GYNECOLOGIC

ONCOLOGY

41, 129-134 (1991)

Detection of Human Papillomavirus Genotypes in Cervical Neoplasia from Korean Women Using Polymerase Chain Reaction’ SUNG EUN NAMKOONG, M.D.,* HUN YOUNG LEE, M.D.,* SEUNG Jo KIM, M.D.,* RICHARD W. DANIEL, B.S. ,t KEERTI V. SHAH, M.D., DR.PHt

JONG SUP PARK, M.D.,*,? *Department

of Obstetrics and Gynecology, Catholic University Medical College, Seoul, Korea; and TDepartment of Immunology Disease, School of Hygiene and Public Health, The Johns Hopkins University, Baltimore, Maryland, 21205

and Infectious

Received October 30. 1990

The histopathologic features of cervical dysplasia (20 cases) and cervical carcinoma (15 cases) in Korean women were correlated with the presence of human papillomaviruses (HPVs) as determined by polymerase chain reaction technology on paraffinprocessed specimens. A segment of the E6 open reading frame of several HPV types (HPV 6, 11, 16, 18, 31, 33, and 35) was amplified using primers which were synthesized to contain 50% G + C, in order to give optimum annealing for the amplification of HPVs. All specimens were also tested for amplification of the cellular j?-globin gene. HPV was found in 19 (54%) cases. The HPV types were HPV 6 in 1 case (3%), HPV 16 in 15 cases (43%), HPV 18 in 2 cases (6%), and HPV 33 in 3 cases (9%). HPV was identified in 16 of 25 (64%) /3-globin-positive and 3 of 10 (30%) /!l-globin-negative tissues. HPV types 11, 31, and 35 were not detected in any of the specimens. HPV 6, 18, and 33 were detected only in preneoplastic lesions, and co-infection with these three viruses was observed in one case of severe dysplasia. HPV type 16 was found in 8 (40%) premalignant lesions and in 7 (47%) invasive carcinomas. These data indicate that HPVs are found along the entire spectrum of cervical neoplasia. HPV 16 was the predominant virus in invasive cancer of the cervix. o 1~91 Academic

33, and 35, have been found to be the predominant viruses in severe dysplasia and cervical carcinoma [4,5]. Numerous studies have examined DNAs extracted from exfoliated cervical cells or from fresh frozen tissues for the presence of HPVs. Viral sequences have been detected in 40 to 90% of the specimens of dysplasia and invasive cervical cancer [6-81. The newly developed, highly sensitive polymerase chain reaction (PCR) allows in vitro enzymatic amplification of specific DNA sequences in clinical samples [9]. This technology has been successfully applied to the study of formalin-fixed archival material to detect viruses in routinely processed pathological specimens [lo]. We report the results of HPV detection in pathological tissues from Korean women using PCR amplification of a segment of the E6 ORF of the virus and employing primers with 50% G + C content. The cervical tissues spanned a pathological spectrum from mild dysplasia to squamous cell carcinoma.

Press, Inc.

MATERIALS

AND METHODS

INTRODUCTION

Tissue Samples During the last decade, the association of human papillomavirus (HPV) infections with the development of genital cervical cancer has been firmly established [l-3]. More than 20 of the 60 distinct HPV types identified infect the genital tract. As a rule, HPV 6 and 11 have been associated with benign and low-grade cervical lesions while HPV 16 and 18, and to a lesser extent HPV 31, ’ This work was supported in part by U.S. Public Health Service Grant PO1AI16959 from the National Institute of Allergy and Infectious Diseases.

Tissue samples were obtained from 20 preinvasive (5 mild dysplasia, 5 moderate dysplasia, 5 severe dysplasia, and 5 carcinoma in situ), 5 microinvasive, and 10 invasive squamous cell cervical carcinomas seen at the Catholic University Medical Center, Seoul, Korea, between March 1987 and March 1989. The ages of patients at the time of diagnosis ranged from 31 to 69 years. The mean age of patients with preinvasive lesions was 44 years as compared to 56 years for those with invasive lesions. Colposcopically directed biopsies and surgically excised spec129 0090~8258/91 $1SO Copyright 0 1991by AcademicPress,Inc. All rights of reproductionin any form reserved.

