Human papillomavirus and herpes simplex virus in vulvar squamous cell carcinoma in situ

Human papillomavirus and herpes simplex virus in vulvar squamous cell carcinoma in situ Raymond H. Kaufman, MD, Jacob Bornstein, MD, Ervin Adam, MD, Joyce Burek, BS, Barbara Tessin, BS, and Karen Adler-Storthz, PhD Houston, Texas Human papillomavirus deoxyribonucleic acid were detected in tissue specimens from 38 of 46 patients (83%) with squamous cell carcinoma in situ of the vulva. Herpes simplex virus type 2-related antigen ICSP 34/35 was demonstrated in 23 of the lesions (50%), and antibodies to herpes simplex virus types 1 and 2 were found in 74% and 65% of the serum samples tested, respectively. Both human papillomavirus deoxyribonucleic acid and herpes simplex virus type 2 antigen were detected in 19 cases (41%). Correlation of human papillomavirus type to the ages of the patients revealed that types 16,18, and 31 are most often seen in older patients, although the frequencies do not differ significantly. No relationship between the presence or absence of herpes simplex virus type 2-related antigen to age of the patient was observed. (AM J OBSTET GVNECOL 1988;158:862-71.)

Key words: Human papillomavirus, herpes simplex virus, vulvar squamous cell carcinoma, in situ hybridization

Squamous cell carcinoma in situ of the vulva (grade 3 vulvar intraepithelial neoplasia) has been associated with herpes simplex virus type 2' and with human papillomavirus." This association and similar findings in squamous neoplasia of the cervix have implicated herpes simplex virus type 2 and human papillomavirus in the etiology of this disease.':" While vulvar infections with human papillomavirus or herpes simplex virus type 2 are frequent, few women develop vulvar neoplasia. For this reason neither virus would appear to be a sole causal agent. Rather, herpes simplex virus type 2 and human papillomavirus may possibly act in synergism or in association with other factors in initiating and promoting a malignant transformation of the epithelium.' Only three cases of squamous cell carcinoma in situ of the vulva have been studied for the presence of both herpes simplex virus type 2 and human papillomavirus "markers," and in none of these cases have both been detected concurrently in the tissue." 6 Hence we felt that a larger study of the relationship of dual infection in this disease was warranted. Tissue specimens from squamous cell carcinoma in situ and from adjacent normal marginal areas were studied From the Departments of Obstetrics and Gynecology and of Virology and Epidemiology, Baylor College ofMedicine, and Department of Microbiology, University of Texas Dental Branch Supported in part by Grant No, ROI DE07008 from the National Institutes of Health and The Women's Fund for Research and Education, Houston, Texas, Presented at the Sixth Annual Meeting ofthe American Gynecological and Obstetrical Society, Phoenix, Arizona, September 10-12, 1987. Reprint requests: Raymond H. Kaufman, MD, Department of Obstetrics and Gynecology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030.

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for evidence of herpes simplex virus type 2-related antigen and human papillomavirus deoxyribonucleic acid (DNA). Results were correlated with the clinical characteristics of the patients. Type-specific antibodies to herpes simplex virus were also targeted for study.

Material and methods Tissue specimens were obtained from 62 patients 21 to 79 years old who had biopsy-proved squamous cell carcinoma in situ of the vulva. In 34 of the patients, two specimens were retrieved at the same time (one from the lesion itself and one from the normalappearing adjacent margin at least 1.5 em distal to the lesion). In three of the patients with later recurrences of vulvar intraepithelial neoplasia, specimens were also obtained from the recurrent lesions. The tissues were snap frozen and kept at - 70° C until tested. Sections (6 uwide) were cut from each of the tissue specimens and placed on slides precoated with 0.3% gelatin and 0.05% chromium potassium sulfate to prevent detachment,of sections. A parallel cut was stained with hematoxylin and eosin stain and submitted under code to one of the authors (R. H. K.) for confirmation of the diagnosis. Sixteen specimens obtained from the 62 patients with carcinoma in situ were excluded from evaluation, for the following reasons: eight specimens contained only normal epithelium; six specimens contained insufficient epithelium for either the herpes simplex virus type 2 or human papillomavirus studies; and two specimens hybridized to pBR322 vector sequences. This left 46 patients for inclusion in the study. The indirect immunoperoxidase method was used to

