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Effect of Human Papillomavirus on Vulvar, Vaginal, and Anal Intraepithelial Lesions and Vulvar

Monika Hampl, MD, Heidi Sarajuuri, MD, Nicolas Wentzensen, MD, Hans G. Bender, MD, and Volkmar Kueppers, MD

OBJECTIVE: Human papillomavirus (HPV) is a necessary women aged younger than 56 years (77%), than in those cause for , and it has been associated with aged 56 years or older (41%). vulvar and and vulvar (VIN) and vaginal CONCLUSION: Based on the data obtained in this study, (VaIN) and anal (AIN) intraepithelial neoplasia. We as- widely-implemented prophylactic HPV vaccination could sessed the prevalence of HPV (and the types) to estimate make an important contribution to the reduction of the the possible effect of a HPV on lower genital tract risk for cervical cancer and could also prevent about half disease prevention. the vulvar carcinomas in younger women and about two METHODS: Two hundred fifty-eight samples of VIN, thirds of the intraepithelial lesions in the lower genital VaIN, AIN, and from 241 women were tract. included in the study. The diagnosis of surgical samples (Obstet Gynecol 2006;108:1361–8) was made using published histomorphologic criteria. The LEVEL OF EVIDENCE: II-3 DNA was extracted for HPV detection and typed using polymerase chain reaction and sequencing. RESULTS: The analyses were performed on 210 intraepi- ulvar cancer is the fourth most common gyneco- thelial neoplasia samples (VIN2/3, VaIN2/3, AIN2/3) and Vlogic cancer, with an incidence of 1.5–2 per 48 vulvar carcinoma samples. Human papillomavirus 100,000 women per year in Germany; it is six times DNA was detected in 92%, 91%, 89%, and 60% of the less frequent than cervical cancer (12 per 100,000 VIN, VaIN, AIN, and vulvar carcinoma samples, respec- women per year). In Germany, 559 women died from tively. High-risk HPV types 16 or 18 were detected in vulvar cancer in 1999. In the it was 76%, 64%, 81%, and 42% of the VIN2/3, VaIN2/3, AIN, estimated that there would be 3,740 new cases and and vulvar carcinoma samples. Women with HPV-posi- 880 deaths in 2005.1 Cigarette , vulvar dys- tive samples were younger than those with HPV-negative trophy, vulvar intraepithelial neoplasia (VIN), persis- samples (46 years compared with 55 years and 51 years tent human papillomavirus (HPV) infection, immu- compared with 61 years, for the VIN2/3 and vulvar nodeficiency syndromes, previous history of cervical carcinoma samples, respectively). Human papillomavi- intraepithelial neoplasia or cancer, and north- rus–positive vulvar carcinoma was more frequent in ern European ancestry have been identified as risk factors for the development of vulvar cancer. Vulvar From the Department of Gynecology and Obstetrics, Institute of , intraepithelial neoplasia are precursor lesions for in- University Hospital, Heinrich Heine University, Duesseldorf, Germany; Depart- vasive squamous carcinoma of the and their ment of Molecular Pathology/Applied Tumor Biology, Institute of Pathology, University of Heidelberg, Heidelberg, Germany; and Clinic, Koenig- incidence is currently estimated to be 2.1 per 100,000 sallee, Duesseldorf, Germany. women per year. The authors thank Gabrielle Breugelmans for help with the data analyses and There has been a striking increase in the inci- Bennett Lee and Margaret Haugh for help in drafting this article. dence of VIN and vulvar carcinoma over the past 30 Corresponding author: Monika Hampl, MD, Department of Gynecology and years, especially in younger women.2–7 The observed Obstetrics, University Hospital of Duesseldorf, Moorenstr. 5, 40 225 Duessel- increase in younger women could be due to a higher dorf, Germany; e-mail: [email protected]. incidence of infection of the lower genital tract with © 2006 by The American College of Obstetricians and Gynecologists. Published by Lippincott Williams & Wilkins. human papillomavirus or it may be explained by ISSN: 0029-7844/06 better diagnostic procedures or both.

