Medicine, Faculty of
Non UBC
Obstetrics and Gynaecology, Department of
Pathology and Laboratory Medicine, Department of
DSpace
Vancouver General Hospital
University of British Columbia. Division of Medical Oncology
Horn, Lars-Christian
Brambs, Christine E.
Alfaraidi, Mona
Gilks, C. Blake
Hoang, Lien N.
Singh, Naveena
Hiller, Grit Gesine Ruth
Hering, Kathrin
McAlpine, Jessica N.
Jamieson, Amy
Aktas, Bahriye
Dornhöfer, Nadja
Höhn, Anne Kathrin
Vulvar carcinoma is a rare disease, meeting the criteria for a “rare cancer”, but its incidence is increasing, especially in women <60 years of age. Squamous cell carcinoma (VSCC) accounts for the overwhelming majority of vulvar carcinomas and is the focus of this review. As with many cancers, the increased understanding of molecular events during tumorigenesis has led to the emergence of the molecular subclassification of VSCC, which is subclassified into tumors that arise secondary to highrisk human papillomavirus infection (HPV-associated, or HPVa) and those that arise independently of HPV (HPVi), most commonly in the setting of a chronic inflammatory condition of the vulvar skin. This latter group of HPVi VSCC arises in most cases secondary to mutations in TP53, but recently, attention has focused on the uncommon TP53 wild-type HPVi VSCC. These three molecular subtypes of VSCC (HPVa, HPVi p53 abnormal, and HPVi p53 wild type), as well as their precursor lesions, cannot be diagnosed based on a routine histopathological examination or immunostaining for p53 and p16 as surrogate markers for TP53 mutation and high-risk HPV infection, respectively, are required. The molecular subtyping of VSCC shows high reproducibility and provides important prognostic information. HPVa VSCC has the most favorable prognosis, while HPVi VSCC with TP53 mutations (p53abn) has the worst prognosis, and HPVi VSCC with wild-type TP53 (p53wt) has an intermediate prognosis. In this review, we discuss the evidence supporting this molecular subclassification and its implications for the diagnosis and treatment of VSCC and its precursors.
Citation: Horn, L.-C.; Brambs, C.E.;Gilks, B.; Hoang, L.; Singh, N.; Hiller,G.G.R.; Hering, K.; McAlpine, J.N.;Jamieson, A.; Alfaraidi, M.; et al.Molecular Subtypes of VulvarSquamous Cell Carcinoma: TheSignificance of HPV-Independent/p53Wild Type. Cancers 2024, 16, 4216.https://doi.org/10.3390/cancers16244216Academic Editor: XavierSastre-GarauReceived: 22 October 2024Revised: 11 December 2024Accepted: 12 December 2024Published: 18 December 2024Copyright: © 2024 by the authors.Licensee MDPI, Basel, Switzerland.This article is an open access articledistributed under the terms andconditions of the Creative CommonsAttribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).ReviewMolecular Subtypes of Vulvar Squamous Cell Carcinoma: TheSignificance of HPV-Independent/p53 Wild TypeLars-Christian Horn 1,* , Christine E. Brambs 2 , Blake Gilks 3, Lien Hoang 3, Naveena Singh 3,Grit Gesine Ruth Hiller 1, Kathrin Hering 4, Jessica N. McAlpine 5 , Amy Jamieson 5, Mona Alfaraidi 3,6,Bahriye Aktas 7 , Nadja Dornhöfer 7 and Anne Kathrin Höhn 11 Division of Gynecologic, Breast and Perinatal Pathology, Institute of Pathology, University Hospital Leipzig,D-04103 Leipzig, Germany; ruth.hiller@uniklinik-leipzig.de (G.G.R.H.);annekathrin.hoehn@medizin.uni-leipzig.de (A.K.H.)2 Department of Obstetrics and Gynecology, Kantonsspital Luzern, 6004 Luzern, Switzerland;christine.brambs@luks.ch3 Department of Anatomical Pathology, Vancouver General Hospital, The University of British Columbia,Vancouver, BC V5Z 1M9, Canada; blake.gilks@vch.ca (B.G.); lien.hoang@vch.ca (L.H.);naveena.singh@vch.ca (N.S.); mona.alfaraidi@vch.ca (M.A.)4 Department of Radiotherapy and Radiation Oncology, University Hospital Leipzig,D-04103 Leipzig, Germany; kathrin.hering@medizin.uni-leipzig.de5 Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, The University of BritishColumbia, Vancouver, BC V5Z 1M9, Canada; jessica.mcalpine@vch.ca (J.N.M.); amy.jamieson@vch.ca (A.J.)6 Prince Sultan Military Medical City, Riyah 12233, Saudi Arabia7 Division of Gynecologic Oncology, Department of Obstetrics and Gynecology, Institute of Trier, UniversityHospital Leipzig, D-04103 Leipzig, Germany; bahriye.aktas@medizin.uni-leipzig.de (B.A.);nadja.dornhoefer@medizin.uni-leipzig.de (N.D.)* Correspondence: hornl@medizin.uni-leipzig.de; Tel.: +49-341-97-150-46; Fax: +49-341-97-15-009Simple Summary: The distinct histopathogenetic pathways of vulvar squamous cell neoplasia haveled to a shift from a morphology-based to an etiology/molecular-based histopathologic classification,adopted in the most recent WHO Classification of Female Genital Tumours. There are two maincategories: HPV-associated (HPVa) and HPV-independent (HPVi) vulvar squamous neoplasia. MostHPVi VSCCs have an underlying TP53 mutation, but attention has recently focused on the uncommonthird molecular subtype: TP53 wild-type HPVi VSCC. We review this shift to the etiology/molecular-based classification of VSCC, emphasizing the diagnostic and treatment implications of this changeand highlighting the third molecular subtype.Abstract: Vulvar carcinoma is a rare disease, meeting the criteria for a “rare cancer”, but its incidenceis increasing, especially in women <60 years of age. Squamous cell carcinoma (VSCC) accounts for theoverwhelming majority of vulvar carcinomas and is the focus of this review. As with many cancers,the increased understanding of molecular events during tumorigenesis has led to the emergence of themolecular subclassification of VSCC, which is subclassified into tumors that arise secondary to high-risk human papillomavirus infection (HPV-associated, or HPVa) and those that arise independentlyof HPV (HPVi), most commonly in the setting of a chronic inflammatory condition of the vulvarskin. This latter group of HPVi VSCC arises in most cases secondary to mutations in TP53, butrecently, attention has focused on the uncommon TP53 wild-type HPVi VSCC. These three molecularsubtypes of VSCC (HPVa, HPVi p53 abnormal, and HPVi p53 wild type), as well as their precursorlesions, cannot be diagnosed based on a routine histopathological examination or immunostainingfor p53 and p16 as surrogate markers for TP53 mutation and high-risk HPV infection, respectively,are required. The molecular subtyping of VSCC shows high reproducibility and provides importantprognostic information. HPVa VSCC has the most favorable prognosis, while HPVi VSCC withTP53 mutations (p53abn) has the worst prognosis, and HPVi VSCC with wild-type TP53 (p53wt)has an intermediate prognosis. In this review, we discuss the evidence supporting this molecularsubclassification and its implications for the diagnosis and treatment of VSCC and its precursors.Cancers 2024, 16, 4216. https://doi.org/10.3390/cancers16244216 https://www.mdpi.com/journal/cancersCancers 2024, 