Risk Factors of Oral Squamous Cell Carcinoma with Special Emphasis on Areca Nut Usage and Its Association with Clinicopathological Parameters and Recurrence

Introduction Oral squamous cell carcinoma (OSCC) is the most prevalent type of head and neck cancer and is associated with high mortality, particularly in Southeast Asian countries. Areca nut usage, smoking, and alcohol consumption are the most common risk factors for OSCC. Areca nut chewing is highly prevalent in Pakistan and has been attributed to an increase in OSCC cases. This study aimed to determine the association between areca nut usage and various clinicopathological features of OSCC and further evaluate the association of clinicopathological parameters of OSCC with tumor recurrence. Materials and Methods The study was conducted using the data of 228 patients with OSCC resected at Liaquat National Hospital, Karachi, Pakistan, over 5 years between 2018 and 2022. Clinicopathological data were collected from hospital archives, and associations between various risk factors and clinicopathological parameters were determined. Results Males were more commonly affected (77.2%), and the most common age group was <50 years (54.4%). Areca nut usage was reported in 59.6% of cases, and the buccal mucosa was the most common site (62.7%). Areca nut usage was significantly associated with male gender, greater tumor size, greater depth of invasion (DOI), higher tumor stage, nodal stage, presence of perineural invasion (PNI), and recurrence. In addition, multivariate analysis revealed that OSCC recurrence was significantly associated with older age, larger tumor size and DOI, nodal metastasis, and areca nut usage. Conclusion Areca nut-related OSCCs were associated with poor prognosis and recurrence in our study population. Furthermore, OSCC recurrence was associated with various clinicopathological parameters, such as larger tumor size, a higher DOI, and nodal metastasis.


Introduction
Oral squamous cell carcinoma (OSCC), which occurs in the oral cavity, is the most common type of head and neck carcinoma and constitutes >90% of all oral cancers [1,2].According to data published by Global Cancer Statistics, a total of 377,713 cases and 177,757 deaths of OSCC were reported globally in 2020, with the majority afecting the Asian population [3].OSCC is associated with high mortality, with the highest mortality rate in developing countries, particularly in India and other Southeast Asian regions [4].
OSCC is more prevalent among males than females, with middle age to elderly age group being the most susceptible age group [5].Te highest incidence of OSCC is at the posterior lateral border of the tongue, accounting for 50% of all OSCC cases [6].Tobacco smoking, betel quid (contains areca nut), and drinking alcohol are some of the most important risk factors for OSCC.Other risk factors include infection with human papilloma virus (HPV) and a diet low in fresh fruits and vegetables [7].Areca nuts are a prominent risk factor for OSCC, and it has been estimated that over 600 million people chew areca nuts globally, and approximately 85% of this population is from Southeast Asian countries [8].Areca nut contains four alkaloids: arecoline, arecaidine, guvacine, and guvacoline, among which arecoline exhibits carcinogenic characteristics [9].
Despite recent advancements in treatment modalities, OSCC remains a healthcare burden due to its adverse prognosis owing to its locally aggressive nature and high incidence of distant metastasis, which contributes to recurrence in approximately 30% of cases [10].Te 5-year survival rate is reported to be 92% in recurrence-free patients, which drops to 30% in patients with recurrence [11].Evaluation of the clinicopathological features of OSCC plays a signifcant role in the diagnosis of the tumor, clinical outcome, and therapeutic course [12].
Te chewing of areca nuts is very common in the Pakistani population and is a major risk factor for the development of OSCC.Although many studies have been conducted to evaluate the clinicopathological characteristics of OSCC, very few studies have been conducted to understand the characteristics of OSCC due to areca nut usage.Tis study aimed to understand the clinicopathological characteristics of areca nut-related OSCC and to determine the association of various clinicopathological parameters with recurrence.

Ethics, Study
Design, and Setting.Tis was a retrospective, cross-sectional study.Te study was conducted at the histopathology department of Liaquat National Hospital, Karachi, Pakistan, over 5 years from 2018 to 2022.Informed consent was obtained from all participants.All procedures were performed in accordance with the Declaration of Helsinki.Tis study was approved by the ethical review committee of Liaquat National Hospital.

Inclusion and Exclusion Criteria.
All biopsy-proven cases of OSCC were included in this study.All patients included after clinical examination and workup including computed tomography (CT) scan underwent surgical treatment at the institute.Cases with missing clinicopathological or surgical data were excluded from the study.Cases with oral tumors of other types and salivary gland tumors were also excluded from the study.Patients who received neoadjuvant chemotherapy or radiotherapy were also excluded.

