Microscopic Extrathyroidal Extension Results in Increased Rate of Tumor Recurrence and Is an Independent Predictor of Patient’s Outcome in Middle Eastern Papillary Thyroid Carcinoma

Background Papillary Thyroid Cancer (PTC) is the most common endocrine malignancy, with recurrence rate as high as 30%. A great deal of controversy surrounds the significance of microscopic extrathyroidal extension (m-ETE) as a prognostic factor. The most recent edition (8th) of American Joint Committee on Cancer (AJCC) staging system has removed m-ETE from the definition of pT3, which suggests that m-ETE may lack prognostic impact in PTC patients. Moreover, data about m-ETE prevalence and clinical impact on Middle Eastern PTC remains unknown. We therefore investigate the prevalence of m-ETE and its clinico-pathological correlation and prognostic impact in Middle Eastern PTC. We also compared the AJCC 7th and 8th staging systems and their prognostic performance. Methods PTCs from 1430 consecutive adult (> 18 years) patients from single tertiary care hospital were included in this study. A retrospective analysis of PTC patients’ survival and recurrence were compared between AJCC 8th and AJCC 7th staging systems using Proportion of Variation Explained (PVE) and Harrell’s C-index. Results Median follow up of the study cohort was 9.3 years. 31.2% (446/1430) of patients had m-ETE. In the overall cohort, m-ETE was associated with multiple adverse features such as older age (p < 0.0001), male sex (p = 0.0245), tall cell variant (p < 0.0001), bilateral tumors (p < 0.0001), multifocality (p < 0.0001), lymphovascular invasion (p < 0.0001), lymph node metastasis (p < 0.0001), distant metastasis (p = 0.0166), tumor recurrence (p < 0.0001), radioactive iodine refractoriness (p < 0.0001), BRAF mutation (p < 0.0001) and reduced recurrence-free survival (RFS; HR = 1.75; 95% CI = 1.30 – 2.35; p < 0.0001) irrespective of tumor size. Of the 611 patients with T3 disease based on AJCC 7th edition, 359 (58.8%) were down-staged in AJCC 8th edition classification. Overall, the prognostic performance of AJCC 8th edition was inferior to AJCC 7th on the basis of lower PVE (3.04% vs. 3.73%) and lower C-index (0.40 vs. 0.48). Conclusions In Middle Eastern PTC, m-ETE is significantly associated with compromised survival and acts as an independent predictor of RFS. Given these findings, m-ETE should be included in the thyroid cancer treatment guidelines.


INTRODUCTION
The incidence of thyroid cancer has rapidly increased over the past two decades (1,2). Papillary thyroid cancer (PTC) is the most common thyroid malignancy and generally carries favorable prognosis (3)(4)(5), whereby low risk PTC patients have excellent outcome with conservative treatment, such as adequate surgery and TSH suppressive therapy (6,7). However, a subset of PTC patients present with aggressive disease and experience recurrence, leading to poor prognosis (8)(9)(10). Therefore, identifying tumors with potentially aggressive behavior and increased likelihood of recurrence is crucial for therapeutic decision-making and appropriate patient management. Interestingly, PTC is one of the most common cancers in Saudi Arabia and is the second commonest cancer affecting Saudi females (11). Middle Eastern PTCs show a relatively higher rate of recurrence than in Western countries (12)(13)(14)(15). Therefore, identifying patients at risk of recurrence is a critical step in the management of high risk PTCs, so that appropriate treatment can be initiated.
A number of prognostic factors, including age, sex, histology, tumor size, vascular invasion, lymph node metastasis and extrathyroidal extension (ETE) have been identified as predictors of recurrence and patient outcome (16)(17)(18)(19)(20). However, there has been considerable controversy regarding microscopic ETE (m-ETE) (as determined histologically using microscopic evaluation) and its prognostic significance, as well as its association with recurrence (21)(22)(23). The American Thyroid Association (ATA) guidelines for predicting recurrence considers patients with m-ETE to be at an intermediate risk for recurrence (6), suggesting that these patients should be treated with more aggressive treatment and radioiodine (RAI) ablation. In agreement with this, the European Thyroid Association is also in favor of RAI ablation for PTC patients presenting with m-ETE (24).
Although ATA guidelines are widely used, the American Joint Committee on Cancer (AJCC) TNM staging remains the most commonly used system for PTC staging. The most recent edition (8 th ) of AJCC staging system has removed m-ETE from the definition of pT3 disease, and minimized the clinical impact of m-ETE, down staging it from T3 to T1/2 classification compared to the 7 th edition (25). These changes reflect doubts on the ability to accurately identify m-ETE by histopathologist and its clinical prognostic impact in PTC. Whether the presence of m-ETE directly impacts clinical outcome and patient management is a matter of strong debate. While some studies have reported that m-ETE does not affect the disease free survival or risk of recurrence (22,(26)(27)(28), others report a negative impact on the clinical outcome of PTCs with m-ETE (21,29,30).
However, none of the above studies were conducted on PTCs from Middle Eastern ethnicity and the clinico-pathological associations as well as prognostic impact of m-ETE still remains unknown in this ethnicity. Therefore, we carried out this study to investigate the incidence and clinical impact of m-ETE as predictor of patient's prognosis in Middle Eastern PTCs treated at our institute whilst also comparing the prognostic performance of AJCC 7 th (pT-7) and AJCC 8 th (pT-8) edition T staging systems.