VOL. 108, NO. 6, DECEMBER 2006 OBSTETRICS & GYNECOLOGY 1361 Vulvar carcinoma can be HPV-positive or HPV- prevalence of HPV infection in women with CIN and negative.8,9 Human papillomavirus–positive tumors cervical . Similar data for VIN, vaginal intra- tend to be nonkeratinizing basaloid or warty tumors, epithelial neoplasia (VaIN), anal lesions (AIN) and often surrounded by bowenoid-type vulvar intraepi- vulvar cancers are sparse, partly because these condi- thelial neoplasia.10–12 Human papillomavirus 16 is the tions are rarer. However, because HPV is known to most frequently observed in these tu- be present in VIN, AIN, and VaIN lesions and in mors.2,10,11,13–16 Human papillomavirus–positive tu- vulvar cancers, it was decided to analyze the HPV mors, which are associated with younger patients, types present in surgical samples from a cohort of 241 have been diagnosed more frequently in recent de- women from two centers in Duesseldorf, Germany. cades and are thought to have a better than The aim was to determine the frequency of high-risk the HPV-negative tumors.2,5,7,11,17–20 HPV types in these lesions, and to use these data to Human papillomavirus–negative tumors are estimate the possible effect of the prophylactic HPV more frequently found in older women, often with a vaccines on the incidence of VIN, VaIN, AIN, and history of nonneoplastic epithelial disorders of the vulvar cancer. vulvar , such as lichen sclerosis.10,11,21 These are differentiated keratinizing MATERIALS AND METHODS and the corresponding precursor lesions are called A total of 258 samples from 241 patients “simplex” or well-differentiated VIN.9,11,21 These lat- diagnosed with lower genital tract dysplastic lesions ter tumors are usually unifocal, more aggressive, and (vulvar [VIN], vaginal [VaIN], anal [AIN], or squa- they tend to recur and metastasize earlier.18,20 mous cell carcinoma of the vulva) were analyzed in At least 70% of women are infected with HPV this study. The specimens were collected between during their lifetime, and this virus is responsible for January 2000 and April 2006 from consecutive the most common sexually transmitted infections women referred to one of the two specialized centers worldwide. However, most HPV infections are in the area of Duesseldorf, Germany, the Dysplasia and clear spontaneously within 12–18 Clinic of the Department of Gynecology and Obstet- months. Less then 10% of women have persistent rics at the University Hospital (center 1) or the infection but this is a for the development Dysplasia Outpatient Clinic, Koenigsallee (center 2). of lower genital tract intraepithelial dysplasia, pre- The study protocol was approved by the institu- dominantly located at the cervix (cervical intraepithe- tional review boards at both sites. All patients under- lial neoplasia: CIN). When untreated, these precursor went surgical treatment consisting of either CO2 lesions can progress to invasive cervical cancer in a vaporization after biopsy, CO2 laser excision or knife gradual process that might take between 10 and 12 excision for vulvar, vaginal, or anal intraepithelial years from primary infection to the development of neoplasia. Vulvar squamous cell carcinomas were invasive cancer.22 treated by wide local excision or , includ- The results from epidemiologic studies of cervical ing complete inguinofemoral lymphadenectomy. Be- cancer specimens show that 99.7% of these tumors are fore , all women gave their signed informed HPV-positive, with HPV 16 and 18 being responsible consent to inclusion in this study. In the majority of for approximately 70% of the tumors.23 Two prophy- women, a -guided biopsy was performed lactic vaccines have been developed to prevent infec- before surgical treatment to confirm the diagnosis. tions with these high-risk HPV types, aiming to Only