Data Collection.
A total of 228 cases of OSCC that fulflled the inclusion criteria were enrolled in the study.Clinicopathological data were retrieved from the hospital archives.Te data included demographic data (gender and age), pathologic data, which included anatomical site (categorized into buccal mucosa, wet mucosa of lips, tongue, and soft palate), histological variant (keratinizing and nonkeratinizing), grade, depth of invasion (DOI) (grouped into <1 cm and >1 cm), perineural invasion (PNI), lymphovascular invasion (LVI), pTNM staging in accordance with the 8th edition of AJCC, and extra-nodal extension.Recurrence and disease-free survival were also monitored in these patients.Te following risk factors were assessed: smoking, alcohol consumption, and areca nut chewing.

Histological Examination.
Te samples collected during surgery were sent to the laboratory after gross examination to determine tumor size and anatomical position.Representative sections were taken from the tumor, and hematoxylin-and eosin-stained slides were prepared.Tese slides were examined by a senior Histopathologist at the institute.Not all cases were reviewed by a second pathologist because all were already biopsy-proven; however, small tumors and cases with atypical histologies (that necessities immunohistochemistry) were reviewed by a second oral pathologist.Histological features like tumor diferentiation, tumor grade, tumor stage, and tumor size were studied.

Data Analysis.
Te collected data were analyzed using the Statistical Package for Social Science (SPSS, Version 26.0;IBM Inc.).Te mean and standard deviation for patient age, tumor size, DOI, smoking duration, areca nut chewing duration, and disease-free survival were calculated.Te frequencies and percentages of all other clinicopathological variables were calculated.Chi-square and Fisher's exact tests were applied to determine the association between clinicopathological parameters and areca nut usage.Binary logistic regression was applied to determine the association between OSCC recurrence and various clinicopathological parameters.

Clinicopathological Characteristics of Study Population.
Table 1 illustrates that OSCC in our study group was more prevalent among men (77.2%) than among women (22.8%).Te mean age of the patients was 50.81 ± 11.77 years, the disease being more common in the younger age group of <50 years (54.4%).Te risk factor analysis showed that the majority (59.6%) of patients were areca nut chewers, with a mean duration of consumption of 15.50 ± 10.55 months.Te other risk factors were fairly uncommon in our study group, with 7% of patients being smokers, with a mean smoking duration of 16.50 ± 7.24 months and alcohol consumption being reported in 1.8% of cases.Te mean diseasefree survival was 27.44 ± 23.51 cm.Recurrence was reported to be present in 57.9% of cases.
Te most common tumor site was the buccal mucosa (62.7%), followed by the tongue (29.8%).Te mean tumor size was 3.43 ± 1.74 cm.In most cases (54.4%), the tumors were of 2.1-4.0 cm in size.Te mean DOI was 1.18 ± 0.77 cm.In 56.1% of cases, the DOI was <1 cm whereas in the 2 International Journal of Surgical Oncology remaining 43.9% of cases the DOI was >1 cm.Nodal metastasis was present in 50.9% of the cases.Te pTNM staging of the cases depicted that the majority, approximately 36.8% cases, were at stage T2, followed by 31.6% at stage T3, whereas a minority of cases reported were at stage T1 and T4 stages (12.3% and 19.3%, respectively).Te majority, approximately 49.1% of cases, were at the N0 stage, followed by 34.6% of cases at the N2b stage.Extra-nodal extension was present in 28.1% of cases.Approximately 52.6% of cases were of the keratinizing type of OSCC.Te most common tumor grade was Grade 2 (moderately diferentiated) in approximately 70.2% of cases.LVI was present in only 2.2% of cases and PNI was present in 19.3% of cases, as shown in Table 2.

