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Original research
Molecular subclassification of vulvar squamous cell carcinoma: reproducibility and prognostic significance of a novel surgical technique
  1. Emily F Thompson1,
  2. Lynn Hoang2,
  3. Anne Kathrin Höhn3,
  4. Andrea Palicelli4,
  5. Karen L Talia5,
  6. Nairi Tchrakian1,6,
  7. Janine Senz7,
  8. Rosebud Rusike8,
  9. Suzanne Jordan8,
  10. Amy Jamieson9,
  11. Jutta Huvila10,
  12. Jessica N McAlpine11,
  13. C Blake Gilks2,
  14. Michael Höckel12,
  15. Naveena Singh13 and
  16. Lars-Christian Horn3
  1. 1Department of Molecular Oncology, BC Cancer Research Center, Vancouver, British Columbia, Canada
  2. 2Department of Pathology and Laboratory Medicine, Vancouver General Hospital, Vancouver, British Columbia, Canada
  3. 3Division of Gynecologic Pathology, University of Leipzig, Leipzig, Germany
  4. 4Pathology Unit, Azienda Unità Sanitaria Locale - IRCCS di Reggio Emilia, Reggio Emilia, Italy
  5. 5Royal Women’s Hospital and VCS Foundation, Melbourne, Victoria, Australia
  6. 6Barts Health NHS Trust, London, UK
  7. 7Department of Molecular Oncology, British Columbia Cancer Research Center, Vancouver, British Columbia, Canada
  8. 8Barts Health NHS Trust, London, London, UK
  9. 9Department of Gynecology and Obstetrics, Division of Gynecologic Oncology, The University of British Columbia, Vancouver, British Columbia, Canada
  10. 10Department of Pathology, University of Turku, Turku, Finland
  11. 11Gynecology and Obstetrics, Division Gynecologic Oncology, University of British Columbia, Vancouver, British Columbia, Canada
  12. 12Leipzig School of Radical Pelvis Surgery, Division of Gynecologic Oncology, University Hospital Leipzig, Leipzig, Germany
  13. 13Cellular Pathology, Barts Health and NHS Trust and Queen Mary University, London, UK
  1. Correspondence to Dr C Blake Gilks, Vancouver General Hospital, Vancouver, BC V5Z 1M9, Canada; blake.gilks{at}vch.ca

Abstract

Objectives Vulvar squamous cell carcinoma is subclassified into three prognostically relevant groups: (i) human papillomavirus (HPV) associated, (ii) HPV independent p53 abnormal (mutant pattern), and (iii) HPV independent p53 wild type. Immunohistochemistry for p16 and p53 serve as surrogates for HPV viral integration and TP53 mutational status. We assessed the reproducibility of the subclassification based on p16 and p53 immunohistochemistry and evaluated the prognostic significance of vulvar squamous cell carcinoma molecular subgroups in a patient cohort treated by vulvar field resection surgery.

Methods In this retrospective cohort study, 68 cases treated by vulvar field resection were identified from the Leipzig School of Radical Pelvic Surgery. Immunohistochemistry for p16 and p53 was performed at three different institutions and evaluated independently by seven pathologists and two trainees. Tumors were classified into one of four groups: HPV associated, HPV independent p53 wild type, HPV independent p53 abnormal, and indeterminate. Selected cases were further interrogated by (HPV RNA in situ hybridization, TP53 sequencing).

Results Final subclassification yielded 22 (32.4%) HPV associated, 41 (60.3%) HPV independent p53 abnormal, and 5 (7.3%) HPV independent p53 wild type tumors. Interobserver agreement (overall Fleiss’ kappa statistic) for the four category classification was 0.74. No statistically significant differences in clinical outcomes between HPV associated and HPV independent vulvar squamous cell carcinoma were observed.

Conclusion Interobserver reproducibility of vulvar squamous cell carcinoma subclassification based on p16 and p53 immunohistochemistry may support routine use in clinical practice. Vulvar field resection surgery showed no significant difference in clinical outcomes when stratified based on HPV status.