surgical specimens of histologically confirmed reduce the worldwide incidence of these tumors.24–26 intraepithelial neoplasia of the , vulva, or anal Initial trials have shown highly efficient protection region or vulvar carcinoma were included in the against infections with HPV 16 and 18. In addition, study. The specimens were examined by an experi- one of the two vaccines is directed against two enced gynecologic pathologist and the diagnosis was low-risk HPV types, 6 and 11, that are responsible for based on published histomorphologic criteria.27 For 90% of genital . At the time that this article was each patient the location and number of lesions, their written, the quadrivalent vaccine (HPV 6/11/16/18) age at diagnosis, smoking habits, prior history of HPV has been granted product licenses for the United induced diseases (ie, cervical HPV-induced disease, States and Australia. Furthermore, in Europe it has condyloma, multicentric HPV-induced lesions) and received a favorable opinion from the European their human virus (HIV) status Medicines Agency’s Committee for Medicinal Prod- were recorded. ucts for Human Use. All histologic and molecular tests were performed Recent studies have provided more data on the under blind conditions. Single-use scalpels were used

1362 Hampl et al HPV in VIN and Vulvar Carcinoma OBSTETRICS & GYNECOLOGY to prevent carry-over contamination in the subse- Positive PCR products were purified using the quent DNA extraction and polymerase chain reac- High Pure PCR product purification (Roche Diag- tion (PCR) amplification. The histologic diagnosis nostics, Mannheim, Germany) according to the man- was made on serial sections of formalin-fixed par- ufacturer’s instructions. The sequence of one strand of affin-embedded tissue samples. Serial sections (5 the purified PCR fragments was determined with the mcm) of intraepithelial neoplastic lesions or inva- Big-Dye Terminator sequencing kit (Applied Biosys- sive vulvar cancer from the paraffin blocks contain- tems) using 3–5 pMol of GP6ϩ or MY09 as sequenc- ing the most disease-specific changes were selected ing primers, and analyzed on an ABI Prism 310 for HPV DNA analysis by PCR and sequencing. automated sequencer (Applied Biosystems). These After deparaffinization, genomic DNA was ex- sequences were compared with the documented virus tracted using the QIAamp DNA Mini Kit (QIA- sequences available in the GenBank databank using GEN, Hilden, Germany) according to the manufac- the BLAST Program (Blast, Pittsboro, NC). When turer’s instructions. The quality of the extracted multiple fluorescent peak patterns were observed in DNA was evaluated by spectrophotometry (Beck- the GENESCAN analyzes, type-specific PCR for the man Coulter, Krefeld, Germany). seven most common HPV types 6, 11, 16, 18, 31, 33 Human papillomavirus DNA was detected using and 51 was performed.31 Stratified analyses were a two-tier PCR-direct sequencing method, modified performed by age, , and center, and a two- as indicated below.28,29 We used the general primers sample Wilcoxon rank sum test, Pearson ␹2 test, or t GP5ϩ/GP6ϩ30 and the consensus primer pair MY09 test, were applied, where applicable, to test for signif- and MY11 for amplification of the corresponding part icant differences. Furthermore, univariable and mul- of the HPV L1 gene, with different fluorescent label- tivariable data analyses were conducted using logistic ing of each of the forward primers. A 20-mcL stan- regression, to examine factors independently associ- dard PCR (3 mcL DNA, 2 mcL 10ϫPCR buffer, 0.2 ated with HPV status (ie, positive or negative for mM dNTPs, 20 pmol primer each, MgCl2 1.5 mM, 1 HPV). All data analyses were conducted using Unit Taq polymerase) was run with 40 cycles of STATA 8.2 (StataCorp, College Station, TX). P values denaturation at 94°C for 20 seconds, and annealing presented are two-tailed and the results were consid- for 20 seconds at 39°C for the general primers and at ered statistically significant at PՅ.05). 