Patient Selection
One thousand four-hundred and thirty consecutive unselected adult PTC patients (> 18 years) diagnosed between 1988 and 2018 at King Faisal Specialist Hospital and Research Centre (Riyadh, Saudi Arabia) were included in the study. Cases were identified based on clinical history followed by fine needle aspiration cytology for confirmation. The Institutional Review Board of the hospital approved this study and the Research Advisory Council (RAC) provided waiver of consent under project RAC # 2110 031 and 2211 168.

Clinico-Pathological Data
Baseline clinico-pathological data were collected from case records and have been summarized in Table 1. Extra-thyroidal extension was further classified, based on previous publications (31)(32)(33), as follows: microscopic ETE was defined as tumor extending beyond the thyroid capsule into the surrounding peri-thyroidal soft tissues of fat and/or skeletal muscle, without visual evidence of this invasion and macroscopic ETE defined as visual evidence of tumor invasion into strap muscles, subcutaneous soft tissue, larynx, trachea, esophagus, recurrent laryngeal nerve or prevertebral fascia. Staging of PTC was performed using the AJCC seventh and eighth edition staging systems. Only structural recurrence (local, regional or distant) was considered for analysis. Recurrence was defined as any newly detected tumor or metastatic lymph node based on ultrasound and/or imaging studies in patients who had been previously free of disease following initial treatment. RAI refractory disease and ATA risk categories were defined based on 2015 ATA guidelines (6).

BRAF Mutation Analysis
BRAF mutation data for the entire PTC cohort was available from our previous study (34).

Follow-Up and Study Endpoint
Patients were regularly followed by both physical examinations and imaging studies to identify tumor recurrence. The median follow-up was 9.3 years (range 1.0 -30.1 years). The primary study endpoint for our analysis was recurrence-free survival (RFS). RFS was defined as the time (in months) from date of initial surgery to the occurrence of any tumor recurrence (local, regional or distant). In case of no recurrence, date of last followup was the study endpoint.

Statistical Analysis
The associations between clinico-pathological variables and extrathyroidal extension was performed using contingency table analysis and Chi square tests. Mantel-Cox log-rank test was used to evaluate recurrence-free survival. Survival curves were generated using the Kaplan-Meier method. Cox proportional hazards model was used for multivariate analysis. Two-sided tests were used for statistical analyses with a limit of significance defined as p value < 0.05. Data analyses were performed using the JMP11.0 (SAS Institute, Inc., Cary, NC) software package.
The relative prognostic performance of each T staging system was evaluated using the Proportion of Variation Explained (PVE) and Harrell's Concordance Index (C-index). The PVE in Cox-proportional hazard model was calculated to compare the relative validity of models with AJCC 7 th and 8 th T stages. The PVE ranges from 0% to 100%, with a higher number indicating better predictability (35). Additionally, we evaluated the predictive capacity of the two models using the Harrell's Cindex. It is commonly used to evaluate risk models in survival analysis (36,37). A model with perfect predictive capacity (sensitivity and specificity of 100%) would have a Harrell's Cindex of 1·00; a category that exhibited a higher Harrell's c-index was considered to exhibit a more accurate predictive capacity. Cindex and PVE were calculated using R version 4.0.1.