  • Vulvar Neoplasms
  • Gynecologic Surgical Procedures

Data availability statement

Data are available upon reasonable request.

https://doi.org/10.1136/ijgc-2021-003251

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    HIGHLIGHTS

    • Vulvar squamous cell carcinoma can routinely be subclassified into one of three molecular subtypes.

    • There is excellent interobserver diagnosis of molecular subtypes of vulvar squamous cell carcinoma.

    • No survival differences are seen in this cohort of patients uniformly treated by vulvar field resection surgery.

    WHAT IS ALREADY KNOWN ON THIS TOPIC

    • HPV associated vulvar squamous cell carcinoma is associated with a more favorable outcome than HPV independent vulvar squamous cell carcinoma.

    WHAT THIS STUDY ADDS

    • Subclassification of vulvar squamous cell carcinoma can be reproducibly accomplished using immunohistochemistry.

    HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE AND/OR POLICY

    • Routine molecular subclassification can inform studies on subtype specific approaches to treatment of vulvar cancer.

    Introduction

    The incidence and death rates for vulvar squamous cell carcinoma have steadily increased since the year 2000 (0.6% and 1.7% per year, respectively, in the USA),1 concurrent with worldwide trends toward more limited surgical excision and increased use of adjuvant radiotherapy.2 3 Conspicuously, these changes in therapeutic management, though they have shown improvements in morbidity, have not led to uniform survival advantages.1–4

    There are divergent underlying etiologies that give rise to vulvar squamous cell carcinoma,5–11 and the prognostic significance of high risk human papillomavirus (HPV) has been demonstrated in multiple cohorts.2 5–7 The World Health Organization (WHO) now classifies vulvar squamous cancer and precancers by HPV status as HPV associated and HPV independent disease.8 The latter can be further subdivided into TP53 mutated and wild type groups, a distinction shown to be prognostically significant.5–7 The combination of immunohistochemistry for p16 and p53 can be used to stratify vulvar squamous cell carcinomas into prognostically distinct groups6 7; the reproducibility of vulvar squamous cell carcinoma subclassification using p16 and p53 immunohistochemistry has not been previously investigated.

    We previously observed that the significantly worse prognosis of HPV independent vulvar squamous cell carcinoma, compared with HPV associated, was only seen after a change in surgical practice in the 1990s, when we moved away from radical vulvectomy with en bloc resection of inguinofemoral lymph nodes, to less radical tumor excision with separate groin incisions.2 In this study, we selected a cohort of patients with vulvar squamous cell carcinoma uniformly managed by a primary radical surgical technique, vulvar field resection, which is based on development derived surgical anatomy related to tissue permissiveness for cancer propagation.12 The aim of this study was to assess the reproducibility of the molecular subclassification for vulvar squamous cell carcinoma using p16 and p53 immunohistochemistry at three specialized cancer centers and evaluate its prognosis significance in a cohort of patients uniformly treated with vulvar field resection surgery.

    Methods

    This study received ethical approval from University of British Columbia Office of Research Ethics H16-02756, 22/1/17 and the Leipzig University Institutional Board (156-2009-06072009). All patients provided written informed consent.

    Case Selection

    This was a retrospective study of 86 cases of primary invasive vulvar squamous cell carcinoma surgically treated between January 2009 and December 2017; in 18 cases no residual tumor was present in the resection specimen, leaving 68 cases from the institutional archive of Leipzig University Hospital. All were surgically excised using the vulvar field resection technique developed by Höckel et al.12 Vulvar field resection is a radical surgical technique in which the extent of resection (partial, total, or extended) and regional lymph node dissection is based on ontogenetic considerations; after careful assessment through physical examination, imaging, and biopsies, each patient’s tumor was assigned a pretreatment ontogenetic stage. Tumors involving just one of three vulvar subcompartments were considered ontogenetic stage oT1, while those involving two or three subcompartments but confined to the vulvar compartment were considered stage oT2. The extent of surgery was then individualized with the goal of resection of all invasive and in situ disease with microscopically negative resection margins.12 Sentinel lymph node biopsy rather than full node dissection was performed for patients with stage oT1 disease starting in 2012. Three patients in this series had metastatic carcinoma (stage IV) disease, with pelvic nodes as the metastatic site in two.

