HPV-related Vulvar Diseases and Perspectives of p16 INK4a Immunochemistry: A Review of the Literature

Abstract

Introduction

Two different types of vulvar intraepithelial neoplasia (VIN), HPV-related and HPV-unrelated, should be considered as two separate entities with different management options. The incidence of HPV-related VIN is increasing worldwide and is implicated in carcinogenesis. Our objective is to investigate the use of p16^INK4a immunostaining or p16^INK4a/p53 double staining for the detection of HPV-related disease to overcome the problem that histological criteria often have significant overlap.

Methods

A systematic literature search was carried out in the online databases PubMed, EMBASE, Cochrane Library, Clincaltrials.gov and Scopus. The key search terms were HPV, VIN, p16^INK4a immunochemistry and p53.

Results

We found that nuclear and cytoplasmic immunostaining for p16^INK4a was intense and diffuse in HPV-associated lesions and weak and focal in normal vulvar epithelium, nondysplastic lesions, lichen sclerosus and keratinizing vulvar squamous cell carcinoma. p53 nuclear immunostaining was always negative in HPV-related disease.

Conclusions

Our findings indicated that p16^INK4a or p16^INK4a/p53 immunoreactivity, along with histological diagnosis, could be a convenient means to adequately classify VIN and its connection to HPV infection. Therefore, the clear recognition of HPV-associated VIN would lead to an appropriate strategy of treatment and follow-up.

Keywords

HPV p53 VIN

Introduction

Vulvar disease is a rare affection of the lower genitalia, with incidence rates of 0.5-2.5 in 100,000 women (4% of gynecological malignancies) (1). Cases of invasive vulvar cancer (IVC) and vulvar intraepithelial neoplasia (VIN) have been rising recently among younger women and the rates of new vulvar cancer events have increased by 0.5% each year over the last 10 years (2, 3). Vulvar squamous cell carcinoma (VSCC) accounts for more than 90% of malignant tumors of the vulva (3), with 2 different etiopathogenic pathways for its development. One is associated with high-risk human papillomavirus (hrHPV) infection and presents with multifocal VIN (warty or basaloid types). The other is not hrHPV related but is more likely associated with chronic granulomatous diseases, squamous hyperplasia, or lichen sclerosus in the adjacent epithelium and may be associated with differentiated-type VIN (4, 5).

In 2004, the International Society for the Study of Vulvar Disease (ISSVD) proposed this VIN classification: HPV-associated usual VIN (uVIN, classic or bowenoid) and HPV-unrelated or differentiated VIN (dVIN, or simplex) (6). HrHPV DNA sequences have been detected in 52%-100% of uVIN and in few cases of dVIN (7–8–9).

HPV-related carcinogenesis is characterized by hrHPV DNA integration into the host DNA (10). As in cervical cancer, p16^INK4a is a marker of HPV integration into the host genome and a useful tool to classify VIN into hrHPV-associated and hrHPV-independent vulvar lesions. The sensitivity and specificity of p16^INK4a immunostaining for detecting uVIN are close to 100%, higher than HPV DNA testing (HC2/PCR) and histological classification (11, 12).

The aim of this review is therefore to collect studies that evaluated p16^INK4a immunostaining in vulvar histological disorders, in order to define the prognosis and management of both uVIN and dVIN.

Materials and Methods

A systematic literature search was conducted to target all scientific publications related to the evaluation of p16^INK4a immunoreactivity versus HPV testing and p53 immunoreactivity in vulvar diseases (VIN and VSCC) from January 2004 to December 2016. PubMed (MEDLINE), EMBASE, the Cochrane Library, Scopus as well as Clincaltrials.gov were systematically and independently screened by 2 authors (Carrone A, Savone D) and then compared to identify the eligible studies. HPV, VIN, p16^INK4a immunochemistry and p53 were used as keywords. We included all English-language retrospective and prospective studies at primary diagnosis assessing p16^INK4a immunostaining or p16^INK4a/p53 staining, with HPV DNA testing (HC2/PCR) as the comparator test, on histological specimens of VIN and VSCC verified with a reference standard. The references of the included studies were independently hand-searched to identify other potentially relevant studies and to eliminate duplicates. Two independent reviewers evaluated all studies to verify the inclusion criteria and make sure the PRISMA guidelines were followed while elaborating this review.

