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Assessment of salivary endothelin-1 in patients with leukoplakia, submucous fibrosis, oral cancer and healthy individuals – a comparative study
J Stomatol Oral Maxillofac Surg 120 (2019) 326–331
Available online at
ScienceDirect www.sciencedirect.com
Original Article
Assessment of salivary endothelin-1 in patients with leukoplakia, submucous fibrosis, oral cancer and healthy individuals – a comparative study K. Ankita a,*, V. Shwetha a, S. Vanitha b, S. Reddy Sujatha a, R. Nagaraju a, K. Tupakula Pavan a a b
Faculty of Dental Sciences, Ramaiah University of Applied Sciences, India M.S Ramaiah Medical College And Hospital, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 January 2019 Accepted 28 February 2019 Available online 9 March 2019
Background: Oral cancer is one of the highly prevalent cancers worldwide being. According to data of GLOBOCAN 2018, the estimated incidence, mortality and 5-year survival rates due to lip, oral cavity and salivary gland cancer in world is (2.0%), (0.5%) and (0.3%) respectively. (Bray, Ferlay and Soerjomataram, 2018). Endothelin-1 (ET-1) is a 21-amino acid peptide; its receptors have been implicated in the growth and progression of both primary and metastatic neoplasms throughout the human body. Studies have shown that ET-1 is expressed in tissue, serum and other body fluids. Aim: To estimate the levels of salivary endothelin-1 in Oral potentially malignant disorders (oral leukoplakia and submucous fibrosis) and oral squamous cell carcinoma. Materials and methods: The study population included 60 subjects and were divided into 4 groups. All patients included in the study are clinically and histopathological diagnosed cases of oral leukoplakia, submucous fibrosis and oral cancer and assessed for salivary ET-1 levels using human ELISA kit. Significant differences between the groups were determined using one-way analysis of variance, LSD and Post HOC, unpaired t test, biserial and spearson’s correlation. Results: The mean levels of salivary Endothelin-1 level in study groups were: 82.78 5.9 pg/mL (OSCC), 65.02 1.8 pg/mL (SMF), 57.76 4.1 pg/mL (LEUKOPLAKIA), 29.72 14.1 pg/mL (CONTROLS). The mean Salivary ET-1 levels among these four groups was compared and the difference was statistically significant (P < 0.001). We also found a significant difference in the means of ET-1 levels among the clinical and histopathological staging of the study groups. Conclusion: Our results demonstrate potential utility of salivary analysis for ET-1 levels to monitor patients at risk for OSCC. Although provides the basis for a larger prospective study to determine the critical levels of salivary ET-1 necessary to diagnose and monitor OPMD’s and its potential to undergo malignant transformation.
C 2019 Elsevier Masson SAS. All rights reserved.
1. Introduction Oral cancer is one of the highly prevalent cancers worldwide being a leading cause of mortality. According to data of GLOBOCAN 2018, the estimated incidence, mortality and 5-year survival rates due to lip, oral cavity and salivary gland cancer in the world is (2.0%), (0.5%) and (0.3%) respectively [1]. Oral cancer is being reported as the third most common type, representing over 30% of all cancers in the nation. Age-balanced rates of oral cancer stated by different oral cancer registries in India change from 8 to 20 for * Corresponding author. E-mail address: [email protected] (K. Ankita). https://doi.org/10.1016/j.jormas.2019.02.024 C 2019 Elsevier Masson SAS. All rights reserved. 2468-7855/
every 100,000 population and are estimated to be high. Regardless of the advantage of early detection of oral cancers, with the available diagnostic aids, most oral cancers are diagnosed late, leading to poor treatment outcomes [2]. This fact emphasizes on the prompt significance of patient education and the need to improve the ability of a clinician to recognize early malignant and premalignant changes in the oral cavity [3]. Since the advent of the ‘omics’ technologies, a plethora of new tools to understand complex biological systems have become available. Promising research is progressing in the field of biomarkers which can aid us in the early diagnosis of OSCC. A cancer biomarker for a specific tumor type can provide vital information needed to successfully treat cancer [4,5]. The ultimate
K. Ankita et al. / J Stomatol Oral Maxillofac Surg 120 (2019) 326–331
goal in the discovery of biomarkers is to increase the survivability of cancer through improved diagnostics and treatment [6]. One such field is the salivary biomarkers. The salivary biomarkers are classified as metabolomics, proteomic and transcriptomic markers [7]. Studying the proteome, i.e. the protein complement of the genome (proteomics), has been proven to be effective in the analysis of protein expression and protein modification in cells/ tissues/organisms. The systematic and simultaneous analysis of proteins expressed by a certain cell type or tissue has become possible with the advent of proteomics. The protein abundances, post-translational modifications (PTMs) and the study of protein structure and interactions can be characterized by these technologies. One such proteomic biomarker is endothelin. Endothelin is a peptide which is composed of 21 amino acids with 2 disulphide bonds between amino acids 1 and 15 and 3 and 11. A resultant big endothelin (with 39 amino acids) is formed by cleavage of the prepro-endothelin precursor, by means of specific endopeptidases. Conversion of big endothelin to endothelin is then carried out by the action of an endothelin-converting enzyme (ECE) [8]. Endothelins comprise a group of three small peptides: ET-1, ET-2 (with similar structure of ET-1) and ET-3 [8,9]. ET-1 activates ETAR which largely contributes to tumour growth and progression, inducing proliferation of cell, survival, angiogenesis and metastatic spread, indicating that ETAR antagonism might improve cancer treatment. ET-1, by acting via ETBR, modulates different stages of neovascularization. ET-1 can also modulate tumour angiogenesis indirectly, through induction of vascular endothelial growth factor (VEGF). Activation of ETAR by ET-1 induces VEGF production by increasing the levels of hypoxia-inducible-factor 1a (HIF 1a) [10]. Endothelin plays a significant role in tumorigenesis and saliva being an important diagnostic tool we planned a study to assess the salivary endothelin levels in patients with oral squamous cell carcinoma and oral potentially malignant disorders. Also ours was the first study to evaluate the levels of salivary ET-1 in patients with submucous fibrosis.
