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Human papillomavirus deoxyribonucleic acid in cervical carcinoma from primary and metastatic sites
Human papillomavirus deoxyribonucleic acid in cervical carcinoma from primary and metastatic sites Wayne D. Lancaster, Ph.D., Carlos Castellano, M.D., Carlos Santos, M.D., Gregorio Delgado, M.D., Robert J. Kurman, M.D., and A. Bennett Jenson, M.D. Washington, D. C., and Lima, Peru Tissue from 13 cervical cancers and pelvic or para-aortic lymph nodes from the same patient were evaluated by deoxyribonucleic acid hybridization with a human papillomavirus type 16 deoxyribonucleic acid probe for the presence of human papillomavirus-related deoxyribonucleic acid sequences. Twelve of the primary malignancies were squamous cancers and one was an adenocarcinoma. Eight of the primary tumors contained human papillomavirus type 16 deoxyribonucleic acid sequences, and five contained viral sequences closely related to human papillomavirus type 16. Histopathologic diagnosis confirmed malignant cells in six of 13 lymph nodes; three of these specimens contained human papillomavirus type 16 sequences while three had human papillomavirus type 16-related sequences. One lymph node that failed to show malignant cells also contained human papillomavirus type 16 deoxyribonucleic acid. The remaining lymph nodes did not contain malignant cells by either histologic examination or deoxyribonucleic acid hybridization. The human papillomavirus deoxyribonucleic acid sequences in the lymph nodes were similar to those in the matched primary cancer in all cases. These data provide further evidence implicating human papillomavirus in the etiology of cervical cancer. (AM J OssTET GvNECOL 1986;154:115-9.)
Key words: Human papillomavirus, cervical cancer, metastases Recently, attention has focused on the role of human papillomavirus (HPV) in cervical neoplasia. This association is based on morphologic, immunocytochemical, and molecular hybridization data showing evidence of HPV in the vast majority of intraepithelial and invasive neoplasms. Morphologic changes consistent with HPV infection can be recognized in 80% of cervical intraepithelial neoplasia,' HPV structural antigens are present in nearly 50% of mild and moderate dysplasia,2· 3 and molecular hybridization studies have revealed HPV deoxyribonucleic acid (DNA) sequences in 73% of all grades of dysplasia! Biopsy specimens of cervical cancers have also been shown to contain HPV DNA sequences. Gissmann et a!.' first demonstrated HPV -11 DNA sequences in five of 27 cervical cancers (three invasive and two carcinoma in situ). The genome of another virus, HPV-16, which was obtained from a biopsy specimen of invasive cervical cancer, has been shown to be present in 19 of 41 (46%) cervical cancers from German, African, and Brazilian patients. 6 In ad-
From the Departments of Obstetrics and Gynecology and Pathology, Vincent T. Lombardi Cancer Research Center, Georgetown University Medical Center, and the Departmento Ginecologia, Instituto Nacional Efermedades Neoplasicas. This work was supported by United States Public Health Service Grants CA32603 and CA32638 and by a special project from the Council for Tobacco Research. Received for publication june 24, 1985; revised October 11, 1985; accepted October 14, 1985. Reprint requests: W. D. Lancaster, Ph.D., Department of Obstetrics and Gynecology, Georgetown University Medical Center, Washington, DC 20007.
clition, DNA of yet another virus, HPV-18, has been molecularly cloned from a cervical cancer and these sequences are present in about 22% (11 of 51) of cervical cancer biopsy specimens from women in Germany, Africa, and BraziJ.1 However, when nonstringent hybridization techniques were used, HPV DNA sequences only distantly related to HPV-16 and HPV-18 could be detected in an additional 14% of cervical cancers. Thus approximately 80% of both intraepithelial and invasive cervical neoplasms have been shown to contain HPV DNA sequences. Since HPV sequences are detectable in biopsy specimens of dysplasia, it is possible that the viral DNA detected in invasive cancers is from adjacent intraepitheliallesions and therefore represents contamination. To clarify this potential problem in the interpretation of molecular hybridization studies, we performed molecular hybridization analysis on DNA extracted from the primary tumor and pelvic or para-aortic lymph nodes from 13 patients with advanced stage cervical cancer. The results indicate that HPV sequences present in biopsy specimens of invasive cervical cancer are not the result of contamination from cervical dysplasia since the identical viral DNA sequences are present in DNA extracted from lymph nodes containing metastatic cervical cancer in the same patient.
