Articles

Prevalence of human papillomavirus DNA and p16INK4a

positivity in vulvar cancer and vulvar intraepithelial
neoplasia: a systematic review and meta-analysis
Zhuang Li*, Penglin Liu*, Ziying Wang, Zhaoyang Zhang, Zhongshao Chen, Ran Chu, Guiju Li, Qiuyue Han, Yong Zhao, Li Li, Jinwei Miao†,
Beihua Kong†, Kun Song†

Summary
Background Human papillomavirus (HPV) DNA and p16INK4a positivity have crucial roles in the pathogenesis of vulvar Lancet Oncol 2023
cancer and vulvar intraepithelial neoplasia. We aimed to examine the pooled prevalence of HPV DNA and p16INK4a Published Online
positivity in vulvar cancer and vulvar intraepithelial neoplasia worldwide. March 15, 2023
https://doi.org/10.1016/
S1470-2045(23)00066-9
Methods In this systematic review and meta-analysis, we searched PubMed, Embase, and the Cochrane Library
*Contributed equally as joint
databases for studies published between Jan 1, 1986, and May 6, 2022, that reported the prevalence of HPV DNA, or first authors
p16INK4a positivity, or both, in histologically verified vulvar cancer or vulvar intraepithelial neoplasia. Studies on a †Contributed equally as joint last
minimum of five cases were included. Study-level data were extracted from the published studies. Random effect authors
models were used to examine the pooled prevalence of HPV DNA and p16INK4a positivity in both vulvar cancer and Department of Obstetrics and
vulvar intraepithelial neoplasia, which were further investigated using stratified analyses by histological subtype, Gynaecology, Qilu Hospital of
geographical region, HPV DNA or p16INK4a detection method, tissue sample type, HPV genotype, publication year, and Shandong University, Jinan,
Shandong, China (Z Li MSc,
age at diagnosis. Additionally, meta-regression was applied to explore sources of heterogeneity. Z Wang MSc, Z Zhang MSc,
Z Chen MD, R Chu MD, G Li Msc,
Findings We retrieved 6393 search results, of which 6233 were excluded for being duplicates or after application of our Q Han MSc, Y Zhao MSc, L Li PhD,
inclusion and exclusion criteria. We also identified two studies from manual searches of references lists. 162 studies Prof B Kong PhD,
Prof K Song PhD); Department
were eligible for inclusion in the systematic review and meta-analysis. The prevalence of HPV in vulvar cancer of Gynecological Oncology,
(91 studies; n=8200) was 39·1% (95% CI 35·3–42·9) and in vulvar intraepithelial neoplasia (60 studies; n=3140) was Beijing Obstetrics and
76·1% (70·7–81·1). The most predominant HPV genotype in vulvar cancer was HPV16 (78·1% [95% CI 73·5–82·3]), Gynaecology Hospital, Capital
followed by HPV33 (7·5% [4·9–10·7]). Similarly, HPV16 (80·8% [95% CI 75·9–85·2]) and HPV33 (6·3% [3·9–9·2]) Medical University, Beijing
Maternal and Child Health Care
were also the most two predominant HPV genotypes in vulvar intraepithelial neoplasia. The distribution of type- Hospital, Beijing, China
specific HPV genotypes in vulvar cancer among geographical regions was different, with HPV16 varying between (P Liu MD, Prof J Miao PhD)
regions, showing a high prevalence in Oceania (89·0% [95% CI 67·6–99·5]) and a low prevalence in South America Correspondence to:
(54·3% [30·2–77·4]). The prevalence of p16INK4a positivity in patients with vulvar cancer was 34·1% (95% CI 30·9–37·4; Dr Kun Song, Department of
52 studies; n=6352), and it was 65·7% (52·5–77·7; 23 studies; n=896) in patients with vulvar intraepithelial neoplasia. Obstetrics and Gynaecology, Qilu
Hospital of Shandong University,
Furthermore, among patients with HPV-positive vulvar cancer, p16INK4a positivity prevalence was 73·3% (95% CI Jinan, Shandong 250012, China
64·7–81·2), compared with 13·8% (10·0–18·1) in HPV-negative vulvar cancer. The prevalence of double positivity for songkun2001226@sdu.edu.cn
HPV and p16INK4a was 19·6% (95% CI 16·3–23·0) in vulvar cancer and 44·2% (26·3–62·8) in vulvar intraepithelial
neoplasia. Most analyses had large heterogeneity (I²>75%).

Interpretation The high prevalence of HPV16 and HPV33 in vulvar cancer and vulvar intraepithelial neoplasia
emphasised the importance of nine-valent HPV vaccination in preventing vulvar neoplasm. Additionally, this study
highlighted the potential clinical significance of double positivity for HPV DNA and p16INK4a in vulvar neoplasm.

Funding Taishan Scholar Youth Project of Shandong Province, China.

Copyright © 2023 Elsevier Ltd. All rights reserved.

Introduction female genital tumours.4 Vulvar cancer can be preceded

Vulvar cancer, as a serious gynaecological malignancy,
accounts for about 5% of all female genital tract cancers.1
Vulvar squamous cell carcinoma is the most predominant
histological subtype of vulvar cancer and accounts for
more than 90% of all cases, including keratinising, non-
keratinising, basaloid, warty, and verrucous carcinoma,2,3
which can further be classified as HPV-associated or
HPV-independent vulvar squamous cell carcinoma
according to the 2020 WHO classification criteria for
by vulvar intraepithelial neoplasia, which is classified as
low-grade squamous intraepithelial lesions, high-grade
squamous intraepithelial lesions, and differentiated
vulvar intraepithelial neoplasia.5–7 Infection with high-risk
human papillomavirus (HPV) is a well established cause
of vulvar cancer and vulvar intraepithelial neoplasia.8
Additionally, low-risk HPV genotypes, such as HPV6 and
HPV11, have been identified as the potential cause of
vulvar cancer and vulvar intraepithelial neoplasia.4

www.thelancet.com/oncology Published online March 15, 2023 https://doi.org/10.1016/S1470-2045(23)00066-9 1