2. Materials and methods Patients visiting the Outpatient department of Oral Medicine and Radiology between the months of February 2018 to September 2018 were included in the study. Clinically and histopathologically confirmed cases of oral leukoplakia (OL), sub mucous fibrosis (SMF), oral cancer (OSCC) comprised the study group. Age and sex matched healthy individuals were taken as controls. A sample size of 60 subjects was divided into 4 groups; 15 in each group (OL, SMF, OSCC and normal healthy controls). Patients with chronic systemic diseases, medically compromised, on corticosteroids or immunosuppressant therapy and patients with conditions affecting the quality and quantity of saliva were excluded from the study. The study was approved by the institutional ethical committee number (UECHT/2016-2018/PGDT/OR02). A written informed consent in English/ vernacular language was obtained from the subjects participating in the study upon fulfilling the inclusion criteria
327
2.1. Saliva collection and preparation Individuals were asked to refrain from eating, drinking, smoking, and oral hygiene procedures for at least 1 hour prior to saliva collection. They were asked to rinse their mouth with water; 5 mL of unstimulated whole saliva sample was collected from each subject using salivary spitting method into sterile uricol containers. The saliva samples were then centrifuged at 5000 rpm for 20 min. at +4degree C to remove cell debris and the supernatants were stored at 80 8C for further analysis. Salivary endothelin 1 levels were quantified using Human endothelin-1 Solid Phase Sandwich ELISA kit (Krishgen biosystems, India). 2.2. Statistical analysis Data was subjected to normality testing using Shapiro-Wilk test and was found to be normally distributed. Hence parametric test (ANOVA) was used. P-values less than 0.05 (P < 0.05) were considered as indicative of significance. Significant differences between the groups were determined using one-way analysis of variance, LSD Post HOC, unpaired t test, and Pearson’s correlation.
3. Results Salivary endothelin 1 levels in OL, SMF, and OSCC patients and in controls were measured using sandwich ELISA test. There were 43 males and 17 females in our study group. The demographic distribution of subjects within the study groups are mentioned in Table 1. All the cases included in the study had association with tobacco and alcohol consumption. Control group was free of any deleterious habit. Table 2 presents the clinical and histopathological data of cases. In our study the mean S.D: Salivary Endothelin-1 level in study groups was: 82.78 5.9 pg/ml in OSCC, 65.02 1.8 pg/mL in SMF, 57.76 4.1 pg/ml in Leukoplakia and 29.72 14.1 pg/mL in healthy controls. The mean Salivary Endothelin-1 levels among these four groups was compared using ANOVA and the difference was statistically significant (F = 113.69, P < 0.001). Therefore, by this analysis it can be inferred that there was a significantly higher expression of salivary ET-1 levels in OSCC followed by OSMF and OL groups respectively. (Table 3, Fig. 1). LSD Post hoc tests were carried out for all the pairs of groups (P < 0.001, adjusted using the Bonferroni correction) which showed a significant difference in salivary endothelin levels between OSCC and SMF (P < 0.001), OSCC and Leukoplakia (P < 0.001), OSCC and Controls (P < 0.001), SMF and Controls (P < 0.001) and Leukoplakia and Controls (P < 0.001). The difference in salivary endothelin levels between SMF and Leukoplakia (P = 0.016) was not statistically significant (Table 4). Correlation of salivary ET-1 levels with habit history was done among the study groups; using Point Biserial Correlation. Within Leukoplakia Group; ET-1 Level with Smokeless form of tobacco showed a positive correlation (rbi = 0.41, P = 0.12) (Table 5). Within SMF and OSCC group, analysis of ET-1 Levels with Smoked form of tobacco showed a negative correlation (rbi = 0.29, P = 0.29) and with male gender
Table 1 Demographic distribution of subjects within the study group. Group Leukoplakia SMF OSCC Controls
Total number (n)
Age
15 15 15 15
20–40 yrs 4 4 1 5
Gender 41–60 yrs 11 11 6 3
61–80 yrs 0 0 5 5
< 80 yrs 0 0 3 2
Male 15 13 8 7
Female 0 2 7 8
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328 Table 2 Clinical and histopathological data of cases. Group
Clinical stage/Type
OSCC
AJCC TNM Staging 1 2 3 4 Kerr et al. Grade 1 Grade 2 Grade 3 Grade 4 WHO Homogeneous Non-homogeneous Proliferative verrucous leukoplakia
SMF
Leukoplakia
Group OSCC
SMF
OL
Histopathological grading Well differentiated Moderately differentiated Poorly differentiated Very early Early Moderately advanced Advanced Epithelial dysplasia (mild, moderate and severe) Hyperkeratosis
Number (%) 9(60) 4(26) 0(0) 2(13) 2(13) 9(60) 4(26) 1(6) 11(73) 3(20) 1(6) Number (%) 6(40) 7(46) 2(13) 2(13) 6(40) 6(40) 1(6) 13(86.6) 2(13)