Material and methods Clinical material. Tissue specimens were obtained from Peruvian women seen at an oncology clinic for abnormal Papanicolaou smears. The patients ranged 115
116 Lancaster et al.
January, 1986 Am J Obstet Gynecol
3
C
LN
C
LN
CASE
NO.
12
15
C
LN
C
17
C LN
LN
18
C LN
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>
Fig. 1. Southern blot hybridization of HPV-16 probe DNA to DNA isolated from cervical (C) or lymph node (LN) biopsy specimens. Cellular DNA was digested with Pstl and approximately equal quantities (10 ~J.g) electrophoresed in parallel and probed for HPV sequences. Virus-specific DNA sequences in the cervical biopsy lanes (C) are indicated by arrows and corresponding sequences are present in lymph node biopsy lanes (LN) for Cases 3, 15, and 18. The restriction enzyme cleavage pattern of Case 1 is identical to the prototype HPV-16 described by Durst et al. 6 Lymph node DNA samples for Cases 1, 12, and 17 did not contain detectable HPV DNA sequences. The size marker (in kilobases) was Hindd111-digested bacteriophage lambda DNA. Autoradiogram exposures were for 48 hours at -70° C in the presence of intensifying screens.
in age from 24 to 63 years with a mean of 42.3 years. The diagnosis of cervical cancer was made on biopsy specimens of the cervix. All patients had clinical and radiologic staging procedure:s and thereafter an exploratory laparotomy with selected sampling of pelvic or para-aortic lymph nodes. 8 All patients had Stage liB or greater disease. Preparation of tissue samples. The preoperative biopsy specimen of cervical tumor was bisected; one portion was fixed in formalin for histologic examination and the portion stored at - 'i'0° C. Clinically questionable lymph nodes were bisected and treated in the same fashion as the cervical biopsy specimen. The tissues were snap-frozen, mounted in water, and sectioned on a cryostat. A representative section from the specimen was stained with hematoxylin and eosin for histologic examination. Total cellular DNA was extracted from the remaining sections for molecular hybridization as previously described.•
Preparation ofradiolabeled HPV-16 DNA. HPV-16 DNA cloned into the bacterial plasmid vector pBR322 was provided by L. Gissmann. Virus DNA was released from flanking plasmid DNA sequences by BamHl restriction enzyme cleavage and the viral sequences electroeluted from agarose gels. DNA was labeled by nick translation with Escherichia coli DNA polymerase and deoxyadenosine triphosphate labeled with radioactive phosphorus to a specific activity of 1 to 2 x 108 cpm per microgram of DNA. 9 Blot transfer hybridization. Cellular DNA was digested with a fourfold excess of the restriction endonuclease Pstl for 4 hours at 37° C and electrophoresed through 0.8% agarose gels. The DNA was transferred to cellulose nitrate membranes essentially as described by Southern.'" After the membranes were baked ina vacuum at 80° C for 2 hours, they were incubated in a solution containing 0.6 mol/L sodium chloride and 0.06 mol/L sodium citrate in 10 X Denhardt's 11 solution
Human papillomavirus DNA in cervical cancer
Volume 154 Number 1
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Table I. Frozen-section diagnoses and HPV-I6 DNA hybridizations to DNA extracted from cervical cancers and lymph nodes from the same patient Cervix Case No.
I
3 5 6 7 8 9 10 II
12 15 17 18
Frozen section
Squamous carcinoma Squamous carcinoma Adenocarcinoma Squamous carcinoma Squamous carcinoma Squamous carcinoma Squamous carcinoma Squamous carcinoma Squamous carcinoma Squamous carcinoma No tumor* Squamous carcinoma Squamous carcinoma
Lymph node
I
HPV DNA
HPV-16 HPV-31 HPV-At HPV-31 HPV-16 HPV-B HPV-16 HPV-16 HPV-16 HPV-16 HPV-16 HPV-16 HPV-C
Frozen section
No tumor* Squamous carcinoma Adenocarcinoma No tumor Squamous carcinoma No tumor No tumor No tumor No tumor* No tumor Squamous carcinoma No tumor Squamous carcinoma
I
HPV DNA
Negative HPV-31 HPV-A Negative HPV-16 Negative Negative HPV-16 HPV-16 Negative HPV-16 Negative HPV-C
*Squamous cell carcinoma was present in the formalin-fixed specimens in these cases. tHPV-A, HPV-B, and HPV-C represent viral DNA that exhibits Pstl cleavage patterns different from the prototype HPV-16 pattern (Fig. I, Case I).
