1981. De Villiers EM, Schneider A, Miklaw H, Papendick U, Wagner D, Wesch H, Wahrendorf J, zur Hausen H: Human papillomavirus infections in women with and without abnormal cervical cytology. Lancet ii: 703, 1987. Dilrst M, Gissmann L, Ikenberg H, zur Hausen H: A new type of papillomaviral DNA from cervical carcinoma and its prevalence in cancer biopsies from different geographic regions. Proc Nat1 Acad Sci (Wash) 50: 3512, 1983. Diirst M, Kleinheinz A, Hotz M, Gissmann L: The physical state of human papillomavirus type 16 DNA in benign
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Kochel et al. and malignant genital tumours. J Gen Virol 66: 1515, 1985. Gissmann L, Diirst M, Oltersdorf T, von Knebel Doeberitz M: Human papillomaviruses and cervical cancer. In: Cancer Cells 5 (eds BM Steinberg, JL Brandsma, LB Taichman) p 275. Cold Spring Harbor Laboratory, New York, 1987. Gissmann L, Schneider A: Human papillomavirus DNA in preneoplastic and neoplastic genital lesions. In: Viral Etiology of Cervical Cancer (eds R Peto, H zur Hauseri) p 217. Cold Spring Harbor Laboratory, New York, 1986. Gissmann L, Wolnik L, Ikenberg H, Koldovsky U, Schnilrch HG, zur Hausen H: Human papillomavirus types 6 and 11 DNA sequences in genital and laryngeal papillomas and in some cervical cancer biopsies. Proc Nat1 Acad Sci (Wash.) 50: 560,1983. Grussendorf-Conen EL, de Viliiers EM, Gissmann L: Human papillomavirus genomes in penile smears of healthy men. Lancet ii: 1092, 1986. Hanahan D: Studies on transformation of Exherichia coli with plasmids. J Mol Biol166: 557, 1983. Kurman RJ, Jenson AB, Lancaster WD: Papillomaviruses and cervical neoplasia: reflections on the past, perceptions of the present and speculations for the future. In: Papillomaviruses: Molecular and Clinical Aspects (eds PM Howley, TR Broker) p 3. Alan R. Liss, New York, 1985. Lehn H, Villa LL, Marziona F, Hilgarth M, Hillemans HG, Sauer G: Physical state and biological activity of human papillomavirus genomes in precancerous lesions of the female genital tract. J Gen Virol69: 157, 1985. Lorincz AT, Lancaster WD, Temple GF: Cloning and characterization of the DNA of a new papillomavirus from a woman with dysplasia of the uterine cervix. J Virol.55: 225, 1986. Lorincz AT, Quinn AP, Lancaster WD, Temple GF: A new type of papillomavirus associated with cancer of the uterine cervix. Virology 1.59: 157, 1987. Maniatis T, Fritsch J, Sambrook J: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, New York, 1982. McCance DI: News on papillomaviruses. Nature 335: 765,1988. McCance DJ: News on papillomaviruses. Nature 333: Singer A: Prevalence of human papillomavirus type 16 DNA sequences in cervical intraepithelial neoplasia and invasive carcinoma of the cervix. Br J Obstet Gynaecol 92: 1101, 1985. Meanwell CA, Cox MF, Blacklegde G, Maitland NJ: HPV 16 DNA in normal and malignant cervical epithel-
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ium: implications for the aetiology and behaviour of cervical neoplasia. Lancet i: 703, 1987. Rigby PWJ, Dieckmann M, Rhodes C, Berg P: Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol 113: 237, 1976. Schneider A: Methods of identification of human papillomaviruses. In: Papillomaviruses and Human Disease (eds K Syrjiinen, L. Gissmann. LG Koss) p 19. Springer-Verlag, Berlin Heidelberg New York, 1987. Schwarz E, Freese UK, Gissmann L, Mayer U, Roggenbuch B, Stremlau A, zur Hausen H: Structure and transcription of human papillomavirus sequences in cervical carcinoma cells. Nature 314: 111, 1985. Shirasawa H, Tomita Y, Kubota K, Kasai T, Sekiya S, Takamizawa H, Simizu B: Detection of human papillomavirus type 16 DNA and evidence for integration into the cell DNA in cervical dysplasia. J Gen Virol 67: 2011, 1986. Shirasawa H, Tomita Y, Sekiya S, Takaniizawa H, Simizu B: Integration and transcription of human papillomavirus type 16 and 18 sequences in cell lines derived from cervical carcinoma. J Gen Virol65: 553, 1987. Smotkin