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Background: Vulvar squamous cell carcinoma (VSCC) arises after an HPV infection or the mutation of p53 or other driver genes and is treated by mutilating surgery and/or (chemo) radiation, with limited success and high morbidity. In-depth information on the immunological make up of VSCC is pivotal to assess whether immunotherapy may form an alternative treatment., Methods: A total of 104 patient samples, comprising healthy vulva (n = 27) and VSCC (n = 77), were analyzed. Multispectral immunofluorescence (15 markers) was used to study both the myeloid and lymphoid immune cell composition, and this was linked to differences in transcriptomics (NanoString nCounter, 1258 genes) and in survival (Kaplan-Meier analyses)., Results: Healthy vulva and VSCC are both well infiltrated but with different subpopulations of lymphoid and myeloid cells. In contrast to the lymphoid cell infiltrate, the density and composition of the myeloid cell infiltrate strongly differed per VSCC molecular subtype. A relative strong infiltration with epithelial monocytes (HLADR - CD11c - CD14 + CD68 - CD163 - CD33 - ) was prognostic for improved survival, independent of T cell infiltration, disease stage or molecular subtype. A strong infiltration with T cells and/or monocytes was associated with drastic superior survival: 5-year survival > 90% when either one is high, versus 40% when both are low (p < 0.001)., Conclusion: A hot myeloid and/or lymphoid infiltrate predicts excellent survival in VSCC. Based on the response of similarly high-infiltrated other tumor types, we have started to explore the potential of neoadjuvant checkpoint blockade in VSCC., (© 2024. The Author(s).)

Background: A profound insight into the immune landscape of vulvar squamous cell carcinoma (VSCC) is lacking. Here, an in-depth interrogation of T cell infiltration, local immune contexture, signaling pathways and checkpoint molecule expression was performed in early-stage and late-stage VSCC., Methods: The type, location, and infiltration pattern of T cells were studied in 109 patients with primary VSCC FIGO stage I-III. RNA expression of genes involved in immune oncology and oncogenic signaling pathways was analyzed in 40 VSCC, matched for prognostic clinicopathological variables, analyzed for HPV and p53 status, and selected based on T cell infiltration., Results: High intraepithelial infiltration with CD4 or CD8 T cells was associated with longer overall and recurrence-free survival and formed an independent prognostic factor, outperforming molecular subtype and stage of the disease. Strong T cell infiltrated VSCC displayed a coordinated immune response reflected by a positive association between T cells and different lymphocyte and myeloid cell subsets. The expression of genes involved in the migration of T cells and myeloid cells, T cell activation and costimulation, interferon (IFN)-γ signaling, cytotoxicity and apoptosis was higher than in low infiltrated tumors. An active immune signaling profile was observed in all inflamed, part of the altered-excluded and not in altered-immunosuppressed or deserted VSCC. While several checkpoint molecules were overexpressed, only PD-L1 expression displayed discriminatory ability and clinical usefulness. High PD-L1 expression was detected in all inflamed and ~60% of the altered-excluded VSCC., Conclusion: An active immune signaling profile is present in 35% of primary FIGO I-III VSCCs, suggesting potential responsiveness to neoadjuvant PD-1/PD-L1 immunotherapy., Competing Interests: Competing interests: The authors (KEK, SJS, MIEvP and SHvdB) have filed a patent relating to the treatment of vulvar squamous cell carcinoma (VSCC) and methods for identifying subjects with VSCC who are likely to benefit from such therapy., (© Author(s) (or their employer(s)) 2021. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Objective: There is great need for better risk stratification in vulvar squamous cell carcinoma (VSCC). Our aim was to define the prognostic significance of stratifying VSCC based on p16 and p53 immunohistochemistry (IHC) as surrogate markers for HPV and TP53 mutations., Methods: A large retrospective cohort of surgically treated women with primary VSCC was used. VSCC were classified into three subtypes: HPV-positive (HPVpos), HPV-negative/p53 mutant (HPVneg/p53mut), and HPV-negative/p53 wildtype (HPVneg/p53wt). Overall survival (OS), relative survival (RS), and recurrence-free period (RFP) were depicted using the Kaplan-Meier method and survival curves for relative survival; associations were studied using univariable and multivariable Cox proportional hazard models., Results: Of the 413 VSCCs, 75 (18%) were HPVpos, 63 (15%) HPVneg/p53wt, and 275 (66%) HPVneg/p53mut VSCC. Patients with HPVneg/p53mut VSCC had worse OS and RS (HR 3.43, 95%CI 1.80-6.53, and relative excess risk (RER) of 4.02; 95%CI 1.48-10.90, respectively, and worse RFP (HR 3.76, 95%CI 2.02-7.00). HPVpos VSCC patients showed most favorable outcomes. In univariate analysis, the molecular subtype of VSCC was a prognostic marker for OS, RS and RFP (p = 0.003, p = 0.009, p < 0.001, respectively) and remained prognostic for RFP even after adjusting for known risk factors (p = 0.0002)., Conclusions: Stratification of VSCC by p16- and p53-IHC has potential to be used routinely in diagnostic pathology. It results in the identification of three clinically distinct subtypes and may be used to guide treatment and follow-up, and in stratifying patients in future clinical trials., Competing Interests: Declaration of Competing Interest None., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Surgical resection with free surgical margins is the cornerstone of successful primary treatment of vulvar squamous cell carcinoma (VSCC). In general reexcision is recommended when the minimum peripheral surgical margin (MPSM) is <8 mm microscopically. Pathologists are, therefore, required to report the minimum distance from the tumor to the surgical margin. Currently, there are no guidelines on how to make this measurement, as this is often considered straightforward. However, during the 2018 Annual Meeting of the British Association of Gynaecological Pathologists (BAGP), a discussion on this topic revealed a variety of opinions with regard to reporting and method of measuring margin clearance in VSCC specimens. Given the need for uniformity and the lack of guidance in the literature, we initiated an online survey in order to deliver a consensus-based definition of peripheral surgical margins in VSCC resections. The survey included questions and representative diagrams of peripheral margin measurements. In total, 57 pathologists participated in this survey. On the basis of consensus results, we propose to define MPSM in VSCC as the minimum distance from the peripheral edge of the invasive tumor nests toward the inked peripheral surgical margin reported in millimeters. This MPSM measurement should run through tissue and preferably be measured in a straight line. Along with MPSM, other relevant measurements such as depth of invasion or tumor thickness and distance to deep margins should be reported. This manuscript provides guidance to the practicing pathologist in measuring MPSM in VSCC resection specimens, in order to promote uniformity in measuring and reporting.