The baseline characteristics of the studies were extracted from each article: the first author, journal and year of publication, and the number of specimens examined in relation to the number of patients. The triage tests were itemized with the commercial triage methods used for each study. The initial findings of the patients and specimens surveyed were also specified. The last items were the outcomes explaining HPV-detection prevalence in association with the immunoreactivity used and the microscopic assessment cutoffs identified for each study to determine the positivity or negativity of the specimens. p16^INK4a positivity levels were considered significant when there was intense and diffuse nuclear and cytoplasmic staining. Basic statistical analysis was conducted using common methods of descriptive statistics with assessment of arithmetic mean, minimum and maximum values and geometric mean to evaluate the sensitivity and specificity percentages of p16^INK4a and HPV DNA, p53 and Ki67.

Studies whose full manuscript or baseline characteristics were not available were excluded from our analysis.

Results

The electronic search yielded 36 eligible studies for the review. Twenty-eight were excluded because patients received radiotherapy (1 study), the language was not English (7 studies), or the results were not compared with HPV testing (20 studies). The remaining 8 studies were included in the review.

The characteristics and technical information of these 8 studies are listed in Table I. One provided data on p16^INK4a immunoreactivity in VIN (13); 2 in VIN plus VSCC (11, 14); another 2 in normal vulvar squamous epithelium, VIN and VSCC (15, 16); the remaining studies (8, 17, 18) evaluated only VSCC. In particular, a retrospective cross-sectional study including 39 countries (14) evaluated HPV DNA and p16^INK4a levels by PCR and immunochemistry, respectively. Five hundred and eighty-seven of the total 2,296 (25.5%) evaluated cases proved to be VIN and 1,709 (74.4%) were identified as IVC. In warty or basaloid type VSCC, positivity for both HPV and p16^INK4a was more frequent. HPV16 was the predominant genotype, followed by HPV18 and HPV33.

Table I

Details and findings of included studies

First author, year (ref.)	Specimens No. pts.	Initial findings	Triage test	Triage methods	Outcomes: immunoreactivity/HPV prevalence	Microscopic evaluation
Santos 2006 (11)	92/92	VIN/VSCC	HPV/p16/p53	p16^INK4A monoclonal Ab clone JC8, Biocare Medical, 1:100//p53 monoclonal Ab BP53-12, Novocastra, dilution 1:50//PCR processor GeneAmp PCR System 9700, Applied Biosystems - PCR fragment (SPF10) primer set 14, Innogenetics Diagnostica	uVIN: HPV+ 100%/p16+ 100%/p53+ 0% dVIN: HPV+ 0%/p16+ 0%/p53+ 90.8% warty/basaloid VSCC: HPV+ 62.5%/p16+ 100%/p53+ 6.2% keratinizing VSCC: HPV+ 37.5%/p16+ 1.3%/p53+ 64.5%	p16+: nuclear and cytoplasmic staining in 25% or more of cells p53+: nuclear staining in 25% or more of cells
Hoevenaars 2008 (13)	120/120	VIN	HPV/p16/p53/Ki67	p16^INK4A clone 16PO4, Neomarkers, 1:500//p53 clone DO7, Dakocytomation, 1:400//MIB1 clone MIB1, Dakocytomation, 1:200//HPV PCR SPF-PCR-LiPA system	uVIN: HPV+ 80%/p16+ 80%/p53+ 7%/MIB1 high+ 98% dVIN: HPV+ 0%/p16+ 0%/p53+ 84%/MIB1 low 96%	p16+: diffuse transepidermal nuclear and cytoplasmic staining p53+: diffuse nuclear staining; Ki67 low+: +cells in the parabasal layers or one third of epithelium; Ki67 high+: +cells in the lower two thirds of epithelium or entire epithelium
De Sanjosé 2013 (14)	2,296/2,296	VIN/VSCC	HPV/p16	p16 CINtec histology kit clone E6H4, Roche mtm laboratories//HPV PCR SPF-PCR-LiPA system	Keratinizing VSCC: HPV+ 13.4%/p16+ 13.3% warty/basaloid VSCC: HPV+ 69.9%/p16+ 76.4% mixed VSCC: HPV+ 30.7%/p16+ 35	p16+: strong and diffuse nuclear and cytoplasmic staining
Riethdorf 2004 (15)	177/177	Normal epithelium and nondysplastic lesions (n = 52)/VIN/VSCC	HPV/p16	p16^INK4A monoclonal antibody clone G175-405, PharMingen; 1.25 g/mL//HPV DNA PCR//HPV16 E6/E7 ISH	Normal epithelium and nondysplastic lesions: HPV+ 0%/p16+ (heterogeneous) 7.6% VIN3: HPV+ 95%/p16+ 82% basaloid/warty VSCC: HPV+ 100%/p16+ 100% keratinizing VSCC: HPV+ 7%/p16+ 4%	p16+: diffuse strong nuclear and cytoplasmic staining
Rufforny 2005 (16)	49/49	Normal epithelium and nondysplastic lesions (n = 11)/VIN/VSCC	HPV/p16	HPV DNA PCR//p16^INK4A monoclonal Ab clone JC8, Biocare Medical, 1:25	VIN1: HPV+ 10%/p16+ 20% VIN2: HPV+ 67%/p16+ 33% VIN3: HPV+ 81%/p16+ 100% VSCC: HPV+ 100%/p16+ 100%	p16+: >10% positivity (2+; 3+)
Hay 2016 (17)	92/92	VSCC	HPV/p16/p53	HPV DNA PCR//p16^INK4A IHC//p53 IHC	Performed on available samples: HPV+ 59% p16+ 54% p53+ 27.7%	p16+: strong, band-like staining involving at least 2/3 tumor thickness p53+: 2+ or 3+ (dark, intense nuclear staining)
Alonso 2011 (8)	98/98	VSCC	HPV/p16/p53/Ki67	HPV DNA PCR//p16^INK4A CINtec histology kit (mtm labs)//Monoclonal antibody BP53-12, Novocastra, 1:50	VSCC HPV+ 45% p16+ 32.6% p53+ 34.3% Ki67+ 53.3%	p16+: diffuse staining (continuous staining of cells of the basal and parabasal layers) p53+: >50% neoplastic cells reactivity (strong) Ki67+: >30% reactivity
Reuschenbach 2013 (18)	186/186	VSCC (anterior fourchette + other locations)	HPV/p16/p53/Ki67	HPV DNA PCR//p16 CINtec histology kit clone E6H4//p53 clone DO7, Dako	VSCC HPV+ 42% p16+ 31% p53+ 37.4%	p16+/Ki67+: diffuse strong nuclear and cytoplasmic staining. Ki67 is p16-associated as dual-staining (at least 1 cell expressing both proteins) p53+: >50% positivity

