Vulvar squamous cell carcinoma arising on human papillomavirus‐independent precursors mimicking high‐grade squamous intra‐epithelial lesion: a distinct and highly recurrent subtype of vulvar cancer

Each category of vulvar squamous cell carcinoma (VSCC), human papillomavirus (HPV)‐associated and HPV‐independent, arises on a specific intra‐epithelial precursor: high‐grade squamous intra‐epithelial lesions (HSIL) and differentiated vulvar intra‐epithelial neoplasia (dVIN), respectively. However, a subset of HPV‐independent VSCC arises on an intra‐epithelial precursor closely mimicking HSIL. We aimed to explore the clinicopathological features of the HPV‐independent tumours with HSIL‐like lesions and compare them with HPV‐independent VSCC with dVIN and HPV‐associated tumours with HSIL.

Vulvar squamous cell carcinoma arising on human papillomavirus-independent precursors mimicking high-grade squamous intra-epithelial lesion: a distinct and highly recurrent subtype of vulvar cancer Aims: Each category of vulvar squamous cell carcinoma (VSCC), human papillomavirus (HPV)associated and HPV-independent, arises on a specific intra-epithelial precursor: high-grade squamous intraepithelial lesions (HSIL) and differentiated vulvar intra-epithelial neoplasia (dVIN), respectively. However, a subset of HPV-independent VSCC arises on an intra-epithelial precursor closely mimicking HSIL. We aimed to explore the clinicopathological features of the HPV-independent tumours with HSIL-like lesions and compare them with HPV-independent VSCC with dVIN and HPV-associated tumours with HSIL. Methods and results: We retrospectively identified 105 cases of surgically treated VSCC with adjacent intra-epithelial precursors. The cases were classified into three groups based on the HPV status and the adjacent precursor identified: (i) HPV-associated VSCC with HSIL (n = 26), (ii) HPV-independent VSCC with dVIN lesions (n = 54) and (iii) HPV-independent VSCC with HSIL-like lesions (n = 25). We analysed the histological and clinical features including the recurrencefree survival and disease-specific survival in the three groups. Patients with HPV-independent VSCC with HSIL-like lesions and with dVIN were older than patients with HPV-associated VSCC (76 and 77 versus 66 years, respectively, P < 0.001). HPV-independent VSCC with HSIL-like lesions recurred more frequently [hazard ratio (HR) = 3.87; P < 0.001] than HPVindependent VSCC with dVIN (HR = 2.27; P = 0.1) and HPV-associated VSCC (HR = 1). In the multivariate analysis, HPV-independent VSCC with HSIL-like lesions remained significant for recurrence. No differences in disease-specific survival were observed between the three groups. Conclusions: Even though VSCC with HSIL-like lesions are not associated with higher mortality, they are more likely to recur and might benefit from more intensive treatment strategies and closer surveillance after treatment.

Introduction
Vulvar squamous cell carcinoma (VSCC) is a rare neoplasm with high morbidity and non-negligible mortality. 1 The 2020 World Health Organisation (WHO) classification has divided VSCC on the basis of its aetiological relationship with the infection by human papillomavirus (HPV) into two distinct types, HPV-associated and HPV-independent. [2][3][4] There is growing evidence indicating that patients with HPVassociated VSCC have a better prognosis than those with HPV-independent tumours. [5][6][7] HPV-associated VSCC typically affects younger women, commonly shows immature, poorly differentiated, basaloid and/or warty histological features and strong, block type staining for p16 in immunohistochemistry (IHC). HPV-associated VSCC usually develops from an HPV-induced intra-epithelial precursor named high-grade squamous cell lesion (HSIL), characterised by immature basal-appearing cells involving the whole thickness of the epithelium, also known as vulvar intra-epithelial neoplasia (VIN) of usual type. 8 As its invasive counterpart, vulvar HSIL typically overexpresses p16. 9,10 In contrast, HPV-independent VSCC affects older women, 3 commonly shows mature, well-differentiated, keratinising features, does not overexpress p16, 3 frequently shows an abnormal p53 IHC pattern 2,11 and commonly arises in the context of chronic inflammatory lesions of the vulvar skin, such as lichen simplex chronicus and lichen sclerosus. 12,13 HPV-independent VSCC develop from an intra-epithelial precursor named differentiated VIN (dVIN), a lesion characterised by atypical basal cells with normal maturation in the superficial layers. dVIN is a subtle lesion with deceptively bland appearance, frequently overlapping with inflammatory dermatoses. 14,15 In spite of its subtle histological features, dVIN has been shown to be a highly oncogenic lesion that rapidly progresses to invasive VSCC. 16 As the invasive tumour, dVIN does not overexpress p16 3 and frequently shows an abnormal p53 IHC pattern. 2,11 Recently, it has been described by our group that approximately 6% of the HPV-independent VSCC arise from an intra-epithelial precursor morphologically indistinguishable from HSIL. 17 Similarly to the true HPV-associated HSIL, this HPV-independent intraepithelial precursor has an immature appearance with whole-thickness abnormal maturation and is composed of cells with basaloid morphology or koilocytotic-like (warty) features. These lesions have been referred to as basaloid dVIN 18 or HSIL-like lesions. 17 In contrast with conventional HSIL, these lesions do not overexpress p16, are negative for highrisk HPV 3,17,18 and frequently show p53 abnormalities, 17 in similarity to conventional dVIN. 15 Due to their recent characterisation, studies analysing the follow-up of VSCC arising on this particular precursor are lacking. Thus, it remains unclear whether HSILlike lesions should be considered as part of the morphological spectrum of dVIN or whether they have any specific clinicopathological and prognostic features.
In this study we evaluated a large series of VSCC with associated intra-epithelial precursors, including HPV-associated HSIL, HPV-independent dVIN and HPV-independent HSIL-like lesion, aiming at analysing the clinicopathological features and the behaviour of these three groups of VSCC.