showed a positive correlation (rbi = 0.36, P = 0.51) (Table 5).There was no statistically significant difference with the association of salivary ET-1 levels and gender in OSCC group (Table 5.5). Association of ET-1 levels with clinical and histopathological staging was done among the study groups. Within Leukoplakia group, using Spearman’s r correlation, salivary ET-1 level with histological staging (P = 0.41, P = 0.13) and clinical staging (P = 0.08, P = 0.78) showed a negative correlation. Within SMF Group, Correlation of ET-1 Level with Histological staging showed a negative correlation (P = 0.18, P = 0.52) whereas salivary ET-1 Levels with Clinical staging showed a positive correlation (P = 0.06, P = 0.82). Also, there was no statistical significant association between age and salivary ET-1 levels among OL and SMF patients (Table 6). Among OSCC group Correlation of ET-1 Level with Histological staging showed a positive correlation (P = 0.08, P = 0.78) and showed a negative correlation (P = 0.1, P = 0.72) with clinical staging. Correlation of ET-1 Level with Age Groups showed a positive correlation (P = 0.16, P = 0.57). None of these correlations were statistically significant (Table 6). 4. Discussion Oral cancer is a global health issue. Oral squamous cell carcinoma (OSCC) is one of the most common epithelial malignancies with significant morbidity and mortality. In spite of diagnostic and therapeutic advances over the decades, the disease still remains a challenge for medical professionals with the
five year survival rate being 30%–50%. Recent observations indicate that the clinical and histological appearance of oral mucosa may not truly depict the damage occurring at the genetic level. An understanding of the molecular mechanisms involved in OSCC is helpful in providing a more complete picture of the ways in which tumor arise and advance and a rationale for novel strategies of cancer detection. The oral cavity is particularly conducive to such strategies, given the ease with which saliva and exfoliated cells can be collected [11].Comprehensive analysis and identification of the salivary proteome may be necessary to understand fully the oral pathophysiology, on one hand, and the possibility of using salivary proteins and peptides as biomarkers of systemic disease, on the other. Previous literature states that expression of endothelins and their receptors is associated with high grade, aggressive colon, colorectal and oesophageal cancers as well as invasion and metastasis and thereby, plays a principle role as a potential biomarker. In the present study, there was a statistically significant increase in the mean salivary ET-1 levels among the OSCC and OPMDs group when compared to normal healthy controls. Similar to our study, Pickering et al. (2007) [12] analysed the salivary ET-1 levels in patients with OSCC prior to the treatment, in comparison to healthy patients. They found a significant increase in the expression of salivary ET-1 levels in OSCC group. Similarly Cheng et al in 2012 [13] stated in their study that salivary ET-1 levels in OSCC patients (P > 0.05) before treatment were significantly higher than the levels found in normal controls (P = 0.004). Tahereh et al. in 2017 [14] reported that the mean of salivary endothelin-1 in patients with OSCC was 163.98 pg/mL and was significantly more than the level of endothelin-1 in control group, which was in accordance with our study. The study also found pairwise correlation of salivary levels of ET-1; the present study also assessed for the pair- wise correlation among the groups which showed a significant difference between OSCC and SMF (P < 0.001), OSCC and leukoplakia (P < 0.001), OSCC and controls (P < 0.001), SMF and controls (P < 0.001) and Leukoplakia and controls (P < 0.001). The difference between OSMF and leukoplakia (P = 0.016) was not statistically significant. Pritam et al. 2015 [15] compared the mean big ET-1 concentrations of the OSCC and control group, the study revealed a significantly higher big ET-1 concentration in OSCC cases when compared to controls (P < 0.0001, t = 11.37).Furthermore, intragroup comparison of the mean levels of serum big ET-1 also revealed statistically significant difference among all OSCC stages. In the present study, we assessed for the intragroup comparison among the histopathological staging of OSCC patients. We also correlated the ET-1 levels with the histopathological staging of SMF and leukoplakia. The mean levels were higher in the advanced stages both in OPMDs and OSCC, which made a meaningful conclusion that salivary ET-1 can be a potential diagnostic marker and it can help us to assess the disease progression. However, the levels were not statistically significant amongst different groups which could be attributed to the small sample size.