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Fig. 1. A, Vulvar carcinoma in situ, hybridized to a biotin-labeled human papillomavirus type 18 probe under stringent conditions. Positive nuclei are clustered throughout the epithelium; they stain dark because of deposits of the oxidized products of diaminobenzidine (light green counterstain ; original magnification x 400). B, Vulvar carcinoma in situ hybridized to human papillomavirus type II under stringent conditions. The nuclei remain unstained, as no positivity is detected (light green counterstain; original magnification x 640).

detect the herpes simplex virus type 2-induced antigen ICSP 34/35. Details of preparation and specificity of this antibody have been reported .t" The primary sera used included rabbit anti-ICSP 34/35 and normal nonimmunized rabbit serum. Conjugation of goat antirabbit IgG with horseradish peroxidase was performed, as previously described.' All antibody reagents, both primary and secondary, were adsorbed with uninfected Hep-2 cells and with mouse liver powder before use . Herpes simplex virus type 2-infected Hep-2 cells were used as the positive control, and uninfected cells served as the negative control. Rabbit antiserum to bovine papillomavirus type 1 (Dako Corporation, Santa Barbara, Calif.) was used as the primary antibody for detection of papillomavirus antigen, and normal rabbit serum was used as the negative control. The avidin-biotin-peroxidase method was used for antibody detection. The peroxidase substrate consisted of 0.0 1% hydrogen peroxide and 0.05 % 3,3/diaminobenzidine tetra-hydrochloride in 0.05 mol/L phosphate-buffered saline solution (pH 7.4). Details of this procedure have been reported ." The genomes of human papillomavirus types 6b, 11, 16, 18, and 31 were supplied cloned into the plasmid pBR322. Each of the plasmids was nick translated by the use of biotin-labeled dUTP (Bethesda Research Laboratories, Gaithersburg, Md.). Biotin-labeled pBR322 DNA was used as a control DNA . Each DNA probe was prepared with 4 J.l.g/ml of biotin-labeled human papillomavirus DNA and 400 J.l.g/ml of salmon sperm DNA in 10% dextran sulfate, 2X standard saline solution citrate (0.3 mol/L NaCI and 0.3 mol/L sodium citrate), and 50% deionized formamide. Before hybridization, the sections were fixed in cold Carnoy's solution (60% ethanol, 30% chloroform, 10%

acetic acid). Endogenous peroxidase activity in the sections was blocked by incubation for 30 minutes in methanol with 0.01 % hydrogen peroxide. After air drying, individual sections were covered with each of the human papillomavirus DNA probes. The slides were then placed over a boiling water bath for 10 minutes to denature both probe and cellular DNA. Hybridization was carried out for 18 hours at 37° C for stringent conditions and at 7° C for nonstringent conditions. After three successive washes , either in 2X saline sodium citrate at room temperature for nonstringent conditions or in 0.2X saline sodium citrate at 37° C for stringent conditions, the sections were tested for hybridized DNA by sequential incubations with 3% normal goat serum, rabbit antibiotin (l: 100; Enzo Biochem, Inc., New York, N. Y.), goat antirabbit IgG conjugated to biotin (1: 200; Vector Laboratories, Burlingame, Calif.), and avidin-biotin-peroxidase reagent (l : 100; Vector Laboratories). Phosphatebuffered saline solution washes were used between all steps. After the last wash, 3,3'-diaminobenzidine tetrahydrochloride substrate (prepared as described above) was added . Slides were washed in water, counterstained with light green, dehydrated, cleared in xylene, and mounted . The positive control tissues used in all experiments were two genital condylomata that had been previously shown to contain human papillomavirus type 6b DNA and papillomavirus antigen. A blood sample was drawn from 23 women at the time grade 3 vulvar intraepithelial neoplasia was diagnosed . The serum samples were stored at - 35° C until they were tested for the presence of herpes simplex virus type-specific antibodies by a microsolid-phase radioimmunoassay, as previously described."

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Table I. Human papillomavirus types of vulvar carcinoma in situ Type

No. of patients*

6 II

5 5

6 + II 16 18 31 Mixed types 6 + 16 6 + II + 16 II + 16 + 18 II + 31 Unidentified type Negative

2 5

Total (%)

Cases with recurrence* (%)

12 (26%)

10

2

17 (37%)

I I I

5 (II %) 4 (9%) 8 (17%)

2

46 (100%)

Total

* In three of the cases with a later recurrence of vulvar carcinoma in situ, an additional specimen was available from the recurrent lesion. One patient had two specimens with human papillomavirus type 16, and one had two specimens with human papillomavirus type 18. The third did not have human papillomavirus DNA in the original specimen, but a biopsy from a recurrent lesion showed human papillomavirus of unidentified type. Only the original specimens are represented in this and other tables.