C A S E S E L E C T I O N
We retrospectively retrieved all VSCC from patients who underwent primary surgery at the Department of Gynaecological Oncology of the Hospital Clinic of Barcelona, Spain, during a 46-year period (February 1975-December 2021). The clinical charts and all the available pathological material were carefully reviewed. The following clinicopathological variables were retrieved from the electronic archives: patient age, type and date/s of treatment/s, tumour size, location, depth of invasion, tumour multifocality, tumour margin status, presence of premalignant lesion in the margin, lymph node involvement, date and site of first cancer recurrence and last follow-up or death.
All available haematoxylin and eosin-stained slides of all cases, including the invasive tumour as well as the adjacent skin, were carefully reviewed by two gynaecological pathologists with expertise in vulvar pathology (N.R. and A.S.), specifically looking for confirmation of the diagnosis of invasive carcinoma and presence of any intra-epithelial precursor in the adjacent skin.
The following exclusion criteria were established: (i) neoadjuvant radiotherapy or chemotherapy, (ii) absence of significant atypia in the adjacent skin (thus, lesions showing only inflammatory or reactive changes or intra-epithelial lesions lacking atypia such as vulvar acanthosis with altered differentiation, or differentiated exophytic vulvar intra-epithelial lesion, were excluded), (iii) insufficient tumour tissue for IHC and HPV DNA testing; and (iv) follow-up time shorter than 6 months.
The institutional ethical approval for this study was obtained (registry reference HCB/2020/1198). Written study consent was obtained from all the patients enrolled into the study.

Histological revision of the invasive tumour and adjacent skin
The histological variant of the invasive VSCC (keratinising, non-keratinising, basaloid, warty and verrucous) 19 and the type of atypical intra-epithelial lesion, including HSIL, dVIN and HSIL-like lesions, were recorded. The diagnosis of dVIN was made in the presence of significant basal atypia with preserved maturation in the upper layers. 20 HSIL and HSIL-like lesions were diagnosed when full-thickness epithelial atypia, high nuclear-to-cytoplasmic ratio and marked nuclear pleomorphism was identified 17,18,20,21 ; these two lesions could not be differentiated on the basis of pure morphological criteria.
In addition to the atypical precursor, other coexisting lesions in the adjacent skin, including nonatypical precursors (vulvar acanthosis with altered differentiation and differentiated exophytic vulvar intra-epithelial lesion) and inflammatory dermatoses (lichen simplex chronicus, lichen sclerosus and lichen planus), was also recorded. These lesions were diagnosed based on classical pathological criteria. 12,13 p16 immunohistochemistry IHC for p16 was performed using a monoclonal antibody (clone E6H4; Roche-mtm-Laboratories, CINtec Histology Kit, Heidelberg, Germany). The staining was classified as either positive (diffuse and block-like staining) or negative (completely negative p16 or patchy staining). 9 p16 IHC was evaluated independently in the invasive tumour and in the premalignant lesion.
HPV DNA detection and genotyping DNA was extracted from whole sections of a representative formalin-fixed paraffin-embedded block from surgical specimens. The analysed tissue included the invasive tumour and the precursor, but no separated analysis of HPV DNA in the invasive tumour and the precursor was conducted.
SPF10 PCR and the LiPA25 system were used for HPV DNA detection and typing (version 1; Labo Biomedical Products, Rijswijk, the Netherlands). A volume of 10 ll of isolated DNA was PCR-amplified using the INNO-LiPA HPV Genotyping Extra II kit (Fujirebio, Ghent, Belgium). This system allows the genotyping of HPV 16,18,31,33,35,