Table 3 The mean S.D: salivary endothelin-1 level in study groups. Group
Mean
Std. deviation
Std. error
95% confidence interval for mean Lower bound
Upper bound
OSCC SMF Leukoplakia Controls
82.78 65.02 57.76 29.72
5.9 1.8 4.1 14.1
1.54 0.46 1.07 3.65
79.46 64.02 55.46 21.89
86.10 66.03 60.06 37.54
ANOVA Between Groups Within Groups
Sum of Squares 21912.64 3589.01
Mean Square 7304.21 64.08
F 113.969
Sig. < 0.001
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Fig. 1. Mean salivary ET-1 levels in OSCC, SMF, OL and CONTROLS.
Table 4 Pair-wise comparison salivary Endothelin-1 level in study groups. Group
Comparison
Mean Difference
Std. Error
Sig.
OSCC
OSMF Leukoplakia Controls Leukoplakia Controls Controls
17.76 25.02 53.06* 7.26 35.30 28.04
2.92 2.92 2.92 2.92 2.92 2.92
< 0.001 < 0.001 < 0.001 0.016 < 0.001 < 0.001
OSMF Leukoplakia
Table 5 Correlation of salivary ET-1 levels with habit history was done among the study groups. Salivary ET-1 level Leukoplakia
Biserial (rbi)
P-value
Smokeless Type OSMF Smoked Type Gender OSCC Smoked Type Gender
0.41 0.4
0.12 0.1
0.29 0.31 0.18
0.29 0.26 0.51
0.26 0.59 0.36
0.35 0.21 0.18
Hoffman et al in his study in 2012 [10] did not find any significant difference between controls and OSCC patients. They stated that several different factors might be related to that discrepancy, including the sample size. Additionally, other factors might be pointed out to explain the differences between their results and that from literature data, such as the presence of periodontal disease, traumatic lesions, dietary factors and the tumor stage. However, different from the data described by Hoffman et al, we had found statistically significant results between the means of salivary ET-1 levels among the study group. 4.1. Endothelin – 1 and age and gender Few studies have assessed for the salivary or serum ET-1 levels among the different age groups and gender. It has been well described that ET-1 level is vastly implicated in vascular related
Table 6 Correlation of salivary ET-1 with age, clinical and histopathological staging of patient’s with leukoplakia, SMF, OSCC. Salivary ET-1 level Leukoplakia
Spearman’s P
P-value
Histological staging Clinical staging Age groups SMF Histological staging Clinical staging Age Groups OSCC Histological staging Clinical staging Age groups
0.41 0.08 0.38
0.13 0.78 0.16
0.18 0.06 0.48
0.52 0.82 0.06
0.08 0.1 0.16
0.78 0.72 0.57
alterations such as hypertension and ageing. Pritam et al. 2015 [15], found in their study that there was no significant difference among groups of different age (P = 0.6064) and gender (P = 0.1734) in relation to the serum big ET-1 value. In our study, salivary ET-1 levels were elevated in elderly age groups, (P = 0.38, P = 0.16), (r = 0.16, P = 0.57) among leukoplakia and OSCC group respectively and in male gender (rbi = 0.36, P = 0.18), (rbi = 0.36, P = 0.51) among OSCC and SMF groups respectively; but the results could not be generalised because of the small sample size included in the study. Concluding that impact of age and gender on salivary ET-1 levels is statistically insignificant. 4.2. Endothelin-1 and systemic diseases In various studies, it has been shown that ET-1 plays a role in the pathogenesis of acute renal failure after renal ischaemia. Also, ET-1 causes potent vasoconstriction and prolonged elevation of blood pressure in experimental models. It has also been noted that plasma endothelin levels are increased in patients with CHF (congestive heart failure). Increased plasma endothelin levels correlate closely with the degree of haemodynamic and functional impairment in patients. In primary pulmonary hypertension there is proliferation of pulmonary arterial smooth muscle and endothelial injury. It has been observed that, endothelin isopeptides can cause either pulmonary vasodilatation or vasoconstriction. Lam et al. in 2004 [16] showed an increase of salivary IR-ET levels
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in patients with gastritis, gastric and duodenal ulcers, which may contribute to the formation of peptic ulcer diseases in humans. Hoffman et al. in 2011 [10] separated their patients according to their sex and hypertension history; there was no significant association with the ET-1 levels. Cheng et al. in 2011 [13] analysed the systemic diseases of the participants in each group; cardiovascular diseases, bone and joint diseases, gastrointestinal diseases, hypothyroidism and diabetes. Due to the small sample size of each group, they did not analyse the statistical correlation between the salivary ET-1 level and the systemic diseases of participants. Looking at the association between hypertension and salivary ET-1 levels; our study did not include cases with a history of any systemic diseases, which can contribute to the limitation of the study. 