To the best of our knowledge, this is the first case series reporting synchronous conventional vulvar intraepithelial lesion and acquired epidermodysplasia verruciformis of the vulva. Previous reports in which classic histologic features of EV were present in vulvar specimens include two cases reported by Pohthipornthawat et al9 and one case by Singh et al.10
In the former study both patients displayed exclusively EV histologic changes, with no concomitant classic vulvar intraepithelial lesion.9 Presumably this can be attributed to iatrogenic acquired EV, considering one patient had received a stem cell transplant and the other was status post multi-organ transplant prior to the vulvar biopsies. Both patients tested positive for β-HPV-5 by PCR performed with DNA extracted from the tissue specimens.
Alternatively, the latter study10 presents a young patient with the inherited form of EV who was diagnosed with high grade vulvar intraepithelial lesion and simultaneous EV changes plus high grade squamous intraepithelial lesion of the uterine cervix. The cervical biopsy showed no EV features. This is consistent with the findings reported in our series, as both patients 2 and 3 had a long history of cervical dysplasia, but none of the cervical specimens reviewed showed the EV features seen in their respective vulvar biopsies. This could be explained by the fact that β-HPV subtypes show stricter cutaneous tropism when compared with α-HPV subtypes that can infect both skin and mucosal surfaces.1,4 In fact, the patient reported by Singh et al was positive for HPV16 and other high-risk α-HPV subtypes by PCR testing on the cervical sample. These would putatively be responsible for the patient’s concurrent cervical and vulvar high grade intraepithelial lesion. No testing for β-HPVs was performed in that study. In our series, both patient 2 and patient 3 were also positive for other high- risk α-HPV subtypes by PCR testing on cervical samples.
While reports of EV histologic features on vulvar biopsies are recent and scarce, DNA of EV-related HPV subtypes was isolated from vulvar specimens as early as 19827, a time in which many HPV genotypes and their causal relationship to human cancer were yet to be characterized. In that study, DNA from an HPV subtype tentatively named “HPV-EV” was isolated from the skin lesions in the hands and legs of a patient with known EV. Homologous “HPV-EV” DNA was extracted from a Bowenoid carcinoma “in situ” of the vulva in the same patient. There was no detailed histologic characterization of this tumor.
Four years later, de Villiers et al16 screened 268 biopsies of a diverse cohort of genital tumors in search of HPV DNA. Three condylomata in that study were found to have unexpected HPV genotypes: two tested positive for HPV 3-related DNA and one tested positive for EV-related HPV DNA. Once again, no morphologic description was provided in the article.
The wide temporal gap between the observations regarding EV and gynecologic pathology likely originates from the fact that α-HPV subtypes, especially high-risk genotypes such as HPV-16 and HPV-18, have been the drivers of research and advancements in the field.2,3 In current clinical practice, routine testing understandably contemplates almost exclusively these high-risk subtypes.
By whole genome sequencing, all four patients in our study demonstrated DNA reads mapping uniquely to both α-HPV and β-HPV reference genomes. While several HPV genotypes could be found, the majority of reads clustered in between 2 to 5 genotypes, which are more likely to represent evidence of actual infection. The “heatmap” distribution used in figure 6 displays most of the HPV subtypes found but emphasizes the greater likelihood of infection by the genotypes most reads come from. The lower number of reads from other HPV subtypes could be attributed to infection with a low number of viral copies, or attributed to noise, considering how similar the DNA sequences between each subtype can be. Nevertheless, in this clinicopathological setting, the WGS findings confirm our hypothesis of co-infection leading to the dual phenotype of conventional high-grade dysplasia (VIN 3) and simultaneous EV histologic changes.
The presence of EV histologic features and DNA from EV-associated β-HPVs in vulvar biopsy samples opens new lanes of investigation. Patient 4, a young female in our series with no clinical history of immune deficiency, represents an illustrative case in terms of uncharted territories. By histology alone, a hyperkeratotic lesion of the vulva displaying focal, yet classic EV features and multinucleated atypia of the vulva (MAV) is a puzzling finding. The patient had no signs of recurrence after a 9-month follow-up period, which may indicate that immunocompetent patients could present with transient or self-limiting, focal EV lesions.