Assignment of HPV status
Both p16 IHC staining and HPV testing were considered for HPV status categorisation in invasive tumours and in the intra-epithelial lesions. Specimens with positive staining for p16 and/or high-risk HPV detected in the molecular analysis were classified as HPV-associated. The inclusion of a tumour as HPV-independent required both negative p16 IHC staining and absence of high-risk HPV DNA. Tumours showing low-risk or unclassifiable HPV types or having an invalid result in the HPV testing with negative p16 staining were considered as HPVindependent.

Classification of the tumours into three groups
All the cases complying with study criteria were further classified into three categories in accordance to the HPV status and type of adjacent premalignant lesions: (i) HPV-associated VSCC with adjacent HSIL; (ii) HPV-independent VSCC with HSIL-like lesions; (iii) HPV-independent VSCC with dVIN. The discrimination between HSIL and HSIL-like lesions was based on positive or negative HPV status of the invasive tumour and p16 IHC positive or negative staining in the premalignant lesion, respectively. p53 immunohistochemistry p53 IHC was performed with the monoclonal antibody CONFIRM (DO-7; Roche, Heildelberg, Germany). The results were evaluated using the pattern-based interpretation framework recently described. 11,22 p53 staining patterns were classified into two major categories: normal (wild-type) and abnormal (mutant). Normal category included scattered and midepithelial staining patterns, whereas abnormal expression included basal or diffuse overexpression, as well as null and cytoplasmic staining.
Results of p53 IHC were not used to categorise either invasive VSCC or vulvar premalignant lesions.

Treatment and follow-up
All patients underwent surgical treatment with vulvectomy or local wide excision of VSCC. The lymph node approach has been evolving from 1975 to the latest version. Before 1998, all women underwent inguinofemoral lymphadenectomy. In 1998, in our centre, we began to perform sentinel lymph node biopsy followed by inguinofemoral lymphadenectomy in order to validate the technique. 23 From 2003, sentinel lymph node biopsy was considered validated and the procedure was performed as the only staging method in all patients with unifocal VSCC measuring less than 4 cm. Patients with a positive sentinel lymph node underwent an ipsilateral inguinofemoral lymphadenectomy. Adjuvant radiotherapy and chemotherapy were indicated in accordance with the clinical guidelines at the time of diagnosis.
Patient follow-up, which included physical examination, was performed every 4-6 months for the first 2 years and annually afterwards. Imaging techniques (magnetic resonance imaging, inguinal ultrasound or computed tomography scan) were periodically conducted in patients with advanced VSCC or when recurrence was suspected. The patient was considered to have local recurrence when the tumour appeared in the same location after a minimum disease-free period of 6 months.
Given that the International Federation of Gynaecology and Obstetrics (FIGO) classification of VSCC has changed during the inclusion period, 24,25 all the patients were retrospectively restaged using the FIGO 2021 criteria 26 in order to use homogeneous staging criteria throughout the study sample. Information on relapse and cause of death was retrieved from the clinical charts.

Statistical analysis
For all data analyses, StataIC version 15.0.591 was used. v 2 tests (categorical data) and analysis of variance (ANOVA; numerical data) were run to compare the clinical and histopathological data between the three study groups.
The endpoints of the study were recurrence-free and disease-specific survival, and were calculated from the date of treatment (primary surgery) to the date of first recurrence or death due to disease, respectively. Survival analyses were conducted using the Kaplan-Meier method and differences between survival curves were calculated using the log-rank test. Univariate Cox regression was performed to evaluate the prognostic role of the three study groups. Multivariate Cox regression was run to confirm the associations after accounting for possible confounding factors, and included all significant variables in univariate analysis. Two-sided P-values < 0.05 were considered statistically significant.
Written study consent was obtained from all the patients enrolled into the study. The study data set is available upon request.