The recent literature has shown that vulvar squamous cell carcinoma (VSCC) can be stratified into two prognostically relevant groups based on human papillomavirus (HPV) status. The prognostic value of p53 for further sub-stratification, particularly in the HPV-independent group, has not been agreed upon. This disagreement is likely due to tremendous variations in p53 immunohistochemical (IHC) interpretation. To address this problem, we sought to compare p53 IHC patterns with TP53 mutation status. We studied 61 VSCC (48 conventional VSCC, 2 VSCC with sarcomatoid features, and 11 verrucous carcinomas) and 42 in situ lesions (30 differentiated vulvar intraepithelial neoplasia [dVIN], 9 differentiated exophytic vulvar intraepithelial lesions [deVIL], and 3 high-grade squamous intraepithelial lesions or usual vulvar intraepithelial neoplasia [HSIL/uVIN]). IHC for p16 and p53, and sequencing of TP53 exons 4-9 were performed. HPV in situ hybridization (ISH) was performed in selected cases. We identified six major p53 IHC patterns, two wild-type patterns: (1) scattered, (2) mid-epithelial expression (with basal sparing), and four mutant patterns: (3) basal overexpression, (4) parabasal/diffuse overexpression, (5) absent, and (6) cytoplasmic expression. These IHC patterns were consistent with TP53 mutation status in 58/61 (95%) VSCC and 39/42 (93%) in situ lesions. Cases that exhibited strong scattered staining and those with a weak basal overexpression pattern could be easily confused. The mid-epithelial pattern was exclusively observed in p16-positive lesions; the basal and parabasal layers that had absent p53 staining, appeared to correlate with the cells that were positive for HPV-ISH. This study describes a pattern-based p53 IHC interpretation framework, which can be utilized as a surrogate marker for TP53 mutational status in both VSCC and vulvar in situ lesions.

