Abstract
Background/Aim: The expression of the cyclin–dependent kinase inhibitor p16 correlates with the presence of human papillomavirus. The purpose of this investigation was to assess the prognostic relevance of p16 expression in patients with vulvar squamous cell carcinoma (VSCC) treated with radical surgery followed by adjuvant (chemo) radiation in selected cases. Patients and Methods: Seventy-eight patients were analyzed retrospectively. Results: Positive p16 immunostaining was detected in 19 (24.4%) patients. Five-year disease-free survival (DFS) and 5-year overall survival (OS) were better in p16-positive compared to p16-negative patients (83.9% versus 37.3% p=0.002 and 91.7% versus 57.6%, p=0.003, respectively). p16 expression retained prognostic relevance at multivariate analysis for both DFS and OS. Conclusion: p16 expression was detected in 24.4% of patients with VSCC and was found to be an independent prognostic variable for both DFS and OS.
GLOBOCAN estimates of the worldwide incidence and mortality for 36 cancers reported 45.240 new cases of vulvar cancer and 17.427 deaths due to this malignancy in 2020 (1). According to the Surveillance, Epidemiology, and End Results (SEER) Program, vulvar cancer represents 0.3% of all new cancer cases, with a rate of 2.6 per 100,000 women per year in the USA (2). Vulvar squamous cell carcinoma (VSCC), which is the most common histological type, has an age dependent incidence ranging from 0.4:100,000 women in their thirties to 20:100,000 in over seventy-year-old (3). Tumor is staged according to the International Federation of Gynecology and Obstetrics (FIGO) staging system (4-6).
Surgery, consisting of tailored radical vulvectomy and inguinofemoral lymphadenectomy or sentinel node biopsy in selected cases, is the primary treatment of early stage disease (2, 7-10). The risk of relapse is mainly dependent on FIGO stage, tumor size, lymph node status, lymph-vascular space involvement (LVSI) and perineural invasion (PNI) (3-6, 11-18). Adjuvant radiotherapy is usually administered to patients with more than one intra-nodal metastasis, with extra-nodal tumor growth, with clinically suspected or fixed ulcerated groin nodes, and with surgical positive margins not amenable of re-excision (8, 9, 14). This adjuvant treatment can also be taken into consideration in patients with nodal involvement regardless of the number of positive nodes, especially in presence of LVSI (19). A large US population-based analysis appeared to suggest that the addition of adjuvant chemotherapy reduced the risk of death in node-positive patients who underwent adjuvant radiotherapy (20). Patients with locally advanced VSCC usually undergo radiation or concurrent chemoradiation either as definitive treatment or neoadjuvant treatment followed by personalized surgery (21).
VSCC can develop through both human papillomavirus (HPV)-associated and HPV-independent pathways, and these variants have different morphological and molecular features, precursor lesions and clinical outcome (22-24). The precursor of HPV-associated VSCC is the usual-type vulvar intraepithelial neoplasia (uVIN), whereas the precursor of HPV-unrelated VSCC is the differentiated vulvar intraepithelial neoplasia (dVIN). The peak of incidence is the 4th-5th decade and 5th-7th decade for HPV-related and HPV–unrelated VSSC, respectively. The expression of the cyclin–dependent kinase inhibitor p16INK4A (p16) closely correlates with the presence of high-risk HPV (25-28). The viral oncoprotein E7 inactivates the protein encoded by the retinoblastoma gene, which exerts a negative feedback on p16. Therefore, p16 expression is a surrogate marker of HPV-dependent neoplasia. Most HPV-unrelated VSCCs are associated with areas of lichen sclerosus and often harbor TP53 mutations (29-32).
The purpose of this retrospective study was to evaluate the incidence and the prognostic relevance of p16 expression in patients with VSCC treated with radical vulvectomy and inguinofemoral lymphadenectomy followed by adjuvant (chemo)radiation in high-risk cases.
Patients and Methods
This investigation retrospectively analyzed 78 patients with VSCC treated with primary radical surgery (June 2004-June 2021), of whom representative formalin-fixed, paraffin-embedded (FFPE) tumor tissue samples were available at our Hospital. Seventy-four patients underwent inguinofemoral lymphadenectomy, and 4 did not. Of the latter, 3 patients had stromal invasion less than 1 mm (FIGO stage IA) and the other one, with stromal invasion of 7 mm, had very old age (91 years) and unfavorable performance status.
Each tumor was retrospectively classified according to the criteria of FIGO 2021 (6).
Representative samples of each case were stained with haematoxylin and eosin for standard morphologic evaluation and histologic features were defined according to FIGO 2021 criteria. FFPE representative tumour samples were stained for p16 (E6H4). The antibody was acquired from Ventana Medical Systems, Inc., (Oro Valley, AZ, USA) and was “ready to use”. Immunostainings were performed on Ventana BenchMark ULTRA System according to the vendor protocols and were evaluated as positive or negative by two independent pathologists (S.P and A.G.N).
Postoperative radiotherapy was usually given to patients with more than one intra-nodal metastasis or with extra-nodal tumor growth, and sometimes to patients with negative nodes but with positive margins not amenable of re-excision. The targets, doses and times of radiotherapy have been detailed in a previous article (18).
Two patients were lost to follow-up. Seventy-six patients were periodically examined until September 2022. Among these the median follow-up of survivors was 52 months (range, 15-194 months).
Statistical methods. Two-tailed Fisher’s exact test was used both to correlate p16 positive immunostaining with patient characteristics and the clinical and histopathological variables with the risk of relapse.