16, 4216 2 of 16Keywords: vulvar cancer; squamous cell carcinoma; p53; p16; human papillomavirus; molecular;classification; prognosis; radiation therapy; surgery1. IntroductionVulvar cancer (VC) is an uncommon disease [1] and meets the criteria for a “rarecancer” according to the definition of the European Society for Medical Oncology, i.e.,cancer with an incidence of <6 per 100,000 per year [2,3]. VC accounts for approximately5.6% of all gynecologic malignancies, with an estimated 6470 new cases in 2023 in theUS [4]. An Australian study reported an 84% increase in VC incidence from 1982 to 1984to 2007–2009 in women younger than 60 years, with no change in incidence in women60+ years [5,6]. Comparing the cancer registry data from 13 high-income countries overa 20-year period, Kang et al. [6] reported an overall increase of 4.6%, with an increase of11.6% in women < 60 years of age but no change in women over 60 years of age.There has been no recent change in the overall survival of vulvar cancer patients [7].Regardless of tumor stage and treatment approaches, an analysis of the SEER datasets be-tween 2000 and 2007 reported a 5-year overall survival of 60.6% (45.7% at 10 years and 33.1%at 15 years) [8]. The vast majority of VCs are squamous cell carcinomas (VSCCs) [8–10].Within this histologic subtype, the most important development in the last 10 years hasbeen the recognition of distinct histopathogenetic pathways [9–12]; these pathways areassociated with distinct precursor lesions, different responses to treatment, and differentoutcomes [11,13–22]. Here, we summarize the clinicopathologic features of the differentmolecular subtypes of VSCC, including their impact on diagnosis and treatment.2. Molecular Subtypes of VSCCAlthough it has been recognized since the early 1990s that VSCC can either be as-sociated with high-risk human papillomavirus infection (HPVa) or independent of HPV(HPVi) [23,24], it has only recently been appreciated that VSCC and squamous cell car-cinomas at other body sites, e.g., the oropharynx [25–27], differ significantly from HPVisquamous cell carcinomas arising at the same body site. It is only in the fifth edition of theWHO Classification of Female Genital Tumours [28] that VSCC and its precursor lesionshave been subclassified as HPVi or HPVa (Figure 1a; [28]).The move from the previous morphology-based classification of VSCC to an etiology/molecular-based classification (Figure 1b) allows for a more precise prognosis character-ization [10,12,14,18]; the same also occurred for cervical squamous cell carcinomas andadenocarcinomas (Figure 1a,b; [28]). This change was subsequently adopted by the In-ternational Collaboration on Cancer Reporting (ICCR; [29]) and the College of AmericanPathologists [29,30], as well as by the most recent update of the guidelines of the EuropeanSociety of Gynaecological Oncology for the management of patients with vulvar cancer [31].The molecular subclassification of VSCC defines at least two distinct subtypes (fordetails and diagnostic approaches (please see Figure 1b below)). Briefly, the first mostcommon and aggressive molecular subtype is HPV-independent and is associated with aTP53 mutation, consequently resulting in an aberrant p53 protein expression on immuno-histochemistry. The second less common and less aggressive subtype is HPV-associatedand is based on an association with high-risk HPV infection, showing a better prognosisand p16 protein overexpression [9,10,14,16].This molecular subclassification has been implemented at sites outside the femalegenital tract where there is HPVa squamous cell carcinoma, e.g., in the penis [32], as wellas in head and neck cancers [27,33–35]. While most HPVi VSCCs have mutations in TP53,associated with a mutant-pattern expression of the p53 protein on immunostaining, Sandet al. [36] evaluated p16 and p53 expression in 18 studies on VSCC and found that 54%of the tumors were HPVi with mutant-pattern p53 expression and that 38% were HPVawith immunohistochemical p16 overexpression; however, 8% of VSCCs were neither p16-Cancers 2024, 16, 4216 3 of 16negative with mutant-pattern p53 nor p16-positive with wild-type 53, thus pointing to athird molecular subtype of VSCC, namely, the HPVi (p16-negative) and p53 wild type.Cancers 2024, 16, x FOR PEER REVIEW 3 of 18 Figure 1. (a) Subdivision of squamous cell carcinomas of the lower female genital tract based on HPV association according to the recommendations of the WHO classification 2020 [28]; (b): Classi-fication of VSCC from morphology-based to morpho-molecular classification, depending on the eti-ology of the disease, using p16 and p53 immunohistochemistry [10,12,14,20]. The molecular subclassification of VSCC defines at least two distinct subtypes (for details and diagnostic approaches (please see Figure 1b below)). Briefly, the first most common and aggressive molecular subtype is HPV-independent and is associated with a i r . ( ) bdivision of squamous cell carcinomas of the lower f male genital tr ct based on HPVassociation according to the recommendations of the WHO classification 2020 [28]; (b): Classificationof VSCC from morphology-based to morpho-molecular classification, depending on the etiology ofthe disease, using p16 and p53 immunohistochemistry [10,12,14,20].Cancers 2024, 16, 4216 4 of 16Nooji et al. [37] were the first to report on this third molecular subtype of VSCC.They performed a targeted NGS analysis of VSCC and its precursors and found a higherfrequency of NOTCH1 and HRAS mutations in HPVi p53wt VSCC than in HPVi p53abnVSCC. They, therefore, suggested that NOTCH1 and HRAS mutations may act as drivers ofmutational events independent of TP53, and this was later supported by another study [15].Furthermore, high rates (up to 71%) of NOTCH1 mutations have been reported to occur inpenile cancer, a male tumor with many similarities to VSCC [38].The prognosis of the three molecular subtypes of VSCC (HPVa, HPVi p53abn, andHPVi p53wt) shows that HPVa VSCC has the most favorable prognosis, HPVi p53abn hasthe worst prognosis, and HPVi p53wt has an intermediate prognosis [12,18,37,39]; thus,molecular analyses have been performed [15,37]. As a result, there is growing acceptanceof the routine classification of VSCC and its precursors into one of these three molecularsubtypes (see Figure 2).Cancers 2024, 16, x FOR PEER REVIEW 4 of 18 TP53 mutation, consequently resulting in an aberrant p53 protein expression on immuno-histochemistry. The second less common and less aggressive subtype is HPV-associated and is based on an association with high-risk HPV infection, showing a better prognosis and p16 protein overexpression [9,10,14,16]. This molecular subclassification has been implemented at sites outside the female genital tract where there is HPVa squamous cell carcinoma, e.g., in the penis [32], as well as in head and neck cancers [27,33–35]. While most HPVi VSCCs have mutations in TP53, associated with a mutant-pattern expression of the p53 protein on immunostaining, Sand et al. [36] evaluated p16 and p53 expression in 18 studies on VSCC and found that 54% of the tumors were HPVi with mutant-pattern p53 expression and that 38% were HPVa with immunohistochemical p16 overexpression; however, 8% of VSCCs were neither p16-neg-ative with mutant-pattern p53 nor p16-positive with wild-type 53, thus pointing to a third molecular subtype of VSCC, namely, the HPVi (p16-negative) and p53 wild type. Nooji et al. [37] were the first to report on this third molecular subtype of VSCC. They performed a targeted NGS analysis of VSCC and its precursors and found a higher fre-quency of NOTCH1 and HRAS mutations in HPVi p53wt VSCC than in HPVi p53abn VSCC. They, therefore, suggested that NOTCH1 and HRAS mutations may act as drivers of mutational events independent of TP53, and this was later supported by another study [15]. Furthermore, high rates (up to 71%) of NOTCH1 mutations have been reported to occur in penile cancer, a male tumor with many similarities to VSCC [38]. The prognosis of the three molecular subtypes of VSCC (HPVa, HPVi p53abn, and HPVi p53wt) shows that HPVa VSCC has the most favorable prognosis, HPVi p53abn has the worst prognosis, and HPVi p53wt has an intermediate prognosis [12,18,37,39]; thus, molecular analyses have been performed [15,37]. As a result, there is growing acceptance of the routine classification of VSCC and its precursors into one of these three molecular subtypes (see Figure 2). Figure 2. Summary of the recently described three-tiered molecular classification of VSCC and its precursors (please see text) (uVIN = usual VIN; dVIN = differentiated VIN; vaVIN = verrucous VIN). Figure 2. Summary of the recently described three-tiered molecular classification of VSCC and itsprecursors (please see text) (uVIN = usual VIN; dVIN = differentiated VIN; vaVIN = verrucous VIN).3. Diagnosis of Molecular Subtypes of VSCCThe molecular subclassification of VSCC is based on the routine use of molecularmarkers, specifically immunostaining for both p53 and p16 in all cases. Ambiguousstaining results may require additional in situ analyses of HPV and/or TP53 sequencing.The immunohistochemical evaluation of p53 is performed as a pattern-based analysisrather than as a form of simple positive or negative staining. The different staining patternsare summarized in Table 1, and when interpreted according to these recommendations,they show a strong correlation with TP53 mutational status.Cancers 2024, 16, 4216 5 of 16Table 1. Pattern-based analysis of p53-immunoexpression in VSCC for molecular classification([22,32,40]; please see text).(1) p53-staining patterns in HPV-high risk related VSCC with p16-overexpression• scattered patternpatchy positivity mostly within basal layers, withheterogeneous nuclear staining intensityCancers 2024, 16, x FOR PEER REVIEW 5 of 18 3. Diagnosis of Molecular Subtypes of VSCC The molecular subclassification of VSCC is based on the routine use of molecular markers, specifically immunostaining for both p53 and p16 in all cases. Ambiguous stain-ing results may require additional in situ analyses of HPV and/or TP53 sequencing. The immunohistochemical evaluation of p53 is performed as a pattern-based analysis rather than as a form of simple positive or negative staining. The different staining pat-terns are summarized in Table 1, and when interpreted according to these recommenda-tions, they show a strong correlation with TP53 mutational status. Table 1. Pattern-based analysis of p53-immunoexpression in VSCC for molecular classification ([22,32,40]; please see text). (1) p53-staining patterns in HPV-high risk related VSCC with p16-overexpression  scattered pattern patchy positivity mostly within basal layers, with heterogeneous nuclear staining intensity  basal sparing/mid‐epithelial nuclear negativity of the outer/basal cell lay-ers of the invasive growing tumor cell nest, often accompanied by increased staining in the mid and central/suprabasal cell layers (2) p53-staining patterns in HPV-high risk negative VSCC without p16-overexpression staining pattern mutational background  basal overexpression strong, homogeneous nuclear staining of ≥80% of basal cells without significant staining of suprabasal cells within the inva-sive growing tumor cell nest Missense muta-tion  parabasal/diffuse overexpression strong, homogeneous nuclear staining of ≥80% of basal cells and of suprabasal cells within the invasive growing tumor cell nest Missense muta-tion • basal sparing/mid-epithelialnuclear negativity of the outer/basal cell layersof the invasive growing tumor cell nest, oftenaccompanied by increased staining in the midand central/suprabasal cell layersCancers 2024, 16, x FOR PEER REVIEW 5 of 18 3. Diagnosis of Molecular Subtypes of VSCC The molecular subclassification of VSCC is based on the routine use of molecular markers, specifically immunostaining for both p53 and p16 in all cases. Ambiguous stain-ing results may require additional in situ analyses of HPV and/or TP53 sequencing. The immunohistochemical evaluation of p53 is performed as a pattern-based analysis rather than as a form of simple positive or negative staining. The different staining pat-terns are summarized in Table 1, and when interpreted according to these recommenda-tions, they show a strong correlation with TP53 mutational status. Table 1. Pattern-based analysis of p53-immunoexpression in VSCC for molecular classification ([22,32,40]; please see text). (1) p53-staining patterns in HPV-high risk related VSCC with p16-overexpression  scattered pattern p chy ositivity mostly within basal layers, wit heterogene us nuclear staining intensity  basal sparing/mid‐epithelial nuclear negativity of the outer/basal cell lay-ers of the invasive growing tumor cell nest, often accompanied by increased staining in the mid nd central/suprabasal cell layers (2) p53-staining patterns in HPV-high risk negative VSCC without p16-overexpression staining pattern mutational background  basal overexpression strong, homogeneous nuclear staining of ≥80% of basal cells without significant staining of suprabasal cells within the inva-sive growing tumor cell nest Missense muta-tion  parabasal/diffuse overexpression strong, homogeneous nuclear staining of ≥80% of basal cells and of suprabasal cells within the invasive growing tumor cell nest Missense muta-tion (2) -staining patterns in HPV-hig risk negative VSCC without p16-overexpressionstaining pattern mutational background• basal overexpressionstrong, homogeneous nuclear stainingof ≥80% of basal cells without significantstaining of suprabasa cells within the invasivegrowing tumor cell nestCancers 2024, 16, x FOR PEER REVIEW 5 of 18 3. Diagnosis of Molecular Subtypes of VSCC The molecular