    Clinicopathologic data, and adjuvant treatment and outcomes (recurrence free survival, disease specific survival, overall survival) were collected. Patients were seen every 3–6 months for 5 years by their personal gynecologists and the oncological status reported to the Leipzig Cancer Center. Also, patients, their close relatives, or their family doctors were telephoned annually for an update about their condition. If there was recurrence or death, the complete clinical documentation from the treating institution was obtained for review. Whole tissue sections (4 µm in thickness) were cut at Leipzig University Hospital and unstained slides were distributed to Vancouver General Hospital and Barts Health NHS Trust/Royal London Hospital for local immunostaining.

    Immunohistochemistry

    Immunohistochemistry was performed at collaborating institutions using their protocols in use for clinical purposes, summarized in online supplemental Table 1). Unstained slides from each case underwent p16 and p53 immunohistochemical staining and an interpretation at each of the three participating centers. Immunohistochemical staining was evaluated independently by seven consultant gynecologic pathologists (CBG, LH, NS, KLT, AP, AKH and LCH) and two gynecologic pathology fellows (JH, EFT). p16 was interpreted as positive if there was continuous basal nuclear and/or cytoplasmic ‘block like’ staining with extension upwards involving at least one third of the epithelial thickness, in accordance with the Lower Anogenital Squamous Terminology13 and the British Association of Gynaecological Pathologists guidelines.14 p53 immunohistochemistry was scored according to a pattern based approach described by Tessier-Cloutier et al and Kortekaas et al.9 10 Immunoexpression for p53 was considered abnormal (mutant pattern) when any of the following patterns were observed: (i) confluent intense uniform nuclear staining of the basal layer, (ii) confluent intense uniform basal and suprabasal nuclear staining, (iii) complete absence of staining (null immunophenotype), and (iv) confluent cytoplasmic staining; p53 was interpreted as wild type when there was either patchy basal non-uniform nuclear staining of variable intensity, or accentuated suprabasal staining with basal sparing. Cases were thus placed into one of four categories based on immunostaining: (i) HPV associated, (ii) HPV independent p53 abnormal, (iii) HPV independent p53 wild type, and (iv) indeterminate where, in the opinion of the reviewing pathologist, further molecular testing was needed to enable categorization.

    Supplemental material

    RNA In Situ Hybridization

    Cases that were interpreted as showing indeterminate p16 immunostaining (ie, weak, mosaic, or atypically confluent) by any of the study pathologists were selected for high risk HPV RNA in situ hybridization. This was performed using Advanced Cell Diagnostics RNA-scope (Newark, California, USA). The high risk HPV probe kit used included HPV types: 16, 18, 26, 31, 33, 35, 39, 45, 51, 52, 53, 56, 58, 59, 66, 68, 73, and 82. Appropriate positive and negative control probes were used, according to the manufacturer’s instructions.

    TP53 Mutation Analysis

    TP53 mutation analysis was undertaken to adjudicate selected cases, including both a random subset of cases where consensus classification was achieved, and those cases where there was interobserver disagreement (to any degree). The coding regions of TP53, exons 2–11, were evaluated using tagged amplicon next generation sequencing using methods previously described.15

    Vulvar Squamous Cell Carcinoma Subclassification

    The final molecular subclass diagnosis for each tumor was defined as consensus agreement of ≥5/9 of evaluators, except in instances where in situ hybridization or TP53 mutation data were available. In situ hybridization results, where available, were given precedence over p16 immunostaining in cases where abnormal p16 and p53 co-expression was observed. TP53 mutational data were interpreted in the context of the immunostaining pattern observed, taking into account that in rare instances TP53 can be mutated and show wild type staining and that some TP53 mutations, particularly large deletions, are not always identified by sequencing but are associated with abnormal (null pattern) immunostaining.16