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Research in context
Evidence before this study
We searched the PubMed, Embase, and Cochrane Library
databases to identify studies published in English between
Jan 1, 1986, and May 6, 2022, using the terms “vulvar cancer”
OR “vulvar intraepithelial neoplasia” AND “p16” OR “HPV”. Four
meta-analyses estimated the pooled prevalence of HPV DNA in
vulvar cancer and vulvar intraepithelial neoplasia. However,
these meta-analyses only included studies published up to
2015, after which the 2020 edition of the WHO classification
criteria for female genital tumours was published and
numerous relevant studies have been published. Furthermore,
to the best of our knowledge, no previous systematic reviews
and meta-analyses have estimated the prevalence of p16INK4a
positivity in vulvar cancer or vulvar intraepithelial neoplasia,
and no meta-analyses have examined the prevalence of double
positivity for HPV DNA and p16INK4a in vulvar intraepithelial
neoplasia or vulvar cancer.
Added value of this study
In this systematic review and meta-analysis, we added more
than 3000 new cases and estimated an HPV DNA prevalence of
39·1% (95% CI 35·3–42·9) in vulvar cancer and 76·1%
(70·7–81·1) in vulvar intraepithelial neoplasia, contributing to a
more robust examination compared with previous meta-
analyses. Among the pooled prevalence of specific HPV
genotypes (HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58),
HPV16 and HPV33 were the two predominant HPV genotypes
The presence of HPV DNA in tumour tissue can be
detected by various methods, including PCR or Hybrid
Capture 2. Integration of HPV DNA into the host
See Online for appendix genome could cause the overexpression of the tumour
suppressor protein p16INK4a, which can be detected by
immuno­histochemical staining and has been proposed
as a surrogate marker of persistent HPV infections.9
The combined detection of HPV DNA and p16INK4a has
been proposed as an increasingly reliable method for
diagnosing true HPV-driven tumours in oropharyngeal
squamous cell carcinomas.10
Since publication of previous meta-analyses,11–14
numerous relevant studies have been published.
Therefore, we aimed to provide updated data on the
pooled prevalence of HPV DNA in vulvar cancer and
vulvar intraepithelial neoplasia, and to examine the
prevalence of p16INK4a positivity, both overall and according
to different HPV statuses. Additionally, we estimated the
prevalence of double positivity for HPV DNA and p16INK4a
in vulvar cancer and vulvar intraepithelial neoplasia.
Methods
Search strategy and inclusion criteria
In this systematic review and meta-analysis, we searched
the PubMed, Embase, and Cochrane Library databases to
identify all English-language studies published between
Jan 1, 1986, and May 6, 2022. All records identified were
2 www.thelancet.com/oncology Published online March 15, 2023 https://doi.org/10.1016/S1470-2045(23)00066-9
in both vulvar cancer and vulvar intraepithelial neoplasia.
Additionally, this was the first study, to our knowledge,
to investigate the pooled prevalence of specific HPV genotypes
according to different geographical regions and report a
disparity in HPV prevalence among regions. To our best
knowledge, this systematic review and meta-analysis was also
the first to estimate the prevalence of p16INK4a positivity in
vulvar cancer and vulvar intra­epithelial neoplasia, with a
prevalence of p16INK4a positivity of 34·1% (95% CI 30·9–37·4) in
vulvar cancer and of 65·7% (52·5–77·7) in vulvar intraepithelial
neoplasia. Furthermore, we also reported the prevalence of
double positivity for HPV DNA and p16INK4a in vulvar cancer
(19·6% [95% CI 16·3–23·0]) and vulvar intraepithelial neoplasia
(44·2% [26·3–62·8]).
Implications of all the available evidence
This meta-analysis reported a high prevalence of HPV DNA in
vulvar cancer (about 40%) and vulvar intraepithelial neoplasia
(about 76%), which was consistent with the findings of four
previous meta-analyses. Owing to the high prevalence of
HPV16 and HPV33 in vulvar cancer and vulvar intraepithelial
neoplasia, multi-valent HPV vaccination covering HPV16 and
HPV33 seems to have a greater clinical potential in preventing
vulvar neoplasm, especially in Africa, Oceania, and Europe.
Additionally, this study highlighted the potential clinical
importance of double positivity for HPV DNA and p16INK4a in
vulvar neoplasm.
imported into the reference manager software Endnote,
and duplicates were removed. Full details of the search
strategies adopted in each database are shown in the
appendix (p 1).
We included studies that reported the prevalence
of p16INK4a positivity, HPV DNA, or both, for a minimum
of five cases of histologically verified vulvar cancer or
vulvar intraepithelial neoplasia. Only studies that applied
PCR or the Hybrid Capture 2 method to detect HPV DNA
and methy­ lation or immunohistochemistry to detect
p16INK4a were eligible for inclusion. Additionally, references
cited in the included publi­cations were investigated to
identify additional relevant articles. Only peer-reviewed
studies were included; we excluded conference abstracts
and case reports. Study populations that were included in
more than one article were enrolled only once, and we
used the data from the article that contained the most
detailed information. We contacted primary study authors
by email for clarifications if needed. The study protocol is
shown in the appendix (p 2).
We did this study in accordance with the PRISMA
guidelines.15
Data analysis
Data were extracted independently by two authors (ZL
and PL), with inconsistencies discussed by two authors
or a third author (ZZ) until a consensus was reached.
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We extracted data on the following variables: first author, primer PCR, and Hybrid Capture 2), tissue type (frozen,
year of publication and sample collection, sample size, fixed, unknown, and exfoliated cells) and publication
geographical region, age at diagnosis, tissue type, type year (1989–99, 2000–09, and 2010–22), were included
of lesion (vulvar intraepithelial neoplasia or vulvar
cancer), histological subtype of vulvar cancer and vulvar
intraepithelial neoplasia, the method and PCR primers 6393 records identified
used for HPV DNA detection, HPV genotypes, and 2230 PubMed
4000 Embase
overall and type-specific HPV prevalence. Furthermore, 163 Cochrane Library
data on the p16INK4a testing method, definition of p16INK4a
positivity, prevalence of p16INK4a positivity (among the
1990 duplicates excluded
whole population, the HPV-negative population, and the
HPV-positive population), prevalence of double positivity
for HPV DNA and p16INK4a were also extracted. In a 4403 screened by title
small observational study,16 which applied both immuno­
histochemistry and methylation methods to test p16INK4a,
3652 non-eligible records excluded
only the evaluation by immunohistochemistry was
included in the analyses.
After data extraction, ZC reviewed the data and relevant 751 abstracts reviewed
full-text papers to correctly classify the method and PCR
primers used for HPV DNA detection. The histological
418 excluded
subtypes of vulvar cancer and vulvar intra­ epithelial 191 had no primary data, or data were not
neoplasia were reclassified on the basis of the WHO peer-reviewed
Classification for Female Genital Tumors4 and the 23 included no patients with vulvar cancer
or vulvar intraepithelial neoplasia
International Society for the Study of Vulvovaginal Disease 143 had no testing for HPV or p16INK4a, or
Terminology of Vulvar Squamous Intraepithelial Lesions.5 HPV test was not PCR or Hybrid Capture 2
7 had no vulvar samples
We did not assess the quality of included studies. 25 fewer than five cases were tested
We used random-effect models to calculate the pooled 8 were not in English
21 included populations with a predisposed
prevalence of HPV DNA and p16INK4a positivity in both risk of HPV-positivity
vulvar intraepithelial neoplasia and vulvar cancer. We
used the DerSimonian–Laird estimator17 to determine
the variance across studies, and applied the arcsine 333 full-text records assessed for eligibility
transformation to stabilise the variance.18 The pooled
prevalence of HPV DNA and p16INK4a positivity in each 173 excluded
study were shown with 95% CIs. Statistical heterogeneity 18 had no primary data, or data were not
between studies was investigated using Higgins’ peer-reviewed
6 included no patients with vulvar cancer or
I² statistics, and the significance of the heterogeneity was vulvar intraepithelial neoplasia
evaluated using Cochran’s Q test.19 Additionally, potential 30 had no testing for HPV or p16INK4a, or HPV
test was not PCR or Hybrid Capture 2
sources of heterogeneity were explored through stratified 2 had no vulvar samples
analyses and meta-regression. In the meta-regression, 9 fewer than five cases were tested
each variable was estimated in a univariable test of a 61 contained data reported in more than
one study
moderator variable, which was based on the χ² test. 7 did not report data separately for benign,
For studies estimating the HPV DNA prevalence premalignant, or malignant conditions
40 included populations with a predisposed
in vulvar intraepithelial neoplasia and vulvar cancer, risk of HPV-positivity
we did subgroup analyses stratified by different histo­
logical subtypes of vulvar cancer (keratinising, non-
2 studies identified through a review of reference
keratinising, warty, basaloid, and verrucous carcinoma), lists
vulvar intra­ epithelial neoplasia (low-grade and high
grade squamous intraepithelial lesions and differ­
entiated vulvar intra­ epithelial neoplasia) and age at 162 studies included in the systematic review
diagnosis (<60 and ≥60 years). The defined variables,
including geographical region (Europe, North America, 162 studies included in the meta-analysis*
South America, Asia, Africa, Oceania, and multicountry 91 on HPV and vulvar cancer
[ie, a population from more than one of these 60 on HPV and vulvar intraepithelial neoplasia
52 on p16INK4a and vulvar cancer
regions]),the method used for HPV DNA detection (ie, 23 on p16INK4a and vulvar intraepithelial neoplasia
consensus primer PCR, type-specific primer PCR,
combination of consensus primers PCR, combination Figure 1: Study selection
of consensus and type-specific primers PCR, unknown HPV=human papillomavirus. *Some studies included data for more than one of the categories listed.