subclassification of VSCC is based on the routine use of molecular markers, specifically immunostaining for both p53 and p16 in all cases. Ambiguous stain-ing results may require additional in situ analyses of HPV and/or TP53 sequencing. The immunohistochemical evaluation of p53 is performed as a pattern-based analysis rather than as a form of simple positive or negative staining. The different staining pat-terns are summarized in Table 1, and when interpreted according to these recommenda-tions, they show a strong correlation with TP53 mutational status. Table 1. Pattern-based analysis of p53-immunoexpression in VSCC for molecular classification ([22,32,40]; please see text). (1) p53-staining patterns in HPV-high ri k r lated VSCC with p16-overexpression  scattered pattern patchy positivity mostly within basal layers, with heterogeneous nuclear staining intensity  basal sparing/mid‐epithelial nuclear negativity of the outer/basal cell lay-ers of the invasive growing tumor cell nest, often accompanied by increased staining in the mid and central/suprabasal cell layers (2) p53-staining patterns in HPV-high risk negative VSCC without p16-overexpression staining pattern mutational background  basal overexpression strong, homogeneous nuclear staining of ≥80% of basal cells without s gnificant staining of suprabasal cells within the inva-sive growing tumor cell nest Missense muta-tion  parabasal/diffuse overexpression strong, homogeneous nuclear staining of ≥80% of basal cells and of suprabasal cells within the invasive growing tumor cell nest Missense muta-tion Missense mutation• parabasal/diffuse overexpressionstrong, homogeneous nuclear stainingof ≥80% of basal cells and of suprabasal cellswithi the invasive growi g tumor cell nestCancers 2024, 16, x FOR PEER REVIEW 5 of 18 3. Diagnosis of Molecular Subtypes of VSCC The molecular subclassification of VSCC is based on the routine use of molecular markers, specifically immunostaining for both p53 and p16 in all cases. Ambiguous stain-ing results may require additional in situ analyses of HPV and/or TP53 sequencing. i munohistochemical evaluation of p53 is p rformed as a pattern-based analysis rather than as a form of simple positive or negative staining. The different staining pat-terns are summarized in Table 1, and whe interpreted according to these r commenda-tions, they show a str ng correlation with TP53 mutational status. Table 1. Pattern-based analysis of p53-immunoexpression in VSCC for molecular classification ([22,32,40]; please see text). (1) p53-staining patterns in HPV-high risk related VSCC with p16-ov rexpression  scattered pattern patchy positivity mostly within basal layers, with hetero en ous uclear staining intensity  basal sparing/mid‐epithelial nuclear negativity of the outer/basal cell lay-ers of the invasive growing tumor cell nest, often accompani d by ncreased staining in the mid and cen ral/suprabasal cell layers (2) p53-staining patterns in HPV-high risk negative VSCC without p16-overexpression staining pattern mutational background  basal overexpression strong, homogeneous nuclear staining of ≥80% of basal cells without significant staining of suprabasal cells within the inva-ive rowing tum r cell nest Missense muta-tion  parabasal/diffuse overexpression strong, homogeneous nuclear staining of ≥80% of basal cells and of suprabasal cells within the invasi growing tumor cell nest Missense muta-tion Missense mutation• absent/null stainingcomplete negative staining of the lesional cellswith positive nuclear staining of adjacent stromalcells/lymphocytes, i.e., internal positive controlCancers 2024, 16, x FOR PEER REVIEW 6 of 18 • absent/null staining complete negative staining of the lesional cells with positive nuclear staining of adjacent stromal cells/lymphocytes, i.e., internal positive control Nonsense or frameshift muta-tion • cytoplasmic positivity clearly visible, most granular staining of the cytoplasm of the le-sional cells with variable nuclear staining Mutation affect-ing the nuclear transportation of the p53-protein Immunohistochemical staining for p16, as a surrogate marker of high-risk HPV in-fection, has been recommended as part of the routine stratification of VSCC by the Inter-national Collaboration on Cancer Reporting (ICCR), the International Federation of Gy-necology and Obstetrics (FIGO), and the WHO [1,14,17,28,29,41,42]. In vulvar intraepithelial neoplasia (VIN) lesions, p16 is considered overexpressed when there is block-like staining, i.e., a strong nuclear and cytoplasmic staining of all le-sional cells from the basal layer upwards through at least one-third of the epithelial thick-ness [43,44]. In invasive or metastatic VSCC, p16 overexpression is recognized when mod-erate or intense nuclear and cytoplasmic staining is detected in 70% or more of the tumor cells [9,44]. This interpretation of p16 staining in invasive carcinoma is based on the rec-ommendations for head and neck squamous cell carcinoma by the American Society of Clinical Oncology and the College of American Pathologists [26]. The foci of keratinization in VSCC should be excluded from consideration (Figure 3, [9]). Figure 3. Patterns of p16 overexpression in non-keratinizing (a) and keratinizing (b) VSCC, so-called block-like staining. Note that the keratinized areas stain negative for a p16 pattern (asterisk; see text). Nonsense or frameshiftmutationCancers 2024, 16, 4216 6 of 16Table 1. Cont.• cytoplasmic positivityclearly visible, most granular staining of thecytoplasm of the lesional cells with variablenuclear stainingCancers 2024, 16, x FOR PEER REVIEW 6 of 18 • absent/null staining complete negative staining of the lesional cells with positive nuclear staining of adjacent stromal cells/lymphocytes, i.e., internal positive control Nonsense or frameshift muta-tion • cytoplasmic positivity clearly visible, most granular staining of the cytoplasm of the le-sional cells with variable nuclear staining Mutation affect-ing the nuclear transportation of the p53-protein Immunohistochemical staining for p16, as a surrogate marker of high-risk HPV in-fection, has been recommended as part of the routine stratification of VSCC by the Inter-national Collaboration on Cancer Reporting (ICCR), the International Federation of Gy-necology and Obstetrics (FIGO), and the WHO [1,14,17,28,29,41,42]. In vulvar intraepithelial neoplasia (VIN) lesions, p16 is considered overexpressed when there is block-like staining, i.e., a strong nuclear and cytoplasmic staining of all le-sional cells from the basal layer upwards through at least one-third of the epithelial thick-ness [43,44]. In invasive or metastatic VSCC, p16 overexpression is recognized when mod-erate or intense nuclear and cytoplasmic staining is detected in 70% or more of the tumor cells [9,44]. This interpretation of p16 staining in invasive carcinoma is based on the rec-ommendations