dVIN = differentiated VIN; HPV = human papillomavirus; IHC = immunohistochemistry; nondysplastic lesions = hyperplasia, lichen sclerosus, lichen simplex chronicus, seborrheic keratosis, condylomas; No. Pts. = number of patients; uVIN = usual VIN; VIN = vulvar intraepithelial neoplasia; VSCC = vulvar squamous cell carcinoma.

Two studies applied p16^INK4a and p53 immunostaining and 3 also applied Ki67 immunostaining. All included studies allowed comparison of immunochemistry triage with HPV DNA PCR test. The positivity criteria for p16^INK4a immunostaining were highly homogeneous in all studies: strong nuclear and cytoplasmic staining in the basal and higher layers of epithelium was considered positive for p16^INK4a expression. In the study by Santos et al (11), p16^INK4a positivity was established when sheets or large clusters of contiguous positive cells representing more than 25% of epithelium were identifiable; also p53 nuclear immunostaining was considered positive when found in 25% or more of cells. For Ki67 protein, immunoreactivity localization in the parabasal layers or in the lower third of the epithelium was considered “low-level”; Ki67 reactivity in the lower two-thirds or in the full-thickness epithelium was considered “high-level”.

Immunohistochemistry was performed in 3,110 specimens with a histological diagnosis of VIN and VSCC, including also 2% (n = 63) of samples of normal epithelium or nondysplastic lesions (hyperplasia, lichen sclerosus, lichen simplex chronicus, seborrheic keratosis, condylomas). Four studies evaluated sample data and percentages for uVIN and dVIN (11, 13–14–15); the other 4 also considered immunostaining in normal epithelium, benign lesions, VIN and VSCC, without distinguishing between uVIN and dVIN (8, 16–17–18).

