Prognostic Value of Perineural Invasion in Oral Tongue Squamous Cell Carcinoma: A Systematic Review and Meta-Analysis

Objectives A significant number of recently published research has outlined the contribution of perineural invasion (PNI) to clinical outcomes in oral tongue squamous cell carcinoma (OTSCC), but some results remain conflicting. This study aimed to determine whether patients with OTSCC with PNI have a worse prognosis than those without PNI. Materials and Methods PubMed, Embase, and the Cochrane Library were queried for potentially eligible articles published up to December 2020. The primary outcomes were the hazard ratio (HR) for locoregional recurrence, overall survival (OS), disease-free survival (DFS), and cancer-specific survival (CSS). The random-effect model was used in all analyses. Results Seventeen studies (4445 patients) were included. Using adjusted HRs, the presence of PNI was associated with a higher risk of locoregional recurrence (HR=1.73, 95%CI: 1.07-2.79, P=0.025, I2 = 33.1%, Pheterogeneity=0.224), worse OS (HR=1.94, 95%CI: 1.39-2.72, P<0.001, I2 = 0.0%, Pheterogeneity=0.838), worse DFS (HR=2.13, 95%CI: 1.53-2.96, P<0.001, I2 = 48.4%, Pheterogeneity=0.071), and worse CSS (HR=1.93, 95%CI: 1.40-2.65, P<0.001, I2 = 25.5%, Pheterogeneity=0.251). PNI had an impact on locoregional recurrence in early-stage OTSCC but not in all stages, and on OS, DFS, and CSS in all-stage and early-stage OTSCC. The sensitivity analyses showed that the results were robust. Conclusion The presence of PNI significantly affects the locoregional recurrence and survival outcomes among patients with OTSCC.


INTRODUCTION
Oral squamous cell carcinoma (OSCC) is the sixth most common type of cancer worldwide (1,2), and, among patients with OSCC, oral tongue squamous cell carcinoma (OTSCC) has been reported as the most common cancer found in the oral cavity (3,4). The prognosis of OSCC is generally poor (1,2,5), and the prognosis of OTSCC is even poorer (6,7).
PNI is usually described in pathological reports, and it may influence the therapeutic management of the patients (12,13). Therefore, this systematic review and meta-analysis aimed to determine whether patients with OTSCC with PNI have a worse prognosis than those without PNI.

Literature Search
This systematic review and meta-analysis was performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines (35).
PubMed, Embase, and the Cochrane Library were queried for potentially eligible articles published up to December 2020, based on the PICO principle (36) and screening according to the eligibility criteria: 1) population: patients with OTSCC; 2) exposure: PNI; 3) outcome: prognostic outcome related to survival with a hazard ratio (HR) reported in a Cox regression model; and 4) language limited to English. Supplementary Table S1 presents the search terms. Multiple studies using the same group of patients were considered duplicated; only the most recent article meeting the eligibility criteria was included.

Data Extraction
Study characteristics (authors, year of publication, country, study design, sample size, and sex and age of the patients), exposurerelated parameters (OTSCC stage and the TMN staging version), and the primary outcome [HR for locoregional recurrence, overall survival (OS), disease-free survival (DFS), and cancerspecific survival (CSS)] were extracted by two investigators (Jiajia Li and Shan Liu) according to a pre-specified protocol. Locoregional recurrence included local and regional recurrence and DFS encompasses all aspects of the disease (local, regional, and distant). Discrepancies were solved by the discussion by referring to the original paper.

Data Synthesis
The HRs and their confidence intervals (CIs) that indicated the prognostic outcome were extracted to summarize the prognostic effects of PNI. Whenever the HRs were reported using univariable and multivariable models, the HRs were extracted from the multivariable model with the most covariables.

Quality of the Evidence
The search ultimately yielded one randomized controlled study (RCT), three prospective cohort studies, and 13 retrospective studies. The level of evidence of RCT and cohort studies was assessed according to the Cochrane Handbook (37), and the Newcastle-Ottawa Scale (NOS) criteria (38), respectively, and Methodological Items for Non-Randomized Studies (MINORS) (39) were used to assess the other studies. Quality assessments were completed independently by two authors (Zhangao Li and Lin Que). Discrepancies in the assessment were resolved through discussion until a consensus was reached. The GRADE method was used to determine the degree of certainty of the outcomes (37,40).
The presence of PNI was reported with a worse CSS in the adjusted analysis (20,26,29,43,45) Figure S1 and Table 2). PNI had an impact on the locoregional       Figure S1 and  Figure S2 and Table 2 Figure S3 and Table 2).

Sensitivity Analyses
The sensitivity analyses showed that the sequential exclusion of each study, in turn, did not affect the results (Supplementary Figure S5). The GRADE analysis suggests that the degree of certainty is high for all four outcomes (Supplementary Table S4).