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ET AL.

TABLE 1 Oligonucleotide Primers and Probes for PCR with HPVs G/C content Nucleotide

sequence

(%)

Amplimer length (base pair)

Primer HPV HPV HJ’V HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV HPV

6-l 6-2 11-l 11-2 16-1 16-2 18-l 18-2 31-1 31-2 33-l 33-2 35-l 35-2

CACCTAAAGGTCCTGTITCG CGGTITGTGACACAGGTAGC GTI-GCTTAGAACTGCAAGGG CGGCTTGTGACACAGGTAAC ACAG’ITACTGCGACGTGAGG TITG’ITCAGGACACAGTGGC TATACCGCATGCTGCATGCC ACGG’I-ITCTGGCACCGCAGG ACACCACACGGAGTGTGTAC ACACAACGGTCI-ITGACACG ACCTITGCAACGATCTGAGG GAACCGCAAACACAGTITAC AGCGGAGTGAGGTATATGAC ATACACCGACCTGTCCACCG

HPV HPV HPV HPV HPV HPV HPV

6 11 16 18 31 33 35

AGGCGGCTATCCATATGCAG GCTGCATATGCACCTACAGT GAGATGGGAATCCATATGCT TTCAGACTCTGTGTATGGAG TGTGTATGGAACAACA-ITAG CCAAATGGATITCCCI CTCT CCACCGATGTTATGGAATCG

50 55 50 55 55 50 55 65 55 50 50 45 50 50

183 134 239 157 162 103 298

Probe

Note. PCR, polymerase

chain reaction;

55 50 45 45 35 45 50

HPV, human papillomavirus.

imens were fixed in neutral phosphate-buffered formalin immediately and were embedded in paraffin. Pathologic diagnosis was made by standard criteria.

water to a 100~~1volume. Type-specific primers were designed to amplify a segment of the E6 region of HPVs and to have approximately 50% G + C content, to provide the optimal annealing condition. The primers amplified 103-298 base pair regions of the E6 open reading Polymerase Chain Reaction with Type-Specific frame (ORF) of HPV 6, 11, 16, 18, 31, 33, and 35 (Table E6 Primers 1). Four sets of amplification reactions were performed To prevent contamination, three physically separate for each specimen: (1) with human P-globin primers [ll] areas were used for the performance of the tests: one for to evaluate the suitability of the DNA for amplification, preparing stock reagents and the master mix, one for (2) with a mixture of HPV 6 and 11 primers, (3) with a processing specimens, and one for processing the ampli- mixture of HPV 16 and 18 primers, and (4) with a mixture fied reaction products. Ten-micrometer sections of par- of HPV 31, 33, and 35 primers. The type-specific oligonuaffin blocks were cut by microtome and placed in Ep- cleotide probes for the E6 PCR products were 20 nupendorf tubes. The sections were deparaffinized with cleotides long and were designed to correspond to unique octane and washed twice with ethanol. The specimens regions in the respective amplified HPV DNA sequences. were resuspended in PCR extraction buffer containing As positive controls for HPVs, 1 pg of HPV plasmid and 100 pg/ml proteinase K (Boehringer-Mannheim), incu- paraffin sections of SiHa (HPV 16) and HeLa (HPV 18) bated at 37°C overnight, and boiled for 7 min to deactivate cell pellets were employed. For positive control for /3proteinase K. Ten microliters of the supernatant was used globin, amounts of placental DNAs equivalent to lo3 and for each polymerase chain reaction. Reaction solutions lo5 copies were used. Five tubes containing only PCR contained templates of tissue samples or of control DNAs, reaction mixture were used in each run for negative con200 PM dNTPs, 50 mM KCl, 1.5 mM MgCl*, 0.01% trols. Forty-five cycles were performed with the following gelatine w/v, 10 mM Tris-HCl, pH 8.3, and oligonu- cycle profile: denaturation at 94°C for 1 min, annealing cleotide primers each at a concentration of 0.2 PM, 2.5 at 45°C for 1 min, extension at 72°C for 1 min. Ampliunits of thermostable Thermus aquaticus (Taq) polymer- fication products were electrophoresed on 3% Nusieve ase (Perkin-Elmer-Cetus Instruments), and distilled and 1% agaiose gel and visualized after ethidium bromide

