GYNECOLOGIC
ONCOLOGY
42, 22-26 (1991)
Human Papillomavirus Type 16 in Vulvar Carcinoma, Vulvar lntraepithelial Neoplasia, and Associated Cervical Neoplasia ULLA HORDING, *,I
SBREN
DAuGAAm,1. ASTRID K. N. IVERSEN,$ JENS KNUDSEN,* JOHANNES E. BOCK,* AND
*Department Denmark;
BODIL NORRILD$
of Obstetrics and Gynecology Y and TDepartment of Pathology, Rigshospitalet, University Hospital, Copenhagen and $DNA Tumor Virus Laboratory, Institute of Medical Microbiology, University of Copenhagen, Copenhagen, Received
September
Vulvar intraepithelial neoplasia (VIN) is becoming more widespread and the patients are becoming still younger. Although progression to invasive vulvar carcinoma is uncommon, local recurrences are frequent and about one-quarter of the patients have multicentric genital disease. The aim of the present study was to search for a possible significant association of human papillomavirus (HPV) infection with vulvar carcinoma, recurrences, and multicentric disease. We used the polymerase chain reaction to examine vulvar and cervical biopsies from 43 patients with vulvar neoplasia for HPV type 16, which is the subtype most often detected in genital malignant or premalignant lesions. HPV 16 DNA sequences were found in 14 of 24 (58%) vulvar squamous carcinomas and in 15 of 19 (79%) VIN lesions. Nine patients (21%) had associated cervical neoplasia and six of these harbored HPV 16 in both lesions. Patients with recurrent intraepithelial neoplasia had a significantly higher incidence of HPV 16-positive lesions. No association was found with regard to the occurrence of multicentric disease or risk of malignant progression. 0 1991 Academic
Press, Inc.
INTRODUCTION About one-quarter of the patients suffering from vulvar intraepithelial neoplasia (VIN) or vulvar carcinoma will also at some time during life have a diagnosis of cervical intraepithelial neoplasia (CIN) or cervical carcinoma. The risk is higher (average 29%) in relatively young VIN patients, while only about 15% of the usually much older patients with invasive vulvar carcinoma have cervical multicentric disease [ 11. Patients with recurrent or multifocal vulvar disease have the highest incidence of associated cervical neoplasia ’ To whom correspondence should be addressed at: Department of Obstetrics and Gynecology Y 4031, Rigshospitalet, Blegdamsvej 9, DK2100 Copenhagen, Denmark.
OG90-8258191 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
DK-2100, Denmark.
17, 1990
[2]. The reason for this connexion could be a generalized genital infection with human papillomavirus (HPV), of which several subtypes, most notably HPV 16, are associated with the development of cervical neoplasia [3]. As a sexually transmitted disease, the cervical HPV infection is most prevalent in younger women and the possibly oncogenic HPV 16 is the commonest subtype [4]. In several studies of vulvar carcinoma or VIN lesions, HPV 16 is also the subtype most often detected [5-91. The present study was carried out to search for a possible prognostic significance of the finding of HPV 16 DNA in vulvar intraepithelial neoplasia and carcinoma, particularly with regard to the occurrence of multicentric lesions, local recurrences, and malignant progression. For the detection of HPV 16, we have applied the polymerase chain reaction (PCR), which is an in vitro method of primer-directed enzymatic amplification of specific DNA sequences [lo], in this case of the E6/E7 region of the viral genome. The method is very sensitive and specific and can be applied on DNA extracts of fresh as well as paraffin-embedded tissues with a sensitivity of 20 viral copies [ll]. MATERIALS
AND METHODS
The Patients During the 3-year period from May 1986 through April 1989, vulvar biopsies were obtained from 43 women referred to Rigshospitalet, University Hospital, for surgical treatment of squamous cell carcinoma of the vulva (24 patients) or vulvar carcinoma in situ (VIN III, 19 patients). Of these, 19 carcinomas and 9 VIN lesions were primary. Fifteen biopsies were from local recurrences. Nine of the forty-three patients also had a diagnosis of cervical carcinoma or CIN.
