Association of Cathepsin B a Salivary Biomarkers in Different Histological Grades of Oral Squamous Cell Carcinoma

Background: Oral cancer is considered a major public health problem due to its high mortality and morbidity rates. Survival rate of OSCC can be signicantly improved by using non -invasive tool such as salivary biomarkers for detection of OSCC which is considered a promising approach. Cathepsin B is a lysosomal cysteine protein, which is present in abundant quantities in lysosome of cells, tissues and different biological uids. Increased expression of Cathepsin B is observed in many malignancies including oral cancer. The present study was designed to determine the salivary levels of Cathepsin B in different histological grades of OSCC. of OSCC of OSCC (poorly differentiated|) and comprises 20 healthy sample was collected from all the four study group and salivary Cathepsin B levels were analyzed by ELISA sandwich technique in duplicate. Results of the present study suggests that Cathepsin B has a great value as a salivary biomarker for diagnosis and monitoring of OSCS in different histological grades of OSCC. It could increase the survival rate and further improve the prognosis of OSCC.


Introduction
Oral cancer is a malignant neoplasm which is considered as a major and alarming health problem of global concern due to increasing prevalence and higher mortality rate 1 . It is the sixth most common and leading cause of mortality worldwide and ranked as the 2nd most common cancer in Pakistan 2, 3 . Oral squamous cell carcinoma (OSCC|) is the most common and frequent type of oral cancer and account for approximately 90% of all oral cancers 4,5 . Approximate annual incidence rate of oral cancer is 300,000 cases and mortality rate is 145,000 cases worldwide 6 . Most common sites of OSCC occurrence are buccal mucosa, tongue and oor of the mouth 7 . The most important and prevalent risk factors responsible for development of OSCC are tobacco, alcohol and smokeless tobacco chewing 8 . The other risk factors include: viral infection HPV16 and HPV18, genetic factors, occupational exposure and nutritional de ciencies such as diet intake of low fruits and vegetables and ultraviolent radiation 9 .The main treatment modalities currently available for OSCC are surgical excision, radiotherapy and chemotherapy 10 .
In spite of the advancement in different treatment regimens, the 5-year survival rate of OSCC is found to be less than 50% 11 . Although the examination of oral cavity is easily assessable but most OSCC are still diagnosed in an advanced stages 12 . Currently, the gold standard for the diagnosis of OSCC is histopathological examination of a biopsy sample 13 . However, biopsy is an invasive procedure and has various drawbacks such as painful, high cost and fear of cancer spread after performing biopsy, which results in late diagnosis of OSCC 14 . Therefore, there is an urgent need to identify new non-invasive methods which will help us in diagnosis and monitoring of OSCC 15 . Diagnosis and monitoring of OSCC by using salivary biomarkers is considered a promising and ideal tool 16 . Using saliva as a diagnostic tool has several advantages as collection of saliva is simple, safe, non-invasive and cost effective in nature and also it has bene ts of ease in sampling, handling and processing 17,18 . Furthermore, due to direct contact between saliva and oral cavity, salivary biomarkers are considered a perfect and classic tool for diagnosis and monitoring of OSCC 19 . Several salivary biomarkers in oral squamous cell carcinoma has been identi ed which can be used as diagnostic and prognostic markers in OSCC 20 .
Cathepsin B (CTSB) is a lysosomal cysteine protease and belongs to a family of cysteine cathepsins. It is present in abundant quantities in lysosomes of cells and different biological uids of body such as saliva and serum 21 . Main physiological functions of Cathepsin B is intracellular degradation of protein and protein turnover 22 . Cathepsin B plays a major and important role in development of cancer and its progression. It is associated with basement membrane dissolution and extracellular matrix degradation, a process which is responsible for tumour progression, growth, metastasis and invasion 23 . Its expression in tissues and serum levels are increased in many types of malignancies including OSCC 24 . However, previously none of the studies have reported Cathepsin B levels in saliva in patients of OSCC. Therefore, by analyzing Cathepsin B levels in saliva in different histological grades may bene t diagnosis and monitoring of OSCC by using non-invasive technique. This will further lead to increase survival rate and improved prognosis of the disease.

Study design and setting
An analytical cross sectional study was conducted during the period of July 2019 to December 2019 after approval from Institutional Review Board of Dow University of Health Sciences (IRB-1223/DUHS/Approval/2019/34). The recruitment of the study participants of Oral Squamous Cell Carcinoma was carried out from the dental OPD of Dow University of Health Sciences. Recruitment of control group participants was carried out from patient's attendant's, friends, family and colleagues.
Informed consent in written was obtained from each research participants.