HPVs IN CERVICAL

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NEOPLASIA

specimens by E6 type-specific primers. The PCR products of the HPV plasmids after gel electrophoresis and ethidium bromide staining are shown in Fig. 1. The bands were of the expected specific sizes (Table 1) and it was possible to differentiate the HPV types by the lengths of 123 the reaction products. We performed cellular p-globin bo gene amplification from the digested specimen to test the suitability of the tissue DNA for PCR. The PCR products were tested by hybridization in a slot blot format with a labeled oligomer probe (Fig. 2). Amplification of the pglobin gene was achieved in 25 of the 35 paraffin sections (71%). Tissues from 35 patients, consisting of 20 cases of dysplasia and 15 cases of invasive carcinoma of the cervix, were studied by PCR for HPV types 6, 11, 16, 18, 31, 33, and 35. HPV DNA could be amplified in 64% (16 of 25) of the tissues from which @-globin gene was amplified and in 30% (3 of 10) of the tissues from which @globin FIG. 1. PCR products after gel electrophoresis and ethidium brogene could not be amplified (P < 0.01) (Table 2). Thus, mide staining. G + C DNA content adjusted primers were designed to amplify a part of the E6 open reading frame for HPVs (Table 1). the samples that failed to give globin amplification were a

b

c

d

e

f

g

h

As positive controls, 1 pg of each HPV DNA was used: Lane a, size marker (123 bp); lane b, HPV 6 (183 bp); lane c, HPV 11 (134 bp); lane d, HPV 16 (239 bp); lane e, HPV 18 (157 bp); lane f, HPV 31 (162 bp); lane g, HPV 33 (103 bp); lane h, HPV 35 (298 bp). Lighter bands in some of the lanes are primer dimers.

staining under uv light. The DNA in the gel was denatured with 0.5 M NaOH and 1.5 M NaCl for 1 hr and neutralized with 1 M Tris-HCl (pH 8.0) for another 1 hr and then transferred to a nylon filter. The products were also tested in the slot blot format without prior electrophoresis. Five microliters of PCR product was denatured in 195 ~1 of 0.5 M NaOH, 25 mM EDTA and was transferred to filters with a slot blot apparatus (Minifold II, Schleicher & Schuell). The blotted DNA was fixed to the filter by exposure to a uv transilluminator for 2 min. The filter was then prehybridized for 1 hr at 42°C in 3 x SSPE, 5 x Denhardt’s solution, 0.5% SDS, and 100 pg/ml tRNA. The products were identified by hybridization with 32P end-labeled virus-specific oligonucleotide probes at 42°C for 1 hr. The filter was washed in succession with 1 x SSPE, 0.1% SDS solution, three times for 5 min at room temperature, and with 5 x SSPE, 0.1% SDS, for 10 min at 60°C. Autoradiography was done at -70°C with Kodak X-AR film overnight.

A

B

C

10 11

GLOBIN

12 13 14 15 16 17 18 19

RESULTS The optimal conditions for PCR were determined by amplifying 1 pg of each HPV DNA with 2.5 units of Taq polymerase and different concentrations of MgC12, dNTPs, and primers. On the basis of the results of the tests, concentrations of 1.5 mM MgCl* and 200 pM dNTPs and primers at 0.2 pM were employed for testing the

20

FIG. 2. Human P-globin PCR on paraffin-embedded tissues. The reaction products from 35 CIN and invasive cancer of cervical tissues (lane A, tissues l-20; lane B, tissues 21-35) were hybridized against a “P end-labeled oligomer probe by slot blot. After autoradiography, the positive controls (lane C: slot 1, lo3 copies and slot 4, lo5 copies of human placental DNA) and 25 of the 35 paraffin sections were positive with strong signals.