HPV
16 IN VULVAR
23
NEOPLASIA
extension at 72°C for 35 sec. In the first cycle, the denaturation step was extended to 2 min at 94°C and in the The vulvar biopsies were bisected. One-half was roufinal cycle the primer extension step was prolonged to 10 tinely processed for conventional light microscopy, while min. In each PCR, two 7-pm sections of heart muscle the other half was frozen at - 70°C for later HPV analysis. were included as HPV-negative controls together with one Formalin-fixed, paraffin-embedded tissues were availpositive control consisting of a 7-pm section from a forable from all nine cervical neoplasms (one carcinoma and malin-fixed, paraffin-embedded pellet of SiHa cells. The eight CIN III lesions). Several 7-pm sections were cut cells of hematoxylin-eosin-stained 3-pm sections of the from each of the paraffin-embedded tissues. The first and pellet were counted by light microscopy, and the 7-pm last sections of each tissue were stained with hematoxylin section was estimated to hold about 200 SiHa cells (equaland eosin for control microscopy, in order to confirm that ling 200-400 copies of the HPV 16 genome). The SiHa the tissue sections used in the HPV analysis contained cells were the gift of professor H. zur Hausen, Deutsches malignant tissue. Krebsforschungszentrum in Heidelberg, Federal Republic All slides, control or routine, were evaluated by one of Germany. pathologist (S.D.). After the PCR, the 120-bp amplification product could be visualized by gel electrophoresis, with a DNA size Sample Preparation marker, followed by inspection of the ethidiumbromidestained gel under uv light. For further confirmation of The frozen biopsies were thawed and subjected to DNA product specificity, 7 ~1 of the amplification product was extraction by phenol and chloroform. Quantitation was spotted unto a GeneScreen Plus membrane through a dotmade by fluorimetric analysis. Most of the vulvar biopsies blot apparatus (Hybridot, Bethesda Laboratories) and were small and the DNA yields were in several cases below 5 pug, so that traditional Southern blotting would fixed to the membrane by alkaline treatment. DNADNA hybridization in aqueous solution [15] was perbe unreliable. We therefore decided to use the recently oligonudeveloped, highly sensitive PCR technique for the de- formed over night using a “P 5’-end-labeled tection of HPV 16 DNA. Aliquots of 1 pg DNA each cleotide probe (28 bases) specific to the internal portion of the amplified sequence. DNA hybridization and subwere subjected to analysis by PCR. Each 7-pm paraffin section was put into a 0.5-ml tube sequent washing of filters was done at 50°C (T,,,, -22°C). Autoradiography was performed for 4 hr on an X-ray film together with a 5% slurry of a chelating agent (Chelex using intensifying screens. 100, Bio-Rad), a resin considered to have a DNA-staFrom each specimen, two aliquots were subjected to bilizing effect [ 121, and was deparaffinized by boiling for analysis by PCR and DNA-DNA hybridization. In the 10 min. After a short centrifugation, the resin-free suinterpretation of results, specimens were considered HPV pernatant was transferred to another 0.5-ml tube and sub16-positive, if one of the results was positive. jected to PCR. Statistical comparisons were made by the x2 test. A P value of less than 0.05 was considered significant. HPV 16 DNA Analysis The Clinical Specimens
The PCR for HPV 16 DNA analysis was performed essentially as described [13] on a DNA thermal cycler (Perkin-Elmer Cetus Instruments). HPV 16-specific oligonucleotide primers were synthesized according to published sequences [14]. The primers each consisted of 20 bases flanking a 120-bp sequence of the E6-E7 region of the viral genome (genomic localization 421-540). For the polymerase chain reaction, 100 pmol of each primer was added to a 100~~1 reaction mixture containing the DNA from biopsies or paraffin sections, 2.5 units of Taq polymerase (Amplitaq, Cetus Corp.), and 200 pmol/liter of each of the four deoxynucleotide triphophates in a buffer solution of 10 mM Tris-HCl at pH 8.3, 50 mM KCl, 2.5 mM MgCl*, and 0.01% (w/v) gelatin. The samples were overlaid with mineral oil to prevent evaporation and subjected to 30 cycles of amplification. Each cycle consisted of a denaturation step at 96°C for 20 set and a primer annealing step at 56°C for 20 set followed bv mimer
RESULTS Patients: Clinical Characteristics Age. The 19 patients of the VIN group were significantly younger at first referral than the 24 patients with invasive carcinoma: median of 37 years (range, 22-55 years) versus median of 71.5 years (range, 34-95 years). Treatment and recurrences. Local recurrences after vulvectomy were seen in 4 patients with carcinoma and 1 of these eventually died from disseminated disease. In the carcinoma patients, the observation period from diagnosis was 2-15 years (median, 3.5 years). In the VIN group, 16 patients had at least one local recurrence during a follow-up period of 2-16 years (median, 5 years). Twelve of the VIN patients had three or more local vulvar excisions performed due to recurrences. A regular vulvectomy was performed in only four VIN
24
HBRDING
ET AL.
TABLE 1 Detection of HPV 16 DNA in the Vulvar and Cervical Lesionsof Nine Patients with Multicentric Genital Malignancy Vulvar neoplasia
Patient
Histology
Age at diagnosis (years)
1 2 3 4 5 6 I 8 9
VIN III VIN III VIN III VIN III VIN III VIN III Carcinoma Carcinoma Carcinomab
33 48 32 40 43 31 45 51 32
Cervical neoplasia
HPV 16 detected
Recurrent disease
Progression
Histology
Age at diagnosis (years)
+ + + + + + + +
No No x4 x2 xl x5 x3 No xl
No No No No Yes No Died No Yes
CIN III CIN III CIN III CIN III CIN III CIN III Carcinoma CIN III CIN III
33 34 25 38 43 34 47 45 30
HPV 16 detected + + + + + +
’ This patient also had a rectum amputation due to an anal squamous cell carcinoma, when she was 39 years old. Examination three different sections from this tumor failed to demonstrate HPV 16 DNA. * The patient was treated for a VIN III lesion when first referred.