Sample size estimation
Open Epi online sample size calculator was used for sample size estimation according to the statistics reported by Yang W et al 25 . A parameter of severity of cancer nodal involvement was reported by author 9.53 times increased in patients if Cathepsin B was present compared to patients in which Cathepsin B was absent. A minimum of 80 sample size was calculated (20 in each group) at 80% power and 95% con dence interval.

Study groups
Total no of 80 research participants were enrolled in our study, which were divided into four groups 20 in each group. Group 1 consists of 20 patients of well-differentiated OSCC, Group 2 consists of 20 patients of moderately differentiated OSCC, Group 3 consists of 20 patients of poorly differentiated OSCC and Group 4 consists of healthy controls. Inclusion criteria for OSCC patients includes: Patients of OSCC of both genders, aged 18-65, newly diagnosed and biopsy proven were selected after clinical examination and further con rmed by histopathological examination. OSCC patients were divided into three respective group of OSCC i.e. well, moderate and poorly differentiated according to Broder's histopathological grading criteria. Patients with underlying systemic illness such as rheumatoid arthritis, osteoarthritis, pancreatitis, chronic atrophic gastritis and periodontitis and other malignant tumour such as breast, ovary, lung, gastric and nasopharyngeal were excluded. Patients with a history of smoking and smokeless tobacco products were excluded from the control group. In a pre-designed questionnaire demographic data and type, frequency, duration of chewing habits and site of lesion was obtained for each participants of OSCC.

Saliva collection method
In a sterilized falcon tube of 15 ml, 2-5 ml of unstimulated whole saliva was collected from each research participant between 9-11 am. Participants were asked to refrain one hour from eating, chewing and drinking before saliva sample collection. After that, the saliva was immediately taken to laboratory and centrifuged for 15 min, 4 ˚C at 8000 rpm. Saliva supernatant was collected and aliquoted into 1.5 ml of Eppendorf tubes and stored at -80°C until further processing of saliva.

Sample analysis
Total protein estimation analysis was performed according to Bradford's method 26 . Salivary levels of Cathepsin B in all the four study groups were determined by Enzyme Linked Immunosorbent Assay (ELISA) sandwich technique according to manufacturer's instructions available in the kit (Bioassay Technology KIT: 35054Hu). Each sample was analyzed in duplicates for better result outcomes.

Statistical analysis
Statistical analysis of all the obtained data was analyzed by SPSS version no 21. For all quantitative variables, mean and standard deviation were recorded and for all categorical variables, percentages and frequencies were recorded. One-way ANOVA test was used for comparison of mean Cathepsin B levels between OSCC groups and control group. Chi square test and Fisher's Exact test was used to determine the association between OSCC groups and control group for patient related factors. For univariate and multivariate analysis linear regression was used as the outcome variable Cathepsin B follows a continuous scale.

Results
Clinicopathological characteristics of all 80 research participants enrolled in our study are summarized in Table 1. Among the total 60 patients of OSCC, males patient comprises 43 (71.66%) and females patients comprises 17 (28.3%) cases. The male to female ratio observed was 2.5:1. With respect to age factors we observed OSCC patients are distributed evenly in all the three groups of OSCC with the minimum age found was 19 years and maximum age found was 65 years. The most common site of OSCC found in our study in all the three groups of OSCC was buccal mucosa 31 cases (51.7%) followed by tongue 10 cases (16.7%), retromolar area 5 cases (8.3%), palate and lower buccal sulcus 4 cases each (6.7%), upper sulcus and lower lip 3 cases each (5%). With regard to tobacco usage of both forms smoking and smokeless tobacco, we observed that usage of tobacco products for greater than 5 years duration causes increase in no of OSCC cases in all three study groups.
The salivary mean protein levels observed in three groups of OSCC and in control group were as follows: well differentiated OSCC 19.5 mg/ml (±14.7), moderately differentiated OSCC 27.3 mg/ml (± 16.2) and poorly differentiated OSCC 15.9 (±9.9) mg/ml and in control group 3.4 mg/ml (± 1.9) as shown in Table 2 and Figure 1. Statistically signi cant difference with p-value (<0.001) was observed in salivary mean protein levels between OSCC and control group. Highest protein concentration was observed in moderately differentiated OSCC followed by well differentiated OSCC and poorly differentiated OSCC. Lowest salivary mean protein levels were observed in control group.
Among the total 60 OSCC patients, Cathepsin B in saliva was detected in 45 (70%) cases where as in the control group salivary Cathepsin B was detected in only 3 (15%) cases. The salivary mean Cathepsin B levels observed in three groups of OSCC were as follows: well differentiated OSCC patients 3.7 (±1.8) ng/ml, moderately differentiated OSCC patients 1.0 (±1.3) ng/ml and poorly differentiated OSCC patients was 3.3 (±3.5) ng/ml. Statistically signi cant difference with p-value (<0.001) was observed in salivary mean Cathepsin B levels between OSCC and control group. Highest salivary Cathepsin B levels were observed in well differentiated OSCC followed by poorly differentiated OSCC and moderately differentiated OSCC as shown in Table 3, Figure 2.
The association of Cathepsin B levels was only found within the four study groups, OSCC and control group. No signi cant association of salivary Cathepsin B levels with age, gender, site, frequency and duration of tobacco intake was observed as shown in Table 5.
Univariate and multivariate analyses was performed to observe the association of salivary Cathepsin B levels with clinicopathological characteristics and study groups. The only variable, which observed signi cant association with Cathepsin B levels, was the OSCC/non-OSCC group. Therefore, multivariate analysis execution was not possible. Interpretations of regression (B) coe cients were as follows: for well-differentiated OSCC patients 3.37 ng/ml, for moderately differentiated OSCC patients 0.63 ng/ml and for poorly differentiated OSCC patients 2.99 ng/ml estimated increase in Cathepsin B levels was observed respectively as shown in Table 6.

