Differential etiopathogenic features of vulvar squamous cell carcinomas in sub‐Saharan Africa and Europe

Abstract Two pathways have been described for vulvar squamous cell carcinomas (VSCC), one associated with human papillomavirus (HPV), and the other HPV‐independent. We compared the etiopathogenic features of a series of VSCC from Mozambique, a sub‐Saharan country with high prevalence of HPV and HIV, with those of Spain, a European country with low prevalence of HPV and HIV. All VSCC diagnosed at the two institutions from January 2018 to December 2020 were included (n = 35 and n = 41, respectively). HPV DNA detection and genotyping, and immunohistochemistry for p16 and p53 were performed. Tumors showing p16 positive staining and/or HPV DNA positivity were considered HPV‐associated. 34/35 tumors (97%) from Mozambique and 8/41 (19%) from Spain were HPV‐associated (P < .001). Mean age of the patients from Mozambique and Spain was 45 ± 12 and 72 ± 14, respectively (P < .001). No differences were found in terms of HPV genotypes or multiple HPV infection rates. 1/35 tumors (3%) from Mozambique and 29/41 (70%) from Spain showed abnormal p53 immunostaining (P < .001). In contrast with the predominance of HPV‐independent VSCC affecting old women in Europe, most VSCC in sub‐Saharan Africa are HPV‐associated and arise in young women. This data may have important consequences for primary prevention of VSCC worldwide.

Project PI20/00368; Caracterizaci on gen omica de los carcinomas de vulva independientes de virus del papiloma humano y de sus precursores", Grant/Award Number: PI20/ 00368 Vulvar squamous cell carcinoma (VSCC) can arise from HPV or non-HPV causes, and that affects prognosis. Here, the authors compared VSCC samples from a population in Mozambique, where HPV prevalence is high, with those from Spain, where it is low. In Mozambique, 97% of VSCC were HPV-associated, based on p16 staining and HPV DNA positivity, compared with only 19% of VSCC in Spain. Mean age of patients in Mozambique was 45, vs 72 in Spain. Vaccination programs, the authors suggest, could therefore help prevent the majority of VSCC in sub-Saharan Africa.

| INTRODUCTION
Human papillomavirus (HPV) is a well-established cause of a variety of human cancers, which include anogenital neoplasms (cervix, anal canal, vulva, vagina and penis) as well as head and neck tumors. 1 In contrast with the uterine cervix, where HPV is etiologically involved in the vast majority of cancers, 1 in other anatomical sites, such as the head and neck, the penis or the vulva, a second, HPV-independent, etiopathogenic pathway is responsible of a significant proportion of tumors. In all these sites, both HPV-associated and HPV-independent tumors are squamous cell carcinomas (SCCs), and this histological type represents over 90% of the malignant neoplasms. In vulvar squamous cell carcinomas (VSCC), it is traditionally accepted that basaloid or warty subtypes are associated with HPV, 2 while keratinizing variants tend to arise independently of HPV infection. 3 However, pure histological criteria have shown limitations in classifying a VSCC as HPVassociated or independent. 4 Interestingly, a number of studies have consistently shown that, in practically all the anatomical sites, HPVassociated tumors have better prognosis than HPV-independent carcinomas. 5,6 Due to this prognostic relevance and the consequences in terms of possible prevention related to HPV vaccination programs, the last revisions of the WHO classifications of cancers of the vulva, 7 vagina, 7 the penis, 8 or the head and neck 9 separate SCCs into two main categories, HPV-associated and HPV-independent. Due to the limitations of pure histological criteria and the assumed sensitivity and specificity of molecular techniques of HPV detection 10 and p16 immunohistochemical (IHC) staining, a surrogate biomarker of HPV status, 11 HPV detection and/or p16 IHC are considered by the World Health Organization (WHO) as essential diagnostic criteria for VSCC.
The proportion of HPV-associated and -independent VSCC is variable depending on the geographic area. 10 Several studies, mainly conducted in Europe and USA have shown that most vulvar tumors arise independently of HPV. These studies show percentages of HPVassociated VSCC that range from 15% to 20% in Europe to 40% to 50% in the USA. 4,10 However, in contrast with the relatively wellknown epidemiology of vulvar cancer in these high-income sites, there is very scant information on the etiopathogenic features of VSCC in many low-income areas and particularly, in sub-Saharan Africa. Indeed, the pivotal study published by de Sanjose et al comprising over 1700 carcinomas of the vulva included only a very minor subset of tumors from sub-Saharan Africa. 10 Remarkably, these regions have marked differences in terms of HPV prevalence, which may result in pronounced variations in the proportion of HPVassociated and -independent tumors. Moreover, some of these regions have a high proportion of people living with HIV frequently associated with severe acquired immunodeficiency syndrome (AIDS), 12 a condition that increases the risk of persistent HPVinfections and HPV-associated carcinomas. 13 In this study, we compared the etiopathogenic features of a series of VSCC from Mozambique, a sub-Saharan country in South-East Africa with high prevalence of HPV and HIV/AIDS, with those of Spain, a country in South-Western Europe with relatively low prevalence of HPV and HIV. from January 2018 to December 2020 were reviewed. All cases diagnosed as VSCC during this period were retrieved, and the available material was reviewed. All cases fulfilling the following inclusion criteria were included in the study: (1) presence of invasive VSCC in the vulva and (2) available material for histological revision and HPV detection by polymerase chain reaction (PCR) and p16 IHC in the invasive tumor.