patients, two of whom later had a recurrence with microinvasive disease (i.e., carcinoma with invasion depth below 3 mm). Malignant progression. Progression to microinvasive vulvar disease occurred in three VIN patients within 35 years from the initial diagnosis. One further patient referred for an invasive carcinoma had been treated for a VIN III lesion by simple vulvectomy 18 years before. Apparently she had no intercurrent malignancy. In these four patients, median age at the diagnosis of invasive vulvar carcinoma was 52.5 years (range, 43-57 years). During the observation period, two patients with vulvar carcinoma died of the disease. No deaths occurred in the VIN group. Multicentric disease. Eight patients had a diagnosis of CIN concomitantly or 2-14 years before the vulvar lesion and one patient had a cervical carcinoma 2 years after treatment for a vulvar carcinoma (Table 1). None of the cervical lesions recurred after treatment. Vulvar epithelial lesions coexistent with carcinoma. Lichen sclerosus et atrophicus lesions were found adjacent to 4/24 vulvar carcinomas and severe dysplasia or carcinoma in situ lesions were found in the edges of 12/24 carcinomas. In the remaining 8 cases, no specific pathology was described outside the carcinoma. HPV 16 Diagnosis
HPV 16 DNA was demonstrated in the vulvar biopsies of 15/19 patients with VIN (79%) and in 14/24 patients with invasive carcinoma (58%). The difference is not statistically significant. Within both groups, the HPV 16-positive patients were slightly younger than the HPV-negative patients. The mean age difference was 12 and 18 months, respectively.
Observation period (years) 2 2 5 5 4 16 16 5 18 by PCR of
HPV 16 DNA was found in 15 of 16 recurrent VIN III lesions (94%), which was significantly more often than in nonrecurring lesions (P < 0.01). Two out of four VIN III lesions progressing to microinvasive carcinoma contained HPV 16 DNA. In the carcinoma patients, 6/11 patients with stage I disease, 4/8 with stage II, 3/4 with stage III, and l/l patient with stage IV disease were HPV 16-positive. Two of four recurrent lesions harbored HPV 16 DNA. HPV 16 DNA was found in 2/4 carcinomas with coexistent lichen sclerosus, in 8/12 carcinomas with adjacent VIN, and in 5/8 carcinomas without other vulvar pathology. In the patients with multicentric disease, HPV 16 DNA was found in eight of nine vulvar lesions, but only six of the nine patients harbored HPV 16 in both the vulvar and the cervical lesions (Table 1). COMMENTS Vulvar squamous cell carcinoma is a disease associated with frequent local recurrences but relatively low mortality [16]. In about 25% of invasive vulvar carcinomas, adjacent carcinoma in situ lesions are found [16-181 and excision of apparently pure VIN lesions reveal foci of microinvasion in 2-6% of cases [2,19]. Consequently, VIN is considered a premalignant disease and lesions are eradicated once they have been detected; no study on the natural story of the disease has been conducted, and the progressive potential of VIN is unknown. Following treatment, the rate of progression seems to be in the order of 2-7% [2,19-211; in all studies, progression occurred in patients who were either older (i.e., over 45 years of age) or immunosuppressed [22]. In accordance with this,
HPV 16 IN VULVAR
3 of 4 patients who in this study developed carcinoma in VIN lesions were over 45 years of age; in the group of VIN patients without malignant progression, only 2 of 1.5 women were over 45 years when entered into the study. In recent years, VIN lesions have been more frequently reported and the patients are becoming still younger [2,22]. One reason for this might be a causal association with vulvar HPV infection, which is probably becoming more widespread [23]. On the basis of histopathological studies, it has been suggested that VIN lesions affect two patient population groups: younger patients with a high incidence of coexistent HPV infection, frequent multifocal and recurrent lesions, and low risk of malignant progression and older patients with unifocal lesions, less frequent association with HPV infection, and higher risk of malignant progression [22,24]. In the present study, however, the patients with HPV 16-containing lesions were only slightly younger than the remainder. Like other authors, we found no consistent relation between multicentric genital disease and the presence of HPV 16 in vulvar lesions [25,26]. Actually, HPV 16 DNA could be demonstrated in both lesions in only two-thirds of the patients with cervical multicentric disease. Furthermore, one patient with HPV 16-containing VIN as well as CIN also had an anal carcinoma from which all biopsies were HPV 16-negative (Table 1). The high incidence of the possibly oncogenic HPV 16 found in this and other studies of vulvar neoplasia [59,261 may implicate some role of this HPV in the development of vulvar neoplasia, although the prevalence of HPV 16 in normal vulvar tissues is at present unknown. A recent case-control study [27] has demonstrated an association between vulvar cancer and a history of genital warts; the study also showed an increased risk of vulvar cancer with the number of lifetime sexual partners and so indicated that cervical and vulvar cancers may share a common etiology. The present study, however, cannot support the theory of HPV 16 as a causative agent in the development of vulvar cancer. The association of HPV 16 infection and recurrent intraepithelial lesions may point rather to a decreased local immunological resistance of the vulvar region or possibly of the entire lower genital tract in these patients. This, in turn, would increase the risk of acquiring dysplastic lesions as well as infection with a widespread genital viral disease. ACKNOWLEDGMENT The study was supported by Grants 88-089 and 90-087 from The Danish Cancer Society.
REFERENCES 1. Choo, Y. C., and Morley, G. W. Double primary epidermoid carcinoma of the vulva and cervix, Gynecol. Oncol. 9,324-333 (1980).