The current understanding of MAV has remained mostly unchanged since the original report was published by McLachlin et al17. MAV is thought to be a reactive change to the keratinocytes in a background of chronic irritation, and previous attempts to detect HPV DNA or evidence of infection by other viruses in such lesions has been unsuccessful17,18,19. Patient 4 in our study demonstrated, by WGS, roughly 55% of reads mapping uniquely to the reference genomes of β-HPVs, namely HPVs 24, 5 and 47, all of which have been previously implicated in EV. It is entirely possible that these findings are coincidental, considering the hyperkeratotic / irritated nature of the patient’s lesion and the fact that cutaneous infection by β-HPVs in immunocompetent humans is thought to be mostly asymptomatic. However, the possibility of EV-related β-HPV subtypes being implicated in MAV needs to be explored.
Furthermore, other lesions previously regarded as HPV-independent may be related to infection by β-HPV subtypes for which testing is not frequently performed. Vulvar vestibulitis or vulvitis, a disputed lesion with relatively vague clinicopathological characteristics20 has been revisited in a 2008 study that reported persistent detection of novel EV-related HPV genotypes21. A possible association between EV-related β-HPV subtypes and vulvo-vaginal melanomas has also been proposed22.
At this point in time, the relationship between β-HPV subtypes and vulvar cancer remains unclear. Is EV another risk factor for cancers of the vulva, at least in the population harboring some form of immune deficiency? Could EV-related HPV subtypes be implicated in the genesis of poorly understood vulvar lesions, such as MAV and vulvitis?
Our study also appears to be the first to report DNA sequencing in patients presenting clinically with the acquired form of EV. Frameshift variants in genes previously reported to be implicated in both classic inherited EV (TMC6)6 and non-classic inherited EV (MST1 and CORO1A)6 were found in three patients in this series.
Additionally, mutations in LCK and DOCK8, found in patient 2 in our study, have been more recently described in patients that appear to harbor non-classic inherited EV via cell-mediated immune deficiency23,24.
Patient 2 in our study also harbored a frameshift variant in CARMIL2, a gene that has never been associated directly with EV skin lesions but is linked to a genetic immune deficiency syndrome that is implicated in a myriad of clinical findings including Epstein Barr Virus-associated smooth muscle tumors, dermatitis, viral and fungal skin infections, and even early onset inflammatory bowel disease25,26,27,28,29.
Unfortunately due to the lack of non-lesion DNA samples from our patients, we cannot establish with absolute certainty which mutations are germline variants and which are somatic mutations. However, apart from the frameshift variant in DOCK8, all other variants found in our study have extremely low frequencies or seem to be “non-existent” in population databases, which could indicate that they might be somatic. The DOCK8 variant found in both samples from patient 2, even though failing the quality filters in P2B, seems to harbor a higher likelihood of representing a germline variant. Additionally, the different mutational profiles found between P2A and P2B may represent evidence that the molecular background in non-classic EV is not static over time.
The WGS findings in our study are intriguing, as they provide initial insight into the possible mechanism for the development of acquired EV. We propose that mutations in genes related to cell-mediated immunity, whether germline or somatic, are either a precondition or a putative risk factor for the development of acquired EV in the setting of an external immune suppressant (whether iatrogenic or infectious). This may partially explain the rarity of the disease, despite an ever-increasing lifespan and absolute number of patients under some form of immune suppression. Further studies are necessary to elucidate the pathogenesis of acquired EV, preferably with long term follow-up and sequencing performed on lesional samples as well as controls.
In summary, we report the first case series in which histologic features of epidermodysplasia verruciformis are seen in tandem with vulvar intraepithelial lesion, coexisting within the same surgical specimen. This phenomenon is caused by co-infection by both α-HPV and β-HPV genotypes and appears to be facilitated by immunosuppression and/or alterations in genes related to cell-mediated immunity. For clinical practitioners and pathologists, we propose that the finding of EV features in vulvar biopsies should prompt investigation of the patient’s immune status.