Table II. Detection of human papillomavirus DNA and herpes simplex virus type 2-related antigen in vulvar carcinoma in situ _____________

Table III. Human papillomavirus DNA and herpes simplex virus type 2 antigen in normal tissue margins adjacent to vulvar carcinoma in situ and recurrence of disease within the first year of follow-up

0-1-

-

-N-O-'

I

patients

%

Human papillomavirus only Herpes simplex virus type 2 only Both Neither

19 4

41 9

19 4

41 9

Total

46

100

The clinical data on all patients were correlated with evidence of viral infection. The significance of differences between groups of patients was determined by the chi-square test. Results Demonstration of human papillomavirus DNA in carcinoma in situ. Thirty-eight of 46 specimens (83%) from patients with neoplasia were found to contain human papillomavirus DNA (Table I). Human papillomavirus types 6, II, or both were detected in 12 of the 46 specimens (26%). Types 16, 18, or 31 were found in I7. tissue specimens (37%), with type 18 the most prevalent (10 of 17). A positive tissue finding and a negative one are shown in Fig. I. A mixture of types of both groups was identified in five cases (II %). Four

Human papillomavirus only Herpes simplex virus type 2 only Neither Total

6 (18%)

3 (50%)

I

0(0%)

(3%) 27 (79%)

6 (22%)

34 (100%)

9 (26%)

*Number in parentheses is the percent of patients with recurrence.

specimens had human papillomavirus DNA when tested under nonstringent conditions, but the type was not identified under stringent hybridization conditions. Eight tissue specimens (17%) were negative for human papillomavirus DNA. Ten of the 46 specimens with carcinoma in situ (22%) contained papillomavirus antigen (data not tabulated). Detection of herpes simplex virus type 2-related antigen. Immunocytochemical staining for the herpes simplex virus type 2-related antigen ICSP 34/35 was positive in 23 specimens (50%) tested. Nineteen of the 46 lesions (41%) studied had evidence of both human papillomavirus DNA and herpes simplex virus type 2 antigens. Only four specimens with vulvar carcinoma in situ demonstrated the presence of herpes simplex virus type 2 antigen alone (9%). Four lesions with carcinoma in situ had no evidence of either human papillomavirus or herpes simplex virus type 2. Thus 91 % of the specimens with carcinoma in situ had evidence of human papillomavirus DNA, herpes simplex virus type 2-related antigens, or a combination of both (Table II). Normal adjacent tissues. Six of the 34 tissues (18%) from the normal adjacent margins available for virologic testing (Table III) contained human papillomavirus DNA similar to that found in the lesion itself (Fig. 2). Only one case was positive for the herpes simplex virus type 2-related antigen ICSP 34/35, and in none were both human papillomavirus DNA and herpes simplex virus type 2 antigen detected. Detection of antibodies to herpes simplex virus. Of the 23 patients tested, 17 (74%) and 15 (65%) patients had antibodies to herpes simplex virus types 1 and 2, respectively. Ten of 13 patients (77%) with ICSP 34/35 in their vulvar lesions whose sera had been tested had serum antibodies to herpes simplex virus type 2. In

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Fig. 2. Normal vulvar tissue removed 2 cm distal to the margin of the same lesion as in Fig. 1, and hybridized to human papillomavirus type 18 under stringent conditions. Positivity is located in the most basal epithelial layer (light green counterstain; original magnification x 400).

each of the other three patients, the detection of the herpes simplex virus type 2 antigen ICSP 34/35 in the lesion was not accompanied by a serologic response to the virus (data not tabulated). Correlations of study findings with clinical variables. The study findings were correlated with certain clinical variables. The distribution of study participants in different age groups and the types of human papillomavirus are shown in Table IV. There were 17 patients aged 20 to 34 years, 19 aged 35 to 49 years, and 10 aged ;;;.50 years. The frequency of a positive finding of human papillomavirus DNA in tissue specimens was similar in all age groups (82%, 79%, and 90%, respectively). Human papillomavirus types 16, 18, and 31 were found more often in women ;;;.35 years than in women aged 20 to 34 years (54% versus 29%), but this difference was not significant (p = 0.13). This trend was similar when specimens with mixed human papillomavirus DNA (types 6/11 and types 16/18/31) were included. The herpes simplex virus type 2-related antigen ICSP 34/35 was found in 41%,58%, and 50% of the women in the different age groups, respectively. Of a total of 23 patients whose specimens were positive for ICSP 34/35, 83% also had concurrent human papillomavirus DNA present. The frequency of this finding was not related to age (data not tabulated). In patients with both viral markers, the detection rate of human papillomavirus types 16, 18, or 31 increased with the women's ages, being found in eight of 14 women over age 35 years (57%). One of five women under age 35 years in this group had evidence of human papillomavirus types 16/18/31 simultaneously with evidence ofICSP 34/35 (p = NS). The distribution of lesions on the vulva is presented