25
NEOPLASIA
Andreasson, B., and Bock, .I. E. Intraepithelial neoplasia in the vulvar region, Gynecof. Oncol. 21, 300-305 (1985). 3 Wright, T. C., and Richart. R. M. Review: Role of human papillomavirus in the pathogenesis of genital tract warts and cancer, Gynecol. Oncol. 37, 151-164 (1990). 4 Hording, U., Iversen, A. K. N., Sebbelov, A., Bock, J. E., and Norrild, B. Prevalence of human papillomavirus types 11, 16 and 18 in cervical swabs. A study of 1362 pregnant women, Eur. J. Obstet. Gynecol. Reprod. Biol. 3.5, 191-198 (1990). 5 Buscema, J., Naghashfar. Z., Sawada, E., Daniel, R., Woodruff, J. D., and Shah, K. The predominance of human papillomavirus type 16 in vulvar neoplasia, Obstet. Gynecol. 71, 601-606 (1988). 6 Pilotti, S., Rotola, A., D’Amato, L., Di Luca, D., Shah, K. V., Cassai, E., and Rilke, F. Vulvar carcinomas: Search for sequences homologous to human papillomavirus and herpes simples virus DNA, Mod. Pathol. 3, 442-448 (1990). 7. Ikenberg, H., Schworer, D., and Pfleiderer, A. Nachweis von humaner Papilloma-Virus (HPV)-DNA in Vulvakarzinomen. Geburtshirfe Frauenheilkd. 48, 776-780 (1988). [Abstract in English] 8 Neill, S. M., Lessana-Leibowitch, M., Pelisse, M., and MoyalBarracco, M. Lichen sclerosus, invasive squamous cell carcinoma, and human papillomavirus (Letter), Am. J. Obstet. Gynecol. 162, 1633-1634 (1990). 9. Gupta, J., Pilotti, S., Shah, K. V., De Palo, G., and Rilke, F. Human papillomavirus-associated early vulvar neoplasia investigated by in situ hybridization, Am. J. Surg. Pathol. 11, 430-434 (1987). 10. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., and Erlich, H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase, Science 239, 487-491 (1988). II. Shibata, D. K., Arnheim, N., and MArtin, W. J. Detection of human papilloma virus in paraffin-embedded tissue using the polymerase chain reaction. J. Exp. Med. 167, 225-230 (1988). 12. Singer-Sam, J., Tanguay, R. L., and Riggs, A. D. Use of Chelex to improve the PCR signal from a small number of cells, Amp/ijications 3, 11 (1989). 13. Saiki, R. K. The design and optimization of the PCR, in PCR technology (H. A. Erlich, Ed.), Stockton Press, New York. p. 8 (1989). 14. Seedorf, K., Krammer, G., Dtirst, M., Suhai, S., and Rowekamp, W. G. Human papillomavirus type 16 DNA sequence, Virology 145, 181-185 (1985). 15. Church, G. M., and Gilbert, W. Genomic sequencing, Proc. Natl. Acad. Sci. USA 81, 1991-1995 (1984). 16. Japaze, H., Garcia-Bunuel, R., and Woodruff, J. D. Primary vulvar neoplasia. A review of in situ and invasive carcinoma, 1935-1972. Obstet. Gynecol. 49, 404-411 (1977). 17. Borgno, G., Micheletti, L., Barbero, M., Preti, M., Cavanna, L., and Ghiringhello, B. Epithelial alterations adjacent to 111 vulvar carcinomas, /. Reprod. Med. 33, 500-502 (1988). 18. Zaino, R. J., Husseinzadeh, N., Nahhas, W., and Mortel, R. Epithelial alterations in proximity to invasive squamous carcinoma of the vulva, Int. J. Gynecol. Pathol. 1, 173-184 (1982). 19. Friedrich, E. G., Wilkinson, E. J., and Fu, Y. S. Carcinoma in situ of the vulva: A continuing challenge, Am. J. Obstet. Gynecol. 2.
136, 830-843
(1980).
20. Woodruff, J. D., Julian, C., Puray, T., Mermut, S., and Katayama, P. The contemporary challenge of carcinoma in situ of the vulva, Am. J. Obstet. Gynecol. 115, 677-686 (1973).
26
HP)RDING
21. Buscema, J., Woodruff, J. D., Parmley, T. H., and Genadry, R. Carcinoma in situ of the vulva, Obstet. Gynecol. 55,225230 (1980). 22. Crum, C. P., Liskow, A., Petras, P., Keng, W. C., and Frick, H. C. Vulvar intraepithelial neoplasia (severe atypia and carcinoma in situ), Cancer 54, 1429-1434 (1984). 23. Chuang, T., Perry, H. O., Kurland, L. T., and Ilstrup, D. M. Condyloma acuminatum in Rochester, Minn, 1950-1978. I. Epidemiology and clinical features, Arch. Dermatol. 120, 469-475 (1984). 24. Husseinzadeh, N., Newman, N. J., and Wesseler, T. A. Vulvar intraepithelial neoplasia: A clinicopathological study of carcinoma in situ of the vulva, Gynecol. Oncol. 33, 157-163 (1989).
ET AL. 25. Bornstein, J., Kaufman, R. H., Adam, E., and Adler-Storthz, K. Multicentric intraepithelial neoplasia involving the vulva. Clinical features and association with human papillomavirus and herpes simplex virus, Cancer 62, 1601-1604 (1988). 26. Beckmann, A. M., Kiviat, N. B., Daling, J. R., Sherman, K. .I., and McDougall, K. K. Human papillomavirus type 16 in multifocal neoplasia of the female genital tract, ht. J. Gynecol. Pathol. 7, 39-47 (1988). 27. Brinton, L. A., Nasca, P. C., Mallin, K., Baptiste, M. S., Wilbanks, G. D., and Richart, R. M. Case-control study of cancer of the vulva, Obstet. Gynecol. 75, 859-866 (1990).