    Statistical Methods

    We calculated interobserver, intralaboratory, and interlaboratory agreement for classification into three and four categories, respectively, based on immunohistochemistry: (i) HPV associated, (ii) HPV independent, p53 wild type, (iii) HPV independent, p53 abnormal, or (iv) indeterminate and requiring further investigation. For the purpose of assessing agreement between three categories, the indeterminate answers were considered correct (ie, they were considered to be concordant with the majority or consensus interpretation for the case). Intralaboratory agreement was calculated using the results of pathologists from each site. Interlaboratory agreement was calculated based on the result rendered by the senior most gynecologic pathologist at each of the three institutions. Cohen kappa was used to assess agreement between any two pathologists and Fleiss kappa when agreement was assessed between three or more pathologists or centers.

    Patient age and tumor size are reported as median (standard deviation (SD)), while categorial variables are reported as absolute and relative frequency. Correlation of HPV status with recurrence free, disease specific, and overall survival was assessed using Kaplan–Meier plots and tested using log rank tests. Survival was calculated starting from the date of surgery. Recurrence free survival was time from surgery to date of clinical diagnosis of recurrent disease, disease specific survival was time from surgery to date of death due to vulvar carcinoma, and overall survival was time from surgery to date of death from any cause. In these analyses, all HPV independent vulvar squamous cell carcinomas were grouped together (due to the low numbers of p53 wild type cases) and outcomes were evaluated against HPV associated vulvar squamous cell carcinomas. Calculation of hazard ratios (HR) for relapse free, disease specific, and overall survival, with 95% confidence intervals, was done for HPV independent versus HPV associated vulvar squamous cell carcinomas using Cox regression analysis, Associations and correlations between clinicopathological parameters and molecular subclasses were assessed using χ2 test or Fisher’s exact test where sample size was small (n<5). The Mann–Whitney U test was used to calculate P value for age at presentation by HPV status. Comparison was done using a two sided test with a 5% level of significance or, p=0,05. Statistical analysis was done using R software (V.4.1.2) and Statistical Product and Service Solutions (SPSS) (V.27).

    Results

    Diagnostic Reproducibility

    For all 68 cases, a four category assessment was made by all nine pathologists (Figure 1). The kappa statistic for interobserver agreement (n=9 observers) was κ=0.74. The kappa statistics for intralaboratory reproducibility were as follows: site 1 κ=0.92, site 2 κ=0.74, and site 3 κ=0.72. The interlaboratory agreement was κ=0.82 (site 1 vs site 2), κ=0.66 (site 1 vs site 3), and κ=0.57 (site 2 vs site 3).

    Figure 1
    Figure 1

    Final subtype assessment and interobserver agreement across four groups of independent evaluators: consultant gynecologic pathologists from sites 1–3 and gynecologic pathology fellows. HPV, human papilloma virus; HPV-A, HPV associated; HPV-I, HPV independent; RNA ISH, RNA in situ hybridization; ABN, abnormal; WT, wild type; NGS, next generation sequencing.

    When assessing the kappa values for three categories (in which scores of indeterminate were considered to agree with the consensus or majority interpretation, based on the assumption that further molecular testing would lead to the correct classification), the interobserver agreement for all nine observers was κ=0.85. The kappa statistics for intralaboratory reproducibility were as follows: site 1 κ=0.92, site 2 κ=0.89, and site 3 κ=0.80. The interlaboratory agreement was κ=0.92 (site 1 vs site 2), κ=0.70 (site 1 vs site 3), and κ=0.64 (site 2 vs site 3),

    Final Vulvar Squamous Cell Carcinoma Subclassification

    HPV in situ hybridization was successfully performed in 12 cases and TP53 sequencing was performed in 23 cases. In three cases where a consensus indeterminate result was reached based on immunostaining, the HPV in situ hybridization result was negative.