HPV positivity and p16^INK4a immunoreactivity detected in uVIN ranged from 80% to 100%, p53 immunostaining was positive in 0%-7% of cases, and MIB1 immunostaining was high in 98% of cases. dVIN was always HPV and p16^INK4a negative, while p53 immunostaining was detected in 84%-90.8% of cases and MIB1 immunostaining was low in 96% of cases. In specimens of warty/basaloid VSCC, HPV positivity ranged from 62.5% to 100%, p16^INK4a immunoreactivity from 76.4% to 100%, and p53 immunostaining was positive in 6.2% of cases. In specimens of keratinizing VSCC, HPV positivity ranged from 1.3 to 13.3% and p53 immunostaining was positive in 64.5% of cases where evaluated.

Results of Individual Studies

By examining each work by its salient characteristics, we can highlight the following findings. In the study performed by Santos et al (11), 92 VSCC surgical specimens, consecutively and retrospectively identified, were examined for HPV DNA, p16^INK4a and p53 positivity. The primary aim was the detection of the immunohistochemical pattern of expression of the 2 types of vulvar cancers and its accuracy: warty/basaloid (HPV-related) and differentiated keratinizing (HPV-independent). Discrepancies were not infrequent in this study. HPV DNA was identified in 16/92 samples (17.4%), while diffuse positivity for p16 and p53 was observed in 100% and 6.2% of HPV-positive tumors and in 2.3% and 64.5% of HPV-negative tumors, respectively. The sensitivity and specificity of p16^INK4a to detect HPV-associated carcinomas were 100% and 98.7%, respectively, which compares favorably to histological criteria (with 93.8% sensitivity and 35.5% specificity) and to p53 negativity (with 62.5% sensitivity and 93.4% specificity). No age differences were found with respect to the other studies evaluated. In the study by Hoevenaars et al (13), VIN specimens of 120 consecutive patients were examined: 75 had dVIN and 45 had uVIN. Most of them showed close areas positive for squamous cell carcinoma (SCC). p16^INK4a, HPV DNA, MIB1 and p53 were examined using immunohistochemical staining, which identified nuclear positivity. A difference in patient age was highlighted: in women with uVIN with adjacent SCC areas the median age was lower (52 years, standard deviation [SD] 13.4 years) than in those with dVIN with associated SCC (74 years, SD 12.5 years). Additionally, women with uVIN and no SCC areas had a still lower median age at the time of diagnosis: 36 years (SD 10.8 years). HPV DNA positivity also was distinguishable between uVIN and dVIN: the first type showed a significantly higher percentage (84%) of hrHPV positivity (also if adjacent SCC areas were present), while 100% of dVIN cases were hrHPV negative. A comparable trend was found for p16^INK4a, thus showing a high correlation between the 2 markers.

In the retrospective study by Riethdorf et al (15), p16^INK4a was assessed by a monoclonal antibody to correlate its positivity levels to those of HPV DNA (PCR amplified) in vulvar cancers compared with benign conditions. One-hundred and seventy-seven samples were considered: 123 cases of VIN, 41 cases of VSCC, and 13 benign squamous epithelium samples. Patient age was unremarkable, with some exceptions: the mean age of women with HPV16 positivity (and the relative E6/E7 transcription) was significantly lower (p<0.001) than that of women with vulvar cancers not linked to HPV DNA.

Rufforny et al (16) in 2005 reported the different expression pattens of p16 for benign vulvar lesions, VIN1, VIN2, VIN3 and VSCC, concluding that in benign cases and in condylomatous lesions, p16^INK4a and HPV DNA (generally the HPV16 genotype) showed no expression, thereby demonstrating the usefulness of p16^INK4a in premalignant and malignant lesions.

The 2016 study by Hay et al (17), one of the most recent studies evaluated, concluded that positivity of HPV DNA and consequently its “surrogate biomarker p16^INK4a” in vulvar cancer marks a less aggressive form, while on the other hand p53 reactivity is related to a worse prognosis and a lower overall survival rate because of recurrence and disease-specific mortality.

In the study by Alonso et al (8), all HPV-positive VSCCs were associated with positive p16^INK4a staining, while only 1.3% of HPV-negative VSCCs were p16^INK4a positive. p53 positivity was, by constrast, of high intensity in HPV-negative tumors, dVIN and squamous cell hyperplasia.

Reuschenbach et al (18) also evaluated VSCC based on HPV DNA, p16^INK4a and p53 expression, with tumor location limited to the anterior vulvar fourchette. They concluded that this location is more frequent in younger women (56.7 years vs. 63.1 for other vulvar sites), but the expression of these markers did not vary from one site to the other.