ONCOLOGY
42, 22-26 (1991)
Human Papillomavirus Type 16 in Vulvar Carcinoma, Vulvar lntraepithelial Neoplasia, and Associated Cervical Neoplasia ULLA HORDING, *,I
SBREN
DAuGAAm,1. ASTRID K. N. IVERSEN,$ JENS KNUDSEN,* JOHANNES E. BOCK,* AND
*Department Denmark;
BODIL NORRILD$
of Obstetrics and Gynecology Y and TDepartment of Pathology, Rigshospitalet, University Hospital, Copenhagen and $DNA Tumor Virus Laboratory, Institute of Medical Microbiology, University of Copenhagen, Copenhagen, Received
September
Vulvar intraepithelial neoplasia (VIN) is becoming more widespread and the patients are becoming still younger. Although progression to invasive vulvar carcinoma is uncommon, local recurrences are frequent and about one-quarter of the patients have multicentric genital disease. The aim of the present study was to search for a possible significant association of human papillomavirus (HPV) infection with vulvar carcinoma, recurrences, and multicentric disease. We used the polymerase chain reaction to examine vulvar and cervical biopsies from 43 patients with vulvar neoplasia for HPV type 16, which is the subtype most often detected in genital malignant or premalignant lesions. HPV 16 DNA sequences were found in 14 of 24 (58%) vulvar squamous carcinomas and in 15 of 19 (79%) VIN lesions. Nine patients (21%) had associated cervical neoplasia and six of these harbored HPV 16 in both lesions. Patients with recurrent intraepithelial neoplasia had a significantly higher incidence of HPV 16-positive lesions. No association was found with regard to the occurrence of multicentric disease or risk of malignant progression. 0 1991 Academic
Press, Inc.
INTRODUCTION About one-quarter of the patients suffering from vulvar intraepithelial neoplasia (VIN) or vulvar carcinoma will also at some time during life have a diagnosis of cervical intraepithelial neoplasia (CIN) or cervical carcinoma. The risk is higher (average 29%) in relatively young VIN patients, while only about 15% of the usually much older patients with invasive vulvar carcinoma have cervical multicentric disease [ 11. Patients with recurrent or multifocal vulvar disease have the highest incidence of associated cervical neoplasia ’ To whom correspondence should be addressed at: Department of Obstetrics and Gynecology Y 4031, Rigshospitalet, Blegdamsvej 9, DK2100 Copenhagen, Denmark.
OG90-8258191 $1.50 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
DK-2100, Denmark.
17, 1990
[2]. The reason for this connexion could be a generalized genital infection with human papillomavirus (HPV), of which several subtypes, most notably HPV 16, are associated with the development of cervical neoplasia [3]. As a sexually transmitted disease, the cervical HPV infection is most prevalent in younger women and the possibly oncogenic HPV 16 is the commonest subtype [4]. In several studies of vulvar carcinoma or VIN lesions, HPV 16 is also the subtype most often detected [5-91. The present study was carried out to search for a possible prognostic significance of the finding of HPV 16 DNA in vulvar intraepithelial neoplasia and carcinoma, particularly with regard to the occurrence of multicentric lesions, local recurrences, and malignant progression. For the detection of HPV 16, we have applied the polymerase chain reaction (PCR), which is an in vitro method of primer-directed enzymatic amplification of specific DNA sequences [lo], in this case of the E6/E7 region of the viral genome. The method is very sensitive and specific and can be applied on DNA extracts of fresh as well as paraffin-embedded tissues with a sensitivity of 20 viral copies [ll]. MATERIALS
AND METHODS
The Patients During the 3-year period from May 1986 through April 1989, vulvar biopsies were obtained from 43 women referred to Rigshospitalet, University Hospital, for surgical treatment of squamous cell carcinoma of the vulva (24 patients) or vulvar carcinoma in situ (VIN III, 19 patients). Of these, 19 carcinomas and 9 VIN lesions were primary. Fifteen biopsies were from local recurrences. Nine of the forty-three patients also had a diagnosis of cervical carcinoma or CIN.