www.thelancet.com/oncology Published online March 15, 2023 https://doi.org/10.1016/S1470-2045(23)00066-9 3

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in the univariate meta-regression. Furthermore, we method (immunohisto­chemistry and methy­lation) and

explored the pooled type-specific prevalence of HPV
genotypes covered by the nine-valent HPV vaccine
(HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58),20 among
patients with HPV-positive vulvar intraepithelial
neoplasia or vulvar cancer, stratifying the analyses to
either single or all HPV infections (ie, single and
multiple infections).
For studies estimating the prevalence of p16INK4a
positiv­
ity in vulvar cancer and vulvar intraepithelial
neoplasia, we did subgroup analyses according to
histological subtype of vulvar cancer and vulvar intra­
epithelial neoplasia. Variables, including geographical
region (Europe, North America, South America, Asia,
Africa, Oceania, and multi­ country), p16INK4a detection
publication year (1998–2005, 2006–14, and 2015–22),
were selected for meta-regression analysis. Additionally,
the pooled p16INK4a positivity prevalence was calcu­lated
among all patients, those who were HPV-negative,
and those who were HPV-positive. We also calculated
the pooled prevalence of p16INK4a positivity according
to the different cutoffs of p16INK4a positivity used in
immunohistochemistry.
To address the risk of bias between studies and to
assess the robustness of pooled estimate, sensitivity
analyses were done by excluding studies that included
populations with an increased risk of HPV infection
(ie, women with HIV). All analyses were done using
the statistical software R (version 4.2.0) with the meta

HPV DNA prevalence p16INK4a positivity prevalence

Studies* Total Patients Pooled I² for p value for Studies* Total Patients Pooled I² for p value for
patients positive for prevalence heterogeneity heterogeneity patients positive prevalence heterogeneity heterogeneity
HPV DNA of HPV for p16INK4a of p16INK4a
DNA positivity
positivity (95% CI)
(95% CI)

Vulvar cancer
All 91 8200 2958 39·1% 90·9% <0·0001 52 6352 1957 34·1% 83·4% <0·0001
(35·3–42·9) (30·9–37·4)
Vulvar 79 5911 2174 37·3% 90·2% <0·0001 50 4538 1426 34·8% 83·8% <0·0001
squamous cell (33·2–41·5) (31·2–38·6)
carcinoma
Keratinising 16 811 142 12·0% 88·1% <0·0001 8 361 45 11·2% 85·8% <0·0001
carcinoma (6·1–19·6) (3·8–21·9)
Non- 7 55 25 31·1% 80·5% <0·0001 4 20 10 50·0% 0·0% 0·7648
keratinising (7·4–62·0) (28·8–71·3)
carcinoma
Basaloid 17 133 109 83·4% 32·2% 0·0984 7 66 54 95·9% 86·0% <0·0001
carcinoma (74·3–90·8) (73·0–100·0)
Warty 13 141 115 86·5% 32·0% 0·1265 7 35 17 85·6% 89·9% <0·0001
carcinoma (76·9–93·8) (31·2–100·0)
Verrucous 7 16 8 58·9% 71·1% 0·0020 3 11 0 0·0% 0·0% 1·0000
carcinoma (14·0–95·9) (0·0–8·5)
Vulvar intraepithelial neoplasia
All 60 3140 2466 76·1% 90·5% <0·0001 23 896 553 65·7% 93·7% <0·0001
(70·7–81·1) (52·5–77·7)
Low-grade 14 147 104 61·1% 85·9% <0·0001 9 110 19 26·6% 80·8% <0·0001
squamous (37·8–82·0) (8·9–49·7)
intraepithelial
lesion†
High-grade 48 1747 1437 83·2% 84·3% <0·0001 19 468 435 97·5% 78·8% <0·0001
squamous (78·3–87·6) (93·2–99·7)
intraepithelial
lesion‡
Differentiated- 12 149 7 2·5% 55·8% 0·0095 10 165 0 0·0% 0·0% 1·0000
type vulvar (0·0–9·2) (0·0–0·6)
intraepithelial
neoplasia
Date are n, % (95% CI), or %. HPV=human papillomavirus.*Studies could contribute cases to more than one histological subtype of vulvar squamous cell carcinoma and vulvar intraepithelial neoplasia.
Furthermore, some studies only contributed to the pooled analysis of vulvar cancer or vulvar intraepithelial neoplasia, as that specific histological subtype was not available. †Includes vulvar intraepithelial
neoplasia grade 1. ‡Includes vulvar intraepithelial neoplasia grade 2 and 3, and usual-type vulvar intraepithelial neoplasia.