for head and neck squamous cell carcinoma by the American Society of Clinical Oncology and the College of American Pathologists [26]. The foci of keratinization in VSCC should be excluded from consideration (Figure 3, [9]). Figure 3. Patterns of p16 overexpression in non-keratinizing (a) and keratinizing (b) VSCC, so-called block-like staining. Note that the keratinized areas stain negative for a p16 pattern (asterisk; see text). Mutation affecting thenuclear transportation of thep53-proteinI unohistochemical staining for p16, as a surrogate marker of high-risk HPV in-fection, has been recommended as part of the routine stratification of VSCC by the In-ternational Collaboratio on Cancer Reporting (ICCR), the I ternational Federation ofGynecology and Obstetrics (FIGO), and the WHO [1,14,17,28,29,41,42].In lvar i traepithelial neoplasia (VI ) lesions, p16 is c si ere erex ressedhen there is block-like staining, i.e., a strong nucle r and cytoplasmic staining of alllesional cells from the basal layer upwards through a least one-third of the epi elialthickness [43,44]. In invasiv or metastatic VSCC, p16 ov rexpre sion is recogniz d whenmoderate or intense nuclear and cytoplasmic staining is detected in 70% or more of thetumor cells [9,44]. This interpretation of p16 stai i g in invasive carcinom is based on therecommendations for head and neck squamous cell carcinoma by the American Society oflinical c l a t e olle of A erica Pathologists [26]. The foci of keratinizationin S sho l be excl e fr i ti ( iCancers 2024, 16, x FOR PEER REVIEW 6 of 18  absent/null staining complete negative staining of the lesional cells with positive nuclear staining of adjacent stro-mal cells/lymphocytes, i.e., internal positive control Nonsense or frameshift muta-tion  cytoplasmic positivity clearly visible, most granular staining of the cytoplasm of the lesional cells with variable nuclear staining Mutation affect-ing the nuclear transportation of the p53-protein Immunohistochemical staining for p16, as a surrogate marker of high-risk HPV in-fection, has been reco mended as part of the routine str tification of VSCC by the Inter-nati al Collaborati n on Cancer Reporting (ICCR), the International Federation of Gy-c logy nd Obstetrics (FIGO), and th WHO [1,14,17,28,29,41,42]. In vulvar intraepi helial neopl sia (VIN) lesions, p16 is considered overexpressed when there is block-like staini g, i.e., a strong nuclear and cytoplasmic staining of all le-sional cells from the basal layer upwards th u h at least o e-third of the epithelial thick-ness [43,44]. In invasiv or met static VSCC, p16 verexpression is recognized when mod-erate or intense nuclear nd cytoplasmic staining is detected i 70% or more of the tumor cells [9,44]. This i t rpretation f p16 staining in i vasive carcinoma is based on the rec-ommendations for ead and neck squamous cell carcinoma by the American Society of Clinical Oncology and the College of American Pathologists [26]. The foci of keratinizati n in VSCC should be excluded from consideration (Figure 3, [9]). Figure 3. Patterns of p16 overexpression in non-keratinizing (a) and keratinizing (b) VSCC, so-called block-like staining. Note that th kerat ized are s sta neg tive for a p16 pattern (asterisk; see text). Figure 3. Patterns of p16 overexpression in non-keratinizing (a) and keratinizing (b) VSCC, so-calledblock-like staining. Note that the keratinized areas stain negative for a p16 pattern (asterisk; see text).4. Accuracy of Molecular Subclassification of VSCCGiven its concordance with in situ detection methods for HPV of up to 100%, p16 overex-pression is considered a reliable surrogate marker for high-risk HPV infection [39,41,45,46].The abovementioned p16 immunostaining has been reported to have a sensitivity of 97.4% to100% and a specificity of 96.6% to 98.7% in the assessment of HPV status [14,42]. Very rarely,the complete absence of p16 overexpression may be seen in HPVa carcinomas of the lowerfemale genital tract [47]; this may be caused by a loss of heterozygosity [48], rarely by pointmutations of the p16 gene or the silencing of the gene by promotor hypermethylation [49].Cancers 2024, 16, 4216 7 of 16In VSCC, p53 immunostaining interpretation has to be performed using a pattern-based analysis, defining two wild-type and four aberrant staining patterns (see Table 1).Aberrant p53 patterns show a strong correlation with TP53 sequencing analyses in VSCC(95% agreement) [16]. Another study reported a 97% accuracy of p53 immunostaining inthe prediction of TP53 mutations in VSCC [50].It is important to note that molecular classification is not possible based on hematoxylinand eosin-stained slides [17,19,42,51]. Therefore, the use of p16 and p53 immunohisto-chemistry is necessary for the correct molecular subtyping of VSCC. Using these twoimmunostains to diagnose the three different molecular subtypes (Figure 2), Yang et al. [14]reported that 95.6% (215/225) of VSCC could be readily classified as one of the three sub-types by using p16 and p53 immunostaining alone. Thompson et al. [20] demonstrated agood interobserver correlation for the immunohistochemical classification of VSCC into oneof three molecular subtypes using p16 and p53 immunohistochemistry, with a kappa valueof 0.74. In cases with ambiguous immunohistochemical staining results, it is sometimesnecessary to examine more than one area of the tumor and more than one tissue block [14].Importantly, immunohistochemistry for p53 and p16 is inexpensive and readily available;it represents a robust method with a standardized technical workup, and the results areusually available within 24 hours [17,19].In cases with ambiguous staining results (<5% of cases), an additional molecularevaluation may be required [14]. An algorithmic approach for use in further moleculartesting (in situ hybridization or sequencing), as suggested by Yang et al. [14], is summarizedin Figure 4.Cancers 2024, 16, x FOR PEER REVIEW 8 of 18 Figure 4. An algorithmic approach for the three-tiered molecular subclassification of VSCC using p16 and p53 immunohistochemistry [14]. ISH = in situ hybridization. One final consideration in the molecular subtyping of VSCC is that HPV high-risk-negative p53abn VSCC may show both an immunohistochemical overexpression of p16 and a mutant-pattern p53 expression, which is so-called double positivity. The frequency of these cases ranges from 0% [22,52] to 4.2% [14] and 6.8% [53] and may be underestimated at this time. With regard to the mechanism of double positivity, p16 is frequently overex-pressed in high-grade serous ovarian cancer, and <1% of these tumors harbor a CDKN2A mutation, so p16 overexpression is thought to be secondary to dysregulation and to un-derlying p53 and/or Rb mutations [54]. In VSCC, Yang et al. [14] reported a CDKN2A (frameshift) mutation in two out of five of their double-positive cases. Clearly, the reason for double-positive staining in VSCC requires further research, but the small number of cases reported to date suggests that the combination of p16 positivity and mutant-pattern p53 staining is usually seen in HPVi p53abn VSCC. The molecular subclassification of VSCC is summarized in Figure 5. 