HPV
16 IN VULVAR
23
NEOPLASIA
extension at 72°C for 35 sec. In the first cycle, the denaturation step was extended to 2 min at 94°C and in the The vulvar biopsies were bisected. One-half was roufinal cycle the primer extension step was prolonged to 10 tinely processed for conventional light microscopy, while min. In each PCR, two 7-pm sections of heart muscle the other half was frozen at - 70°C for later HPV analysis. were included as HPV-negative controls together with one Formalin-fixed, paraffin-embedded tissues were availpositive control consisting of a 7-pm section from a forable from all nine cervical neoplasms (one carcinoma and malin-fixed, paraffin-embedded pellet of SiHa cells. The eight CIN III lesions). Several 7-pm sections were cut cells of hematoxylin-eosin-stained 3-pm sections of the from each of the paraffin-embedded tissues. The first and pellet were counted by light microscopy, and the 7-pm last sections of each tissue were stained with hematoxylin section was estimated to hold about 200 SiHa cells (equaland eosin for control microscopy, in order to confirm that ling 200-400 copies of the HPV 16 genome). The SiHa the tissue sections used in the HPV analysis contained cells were the gift of professor H. zur Hausen, Deutsches malignant tissue. Krebsforschungszentrum in Heidelberg, Federal Republic All slides, control or routine, were evaluated by one of Germany. pathologist (S.D.). After the PCR, the 120-bp amplification product could be visualized by gel electrophoresis, with a DNA size Sample Preparation marker, followed by inspection of the ethidiumbromidestained gel under uv light. For further confirmation of The frozen biopsies were thawed and subjected to DNA product specificity, 7 ~1 of the amplification product was extraction by phenol and chloroform. Quantitation was spotted unto a GeneScreen Plus membrane through a dotmade by fluorimetric analysis. Most of the vulvar biopsies blot apparatus (Hybridot, Bethesda Laboratories) and were small and the DNA yields were in several cases below 5 pug, so that traditional Southern blotting would fixed to the membrane by alkaline treatment. DNADNA hybridization in aqueous solution [15] was perbe unreliable. We therefore decided to use the recently oligonudeveloped, highly sensitive PCR technique for the de- formed over night using a “P 5’-end-labeled tection of HPV 16 DNA. Aliquots of 1 pg DNA each cleotide probe (28 bases) specific to the internal portion of the amplified sequence. DNA hybridization and subwere subjected to analysis by PCR. Each 7-pm paraffin section was put into a 0.5-ml tube sequent washing of filters was done at 50°C (T,,,, -22°C). Autoradiography was performed for 4 hr on an X-ray film together with a 5% slurry of a chelating agent (Chelex using intensifying screens. 100, Bio-Rad), a resin considered to have a DNA-staFrom each specimen, two aliquots were subjected to bilizing effect [ 121, and was deparaffinized by boiling for analysis by PCR and DNA-DNA hybridization. In the 10 min. After a short centrifugation, the resin-free suinterpretation of results, specimens were considered HPV pernatant was transferred to another 0.5-ml tube and sub16-positive, if one of the results was positive. jected to PCR. Statistical comparisons were made by the x2 test. A P value of less than 0.05 was considered significant. HPV 16 DNA Analysis The Clinical Specimens
The PCR for HPV 16 DNA analysis was performed essentially as described [13] on a DNA thermal cycler (Perkin-Elmer Cetus Instruments). HPV 16-specific oligonucleotide primers were synthesized according to published sequences [14]. The primers each consisted of 20 bases flanking a 120-bp sequence of the E6-E7 region of the viral genome (genomic localization 421-540). For the polymerase chain reaction, 100 pmol of each primer was added to a 100~~1 reaction mixture containing the DNA from biopsies or paraffin sections, 2.5 units of Taq polymerase (Amplitaq, Cetus Corp.), and 200 pmol/liter of each of the four deoxynucleotide triphophates in a buffer solution of 10 mM Tris-HCl at pH 8.3, 50 mM KCl, 2.5 mM MgCl*, and 0.01% (w/v) gelatin. The samples were overlaid with mineral oil to prevent evaporation and subjected to 30 cycles of amplification. Each cycle consisted of a denaturation step at 96°C for 20 set and a primer annealing step at 56°C for 20 set followed bv mimer
RESULTS Patients: Clinical Characteristics Age. The 19 patients of the VIN group were significantly younger at first referral than the 24 patients with invasive carcinoma: median of 37 years (range, 22-55 years) versus median of 71.5 years (range, 34-95 years). Treatment and recurrences. Local recurrences after vulvectomy were seen in 4 patients with carcinoma and 1 of these eventually died from disseminated disease. In the carcinoma patients, the observation period from diagnosis was 2-15 years (median, 3.5 years). In the VIN group, 16 patients had at least one local recurrence during a follow-up period of 2-16 years (median, 5 years). Twelve of the VIN patients had three or more local vulvar excisions performed due to recurrences. A regular vulvectomy was performed in only four VIN
24
HBRDING
ET AL.
TABLE 1 Detection of HPV 16 DNA in the Vulvar and Cervical Lesionsof Nine Patients with Multicentric Genital Malignancy Vulvar neoplasia
Patient
Histology
Age at diagnosis (years)
1 2 3 4 5 6 I 8 9
VIN III VIN III VIN III VIN III VIN III VIN III Carcinoma Carcinoma Carcinomab
33 48 32 40 43 31 45 51 32
Cervical neoplasia
HPV 16 detected
Recurrent disease
Progression
Histology
Age at diagnosis (years)
+ + + + + + + +
No No x4 x2 xl x5 x3 No xl
No No No No Yes No Died No Yes
CIN III CIN III CIN III CIN III CIN III CIN III Carcinoma CIN III CIN III
33 34 25 38 43 34 47 45 30
HPV 16 detected + + + + + +
’ This patient also had a rectum amputation due to an anal squamous cell carcinoma, when she was 39 years old. Examination three different sections from this tumor failed to demonstrate HPV 16 DNA. * The patient was treated for a VIN III lesion when first referred.
