Prognostic Significance of P16INK4a Expression in Penile Squamous Cell Carcinoma: A Meta-Analysis with Trial Sequential Analysis

Background Recently, P16INK4a expression has been shown to be correlated with cancer-specific survival (CSS) in penile squamous cell carcinoma (SCC). Therefore, we performed this meta-analysis to clarify the prognostic value of P16INK4a for penile SCC. Methods A systematic search was performed in PubMed, Embase, and Web of Science to identify all relevant articles up to May 25, 2017. Hazard ratios (HRs) and corresponding 95% confidence intervals (CIs) of included studies were pooled to estimate the prognostic value. Trial sequential analysis (TSA) was performed to assess the quantity and strength of survival evidence. Results Five retrospective studies consisting of 323 cases were finally included. We found that P16INK4a positive expression was significantly associated with a better CSS of penile SCC (HR=0.45, 95%CI: 0.30-0.67, P<0.001). No heterogeneity or publication bias was noted among the included studies. Furthermore, TSA demonstrated that the findings were based on sufficient evidence. Conclusions P16INK4a positive expression is independently associated with improved CSS for patients with penile SCC.


Introduction
Penile cancer is an uncommon male cancer, with a very low incidence in Western countries (under 1/100 000 men in the United States and 0.1-0.9/100 000 men in Europe). However, its prevalence in the developing countries of Africa, Asia, and Latin America is greater (up to 3.7/100 000 men), and the cancer-related mortality remains significant in these areas [1,2]. The differences in prevalence are likely explained by the variance in risk factors such as human papillomavirus (HPV) infection, living in rural regions, poor genital hygiene habits, and the practice of neonatal circumcision [3]. HPV infection has been demonstrated to play an important role in the pathogenesis of penile cancer and is associated with certain histological subtypes, in particularly squamous cell carcinoma (SCC), the most common histological subtype of penile cancer [4,5].
In recent years, overexpression of P16 INK4a has been reported in HPV-related tumors such as the head and neck SCC and cervical cancer [6], which is considered a reliable marker for the presence of high-risk HPV DNA. However, limited data are available regarding P16 INK4a detection and its prognostic significance in penile SCC. P16 INK4a , known as a tumor suppressor, is shown to induce cell cycle arrest and prevent cell division by cyclin-dependent kinase 4 (CDK4) and cyclin-dependent kinase 6 (CDK6) inhibition, so as to control disease progression by mediating G1 arrest [7]. Besides, P16 INK4a is involved in retinoblastoma (Rb) inactivation by the viral E7 protein [8]. Recent studies reported deregulated P16 INK4a expression in different populations with penile SCC, and P16 INK4a expression has been shown to be correlated with an improved cancer-specific survival (CSS) in penile SCC [1,[9][10][11][12]. These evidences support the hypothesis  that P16 INK4a might be a prognostic marker for patients with penile SCC, which has not been conducted for a metaanalysis to date. Therefore, we searched relevant researches and performed this quantitative meta-analysis to clarify the prognostic value of P16 INK4a expression in penile cancer.

Materials and Methods
This meta-analysis was conducted according to the guidelines of the PreferredReporting Items for Systematic Reviews and Meta-Analyses (PRISMA) [13].  Figures 1 and 2). A flow diagram with details of the study selection process is presented in Figure 3.

Data Extraction.
All eligible studies were reviewed according to the inclusion criteria by two independent reviewers (JYZ and HCZ), and uncertain data were reassessed by NHS. The following elements were extracted from each literature: (1) the first author and publication year, (2) characteristics of the studied population, (3) the number of patients, (4) detection method of P16 INK4a and cut-off definition, (5) follow-up time, and (6) HRs along with their 95% CIs and P values. If HRs and 95% CIs were not directly reported in publications, survival data were extracted from Kaplan-Meier (K-M) curves by using previously described methods [14,15]. Data from graphical survival plots were independently evaluated by two researchers (JYZ and HCZ) using Engauge Digitizer v.5.1 (license type: GPL, developed by Mark Mitch; Category: C:\Science/CAD). All the above-mentioned data are presented with details in Table 1.