D, Wettstein FO: The major human papillomavirus protein in cervical cancers is a cytoplasmic phosphoprotein. J Virol62: 1686,1987. Syrjiinen K, de Villiers EM, Saarikoski S, Castren 0, VPyrynen M, Miintyjarvi R, Parkkinen S: Cervical papillomavirus infection progressing to invasive cancer in less than three years. Lancet i: 510,1985. Syrjgnen K, Mtintyjiirvi R, Parkkinen S, Vtiyrynen M, Saarikoski S, Syrjiinen S, Castren 0: Prospective followup in assessment of the biological behaviour of cervical HPV-associated dysplastic lesions. In: Viral Etiology of Cervical Cancer (eds R Peto, H zur Hausen) p 167. Cold Spring Harbor Laboratory, New York, 1986. Wickenden C, Malcolm ADB, Steele A, Coleman DV: Screening for wart virus infection in normal and abnormal cervices by DNA hybridisation of cervical scrapes. Lancet i: 65, 1985.
Address for reprints: H.G. Kikhel Center of Hygiene and Human Genetics of the University Department of Medical Microbiology Kreuzbergring 57 D-3400 Gttttingen FRG
Int. J. Gynecol. Obstet., 1990,31: 145-152 International Federation of Gynecology and Obstetrics
Occurrence of human papillomavirus DNA types 16 and 18 (HPV-16/l@ in cervical smears as compared to cytological findings H.G. KiicheP, A. Teichmannb,
N. Eckardt”,
P. Arendtb, W. Kuhnb and R. Thomssena
OCenterof Hygiene and Human Genetics of the University, Department of Medical Microbiology, Kreuzbergring 57, D-3400 Giittingen and “Department of Obstetrics and Gynecology of the University, Robert-Koch-Strasse 40, D-3400 Gtittingen (FRG) (Received June 27th, 1988) (Revised and accepted January 30th. 1989)
Abstract
Cervical smears from 2336 women were examined for the presence of HPV-16118 by dot-blot hybridization using 32P-labeled HPV-X6/18 DNA under high stringency conditions. The hybridization data were compared with cytological findings classified according to Papanicolaou. The ages of the patients ranged from under 20 to over 70 years. Ninety-eight (4.40/o) of the 2237 cytologically normal cervical samples (Pap I and II) were HPV-16/18 positive. Thirteen out of 32 (40.6%) samples showing signs of mild and moderate dysplasia (Pap IUD) were found to be HPV-16118positive. In 5 out of 7 (71.4%) samples from women with severe dysplasia or carcinoma in situ (Pap IV) and in 9 out of 25 (32. I Yo) samples from patients with invasive cervical carcinoma (Pap V) HPV-16/18 DNA was detected. Thirty-two smears were from women with severe unspecific cervical inflammation (Pap III). Two (6.2Yo) out of them were HPV-Id/18 positive. Normal smears showed an apparent age-dependent pattern of HPV-16/18 positivity with a peak prevalence of 10.6% among women younger than 20 years old. The majority of premalignant lesions was detected among women younger 0020-7292/90/$03.50
0 1990 International Federation of Gynecology and Obstetrics Published and Printed in Ireland
than 40 years old; whereas all invasive lesions were from women older than 39 years. Compared to the HPV-16/18 prevalence rate in normal smears, abnormal smears harbored HPV-/-16/18 DNA approximately 9 times more frequently. This finding supports the hypothesis that HPV-16/18 may be involved in the development of cervical cancer. Keywords: Cervical smears; Papanicolaou cytology; Cervical dysplasia; Cervical carcinoma; HPV-16/18; Dot-blot hybridization. Introduction
Epidemiologic studies support the involvement of a sexually transmitted agent in cervical carcinogenesis. Several attempts have been made to identify the nature of this infectious agent. Recently, attention has been focused on human papillomaviruses (HPV) as possible candidates. Of more than 50 different HPV types [20], only a few have been identified in tissue originating from cervical and genital lesions. It has been shown by DNA hybridization techniques that HPV-6 and -11 are predominantly present in genital warts and low grade dysplasias Clinical and Clinical Research
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Kochel et al.