in Table V. The frequency of unifocal lesions was related to age. This frequency increased from 12% of women aged 20 to 34 years to 50% of the women in the oldest age group (p < 0.05; one sided test). The frequency of human papillomavirus DNA detected in patients with unifocal and multifocal vulvar intraepithelial neoplasia was similar. Patients with unifocal disease had a somewhat lower rate of herpes simplex virus infection (42%) than those patients with multifocal disease (53%), but this difference was not significant. No case of unifocal vulvar intraepithelial neoplasia was associated with herpes simplex virus type 2 antigen alone. Local recurrence of disease occurred in nine of 43 women within 1 year of initial treatment (Table VI). Adequate follow-up data were not available for three other women. There was no significant difference in human papillomavirus types or in the presence or absence of herpes simplex virus antigen between patients with or without a recurrence of disease. Three of six women (50%) with human papillomavirus DNA in normal skin adjacent to vulvar carcinoma in situ had recurrences of disease (Table III), while six of27 women (22%) with neither human papillomavirus DNA or ICSP 34/35 in normal adjacent skin developed recurrences (difference was not significant), Recurrent carcinoma in situ did not occur in the one patient found to have herpes simplex virus type 2 antigen alone in the normal adjacent skin. Comment

During the past two decades, the frequency of squamous cell carcinoma in situ of the vulva has increased. This increase has been seen most prominently in

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April 1988 Am J Obstet Gynecol

Table IV. Detection of human papillomavirus DNA and herpes simplex virus type 2 antigen in vulvar carcinoma in situ by age of the patient Type of HPVt Age group (yr)

No. of patients tested

Positive forHPV DNA *

6,11, or 6+1J:t

20-34 35-49 2:50

17 19 10

14 (82%) 15 (79%) 9 (90%)

7 (50%) 2 (13%) 3 (33%)

Total

46

38 (83%)

12 (32%)

HSV type 2 antigen ICSP 34135*·t (%)

Mixture of 6111 and 16118131

Not identified

4 (29%) 8 (53%) 5 (55%)

2 2

3

I

o

7 (41%) II (58%) 5 (50%)

17 (45%)

5

4

23 (50%)

16,18, or 3J:t

I

HPV, Human papillomavirus; HSV, herpes simplex virus. *Number in parentheses is the percent of positive cases of all patients of same age group. tNegative findings are not tabulated. *Number in parentheses is the percent of patients with a specific human papillomavirus type of the positive cases in the same age group.

Table V. Detection of human papillomavirus DNA in vulvar carcinoma in situ according to the distribution of the disease on the vulva No. of patients in each age group*

Type of HPV:t

No. of patients tested

20-34 yr

35-49 yr

2:50 yr

No. positive for HPV DNAt

Unifocal (n = 12) Multifocal (n = 34) Total (N = 46)

2 (12%) 15 (88%) 17 (100%)

5 (26%) 14 (74%) 19 (100%)

5 (50%) 5 (50%) 10 (100%)

10 (83%) 28 (82%) 38 (83%)

6, 11 or 6 + 11§

16,18, or 31§

2 (20%) 10 (36%) 12 (32%)

5 (50%) 12 (43%) 17 (45%)

Mixture of 6111 and 16118131

Not identified

I 4

2 2

5

4

HPV, Human papillomavirus. *Number in parentheses is the percent of patients of same age group of all patients in the same age group. tNumber in parentheses is the percent of positive cases of all patients tested with the same distribution of disease. *Negative findings are not tabulated. §Number in parentheses is the percent of patients with a specific human papillomavirus type of all positive cases with the same distribution of disease.