Statistical Analysis.
In the present meta-analysis, HRs and corresponding 95% CIs of included studies were combined to calculate the prognostic value of P16 INK4a in penile SCC. A pooled HR of <1.0 represented an improved prognosis, and an HR of >1.0 correlated with poorer prognosis. Heterogeneity among studies was identified by the Cochran Q test and was quantified by the Higgins I 2 statistic. Quantification of heterogeneity has been assigned low, moderate, and high to I 2 values of 25%, 50%, and 75%. Besides, Galbraith plot and sensitivity analysis for included studies were, respectively, implemented to identify the source of heterogeneity. The random-effects model was used when significant heterogeneity was observed (P<0.10 or I 2 >  50%); otherwise, the fixed-effects model was applied [16]. Publication bias was assessed by funnel plot visual inspection and was statistically evaluated by Begg's test and Egger's test, and P<0.05 was considered statistically significant. All above-mentioned statistical analyses were performed by Stata V.12.0 (StataCorp LP, College Station, Texas, USA) and Microsoft Excel (v.2010, Microsoft Corporation, Redmond, Washington, USA).

Trial Sequential Analysis.
Meta-analyses may result in type I errors due to random error from the studies included in the meta-analysis which had a small sample size, publication bias, and low quality, and studies whose conclusions tended to be changed by later studies with a larger sample size [17]. We did trial sequential analysis (TSA) for final included studies in the meta-analysis to estimate and correct these limitations and determine whether cumulative evidence is enough reliable. Our assumptions included two-sided testing with a type I error of 5%, and a type II error of 20% (power of 80%). The main results of TSA were showed in a cumulative Z-curve graph, and the monitoring boundary of required information size in the graph was determined according to O'Brien-Fleming spending function [18]. Besides, the futility boundary was set on the basis of O'Brien-Flemingspending function. When the cumulative Z-curve crosses the trial sequential monitoring boundary or enters the futility area, a sufficient level of evidence may have been reached, and no further trials are needed. If the cumulative Z-curve does not cross any of the boundaries, and the required information size has not been reached, there is insufficient evidence to reach a conclusion. TSA was carried out by the statistical software, TSA version 0.9 beta (User Manual for TSA, Copenhagen Trial Unit 2011, http://www.ctu.dk/tsa). Figure 3, a total of 184 articles were identified through the primary comprehensive online search. Then 169 records were screened by title and abstract, after 15 duplicates were removed. Furthermore, 154 studies were excluded by preliminary review, and 10 studies with insufficient survival data were considered ineligible by fulltext screening. Finally, a total of five retrospective studies were included in the meta-analysis [1,[9][10][11][12].

Eligible Trials. As shown in
The main characteristics of the included studies are summarized in Table 1. A total of 323 participants from Canada, Germany, Spain, and the United States were enrolled, with the numbers of cases of P16 INK4a positive and P16 INK4a negative. The mean or median age of included patients ranged from 60.0 to 69.0 years. All of the studies performed the detection of P16 INK4a expression on tissue samples for immunohistochemical staining. Among the five studies, three directly reported the HR values [1,9,11], and HRs with 95% CIs of the other two were extracted from the K-M curves [10,12]. The mean or median follow-up time among these studies ranged from 1.8 to 3.9 years.

Quantitative Synthesis Results.
We found no heterogeneity among the included studies (I 2 = 0%, P=0.764) (Figure 4), which was also confirmed by sensitivity analysis and Galbraith plot ( Figures 5 and 6). Survival outcomes of the five eligible studies were performed on a fixed-effects model, and we found that P16 INK4a positive expression was significantly associated with a better CSS of penile SCC (HR=0.45, 95%CI: 0.30-0.67, P<0.001).
A random-effects model (DL) was applied in the TSA for the five enrolled studies. The heterogeneity-adjusted required information size to demonstrate or reject a 54.3 % relative risk reduction (low-bias risk trial estimate) of P16 INK4a positive patients (with value of 5% and a value of 20%) was 217 (Figure 7). The cumulative Z-curve crossed both the conventional boundary for benefit and the required information size, indicating that the evidence was sufficiently conclusive and no further trials were required.