[6,12]; whereas HPV-16/18 are associated with the majority of cervical carcinomas and of high grade dysplastic lesions [2,5,8]. In rare cases DNA of HPV-3 1 [17], -33 [l] and -39 [ 181has also been detected. The genomes of HPV-16 and -18 have been usually found to be integrated into the cellular genomes of cancer cells [9] and of cell lines derived from cervical carcinomas [25]. The presence of HPV genomes has also been demonstrated for corresponding metastatic tissue [ 151. In cancer cells the viral DNA is not only transcriptionally active [25,27], but also specific early proteins have been identified [28]. This suggests that the presence of the viral genome and the permanent expression of certain viral functions may be necessary for the induction and maintenance of the malignant phenotype. Because of their association with either benign or malignant cervical lesions the HPV types mentioned above may be classified as low risk (HPVd/ll) and high risk types (HPV-16/18 and other related HPV-types). The early detection of the so-called high-risk types may help to identify women at risk for developing cervical carcinoma. However, up to now an etiological relationship between a HPV-16/18 infection and cervical cancer has not been established. For this purpose we have initiated a follow-up study on a large cohort of patients. In order to specify the number of patients necessary for this followup investigation, we have attempted to determine the prevalence rates of HPV-16/18 in normal and abnormal cervical epithelia. We examined 2336 cervical samples from women between under 20 and over 70 years of age living in and around Gottingen (Northern West Germany). In addition to exfoliative (Papanicolaou) cytology the analysis was performed by dot-blot and in some selected cases by Southern-blot hybridization. The DNA assay and the cytological classification were performed independently (double blind). This report will show that the presence of HPV-16/18 correlates well with cervical dysInt J Gynecol Obstet 31
plasia and carcinoma. It will also demonstrate that HPV-16/18 are widespread in women with normal cytology irrespective of the age of the patients. Materials and methods Sample collection Cervical samples were obtained from women being treated by the outpatient clinic of the University Women’s Hospital of Gottingen for their routine Pap-smear. A combined ecto- and endocervical sample was taken with cotton swabs. After preparation of the Pap-smear the cotton swabs were transferred to 4 ml of phosphate buffered saline (PBS, pH 7.4) in 10 ml conical plastic tubes Nuembrecht, Germany). This (Sarstedt, procedure ensured that the same sample was used for the cytological examination as for hybridization analysis. The samples were either immediately processed or stored overnight at 4 OCprior to processing. Extraction of DNA The plastic tubes containing the swabs were vigorously shaken on a Vortex mixer. The swab was removed and the suspended cervical cells were collected by centrifugation at 3000 x g for 5 min. The supernatant was discarded, the cells resuspended in the remaining PBS and transferred to 1.5 ml Eppendorf tubes. After a second centrifugation step, the PBS was removed and the cells were lysed in 300 ~1 of 50 mM Tris-Cl (pH 7.4), 10 mM EDTA, 50 mM NaCl, 2% SDS in the presence of 200 pg/ml proteinase K. Incubation was at 50°C for at least 2 h. Lysates were extracted with an equal volume of phenol/chloroform After centrifugation the (50: 50, w/v). aqueous supernatants were transferred to new Eppendorf tubes and the DNA was ethanol precipitated. The DNA was pelleted by centrifugation and dissolved in 300 ~1 of TE (pH 7.4). One hundred ~1 of the DNA solutions were stored at - 20°C for further analysis, such as Southern-blot hybridization. The remaining 200 ~1 DNA solution were processed as described below.