women under age 40 years. One possible explanation for this is the increased incidence of sexually transmitted diseases, such as human papillomavirus and herpes simplex virus infections. I. 11 It has been suggested that one or the other of these viruses-alone, together, or in association with other factors-might be implicated in the genesis of lower genital tract neoplasia" The present study was undertaken to look for a possible association of human papillomavirus and herpes simplex virus viruses with squamous cell carcinoma in situ of the vulva. Human papillomavirus DNA was detected in 38 of 46 (83%) tissue specimens of women with squamous cell carcinoma in situ of the vulva. The in situ hybridization used to detect human papillomavirus DNA reveals the precise epithelial location of the DNA (Figs. I and 2) and, therefore, differentiates human papillomavirus DNA within the vulvar intraepithelial neoplasia from that which might be present in a nearby condyloma acuminatum that conceivably could have

been removed by the same biopsy procedure. 12, 13 Prior studies have looked for the presence of human papillomavirus DNA under nonstringent conditions in vulvar carcinoma in situ 6 • 14 (Buscema ], personal communication). One of these studies included only one positive case." A second study presented data on eight cases, four of which (50%) were positive." In a third study, lesions were examined from 24 patients, of which 84% were human papillomavirus positive (Buscerna ], personal communication). Several other investigators looking primarily for the presence of human papillomavirus type 16 have reported the presence ofthis type in 33% to 100% of specimens examinedv ":" (Smotkin D, personal communication). The differences in these findings may be attributed either to the small number of cases studied or to geographic variations in the prevalence of certain human papillomavirus types. In the present study, the most common human papillomavirus type detected was type 18, with the other types (6, 11, and 16) found in smaller but equal num-

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Table VI. Detection of human papillomavirus and herpes simplex virus type 2 in vulvar carcinoma in situ according to local recurrence within the first year after treatment in 43 patients with I year or more of follow-up Type of HPVt

No. of patients tested

Positive forHPV DNA *

Mixture of 6111 and 6/18131

6, 11, or 6 + ll ;

16,18, or 3 J:j:

Recurrence (n = 9) No recurrence (n = 34) Total (N = 43)

8 (89%) 28 (82%) 36 (84%)

3 (37%) 9 (32%) 12 (33%)

2 (25%) 15 (54%) 17 (47%)

2

2 4

Not identified

PapillomavIrus antigen*·t

HSV type 2 antigen ICSP 34135*·t

I 2 3

I (II %) 9 (26%) 10 (23%)

5 (56%) 17 (50%) 22(51%)

HPV, Human papillomavirus; HSV, herpes simplex virus. *Number in parentheses is the percent of positive cases of the patients with or without recurrence. tNegative findings are not tabulated. :j:Number in parentheses is the percent of patients with a specific human papillomavirus type of the positive cases with or without recurrence.

bers. Five of the 38 human papillomavirus-positive lesions (13%) were found to have mixed types. McCance et al." have reported similar findings. Our results suggest that a variety of human papillomavirus types may be associated with vulvar carcinoma in situ. A similar finding has been documented with invasive vulvar carcinoma" 16. 17 (Roman A, personal communication). The high frequency of antibodies to herpes simplex virus type 2 found in our study population, as well as the finding of herpes simplex virus type 2-related antigen in a large number of women with intraepithelial neoplasia is similar to previous findings in neoplasia of the cervix. Although an association of herpes simplex virus type 2-related antigens has been previously reported, I. 22 in the present study this association was not as strong as was the association of human papillomavirus with this disease. In our study, only human papillomavirus DNA was detected in 41 % of women studied and only herpes simplex virus type 2 antigen was detected in 9%. Half of the patients with vulvar carcinoma in situ had evidence of single viral infection; 41 % had evidence of dual infection. Although the frequency of human papillomavirus was dominant, the actual role of the viruses in the pathogenesis of genital neoplasia remains questionable. Both viruses are sexually transmitted, and it is known that early sexual activity is associated with a higher frequency of sexually transmitted diseases, including herpes simplex virus infections. This may also be valid for infection with the sexually transmitted human papillomavirus. However, smoking, another suspected carcinogen, is also known to be associated with sexual habits. Therefore, it remains speculative what role, if any, viruses play in the development of neoplasia. Human papillomavirus and herpes simplex virus markers were not detected simulatenously in the three cases reported elsewhere in which both viral types were examined.v" but evidence