Table 1: Pooled prevalence of HPV DNA and p16INK4a positivity in vulvar cancer and vulvar intraepithelial neoplasia stratified by histological subtypes

4 www.thelancet.com/oncology Published online March 15, 2023 https://doi.org/10.1016/S1470-2045(23)00066-9

 Vulvar cancer Vulvar intraepithelial neoplasia

Studies Total Patients Pooled prevalence p value I² for p value for Studies Total Patients Pooled prevalence p value I² for p value for
patients positive of HPV DNA (univariable heterogeneity heterogeneity patients positive of HPV DNA (univariable heterogeneity heterogeneity
for HPV positivity (95% CI) test of for HPV positivity (95% CI) test of
DNA moderator DNA moderator
variable) variable)
Geographical <0·0001 0·6800
location
Europe 47 4171 1401 30·8% (26·1–35·7) ·· 90·6% <0·0001 34 1405 1067 76·3% (67·6–83·9) ·· 92·1% <0·0001
North America 19 911 492 52·9% (44·7–61·1) ·· 82·5% <0·0001 10 543 408 68·3% (51·3–83·1) ·· 92·3% <0·0001
South America 2 266 82 32·1% (17·1–49·3) ·· 87·8% 0·0041 2 41 22 57·4% (32·0–80·9) ·· 57·2% 0·1265
Asia 16 756 353 48·4% (39·2–57·8) ·· 79·3% <0·0001 9 391 320 82·3% (72·7–90·2) ·· 66·2% 0·0026
Africa 2 104 65 66·0% (45·5–83·8) ·· 74·3% 0·0485 0 ·· ·· ·· ·· ·· ··
Oceania 4 283 77 44·4% (20·6–69·7) ·· 93·5% <0·0001 3 111 88 77·3% (50·6–95·5) ·· 88·7% 0·0001
Multicountry* 1 1709 488 28·6% (26·4–30·8) ·· ·· ·· 2 649 561 86·5% (83·7–89·0) ·· 0·0% 0·5476

HPV test and 0·1148 0·4698

primers
PCR, consensus 50 5516 1824 35·6% (30·9–40·5) ·· 91·5% <0·0001 32 1991 1537 74·3% (66·0–81·9) ·· 92·7% <0·0001
primers
PCR, type- 11 613 228 40·6% (30·2–51·4) ·· 83·9% <0·0001 8 207 169 81·2% (67·4–91·9) ·· 77·8% <0·0001
specific primers
PCR, 9 1040 526 52·9% (40·9–64·7) ·· 91·4% <0·0001 5 200 182 89·1% (78·6–96·3) ·· 71·9% 0·0066
combination of
consensus
primers
PCR, 17 882 304 38·7% (29·6–48·3) ·· 87·2% <0·0001 9 442 352 73·2% (59·7–84·7) ·· 87·8% <0·0001
combination of

www.thelancet.com/oncology Published online March 15, 2023 https://doi.org/10.1016/S1470-2045(23)00066-9

consensus and
type-specific
primers
PCR, unknown 3 114 61 53·5% (44·4–62·6) ·· 0·0% 0·6554 2 174 149 85·7% (80·1–90·5) ·· 0·0% 0·6257
primers
Hybrid Capture 2 1 35 15 42·9% (26·3–60·6) ·· ·· ·· 4 126 77 63·6% (43·8–81·2) ·· 80·1% 0·0018
Tissue sample type 0·2032 0·2840
Frozen 14 507 249 46·6% (34·9–58·6) ·· 85·1% <0·0001 10 226 181 84·7% (66·1–96·7) ·· 90·3% <0·0001
Fixed 73 7527 2665 38·1% (34·1–42·3) ·· 91·5% <0·0001 39 2364 1841 72·7% (65·7–79·2) ·· 91·6% <0·0001
Unknown 4 166 44 30·8% (16·6–47·3) ·· 76·7% 0·0050 9 456 379 82·1% (71·4–90·8) ·· 84·3% <0·0001
Exfoliated cells 0 ·· ·· ·· ·· ·· ·· 2 94 65 69·2% (59·5–78·0) ·· 0·0% 0·9521
Publication year 0·9740 0·0182
1989–99 20 876 303 37·8% (30·8–45·0) ·· 77·2% <0·0001 12 557 423 80·8% (69·4–90·1) ·· 86·8% <0·0001
2000–09 22 823 367 38·5% (26·8–50·8) ·· 91·7% <0·0001 26 770 509 66·3% (53·8–77·8) ·· 91·8% <0·0001
2010–22 49 6501 2288 39·2% (34·6–43·9) ·· 92·4% <0·0001 22 1813 1534 83·1% (77·9–87·7) ·· 84·5% <0·0001
Date are n, % (95% CI), or %. If only one study contributed to the analysis, then no pooled prevalence was estimated and the actual proportion reported in the study was provided. HPV=human papillomavirus. *Multicountry indicates a population from
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two or more of the listed regions.

Table 2: Pooled prevalence of HPV DNA in vulvar cancer and vulvar intraepithelial neoplasia, by stratified variable

5
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Pooled prevelance Pooled prevelance Pooled prevelance

(95% CI) (95% CI) (95% CI)
A Global B Africa* C Asia

HPV6 0·6% (0·2–1·3) ·· 2·5% (0·0–9·6)

0·4% (0·1–0·9) ·· 1·8% (0·0–10·3)

HPV11 0·1% (0·0–0·5) ·· 0·3% (0·0–3·1)

0·0% (0·0–0·1) ·· 0·0% (0·0–1·8)

HPV16 78·1% (73·5–82·3) 72·4% (51·4–89·3) 66·4% (54·4–77·4)

78·6% (73·2–83·5) 60·9% (40·6–79·4) 64·7% (51·5–76·9)

HPV18 4·4% (2·5–6·8) 0·9% (0·0–8·6) 10·3% (2·6–22·2)

3·0% (1·4–5·2) 4·4% (0·0–16·2) 9·8% (2·2–22·0)
HPV genotype

HPV31 0·7% (0·2–1·5) 3·1% (0·0–26·4) 1·8% (0·0–9·7)

0·5% (0·1–1·3) 13·0% (2·7–29·5) 1·1% (0·0–10·3)

HPV33 7·5% (4·9–10·7) 9·1% (3·4–17·2) 3·4% (0·0–18·4)

5·2% (2·8–8·2) 13·0% (2·7–29·5) 1·8% (0·0–17·8)

HPV45 0·4% (0·1–0·8) 2·0% (0·0–17·7) 0·5% (0·0–3·8)

0·3% (0·1–0·9) 8·7% (0·9–23·3) 0·0% (0·0–2·2)

HPV52 0·5% (0·1–1·1) 1·5% (0·0–10·9) 0·3% (0·0–2·5)

0·2% (0·0–0·6) 0·0% (0·0–4·1) 0·5% (0·0–4·0)

HPV58 0·1% (0·0–0·4) 0·0% (0·0–1·5) 1·5% (0·1–4·9)

0·1% (0·0–0·4) 0·0% (0·0–4·1) 1·0% (0·0–4·2)

D Europe E North America F Oceania

HPV6 0·3% (0·0–0·9) 1·7% (0·1–5·1) 0·0% (0·0–5·9)

0·2% (0·0–0·9) 0·9% (0·0–3·0) 0·0% (0·0–6·3)

HPV11 0·2% (0·0–0·9) 0·0% (0·0–0·5) 0·0% (0·0–5·9)

0·0% (0·0–0·2) 0·0% (0·0–0·6) 0·0% (0·0–6·3)