HPV-16/18
Probe preparation Human papillomavirus types 16 and 18 DNAs cloned in pBR 322 were kindly provided by Dr. zur Hausen, DKFZ, Heidelberg, Germany. Viral DNA from HPV-16 and HPV-18 was isolated from the plasmids by restriction digestion with BamHI and EcoRI, respectively. HPV DNA was separated from the vector by agarose gel electrophoresis and subsequent electroelution in dialyse bags. DNA was radiolabeled by nicktranslation [23] in the presence of [a32P]dCTP (Amersham, Braunschweig, Germany). Specific activity of the radiolabeled DNA was between 1 and 3 x lo8 cpm/ c(g. For the determination of the amount of cellular DNA a human repetitive DNA sequence was prepared in the following manner. Human DNA extracted from the liver was digested with BamHI and cloned into the BamHI site of pBR 322. E. coli cells were transformed [ 141 and resulting ampicillin resistant bacterial colonies were hybridized in situ with 32P-labeled total human DNA. One positive clone was selected and its plasmid DNA was isolated by routine methods [19]. This plasmid DNA was 32P-labeled and characterized by Southern-blot hybridization against BamHI restricted DNA from humans, guinea pigs and the fungus Candida albicans. The plasmid called pNANB 2/63 reacted specifically with human DNA producing a continuous smear and some minor bands on the autoradiogram. This repetitive DNA was not further characterized. Prior to use, it was 32P-labeled as described above. Dot blot hybridization To the remaining 200 ~1 of the DNA solution l/10 volume of 3 M NaOH was added and the DNA denatured at 60°C for 1 h. After being neutralized by the addition of an equal volume of 2 M ammonium acetate, the DNA was bound to a nitrocellulose membrane (BA 85, Schleicher and Schuell, Dassel, Germany) by means of a manifold filtration device (Minifold SRC 96, Schleicher and
in cervicalsmears
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Schuell, Dassel, Germany). As a control a standard series of HPV-16/18 DNA ranging from 0.5 to 100 pg was additionally applied to the membranes. In order to determine the relative amount of cellular DNA, l/10 aliquots of the DNA solutions were filtered through a separate membrane in the same order. One to 5 pg of DNA from human placenta served as a control. Prior to filtration the membranes were soaked in 1 M ammonium acetate for 10 min. After filtration the membranes were baked for 1 h at 80°C in a vacuum oven and then washed in prewash solution 1191at 42OC for 1 h. Before hybridization, the membranes were prehybridized in 100~l/cm20f 50% formamide, 5 x SSC, 5 x Denhardt’s solution, 0.25% lithium dodecylsulfate and 100 pg/ml of sheared and denatured salmon sperm DNA at 42OC overnight. Hybridization was conducted at 42OC for 36 h in 100 Ccl/cm2of the same mixture described above except that 1 x lo6 cpm/ml of 32Plabeled HPV-16/18 DNA was added. To the filters used for the determination of the amount of human DNA lo5 cpm/ml of the 32P-labeled human repetitive element was added. Southern-blot hybridization Selected samples were also tested for the presence of HPV-16118 DNA by Southernblot hybridization. For this purpose the DNA extracted from cervical cells was digested with Hind111 and subjected to agarose gel electrophoresis. After alkali denaturation and subsequent neutralization, the DNA was transferred onto nitrocellulose membranes and hybridized as described above. After hybridization all filters were subjected to three consecutive washings under stringent conditions (0.5 X SSC, 0.1% SDS, 65OC). Filters were exposed to Kodak XAR X-ray films for l-7 days. Autoradiograms were evaluated by visual inspection and comparison with the standard series. DNA samples which did not hybridize with the repetitive element were excluded from this study. Clinical and Clinical Research