DISCUSSION
In the era of personalized medicine and risk-tailored management, risk stratification is necessary to provide appropriate therapy and predict response to the initial treatment. Recent research has indicated that m-ETE only exerts minor effect on patient prognosis and therapy decisions (28,38,39). The updated 8 th edition of the AJCC staging system removed the sub classification of m-ETE, resulting in down staging of T3 tumors. Therefore, we conducted this study to evaluate, for the first time, the clinico-pathological characteristics and clinical impact of m-ETE on patient outcome in a large cohort of Middle Eastern PTC.
In our study, the overall incidence of m-ETE was 31.2% in PTCs. Interestingly, m-ETE was associated with several adverse clinico-pathological factors such as older age group, tall cell variant, multifocality, lymphovascular invasion, regional lymph node metastasis, and radioiodine therapy refractiveness. Moreover, strong correlation between the presence of m-ETE and BRAF mutation was noted, with 72.7% of m-ETE PTC having concurrent BRAF mutations.
There is considerable controversy regarding the prognostic role of m-ETE. We found m-ETE to be associated with poor OS in univariate analysis only. Previous studies have found contrasting results, with some showing an unfavorable impact of m-ETE on OS (33,40), whereas others found no association   (28). Interestingly, in the present research, m-ETE was associated with higher risk of recurrence compared to PTCs without m-ETE and multivariate analysis revealed that m-ETE was an independent prognostic marker for poor RFS. This raises the likelihood that patients presenting with m-ETE might have more aggressive disease and are therefore less likely to respond well to initial treatment. With a median follow-up of 9.3 years, the present study revealed more recurrent disease among patients with m-ETE. Indeed, we showed that the presence of m-ETE conferred a 2.5-fold increased risk of recurrence for all ages (p < 0.0001; Table 2 Although the AJCC staging system is primarily used as a predictor of mortality, recent studies have shown the utility of this staging system as a novel tool for predicting PTC recurrence (42,45,46). In addition, we were intrigued by the exclusion of m-ETE from the AJCC 8th edition staging system and hence sought to determine which of the two staging systems (AJCC 7 or AJCC 8 T stage) was a better predictor of recurrence. Therefore, we further compared the prognostic performance of the AJCC 7 th and 8 th edition staging system in the whole cohort and found the ability of pT-7 to predict RFS was superior to pT-8 based on different models' performance regardless of age or the presence of distant metastasis. The superiority of the prognostic impact of pT-7 and pT-8 was highlighted in a previous study (42), where however, it was seen only in PTC patients ≥ 55 years old without distant metastasis. Another important highlight of this study is the significant association between m-ETE and poor response to RAI, which could reflect the potential influence of m-ETE on treatment decision. However, this needs to be confirmed by further studies, since our data only showed this correlation on univariate analysis. Collectively, our results support the inclusion of m-ETE in risk stratification, as in previous AJCC TNM editions and the ATA risk of recurrence guidelines.
Despite the obvious strengths of our study, including the use of more than 1400 PTCs from a unique ethnicity, the presence of comprehensive clinical and follow-up data and the finding of m-ETE to be a robust independent prognostic factor for RFS, irrespective of tumor size, our study should be viewed in light of a few limitations. Our study was a retrospective and single center study, which could carry selection bias, and hence more prospective multicenter studies in Middle Eastern population are needed. Additionally, we do acknowledge that inter-observer variability for the interpretation of m-ETE is a source of debate (47). However, the histopathologic sections have been reviewed by at least two pathologists to minimize the inter-observer and intraobserver variability. Despite our efforts, we cannot deny the effect of inter-observer and intra-observer variability in our study. Therefore, our conclusions should be interpreted with caution.
In conclusion, our study shows that m-ETE plays an important role in PTC patients from Middle Eastern ethnicity. We found that m-ETE alone is associated with aggressive PTC markers and is an independent marker for poor RFS. Thus omitting minimal ETE from the definition of T3 disease could compromise patient care and management, resulting in these patients being less likely to undergo RAI therapy. Therefore, our results support the inclusion of m-ETE in risk stratification

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.

ETHICS STATEMENT
The Institutional Review Board of King Faisal Specialist Hospital and Research Centre approved this study and the Research Advisory Council (RAC) provided waiver of consent under project RAC # 2110 031 and 2211 168. Written informed consent for participation was not required for this study in accordance with the national legislation and the institutional requirements.

AUTHOR CONTRIBUTIONS
SP and AS analyzed the clinical data, designed and wrote the manuscript. ZQ and KS performed statistical analysis. FD performed clinical data abstraction. SA-S and FA-D contributed samples and analyzed clinical data. KA-K designed, implemented the study, wrote and critically reviewed the manuscript. All authors contributed to the article and approved the submitted version.