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TABLE 2 Correlation between j%Globin and HPV DNA Amplification by Polymerase Chain Reaction in 35 Paraffin Sections from Cervical Neoplastic Lesions Number of positive HPV DNA amplification (%I

Number of tissues (tested)

/3-Globin gene amplification Positive Negative

25 10

16 (W

Total

35

19 (54)

3 (30)

Note. HPV, human papillomavirus.

not necessarily negative for HPV amplification. HPV was detected in 19 of 35 patients (54%) (Table 3). Of the 20 cases of dysplasia, 12 were positive for HPVs; 1 case for HPV 6, 8 cases for HPV 16 (Fig. 3), 2 cases for HPV 18, and 3 cases for HPV 33. One case of dysplasia was positive for three HPV types, 6, 18, and 33. HPV 16 was the only type recovered from carcinoma tissues and was found in 7 of 15 cancers: in 3 of 5 cases of microinvasive carcinoma and in 4 of 10 cases of invasive carcinoma. As compared to other HPV types, HPV 16 was present in specimens spanning the entire histologic spectrum of cervical neoplastic lesions, from mild dysplasia to invasive cervical carcinoma. Specific HPV sequences were routinely identified in positive controls of SiHa cells, HeLa cells, and plasmid HPV DNAs. No HPV sequences were identified in the negative controls. DISCUSSION Cervical cancer is the most common female genital malignancy in Korea as it is in many Asian and Latin Amer-

FIG. 3. Slot and Southern blot hybridization of PCR products using HPV 16 E6 type-specific primers. PCR products of 35 tissue samples of CIN and invasive cancer of the cervix were transferred to filters by slot blot (lane A, tissues l-20; lane B, tissues 21-35) and Southern blot (lane I, tissues l-10; lane II, tissues 11-20; lane III, tissues 2130; and lane IV, tissues 31-35). Hybridization to HPV 16 can be seen in 15 of 35 cases. Positive controls: PCR products from paraffin section of SiHa cell line (1-2 copies of HPV 16 DNA per cell) (v) and 1 pg of HPV 16 plasmid ( J ). Corresponding Southern blots are shown in the two panels on the right. Doublets in some of the lanes probably represent single and double strands of amplified target DNA.

ican countries. The association of human papillomaviruses with cervical cancer has been extensively studied in recent years [12,13]. There is abundant evidence that some HPV types are preferentially distributed in cervical cancers, whereas almost all HPV types are found in low-grade dysplasia [14,15]. HPV 16 and 18 are the most predom-

TABLE 3 HPV Types and Severity of Lesions in the Neoplastic Tissues of Uterine Cervix from Korean Women Distribution of HPV type Number of HPV positive

Diagnosis

11

16

18

31

33

35

Mild dysplasia (n = 5) Moderate dysplasia (n = 5) Severe dysplasia (n = 5) Carcinoma in situ (n = 5)

2 3 4” 3

0 0 0 0

1 3 1 3

1 0 1 0

0 0 0 0

0 0 3 0

0 0 0 0

Microinvasion (n = 5) Invasive cancer (n = 10)

3 4

0 0

3 4

0 0

0 0

0 0

0 0

19

0

15

2

0

3

0

Total

35

’ Three HPV types were amplified from one case. Note. HPV, human papillomavirus.