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Results Sensitivity and specificity of the method In order to evaluate the method employed different amounts (0.1-5 pg) of DNA extracted as described above from HeLa cells harboring integrated HPV-18 DNA [25], and from a HPV negative human cell line were applied to four separate nitrocellulose filters. A standard series of each of the HPV types 6, 11, 16 and 18 served as a control. Filters were separately hybridized with purified and 32P-labeled HPV-6, 11, 16 or 18 DNA. Under the described conditions HeLa cell DNA hybridized to HPV-18 only. No crosshybridization was visible between the different HPV types and human cellular DNA. In addition, the different HPV types did not normally hybridize with each other. An exeption was HPV-6 and -11 which did cross-hybridize; this is due to their striking sequence homology. When HPV-16/18 DNAs were hybridized with their homologeous counterparts the detection limit was 0.5 pg. However, when DNA from cervical smears was examined it was often impossible to decide whether those weak hybridization signals were due to small amounts of HPV16118 DNA or to large amounts of cellular DNA. This was especially the case after prolonged exposure of the autoradiograms. Therefore, exposure times were limited to a maximum of 3 days and the samples causing signals equivalent to less than 1 pg of HPV DNA were considered HPV- 16/ 18 negative. HP V-l 6/l 8 prevalence in cervical smears
Of the 2336 cervical smears which were examined for the presence of HPV-16/18, 2237 appeared cytologically normal (Pap I/ II). Ninety-eight (4.4%) of these were HPV16118 positive (Table I). The distribution of HPV- 16/l 8 positive cases within the normal group seemed to be age-dependent. The highest prevalence rates were found among women of less than 20 (10.6%) and between 20 and 29 years of age (7.7%) (Table II). The Int J Gynecol Obstet 31
Table 1.
Prevalence of HPV-16/18 in relation to cervical cytology (N,, number of investigated smears; N+ , number of HPV-16/18 positive smears; %total, percentage of N, in relation to the total number of investigated smears; %N,, percentage of smears positive for HPV-16118). Cytological diagnosis
HPV-16/18 prevalence
Pap
N
%total
I/II
III IUD IV V
2237 32 32 I 28
95.1 1.4 1.4 0.3 1.2
Total
2336
N+
%N,
98 2 13 5 9
4.4 6.2 40.6 71.4 32.1
127
5.4
other age groups showed a relatively uniform distribution of HPV-16118 positivity ranging from 2.5 to 4.2%. Thirty-two women had signs of mild or moderate dysplasia (Pap IIID). Thirteen (40.6%) were HPV-16/18 positive. Seven women had severe dysplasia or carcinoma in situ (Pap IV); HPV-16/18 being detected in 5 women (71.4%). Twentyeight smears came from cancer patients (Pap V); 9 of them (32.1%) were associated with HPV-16/18. As a control we extracted DNA as described above from 17 cervical carcinoma biopsies and found 10 (59%) positive for HPV-16/18 (not shown in tables). The age distribution of cervical carcinoma patients differed from that of women with premalignant lesions. Pap IIID and IV were most common among women between under 20 and 39 years of age; whereas all Pap V smears were from women over 39 years of age. The category Pap III included smears from women suffering from severe unspecific cervical inflammation or a cervical carcinoma which could not definitively be identified from Pap smears. One hundred four samples from women between 20 and 29 years of age (same cohort) were also tested for the presence of HPVd/ 11. Ninety women had normal smears; eleven were HPV-16/18 and 1 HPV-6/11 positive.
HPV-16/18
Cytological findings and HPV-16/18 prevalence in relation to age of the women (N, number of smears; N + positive smears; % + , percentage of smears positive for HPV-16118).