4.3. Endothelin-1 and habit history Lam et al. 2004 [16] assessed the levels of ET-1 in patients with a history with smoking. In their study, the smokers show significantly higher salivary levels of IR-ET than those of the non-smokers. Very few studies have analysed for the habit history and its correlation with ET-1 levels in saliva of the participants. In our study, within the study group, there was a positive correlation (rbi = 0.41, P = 0.12) of salivary ET-1 level with Smokeless form of tobacco in the leukoplakia group. None of the cases in this group had a history of consuming smoked form of tobacco, therefore we couldn’t comment on its association with salivary ET-1. 4.4. Endothelin -1 and periodontitis Yamamoto et al. in 2003 [17] found that the expression of ET-1 in gingival cells was strongly induced by the P. gingivalis infection. However, Pradeep et al. in 2008 [18] conducted a study where it was found that the gingival crevicular fluid samples in all the groups tested negative for the endothelin-1 molecule. These results were not in accordance with those of a previous study by Yamamoto et al. in 2003 [19]. So to conclude as mentioned by Hoffman et al. [10, the levels of ET-1 in periodontitis might affect its mean levels in the result data. However, our study did not assess for the periodontal status of the patients for evaluation of ET-1 levels, which accounts for a drawback. 4.5. Endothelin- 1 and oral fibroblasts Hinsley et al. in 2012 [20] stated that the significance of the tumour stroma in carcinogenesis and progression of disease is increasingly recognised, and represents an attractive target for emerging therapeutic approaches. The predominant cell type in the stroma, fibroblasts, frequently undergo a number of changes in response to signals released by malignant epithelial cells including increased proliferation, migration and acquisition of a contractile, myofibroblastic phenotype. In OSCC, the presence of myofibroblasts in the tumour stroma was recently identified as the strongest negative prognostic indicator, and myofibroblasts were shown to promote cancer cell invasion. They showed that ET-1 stimulation of fibroblasts increases their migration and confers on them a more contractile phenotype. This is associated with an increased ability to stimulate oral cancer cell invasion but is not concomitant with the acquisition of markers of myofibroblastic transdifferentiation, suggesting ET-1may synergise with other factors to generate the reactive, myofibroblast rich stroma associated with aggressive oral malignancies. Lieu et al in 2016 [21] found that the proteome analysis in saliva of normal buccal mucosa, SMF and OSCC revealed a number of potential biomarker and they seem to have large prognostic value for patient survival, which suggest that SMF and OSCC biomarkers
are potential targets for therapeutical intervention. Therefore, ours was the first study to hypothesize that ET-1 levels can be expressed in SMF patients and the mean levels of salivary ET-1 were significantly high when compared to that of normal controls. The limitation of the present study is that, lesser number of samples was being considered for analysis. Although significant levels have been found in assessing salivary ET-1 levels in OPMD and OSCC, the findings of the study cannot be generalised owing to smaller sample size. Studies on larger populations to validate the results of the present pilot study are therefore necessary. 5. Conclusions and future directions In the present study, salivary ET-1 levels were highly expressed in patients with OSCC, SMF and OL as compared to normal healthy controls. Also, the salivary levels of ET-1 were highly expressed in initial clinical stages of OSCC, implicating the role of ET-1 as a growth factor and tumour initiation factor. It was found that the levels of ET-1 were highly expressed in all stages of SMF, which may indicate its role in oral fibroblast proliferation and disease progression. Therefore, our results demonstrate potential utility of salivary analysis for ET-1 levels to monitor patients at risk for OSCC. This study provides the basis for a larger prospective study to determine the critical levels of salivary ET-1 necessary to diagnose and monitor OPMD’s and its potential to undergo malignant transformation. More research in the direction of including other oral potentially malignant disorders will help us to estimate the levels of salivary ET-1, thereby reducing the incidence of OPMDs and consequently preventing oral carcinogenesis. Disclosure of interest The authors declare that they have no competing interest.