4. n algorithmic ap roach for the three-tiered molecular subclassification f VSCC using p16and p53 immunohistochemistry [14]. ISH = in situ hybridization.l i i i l l i i i i -ti p53abn VSCC may show both an im unohistochemical overexpression of p16 andmutant-pattern p53 expression, which is so-called double positivity. The frequency of thesecases ranges from 0% [22,52] to 4.2% [14] and 6.8% [53] and may be underestimated at thistime. With regard to the mechanism of double positivity, p16 is frequently overexpressedCancers 2024, 16, 4216 8 of 16in high-grade serous ovarian cancer, and <1% of these tumors harbor a CDKN2A mutation,so p16 overexpression is thought to be secondary to dysregulation and to underlying p53and/or Rb mutations [54]. In VSCC, Yang et al. [14] reported a CDKN2A (frameshift)mutation in two out of five of their double-positive cases. Clearly, the reason for double-positive staining in VSCC requires further research, but the small number of cases reportedto date suggests that the combination of p16 positivity and mutant-pattern p53 staining isusually seen in HPVi p53abn VSCC. The molecular subclassification of VSCC is summarizedin Figure 5.Cancers 2024, 16, x FOR PEER REVIEW 9 of 18 Figure 5. Immunohistochemical classification of VSCC into its molecular subtypes (for details, please see the text and Figures 4 and 6 and Table 1). Figure 6. Algorithmic approach in routine pathology workup for the three-tiered molecular classi-fication of VSCC [10,14,40]. 5. Prognostic Impact of Molecular Subtyping It is now accepted that the HPV status of VSCC has a highly significant impact on prognosis [17,19,20,36,52,53,55,56], and it has been the subject of two meta-analyses. Cao et al. [57] reported a higher overall survival (RR = 0.53, (95% CI [0.35–0.80]; p = 0.003) for HPVa/p16-positive tumors. Sand et al. [36] reported a pooled hazard ratio of 0.40 (95% CI Figure 5. Immunohistochemical classification of VSCC into its molecular subtypes (for details, pleasesee th t xt and Figures 4 and 6 T ble 1).Cancers 2024, 16, x FOR PEER REVIEW 9 of 18 Figure 5. Immunohistochemical classification of VSCC into its molecular subtypes (for details, please see the text and Figures 4 and 6 and Table 1). Figure 6. Algorithmic approach in routine pathology workup for the three-tiered molecular classi-fication of VSCC [10,14,40]. 5. Prognostic Impact of Molecular Subtyping It is now accepted that the HPV status of VSCC has a highly significant impact on prognosis [17,19,20,36,52,53,55,56], and it has been the subject of two meta-analyses. Cao et al. [57] reported a higher overall survival (RR = 0.53, (95% CI [0.35–0.80]; p = 0.003) for HPVa/p16-positive tumors. Sand et al. [36] reported a pooled hazard ratio of 0.40 (95% CI Figure 6. Algorithmic approach in routine pathology workup for the three-tiered molecular classifica-tion of VSCC [10,14,40].Cancers 2024, 16, 4216 9 of 165. Prognostic Impact of Molecular SubtypingIt is now accepted that the HPV status of VSCC has a highly significant impact onprognosis [17,19,20,36,52,53,55,56], and it has been the subject of two meta-analyses. Caoet al. [57] reported a higher overall survival (RR = 0.53, (95% CI [0.35–0.80]; p = 0.003)for HPVa/p16-positive tumors. Sand et al. [36] reported a pooled hazard ratio of 0.40(95% CI [0.29–0.55]) for p16-positive VSCC and 1.81 (95% CI [1.22–2.68]) for tumors withmutant-pattern p53 expression.McAlpine et al. [19] reported significantly worse survival in HPVi and p53-drivenVSCC in the era following the move away from en bloc radical vulvectomy. Their study(n = 197) compared two surgical approaches: radical vulvectomy with en bloc resectionof the vulva and groins (called long-horn excision; group 1) versus a localized radicalvulvectomy with separate incisions for the vulva and both groins (so-called triple incision;group 2). HPVa tumors were shown to have a superior progression-free (HR 0.17; p < 0.001),disease-specific (HR 0.08; p < 0.001), and overall survival (HR 0.24; p < 0.001) in the grouptreated with less radical surgery. In contrast, HPV status had no prognostic relevance inthe group treated with the more radical surgical approach. Furthermore, 30–40% of thepatients in both groups received adjuvant radiation therapy, with no reported inter-groupdifferences in the radiation technique. In the radical surgical technique of a vulvar fieldresection (VFR), the extent of the resection (partial, total, or extended) and regional lymphnode sampling is based on the understanding of embryonic and fetal development, aimingto resect the ontogenetic cancer field. Early evidence showed that VFR without adjuvantradiotherapy compares favorably to standard multimodal treatment [58]. A study byThompson et al. [20] reported equivalent outcomes in HPVa and HPVi p53abn VSCCstreated with the VFR-based surgical approach. These results are consistent with those ofthe study by McAlpine et al. [19], leading to the hypothesis that a radical primary resectionleads to an improved outcome for patients with a high-risk molecular subtype of VSCC,i.e., HPVi p53abn. Ultimately, however, a prospective clinical trial of different surgicalapproaches is needed to adequately test the hypotheses that more radical surgery leadsto an improved outcome for patients with HPVi VSCC and that equivalently favorableoutcomes can be achieved in select cases of younger patients with HPVa VSCC throughless aggressive surgery with adjuvant radiotherapy.With regard to adjuvant radiotherapy, Dohopolski et al. [59] examined 39 patients withadjuvant external-beam (chemo-) RT and reported an in-field relapse rate of 72.2% for HPViand 59.1% for HPVa tumors (p = 0.062), as well as a five-year progression-free survivalof 22.1% versus 63.5% (p = 0.072). Two other studies mainly included patients treatedwith adjuvant radiation only: Yap et al. ([60]; 26/40 with adjuvant RT only) described alocoregional relapse rate of 81.2% for HPVi versus 15.4% for HPVa VSCC (p = 0.003), andsimilar results were reported by Lee et al. ([61]; 28/57 with adjuvant RT only), with a localrecurrence rate of 75% versus 19% (p < 0.01). In a study by Lindell et al. [62], 24 of 75 patientsunderwent adjuvant RT, showing a significantly better recurrence-free, disease-specific,and overall survival in those with HPVa tumors (p ≤ 0.03). In a cohort of 73 patients with amedian tumor size of 5.0 cm treated initially with radiotherapy, a complete clinical responserate of 63.6% was reported in HPVa versus 35.0% in HPVi VSCC [53]. In patients treatedwith a neoadjuvant treatment approach (n = 