55°C for the MY09, MY11, and ␤-globin primers, followed by an extension at 72°C for 40 seconds. The RESULTS initial denaturation was performed at 94°C for 5 During our study revised guidelines for VIN termi- minutes, and the final extension was performed at nology from the International Society for the Study of 72°C for 5 minutes. The integrity of human genomic Vulvovaginal Disease were published. Before this, DNA was verified by PCR amplification of a 268 squamous vulvar intraepithelial neoplasia were clas- base-pair fragment of the ␤-globin gene. The DNA sified as either low-grade lesions (VIN 1), medium- extracted from HPV 16-positive Caski cells was used grade lesions (VIN 2), or high-grade lesions, including as a positive control, and for the negative control, (VIN 3) In the new guidelines, the water was used as template instead of DNA. The term VIN 1 was no longer recommended for use, and lower limit of the number of viral copies to result in a the term “VIN” now encompasses the former VIN 2 positive test was estimated to be 300–400 copies of and VIN 3 lesions. We, therefore, eliminated the HPV depending on the type. samples that were classified as VIN 1 (nϭ14), corre- An aliquot (0.8 mcL) of each of the three PCR sponding to 13 women.32 We combined the samples products was mixed with 10 mcL formamide and 0.5 classified as VIN 2 and VIN 3 from 168 women into ␮L internal standard (GENESCAN 500-ROX; Ap- a single diagnostic category termed VIN2/3 (nϭ183). plied Biosystems, Foster City, CA). After denatur- We also combined the VaIN 2 and VaIN 3 samples ation at 90°C for 2 minutes and cooling, the PCR from 11 women into one category termed VaIN2/3 products were analyzed on an automated ABI 310 (nϭ11), and the AIN2/3 samples from 14 women genetic analyzer (Applied Biosystems). The PCR were analyzed separately (nϭ16). Thus, 210 sam- product sizes were determined using GENESCAN ples of histologically confirmed nondifferentiated 2.1.1 software (Applied Biosystems). We controlled intraepithelial neoplastic lesions of the lower geni- for inter- and intra-assay variation by monitoring the tal tract from 193 women (17 women had two fluorescence in the positive controls in each run and lesions) and 48 specimens of microinvasive and by duplicate testing of randomly selected samples in invasive vulva carcinoma from 48 women were one run. included in the analysis.

VOL. 108, NO. 6, DECEMBER 2006 Hampl et al HPV in VIN and Vulvar Carcinoma 1363 Multifocal disease was observed in 27% (70 of Table 2. Human papillomavirus infections in 258) of the samples. Of the 48 samples of invasive vulvar cancer samples as a function of vulva carcinoma, 11 were diagnosed as microinvasive age at diagnosis carcinoma (depth of infiltration less than 1 mm) HPV- HPV- arising from VIN 3. Only two patients were seropos- Age at diagnosis (y) Positive Negative Total itive for HIV, and none of the other women had any Younger than 56 20 (77)* 6 (23) 26 (100) immunodeficiency conditions. The two HIV-infected 56 or older 9 (41) 13 (59) 22 (100) patients were aged 21 and 41 years. The 21 year old Total 29 (60) 19 (40) 48 (100) had multicentric disease with a HPV 16–positive VIN HPV, human papillomavirus. 