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Original Article
Assessment of salivary endothelin-1 in patients with leukoplakia, submucous fibrosis, oral cancer and healthy individuals – a comparative study K. Ankita a,*, V. Shwetha a, S. Vanitha b, S. Reddy Sujatha a, R. Nagaraju a, K. Tupakula Pavan a a b
Faculty of Dental Sciences, Ramaiah University of Applied Sciences, India M.S Ramaiah Medical College And Hospital, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 25 January 2019 Accepted 28 February 2019 Available online 9 March 2019
Background: Oral cancer is one of the highly prevalent cancers worldwide being. According to data of GLOBOCAN 2018, the estimated incidence, mortality and 5-year survival rates due to lip, oral cavity and salivary gland cancer in world is (2.0%), (0.5%) and (0.3%) respectively. (Bray, Ferlay and Soerjomataram, 2018). Endothelin-1 (ET-1) is a 21-amino acid peptide; its receptors have been implicated in the growth and progression of both primary and metastatic neoplasms throughout the human body. Studies have shown that ET-1 is expressed in tissue, serum and other body fluids. Aim: To estimate the levels of salivary endothelin-1 in Oral potentially malignant disorders (oral leukoplakia and submucous fibrosis) and oral squamous cell carcinoma. Materials and methods: The study population included 60 subjects and were divided into 4 groups. All patients included in the study are clinically and histopathological diagnosed cases of oral leukoplakia, submucous fibrosis and oral cancer and assessed for salivary ET-1 levels using human ELISA kit. Significant differences between the groups were determined using one-way analysis of variance, LSD and Post HOC, unpaired t test, biserial and spearson’s correlation. Results: The mean levels of salivary Endothelin-1 level in study groups were: 82.78 5.9 pg/mL (OSCC), 65.02 1.8 pg/mL (SMF), 57.76 4.1 pg/mL (LEUKOPLAKIA), 29.72 14.1 pg/mL (CONTROLS). The mean Salivary ET-1 levels among these four groups was compared and the difference was statistically significant (P < 0.001). We also found a significant difference in the means of ET-1 levels among the clinical and histopathological staging of the study groups. Conclusion: Our results demonstrate potential utility of salivary analysis for ET-1 levels to monitor patients at risk for OSCC. Although provides the basis for a larger prospective study to determine the critical levels of salivary ET-1 necessary to diagnose and monitor OPMD’s and its potential to undergo malignant transformation.
C 2019 Elsevier Masson SAS. All rights reserved.
1. Introduction Oral cancer is one of the highly prevalent cancers worldwide being a leading cause of mortality. According to data of GLOBOCAN 2018, the estimated incidence, mortality and 5-year survival rates due to lip, oral cavity and salivary gland cancer in the world is (2.0%), (0.5%) and (0.3%) respectively [1]. Oral cancer is being reported as the third most common type, representing over 30% of all cancers in the nation. Age-balanced rates of oral cancer stated by different oral cancer registries in India change from 8 to 20 for * Corresponding author. E-mail address: [email protected] (K. Ankita). https://doi.org/10.1016/j.jormas.2019.02.024 C 2019 Elsevier Masson SAS. All rights reserved. 2468-7855/
every 100,000 population and are estimated to be high. Regardless of the advantage of early detection of oral cancers, with the available diagnostic aids, most oral cancers are diagnosed late, leading to poor treatment outcomes [2]. This fact emphasizes on the prompt significance of patient education and the need to improve the ability of a clinician to recognize early malignant and premalignant changes in the oral cavity [3]. Since the advent of the ‘omics’ technologies, a plethora of new tools to understand complex biological systems have become available. Promising research is progressing in the field of biomarkers which can aid us in the early diagnosis of OSCC. A cancer biomarker for a specific tumor type can provide vital information needed to successfully treat cancer [4,5]. The ultimate
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goal in the discovery of biomarkers is to increase the survivability of cancer through improved diagnostics and treatment [6]. One such field is the salivary biomarkers. The salivary biomarkers are classified as metabolomics, proteomic and transcriptomic markers [7]. Studying the proteome, i.e. the protein complement of the genome (proteomics), has been proven to be effective in the analysis of protein expression and protein modification in cells/ tissues/organisms. The systematic and simultaneous analysis of proteins expressed by a certain cell type or tissue has become possible with the advent of proteomics. The protein abundances, post-translational modifications (PTMs) and the study of protein structure and interactions can be characterized by these technologies. One such proteomic biomarker is endothelin. Endothelin is a peptide which is composed of 21 amino acids with 2 disulphide bonds between amino acids 1 and 15 and 3 and 11. A resultant big endothelin (with 39 amino acids) is formed by cleavage of the prepro-endothelin precursor, by means of specific endopeptidases. Conversion of big endothelin to endothelin is then carried out by the action of an endothelin-converting enzyme (ECE) [8]. Endothelins comprise a group of three small peptides: ET-1, ET-2 (with similar structure of ET-1) and ET-3 [8,9]. ET-1 activates ETAR which largely contributes to tumour growth and progression, inducing proliferation of cell, survival, angiogenesis and metastatic spread, indicating that ETAR antagonism might improve cancer treatment. ET-1, by acting via ETBR, modulates different stages of neovascularization. ET-1 can also modulate tumour angiogenesis indirectly, through induction of vascular endothelial growth factor (VEGF). Activation of ETAR by ET-1 induces VEGF production by increasing the levels of hypoxia-inducible-factor 1a (HIF 1a) [10]. Endothelin plays a significant role in tumorigenesis and saliva being an important diagnostic tool we planned a study to assess the salivary endothelin levels in patients with oral squamous cell carcinoma and oral potentially malignant disorders. Also ours was the first study to evaluate the levels of salivary ET-1 in patients with submucous fibrosis.