patients, two of whom later had a recurrence with microinvasive disease (i.e., carcinoma with invasion depth below 3 mm). Malignant progression. Progression to microinvasive vulvar disease occurred in three VIN patients within 35 years from the initial diagnosis. One further patient referred for an invasive carcinoma had been treated for a VIN III lesion by simple vulvectomy 18 years before. Apparently she had no intercurrent malignancy. In these four patients, median age at the diagnosis of invasive vulvar carcinoma was 52.5 years (range, 43-57 years). During the observation period, two patients with vulvar carcinoma died of the disease. No deaths occurred in the VIN group. Multicentric disease. Eight patients had a diagnosis of CIN concomitantly or 2-14 years before the vulvar lesion and one patient had a cervical carcinoma 2 years after treatment for a vulvar carcinoma (Table 1). None of the cervical lesions recurred after treatment. Vulvar epithelial lesions coexistent with carcinoma. Lichen sclerosus et atrophicus lesions were found adjacent to 4/24 vulvar carcinomas and severe dysplasia or carcinoma in situ lesions were found in the edges of 12/24 carcinomas. In the remaining 8 cases, no specific pathology was described outside the carcinoma. HPV 16 Diagnosis
HPV 16 DNA was demonstrated in the vulvar biopsies of 15/19 patients with VIN (79%) and in 14/24 patients with invasive carcinoma (58%). The difference is not statistically significant. Within both groups, the HPV 16-positive patients were slightly younger than the HPV-negative patients. The mean age difference was 12 and 18 months, respectively.
Observation period (years) 2 2 5 5 4 16 16 5 18 by PCR of
HPV 16 DNA was found in 15 of 16 recurrent VIN III lesions (94%), which was significantly more often than in nonrecurring lesions (P < 0.01). Two out of four VIN III lesions progressing to microinvasive carcinoma contained HPV 16 DNA. In the carcinoma patients, 6/11 patients with stage I disease, 4/8 with stage II, 3/4 with stage III, and l/l patient with stage IV disease were HPV 16-positive. Two of four recurrent lesions harbored HPV 16 DNA. HPV 16 DNA was found in 2/4 carcinomas with coexistent lichen sclerosus, in 8/12 carcinomas with adjacent VIN, and in 5/8 carcinomas without other vulvar pathology. In the patients with multicentric disease, HPV 16 DNA was found in eight of nine vulvar lesions, but only six of the nine patients harbored HPV 16 in both the vulvar and the cervical lesions (Table 1). COMMENTS Vulvar squamous cell carcinoma is a disease associated with frequent local recurrences but relatively low mortality [16]. In about 25% of invasive vulvar carcinomas, adjacent carcinoma in situ lesions are found [16-181 and excision of apparently pure VIN lesions reveal foci of microinvasion in 2-6% of cases [2,19]. Consequently, VIN is considered a premalignant disease and lesions are eradicated once they have been detected; no study on the natural story of the disease has been conducted, and the progressive potential of VIN is unknown. Following treatment, the rate of progression seems to be in the order of 2-7% [2,19-211; in all studies, progression occurred in patients who were either older (i.e., over 45 years of age) or immunosuppressed [22]. In accordance with this,
HPV 16 IN VULVAR
3 of 4 patients who in this study developed carcinoma in VIN lesions were over 45 years of age; in the group of VIN patients without malignant progression, only 2 of 1.5 women were over 45 years when entered into the study. In recent years, VIN lesions have been more frequently reported and the patients are becoming still younger [2,22]. One reason for this might be a causal association with vulvar HPV infection, which is probably becoming more widespread [23]. On the basis of histopathological studies, it has been suggested that VIN lesions affect two patient population groups: younger patients with a high incidence of coexistent HPV infection, frequent multifocal and recurrent lesions, and low risk of malignant progression and older patients with unifocal lesions, less frequent association with HPV infection, and higher risk of malignant progression [22,24]. In the present study, however, the patients with HPV 16-containing lesions were only slightly younger than the remainder. Like other authors, we found no consistent relation between multicentric genital disease and the presence of HPV 16 in vulvar lesions [25,26]. Actually, HPV 16 DNA could be demonstrated in both lesions in only two-thirds of the patients with cervical multicentric disease. Furthermore, one patient with HPV 16-containing VIN as well as CIN also had an anal carcinoma from which all biopsies were HPV 16-negative (Table 1). The high incidence of the possibly oncogenic HPV 16 found in this and other studies of vulvar neoplasia [59,261 may implicate some role of this HPV in the development of vulvar neoplasia, although the prevalence of HPV 16 in normal vulvar tissues is at present unknown. A recent case-control study [27] has demonstrated an association between vulvar cancer and a history of genital warts; the study also showed an increased risk of vulvar cancer with the number of lifetime sexual partners and so indicated that cervical and vulvar cancers may share a common etiology. The present study, however, cannot support the theory of HPV 16 as a causative agent in the development of vulvar cancer. The association of HPV 16 infection and recurrent intraepithelial lesions may point rather to a decreased local immunological resistance of the vulvar region or possibly of the entire lower genital tract in these patients. This, in turn, would increase the risk of acquiring dysplastic lesions as well as infection with a widespread genital viral disease. ACKNOWLEDGMENT The study was supported by Grants 88-089 and 90-087 from The Danish Cancer Society.
REFERENCES 1. Choo, Y. C., and Morley, G. W. Double primary epidermoid carcinoma of the vulva and cervix, Gynecol. Oncol. 9,324-333 (1980).