C H A R A C T E R I S T I C S O F T H E S T U D Y C O H O R T
Two hundred and four VSCC were identified during the study period. Five patients (2.4%) had advanced tumours treated with initial radiation and/or chemotherapy and were consequently excluded from the study. Eighty-eight patients (43.1%) were excluded due to the absence of atypical premalignant lesion in the skin. No case was excluded due to insufficient tumour tissue for IHC and HPV DNA testing. Finally, six patients (2.9%) were discarded because of insufficient follow-up time (less than 6 months). Thus, the final study cohort included 105 patients with VSCC.
The mean age at diagnosis was 72.6 years (range = 30.7-95.4) and the mean follow-up time was 57.8 AE 43.2 months. Forty-eight patients (45.7%) were treated with vulvectomy and 57 (54.3%) with wide local excision. Eighty-eight patients (83.8%) underwent surgical lymph node evaluation either by sentinel lymph node biopsy (n = 39), inguinofemoral lymphadenectomy (n = 28) or both sentinel and inguinofemoral dissection (n = 21). Five patients had IA FIGO stage VSCC, thus negativity of lymph nodes was assumed. Twelve patients (11.4%) did not undergo surgical lymph node evaluation because of poor performance status. Twenty-eight patients (26.6%) received adjuvant radiation therapy after the initial surgical treatment and four (3.8%) received adjuvant chemotherapy.

C L A S S I F I C A T I O N O F T H E T U M O U R S I N T O T H R E E G R O U P S
Twenty-six VSCC (24.8%) were classified as HPVassociated. All these tumours were p16-positive, and 23 (88.5%) tested positive for high-risk HPV. All the 26 VSCC had HSIL in the adjacent skin, which stained positive for p16.
No differences in the surgical treatment (vulvectomy versus wide excision or type of lymph node evaluation), in the proportion of patients without nodal evaluation or in the proportion of patients receiving adjuvant treatment were seen between study groups (Supporting information, Table S1).
The mean age at diagnosis was 61.4 AE 16.0 years for the patients with HPV-associated VSCC with HSIL, 76.8 AE 11.5 years for HPV-independent VSCC arising on dVIN and 75.2 AE 9.9 years for the HPVindependent VSCC arising on HSIL-like lesions (P < 0.001). Figure 1 shows the clinical appearance, histological and immunohistochemical features of VSCC with adjacent HSIL, HSIL-like and dVIN.  Table 1 shows the histological subtypes, p53 IHC staining and other associated inflammatory lesions of the three groups included into the study. Most of the invasive tumours in the groups of HPV-associated VSCC with HSIL and HPV-independent VSCC with HSIL-like lesions displayed basaloid, warty or nonkeratinising features, while HPV-independent VSCC were mostly keratinising (P < 0.001). The histological features were identical in HPV-associated VSCC tumours with HSIL and HPV-independent VSCC with HSIL-like lesion, both in invasive tumour and adjacent lesion: architectural disarray was prominent, mitotic rate was high, atypical mitoses were readily identifiable and koilocytotic changes were common. No cases with coexistent HSIL, dVIN or HSIL-like lesions and other non-atypical precursors (vulvar acanthosis with altered differentiation and differentiated exophytic vulvar intra-epithelial lesion) were identified. Figure 2 shows the morphological spectrum of HSIL-like lesions and of the invasive VSCC developing in the background of these precursors.
The majority of the HPV-associated VSCC showed a wild-type pattern of expression of p53, whereas most of the HPV-independent VSCC (with dVIN or HSILlike) showed an abnormal pattern of p53 IHC staining (P < 0.001). Among patients with abnormal p53 staining, 53 (71.6%) showed a diffuse overexpression pattern, 13 (17.8%) showed null staining, five (6.8%) showed a basal overexpression pattern and three (4.1%) showed cytoplasmatic staining. Twenty-three (74.2%) patients with p53 normal staining showed a scattered pattern and eight (25.8%) a mid-epithelial pattern of staining. Inflammatory lesions coexisted in more than one-third of patients with HPVindependent VSCC arising on dVIN or HSIL-like lesions, while no inflammatory lesions were identified in HPV-associated VSCC with adjacent HSIL (P < 0.001). Table 2 shows the pathological prognostic features and the FIGO staging of the three groups of patients with VSCC. HPV-independent tumours with dVIN were larger and more deeply invasive than HPV-associated tumours with HSIL and HPV-independent VSCC with HSIL-like lesions (P = 0.004 and P = 0.003, respectively). No differences were observed between the groups in terms of location, multifocality, presence of premalignant lesion in surgical margin, lymphovascular invasion or lymph node metastases. A higher proportion of patients with surgical margin affected by invasive carcinoma was seen in HPVassociated VSCC (23.1%, P = 0.029). More than 75% of patients within each of the three groups showed early FIGO stage, with no differences between them (P = 0.456). No changes in FIGO 2021 staging category (early versus advanced stage VSCC) were identified as a result of implementing FIGO 2021 criteria for VSCC staging.