(0.2% each Ficoll polyvinylpyrrolidone, and bovine serum albumin) with 50 !Lg/ml of sonicated and denatured human lymphocyte DNA. Hybridizations were carried out in a solution containing I to 5 X I 0" cpm of a heat-denatured probe, I moi/L sodium chloride, 0.15 moi/L tris(hydroxymethyl)-2-aminoethanesulfonic acid, pH 7.5, 40% formamide, and 2 X Denhardt's solution with 50 j.Lg/ml of sonicated, heat-denatured human lymphocyte DNA at 37° C for 24 hours. The membranes were washed extensively in 0.3 moi/L sodium chloride and 0.03 moi/L sodium citrate at 60° C and exposed to x-ray film for autoradiography. The conditions of hybridization were equivalent to 32° C below the melting temperature of the DNA (about 22% allowable base mismatch). 12 Results
Histopathology. Histologic examination of the portions of the 13 cervical biopsy specimens fixed in formalin revealed squamous carcinoma in 12 of the cases and adenocarcinoma in one. Frozen-section diagnosis of the cervical tissue used for molecular hybridization revealed invasive carcinoma in all cases except one (Case I5), in which only normal squamous epithelium was present. Histologic examination of the formalinfixed lymph nodes revealed squamous carcinoma in six, adenocarcinoma in one, and no tumor in the remaining six cases. Microscopic examination of the frozen tissue used for molecular hybridization was concordant in I1 instances. In the remaining two cases, tumor was not identified in the frozen tissue but was present in the formalin-fixed specimens (Cases 1 and I1). Molecular hybridization. With the use of an HPV16 probe under moderately stringent conditions, HPV DNA sequences were detected in all of the primary
cervical cancers and in seven lymph nodes. Representative examples of the hybridization reactions are shown in Fig. I. Analysis of the cleavage products of the viral sequences in the cervical cancer biopsy specimens indicated that eight of 13 were similar or identical to the Pst1 digestion pattern of the prototype HPV-I6 described by Durst et aJ.6 Two cancers contained sequences that had fragments in common with HPV-16 and may have lost some Pst1 recognition sequences (HPV-A and HPV-B, data not shown). Viral sequences in two of the cervical biopsy specimens (represented by Case 3, Fig. 1) contained a restriction pattern representative of a new HPV type (HPV -31) that has a limited amount of sequence homology to the HPV-16 probe (Lorincz A, eta!., unpublished data). One biopsy specimen (Case 18) contained sequences with only limited homology to HPV-16 (HPV-C). The sum of the sizes of the restriction fragments of this HPV genome is significantly less than expected for papillomavirus DNA, suggesting that additional sequences may be present that have a low degree of homology to the HPV -16 probe under the conditions of hybridization employed. Thus these viral sequences may represent a new HPV type. Variations in the sizes of some of the restriction fragments of HPV-16-containing lesions were also observed. In many instances extra bands were detected that could represent either incomplete restriction enzyme digestion or virus-cell DNA junction fragments resulting from integration. Although the lanes in the autoradiogram in Fig. 1 contained approximately equal quantities of cell DNA, there was considerable variation in the amount of HPV -16 detected almost to the point of obliteration of the signal (compare Case 1 with Cases
118 Lancaster et al.
12, 15, and 17). Similar variations in viral genome copy number have been noted in other papillomavirus-induced tumors and transformed cells. 13 Correlation of histopathologic examination and molecular hybridization. The results of histopathologic findings and DNA hybridization reactions are summarized in Table I. Of the seven lymph nodes shown to be positive for HPV DNA sequences, five contained metastatic cancer upon frozen-section evaluation. Two lymph nodes that contained HPV DNA sequences failed to reveal cancer cells in the frozen section used for histopathologic examination; however, one of these lymph nodes was shown to contain cancer cells in the permanent sections. Thus HPV DNA sequences were detected in each lymph node shown to contain cancer cells by histopathologic examination. In addition, the viral DNA present in each of the seven cases with a lymph node containing HPV DNA had restriction patterns similar to the viral sequences in the cervical cancer from the same patient. There was no correlation between the presence of metastases and virus type (or a particular Pstl cleavage pattern) nor was there an association between viral sequences, clinical stage of disease, or histologic type. All of the cancers in this study, however, contained either HPV -16 sequences or HPV sequences that exhibited homology to the HPV-16 probe.