Discussion
Oral squamous cell carcinoma worldwide is one of the most common malignant tumour, which has high rates of morbidity and mortality 27 . The main reason behind this is that most OSCC when diagnosed are in advanced stages 28 . The diagnosis and monitoring of OSCC by using salivary biomarkers is considered a promising approach for lowering oral cancer burden and increases the overall survival rate 29  In our study, we observed OSCC patients are evenly distributed in all age groups. Prevalence of OSCC is high in male patients as compared to female patients. Buccal mucosa as the most common site of OSCC occurrence is observed in our study. We also observed tobacco consumption of both forms smoking and smokeless tobacco for greater than 5 years duration causes increase in no of OSCC cases.
In our study, Cathepsin B was observed in 45 (70%) patients out of total 60 OSCC patients and in control group Cathepsin was detected in only 3(15%) of research participants. Mean salivary total protein levels are increased signi cantly with p-value (<0.001) in patients of OSCC as compared to control group. Kavya Prabhu M Sayeeda Mussavira et al., and Hivashankara AR has also reported in there studies that salivary protein levels are increased signi cantly in OSCC patients in comparison to control group 34,35 . Proteins are accountable for most functions of saliva such as physical protection, lubrication, buffering, tooth integrity maintenance and antibacterial activity 36 . Total salivary proteins are increased in patients of OSCC probably due to the ongoing in ammatory response, which triggers the sympathetic activity and increases the synthesis and production of some proteins to increase the shielding effect of saliva and provide protective function to combat against OSCC 36 . Furthermore, increase in salivary total proteins occurs to combat the violation and aberration in capillaries and mucosal lining as a result of in ammation in OSCC 37 . In our study, we observed highest total salivary protein levels in moderately differentiated OSCC group followed by well differentiated and poorly differentiated group of OSCC. This could be due to the fact that total salivary protein levels in patients of OSCC is dependent on several factors relatable to patients such as (diet, gender and age) and factors related to disease such as (infection, metastasis and lymph node invasion) 38 .
We determined the levels of salivary

Declarations Acknowledgments
We are very grateful and like to thank to all the research participants for their cooperation and giving precious time and consent for participating in our study.

Authors contribution
AS: contributed in conceptualizing the overall study, data collection, sample processing and drafting of manuscript. H.W. and S.H supervised the study and contributed in conceptualizing the overall study, data collection, sample processing, drafting and editing of manuscript S.S contributed in data collection and helped in assessments of patients of Oral Squamous Cell Carcinoma.

Funding
Self-funded research, no funding for the current study was received by any organization.
Availability of data and materials All data used, generated and analyzed in this current study is available on reasonable request from authors.

Ethics approval and consent to participate
The present study is completed after approval from Institutional Review Board of Dow University of Health Sciences IRB Number: IRB-1223/DUHS/Approval/2019/34. Informed consent in written from all the research participants was obtained.

Consent for publication
The current study does not contain any images or videos related to any research participant. The consent for publication is obtained from all research participants included in the current study. Box plot of mean salivary cathepsin B levels of OSCC and control group.

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