3 and CIN 3, and the 41 year old presented with a Data are n (%). * Vulvar cancers in younger women (younger than 56 years at HPV 16–positive VIN3 with microinvasive cancer, diagnosis) are four times more often associated with HPV depth of invasion less then 1 mm. No evidence of infection than those occurring in older women (older than 56 PCR amplification was observed for the negative years) (odds ratio 4.3, 95% confidence interval 1.2–15.4), inde- control samples run in each PCR test, and HPV 16 pendent of the center where they were treated. DNA was detected in the positive control samples (HPV 16-positive Caski cells). Females in center 2 with a VIN2/3 were, inde- The mean age of the patients is listed in Table 1, pendent of age, more likely to have HPV-positive as well as the number of HPV positive samples (92%, samples compared with center 1 (Pϭ.03). This differ- 91%, 88%, and 60% of the VIN2/3, VaIN2/3, ence in HPV positivity between centers was not seen AIN2/3, and vulvar carcinoma samples, respectively). in women with vulvar carcinoma, VaIN2/3, or Women with VIN2/3 and vulvar carcinoma HPV- AIN2/3. Testing for effect modification between HPV positive samples were significantly younger than positivity, age, and center did not show any significant those with HPV-negative samples (PϽ.05; Table 1). differences. This was observed in both treatment centers. As The majority of the HPV-positive samples (91%, expected, HPV-positive vulvar carcinoma was more 90%, 71% ,and 83%, for the VIN2/3, VaIN2/3, AIN, frequent in women aged younger than 56 years (77%), and vulvar carcinoma samples, respectively) con- than in those aged 56 years or older (41%); adjusting tained only high-risk HPV types (single or multiple for center, this difference was statistically significant infections: Table 3). Low-risk HPV types only were (odds ratio [OR] 4.3, 95% confidence interval [CI] observed in less than 5% of the samples. Infections 1.2–15.4) (Table 2). However, we identified a HPV- with two or more different HPV types were present in positive vulva carcinoma (HPV 6, 31, and 68) in an 13%, 10%, 21%, and 13% of the VIN2/3, VaIN2/3, 83-year-old and a HPV-negative microcarci- AIN, and vulvar carcinoma samples, respectively. noma with 1.2-mm infiltration and without surround- The distribution of HPV types is summarized in ing in a 32-year-old woman. Table 3. High-risk HPV types 16 or 18 were found in

Table 1. Human Papillomavirus Infections in Samples From Women With Vulvar, Vaginal, and Anal Intraepithelial Neoplasia and Vulvar Carcinoma* VIN2/3 VAIN2/3 AIN2/3 Vulvar Carcinoma Women (Nϭ241) 168 11 14 48 Samples (Nϭ258) 183 11 16 48 Mean age at diagnosis (y) 47 (Ϯ12.3) 49 (Ϯ14.3) 43 (Ϯ15.2) 55 (Ϯ15.5) Women current or previous smokers 141 5 10 25 Women with multifocal disease 60 1 6 3 Women with HPV-induced multicentric disease* 89 2 7 6 White 168 11 14 48 Immunodeficiency syndrome 1 0 0 1 HPV-positive 169 (92.3) 10 (90.9) 14 (87.5) 29 (60.4) Mean age at diagnosis (y) 46 (Ϯ11.7) 48 (Ϯ14.0) 46 (Ϯ14.3) 51 (Ϯ15.4) HPV-negative 14 (7.6) 1 (9.1) 2 (12.5) 19 (39.6) Mean age at diagnosis (y) 55 (Ϯ15.7) 65 (—) 28 (Ϯ14.1) 61 (Ϯ13.9) VIN2/3, vulvar intraepithelial neoplasia; VaIN2/3, vaginal intraepithelial neoplasia; AIN2/3, anal intraepithelial neoplasia; HPV, human papillomavirus. Data are n, n (Ϯstandard deviation), or n (%). * Multicentric HPV-induced disease includes history of or concordant condyloma, cervical, vaginal or anal intraepithelial neoplasia.

1364 Hampl et al HPV in VIN and Vulvar Carcinoma OBSTETRICS & GYNECOLOGY Table 3. Distribution of Human Papillomavirus Types Within the Different Sample Classifications VIN 2/3 VAIN 2/3 AIN2/3 Vulvar Carcinoma Total number of samples (Nϭ258) 183 11 16 48 HPV positive (nϭ222) 169 10 14 29 Only high-risk HPV (type) 154 9 10 24 HPV 16 134 7 13 19 HPV 33 18 0 0 4 HPV 31 7 0 0 2 HPV 18 5 0 0 1 Only low-risk HPV 8 0 0 2 Rare or unknown-risk HPV 1 0 2 1 Single HPV infection 147 9 11 25 Single high-risk HPV infection 139 9 10 22 Single low-risk HPV infection 7 0 0 2 Multiple HPV infection 22 1 3 4 VIN2/3, vulvar intraepithelial neoplasia; VaIN2/3, vaginal intraepithelial neoplasia; AIN2/3, anal intraepithelial neoplasia; HPV, human papillomavirus. Data are number of samples with each HPV type.