Comment All 13 cervical carcinomas examined in this study contained HPV DNA sequences. Likewise, HPV DNA sequences were detected in lymph nodes from six cases containing metastatic tumor. The viral sequences had the same restriction enzyme cleavage pattern as those in the primary cervical cancers. This is significant since four different cleavage patterns were represented among this group of matched primary and metastatic cancers. Moreover, all of the viral DNA found in the lesions represented sequences closely related to or identical to HPV -16, one of the virus types preferentially associated with cervical cancer. 6 In one case the lymph node sample contained HPV sequences but metastatic tumor was not identified. Since only a limited number of frozen sections can be evaluated from tissue from which DNA is extracted, it is likely that sampling error could account for this discrepancy. Similarly, the frozen-section evaluation of one of the cervical cancers showed normal squamous epithelium although HPV DNA sequences were readily detectable in the tissue DNA preparation and tumor was present in the formalin-fixed specimen. There are several possible explanations for the finding of HPV DNA sequences in lymph nodes of patients with cervical cancer. First, the lymph nodes may contain intact HPV filtered during viremia. This is unlikely
January, 1986 Am J Obstet Gynecol
since lymph nodes with normal histologic features in five patients failed to show HPV sequences and infectious virus has never been detected by immunologic techniques either in the serum or in the primary cervical lesion (Jenson AB, et al., unpublished results). Second, viral sequences, either free or cell-associated, could reach lymph nodes by lymphatic drainage from the site of the carcinoma. This too is unlikely since all but one of the cases with HPV DNA sequences in the lymph nodes occurred in nodes that contained histologic evidence of metastases. Furthermore, free DNA sequences would probably be degraded by enzymes released by necrotic cancer cells. The most likely explanation for the presence of HPV DNA in lymph nodes containing metastatic tumor is that the HPV DNA sequences are present in the tumor cells themselves. This conclusion is supported by the concordance of the molecular hybridization and histopathologic data. There is precedence for the association of HPV DNA sequences in primary and metastatic lesions of squamous carcinomas arising in humans. This has been reported in patients with epidermodysplasia verruciformis, a rare autosomal recessive disease characterized by varying degrees of decreased cell-mediated immunity and increased susceptibility to HPV infection. 14 In approximately 25% of patients with epidermodysplasia verruciformis, malignant conversion occurs in pityriasis rosea-like warts exposed to sunlight. These lesions progress through increasing degrees of dysplasia, histologically similar to cervical dysplasias, before developing into carcinoma and eventually invasive cancer. These tumors may occasionally metastasize. Of the numerous HPV types preferentially associated with epidermodysplasia verruciformis, HPV-5 was identified in both primary and metastatic cancers of a patient with this disease. 15 Thus it appears that the role of HPV -5 in dysplastic and neoplastic lesions of epidermodysplasia verruciformis may be analogous to the role of HPV16 and related HPVs in cervical carcinomas with distant metastases. In this study as well as previous studies demonstrating HPV DNA sequences in cervical cancer biopsies, the possibility that viral sequences were derived from adjacent premalignant lesions (dysplasias) cannot be ruled out. Our finding of similar HPV DNA sequences in lymph nodes with metastatic carcinoma as well as the primary cervical tumor rules out this possibility and is the most compelling evidence to date implicating HPV in the pathogenesis of cervical cancer. The expert assistance of Donald C. F. Hay and Marsha Warda is greatly appreciated. REFERENCES l. Kurman RJ, Jenson AB, Lancaster WD. Papillomavirus
infection of the cervix. II. Relationship to intraepithelial
Human papillomavirus DNA in cervical cancer
Volume 154 Number I
2.
3.
4.
5.
6.