    There were four tumors with consensus classification as HPV independent p53 abnormal vulvar squamous cell carcinoma based on immunostaining, in which no TP53 mutation was identified on sequencing. In one case, the abnormal staining pattern observed was null type and in one case the area sampled for sequencing did not contain sufficient tumor cellularity, on review of the post core slide; thus both of these were interpreted as false negative TP53 next generation sequencing results. In the remaining two tumors, there was basal staining for p53 that was stronger than in adjacent benign squamous epithelium (Figure 2) and a majority of observers (5 and 6 of 9 observers, respectively, for cases 64 and 65 (Figure 1)) considered this to be p53 abnormal or mutant pattern p53 staining, but TP53 sequencing was negative for a mutation and these were considered false positive immunostaining results. Thus in 2 of 23 (8.7%) cases where TP53 next generation sequencing was performed, the classification based on p53 immunostaining was changed, from p53 abnormal to p53 wild type.

    Figure 2
    Figure 2

    High wild type basal p53 immunohistochemical staining (A–D) mimicking true TP53 mutational associated basal overexpression (E–F). (A–D) Human papilloma virus (HPV) independent p53 wild type vulvar squamous cell carcinomas with accentuated wild type p53 staining in proliferating basal/peripheral–most cells of infiltrating tumor nests (see Figure 1, heat map No 64 and No 65). (B, D) p53 staining which is strong but non-uniform and confined to the basal/peripheral–most layer; here only very occasional and non-uniform scattered suprabasal staining is observed. By contrast (E, F) depict morphologic and immunohistochemical features of HPV independent p53 abnormal vulvar squamous cell carcinoma with true TP53 mutational associated basal overexpression. Note the uniformity of staining and suprabasal extension of p53 immunostaining in (F).

    In summary, the final carcinoma subclassification based on immunostaining, with HPV in situ hybridization and TP53 sequencing performed in a subset of cases, was 22 HPV associated, 41 HPV independent p53 abnormal, and 5 HPV independent p53 wild type.

    Clinicopathologic Data

    Clinicopathologic data for the patient cohort are summarized in Table 1. Complete clinicopathologic data were available for 68 patients. Median follow-up time was 2.2 years (range 0.01–8.4). Median age for patients with HPV associated vulvar squamous cell carcinoma was 60.5 years (SD 16.0 years); women with HPV associated carcinomas were significantly younger than those with HPV independent carcinomas (p=0.003). Median age for patients with HPV independent p53 wild type vulvar squamous cell carcinoma (n=5, 77.0 years, SD 7.9 years) was comparable with that of patients with HPV independent p53 abnormal disease (n=41, 74.0 years, SD 14.1 years). Stage distribution was evenly divided between organ confined and node positive/advanced stage disease (International Federation of Gynecology and Obstetrics (FIGO) stages IA=1, IB=34, II=4, III=26, and IV=3).

    View this table:
    Table 1

    Clinicopathologic characteristics and prognostic variables for the study cohort, stratified according to human papillomavirus and TP53 mutational status

    Among carcinomas classified as HPV associated (n=22, 32.4%), node positivity was observed in eight patients (36.4%). Patients with HPV independent carcinoma (n=46, 67.6%) were predominantly those with TP53 mutated disease (n=41, 60.3% of total cohort). Lymph node involvement was observed in 20 (48.8%) of those with HPV independent p53 abnormal vulvar squamous cell carcinoma. The difference in node positivity between HPV associated and HPV independent p53 abnormal vulvar carcinomas was not statistically significant (p=0.26). Patients with HPV independent p53 wild type vulvar squamous cell carcinoma (n=5, 7.4% of total cohort) presented predominantly with organ confined disease (n=4). Two patients received adjuvant radiation treatment outside of the vulvar field resection protocol, which specifically precludes radiotherapy as part of the primary treatment, one with HPV associated and one with HPV independent p53 abnormal carcinoma.