2. Materials and methods Patients visiting the Outpatient department of Oral Medicine and Radiology between the months of February 2018 to September 2018 were included in the study. Clinically and histopathologically confirmed cases of oral leukoplakia (OL), sub mucous fibrosis (SMF), oral cancer (OSCC) comprised the study group. Age and sex matched healthy individuals were taken as controls. A sample size of 60 subjects was divided into 4 groups; 15 in each group (OL, SMF, OSCC and normal healthy controls). Patients with chronic systemic diseases, medically compromised, on corticosteroids or immunosuppressant therapy and patients with conditions affecting the quality and quantity of saliva were excluded from the study. The study was approved by the institutional ethical committee number (UECHT/2016-2018/PGDT/OR02). A written informed consent in English/ vernacular language was obtained from the subjects participating in the study upon fulfilling the inclusion criteria
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2.1. Saliva collection and preparation Individuals were asked to refrain from eating, drinking, smoking, and oral hygiene procedures for at least 1 hour prior to saliva collection. They were asked to rinse their mouth with water; 5 mL of unstimulated whole saliva sample was collected from each subject using salivary spitting method into sterile uricol containers. The saliva samples were then centrifuged at 5000 rpm for 20 min. at +4degree C to remove cell debris and the supernatants were stored at 80 8C for further analysis. Salivary endothelin 1 levels were quantified using Human endothelin-1 Solid Phase Sandwich ELISA kit (Krishgen biosystems, India). 2.2. Statistical analysis Data was subjected to normality testing using Shapiro-Wilk test and was found to be normally distributed. Hence parametric test (ANOVA) was used. P-values less than 0.05 (P < 0.05) were considered as indicative of significance. Significant differences between the groups were determined using one-way analysis of variance, LSD Post HOC, unpaired t test, and Pearson’s correlation.
3. Results Salivary endothelin 1 levels in OL, SMF, and OSCC patients and in controls were measured using sandwich ELISA test. There were 43 males and 17 females in our study group. The demographic distribution of subjects within the study groups are mentioned in Table 1. All the cases included in the study had association with tobacco and alcohol consumption. Control group was free of any deleterious habit. Table 2 presents the clinical and histopathological data of cases. In our study the mean S.D: Salivary Endothelin-1 level in study groups was: 82.78 5.9 pg/ml in OSCC, 65.02 1.8 pg/mL in SMF, 57.76 4.1 pg/ml in Leukoplakia and 29.72 14.1 pg/mL in healthy controls. The mean Salivary Endothelin-1 levels among these four groups was compared using ANOVA and the difference was statistically significant (F = 113.69, P < 0.001). Therefore, by this analysis it can be inferred that there was a significantly higher expression of salivary ET-1 levels in OSCC followed by OSMF and OL groups respectively. (Table 3, Fig. 1). LSD Post hoc tests were carried out for all the pairs of groups (P < 0.001, adjusted using the Bonferroni correction) which showed a significant difference in salivary endothelin levels between OSCC and SMF (P < 0.001), OSCC and Leukoplakia (P < 0.001), OSCC and Controls (P < 0.001), SMF and Controls (P < 0.001) and Leukoplakia and Controls (P < 0.001). The difference in salivary endothelin levels between SMF and Leukoplakia (P = 0.016) was not statistically significant (Table 4). Correlation of salivary ET-1 levels with habit history was done among the study groups; using Point Biserial Correlation. Within Leukoplakia Group; ET-1 Level with Smokeless form of tobacco showed a positive correlation (rbi = 0.41, P = 0.12) (Table 5). Within SMF and OSCC group, analysis of ET-1 Levels with Smoked form of tobacco showed a negative correlation (rbi = 0.29, P = 0.29) and with male gender
Table 1 Demographic distribution of subjects within the study group. Group Leukoplakia SMF OSCC Controls
Total number (n)
Age
15 15 15 15
20–40 yrs 4 4 1 5
Gender 41–60 yrs 11 11 6 3
61–80 yrs 0 0 5 5
< 80 yrs 0 0 3 2
Male 15 13 8 7
Female 0 2 7 8
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328 Table 2 Clinical and histopathological data of cases. Group
Clinical stage/Type
OSCC
AJCC TNM Staging 1 2 3 4 Kerr et al. Grade 1 Grade 2 Grade 3 Grade 4 WHO Homogeneous Non-homogeneous Proliferative verrucous leukoplakia
SMF
Leukoplakia
Group OSCC
SMF
OL
Histopathological grading Well differentiated Moderately differentiated Poorly differentiated Very early Early Moderately advanced Advanced Epithelial dysplasia (mild, moderate and severe) Hyperkeratosis