Recurrence of OSCC and Its Association with Various
Clinicopathological Features.Table 5 illustrates OSCC recurrence and its association with clinical features and risk factors by univariate and multivariate analyses.Te study showed a statistically signifcant (p value <0.05) association of recurrence with age and history of areca nut usage.Keeping 95% CI and adjusting variables, we found that age >50 years and tumor size >4 cm were associated with a higher recurrence rate in both the unadjusted and adjusted International Journal of Surgical Oncology groups.History of areca nut usage increased the risk of recurrence 2.0 times in the unadjusted group and 2.586 times in the adjusted group.Table 6 depicts the association of recurrence with pathological features.We found a statistically signifcant association of recurrence with tumor size, DOI, nodal metastasis, tumor stage, nodal stage, and extra-nodal extension.A DOI of >1 cm was associated with a higher recurrence rate than a DOI of <1 cm (OR of 1 vs. 0.3, respectively) in the unadjusted group.Nodal metastasis was associated with recurrence with an OR of 3.5 (2.013-6.085) in the unadjusted group and an OR of 4.45 (1.853-10.723) in the adjusted group.Higher tumor stage was positively associated with recurrence (OR: T1-0.296,T2-0.111, and T4-1).Similarly, higher nodal stage was associated with recurrence, i.e., N2b showed 4.211 times and N2c showed 10.667 times greater risk of recurrence in the unadjusted group.No statistically signifcant association was established between OSCC recurrence and tumor site, keratinizing type, histological grade, LVI, or PNI.

Comparison of Recurrence-Free Survival between Patients
with and without a History of Areca Nut Use. Figure 1 demonstrates the survival curve using the Kaplan-Meier graph, which shows that recurrence-free survival among patients with a history of areca nut use was signifcantly lower than that among patients with no areca nut use.

Association of Duration of Areca Nut
Use with Clinicopathological Parameters.Table 7 shows the mean comparison of the duration of areca nut usage with the main prognostic parameters, including recurrence, tumor stage, nodal metastasis, PNI, and DOI.No signifcant association was noted between the duration of areca nut usage and these parameters.