139 of 183 VIN 2/3 (76%), 7 of 11 VAIN2/3 (64%), in four VIN2/3 lesions would be prevented, because and 13 of 16 AIN 2/3 specimens (81%). In addition, the vaccine has been shown to prevent 100% of 20 of 48 (42%) vulvar cancer specimens tested posi- infections by HPV 16 and 18. In addition, HPV 16 tive for HPV 16 or 18, and therefore a total of 179 of and 18 were detected in 58% (15/26) of the vulvar 258 lesions included in this study (69%) were HPV 16 carcinoma samples in the subgroup of younger or 18 induced. The low-risk HPV types 6 and 11 were women (younger than 56 years); and therefore, wide- only found in 14 samples (5%). High-risk HPV types spread vaccination could prevent more than one half 16/18 were detected in 76%, 70%, 93%, and 42% of of the vulvar carcinomas in these women. the VIN2/3, VaIN2/3, AIN2/3, and vulvar carci- noma samples. Human papillomavirus 16 (nϭ134) DISCUSSION and 33 (nϭ18) were the most common HPV types Here we report the results from a large case series identified in the VIN2/3 samples. Human - from Du¨ sseldorf, Germany in which sensitive HPV virus 16 was the only high-risk type found in the DNA-detection techniques (PCR and DNA sequenc- VaIN2/3 and AIN samples. The most frequent HPV ing) were used. In this case series of 258 samples of type in the vulvar carcinoma samples was HPV 16 intraepithelial neoplasia of the vagina, vulva, and (nϭ19; 66%), followed by HPV 33 DNA (nϭ4; 14%). anus and invasive carcinoma from 241 women, the Independent of treatment center, younger women median age was 48 years, which is young, but pro- (younger than 56 years) with vulvar carcinoma were vides further evidence of the increasing frequency of 4.4 times more likely to be infected with HPV type16 these lesions in younger women in recent years.6,33 compared with older women (OR 4.4, 95% CI 1.19– A literature search performed in PubMed (http:// 16.36). This difference was not observed in women www.ncbi.nlm.nih.gov) using the search terms vulvar with VIN2/3, VAIN 2/3, or AIN 2/3. carcinoma, HPV, and pathogenesis from 1970 to May Human papillomavirus 18 was identified in only 2006 with no language restrictions showed that pres- 6 samples (5 VIN2/3 and 1 vulvar carcinoma). Only ence of HPV DNA in VIN and vulvar cancerous rare HPV types or HPV with unknown risk (eg, HPV lesions has been reported to vary from 0% to 40, 53, 55, 66, 67, 91, and HPV IA 18) were identified 89%.2,8–17,20,34–47 The published studies have generally in one sample each of the VIN2/3, AIN2/3, and vulvar involved relatively few samples and the methods used carcinoma samples. In total, 26 different HPV types for HPV DNA detection have had different sensitiv- (6,11,16,18,31,33,35,40,42,44,45,51,52,53,55,56,58,61, ities. This wide variation is probably due to the small 66,67,68,73,74,91,IS887/MM4, and HPVIA18) were sample sizes, differences in patients recruited and identified in the samples examined. HPV detection methods used, and the year of Human papillomavirus 16 and 18 were detected publication. in 76% of the VIN2/3 lesions (139 of the 183 lesions In our study, HPV DNA was detected in 86% of analyzed); therefore, if the prophylactic HPV vaccine all samples (Table 1), which seems to be high com- were to be implemented, we could expect that three pared with the percentages reported in other studies.