HPV16 81·3% (74·6–87·1) 79·8% (72·9–86·0) 89·0% (67·6–99·5)

83·6% (76·8–89·4) 83·1% (75·2–89·8) 79·2% (66·0–89·9)

HPV18 2·6% (0·9–5·2) 5·2% (1·7–10·6) 2·1% (0·0–15·5)

1·2% (0·2–3·1) 3·5% (0·8–8·0) 1·3% (0·0–8·6)
HPV genotype

HPV31 0·6% (0·1–1·6) 0·1% (0·0–1·2) 0·0% (0·0–5·9)

0·4% (0·0–1·2) 0·2% (0·0–1·4) 0·0% (0·0–6·3)

HPV33 8·3% (4·9–12·3) 7·1% (2·6–13·5) 12·5% (1·4–32·5)

6·3% (3·2–10·4) 2·3% (0·0–7·8) 13·3% (1·5–34·4)

HPV45 0·0% (0·0–0·3) 1·1% (0·0–4·7) 0·0% (0·0–5·9)

0·0% (0·0–0·4) 0·9% (0·0–3·5) 0·0% (0·0–6·3)

HPV52 0·2% (0·0–0·7) 1·2% (0·1–3·7) 6·3% (0·0–22·8)

0·1% (0·0–0·5) 0·6% (0·0–2·5) 6·7% (0·0–24·2)

HPV58 0·2% (0·0–0·7) 0·0% (0·0–0·6) 0·0% (0·0–5·9)

0·1% (0·0–0·5) 0·0% (0·0–0·7) 0·0% (0·0–6·3)
0 20 40 60 80 100 0 20 40 60 80 100
G South America Prevalence (%) Prevalence (%)

Among patients with vulvar cancer and HPV infection

HPV6 0·0% (0·0–2·2)
0·0% (0·0–2·7) Among patients with vulvar cancer and a single HPV genotype infection

HPV11 0·0% (0·0–2·2)

0·0% (0·0–2·7)

HPV16 54·3% (30·2–77·4)

50·3% (25·2–75·4)

HPV18 22·0% (1·9–55·5)

17·2% (0·4–50·9)
HPV genotype

HPV31 0·6% (0·0–5·1)

0·0% (0·0–1·4)

HPV33 7·6% (0·0–53·9)

3·7% (0·0–29·5)

HPV45 2·4% (0·2–6·9)

2·9% (0·3–8·2)

HPV52 0·0% (0·0–1·2)

0·0% (0·0–1·4)

HPV58 0·0% (0·0–1·2)

0·0% (0·0–1·4)
0 20 40 60 80 100
Prevalence (%)

Figure 2: Pooled prevalence of specific HPV genotypes in vulvar cancer, by geographical region
HPV=human papillomavirus. *Data on the prevalence of HPV6 and HPV11 were not available in the studies from Africa.

6 www.thelancet.com/oncology Published online March 15, 2023 https://doi.org/10.1016/S1470-2045(23)00066-9

 Articles

package,21 and a two-sided p value of less than 0·05 was

Date are n, % (95% CI), or %. If only one study contributed to the analysis, then no pooled prevalence was estimated and the actual proportion reported in the study was provided. *Multicountry indicates a population from two or more of the listed regions.
heterogeneity
considered to be statistically significant.

p value for

<0·0001
<0·0001
<0·0001

<0·0001
<0·0001
0·0303

0·0638
0·0036
Role of the funding source

··

··
··
··
The funder of the study had no role in the study design,

heterogeneity
data collection, data analysis, data interpretation, or
writing of the report.

96·8%

88·2%

94·2%

95·4%
91·6%

49·7%
95·7%
78·7%
I² for

··
··
··

··
Results

moderator
A total of 6393 records were identified in the PubMed,

variable)
variable

0·4988

0·9048
0·4582
Pooled prevalence of p value

test of
Embase, and Cochrane Library database search. After the

(uni-

··
··

··

··
··
··
··

··
··
··

··
··
removal of 1990 duplicates, 4403 records were assessed by
title for eligibility and 3652 were excluded (figure 1). We

92·3% (43·7–100·0)

67·8% (44·6–87·1)
60·3% (27·8–88·3)
68·7% (60·0–76·7)
66·8% (51·7–80·3)
61·1% (24·9–91·4)

79·1% (67·4–88·1)

49·7% (17·7–81·8)
50·0% (27·2–72·8)

67·2% (53·2–79·7)
21·4% (8·3–41·0)
reviewed 751 abstracts and excluded 418. We assessed

p16INK4a positivity
333 full-text records and excluded 173, leaving 160 relevant
studies were eligible for inclusion. Two additional relevant
(95% CI)
studies were retrieved after reviewing the reference lists

··
of the included studies. Thus, 162 studies, published
Vulvar intraepithelial neoplasia

Patients
positive

between March, 1989, and May, 2022, were included in

p16INK4a

266
528
338

104
183
10
53

25
122
6
24
this meta-analysis. Details and references of all included
for

··
studies are shown in the appendix (pp 3–18; 20–29).
Patients

The prevalence of HPV DNA in vulvar cancer was

408
534
220

851

261
20
67

45
28
27

227
examined in 91 studies (n=8200). The prevalence of HPV

··
DNA ranged from 0% to 88·9%, with an overall prevalence
Studies

of 39·1% (95% CI 35·3–42·9) and a large heterogeneity

1
1

21

2
12
6
1

7
2
0

5
11
(I²=90·9%; p<0·0001; table 1; appendix p 31). For the

Table 3: Pooled prevalence of p16INK4a positivity in vulvar cancer and vulvar intraepithelial neoplasia, by stratified variable
sensitivity analysis, we excluded two studies22,23 that
heterogeneity
p value for

included populations with an increased risk of HPV

<0·0001
<0·0001

<0·0001
<0·0001
<0·0001
0·0478

0·0047
0·0030
0·7003
0·2849

infection (ie, women with HIV) and the pooled prevalence

··
··

of HPV DNA (38·4% [95% CI 34·7–42·3]) changed very

little, with an I² of 90·8% (p<0·0001). In the stratified
heterogeneity

analyses by the histological subtype of vulvar squamous

cell carcinoma, HPV DNA prevalence was highest in

80·0%
62·4%

83·4%

85·4%
79·5%
82·1%

12·6%

81·3%
67·1%
0·0%
I² for

··
··

warty carcinoma (86·5% [95% CI 76·9–93·8]) and lowest

in keratinising carcinoma (12·0% [6·1–19·6]; table 1).
moderator
variable)

When stratified by geographical region, the univariable

variable

0·0002

0·2016
0·2935
p value

test of
(uni-

meta-regression showed a significant difference in the

··

··

··
··

··
··
··
··

··
··
··

··
pooled prevalence of HPV DNA among vulvar cancer
36·2% (28·4–44·4)
34·5% (26·8–42·6)
52·9% (43·6–62·2)
73·3% (44·9–92·2)
43·5% (29·7–58·0)

41·9% (30·8–53·5)
27·8% (21·6–34·4)
43·9% (37·8–50·1)
positivity (95% CI)