HPVs IN CERVICAL

inant types in invasive cervical cancer and together are found in 40 to 70% of the cases. This prevalence suggests that HPV 16 and HPV l&associated lesions progress from dysplasia to carcinoma. In this study of Korean cases of dysplasia and invasive cancer of the cervix, 54% of the cases were positive for HPV DNA by PCR. This detection rate is comparable to the results of previous studies which employed different techniques [3-61. It is noteworthy that HPV 18 and HPV 33 were found in dysplastic lesions, but not in invasive tissues. HPV 16 was found across the entire spectrum of histologic lesions from mild dysplasia to invasive carcinoma and was the only type recovered from invasive cancer of the cervix. These findings suggest that HPV 16associated dysplasia in Korean women is the precursor of cervical cancer. Until recently, HPV typing has been done by Southern hybridization, which requires relatively large amounts of fresh tissue [7,8]. In addition, HPV diagnosis has been made by in situ hybridization of paraffin sections of fixed tissues [16-181. However, both of these techniques are insensitive and labor intensive. The newly developed PCR technology is exquisitely sensitive because target DNA sequencesin the test sample are selectively amplified a million-fold or more by repeated cycles of enzyme-directed extension of primers. In our study, an estimated lo3 SiHa cells in paraffin sections (equivalent to about lo3 HPV 16 genomes) were routinely positive for HPV 16. The type-specific primers were designed to amplify different lengths of the E6 open reading frame of the HPVs to allow differentiation of the viral types by gel electrophoresis. However, we found that hydridization was necessary for definitive identification of the viral types. The specific sequences for primers were selected to contain about 50% G + C in order to optimize the annealing reaction. We chose to amplify a short segment (103-298 base pair) of the E6 ORF because the longer segment (450 base pair) of Ll ORF is not efficiently amplified from fixed paraffin-processed material [ 191. We used the PCR to amplify human P-globin gene to evaluate the accessibility of the DNA in the paraffinembedded tissues for amplification. Amplification of globin gene was possible in 71% of the tissues. The samples that failed to give P-globin amplification were not necessarily negative for HPV. Thus, HPV was detected in 30% of tissues from which globin gene could not be amplified, as compared to from 64% of the tissues from which globin gene was amplified. In limited studies, the lack of globin amplification could not be attributed to the presence of inhibitors in sample extracts; addition of globin-negative extracts did not inhibit the amplification of a A genomic sequence. Some archival materials may be unsuitable for DNA amplification. It is known that time

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between surgical removal and fixation, type of fixative, duration of fixation, length of storage, and levels of nuclease in the different tissues affect the accessibility of DNA in fixed tissue [20-231. We used a simple proteinase K extraction procedure on a single section to process the tissue for amplification. Tissue DNA purified by phenolchloroform extraction may be expected to improve the accessibility of the tissue DNA for amplification but we avoided this extraction procedure in order to reduce the possibility of contamination. The results of this study show that HPV 16 is the virus predominantly associated with cervical carcinoma in Korean women. The PCR technology was useful in retrospective diagnosis of routinely collected pathologic material. Analysis of routinely collected, well-characterized pathological specimens by HPV PCR is likely to contribute to the understanding of the role of HPVs in the development of cervical cancer. REFERENCES zur Hausen, H. Papillomaviruses as carcinomaviruses, Adv. Viral Oncol. 8, 1-26 (1989). zur Hausen, H. Papillomaviruses in anogenital cancer as a model to understanding the role of viruses in human cancers, Cancer Res. 49, 4677-4681 (1989). Shah, K. V., and Howley, P. M. Papihomaviruses, in Virology (B. N. Fields and D. M. Knipe, Eds.), Raven Press, New York, pp. 1651-1675 (1990). zur Hausen, H., and Schneider, A. The role of papillomaviruses in human anogenital cancer, in The papovaviridae. 2. The papillomaviruses (N. P. Salzmann and P. M. Howley, Eds.), Plenum, New York, pp. 245-263 (1987). Jenson. A. B.. and Lancaster. W. D. Association of human papillomaviruses with benign, premahgnant, and malignant anogenital lesions, in Human papillomaviruses and human cancer (H. Pfister Ed.), CRC Press, Boca Raton, FL, pp. 11-43 (1990). 6. de Vilhers, E. M., Wagner, D., Schneider, A., et al. Human papillomavirus infections in women with and without abnormal cervical cytology, Lancer 2, 703-706 (1987). 7. Reid, R., Greenberg, M., Jenson, A. B., et al. Sexually transmitted papillomaviral infections. I. The anatomic distribution and pathologic grade of neoplastic lesions associated with different viral types, Am. 1. O&et. GynecoL 156, 212-222 (1987). 8. Lorincz, A. T., Temple, G. F., Kurman, R. J., Jenson, A. B., and Lancaster, W. D. Oncogenic association of specific human papillomavirus types with cervical neoplasia, J. Natl. Cancer Inst. 79, 671-677 (1987). 9. Saiki, R. K., Gelfand, D. H., Stoffel, S., et al. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase, Science 239, 487-491 (1988). 10. Shibata, D. K., Arnheim, N., and Martin, W. J. Detection of human papillomavirus in paraffin-embedded tissue using the polymerase chain reaction, J. Exp. Med. 167, 225-230 (1988). 11. Saiki, R. K., Scharf, S., Faloona, F., et al. Enzymatic amplification of P-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia, Science 230, 1350-1354 (1985). 12. Schwarz, E., Freese, K. U., Gissmann, L., et al. Structure and I.