Table II. HPV-16/18 Age
in cervicalsmears
Pap I/II
Pap IV
149
, number of
Total
Pap V
Pap III
Pap IIID
N
N+
N
N+
%+
N
N+
N
N+
%+
N
N-k
% +
-
4 11
2 6
50.0 54.5
4
3
-
-
-
71 343
9 34
12.6 9.9
-
N
N+
%+
70
229
f
3.1
8
2
-
-
1
-
10
4
40.0
248
13
5.2
-
Four samples contained cells consistent with mild or moderate dysplasia; three harbored HPV-16/18 and one HPVd/l 1. One patient with severe dysplasia was neither HPV-16/18 nor HPVd/l 1 positive. In total 13% of women of this patient group were HPV-16118 and 2% HPV-6/11 positive (not shown in tables). Southern-blot analysis Because dot-blot hybridization cannot provide information on the physical state of the viral DNA, selected samples from different Pap-groups were additionally examined by Southern-blot hybridization. However, a detailed analysis was precluded by limited amounts of material. Extracted cellular DNA was digested only with the restriction enzyme Hind111 which does not cleave either HPV-16 or -18. If the HPV-DNA has become integrated into the cellular genome, the digestion with Hind111 will result in autoradiographic bands appearing above the position of the monomeric episomal form II (open circular). Otherwise, the three isomeric forms of episomal HPV DNA should be visible. However, if only Hind111 is used, integrated DNA and oligomerized forms of DNA episomal cannot be reliably distinguished. In all 12 samples which were examined, episomal forms of HPV-16/18 DNA were
present (Fig. 1, FI, covalently closed circular; FII, open circular; FIII, linearized form). Additional high molecular weight bands which may be indicative of integrated viral DNA were almost exclusively detected in premalignant and malignant lesions (Pap IIID, IV, V, Fig 1, lanes 5, 6, 8, 10, 11, 12). Viral DNA of high molecular weight was also found in a Pap III smear from a women 70 years of age (Fig. 1, lane 3). Discussion
The presence of HPV-16/18 DNAs in cervical cancer cells cannot provide evidence of an etiological role for these viruses in the development of cervical carcinoma. The possibility that they are passengers rather than causative agents cannot be excluded. We have therefore initiated a follow up study which will attempt to answer the question whether papillomaviruses are able to induce and maintain transformation of the normal cervical epithelium. First, the prevalence rate of HPV-16/18 in cervical smears from women of different age groups was examined in relation to their cytological classification. Using dot-blot hybridization for this purpose, we found an overall HPV-16/18 positivity of 5.4% irrespective of the cytological diagnosis. When correlated to different age groups, the HPV- 16/l 8 prevalence rate Clinical and Clinical Research
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Kochel et al.
-FII -FIII
--FI
Fig. 1. Southern-blot analysis of HPV-16/18 DNA sequences in cervical smears of different Pap groups. DNA samples were digested with Hind111 and separated on a 0.7% agarose gel, transferred to nitrocellulose, and hybridized under stringent conditions as described in Materials and methods. Lanes 1 and 2, Pap II; lanes 3 and 4, Pap III; lanes S-10, Pap IIID; lane 11, Pap IV; lane 12, Pap V; lane 13, positive control, i.e. 20 pg of covalently closed circular plasmid DNA consisting of HPV-16 DNA and pBR 322. HPV-16/18 specific signals were visualized after exposure to X-ray film for 7 days. Positions of the monomeric episomal forms (FI, FII, FIII) of HPV-16/18 genomes are indicated by arrows.