7.
neoplasia based on the presence of specific viral structural proteins. Am J Surg Pathol 1983;7:39. Kurman RJ, Shah KH, Lancaster WD, Jenson AB. Immunoperoxidase localization of papillomavirus antigens in cervical dysplasia and vulvar condylomas. AMJ OBSTET GYNECOL 1981;40:931. Kurman RJ, Sanz LE,Jenson AB, Perry S, Lancaster WD. Papillomavirus infection of the uterine cervix. I. Correlation of histology with specific structural antigens and DNA sequences. lntJ Gynecol Patholl982;1:17. Lancaster WD, Kurman RJ, Sanz LE, Perry S,Jenson AB. Human papilloma virus: detection of viral DNA sequences and evidence for molecular heterogeneity in metaplasias and dysplasias of the uterine cervix. Intervirology 1983; 20:202. Gissmann L, Wolnick L, Ikenberg H, Koldovsky U, Schnurch HG, zur Hausen H. Human papillomavirus type 6 and II DNA sequences in genital and layrngeal papillomas and in some cervical cancers. Proc Natl Acad Sci USA 1983;80:560. Durst M, Gissmann L, Ikenberg H, zur Hausen H. A papillomavirus DNA from a cervical carcinoma and its prevalence in cancer biopsies from different geographic regions. Proc Natl Acad Sci USA 1983;80:3812. Boshart M, Gissmann L, Ikenberg H, Kleinheinz A, Scheurlen W, zur Hausen H. A new type of papillomavirus DNA, its presence in genital cancer biopsies and in
8. 9.
10. 11. 12. 13. 14. 15.
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cell lines derived from cervical cancer. EMBO J 1984; 3:1151. Delgado G, Chun BK, Caglar H, Bepko F. Para-aortic lymphadenectomy in gynecologic malignancies confined to the pelvis. Obstet Gynecol 1977;50:418. Rigby P, Rhodes D, Dieckmann M, Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Bioi 1977; 113:237. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Bioi 1975;98:503. Denhardt DT. A membrane filter technique for the detection of complementary DNA. Biochem Biophys Res Commun 1966;23:641. Law M-F, Lancaster WD, Howley PM. Conserved polynucleotide sequences among the genomes of papillomaviruses. J Virol 1979;32: 199. Lancaster WD, Olson C. Animal papillomaviruses. Microbioi Rev 1982;46:191. Pfister H. Biology and biochemistry of papillomaviruses. Rev Physiol Biochem Pharmacol 1984;99: Ill. Ostrow RS, Bender M, Niimura M, et al. Human papillomavirus DNA in cutaneous primary and metastasized squamous cell carcinomas from patients with epidermodysplasia verruciformis. Proc Natl Acad Sci USA 1982; 79:1634.
Key words: Human papillomavirus, cervical cancer, metastases Recently, attention has focused on the role of human papillomavirus (HPV) in cervical neoplasia. This association is based on morphologic, immunocytochemical, and molecular hybridization data showing evidence of HPV in the vast majority of intraepithelial and invasive neoplasms. Morphologic changes consistent with HPV infection can be recognized in 80% of cervical intraepithelial neoplasia,' HPV structural antigens are present in nearly 50% of mild and moderate dysplasia,2· 3 and molecular hybridization studies have revealed HPV deoxyribonucleic acid (DNA) sequences in 73% of all grades of dysplasia! Biopsy specimens of cervical cancers have also been shown to contain HPV DNA sequences. Gissmann et a!.' first demonstrated HPV -11 DNA sequences in five of 27 cervical cancers (three invasive and two carcinoma in situ). The genome of another virus, HPV-16, which was obtained from a biopsy specimen of invasive cervical cancer, has been shown to be present in 19 of 41 (46%) cervical cancers from German, African, and Brazilian patients. 6 In ad-
From the Departments of Obstetrics and Gynecology and Pathology, Vincent T. Lombardi Cancer Research Center, Georgetown University Medical Center, and the Departmento Ginecologia, Instituto Nacional Efermedades Neoplasicas. This work was supported by United States Public Health Service Grants CA32603 and CA32638 and by a special project from the Council for Tobacco Research. Received for publication june 24, 1985; revised October 11, 1985; accepted October 14, 1985. Reprint requests: W. D. Lancaster, Ph.D., Department of Obstetrics and Gynecology, Georgetown University Medical Center, Washington, DC 20007.
clition, DNA of yet another virus, HPV-18, has been molecularly cloned from a cervical cancer and these sequences are present in about 22% (11 of 51) of cervical cancer biopsy specimens from women in Germany, Africa, and BraziJ.1 However, when nonstringent hybridization techniques were used, HPV DNA sequences only distantly related to HPV-16 and HPV-18 could be detected in an additional 14% of cervical cancers. Thus approximately 80% of both intraepithelial and invasive cervical neoplasms have been shown to contain HPV DNA sequences. Since HPV sequences are detectable in biopsy specimens of dysplasia, it is possible that the viral DNA detected in invasive cancers is from adjacent intraepitheliallesions and therefore represents contamination. To clarify this potential problem in the interpretation of molecular hybridization studies, we performed molecular hybridization analysis on DNA extracted from the primary tumor and pelvic or para-aortic lymph nodes from 13 patients with advanced stage cervical cancer. The results indicate that HPV sequences present in biopsy specimens of invasive cervical cancer are not the result of contamination from cervical dysplasia since the identical viral DNA sequences are present in DNA extracted from lymph nodes containing metastatic cervical cancer in the same patient.