29·5% (25·6–33·5)

41·7% (21·2–63·8)

32·1% (28·3–35·9)
33·7% (30·4–37·0)
Pooled prevalence

between geographical regions, which ranged from 30·8%

(95% CI 26·1–35·7) in Europe to 66·0% (45·5–83·8) in
of p16INK4a

Africa (p<0·0001; table 2). When stratified by age, the

pooled prevalence of HPV DNA in patients with vulvar
cancer who were younger than 60 years was 66·3%
Patients
positive

(95% CI 56·0–78·9) and was 30·9% (22·0–40·7) in those

p16INK4a

557

1913

44
935

165
316
52
23

265
11
63

1527

aged 60 years or older (appendix p 19).

for

The prevalence of HPV DNA in patients with

Patients

vulvar intraepithelial neoplasia was assessed in

6248
3404

1821

104

459
753
187
53

801
5092
15
119
Vulvar cancer

60 studies (3140 patients). The pooled prevalence of

HPV DNA in vulvar intraepithelial neoplasia was
Studies

76·1% (95% CI 70·7–81·1; appendix p 32), with a higher

49
3

3
31

7
11
3
2

15
1
1

30

prevalence in vulvar high-grade squamous intra­

p16INK4a methylation

epithelial lesions (83·2% [78·3–87·6]) and low-grade

Geographical region

p16 immuno-
South America
North America

histochemistry
Multicountry*

squamous intra­epithelial lesions (61·1% [37·8–82·0])

Publication year
1998–2005

than in differentiated vulvar intraepithelial neoplasia

p16INK4a test

2006–14
2015–22
Oceania
Europe

INK4a

(2·5% [0·0–9·2]; table 1). When stratified by age,

Africa
Asia

the pooled prevalence of HPV DNA in patients with

vulvar intraepithelial neoplasia who were younger

www.thelancet.com/oncology Published online March 15, 2023 https://doi.org/10.1016/S1470-2045(23)00066-9 7

 Articles

A Patients who are HPV-positive

Events (n/N) Prevalence (95% CI)

Gasco et al (2002) 24
11/13 84·6% (54·6–98·1)
Osakabe et al (2007)25 4/5 80·0% (28·4–99·5)
Aulmann et al (2008)26 5/5 100·0% (47·8–100·0)
de Koning et al (2008)27 11/13 84·6% (54·6–98·1)
Alonso et al (2011)28 19/19 100·0% (82·4–100·0)
Lavorato-Rocha et al (2013)29 11/33 33·3% (18·0–51·8)
Reuschenbach et al (2013)30 39/74 52·7% (40·7–64·4)
Lee et al (2016)31 14/15 93·3% (68·1–99·8)
Sznurkowski et al (2016)32 25/38 65·8% (48·6–80·4)
Wakeham et al (2017)33 23/32 71·9% (53·3–86·3)
Pils et al (2017)34 32/40 80·0% (64·4–90·9)
Halec et al (2017)35 395/476 83·0% (79·3–86·3)
Hinten et al (2018)36 53/73 72·6% (60·9–82·4)
Arians et al (2019)37 13/16 81·2% (54·4–96·0)
Stefansson et al (2019)38 22/36 61·1% (43·5–76·9)
Thomas et al (2020)39 6/10 60·0% (26·2–87·8)
Lérias et al (2020)40 9/26 34·6% (17·2–55·7)
Allo et al (2020)41 30/33 90·9% (75·7–98·1)
Preti et al (2020)42 7/17 41·2% (18·4–67·1)
Gensthaler et al (2020)43 20/30 66·7% (47·2–82·7)
Kolitz et al (2022)44 15/22 68·2% (45·1–86·1)
Pooled prevalence 764/1026 73·3% (64·7–81·2)
Heterogeneity: I2=84·9%, p<0·0001

B Patients who are HPV-negative

Gasco et al (2002)24 12/23 52·2% (30·6–73·2)

van der Avoort et al (2006)45 2/16 12·5% (1·6–38·3)
Osakabe et al (2007)25 7/16 43·8% (19·8–70·1)
Aulmann et al (2008)26 15/23 65·2% (42·7–83·6)
de Koning et al (2008)27 1/26 3·8% (0·1–19·6)
Alonso et al (2011)28 1/79 1·3% (0·0–6·9)
Lavorato-Rocha et al (2013)29 18/68 26·5% (16·5–38·6)
Reuschenbach et al (2013)30 18/101 17·8% (10·9–26·7)
Lee et al (2016)31 6/41 14·6% (5·6–29·2)
Sznurkowski et al (2016)32 10/47 21·3% (10·7–35·7)
Wakeham et al (2017)33 2/27 7·4% (0·9–24·3)
Pils et al (2017)34 6/136 4·4% (1·6–9·4)
Halec et al (2017)35 103/1194 8·6% (7·1–10·4)
Hinten et al (2018)36 32/245 13·1% (9·1–17·9)
Arians et al (2019)37 6/59 10·2% (3·8–20·8)
Stefansson et al (2019)38 4/61 6·6% (1·8–15·9)
Thomas et al (2020)39 4/45 8·9% (2·5–21·2)
Lérias et al (2020)40 3/58 5·2% (1·1–14·4)
Allo et al (2020)41 12/77 15·6% (8·3–25·6)
Preti et al (2020)42 4/85 4·7% (1·3–11·6)
Gensthaler et al (2020)43 12/105 11·4% (6·0–19·1)
Kolitz et al (2022)44 5/14 35·7% (12·8–64·9)
Pooled prevalence 283/2546 13·8% (10·0–18·1)
Heterogeneity: I2=84·2%, p<0·0001

0 20 40 60 80 100
Prevalence (%)

Figure 3: Pooled prevalence of p16INK4a positivity in vulvar cancer globally, by HPV status
Grey squares represent the point estimate of the prevalence of p16INK4a positivity and horizontal lines represent 95% CIs. Details and references of all included studies
are shown in the appendix (pp 3–18; 20–29). HPV=human papillomavirus.