for coexistence of both viruses has been documented in cervical neoplasms.";" Analysis of the clinical variables and their correlation with our laboratory findings revealed that the herpes simplex virus type 2 antigen ICSP 34/35 and human papillomavirus DNA were detected with similar frequencies in different age groups. The prevalence of HPV types 16, 18, or 31, however, appeared to be age related, increasing gradually (although not significantly) from 29% in the younger patients to 55% in the oldest age group. This confirms the findings of two recent studies."-" Meanwell et al." reported that human papillomavirus type 16 positivity was found more often in patients older than 40 years than in younger women. This was noted in patients with cervical cancer and in normal control tissues. The second report demonstrated that positivity of human papillomavirus types 16 or 18 in endocervical smears from healthy women gradually increased with age, from 22% in women below age 30 years to 56% in women above age 50 years." The higher frequency of detection of human papillomavirus type 16 in older ages may be explained either by the increased likelihood of exposure to the virus with age or by a superior potential for long-term latency of human papillomavirus types 16, 18, or 31. Squamous cell carcinoma in situ tends to be unifocal on the vulva in the older patient. This trend was demonstrated in our study, in which the frequency of unifocallesions significantly increased with age (Table V). To analyze whether the increased frequencies of human papillomavirus types 16, 18, and 31 with age were related to the distribution of the disease on the vulva, we compared the frequencies of the different viral types in the group of patients with unifocallesions with those patients with multifocallesions. We observed that the frequency of viral types was not related to the distribution of the disease on the vulva, and that the fre-

868 Kaufman at al. Am J

quencies of the different human papillomavirus types were similar in the two groups. After initial surgical treatment, 43 of the patients were followed for at least 1 year, during which time nine of them (21%) presented with recurrent vulvar intraepithelial neoplasia. We did not find any significant relationship between human papillomavirus type and recurrence of disease. This is in accordance with three recent prospective studies on patients with squamous cervical neoplasia" (lkenberg H, personal communication; Syrjanen K, personal communication). This finding contradicts earlier publications that introduced the concept of an association of human papillomavirus types 16 and 18 with a more malignant course of cervical neoplasia than that of human papillomavirus types 6 or 11.2 • 27 Furthermore, human papillomavirus types 6 and 11 have recently been associated with recurrent and invasive vulvar neoplasia" (Roman A, personal communication; Kasher MS, personal communication). This contradiction could be related to a difference in the biologic behavior of cervical and vulvar neoplasia, to variations in the extent of the initial treatment practiced at different centers, and/or to the presence of other factors, such as smoking, in some patients." Human papillomavirus DNA has previously been detected in seven specimens (35%) of "normal" skin margins from 20 patients with condylomata acuminata." Six of these seven patients developed recurrences of their disease. This finding has been proposed as evidence of a latent phase of human papillomavirus infection. Di Luca et al. 16 reported on the findings in six specimens of "normal" skin adjacent to vulvar intraepithelial neoplasia. All were negative for human papillomavirus DNA. We looked for evidence of both human papillomavirus DNA and herpes simplex virus type 2 antigen in normal tissues adjacent to squamous cell carcinoma in situ of the vulva in 34 cases. Eighteen percent of the specimens were positive for human papillomavirus DNA, while herpes simplex virus type 2 antigen was detected in only one of the 34 cases. Three of six women (50%) found to have human papillomavirus DNA in normal skin adjacent to vulvar carcinoma in situ developed recurrent disease. Six of 27 women (27%) with no evidence of human papillomavirus DNA in normal adjacent skin subsequently were found to have recurrent disease. Our findings suggest a strong association between human papillomavirus infection and vulvar carcinoma in situ. Human papillomavirus DNA was detected in 38 (83%) of 46 tested tissue specimens. The herpes simplex virus type 2-related antigen lCSP 34/35 was detected in 23 (50%) of the women, while antibodies to types 1 and 2 were demonstrated in 74% and 65% of 23 tested women, respectively. Dual infection, as