Material and methods Clinical material. Tissue specimens were obtained from Peruvian women seen at an oncology clinic for abnormal Papanicolaou smears. The patients ranged 115
116 Lancaster et al.
January, 1986 Am J Obstet Gynecol
3
C
LN
C
LN
CASE
NO.
12
15
C
LN
C
17
C LN
LN
18
C LN
kb
23.19.46.64.4-
>
0.5> >
>
Fig. 1. Southern blot hybridization of HPV-16 probe DNA to DNA isolated from cervical (C) or lymph node (LN) biopsy specimens. Cellular DNA was digested with Pstl and approximately equal quantities (10 ~J.g) electrophoresed in parallel and probed for HPV sequences. Virus-specific DNA sequences in the cervical biopsy lanes (C) are indicated by arrows and corresponding sequences are present in lymph node biopsy lanes (LN) for Cases 3, 15, and 18. The restriction enzyme cleavage pattern of Case 1 is identical to the prototype HPV-16 described by Durst et al. 6 Lymph node DNA samples for Cases 1, 12, and 17 did not contain detectable HPV DNA sequences. The size marker (in kilobases) was Hindd111-digested bacteriophage lambda DNA. Autoradiogram exposures were for 48 hours at -70° C in the presence of intensifying screens.
in age from 24 to 63 years with a mean of 42.3 years. The diagnosis of cervical cancer was made on biopsy specimens of the cervix. All patients had clinical and radiologic staging procedure:s and thereafter an exploratory laparotomy with selected sampling of pelvic or para-aortic lymph nodes. 8 All patients had Stage liB or greater disease. Preparation of tissue samples. The preoperative biopsy specimen of cervical tumor was bisected; one portion was fixed in formalin for histologic examination and the portion stored at - 'i'0° C. Clinically questionable lymph nodes were bisected and treated in the same fashion as the cervical biopsy specimen. The tissues were snap-frozen, mounted in water, and sectioned on a cryostat. A representative section from the specimen was stained with hematoxylin and eosin for histologic examination. Total cellular DNA was extracted from the remaining sections for molecular hybridization as previously described.•
Preparation ofradiolabeled HPV-16 DNA. HPV-16 DNA cloned into the bacterial plasmid vector pBR322 was provided by L. Gissmann. Virus DNA was released from flanking plasmid DNA sequences by BamHl restriction enzyme cleavage and the viral sequences electroeluted from agarose gels. DNA was labeled by nick translation with Escherichia coli DNA polymerase and deoxyadenosine triphosphate labeled with radioactive phosphorus to a specific activity of 1 to 2 x 108 cpm per microgram of DNA. 9 Blot transfer hybridization. Cellular DNA was digested with a fourfold excess of the restriction endonuclease Pstl for 4 hours at 37° C and electrophoresed through 0.8% agarose gels. The DNA was transferred to cellulose nitrate membranes essentially as described by Southern.'" After the membranes were baked ina vacuum at 80° C for 2 hours, they were incubated in a solution containing 0.6 mol/L sodium chloride and 0.06 mol/L sodium citrate in 10 X Denhardt's 11 solution
Human papillomavirus DNA in cervical cancer
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Table I. Frozen-section diagnoses and HPV-I6 DNA hybridizations to DNA extracted from cervical cancers and lymph nodes from the same patient Cervix Case No.