appeared to be age dependent. Women younger than between 20 and 29 years of age showed seemingly higher HPV- 16/ 18 infection rates than older women. This higher rate of HPV-16/18 positivity among young women correlates well with the prevalence rates of HPV in penile swabs from healthy men [ 131. Highest positivity rates (7.5%) were found among men between 16 and 35 years old; whereas among older men the HPV positivity declined to 1% . In cases of dysplasia the HPV-16/18 prevalence rates seemed to increase with the severity of the lesions (Pap IIID, 40.6%; Pap IV, 7 1.4%) but the difference was not statistically significant. However, other reports also describe a higher prevalence of HPV-16/18 in more severe dysplastic lesions [4,30]. This appears to support the hypothesis of an increase in HPV-16/18 prevalence with increasing lesion severity. Int J Gynecol Obstet 31
It has been demonstrated by molecular hybridization studies that about 65% of cervical carcinoma biopsies contained HPV- 16/ 18 DNA with some geographical fluctuations [ 111. In this study only 32.1% of smears from cancer patients were HPV- 16/l 8 positive, because smears from cervical carcinomas may be inappropriate for the detection of HPV due to the heterogeneous cell composition of the tumor and/or low copy numbers of HPV DNA in cancer cells [22]. This inadequacy is supported by the finding that approximately 60% of carcinoma biopsies which we examined were HPV- 16/ 18 positive. Previous studies dealing with the HPV-16/ 18 prevalence rates were mostly based on small numbers of patients and different hybridization techniques. However, de Villiers et al. [7] recently published a corresponding report on more than 9000 patients. Although they used the filter in-situ hybridi-
HPV-16/18 in cervicalsmears
zation method and pooled their HPV-6/11 and HPV-16/ 18 data, there are remarkable concordances between their results and ours. In particular, they described the same agedependent pattern of HPV positivity among women with normal smears. In agreement with our results premalignant and malignant lesions showed no consistent age-dependent pattern of HPV positivity. Without further documentation they also mentioned that HPV-16/18 were more prevalent in cervical smears than HPV-6/11. The infection rates for HPV-16/18 and HPV-6111 which we found in a small group of women aged between 20 and 29 years were 13% and 2%) respectively. However, these data have to be substantiated by screening larger groups of patients. De Villiers et al. [7] emphasized that their results probably underestimate the real HPV16/ 18 infection rate by a factor of 2 to 3. This could also be true for our examination. The problems regarding the sensitivity and applicability of the different hybridization methods have been widely discussed by Schneider 1241 and Gissmann et al. [lo]. The 9 times higher HPV- 16/ 18 positivity of abnormal smears in comparison to normal smears implies, but does not necessitate, that HPV-16/18 may be involved in the development of cervical carcinoma. Namely, the possibility that HPV DNA is more readily detected in abnormal than in normal tissue or that abnormal tissue is more prone to HPV infections cannot be disregarded. Therefore, a general screening of cervical scrapes for HPV infections as suggested by some authors [3,31] is not required at present. However, other authors observed a difference in the progression rate of mild dysplastic lesions to severe lesions depending on the HPV type [4,29,30]. Progression rates have been higher among women infected with HPV-16/18 than among those in whom HPV6111 was present. Whether the presence of HPV-16/18 DNA in cervical cells especially of its integrated form in premalignant lesions, which was described in this and in other
151