R E C U R R E N C E -F R E E A N D D I S E A S E -S P E C I F I C S U R V I V A L
More than half (64.0%) of the patients with HPVindependent VSCC with HSIL-like lesions recurred, whereas recurrence was documented in only 35.0% of HPV-independent VSCC with dVIN and 19.2% of HPV-associated VSCC with HSIL. A large amount of the recurrences in all the three groups occurred locally, in the same area as the previous tumour: 40% (two of five) of recurrences in HPV-associated VSCC with HSIL, 68.4% (13 of 19) of the recurrences in HPV-independent VSCC with dVIN and 68.8% (11 of 16) in the HPV-independent VSCC with HSIL-like lesions. Table 3 shows the prognostic features of the three main groups of patients. Figure 3 shows the Kaplan-Meier curves for recurrence-free and disease-specific survival rates for the three groups. With a median recurrence-free survival time of 28.6 months, patients with HPVindependent VSCC and HSIL-like adjacent lesions showed a significantly higher tendency to recurrence (P = 0.01). No differences in disease-specific survival were identified between the three groups. Table 4 shows the results of univariate and multivariate Cox regression analysis for recurrence-free and disease-specific survival. HPV-independent VSCC with HSIL-like lesion and the presence of premalignant lesion in contact with the surgical margins were independent factors for recurrence. Regarding disease-specific survival, no variables reached the preset 0.05 level of significance in univariate nor multivariate analysis. FIGO stage and the presence of lymph node metastases showed the highest hazard ratio for mortality (3.31 and 3.68 respectively, P = 0.06), with wide confidence intervals. In spite of its association with recurrence, HPV-independent VSCC with HSIL-like lesion did not have a significant effect on disease-specific survival.