63), the pathologic complete response rate was53.8% versus 31.4% (p = 0.067, HPVa/p16 + ve versus HPVi/p16-ve, respectively). Proctoret al. [55] reported a higher complete clinical response rate in 48 patients with definitive(chemo-) radiation for HPVa (72.2%) versus 42.9% in HPVi VSCC. A case of HPVa VSCCwith a dramatic clinical response to chemoradiation is illustrated in Figure 7. It should benoted that these studies, while strongly suggesting a difference in radiosensitivity betweenHPVa and HPVi VSCC, are retrospective, and there are no randomized prospective studiesof VSCC where the molecular subtype is determined.Cancers 2024, 16, 4216 10 of 16Cancers 2024, 16, x FOR PEER REVIEW 11 of 18 Figure 7. A 57-year-old woman with a poorly differentiated squamous cell carcinoma ((a); FIGO stage IVA) with p16 block-type staining overexpression (b) and p53 wild-type expression staining pattern (scattered; (c)) on immunohistochemistry, treated using hypofractionated radiation with a total dose of 38 Gy and sequential chemotherapy consisting of 5 cycles of carboplatin AUC 5 and paclitaxel 175 mg/m2. (d) Prior treatment: 25 cm × 10 cm exophytic superficially ulcerated tumor. (e) Partial clinical response after three months of treatment. (f) Complete local clinical response after 10 months. There was no local recurrence within 363 months of follow-up. 6. HPV-Independent, p53 Wild-Type VSCC: Clinical Features Most studies exploring the prognostic impact of molecular subtypes in VSCC have not subdivided the HPVi VSCC into p53abn and p53wt, so the data comparing all three molecular subtypes are still limited. Some studies excluded HPVi p53wt tumors from the analyses because of their rarity [20]. Both Hinten et al. [11] and Barlow et al. [18] reported details of the HPVi p53wt tumor molecular subtype only in the Supplementary Material and not in the main analyses. As previously noted, HPVi p53wt is the least common of the three subtypes (see Table 2). Table 2. Frequency of the different molecular subtypes reported in the literature. Case Number Treatment HPV-Associated HPV-Independend/p53 mut HPV-Independend/p53 wt Horne et al. (2018) * [53] 73 RX/RCX 38.3% 31.5% 23.3% Barlow et al. (2020) [15] 119 OP + adj. RX 52.9% 37.0% 10.1% Kortekaas et al. (2020a) [12] 413 OP + adj. RCX 18.1% 66.6% 15.2% Figure 7. A 57-year-old woman with a poorly differentiated squamous cell carcinoma ((a); FIGOstage IVA) with p16 block-type staining overexpression (b) and p53 wild-type expression stainingpattern (scattered; (c)) on immunohistochemistry, treated using hypofractionated radiation with atotal dose of 38 Gy and sequential chemotherapy consisting of 5 cycles of carboplatin AUC 5 andpaclitaxel 175 mg/m2. (d) Prior treatment: 25 cm × 10 cm exophytic superficially ulcerated tumor.(e) Partial clinical response after three months of treatment. (f) Complete local clinical response after10 months. There was no local recurrence within 363 months of follow-up.6. HPV-Independent, p53 Wild-Type VSCC: Clinical FeaturesMost studies exploring the prognostic impact of molecular subtypes in VSCC havenot subdivided the HPVi VSCC into p53abn and p53wt, so the data comparing all threemolecular subtypes are still limited. Some studies excluded HPVi p53wt tumors from theanalyses because of their rarity [20]. Both Hinten et al. [11] and Barlow et al. [18] reporteddetails of the HPVi p53wt tumor molecular subtype only in the Supplementary Materialand not in the main analyses.As previously noted, HPVi p53wt is the least common of the three subtypes (seeTable 2).To illustrate the clinicopathologic findings of the three-tiered molecular classificationof VSCC, Table 3 summarizes the results of two recent large studies analyzing n = 413cases [12] and n = 411 cases [39], respectively.Nooji et al. [37] evaluated a series of 236 VSCCs and reported a three times higherrate of local recurrence in HPVi p53wt n a four times higher recurrence rate in HPVip53abn VSCC than in HPVa cancers (p = 0.044). A study by Kortekaas et al. [12] reportedbetter five-year overall survival in HPVa VSCC than in HPVi p53wt (HR 2.16; p = 0.049)and HPVi p53abn VS C (HR 3.43; p < 0.001). Comparing the two HPVi molecular subtypes,p53wt VSCC also had a better five-year overall survival than p53abn tumors (HR 0.63;p = 0.06), and p53wt tumors showed a longer recurrence-free period than p53abn VSCC(HR 0.43; p = 0.003; [12]). In a multivariate analysis, the three-tiered molecular classifi-cation of VSCC significantly correlated with the risk of recurrence irrespective of otherclinicopathologic factors (e.g., tumor stage, tumor size, the depth of invasion, and inguinalCancers 2024, 16, 4216 11 of 16lymph node involvement). Interestingly, recurrent disease occurred locally in all threemolecular subtypes.Table 4 summarizes the prognostic data obtained from the literature for the three-tieredmolecular classification of VSCC.Table 2. Frequency of the different molecular subtypes reported in the literature.Case Number Treatment HPV-AssociatedHPV-Independend/p53mutHPV-Independend/p53wtHorne et al. (2018) * [53] 73 RX/RCX 38.3% 31.5% 23.3%Barlow et al. (2020) [15] 119 OP + adj. RX 52.9% 37.0% 10.1%Kortekaas et al.(2020a) [12] 413 OP + adj. RCX 18.1% 66.6% 15.2%Woelber et al. (2021a) [39] 411 mixed 32.1% 39.7% 28.2%Thompson et al.(2022) [20] 68 VFR 32.3% 60.3% 7.4%Corey et al. (2023) [22] 105 N.A. 37.1% 49.5% 13.4%Carreras-Dieguez et al.(2023a) [21] 190 OP + adj. R(C)X 17.4% 72.1% 10.5%pooled 1.379 32.6% 50.9% 15.4%* p16+/p53+: 5/73 = 6.8%; N.A. = not applicated.Table 3. Clinicopathologic characteristics of the three-tiered molecular classification of VSCC [12,39].HPV-Associated HPV-Independent/p53WildtypeHPV-Independent/p53Abnormal p-ValueFrequencyKortekaas et al. (2020a) [12] 18.1% (75/413) 15.2% (63/413) 66.7% (275/413)Woelber et al. (2021a) [39] 32.1% (132/411) 28.2% (116/411) 39.7% (163/411)AgeKortekaas et al. (2020a) [12]—median 59 years 73 years 75 years p < 0.0001Woelber et al. (2021a) [39]—mean 63.5 years 68.9 years 71.6 years p < 0.001Tumor stageKortekaas et al. (2020a)[12]—FIGO ≥ II 21.30% 22.20% 47.30% p < 0.0001Woelber et al. (2021a) [39]—pT1b 33.30% 28.40% 23.90% p = 0.15Tumor SizeKortekaas et al. (2020a) [12]median 1.9 cm 2.5 cm 2.5 cm p = 0.25≤2 cm 52.00% 38.10% 36.40% N.A.Woelber et al. (2021a) [39]mean 3.9 cm 3.5 cm 3.4 cm p = 0.51Depth of invasionKortekaas et al. (2020a) [12]—median 3.0 mm 4.5 mm 6.0 mm p = 0.001Woelber et al. (2021a) [39]—mean 6.9 mm 8.4 mm 9.7 mm p = 0.13LVSIKortekaas et al. (2020a) [12] 9.30% 9.50% 16.00% p = 0.03Nodal statusWoelber et al. (2021a) [39]—pN+ 28.00% 30.20% 42.30% p = 0.050Cancers 2024, 16, 4216 12 of 16Table 4. Prognostic impact of the three-tiered molecular classification of VSCC [11,12,18,39,63].Outcome HPV-Associated HPV-IndependentVariables (p16+ve/p53wt) (p16−ve/p53abn) (p16−ve/p53wt) p-ValueNooji et al. (2017) [37] N = 236 N = 38 (16.1%) N = 155 (65.7%) N = 43 (18.2%)recurrence≤ 2 yrs 5.30% 22.60% 16.30% p = 0.044 *overall survival 75.00% 67.20% 56.30% p = 0.296D.O.D. 21.60% 45.90% 39.50% p = 0.043Hinten et al. (2018) [11] N = 347 N = 55 (15.5%) N = 263 (75.8%) N = 29 (8.4%)recurency rate 20% 36% 21% p = 0.024D.O.D. 12.70% 34.20% 31.00% NAKortekaas et al.