Publication Bias.
Begg's test and Egger's test were, respectively, conducted to evaluate the publication bias. As expected, the funnel plot of Begg's test exhibited symmetricalness with a P value of 0.462, and the P value of Egger's test was 0.249 ( Figure 8). Therefore, no evidence of publication bias in this meta-analysis was observed.

Discussion
As a cancer of male sex organ, penile cancer and its treatment can seriously impact sexuality and intimacy, body image, urinary function, mental health, and life quality [19]. Historically (partial) penectomy and penile sparing were useful surgical treatment of penile cancer; however, there remained a potential possibility of recurrence. Therefore, a reliable biomarker that can predict survival outcome for penile cancer is urgently required. Alterations in the P16/CyclinD1/Rb and ARF/Mdm2/P53 pathways are frequent events in the pathogenesis of SCC, especially the upregulation of P16 INK4a at an early stage of tumorigenesis [20]. P16 INK4a induces cell cycle arrest and prevents cell division by inhibition of CDK4 and CDK6 and inhibition of CDK-mediated phosphorylation of the Rb gene [21]. Positive and negative regulation of tumor suppressor P16 INK4a expression has been described in penile carcinoma [22], and thus the mechanisms and the predictive value of P16 INK4a in penile SCC requires further investigation.
Our meta-analysis found that P16 INK4a was an independent prognostic factor for CSS in penile SCC. In the TSA, the pooled sample size was 323, which has surpassed the required information size of 217. Besides, the cumulative  Z-curve has already crossed the conventional boundary and the -spending monitoring boundary, suggesting that our meta-analysis was of sufficient evidence. In addition, there existed several advanced points in our meta-analysis. First, we strictly followed the inclusion criteria for articles, so as to ensure the quality of enrolled literatures. Second, the pooled sample size of our study was larger than that of any individual study, making the effect estimation more precise. Third, there was no statistical heterogeneity among the included studies (I 2 = 0%, P=0.764), and thus a fixed-effects model was utilized. Fourth, the pooled HR of P16 INK4a for CSS was 0.45, which was statistically significant (P<0.001) and was strong enough. Empirically, a predictive factor is considered to be strong when the value of HR is less than 0.5 or more than 2.0 [23]. Moreover, no publication bias was observed in this meta-analysis. However, several limitations remained to be further refined. First of all, there were five eligible articles included     drawbacks such as different median or mean follow-up times among included studies were observed.
In conclusion, P16 INK4a positive expression is independently associated with improved CSS for patients with penile SCC, and TSA has been conducted for the first time to assess the quantity and strength of current evidence. In addition, a number of studies, which focus on the relationship between P16 INK4a and penile cancer prognosis in Asian population, are required for further investigation in the future.

Data Availability
The hazard ratio data and trial sequential analysis data used to support the findings of this study are included within the article. The data supporting this meta-analysis are from previously reported studies and datasets, which have been cited.

Conflicts of Interest
The authors have no conflicts of interest to declare.

Authors' Contributions
Conception and design were by Min Gu and Ninghong Song. Provision of study materials or patients was performed by Ninghong Song. Collection and assembly of data were conducted by Hengcheng Zhang and Hong Cheng. Data analysis and interpretation were done by Jiayi Zhang and Yanyan Xiu. Manuscript writing was performed by Jiayi Zhang and Hengcheng Zhang. Final approval of manuscript was by all authors. Jiayi Zhang, Hengcheng Zhang, and Yanyan Xiu contributed equally to this work.