Discussion
OSCC is the most common malignant tumor of the head and neck and is a global health issue because of its aggressive nature [13].Tis study determined the clinicopathological features of the disease in our study population, its recurrence, and the association of clinicopathological features with areca nut.
A study conducted in Qatar by Elaiwy et al. [14] to determine the pathological features of OSCC concluded that similar to our study, males were predominantly more affected than females.Te mean age in their study was reported to be 46.93 years, whereas in our study the mean age was 50.81 ± 11.77 years which corroborates with the data reported previously that most OSCC patients are >45 years (median 62 years) [15].Te most common site in their study was tongue (50%), followed by the buccal mucosa, whereas in our study, the buccal mucosa was the most common site of OSCC (62.7%).Te mean DOI in their study was 8.8 mm, whereas in our study the mean DOI was slightly greater 1.18 ± 0.77 cm.Similar to our fndings, the most common histological grade was Grade 2, and most cases showed no LVI or PNI.Tis study was conducted to evaluate the clinicopathological features associated with areca nut-related OSCC.We concluded that areca nut usage was signifcantly associated with male gender, greater tumor size (2.0->4 cm), DOI of >1 cm (52.9%), higher tumor stage (T3 and T4-32.4% and 26.5%, respectively), higher nodal stage (N2b-38.2%),and a higher histological grade (Grade 2 and Grade 3-61.8% and 8.8%, respectively).Areca nut-related OSCC was more likely to show PNI (23.5%) than nonareca nutrelated OSCC (13%).Te recurrence rate of areca nut-related OSCC in our study was 64.7%, which was much higher than that of OSCC not related to areca nut (47.8%).
Clinicopathological features associated with areca nut use remain largely unexplored.A few studies have been conducted to determine the association between various International Journal of Surgical Oncology  A similar study was conducted in China to determine the correlation between betel nut chewing and clinicopathological factors of OSCC.In contrast to our study, they reported no signifcant correlation between betel nut chewing and gender, age, location, pathological T stage, and cervical lymph node metastasis [16].
In accordance with our study, a study conducted in Pakistan to determine the role of chewing habits in the diferentiation of OSCC found that patients with chewing habits were associated with poorly diferentiated (Grade 3) tumors and were of younger age [17].In our study, no association was found between age and areca nut chewing.
Another study conducted in Northern Pakistan, similar to our study, reported an association between male gender and betel nut chewing [18].
A study conducted by Li et al. on multifaceted mechanism of areca nut in oral carcinogenesis, proposed that males with areca nut chewing habits were more likely to develop OSCC (especially of buccal mucosa), and are of aggressive phenotype, with a greater risk of metastasis, a higher recurrence rate, and a poor survival rate [19].Tese fndings were supported by our study, which reported that areca nut usage was signifcantly associated with male gender and a high recurrence rate, with a recurrence rate of 64.7%.Another study by Liao et al. [20] also corroborated that OSCC in habitual areca nut chewers showed an aggressive International Journal of Surgical Oncology clinical course.Similar to our fnding, a study conducted in Taiwan reported that 49.3% of OSCC patients who consumed areca nut usage presented with a later stage tumor (T3-T4) [21].
Local recurrence is an important prognostic factor in OSCC [10].Our study also evaluated the association of OSCC recurrence with clinicopathological parameters, and we concluded that OSCC recurrence showed a strong association with age of >50 years, tumor size of >4 cm, DOI of >1 cm, nodal metastasis, higher tumor and nodal stage, and history of pan.
PNI is strongly associated with recurrence and distant metastasis in OSCC [22].Tis study failed to establish a statistically signifcant association between PNI and OSCC recurrence.Wang et al. [10] reported a signifcant association between tumor grade and recurrence, which our study failed to establish.A previous study reported that margin status was the only independent predictor of recurrence, whereas DOI and nodal status were important prognostic factors of survival [23].Wang et al. [10] also concluded that T stage, nodal stage, and degree of diferentiation were independent factors of recurrence.Tumor and nodal stages are important factors afecting recurrence, which is also supported by a study by Ebrahimi et al. [24].
Apart from areca-nut use, various other risk factors are also established in OSCC pathogenesis, including the emerging use of e-cigarettes/vaping.Several known carcinogens are found in e-cigarettes, and studies have shown certain molecular alterations associated with e-cigarettes, including DNA strand breaks, that are potentially linked to oral cancers [25].In addition to nicotine in its various forms, human papilloma virus (HPV) is another strongly associated risk factor involved in OSCC.Approximately 20% of OSCCs are attributed to HPV.Conversely, HPV-associated OSCCs are associated with better prognostic features than nicotine/ betal-nut-associated OSCC [13].
As established in our study and supported by other reports, it is evident that areca-nut use is one of the most important risk factors for OSCC in Southeast Asia.Terefore, it is imperative to devise measures to spread awareness in the public to restrict its use, maintain good oral hygiene, and seek early medical advice in cases with any oral ulcer or nodule.
4.1.Limitations.We recognized a few limitations of this study.First, this was a single institute-centered study; hence, the sample size was limited.Tis study lacks a comprehensive molecular study.Te study is retrospective.A multicenter prospective study is imperative to understand the disease and to modulate newer treatment modalities to improve overall survival and prognosis.
Because of the retrospective study design, certain aspects of the link between OSCC occurrence and areca nut usage could not be uncovered in our study.First, in this study, chronically addicted areca-nut patients were labeled as areca nut users, and this history was obtained when these patients presented in the ENT OPD.Te degree of areca nut usage was not quantifed; however, the duration of use was evaluated, and it was noted that the mean duration of area nut usage in our study was 15.50 ± 10.55 months.Tis specifes that unlike other risk factors, such as smoking, even a small duration of areca nut usage can cause ORCC.However, how much areca nut could be safe could not be determined by our study design, as there was no control group (without OSCC) in our study.A prospective cohort study is required to further establish the risk of OSCC associated with areca-nut use.
Similarly, it is also important to understand the molecular alterations that are specifcally associated with arecanut use to devise new treatment modalities for ORCC in this part of the world.

Conclusion
We found that areca nut-related OSCC was signifcantly associated with various clinicopathological features, including gender, tumor size, DOI, T stage, nodal stage, PNI, and recurrence.Furthermore, we concluded that age, tumor size, DOI, nodal metastasis, tumor stage, nodal stage, and history of areca nut usage were important factors for recurrence.

Table 1 :
Clinical parameters and risk factors of population under study.

Table 2 :
Pathological parameters of population under study.

Table 3 :
Association of clinical parameters and other risk factors with areca nut usage.

Table 4 :
Association of pathological parameters with areca nut usage.

Table 5 :
Recurrence of oral squamous cell carcinoma, comparison with groups based on clinical features/risk factors: univariate and multivariate analysis.Binary logistics regression was applied.CI: confdence interval, ® reference group.* * * Signifcant at 0.05 level.

Table 6 :
Recurrence of oral squamous cell carcinoma, comparison with diferent pathological groups: univariate and multivariate analysis.

Table 6 :
Continued.Figure 1: Survival analysis of oral carcinoma patients (areca nut usage vs. no areca nut use) by the Kaplan-Meier method.