(2020a) [12] N = 413 N = 75 (18.1) N = 275 (66.6%) N = 63 (15.3%)recurrency rate 14.70% 43.30% 25.40% p < 0.0001D.O.D. 18.70% 65.10% 46.90% p < 0.001Barlow et al. (2020) [18] N = 119 N = 63 (52.9%) N = 44 (37.0%) N = 12 (10.1%)disease specific 89% 75% 83% NAsurvivalWoelber et al.(2021) [39] N = 411 N = 132 (32.1%) N = 163 (39.7%) N = 116 (28.2%)recurrent disease 22.70% 34.90% 30.20% NA2-year disease freeSurvival 63.90% 47.10% 60.20% p < 0.0012-year overallSurvival 82.50% 70.40% 75.40% p = 0.002D.O.D. = died of the disease, NA = not applicated. * No significant difference between the local recurrence rate bycomparing p53mut and HPV/p16-/p53wt cases (p = 0.246).7. Future DirectionsDifferent targeted treatment approaches have been reported for VSCC (e.g., targetingEGFR (erlotinib), VEGF (bevacizumab), and PD-L1 (pembrolizumab; [64])). However,the results of targeted therapy treatment in VSCC are currently too limited to determinewhether the molecular subtype can guide targeted therapy choices [31,58,64]. Evaluatingbiomarkers related to immune checkpoint therapy, Corey et al. [22] reported no significantdifferences in mismatch repair deficiency (dMMR/MSI-H), tumor mutational burden, andPD-L1 expression between the three different molecular subtypes.Some results have shown that the NOTCH pathway may serve as a potential therapeu-tic target [38] through its inhibition by sarco-endoplasmic reticulum Ca2+-ATPase (SERCA)modulators [65]. The recently described third molecular pathway of VSCC may, therefore,be targetable by SERCA modulators. Additionally, HRAS mutations are prevalent in themolecular pathway of HPVi p53wt VSCC [37]. HRAS is involved in the RTK/RAS/PIK3-CApathway and is targetable by selective mTOR inhibitors [66]. Treatment recommendationsbased on molecular subtype are not in the most recent update of the guidelines of theEuropean Society of Gynaecological Oncology for the management of patients with vulvarcancer [31]; for this to happen, the molecular subtyping of VSCC will first have to beincorporated into future studies.8. ConclusionsIn more than 95% of cases, VSCC can be reliably subclassified into one of three differentmolecular subtypes using immunohistochemistry alone [14,40,50]. Immunostaining forboth p16 and p53 is required for molecular subtype diagnosis [14,17], and attention torecent improvements in the interpretation of both of these immunomarkers is critical (seeTable 1 and Figures 5 and 6) [40,41,67,68]. While the molecular subtypes of VSCC are ofprognostic significance, more data are needed to assess whether they can guide the extentof surgery or choice of adjuvant therapy, as is the case with the molecular classification ofother tumor types, such as breast and endometrial carcinoma. The generation of such datawill be facilitated by a routine and accurate molecular classification of all VSCCs; this isparticularly critical for cases included in research studies/clinical trials. The impact of thethree-tiered molecular subclassification of VSCC is schematically summarized in Figure 8.Cancers 2024, 16, 4216 13 of 16Cancers 2024, 16, x FOR PEER REVIEW 14 of 18 8. Conclusions In more than 95% of cases, VSCC can be reliably subclassified into one of three dif-ferent molecular subtypes using immunohistochemistry alone [14,40,50]. Immunostain-ing for both p16 and p53 is required for molecular subtype diagnosis [14,17], and attention to recent improvements in the interpretation of both of these immunomarkers is critical (see Table 1 and Figures 5 and 6) [40,41,67,68]. While the molecular subtypes of VSCC are of prognostic significance, more data are needed to assess whether they can guide the extent of surgery or choice of adjuvant therapy, as is the case with the molecular classifi-cation of other tumor types, such as breast and endometrial carcinoma. The generation of such data will be facilitated by a routine and accurate molecular classification of all VSCCs; this is particularly critical for cases included in research studies/clinical trials. The impact of the three-tiered molecular subclassification of VSCC is schematically summa-rized in Figure 8. Figure 8. Summary of the three-tiered molecular classification of VSCC. Author Contributions: Conceptualization, L.-C.H., A.K.H., G.G.R.H., C.E.B.; methodology, B.G., L.-C.H., A.K.H., G.G.R.H., C.E.B.; formal analysis, L.-C.H., C.E.B., N.S., M.A., B.G., L.H., K.H., A.K.H., G.G.R.H.; data curation, L.-C.H., C.E.B., N.S., M.A., B.G., L.H., K.H., A.K.H., G.G.R.H.; writ-ing—original draft preparation, L.-C.H., A.K.H., G.G.R.H., C.E.B.; writing—review and editing, L.-C.H., A.K.H., B.G., A.J., J.N.M., C.E.B.; visualization, L.-C.H., K.H., B.G.; supervision, B.G., B.A., N.D.; project administration, L.-C.H., B.G., L.H. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conflicts of Interest: The authors declare no conflict of interest. References 1. Olawaiye, A.B.; Cuello, M.A.; Rogers, L.J. Cancer of the vulva: 2021 update. Int. J. Gynaecol. Obstet. 2021, 155 (Suppl. S1), 7–18. https://doi.org/10.1002/ijgo.13881. 2. Gatta, G.; Capocaccia, R.; Trama, A.; Martínez-García, C. The burden of rare cancers in Europe. Adv. Exp. Med. Biol. 2010, 686, 285–303. https://doi.org/10.1007/978-90-481-9485-8_17. Figure 8. Summary of the three-tiered molecular classification of VSCC.Author Contributions: Conceptualization, L.-C.H., A.K.H., G.G.R.H., C.E.B.; methodology, B.G.,L.-C.H., A.K.H., G.G.R.H., C.E.B.; formal analysis, L.-C.H., C.E.B., N.S., M.A., B.G., L.H., K.H., A.K.H.,G.G.R.H.; data curation, L.-C.H., C.E.B., N.S., M.A., B.G., L.H., K.H., A.K.H., G.G.R.H.; writing—original draft preparation, L.-C.H., A.K.H., G.G.R.H., C.E.B.; writing—review and editing, L.-C.H.,A.K.H., B.G., A.J., J.N.M., C.E.B.; visualization, L.-C.H., K.H., B.G.; supervision, B.G., B.A., N.D.;project administration, L.-C.H., B.G., L.H. All authors have read and agreed to the published versionof the manuscript.Funding: This research received no external funding.Conflicts of Interest: The authors declare no conflict of interest.References1. Olawaiye, A.B.; Cuello, M.A.; Rogers, L.J. Cancer of the vulva: 2021 update. Int. J. Gynaecol. Obstet. 2021, 155 (Suppl. S1), 7–18.[CrossRef]2. 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vulvar cancer
squamous cell carcinoma
p53
p16
human papillomavirus
molecular
classification
prognosis
radiation therapy
surgery
Molecular Subtypes of Vulvar Squamous Cell Carcinoma: The Significance of HPV-Independent/p53 Wild Type
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