Aims: The most commonly mutated gene in vulvar squamous cell carcinoma (VSCC) is TP53 and its prognostic value, particularly in HPV-independent VSCC, is uncertain. In other tumours, p53 immunohistochemistry (IHC) is an excellent surrogate marker for TP53 mutations. In order to study this in VSCC, we assigned six p53 IHC patterns into two final classes: 'wild-type' or 'mutant'. We determined the performance and interobserver variability of this pattern-based p53 IHC approach., Methods and Results: Two experienced gynaecological pathologists scored the predefined p53 IHC patterns of 59 VSCC, independently and blinded for molecular data. Agreement was calculated by Cohen's kappa. All disagreements regarding p53 IHC patterns were resolved by a consensus meeting. After DNA isolation, the presence of pathogenic TP53 variants was determined by next-generation sequencing (NGS). Sensitivity, specificity and accuracy of p53 IHC as a surrogate marker for TP53 mutation status were calculated. Initial p53 IHC pattern interpretation showed substantial agreement between both observers (k = 0.71, P < 0.001). After consensus, 18 cases (30.5%) were assigned a final p53 IHC class as TP53 wild-type and 41 cases (69.5%) as mutant. The accuracy between the p53 IHC class and TP53 mutation status, after the consensus meeting, was 96.6%. Moreover, the sensitivity and specificity were high 95.3% [95% confidence interval (CI) = 82.9-99.1% and 100% (95% CI = 75.9-100%)]., Conclusions: Pattern-based p53 IHC classification is highly reproducible among experienced gynaecological pathologists and accurately reflects TP53 mutations in VSCC. This approach to p53 IHC interpretation offers guidance and provides necessary clarity for resolving the proposed prognostic relevance of final p53 IHC class within HPV-independent VSCC., (© 2020 The Authors. Histopathology published by John Wiley & Sons Ltd.)

Background: Vulvar squamous cell carcinoma (VSCC) has been suggested to consist of three subtypes; HPV-positive, HPV-negative mutated TP53 or HPV-negative TP53 wildtype, with different clinical courses. To analyze the immune infiltrate in these molecular subtypes and its impact on clinical outcome, an in-depth study of the tumor immune microenvironment was performed., Methods: Sixty-five patients with invasive VSCC matched for age, FIGO stage and treatment modality, were grouped according to the presence of HPV and p53 protein expression status. Archived tissues were analyzed for intraepithelial and stromal expression of CD3, CD8, Foxp3, PD-1, and pan-keratin in randomly selected areas using immunofluorescence. Additional phenotyping of T cells was performed ex-vivo on VSCC (n = 14) and blood samples by flow cytometry. Healthy vulvar samples and blood served as controls., Results: Based on T-cell infiltration patterns about half of the VSCC were classified as inflamed or altered-excluded while one-third was immune-deserted. High intraepithelial helper T cell infiltration was observed in 78% of the HPV-induced VSCC, 60% of the HPVnegVSCC/p53wildtype and 40% of the HPVnegVSCC with abnormal p53 expression. A high intraepithelial infiltration with activated (CD3 + PD-1 + ), specifically helper T cells (CD3 + CD8 - Foxp3 - ), was associated with a longer recurrence-free period and overall survival, irrespective of HPV and p53 status. Flow cytometry confirmed the tumor-specific presence of activated (CD4 + PD-1 ++ CD161 - CD38 + HLA-DR + and CD8 + CD103 + CD161 - NKG2A +/- PD1 ++ CD38 ++ HLA-DR + ) effector memory T cells., Conclusion: This is the first study demonstrating an association between intraepithelial T cells and clinical outcome in VSCC. Our data suggest that abnormal p53 expressing VSCCs mostly are cold tumors whereas HPV-driven VSCCs are strongly T-cell infiltrated.

Introduction: Vulvar squamous cell carcinoma (VSCC) develops via two different pathways: TP53 mutations in a background of lichen sclerosus or a persistent infection with high-risk human papilloma virus (HPV). The latter group of tumor responds better to treatment than the non-virally induced VSCC. This may be explained by a difference in the tumor immune microenvironment (TME)., Areas Covered: This review summarizes literature on TME of VSCC and its precursors, and extrapolates this to foster the development of new therapeutic strategies., Expert Opinion: Both types of VSCC and their precursors are infiltrated with variable numbers of M2 macrophages, regulatory T cells and CD8+ T cells, indicating that they express targetable tumor antigens. Type 1 T cell immunity in precursor lesions is associated with fewer recurrences and better clinical responses to immunotherapy. Escape of these lesions and progression toward VSCC is associated with the downregulation of HLA Class I, increased expression of co-inhibitory molecules, infiltration with immunosuppressive cells and the local production of immunosuppressive enzymes and cytokines. More in-depth studies of the VSCC TME are required to fully comprehend the impact of the immune system on VSCC, and subsequently to identify patients who will benefit from immunotherapeutic strategies.