    Patient Outcomes

    Nine (13.2%) patients experienced disease recurrence with a median recurrence free survival of 2.1 years (range 0.1–8.4). There were 11 (16.2% of total study cohort) deaths classified as disease related. Disease specific deaths occurred in two patients with HPV associated vulvar squamous cell carcinoma (stages IIIC and IVB at presentation) and in nine patients with HPV independent vulvar squamous cell carcinoma (of whom two presented with stage IB disease, the remaining were stages IIIA–IIIC at presentation). Of those with recurrent disease, two were classified as HPV associated (stages IA and IIIB at presentation) and seven were classified as HPV independent (all stages III–IV). We observed no statistically significant differences in recurrence free survival, disease specific survival, or overall survival when comparing HPV associated versus HPV independent disease (Figure 3). Subset analysis for HPV independent vulvar squamous cell carcinoma (ie, p53 abnormal vs p53 wild type) was not performed because of the small number of HPV independent p53 wild type tumors (n=5). The hazard ratios (95% confidence intervals) for HPV independent vulvar squamous cell carcinoma compared with HPV associated vulvar squamous cell carcinoma, for recurrence free, disease specific, and overall survival were: recurrence free survival HR 1.98 (0.41 to 9.55), disease specific survival HR 2.40 (0.52 to 11.1), and overall survival HR 2.23 (0.74 to 6.75). This was for all patients and no subset analyses were undertaken for stages I–II vulvar squamous cell carcinoma because of the very small number of events in these patients.

    Figure 3
    Figure 3

    Kaplan–Meier survival curves for human papillomavirus (HPV) independent and HPV associated vulvar squamous cell carcinoma. The x axis is survival time in years. The survival plots in (A) depict outcomes for all stages, whereas (B) depicts oiutomes for patients with organ confined disease (International Federation of Gynecology and Obstetrics (FIGO) stages I–II only) . OS, overall survival; DSS, disease specific survival; RFS, recurrence free survival.

    Discussion

    Summary of Main Results

    The main study results relate to reproducibility of assignment of vulvar squamous cell carcinoma into one of three molecular subclasses, based on p16 and p53 immunostaining. For nine observers with a range of professional experience, after allowing for indeterminate results to be resolved with further molecular testing, namely in situ hybridization for HPV or TP53 mutational analysis, there was excellent interobserver agreement (κ=0.85). We also assessed survival based on vulvar squamous cell carcinoma subclass but the study was underpowered and no conclusions could be drawn regarding the prognostic significance of the molecular subclass in this cohort of patients treated with vulvar field resection surgery.

    Results in the Context of Published Literature

    Subclassification of Vulvar Squamous Cell Carcinoma Using p16 and p53 Immunohistochemistry

    For the treatment of individual patients to be determined based on molecular subclassification, there must be robust, reproducible, affordable, and widely available testing. We showed that consistent subclassification using a three tiered approach is achievable, as evidenced by strong interobserver agreement and high diagnostic accuracy observed in different medical centers and across a range of evaluators. We were also able to make the following specific observations about molecular subclassification. The challenges in p16 interpretation were predominantly from one of the participating centers (Figure 1), and related to failure to recognize p16 positivity in those cases with weaker staining confined to the lower third of the squamous epithelium (Figure 2), a pattern that is relatively common in HPV associated vulvar squamous cell carcinoma.14 The presence of suprabasal p53 immunostaining can help guide correct interpretation of the p16 immunostaining as being indicative of HPV in such cases. Suprabasal p53 expression pattern is, to the best of our knowledge, exclusively associated with high risk HPV and was at least focally present in 15 of 22 HPV associated vulvar squamous cell carcinoma in this study. A heatmap of individual immunoscores for p53 and p16 (Figure 1) showed that most of the challenges in p53 assessment related to the boundary between high wild type versus mutant pattern with overexpression.