The time from surgery to first relapse or death from any cause without relapse was defined as disease-free survival (DFS). The time from surgery to death or last observation was defined as overall survival (OS).
The cumulative probability of DFS and OS was estimated according to the Kaplan-Meier product-limit method. The log-rank test was used to compare the homogeneity of DFS and OS functions across strata defined by categories of prognostic variables. A multiple regression analysis based on the Cox proportional hazard model was used to test jointly the relative importance of variables as predictors of survival times. Significance level was set to 5%, and the software IBM SPSS ver. 13 (SPSS, Chicago, IL, USA) was used to carry out the analysis.
Results
Patient characteristics are shown in Table I. At presentation, median age of patients was 74 years old (range, 35-91 years). As shown in the Table II, p16 positive immunostaining rate was significantly higher in patients with early stage (p=0.015), with tumor size ≤2 cm (p=0.024), with stromal invasion ≤5 mm (p=0.003), with negative nodes (p=0.015), without LVSI (p=0.031) and without PNI (p=0.031).
Patient characteristics (n=78).
p16 positive immunostaining by patient characteristics.
Nineteen patients underwent adjuvant radiotherapy, and adjuvant cisplatin–based concurrent chemotherapy was added in 9 of these. One patient received adjuvant chemotherapy with cisplatin 50 mg/m2 + paclitaxel 175 mg/m2 every 3 weeks for 5 cycles for the presence of 9 histologically proven positive groin nodes and extensive LVSI on the surgical samples.
Follow-up data were available for 76 patients. Forty (51.3%) patients relapsed after a median time of 12.0 months after surgery (range, 2-147 months). The failure was local in 23 (57.5%) patients, inguinal in 5 (12.5%), distant in 3 (7.5%) (lung in 2 and pelvic nodes in 1), local + inguinal in 3 (7.5%), local + distant in one (2.5%) (para-aortic and pelvic nodes), local + inguinal + distant in one (2.5%) (pelvic nodes), and inguinal + distant in 4 (10.0%) (pelvic nodes in 3 and para-aortic + mediastinal nodes in one).
The relapse rate significantly correlated with FIGO stage (p=0.037), tumor size (p=0.013), nodal status (p=0.037), LVSI (p=0.034) and p16 expression (p=0.003) (Table III). At present, 37 patients (48.7%) are alive with no evidence of tumor and 4 (5.3%) are alive with tumor relapse. Twenty-nine patients (38.1%) died of tumor and 6 patients (7.9%) died for intercurrent disease with no evidence of tumor.
Relapse rate by prognostic variables (n=76)*.
At univariate analysis, DFS correlated with FIGO stage (p=0.001), tumor size (p=0.01), stromal invasion (p=0.02), nodal status (p=0.001), LVSI (p<0.001), PNI (p=0.009) and p16 expression (p=0.002) (Table IV) (Figure 1). OS was significantly related to FIGO stage (p<0.001), tumor grade (p=0.039), stromal invasion (p=0.015), nodal status (p<0.001), LVSI (p<0.001), PNI (p=0.006) and p16 expression (p=0.003) (Table V), (Figure 2).
Disease-free survival by prognostic variables (Univariate analysis).
Disease–free survival of patients with squamous cell carcinoma of the vulva according to p16 expression.
Overall survival by prognostic variables (Univariate analysis).
Overall survival of patients with squamous cell carcinoma of the vulva according to p16 expression.
At multivariate analysis, LVSI and p16 immunoreactivity were independent prognostic variables for both DFS and OS (Table VI).
Variables predictive of disease-free survival and overall survival by multivariate analysis.
Discussion
uVIN is usually p16 positive and p53 negative on immunohistochemistry, and has a lower chance to progress to VSCC compared with dVIN, whereas the latter is usually p16 negative and p53 positive (33-35). HPV-positive VSCCs develop from uVIN, and HPV–related oncogenic pathway accounts for 20-40% of VSCC cases (23, 30).
In the present investigation positive p16 immunostaining, which is a surrogate marker of HPV-dependent neoplasia, was detected in 24.4% of patients, and it was found to be more frequent in those with early FIGO stage, with small tumor size, with stromal invasion ≤5 mm, with negative nodes, without LVSI and without PNI. At univariate analysis, FIGO stage, stromal invasion, nodal status, LVSI and PNI were significantly related to the clinical outcome of patients, in agreement with the literature (3-6, 11-18). p16 expression correlated with DFS and OS both at univariate and multivariate analysis. It is noteworthy that patients with p16 negative immunostaining had a 3.030-fold higher risk of relapse and a 4.827-fold higher risk of death compared to those with p16 positive immunostaining.
It is worth noting that HPV-positive head and neck tumors appear to be more responsive to radiotherapy and chemotherapy than HPV-negative tumors, and that p16 expression is associated with better DFS and OS (36-39).
Our data confirm the prognostic relevance of p16 expression in patients with VSCC treated with radical surgery followed by adjuvant radiotherapy or concurrent chemoradiation in high-risk cases. We must take into consideration that the 2021 revision of the FIGO staging strongly recommends specifying the HPV status (HPV-related or HPV-unrelated) of VSCC, that can be assessed with p16 immunoreactivity and/or molecular testing for HPV (5, 6).
Footnotes
Authors’ Contributions
Conceptualization, writing-original draft: AG; data curation, formal analysis, methodology: AG, ES, SC, VD, SP. writing-review and editing: AG, ES, SC, AF, VD, CL, AGN, SP.
Conflicts of Interest
The Authors declare no conflict of interest in relation to this study.
- Received January 19, 2023.
- Revision received January 30, 2023.
- Accepted February 1, 2023.
- Copyright © 2023 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
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