25
NEOPLASIA
Andreasson, B., and Bock, .I. E. Intraepithelial neoplasia in the vulvar region, Gynecof. Oncol. 21, 300-305 (1985). 3 Wright, T. C., and Richart. R. M. Review: Role of human papillomavirus in the pathogenesis of genital tract warts and cancer, Gynecol. Oncol. 37, 151-164 (1990). 4 Hording, U., Iversen, A. K. N., Sebbelov, A., Bock, J. E., and Norrild, B. Prevalence of human papillomavirus types 11, 16 and 18 in cervical swabs. A study of 1362 pregnant women, Eur. J. Obstet. Gynecol. Reprod. Biol. 3.5, 191-198 (1990). 5 Buscema, J., Naghashfar. Z., Sawada, E., Daniel, R., Woodruff, J. D., and Shah, K. The predominance of human papillomavirus type 16 in vulvar neoplasia, Obstet. Gynecol. 71, 601-606 (1988). 6 Pilotti, S., Rotola, A., D’Amato, L., Di Luca, D., Shah, K. V., Cassai, E., and Rilke, F. Vulvar carcinomas: Search for sequences homologous to human papillomavirus and herpes simples virus DNA, Mod. Pathol. 3, 442-448 (1990). 7. Ikenberg, H., Schworer, D., and Pfleiderer, A. Nachweis von humaner Papilloma-Virus (HPV)-DNA in Vulvakarzinomen. Geburtshirfe Frauenheilkd. 48, 776-780 (1988). [Abstract in English] 8 Neill, S. M., Lessana-Leibowitch, M., Pelisse, M., and MoyalBarracco, M. Lichen sclerosus, invasive squamous cell carcinoma, and human papillomavirus (Letter), Am. J. Obstet. Gynecol. 162, 1633-1634 (1990). 9. Gupta, J., Pilotti, S., Shah, K. V., De Palo, G., and Rilke, F. Human papillomavirus-associated early vulvar neoplasia investigated by in situ hybridization, Am. J. Surg. Pathol. 11, 430-434 (1987). 10. Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., and Erlich, H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase, Science 239, 487-491 (1988). II. Shibata, D. K., Arnheim, N., and MArtin, W. J. Detection of human papilloma virus in paraffin-embedded tissue using the polymerase chain reaction. J. Exp. Med. 167, 225-230 (1988). 12. Singer-Sam, J., Tanguay, R. L., and Riggs, A. D. Use of Chelex to improve the PCR signal from a small number of cells, Amp/ijications 3, 11 (1989). 13. Saiki, R. K. The design and optimization of the PCR, in PCR technology (H. A. Erlich, Ed.), Stockton Press, New York. p. 8 (1989). 14. Seedorf, K., Krammer, G., Dtirst, M., Suhai, S., and Rowekamp, W. G. Human papillomavirus type 16 DNA sequence, Virology 145, 181-185 (1985). 15. Church, G. M., and Gilbert, W. Genomic sequencing, Proc. Natl. Acad. Sci. USA 81, 1991-1995 (1984). 16. Japaze, H., Garcia-Bunuel, R., and Woodruff, J. D. Primary vulvar neoplasia. A review of in situ and invasive carcinoma, 1935-1972. Obstet. Gynecol. 49, 404-411 (1977). 17. Borgno, G., Micheletti, L., Barbero, M., Preti, M., Cavanna, L., and Ghiringhello, B. Epithelial alterations adjacent to 111 vulvar carcinomas, /. Reprod. Med. 33, 500-502 (1988). 18. Zaino, R. J., Husseinzadeh, N., Nahhas, W., and Mortel, R. Epithelial alterations in proximity to invasive squamous carcinoma of the vulva, Int. J. Gynecol. Pathol. 1, 173-184 (1982). 19. Friedrich, E. G., Wilkinson, E. J., and Fu, Y. S. Carcinoma in situ of the vulva: A continuing challenge, Am. J. Obstet. Gynecol. 2.
136, 830-843
(1980).
20. Woodruff, J. D., Julian, C., Puray, T., Mermut, S., and Katayama, P. The contemporary challenge of carcinoma in situ of the vulva, Am. J. Obstet. Gynecol. 115, 677-686 (1973).
26
HP)RDING
21. Buscema, J., Woodruff, J. D., Parmley, T. H., and Genadry, R. Carcinoma in situ of the vulva, Obstet. Gynecol. 55,225230 (1980). 22. Crum, C. P., Liskow, A., Petras, P., Keng, W. C., and Frick, H. C. Vulvar intraepithelial neoplasia (severe atypia and carcinoma in situ), Cancer 54, 1429-1434 (1984). 23. Chuang, T., Perry, H. O., Kurland, L. T., and Ilstrup, D. M. Condyloma acuminatum in Rochester, Minn, 1950-1978. I. Epidemiology and clinical features, Arch. Dermatol. 120, 469-475 (1984). 24. Husseinzadeh, N., Newman, N. J., and Wesseler, T. A. Vulvar intraepithelial neoplasia: A clinicopathological study of carcinoma in situ of the vulva, Gynecol. Oncol. 33, 157-163 (1989).
ET AL. 25. Bornstein, J., Kaufman, R. H., Adam, E., and Adler-Storthz, K. Multicentric intraepithelial neoplasia involving the vulva. Clinical features and association with human papillomavirus and herpes simplex virus, Cancer 62, 1601-1604 (1988). 26. Beckmann, A. M., Kiviat, N. B., Daling, J. R., Sherman, K. .I., and McDougall, K. K. Human papillomavirus type 16 in multifocal neoplasia of the female genital tract, ht. J. Gynecol. Pathol. 7, 39-47 (1988). 27. Brinton, L. A., Nasca, P. C., Mallin, K., Baptiste, M. S., Wilbanks, G. D., and Richart, R. M. Case-control study of cancer of the vulva, Obstet. Gynecol. 75, 859-866 (1990).