| Histological revision and review of the clinical charts
Hematoxylin and eosin sections of all tumors were reviewed. In the histological revision, the presence of invasive carcinoma was confirmed, the histological variant of the tumor was assessed (keratinizing, nonkeratinizing, basaloid, warty, verrucous, etc.) and the most representative and well-preserved paraffin-embedded block was selected for HPV detection and IHC staining.
All the histological slides were evaluated by two gynecological pathologists with expertise in vulvar pathology (NR, JO). The observers were aware of the geographical origin of the cases but were blind to the HPV testing results.
The clinical charts form all patients were reviewed.

| p16 and p53 IHC
All cases were stained with a p16 monoclonal antibody using the CINtec Histology Kit (clone E6H4; Roche-mtm-Laboratories, Heidelberg, Germany). Tumors showing strong and diffuse block-like staining were considered as positive (p16 upregulation), whereas patchy or completely negative p16 staining was considered as p16 negative. 11,14 All cases were stained for p53 with the monoclonal antibody (clone DO-7; Dako, Carpinteria, CA). The IHC staining was evaluated in the invasive tumor following the recent p53 pattern-based interpretation framework, 15,16 which includes six major categories: two normal (wild-type) and four abnormal (mutant) patterns.
Normal patterns, suggestive of wild-type p53 protein included:    Table 2 shows the histological variant, HPV-DNA result, p16 and p53 IHC of the tumors from Spain. Figure 1 illustrates histological and IHC features of each of the two types of VSCC, HPV-associated and HPV-independent.
No significant differences were found in terms of percentage of HPV genotypes between the two countries.

| DISCUSSION
Our study provides relevant data on the epidemiology of VSCC in sub-Saharan Africa, a geographical region where this information is almost nonexistent. Our series highlight the exceptionally high prevalence of HPV in VSCC in Mozambique and confirms that marked etiopathogenic differences exist between VSCC in sub-Saharan Africa and Europe. Thus, over 95% of the VSCC in Mozambique but only 19% of the VSCC from Spain were HPV-associated. In accordance with these etiopathogenic differences, the clinical presentation was also markedly different, with patients from Spain being almost three decades older in average than patients from Mozambique.  Although most of the cases showing p53 IHC abnormal patterns of staining suggestive of mutation were HPV-independent VSCC, a small subset of HPV-associated tumors showed these abnormal patterns of p53 staining. This finding has already been reported in studies based on IHC, as well as in studies analyzing genetic mutations. 4,15,16 Indeed, almost all the mutational differences between HPV-associated and HPV-independent VSCC are quantitative and not qualitative, as almost all mutations have been described in the two types of tumors, but with different frequencies.
In conclusion, the majority of VSCC in Mozambique, a country in

FUNDING INFORMATION
Project PI17/00772 funded by Instituto de Salud Carlos III and co-funded by the European Union (ERDF) "A way to make Europe."

CONFLICT OF INTEREST
The authors declare no conflicts of interest.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available from the corresponding author upon reasonable request.

ETHICS STATEMENT
The study was approved by the Ethics Committees of the Faculty of