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Obstet Gynecol

judged by the finding of both human papillomavirus DNA and the herpes simplex virus type 2-related antigen lCSP 34/35 in the same tissue specimen, was found in 19 of the 46 (41%) women. Either human papillomavirus DNA, herpes simplex virus type 2 antigen, or both were found in 91 % of the specimens examined. There was an indication that human papillomavirus types 16, 18, or 31 were detected with gradually increasing frequency with increased age of the patients. The local distribution of the lesions on the vulva was also associated with age, with unifocal disease found significantly more often in patients above age 50 years. Recurrence of vulvar intraepithelial neoplasia during the first year of follow-up was not associated with any specific human papillomavirus type. The presence of human papillomavirus DNA in the normal skin margins was associated with a somewhat higher local recurrence rate than observed in the presence of negative margins. Based on these findings, we conclude that all types of human papillomavirus are associated with vulvar intraepithelial neoplasia, and that the role of individual or multiple infection with both human papillomavirus and herpes simplex virus type 2 in the pathogenesis of neoplasia needs to be elucidated further. We thank Dr. Harald zur Hausen of the Deutches Krebsforschungszentrum, Heidelberg, Germany, for supplying the genomes of human papillomavirus 6, 11, 16, and 18, and Dr. Wayne Lancaster of Georgetown University Medical Center, Washington, D. C., for supplying the genome of human papillomavirus 31. We also appreciate Ms. Socorro Zermeno, Ms, Irma Phillips, Ms. Virginia Ramirez, Ms. Jeryl Silverman, and Ms. Charlotte Haris for their help in preparing the manuscript. REFERENCES 1. Kaufman RH, DreesmanGR, Burek], et aI. Herpesvirusinduced antigens in squamous-cell carcinoma in situ of the vulva. N Engl] Med 1981;305:483. 2. Winkler B, Richart RM. Human papillomavirus and gynecologic neoplasia. Curr Prob Obstet Gynecol Fertil 1987; 10:49.

3. Rawls WE, Adam E. Herpes simplex viruses and human malignancies. Cold Spring Harbor Laboratory: Cold Spring Harbor Conference on Cell Proliferation, 1977; 4:1133. 4. zur Hausen H. Human genitalcancer: Synergism between

twovirusinfectionsor synergismbetweena virusinfection and initiating events. Lancet 1982;2;1370. 5. Manservigi R, Cassai E, Deiss LP, DiLuca D, Segala V, Frenkel N. Sequenceshomologous to two separate transforming regions of herpes simplexDNAare linked in two human genital tumors. Virology 1986;155:192. 6. Macnab]CM, Walkinshaw SA,Cordiner]W, Clements]B. Human papillomavirus in clinically and histologically normal tissue of patients with genital cancer. N Engl] Med 1986;315:1052.

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by herpes simplex virus type 2 in Hep-2 cells. J Virol 1976;19:717. 8. Powell KL, Purifoy DJM. DNA-binding proteins of cells infected by herpes simplex virus type I and type 2. Intervirol 1976;7:225. 9. Adler-Storthz K, Newland JR, Tessin BA, Yeudall WA, Shillitoe EJ. Identification of human papillomavirus types in oral verruca vulgaris. J Oral Pathol 1986;15:230. 10. Matson D, Adler-Storthz K, Adam E, et al. A micro solidphase radioimmunoassay for detection of herpes virus type-specific antibody: Parameters involved in standardization . .J Virol Methods 1983;6:71. 11. Gardner HL, Kaufman RH. Benign diseases ofthe vulva and vagina. Boston: GK Hall, 1981:195-216. 12. Ostrow RS, Marrias DA, Clark BA, Okagaki T, Twiggs LB, Faras AJ. Detection of human papillomavirus DNA in invasive carcinomas of the cervix by in situ hybridization. Cancer Res 1987;47:649. 13. Stoler MH, Broker TR. In situ hybridization detection of human papillomavirus DNAs and messenger RNAs in genital condylomas and a cervical carcinoma. Human Pathol 1986; 17:1250. 14. Zachow KR, Ostrow RS, Bender M, et al. Detection of human papillomavirus DNA in anogenital neoplasia. Nature 1982;300:771. 15. Beaudenon S, Kremsdorf D, Croissant 0, jablonska S, Wain-Hobson S, Orth G. A novel type of human papillomavirus associated with genital neoplasias. Nature 1986; 321:246. 16. di Luca D, Pilotti S, Stefanon B, et al. Human papillomavirus type 16 DNA in genital tumors: a pathological and molecular analysis. J Gen Virol 1986;67:583. 17. de Villires EM, Schneider A, Gross G, zur Hausen H. Analysis of benign and malignant urogenital tumors for human papillomavirus infection by labelling cellular DNA. Med Microbiol ImmunoI1986;174:287. 18. Gross G, Ikenberg H, Hagedorn M, Gissmann L. Nachwei's von papillomavirus capsidantigenen und human papillomavirus (HPV) 16 verwaandter DNS in bowenoider papulose und morbus bowen. Z Hantkr 1984; 59:1084. 19. McCance DJ, Clarkson PK, Dyson JL, Walker PG, Singer A. Human papaillomavirus types 6 and 16 in multifocal intraepithelial neoplasiaas of the female lower genital tract. Br J Obstet Gynaecol 1985;92: 1093. 20. Pfister H, Haneke E. Demonstration of human papillomavirus type 2 DNA in Bowen's disease. Arch Dermatol Res 1984;276: 123. 21. Gupta J, Pilotti S, Rilke F, Shah K. Association of human papillomavirus type 16 with neoplasitc lesions of the vulva and other gnital sites by the in situ hybridization. Am J Pathol 1987;127:206. 22. Cabral GA, Marciano-Cabral F, Fry D, Lumpkin CK, Mercer L, Goplerud D. Expression of herpes simplex virus type 2 antigens in premalignant and malignant human vulvar cells. AM J OBSTET GYNECOL 1982;143:611. 23. Alonio LV, Dalbert D, Mural J, et al. Herpes simplex virus type 2 and papillomavirus antigens in different lesions of the uterine cervix. Cervix Lower Fern Genital Tract 1986;4:47. 24. Prakash SS, Reeves WC, Sisson GR, et al. Herpes simplex virus type 2 and human papillomavirus type 16 in cervicitis, dysplasia, and invasive cervical carcinoma. Int J Cancer 1985;35:51. 25. Meanwell CA, Blackledge G, Cox MF, Maitland NJ. HPV 16 DNA in normal and malignant cervical epithelium: implications for the aetiology and behaviour of cervical neoplasia. Lancet 1987;1:703. 26. Reeves WC, Caussy D, Brinton LA, et al. Case-control study of human papillomaviruses and cervical cancer in Latin America. Int J Cancer (in press). 27. Pfister H. Biology and biochemistry of papillomaviruses. Rev Physiol Biochem Pharmacol 1984;99: Ill.