I
3 5 6 7 8 9 10 II
12 15 17 18
Frozen section
Squamous carcinoma Squamous carcinoma Adenocarcinoma Squamous carcinoma Squamous carcinoma Squamous carcinoma Squamous carcinoma Squamous carcinoma Squamous carcinoma Squamous carcinoma No tumor* Squamous carcinoma Squamous carcinoma
Lymph node
I
HPV DNA
HPV-16 HPV-31 HPV-At HPV-31 HPV-16 HPV-B HPV-16 HPV-16 HPV-16 HPV-16 HPV-16 HPV-16 HPV-C
Frozen section
No tumor* Squamous carcinoma Adenocarcinoma No tumor Squamous carcinoma No tumor No tumor No tumor No tumor* No tumor Squamous carcinoma No tumor Squamous carcinoma
I
HPV DNA
Negative HPV-31 HPV-A Negative HPV-16 Negative Negative HPV-16 HPV-16 Negative HPV-16 Negative HPV-C
*Squamous cell carcinoma was present in the formalin-fixed specimens in these cases. tHPV-A, HPV-B, and HPV-C represent viral DNA that exhibits Pstl cleavage patterns different from the prototype HPV-16 pattern (Fig. I, Case I).
(0.2% each Ficoll polyvinylpyrrolidone, and bovine serum albumin) with 50 !Lg/ml of sonicated and denatured human lymphocyte DNA. Hybridizations were carried out in a solution containing I to 5 X I 0" cpm of a heat-denatured probe, I moi/L sodium chloride, 0.15 moi/L tris(hydroxymethyl)-2-aminoethanesulfonic acid, pH 7.5, 40% formamide, and 2 X Denhardt's solution with 50 j.Lg/ml of sonicated, heat-denatured human lymphocyte DNA at 37° C for 24 hours. The membranes were washed extensively in 0.3 moi/L sodium chloride and 0.03 moi/L sodium citrate at 60° C and exposed to x-ray film for autoradiography. The conditions of hybridization were equivalent to 32° C below the melting temperature of the DNA (about 22% allowable base mismatch). 12 Results
Histopathology. Histologic examination of the portions of the 13 cervical biopsy specimens fixed in formalin revealed squamous carcinoma in 12 of the cases and adenocarcinoma in one. Frozen-section diagnosis of the cervical tissue used for molecular hybridization revealed invasive carcinoma in all cases except one (Case I5), in which only normal squamous epithelium was present. Histologic examination of the formalinfixed lymph nodes revealed squamous carcinoma in six, adenocarcinoma in one, and no tumor in the remaining six cases. Microscopic examination of the frozen tissue used for molecular hybridization was concordant in I1 instances. In the remaining two cases, tumor was not identified in the frozen tissue but was present in the formalin-fixed specimens (Cases 1 and I1). Molecular hybridization. With the use of an HPV16 probe under moderately stringent conditions, HPV DNA sequences were detected in all of the primary
cervical cancers and in seven lymph nodes. Representative examples of the hybridization reactions are shown in Fig. I. Analysis of the cleavage products of the viral sequences in the cervical cancer biopsy specimens indicated that eight of 13 were similar or identical to the Pst1 digestion pattern of the prototype HPV-I6 described by Durst et aJ.6 Two cancers contained sequences that had fragments in common with HPV-16 and may have lost some Pst1 recognition sequences (HPV-A and HPV-B, data not shown). Viral sequences in two of the cervical biopsy specimens (represented by Case 3, Fig. 1) contained a restriction pattern representative of a new HPV type (HPV -31) that has a limited amount of sequence homology to the HPV-16 probe (Lorincz A, eta!., unpublished data). One biopsy specimen (Case 18) contained sequences with only limited homology to HPV-16 (HPV-C). The sum of the sizes of the restriction fragments of this HPV genome is significantly less than expected for papillomavirus DNA, suggesting that additional sequences may be present that have a low degree of homology to the HPV -16 probe under the conditions of hybridization employed. Thus these viral sequences may represent a new HPV type. Variations in the sizes of some of the restriction fragments of HPV-16-containing lesions were also observed. In many instances extra bands were detected that could represent either incomplete restriction enzyme digestion or virus-cell DNA junction fragments resulting from integration. Although the lanes in the autoradiogram in Fig. 1 contained approximately equal quantities of cell DNA, there was considerable variation in the amount of HPV -16 detected almost to the point of obliteration of the signal (compare Case 1 with Cases