8 www.thelancet.com/oncology Published online March 15, 2023 https://doi.org/10.1016/S1470-2045(23)00066-9


than 60 years was 85·5% (95% CI 79·1–90·9) and
46·8% (22·3–72·2) in those aged 60 years or older
(appendix p 19).
The pooled prevalence of specific HPV genotypes (HPV
types 6, 11, 16, 18, 31, 33, 45, 52, and 58), globally and by
geographical region, was examined among patients with
HPV-positive vulvar cancer (figure 2). Among all patients
with HPV DNA-positive vulvar cancer, HPV16 was the
most frequent genotype, with a pooled prevalence of
78·1% (95% CI 73·5–82·3), followed by HPV33 (7·5%
[4·9–10·7]), and HPV18 (4·4% [2·5–6·8]). When stratified
by geographical regions, HPV16 was also the most
predominant genotype in all regions, varying substantially
between regions, with a high prevalence in Oceania
(89·0% [95% CI 67·6–99·5]) and a low prevalence in
South America (54·3% [30·2–77·4]). However, despite
HPV33 being the second most predominant type in
vulvar cancer worldwide, HPV18 was the second most
frequent type in South America and Asia. A reasonably
high pooled prevalence of HPV52 was observed
in Oceania.
Among all patients with HPV DNA-positive vulvar
intraepithelial neoplasia, HPV16 was the most common
genotype, with a pooled prevalence of 80·8% (95% CI
75·9–85·2; appendix p 33). Additionally, the prevalence
of HPV33 was 6·3% (95% CI 3·9–9·2), and HPV6
accounted for 2·9% (95% CI 0·8–6·3). When stratified
by geographical region, HPV16 was also the most
predominant genotype in all regions. Additionally,
HPV33 was the second most prevalent genotype in
Europe and North America, whereas HPV18 was the
second most common genotype in Asia (appendix p 33).
52 studies (6352 patients) detected the prevalence of
p16INK4a positivity in vulvar cancer. The pooled prevalence of
p16INK4a positivity in vulvar cancer was 34·1% (95% CI
30·9–37·4), with a considerable heterogeneity (I²=83·4%;
p<0·0001; table 1; appendix p 34). In the stratified analyses
according to the histological subtype of vulvar squamous
cell carcinoma, the pooled prevalence of p16INK4a positivity
differed among the different histologic subtypes, with the
highest prevalence of 95·9% (95% CI 73·0–100·0) in
basaloid carcinoma and the lowest prevalence of 0%
(0·0–8·5) in verrucous carcinoma (table 1). In the
meta-regression, the pooled prevalence of p16INK4a positivity
in vulvar cancer was significantly different between
geographical regions (p=0·0002; table 3). Additionally, the
prevalence of p16INK4a positivity was similar among studies
that used different cutoffs to define p16INK4a positivity
(appendix p 30). When estimating the prevalence of p16INK4a
positivity on the basis of HPV status of vulvar cancer,
we observed a much higher pooled p16INK4a positivity
prevalence among HPV-positive vulvar cancer (73·3%
[95% CI 64·7–81·2]) than in HPV-negative vulvar cancer
(13·8% [10·0–18·1]; figure 3).
We included 23 studies (896 patients) that estimated
the prevalence of p16INK4a positivity in vulvar intra­
epithelial neoplasia. The pooled prevalence of p16INK4a
www.thelancet.com/oncology Published online March 15, 2023 https://doi.org/10.1016/S1470-2045(23)00066-9 9
Articles
positivity in vulvar intraepithelial neoplasia was 65·7%
(95% CI 52·5–77·7; appendix p 35), with a higher
prevalence in high-grade squamous intraepithelial
lesions (97·5% [93·2–99·7]) and low-grade squamous
intraepithelial lesions (26·6% [8·9–49·7]) than in
differ­
entiated vulvar intraepithelial neoplasia (0%
[0·0–0·6]; table 1). When estimating the pooled
prevalence of p16INK4a positivity on the basis of the HPV
status of vulvar intraepithelial neoplasia, HPV-positive
vulvar intraepithelial neoplasia had a higher prevalence
of p16INK4a positivity (92·7% [95% CI 80·2–99·3]) than
HPV-negative vulvar intra­epithelial neoplasia (42·6%
[20·6–66·3]; appendix p 36).
22 studies (3572 patients) estimating the prevalence of
double positivity for HPV DNA and p16INK4a in vulvar
cancer were included. The overall prevalence of double
positivity for HPV DNA and p16INK4a in vulvar cancer
ranged from 0% to 41·7%, yielding an overall pooled
prevalence of 19·6% (95% CI 16·3–23·0; appendix p 37).
Additionally, eight studies (381 patients) estimated the
prevalence of double positivity for HPV DNA and p16INK4a
in vulvar intraepithelial neoplasia. The overall prevalence
of double positivity for HPV DNA and p16INK4a in vulvar
intraepithelial neoplasia was 44·2% (95% CI 26·3–62·8;
appendix p 37).
Discussion
In this systematic review and meta-analysis, we estimated
a pooled HPV DNA prevalence of approximately 40% in
vulvar cancer and 76% in vulvar intraepithelial neoplasia.
This meta-analysis included numerous new studies and
cases, contributing to increasingly robust and reliable
findings. To our knowledge, this study was the first to
estimate an overall p16INK4a positivity prevalence of about
34% in vulvar cancer and 66% in vulvar intraepithelial
neoplasia. When stratified by age, we observed a higher
pooled prevalence of HPV DNA in patients younger
than 60 years than in those aged 60 years or older, in
both vulvar cancer and vulvar intraepithelial neoplasia.
Furthermore, we also were the first to report a pooled
double positivity prevalence of HPV DNA and p16INK4a,
of about 20% in vulvar cancer and 44% in vulvar
intraepithelial neoplasia.
The most recent related meta-analysis was published
in 2017.11 Our analysis included an additional 27 new
studies concerning the prevalence of HPV DNA in vulvar
cancer, adding about 3200 patients to the pooled estimate.
The pooled HPV DNA prevalence in our meta-analysis
was in agreement with the results of previous meta-
analyses.11–13 When stratified by geographical regions, we
found significant variation in the pooled prevalence of
HPV DNA in vulvar cancer across regions, with a high
preva­lence in Africa (66·0%). To our knowledge, the
prevalence of HPV DNA in vulvar cancer in Africa was
examined for the first time in our meta-analysis, and the
high prevalence showed the necessity of preventing
HPV infection in Africa. Regarding the prevalence of
 Articles
HPV DNA in vulvar intraepithelial neoplasia, we found a
similar pooled prevalence (76·1%) to that in the previous
meta-analysis (76·3%).11
In our meta-analysis, the type-specific HPV DNA
preva­lence was estimated separately in both vulvar
cancer and vulvar intraepithelial neoplasia, according to
the genotypes covered by the nine-valent HPV vaccine.
HPV16 and HPV18 are widely recognised as the crucial
oncogenic genotypes for cervical neoplasm,46,47 and
HPV16 and HPV33 are frequently associated with
vulvar neoplasm.3,48 Similarly, we found that HPV16 and
HPV33 were the two most prevalent HPV types in
vulvar cancer and vulvar intraepithelial neoplasia, which
emphasised that the regular screening of the vulva for
precancerous lesions should not be ignored among
women with HPV16 or HPV33 infection. It is well
established that high-risk HPV is a causative agent in
vulvar carcinogenesis, and the pooled prevalence of
HPV (for both high-risk and low-risk HPV) in our study
might lead to the overestimation of oncogenic effect of
HPV on the vulvar neoplasm. Notably, we found that the
prevalence of HPV6 and HPV11 was low in vulvar
cancer and vulvar intraepithelial neoplasia; however,
some patients with vulvar cancer or vulvar intraepithelial
neoplasia were only infected by HPV6 or HPV11,
which indicated that the potential carcinogenic effect
of HPV6 and HPV11 in vulvar neoplasm requires
further investigation.
To our knowledge, this was the first meta-analysis to
explore the pooled type-specific HPV DNA prevalence in
vulvar cancer stratified by geographical region. Despite
HPV16 being the most predominant genotype in every
geographical region, the prevalence of other type-specific
HPV genotypes varied substantially between regions,
with the second most frequent genotype being HPV18 in
Asia and South America, and HPV33 in the other
regions. We also observed a relatively high prevalence of
HPV52, HPV45, and HPV31 in Oceania and Africa
compared with other regions. Our findings reported
a different pooled type-specific HPV distribution in
different regions and underlined the possibly greater
potential of nine-valent HPV vaccination in preventing
vulvar neoplasm.
This was the first meta-analysis to report the pooled
prevalence of p16INK4a positivity in vulvar cancer and
vulvar intraepithelial neoplasia. Notably, the prevalence
of p16INK4a positivity across geographical regions in
this meta-analysis should be compared with caution,
because most studies on the prevalence of p16INK4a
positivity in vulvar cancer and vulvar intraepithelial
neoplasia originated from Europe and North America,
with only a small number of studies from other regions.
As we expected, a much higher pooled prevalence
of p16INK4a positivity was observed in HPV DNA-
positive vulvar cancer compared with HPV DNA-
negative vulvar cancer (73·3% vs 13·8%), suggesting the
active involvement of HPV in the carcino­ genesis of
10 www.thelancet.com/oncology Published online March 15, 2023 https://doi.org/10.1016/S1470-2045(23)00066-9
vulvar cancer. In this meta-analysis, the definition
of p16INK4a positivity cutoff varied across studies, with
no signifi­ cant differences in the pooled prevalence
of p16INK4a positivity.
When stratified by different subtypes of vulvar
squamous cell carcinoma, the prevalence of HPV DNA
was higher in basaloid and warty carcinoma than in
keratinising carcinoma. Additionally, the pooled HPV
DNA prevalence (86·5%) among warty carcinoma in
this study was higher than that (76·5%) in the previous
meta-analysis.11 Nevertheless, the additional studies
included in this meta-analysis might lead to increasingly
robust results. We found a higher prevalence of p16INK4a
positivity in basaloid and warty carcinoma than in
keratinising carcinoma. Furthermore, verrucous carcin­
oma (categorised as a kind of HPV-independent vulvar
carcinoma according to the latest WHO classification
criteria for female genital tumours, 2020),4 showed a
high HPV DNA prevalence of 58·9% and a low pooled
p16INK4a positivity prevalence of 0%. These results were
consistent with a study of penile cancer49 that reported a
somewhat high prevalence of HPV DNA (21·7%) and
a low pooled prevalence of p16INK4a positivity (4·8%).
However, these findings regarding verrucous carcinoma
should be interpreted with caution, owing to the small
number of cases.
HPV E6 and E7 overexpression is a well established
marker of the tumourigenesis driven by HPV.50
Nevertheless, the detection of HPV E6 and E7 gene
products (ie, proteins or mRNA) from tumour tissue
might not be easy to accomplish in routine diagnostic
laboratories. Hence, alternative markers of HPV
infection, such as the presence of HPV DNA or p16INK4a
overexpression, provide potential alternatives despite
their respective concomitant drawbacks. However, the
existence of HPV DNA could reflect a transient infection
rather than genuine HPV-driven carcinogenesis, which
might lead to the overestimation of the proportion of
truly HPV-driven tumours.51 Additionally, despite p16INK4a
overexpression being regarded as a surrogate marker
for HPV-induced transformation, it can also be caused
by an event other than HPV transformation.3,52,53 To
increase the specificity with minimal decreases in
sensitivity, some scholars have proposed that double
positivity for HPV DNA and p16INK4a could be a well
established marker for HPV-induced head and neck
squamous cell carcinoma.54
The double positivity for HPV DNA and p16INK4a is
regarded as a better prognostic marker in patients with
tonsillar and base of tongue cancer, compared with
either HPV or p16INK4a positivity alone.55 However, the
clinical value of double positivity for HPV DNA and
p16INK4a in vulvar cancer has yet to be widely investigated.
On the basis of previous findings in tonsillar and base
of tongue cancer, we speculate that patients with vulvar
cancer who have double positivity for HPV and p16INK4a
might correlate with a better prognosis than those who