DISCUSSION
A significant number of recently published research has outlined the contribution of PNI to clinical outcome in OTSCC (10,(19)(20)(21)(25)(26)(27)(28)(29)(30)(31)(32), but the results remain conflicting. Therefore, this meta-analysis aimed to determine whether patients with OTSCC with PNI have a worse prognosis than those without PNI. The results indicate that the presence of PNI significantly affects the locoregional recurrence and survival outcomes among patients with OTSCC.
PNI results from the complex interaction between invading tumor cells and the particular perineural niche (8)(9)(10)(11). PNI is defined by tumor cells invading perineural tissues, tracking along nerves; since nerves travels across a wide number of structures in the head and neck area, the tumor cells can invade a large area (12,13). The currently proposed mechanisms suggest that the perineural microenvironment is favorable to tumor cell growth and mobility. Indeed, the nerve microenvironment includes blood supply and numerous cell types that maintain and support the surrounding neurons, but that can also maintain and support tumor cells (51,52). Several chemokines might also be involved, but their involvement might vary according to cancer type (53,54).
PNI has been reported to be associated with cancer outcomes in various types of cancer (8)(9)(10)(11), including HNSCC (19)(20)(21) and OTSCC (20,27,28,30,43,48,50), but the findings in OTSCC are not unanimous (25,26,31,32,44,46). Nevertheless, when synthesized using the meta-analysis methodology, these conflicting studies support that PNI is associated with locoregional recurrence and poor OS, DFS, and CSS. This is supported by a previous meta-analysis of PNI in HNSCC, in which PNI was associated with OS, DFS, and CSS (55). The present study refines the results of the previous meta-analysis by showing that the associations remain true in OTSCC, which is a particularly aggressive subtype of HNSCC (5,27). In head and  neck adenoid cystic carcinoma, PNI is independently associated with a poor prognosis, according to a meta-analysis by Ju et al. (56), and the prognosis of PNI was worst in males and young patients, but less definitive results were found by other systematic reviews (57,58). A previous meta-analysis indicated that PNI is a strong factor predicting local recurrence and survival in colorectal cancer and that the prognostic value of PNI was similar to that of the depth of invasion, tumor differentiation, positive lymph nodes, and lymphatic and extramural invasion (9). In invasive cervical carcinoma, PNI is associated with OS but not with DFS (59). Previous meta-analyses also reported similar results for esophageal carcinoma (60), gastric cancer (61,62), and rectal carcinoma (63). The present study provides further evidence that PNI is also associated with the outcomes of OTSCC. Of course, the magnitude of the association might vary among different types of cancer. Future studies could aim at quantifying these differences. In addition, the characteristics of PNI (i.e., size of the involved nerves, number of foci and involved nerves, and intratumoral or peritumoral localization) influence the prognostic significance of PNI (64)(65)(66). In the present metaanalysis, the characteristics of PNI were not consistently reported among the included studies, and the number of studies was too small for stratified analyses. Future studies should examine the characteristics of PNI more closely. Hence, PNI is a prognostic marker in OTSCC. PNI could be used as a marker for more aggressive management in patients with OTSCC. Indeed, the presence of PNI has been suggested to guide the management of various cancers, like skin squamous and basal cell carcinoma (67), oral cancer (68), and colorectal cancer (69). Of note, Yang et al. (43) showed that elective neck dissection did not improve the prognosis in patients with OTSCC and PNI. This implies that surgery, which aims at macroscopic disease, might not be adequate for this type of OTSCC and that modalities targeting the microscopic disease, like radiotherapy and systemic therapy, might be more appropriate. Still, it will have to be examined in future studies.
This study has limitations. Firstly, even though 4445 patients were included in this study, the number of prospective studies was small, indicating that the results might be influenced by the biases inherited from the retrospective studies. In such instances, the quality of evidence of the analysis would be undermined. Secondly, we were interested in examining the impact of PNI on the survival outcomes among patients with OTSCC of different stages as we assumed that PNI could be incorporated as a reliable predictor into the current staging guidelines. Although a subgroup analysis for the stage was performed, there are a few points that need to be addressed before concluding about the assumption that PNI is associated with survival. First, only a few numbers of studies investigated early-stage OTSCC patients, resulting in low power. Besides, the guidelines used for defining cancer stages varied from the 6 th to the 8 th edition of the AJCC staging system, which might cause significant disparity among studies and affect the results. In addition, the publication bias could not be assessed because the number of studies included in each quantitative analysis was <10 (37,42). Finally, the adjusted HRs were analyzed, but the covariates used for adjustment varied considerably among the included studies, probably contributing to heterogeneity and suggesting that the results should be taken with caution.
In conclusion, the presence of PNI significantly affects the locoregional recurrence and survival outcomes among patients with OTSCC. Extensive prospective studies should thoroughly investigate the impact of PNI in OTSCC as it is strongly affecting OTSCC locoregional recurrence and patient survival. The heterogeneity of the prognostic outcomes according to PNI among different cancer stages should be analyzed and discussed in the future.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding author.

AUTHOR CONTRIBUTIONS
JL conceived and coordinated the study, designed, performed, and analyzed the experiments, wrote the paper. JL and SL carried out the data collection, data analysis and revised the paper. All authors contributed to the article and approved the submitted version.

ACKNOWLEDGMENTS
We would like to thank all the contributors for their contributions to this study.