Discussion
In this study, we have explored the prognostic features of patients with HPV-independent VSCC arising on HSIL-like lesions in a relatively large cohort of VSCC surgically treated in a single institution during a 46-year-long period. To our knowledge, this is the first study to assess the prognostic implications of this unusual subset of patients with HPV-independent VSCC arising on a precursor that closely mimics the HPV-associated precursor, HSIL. Although our group first described this lesion in 2009, 18 and further characterised its histological features in a larger series of cases in 2020, 17 the possible prognostic implications of these lesions remained unknown due to the absence of follow-up in both studies. 17,18 The key finding of this study is that HPV-independent VSCC with adjacent HSIL-like lesions have a strong tendency towards recurrence. Importantly, this observation was maintained after adjustment for confounding factors. These data could have major clinical implications in terms of recurrence risk stratification.
The HPV-independent HSIL-like lesions were initially described as an unusual, basaloid variant of dVIN 18 and briefly mentioned in the last 2020 WHO classification in the dVIN chapter. 19 However, herein we show that VSCC arising on these lesions have a number of distinctive clinicopathological features. First, HPV-independent VSCC with HSIL-like lesions are histologically indistinguishable from HPVassociated VSCC both in the invasive tumour and in the adjacent skin precursor, showing mainly basaloid, warty or non-keratinising histology. Secondly, in similarity to HPV-associated VSCC, these tumours tended to be smaller and more superficially invasive than those of dVIN group. Finally, and most remarkably, in spite of being small and superficially invasive tumours, more than half of the patients with HPVindependent VSCC with HSIL-like lesions showed recurrences.
A possible explanation for the smaller size and more superficial invasion of HPV-associated VSCC and HPV-independent VSCC with HSIL-like lesions is their clinical presentation. HSIL and HSIL-like lesions tended to be raised, coalescent lesions, probably causing significant discomfort to the patient and prompting early medical consultation. In contrast with lesions with HSIL features, dVIN lesions were smaller and poorly defined, which might justify a delay in consultation and diagnosis.
In addition to the features similar to HPVassociated tumours with conventional HSIL described above, HPV-independent VSCC with HSIL-like lesions show a number of similarities with HPV-independent VSCC arising on dVIN. First, as expected from any HPV-independent VSCC, 2 both groups share high rates of p53 alterations (84.0 and 94.4%) and frequently coexist with adjacent inflammatory lesions (44.0 and 92.6%, respectively), which contrasts with the almost constant wild-type pattern of p53 IHC staining and the absence of inflammatory lesions in HPV-associated tumours. In addition, the patients from the two HPV-independent groups show similar ages (mean age = 75 and 77 years), while patients with HPV-associated VSCC were more than 10 years younger (mean age = 61 years). Even though we identified no differences in terms of disease-specific survival, HPV-independent tumours with HSIL-like lesions showed a higher recurrence rate in both the univariate and the multivariate analyses. The factors involved in the high recurrence rate of these tumours should be explored in subsequent  studies. Strikingly, most of these tumours recurred despite being at initial FIGO stage at diagnosis and with low rates of lymph node involvement, factors usually associated with improved prognosis. 27 Curiously, in a series of patients with superficially invasive VSCC, Preti et al. 28 describe a higher recurrence rate in smaller tumours; the authors hypothesise that the potential presence of microscopic satellite lesions not clinically visible could be the cause of this observation. Of note, basaloid histology, which was frequent in tumours with HSIL-like adjacent lesion, has been previously associated with aggressive clinical behaviour in other cancer types, 29-31 especially in HPV-negative head and neck squamous cancers. 32 Indeed, basaloid laryngeal squamous cell carcinoma, a predominantly HPV-negative tumour, 33 shows higher rates of recurrence than conventional keratinising tumour. 34 Although morphological overlap between HPVassociated and -independent VSCC precursors has been described by several authors, 17,18,35,36 a standard terminology for HSIL-like lesions related to HPVindependent VSCC has not been defined due to their poor characterisation and the lack of evidence on their clinical and prognostic implications. The 2021 WHO classification states that HPV-independent precursor lesions can show a basaloid or warty/condylomatouslike morphology simulating HPV-associated HSIL. Due to their morphology closely mimicking HPVassociated HSIL, we prefer the term HSIL-like for the definition of these lesions.
In spite of their HSIL-like morphology, our results suggest HPV-independent VSCC arising on HSIL-like lesions have distinctive clinical behaviour. These tumours can be identifiable using routine haematoxylin and eosin staining and p16/p53 IHC, which are widely available tools in pathology laboratories in settings where HPV-independent VSCC are predominant. Our findings highlight the importance of using basic molecular biomarkers (p53 and p16) for characterisation of VSCC and its precursors. The main strength of this study is the high number of patients with VSCC included, all of them tested for the presence of HPV and stained with p16 and p53 IHC, and especially the accurate clinical information and the long-term follow-up available. The limitations include the retrospective design of the study, the possible selection bias due its single institutional nature and the heterogeneity of treatment strategy related to the long period of inclusion, with different surgical strategies applied in different periods (more radical resection in the earlier cases). Finally, due to the low mortality rate of this cancer, the cohort is probably too small to ascertain statistical differences for disease-specific survival between the three groups.

Conclusion
In conclusion, we show for the first time that HPVindependent VSCC arising on HSIL-like lesions are infrequent but have distinctive clinical, pathological and behavioural features. They are highly recurrent tumours which warrant closer surveillance after surgery. Further prospective studies are needed to determine if this subgroup of patients might benefit from more intensive treatment strategies (i.e. more radical excisions or adjuvant treatment). Importantly, these unusual tumours can be easily identifiable at the premalignant stage using routine haematoxylin and eosin staining and p16/p53 IHC. As these tools are widely available in pathology laboratories in settings where HPV-independent VSCC are predominant, 37 these tumours should be routinely reported by pathologists. Advanced age of the patients and the presence of concomitant inflammatory lesions in the adjacent skin should raise the suspicion of this entity to the pathologist. Evaluation of clinicopathological features of these tumours in a larger number of VSCC patients may yield more definitive prognostic information, especially for disease-specific survival.
investigation, writing and editing; A.G.: investigation, writing and editing, L.M.: investigation, methodology, writing and editing; S. D-M.: investigation, writing and editing; P. F.: investigation, writing and editing; M. T. R.-C.: investigation, writing and editing, N.V.: investigation, methodology, writing and editing, A.T.: study design, resources, investigation, methodology, writing and editing, project management; N.R.: study design, resources, investigation, methodology, statistical analysis, writing of the first draft, writing and editing, project management.

Patient consent statement
Written study consent was obtained from all the patients enrolled in the study.

Data availability statement
The study dataset is available upon request.