Discussion

The role of p16^INK4a expression in uterine cervical cancer is well established. However, its expression in vulvar carcinomas, which have a diverse pathogenesis, has not been extensively studied and only a few studies were reported in the scientific literature.

Despite the bias of few comparable studies, our findings indicated that p16^INK4a immunoreactivity could be a helpful tool to correctly classify VIN and VSCC according to their connection to hrHPV infection, and to overcome the problem that histological criteria often overlap. This overlapping may lead to a certain level of subjectivity, as reported by some authors (19, 20). Keratinization in basaloid carcinomas may sometimes complicate the differential diagnosis with poorly differentiated keratinizing VSCC; in particular, it can be arduous to discriminate dVIN from a benign vulvar lesion or from uVIN (21).

HrHPV-associated premalignant lesions and carcinomas showed intense and diffuse nuclear and cytoplasmic immunostaining for p16^INK4a, while no expression of p16^INK4a or heterogeneous expression was found in normal vulvar epithelium and nondysplastic lesions.

A risk of bias across the assessed studies was due to the not perfectly homogeneous interpretation of p16^INK4a expression and the different kits used for immunostaining; thus, it will be necessary to standardize the nuclear and cytoplasmic scoring system and the immunohistochemistry technique. The evaluated studies showed comparable p16^INK4a immunoreactivity and HPV positivity in HPV-related uVIN. For HPV-related basaloid/warty VSCC, p16^INK4 immunostaining was able to distinguish transforming HPV infections better than HPV DNA detection and with higher interobserver agreement. Technical problems, such as the varying sensitivity or the possibility of contamination, and the high cost of the molecular test employed for HPV investigation (HC2 or PCR assay) could be overcome by applying p16^INK4a, p53 and/or Ki67 immunostaining. Normal vulvar epithelium, nondysplastic lesions and HPV-related uVIN and basaloid/warty VSCC were negative for p53, while nuclear and diffuse p53 immunostaining was detected in dVIN and keratinizing VSCC. Also, Ki67 immunostaining showed high, diffuse and nuclear positivity in uVIN, while it was absent or confined to the parabasal epithelial layer in dVIN and it was absent in normal epithelium. Like in cervical immunochemistry, p16^INK4a or p16^INK4a/p53 or p16^INK4a/Ki67 dual staining could improve the identification of uVIN and increase the sensitivity of histological interpretation.

Differently from dVIN, there is no agreement on the gold-standard treatment for uVIN, which often presents multicentric and multifocal involvement. High morbidity and recurrence rates after surgery make less invasive treatments highly advisable. However, combined modalities with topical treatment (imiquimod or cidofovir) and a surgical approach may represent the best treatment for this multiform disease (13, 19, 20, 22–23–24). Therefore, the clear recognition of HPV-related lesions should lead to an appropriate strategy of treatment and follow-up.

Review studies indicated that p16^INK4a or p16^INK4a/p53 immunoreactivity along with histological diagnostics could adequately classify VIN and its connection to HPV infection. Lee et al (25) evaluated the presence of HPV or its surrogate, p16 immunostaining, as an independent prognostic factor for in-field relapse and survival in women with VSCC treated with radiotherapy. Hay et al (17) found that patients with p16 and HPV positivity were, in both cases, more protected against vulvar cancer recurrences and against death from the disease, while the opposite was true for p53 positivity, which was associated with a 3-fold greater risk of recurrence and a 7-fold greater risk of death from the disease. These findings could be useful to personalize surgical and adjuvant treatments basing on different molecular profiles, but in the patient series studied by Alonso et al (8) HPV/p16-positive VSCC showed a similar prognosis to HPV/p16-negative VSCC. Also Reuschenbach et al (18) highlighted that hrHPV/p16 positivity is frequent in young women, irrespective of tumor site (anterior fourchette or other vulvar locations). This is in agreement with the findings of Rufforny et al (16), who demonstrated that HPV-related VIN is a “younger” pathology than HPV-negative vulvar tumors.

Further, larger and controlled studies are needed to obtain consensus diagnostic results in order to use these markers in everyday clinical practice as a valid, effective and economically convenient instrument.

Footnotes

Abbreviations

Financial support: None.

Conflict of interest: The authors declare they have no competing interests related to this article.

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