are positive for either HPV or p16INK4a alone. Therefore,
patients with vulvar cancer who are positive for either
HPV or p16INK4a alone might need more intensive
treatment strategies and stricter follow-up surveillance
compared with those with double positivity for HPV
and p16INK4a. Further prospective randomised controlled
trials are warranted to investigate the association of
HPV and p16INK4a positivity with prognosis in patients
with vulvar cancer.
Our study had several limitations. First, considerable
heterogeneity among studies was observed, and despite
the investigation of sources of heterogeneity through
stratified analyses and meta-regressions, the sources
of heterogeneity could not be explained, which
might affect the generalisability of our findings.
Second, some of the estimates were based on small
samples and should be interpreted cautiously. Third,
most of the studies included in this meta-analysis
reported a mixed prevalence of both high-risk and low-
risk HPV, which limited the evaluation of pooled
prevalence for high-risk and low-risk HPV, respectively.
Finally, we included only English-language studies,
although it has been suggested that pooled estimates
do not seem to be affected by language restrictions in
meta-analyses.56
In conclusion, we found a high pooled prevalence of
HPV DNA in vulvar intraepithelial neoplasia and
vulvar cancer, with HPV16 and HPV33 being the most
predominant specific HPV genotypes, highlighting that
the regular screening of vulvar precancerous lesions
should be enhanced among women with HPV16 or
HPV33 infection. Different distribution of specific HPV
genotypes among geographical regions was observed in
this meta-analysis. These findings emphasised the great
value of prophylactic nine-valent HPV vaccination in
preventing vulvar cancer and vulvar intraepithelial
neoplasia, particularly in Africa, Oceania, and Europe.
Additionally, this study highlighted the potential clinical
importance of double positivity for HPV DNA and p16INK4a
in vulvar neoplasm.
Contributors
KS, BK, and JM conceptualised and designed the study. ZL and PL wrote
the original manuscript. ZL, PL, ZW, and ZZ collected the data. ZC, RC,
and GL did the statistical analyses. ZL, PL, QH, and YZ accessed and
verified the data. LL offered valuable suggestions and consultations on
study design and data analyses. All authors reviewed and edited the
manuscript and approved the final version. All authors had full access to
all the data in the study and had final responsibility for the decision to
submit for publication.
Data sharing
This systematic review and meta-analysis used data that were available
in previously published studies.
Declaration of interests
We declare no competing interests.
Acknowledgments
This study was supported by the Taishan Scholar Youth Project of
Shandong Province (grant number tsqn201812130). We acknowledge all
the authors whose papers have been included in this meta-analysis for
their work, which made this analysis possible.
www.thelancet.com/oncology Published online March 15, 2023 https://doi.org/10.1016/S1470-2045(23)00066-9 11
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