    Two tumors were interpreted as p53 abnormal by the majority of observers, in the context of non-uniform basal accentuation of p53 immunostaining seen in the tumor, compared with adjacent benign squamous epithelium (Figure 4). In retrospect, such staining, which is uncommon, should be considered indeterminate and trigger TP53 mutational analysis. With regards to the three participating centers, the site with the most experience in molecular subclassification of vulvar squamous cell carcinoma (site 1) had the greatest reproducibility, and this was true even for less experienced practitioners (ie, fellows), suggesting that improvements in performance are possible through use of educational materials, such as the guidance document on interpretation of p16 available on the British Association of Gynaecological Pathologists website.14

    p16 immunostaining has emerged relatively recently as a surrogate marker for high risk HPV associated neoplasia of the vulva (and other anatomic sites), as criteria for interpretation were developed that resulted in very high levels of agreement between p16 immunohistochemistry and molecular assays for HPV7 17 18 The use of p16 as a marker to subclassify vulvar squamous cell carcinoma into HPV associated and HPV independent is now recommended in the WHO Classification of Female Genital Tumors.8 The emergence of p53 immunostaining as a surrogate for TP53 mutation status is even more recent for vulvar squamous cell carcinoma10 but has been well established for other anatomic sites.16 In this study, we demonstrated the reproducibility of these markers when used by a large number of interpreting pathologists (n=9) from multiple cancer centers (n=3).

    Figure 4
    Figure 4

    p16 and p53 immunohistochemical staining patterns observed in human papilloma virus (HPV) associated vulvar squamous cell carcinoma. (A, B) p16 and p53 immunohistochemical staining for the case shown in Figure 1, heat map No 15. (C, D) p16 and p53 immunostaining for the case depicted as heat map No 16.

    Clinical Outcomes of HPV Associated and HPV Independent Vulvar Squamous Cell Carcinoma Treated With Radical Surgery

    Most but not all studies that have evaluated vulvar squamous cell carcinoma outcomes by etiologic subtype have observed prognostic significance based on HPV status (literature summarized in Online Supplemental Table 2). Studies that have failed to demonstrate divergent outcomes in HPV independent vulvar squamous cell carcinoma (the majority of which are TP53 mutated) versus HPV associated vulvar squamous cell carcinoma have (a) studied cohorts heavily weighted with stage III–IV disease,19 (b) used p53 immunostaining interpretation criteria that do not reflect TP53 mutation status in vulvar squamous cell carcinoma,19 20 or (c) had significantly different subgroup cohorts in terms of stage distribution and margin status.21 One frequently cited study demonstrating no significant difference in outcomes was in a cohort predominantly (67.3%) treated by radical vulvectomy.20 To date, few studies have evaluated outcomes of patients with vulvar squamous cell carcinoma subclassified into three groups based on HPV and TP53 mutational status, and to the best of our knowledge, none has done so in a uniformly treated patient cohort.

    Supplemental material

    We previously observed that a significant difference in outcomes between HPV independent and HPV associated vulvar squamous cell carcinoma was only seen in patients treated after 1995, when treatment protocols in our center had changed to less radical surgery (local radical excisions with separate groin incisions for lymph node assessment); no significant difference was seen in patients treated prior to 1995.2 We therefore hypothesized that primary radical surgery is critically important in HPV independent vulvar squamous cell carcinoma and that returning to a practice of more radical excision will improve outcomes for patients with HPV independent vulvar squamous cell carcinoma such that there is no longer a difference in prognosis compared with HPV associated vulvar squamous cell carcinoma. While the current study is underpowered to adequately test this hypothesis (see discussion of study weaknesses below), it was undertaken based on its potential to provide a stronger rationale for proceeding to a formal test of this hypothesis, through study of this cohort of uniformly treated patients.

    Vulvar field resection is a radical surgical technique in which the extent of resection (partial, total, or extended) and regional lymph node dissection is based on our understanding of embryonic and fetal development, following the concept of cancer field theory.12 This novel technique was developed at the Leipzig School of Radical Pelvic Surgery following successful application of ontogenetic cancer field resection for cervical cancer.22 Although vulvar field resection has not been evaluated against the standard of care on a randomized phase III trial, data from a single arm prospective study suggest encouraging outcomes that seem similar to those reported with standard multimodal treatment.12 23 A prospective clinical trial of different surgical approaches would be needed to adequately test the hypothesis that a change in surgical approach leads to improved outcomes for patients with HPV independent vulvar squamous cell carcinoma, while equivalently favorable outcomes can be achieved in the younger patients with HPV associated vulvar squamous cell carcinoma through conventional wide excision with adjuvant radiotherapy in selected cases.