reports [16,26], represents a valuable diagnostic parameter remains to be defined. To establish the etiological relationship between HPV infections and cervical cancer, large prospective cytologic, colposcopic and virologic studies are necessary. However, the assessment of this relation will be made more difficult by the high HPV-16/18 prevalence rates among young women with normal cytology, particularly if an underestimation of the actual infection rate by a factor of 2 to 3 [7] is assumed. Acknowlegdment This study was supported by the BtittnerStiftung of the University of Gottingen. References Beaudenon S, Kremsdorf D, Croissant 0, Jablonska S, Wain-Hobson S, Orth G: A novel type of papillomavirus associated with genital neoplasias. Nature 321: 246, 1986. Boshardt M, Gissmann L, Ikenberg H, zur Hausen H: A new type of papillomavirus DNA, its presence in genital cancer biopsies and in cell lines derived from cervical cancer. EMBO 53: 1151, 1984. Burk RD, Kadish AS, Calderin S, Romney SL: Human papillomavirus infection of the cervix detected by cervicovaginal lavage and molecular hybridization: correlation with biopsy results and Papanicolaou smear. Am J Obstet Gynecoll54: 982, 1986. Campion MJ, Cuzich J, McCarice DJ, Singer A: Progressive potential of mild cervical atypia: Prospective cytological, colposcopic and virological study. Lancet ii: 937, 1986. Crum CP, Ikenberg H, Richart RM, Gissmann L: Human papillomavirus type 16 and early cervical neoplasia. N Engl J Med 310: 550, 1984. De Villiers EM, Gissmann L, zur Hausen H: Molecular cloning of viral DNA from genital warts. J Viral 40: 932, 1981. De Villiers EM, Schneider A, Miklaw H, Papendick U, Wagner D, Wesch H, Wahrendorf J, zur Hausen H: Human papillomavirus infections in women with and without abnormal cervical cytology. Lancet ii: 703, 1987. Dilrst M, Gissmann L, Ikenberg H, zur Hausen H: A new type of papillomaviral DNA from cervical carcinoma and its prevalence in cancer biopsies from different geographic regions. Proc Nat1 Acad Sci (Wash) 50: 3512, 1983. Diirst M, Kleinheinz A, Hotz M, Gissmann L: The physical state of human papillomavirus type 16 DNA in benign
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Kochel et al. and malignant genital tumours. J Gen Virol 66: 1515, 1985. Gissmann L, Diirst M, Oltersdorf T, von Knebel Doeberitz M: Human papillomaviruses and cervical cancer. In: Cancer Cells 5 (eds BM Steinberg, JL Brandsma, LB Taichman) p 275. Cold Spring Harbor Laboratory, New York, 1987. Gissmann L, Schneider A: Human papillomavirus DNA in preneoplastic and neoplastic genital lesions. In: Viral Etiology of Cervical Cancer (eds R Peto, H zur Hauseri) p 217. Cold Spring Harbor Laboratory, New York, 1986. Gissmann L, Wolnik L, Ikenberg H, Koldovsky U, Schnilrch HG, zur Hausen H: Human papillomavirus types 6 and 11 DNA sequences in genital and laryngeal papillomas and in some cervical cancer biopsies. Proc Nat1 Acad Sci (Wash.) 50: 560,1983. Grussendorf-Conen EL, de Viliiers EM, Gissmann L: Human papillomavirus genomes in penile smears of healthy men. Lancet ii: 1092, 1986. Hanahan D: Studies on transformation of Exherichia coli with plasmids. J Mol Biol166: 557, 1983. Kurman RJ, Jenson AB, Lancaster WD: Papillomaviruses and cervical neoplasia: reflections on the past, perceptions of the present and speculations for the future. In: Papillomaviruses: Molecular and Clinical Aspects (eds PM Howley, TR Broker) p 3. Alan R. Liss, New York, 1985. Lehn H, Villa LL, Marziona F, Hilgarth M, Hillemans HG, Sauer G: Physical state and biological activity of human papillomavirus genomes in precancerous lesions of the female genital tract. J Gen Virol69: 157, 1985. Lorincz AT, Lancaster WD, Temple GF: Cloning and characterization of the DNA of a new papillomavirus from a woman with dysplasia of the uterine cervix. J Virol.55: 225, 1986. Lorincz AT, Quinn AP, Lancaster WD, Temple GF: A new type of papillomavirus associated with cancer of the uterine cervix. Virology 1.59: 157, 1987. Maniatis T, Fritsch J, Sambrook J: Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, New York, 1982. McCance DI: News on papillomaviruses. Nature 335: 765,1988. McCance DJ: News on papillomaviruses. Nature 333: Singer A: Prevalence of human papillomavirus type 16 DNA sequences in cervical intraepithelial neoplasia and invasive carcinoma of the cervix. Br J Obstet Gynaecol 92: 1101, 1985. Meanwell CA, Cox MF, Blacklegde G, Maitland NJ: HPV 16 DNA in normal and malignant cervical epithel-
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Address for reprints: H.G. Kikhel Center of Hygiene and Human Genetics of the University Department of Medical Microbiology Kreuzbergring 57 D-3400 Gttttingen FRG