VOL. 108, NO. 6, DECEMBER 2006 Hampl et al HPV in VIN and Vulvar Carcinoma 1365 However, many of these previous studies were con- 98%, with a persistent antibody titer for at least 5 years. ducted more than 10 years ago. In addition, our The efficacy for preventing HPV 6-, 11-, 16- and 18- results, which show that 60% of the vulvar carcinomas related genital warts, VIN, and vaginal or vulvar carci- and 92% of the VIN2/3, VaIN2/3, and AIN samples noma after 2 years follow-up in 2,261 vaccine and 2,279 were HPV-positive (Table 1), are similar to results placebo recipients was 100% (95% CI 88–100%): 40 from the most recent studies, which reported 60–85% histologically confirmed events in the placebo group HPV-positive vulvar carcinomas2,20 and 71–80% and none in the vaccine group (Villa et al. Oral presen- HPV-positive high-grade VIN.39,48 tation at EUROGIN 2006, Paris, France, April 24–26, Many epidemiologic studies assessing the presence 2006). Thus, a prophylactic vaccine for HPV 16 and 18 of HPV in lower genital tract carcinomas have focused might be effective in reducing the incidence of high- on cervical carcinoma. Their results show that HPV grade VIN, VaIN, and AIN, because HPV 16 and 18 DNA can be detected in more than 99% of the lesions, were detected in 76% of the VIN2/3, VaIN2/3, and thus establishing the role of HPV infection as a neces- AIN2/3 lesions (159 of a total of 210 lesions analyzed). sary cause of these carcinomas.23,49 In contrast, until In addition, although HPV 16 and 18 were detected in recently, few studies have examined the role of HPV 42% of the vulvar carcinoma samples, independent of infection in vulvar carcinoma.2,8–17,20,33,34,36–44 the patient’s age (20 of 48 samples analyzed), in the The number of HPV-positive invasive vulvar subgroup of younger women (younger than 56 years; carcinoma in younger woman has increased in the last nϭ26), HPV 16 and 18 were detected in 58% (15/26) of decade from about 2–5% to more than 20% of all the samples. cases of invasive vulvar carcinoma.2,5,7,19 We observed Although the incidence of vulvar cancer has a similar trend toward younger age in our cohort of increased over the past 30 years4,6 and is still increas- patients with invasive vulvar carcinoma. The increase ing (in the United States it is estimated to be increas- in the incidence of VIN with microinvasion, and of ing at a rate of 1.2% per year1) vulvar lesions remain invasive carcinoma in younger woman seems to be less frequent than cervical lesions. Although the re- associated with the increased incidence of HPV infec- sults from a recent systematic review suggest that tion of the lower genital tract. although some VIN lesions do progress to invasive In the majority of women with basaloid and vulvar carcinoma, the progression rate is low.53 How- warty type of vulvar cancer (more frequent among ever, the treatment of vulvar lesions is often mutilat- younger women),11,50 HPV 16, 18, 31, and 33 are the ing and traumatic for women, and the high-grade most frequent HPV types detected, whereas HPV 45, lesions have an elevated risk of recurrence. Therefore, 6, and 11 are rarely detected.2,7,13–16,20,35,39,44,51 Our data prevention of these lesions would reduce the number are consistent with these findings, with HPV 16 being, of multiple treatment sessions, mutilating operations, by far, the most common type (76% of the HPV- and trauma in women with these lesions. Prophylactic positive samples) followed by HPV 33 (10%). HPV vaccination might have a large effect on the This analysis has been performed on one of the control of HPV-associated human cancers, and could largest series of samples, using PCR techniques that make a significant contribution to the reduction of the are very sensitive for detecting HPV. However, in our risk for cervical as well as that for vulvar cancers in study, the samples were paraffin-embedded, and women. We conclude that if prophylactic HPV vac- therefore the PCR technique is not as sensitive as with cination is widely implemented, approximately one non–paraffin-embedded samples. We estimate that half of the vulvar carcinomas in younger women the sensitivity is between 100 and 300 , de- (younger than 56 years) (current incidence: approxi- pending on the HPV type, compared with sensitivity mately 1 per 100,000 women per year), and approx- reported in the original publication of the method, imately two thirds of the intraepithelial precursor which was 10 viruses. Therefore, our analysis may lesions in the lower genital tract (current incidence: underestimate the presence of HPV in our samples. 2.1 per 100,000 women per year) might be prevented. Despite this limitation, our results confirm those 2,8–17,20,33–46 obtained in the previously reported studies. REFERENCES Prophylactic HPV vaccines have been reported to 1. Ries LAG, Harkins D, Krapcho M, Mariotto A, Miller BA, be highly immunogenic and well-tolerated with no Feuer EJ, et al, editors. SEER Cancer Statistics Review, severe adverse events.24,26,52 Results from phase III stud- 1975-2003. 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