    Not depending on the efficacy of adjuvant radiotherapy may have contributed to better outcomes observed in HPV independent vulvar squamous cell carcinomas in our study. Significantly worse disease specific outcomes in radiation treated HPV independent vulvar squamous cell carcinomas have been demonstrated in several cohorts,24–27 consistent with poor outcomes observed across a range of studies evaluating irradiated p53 mutated cancers within and beyond the gynecologic tract,.28–34 Given the low radiosensitivity of TP53 mutated cancers, agnostic of tissue origin, HPV independent vulvar squamous cell carcinoma may be best managed by complete surgical excision. By contrast, HPV associated vulvar squamous cell carcinoma may be adequately treated using less radical and less morbid surgical procedures and radiotherapy, as is done for HPV associated disease at other disease sites (eg, oropharyngeal squamous cell carcinoma).

    Strengths and Weaknesses

    Strengths of this study include having a cohort of uniformly surgically treated patients from a single center, ensuring uniform specimen handling (fixation, prosection). Another strength is that three geographically separate centers participated in assessment of reproducibility of molecular based subclassification of vulvar squamous cell carcinoma, with all staining performed independently at these sites, and with multiple interpreters, with a range of experience, independently assessing the slides at each center. The major weakness of this study relates to the small sample size, such that it does not allow for testing of a hypothesis regarding survival differences between the molecular subtypes in this cohort. Another weakness is the limited duration of follow-up available for these patients (median 2.2 years).

    Implications for Practice and Future Research

    While the results can help inform power calculations for an appropriate randomized multicenter clinical trial, as described above, they do not provide evidence to support a change in clinical management. The interlaboratory and interobserver reproducibility results do support the generalizability of this approach to subclassification, which is advocated in the WHO Classification of Female Genital Tumors8 in independent centers, providing evidence of reproducibility in classification and highlighting some minor adjustments that can be made to improve performance further.

    Conclusions

    Reproducible molecular subclassification is achievable using p16 and p53 immunostaining alone, with use of additional molecular testing in a small subset of cases where immunohistochemistry results are inconclusive. There were no significant differences in outcome between HPV associated and HPV independent vulvar squamous cell carcinomas treated by vulvar field resection surgery; this supports proceeding to a clinical trial where the impact of a change of surgical approach on outcomes of patients with the more aggressive HPV independent vulvar squamous cell carcinoma can be assessed.

    Data availability statement

    Data are available upon reasonable request.

    Ethics statements

    Patient consent for publication

    Ethics approval

    This study involves human participants and was approved by University of British Columbia Office of Research Ethics H16-02756, 22/1/17 and the Leipzig University Institutional Board (156-2009-06072009). Participants gave informed consent to participate in the study before taking part.

    Acknowledgments

    Funding for this study was provided by the Sumiko Kobayashi Marks Memorial OVCARE Research Grants supported by the VGH and UBC Hospital Foundation.

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    Footnotes

    • Contributors ET: investigation, data curation, visualization, writing-original draft, and funding acquisition. LH: writing-review and editing. AKH: investigation and data curation. AP: investigation. KT: investigation, and writing-review and editing. NT: investigation. JS: investigation. RR: investigation. SJ: investigation. AJ: writing-review and editing. JH: investigation, data curation, formal analysis, visualization, and writing-review and editing. JNM: writing-review and editing. CBG: conceptualization, methodology, investigation and guarantor, writing-review and editing, and supervision. MH: writing-review and editing. NS: conceptualization, methodology, investigation, data curation, writing-review and editing, and supervision. L-CH: conceptualization, methodology, investigation, data curation, writing-review and editing, and supervision.

    • Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial, or not-for-profit sectors.

    • Competing interests None declared.

    • Provenance and peer review Not commissioned; externally peer reviewed.

    • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.