118 Lancaster et al.
12, 15, and 17). Similar variations in viral genome copy number have been noted in other papillomavirus-induced tumors and transformed cells. 13 Correlation of histopathologic examination and molecular hybridization. The results of histopathologic findings and DNA hybridization reactions are summarized in Table I. Of the seven lymph nodes shown to be positive for HPV DNA sequences, five contained metastatic cancer upon frozen-section evaluation. Two lymph nodes that contained HPV DNA sequences failed to reveal cancer cells in the frozen section used for histopathologic examination; however, one of these lymph nodes was shown to contain cancer cells in the permanent sections. Thus HPV DNA sequences were detected in each lymph node shown to contain cancer cells by histopathologic examination. In addition, the viral DNA present in each of the seven cases with a lymph node containing HPV DNA had restriction patterns similar to the viral sequences in the cervical cancer from the same patient. There was no correlation between the presence of metastases and virus type (or a particular Pstl cleavage pattern) nor was there an association between viral sequences, clinical stage of disease, or histologic type. All of the cancers in this study, however, contained either HPV -16 sequences or HPV sequences that exhibited homology to the HPV-16 probe.
Comment All 13 cervical carcinomas examined in this study contained HPV DNA sequences. Likewise, HPV DNA sequences were detected in lymph nodes from six cases containing metastatic tumor. The viral sequences had the same restriction enzyme cleavage pattern as those in the primary cervical cancers. This is significant since four different cleavage patterns were represented among this group of matched primary and metastatic cancers. Moreover, all of the viral DNA found in the lesions represented sequences closely related to or identical to HPV -16, one of the virus types preferentially associated with cervical cancer. 6 In one case the lymph node sample contained HPV sequences but metastatic tumor was not identified. Since only a limited number of frozen sections can be evaluated from tissue from which DNA is extracted, it is likely that sampling error could account for this discrepancy. Similarly, the frozen-section evaluation of one of the cervical cancers showed normal squamous epithelium although HPV DNA sequences were readily detectable in the tissue DNA preparation and tumor was present in the formalin-fixed specimen. There are several possible explanations for the finding of HPV DNA sequences in lymph nodes of patients with cervical cancer. First, the lymph nodes may contain intact HPV filtered during viremia. This is unlikely
January, 1986 Am J Obstet Gynecol
since lymph nodes with normal histologic features in five patients failed to show HPV sequences and infectious virus has never been detected by immunologic techniques either in the serum or in the primary cervical lesion (Jenson AB, et al., unpublished results). Second, viral sequences, either free or cell-associated, could reach lymph nodes by lymphatic drainage from the site of the carcinoma. This too is unlikely since all but one of the cases with HPV DNA sequences in the lymph nodes occurred in nodes that contained histologic evidence of metastases. Furthermore, free DNA sequences would probably be degraded by enzymes released by necrotic cancer cells. The most likely explanation for the presence of HPV DNA in lymph nodes containing metastatic tumor is that the HPV DNA sequences are present in the tumor cells themselves. This conclusion is supported by the concordance of the molecular hybridization and histopathologic data. There is precedence for the association of HPV DNA sequences in primary and metastatic lesions of squamous carcinomas arising in humans. This has been reported in patients with epidermodysplasia verruciformis, a rare autosomal recessive disease characterized by varying degrees of decreased cell-mediated immunity and increased susceptibility to HPV infection. 14 In approximately 25% of patients with epidermodysplasia verruciformis, malignant conversion occurs in pityriasis rosea-like warts exposed to sunlight. These lesions progress through increasing degrees of dysplasia, histologically similar to cervical dysplasias, before developing into carcinoma and eventually invasive cancer. These tumors may occasionally metastasize. Of the numerous HPV types preferentially associated with epidermodysplasia verruciformis, HPV-5 was identified in both primary and metastatic cancers of a patient with this disease. 15 Thus it appears that the role of HPV -5 in dysplastic and neoplastic lesions of epidermodysplasia verruciformis may be analogous to the role of HPV16 and related HPVs in cervical carcinomas with distant metastases. In this study as well as previous studies demonstrating HPV DNA sequences in cervical cancer biopsies, the possibility that viral sequences were derived from adjacent premalignant lesions (dysplasias) cannot be ruled out. Our finding of similar HPV DNA sequences in lymph nodes with metastatic carcinoma as well as the primary cervical tumor rules out this possibility and is the most compelling evidence to date implicating HPV in the pathogenesis of cervical cancer. The expert assistance of Donald C. F. Hay and Marsha Warda is greatly appreciated. REFERENCES l. Kurman RJ, Jenson AB, Lancaster WD. Papillomavirus
infection of the cervix. II. Relationship to intraepithelial
Human papillomavirus DNA in cervical cancer
Volume 154 Number I
2.
3.
4.
5.
6.
7.
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