28. Rando RF, Groff DE, Cirikjian JG, Lancaster WD. Isolation and characterization of a novel human papillomavirus type 6 DNA from an invasive vulvar carcinoma. J Virol 1986;57:353. 29. Mabucki K, Bross DS, Kessler II. Epidemiology of cancer of the vulva. A case control study. Cancer 1985;55:1843. 30. Ferenczy A, Mital M, Nagai N, Silverstein SJ, Crum CP. Latent papillomavirus and recurring genital warts. N Engl J Med 1985;313:784.

Discussion DR. GEORGE D.

WILBANKS, Chicago, Illinois. Dr. Kaufman and his associates have been interested in vulvar neoplasia and viral disease for many years. This is an important report of their current findings in carcinoma in situ of the vulva related to human papillomavirus and herpes simplex virus as possible causative agents. Most researchers agree that the epithelial malignancies of the lower genital tract are probably not caused by a single agent but by some combination of agents or factors. This is the largest series of such a combination of data. My remarks are directed to three areas: first, the patients; second, the methods; and finally, the results. They initially studied 62 patients from 21 to 79 years of age with 16 "quality control" patients excluded. Although this seems to be a large number of exclusions (25%), each seems appropriate. Eight patients had only normal epithelium in the biopsy specimen, six had insufficient epithelium for either herpes simplex virus or human papiIIomavirus analyses, and two lesions reacted with the plasmid vector. Is there any significance of the hybridization to the plasmid and has this occurred in other tissues studied? Of the remaining 46 patients, 34 patients had two specimens, that is, one from the carinoma in situ and one from normal skin 1.5 em distal to the lesion. There were a total of nine recurrences in 43 patients who had been followed up for ~ 1 year, and three patients were not available for follow-up. The methods seem rather standard and the Kaufman group have expertise in these various methodologies. Parallel specimens taken for hematoxylin and eosin examination were used to control the type of epithelium in the immunologic tests. The materials were "snap frozen until tested." Were the analyses of all of the specimens for one type of test, i.e., herpes simplex virus type 2, human papillomavirus type 1, etc., performed at the same time? This has to do with some variance in the tests resulting from different batches of antigens or other variables included in the test. In situ hybridization allows localization ofthe antigenantibody reaction to specific sites but is not as sensitive as extraction procedures such as Southern hybridization. Are the results reasonably parallel to what Dr. Kaufman would expect from Southern hybridization? The herpes simplex virus type 2 CSP-34/35 antigen is related to herpes simplex virus type 2. A certain number of genital herpes infections are caused by herpes simplex virus type 1. Would Dr. Kaufman com-