Discussion
The prevalence of HPV-positive tumors in our series was nearly 44.7 %. Previous studies on vSCC have reported highly variable numbers of HPV-positive cases that have ranged between 0 and 66 % depending on the method of HPV detection and the histological types of vSCC analyzed [
12‐
15].
In 2013, de Sanjosé S et al. reported 28.6 % HPV-positive cases among 1709 invasive vulvar cancers (IVC) [
5]. An overestimation of HPV positivity in our cohort could be generally explained by different case selection, as both studies were conducted in the same Laboratory (DDL Diagnostic Laboratory in Rijswijk, The Netherlands) with an identical protocol using SPF-10 broad-spectrum primers and genotyping with a reverse hybridization line probe assay (LiPA25).
Indeed, all of our cases come from Poland (Europe) while only 49.8 % of women with IVC included in de Sanjosé’s study were European. The prevalence of HPV-DNA was found to be higher among European women than women living in other geographical regions [
5].
Additionally, the number of well differentiated tumors was higher in our group than in IVC cohort described by Sanjosé S et al. (76 % vs. 71 %).
Indeed, the proportion of histological types is crucial for cohort prevalence, as it was shown that HPV-positivity among warty-basaloid and keratinizing vSCC tumors varies, and it was described as 69 and 11.5 % of cases, respectively [
5].
Our study confirmed the predominant contribution of HPV-16 to the etiology of HPV-related vulvar cancer and suggested that other HPV types, such as HPV-33, HPV-18 and HPV-56, which are common in cervical cancer, are also important to vulvar carcinogenesis, although to a lesser degree.
In 28.6 % of p16
ink4a-positive tumors, a lack of (hr)HPV-DNA was observed, and in 24.0 % of p16
ink4a-negative tumors, (hr)HPV-DNA was detected. The substantial mismatch between p16
ink4a-overexpression and HPV-status reported here was also observed in the largest cohort of 1709 vSCC cases [
5]. Seventeen percent of tumors expressing p16
ink4a lack (hr)HPV-DNA, and 9.4 % of tumors lack p16
ink4a-overexpression despite the presence of (hr)HPV-DNA [
5].
A lack of (hr)HPV-DNA in p16-positive tumors could be explained by the fact that the HPV oncoprotein, E7, which functionally inactivates RB, is not the only thing responsible for the increases in p16
ink4a expression [
16]. Although it is believed that RB inactivation is a requisite for the elevation of p16
ink4a expression in cancer [
2,
3], aberrations in the RB pathway are not obvious in every tumor. The RB checkpoint is deregulated by multiple mechanisms independent of
RB1 mutation, deletion or methylation. The viral oncogene expression represents just one potential form of multiple possible ways of RB inactivation [
16].
Several findings have proven the strong association between age-promoting, ‘gerontogenic’ signals and p16
ink4 expression [
16]. Thus, the impact of senescence and inflammation on p16
ink4 expression in our older age cohort of vSCC patients should also be considered.
There remains the possibility that a certain fraction of HPV-negative samples were false negatives. However, during the testing, we checked all the samples for amplifiable human genomic DNA. All samples showed the presence of human DNA by PCR (RNAseP gene). The size of the PCR fragment in this test is also 65 base pairs, and therefore, it is most sensitive PCR for formalin-fixed paraffin-embedded/FFPE/tissue samples.
(hr)HPV-DNA-positive cancer cases without marked p16-overexpression could be explained by the fact that close to half of all human cancers show p16
Ink4a-inactivation, ranging from 25 to 70 % [
17]. Such an event could exist parallel to functional inactivation of RB by the E7 protein. Some of the HPV-positive samples could also be false positive. By performing Laser Capture Microdissection [
18], it is possible to assign HPV types to the lesional cells themselves; however, it was not performed in the current study. Therefore, we cannot exclude the possibility of contamination of the cancer samples by HPV virions from the surrounding vulvar epithelium.
Taking these facts together, we postulate not to treat p16
ink4a-overexpression as a surrogate marker for (hr)HPV infection in vSCC. The correlation between p16
ink4a and (hr)HPV-DNA varies in squamous cell carcinomas. In cervical cancer, p16
ink4a overexpression and (hr)HPV status are quite well correlated [
19], while in oral cancer, a lack of concordance is frequently reported [
20].
The combined presence of (hr)HPV-DNA and p16
ink4a-overexpression was detected in 25 of the 85 cases (29.4 %). This result is in the range of the series reported by de Sanjosé S et al., who have reported 22.4 % HPV-driven cases out of 1709 vSCCs [
5]. Probably, this is the real contribution of the HPV infection to vSCC development.
In the following analyses, we assessed the prognostic significance of (hr)HPV-DNA status and p16 overexpression separately. The (hr)HPV-DNA status of the primary tumor has no impact on the survival of vSCC patients. P16-overexpression was found to be prognostic, and also predicted a better response to radiochemotherapy.
Several reports investigating the relationship between HPV DNA and vSCC prognosis have produced conflicting results [
6,
21‐
24]. Two old studies from the early 1990s [
23,
24] reported a better survival in DNA HPV-positive patients, but their results are both hampered by the limited number of cases included (55 and 60, respectively) and the tests used for HPV detection. In recent years, one paper (with a median follow up of 42 months) confirmed a prolonged survival in patients with vSCC tumors positive for high risk DNA HPV [
22], but two others (with a longer follow up) denied the prognostic significance of HPV DNA within cancer tissue [
6,
21].
We identified only two studies that utilized p16 expression for the survival analysis of vSCC patients, and they reported contradictory results [
6,
25]. Our results were consistent with the findings of Tringler et al. [
25], but they were in opposition to the results of Alonso et al. [
6], who did not identify p16 status as a prognostic indicator of vSCC. The low prevalence of p16-positive tumors (19 % [19/98]) might explain the lack of prognostic significance of p16 status in the Alonso et al. study, whereas the percentage of p16-positive cases in our study and that by Tringler et al. [
25] was 41 % (35/85) and 43 % (34/80), respectively. The median follow-up in the Alonso et al. cohort was only 45 months [
6], while it was 52 and 89 months in the Tringler study [
25] and our study, respectively.
The conducted univariate and multivariate analyses revealed that p16-overexpression is an independent prognostic factor with respect to survival.
RB inactivation releases p16
Ink4a from its negative feedback control, causing a paradoxical increase in the levels of this protein, which attempts to inhibit uncontrolled cellular replication [
26]. Thus, it is not surprising that p16
ink4a-overexpression itself (not the HPV virus) has a protective role in HPV-related malignancies.
It was shown that cancers that present p16
Ink4a-overexpression are very sensitive to radiotherapy, and they have a better prognosis [
27]. A better response to radiochemotherapy has been associated with a more favorable prognosis of HPV-positive head and neck cancers [
28‐
32]. Indeed, we also confirmed that p16
Ink4a-overexpression predicts a better clinical outcome among patients requiring adjuvant radiotherapy. The two compared groups (irradiated p16-positive and p16-negative) were similar in terms of the number of positive nodes and the presence of extracapsular spread, which strongly supports this conclusion.
This study has the traditional weaknesses of a retrospective design, and the results obviously represent a small cohort. Lack of information on smoking and cause of death potentially limit the prognostic analysis. The strengths of the study include the treatment of patients according to uniform standards and a sufficient follow-up duration to reveal recurrences and to allow for the reliable assessment of the prognostic significance of all analyzed biomarkers. Data on (hr)HPV-DNA prevalence were provided by highly experienced sources in the HPV-DNA detection laboratory.