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transcription of human papillomavirus sequences in cervical cancer cells, Nuture 314, 111-114 (1985). 13. Fuchs, P. G., Girardi, F., and Pfister, H. Human papillomavirus DNA in normal, metaplastic, preneoplastic and neoplastic epithelia of the cervix uteri, Ztrt. J. Cancer 41, 41-45 (1988). 14. zur Hausen, H. Papillomaviruses in human cancer, Cancer 59,16921696 (1987). 15. Shah, K. V., and Buscema, J. Genital warts, papillomaviruses, and genital malignancies, Ann. Rev. Med. 39, 371-379 (1988). 16. Stoler, M. H., and Broker, T. R. In situ hybridization detection of human papillomavirus DNAs and messenger RNAs in genital condylomas and a cervical carcinoma, Human Puthol. 17, 1250-1258 (1986). 17. Nagai, N., Nuovo, G., Friedman, D., and Crum, C. P. Detection of papillomavirus nucleic acids in genital precancers with the in situ hybridization technique, Int. J. Gynecol. Pathol. 6,366-379 (1987). 18. Gupta, J. W., Saito, K., Saito, A., Fu, Y. S., and Shah, K. V. Human papillomaviruses and the pathogenesis of cervical neoplasia. A study by in situ hybridization, Cancer 64, 2104-2110 (1989).

19. Park, J. S., Jones, R. W., McLean, M. R., et al. Possible etiologic heterogeneity of vulvar intraepithelial neoplasia: A correlation of pathologic characteristics with human papillomavirus detection by in situ hybridization and polymerase chain reaction, Cancer 67, 1599-1607 (1991). 20. Goelz, S. E., Hamilton, S. R., and Vogelstein, B. Purification of DNA from formalin fixed and paraffin embedded human tissue, Biochem. Biophys. Res. Commun. 130, 118-126 (1985). 21. Dubeau, L., Chandler, L. A., Gralow, J. R., Nicholas, P. W., and Jones, P. A. Southern blot analysis of DNA extracted from formalin-fixed pathology specimens, Cancer Res. 46,2964-2969 (1986). 22. Nuovo, G. J., and Silverstein, S. J. Comparison of formalin, buffered formalin, and Bouin’s fixation on the detection of human papillomavirus deoxyribonucleic acid from genital lesions, Lab. Znvest. 59, 720-724 (1988). 23. Bramwell, N. H., and Burns, B. F. The effects of fixative type and fixation time on the quantity and quality of extractable DNA for hybridization studies on lymphoid tissue, Exp. Hematol. 16, 730732 (1988).

Detection of human papillomavirus genotypes in cervical neoplasia from Korean women using polymerase chain reaction.

The histopathologic features of cervical dysplasia (20 cases) and cervical carcinoma (15 cases) in Korean women were correlated with the presence of h...
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