Number (%) 9(60) 4(26) 0(0) 2(13) 2(13) 9(60) 4(26) 1(6) 11(73) 3(20) 1(6) Number (%) 6(40) 7(46) 2(13) 2(13) 6(40) 6(40) 1(6) 13(86.6) 2(13)
showed a positive correlation (rbi = 0.36, P = 0.51) (Table 5).There was no statistically significant difference with the association of salivary ET-1 levels and gender in OSCC group (Table 5.5). Association of ET-1 levels with clinical and histopathological staging was done among the study groups. Within Leukoplakia group, using Spearman’s r correlation, salivary ET-1 level with histological staging (P = 0.41, P = 0.13) and clinical staging (P = 0.08, P = 0.78) showed a negative correlation. Within SMF Group, Correlation of ET-1 Level with Histological staging showed a negative correlation (P = 0.18, P = 0.52) whereas salivary ET-1 Levels with Clinical staging showed a positive correlation (P = 0.06, P = 0.82). Also, there was no statistical significant association between age and salivary ET-1 levels among OL and SMF patients (Table 6). Among OSCC group Correlation of ET-1 Level with Histological staging showed a positive correlation (P = 0.08, P = 0.78) and showed a negative correlation (P = 0.1, P = 0.72) with clinical staging. Correlation of ET-1 Level with Age Groups showed a positive correlation (P = 0.16, P = 0.57). None of these correlations were statistically significant (Table 6). 4. Discussion Oral cancer is a global health issue. Oral squamous cell carcinoma (OSCC) is one of the most common epithelial malignancies with significant morbidity and mortality. In spite of diagnostic and therapeutic advances over the decades, the disease still remains a challenge for medical professionals with the
five year survival rate being 30%–50%. Recent observations indicate that the clinical and histological appearance of oral mucosa may not truly depict the damage occurring at the genetic level. An understanding of the molecular mechanisms involved in OSCC is helpful in providing a more complete picture of the ways in which tumor arise and advance and a rationale for novel strategies of cancer detection. The oral cavity is particularly conducive to such strategies, given the ease with which saliva and exfoliated cells can be collected [11].Comprehensive analysis and identification of the salivary proteome may be necessary to understand fully the oral pathophysiology, on one hand, and the possibility of using salivary proteins and peptides as biomarkers of systemic disease, on the other. Previous literature states that expression of endothelins and their receptors is associated with high grade, aggressive colon, colorectal and oesophageal cancers as well as invasion and metastasis and thereby, plays a principle role as a potential biomarker. In the present study, there was a statistically significant increase in the mean salivary ET-1 levels among the OSCC and OPMDs group when compared to normal healthy controls. Similar to our study, Pickering et al. (2007) [12] analysed the salivary ET-1 levels in patients with OSCC prior to the treatment, in comparison to healthy patients. They found a significant increase in the expression of salivary ET-1 levels in OSCC group. Similarly Cheng et al in 2012 [13] stated in their study that salivary ET-1 levels in OSCC patients (P > 0.05) before treatment were significantly higher than the levels found in normal controls (P = 0.004). Tahereh et al. in 2017 [14] reported that the mean of salivary endothelin-1 in patients with OSCC was 163.98 pg/mL and was significantly more than the level of endothelin-1 in control group, which was in accordance with our study. The study also found pairwise correlation of salivary levels of ET-1; the present study also assessed for the pair- wise correlation among the groups which showed a significant difference between OSCC and SMF (P < 0.001), OSCC and leukoplakia (P < 0.001), OSCC and controls (P < 0.001), SMF and controls (P < 0.001) and Leukoplakia and controls (P < 0.001). The difference between OSMF and leukoplakia (P = 0.016) was not statistically significant. Pritam et al. 2015 [15] compared the mean big ET-1 concentrations of the OSCC and control group, the study revealed a significantly higher big ET-1 concentration in OSCC cases when compared to controls (P < 0.0001, t = 11.37).Furthermore, intragroup comparison of the mean levels of serum big ET-1 also revealed statistically significant difference among all OSCC stages. In the present study, we assessed for the intragroup comparison among the histopathological staging of OSCC patients. We also correlated the ET-1 levels with the histopathological staging of SMF and leukoplakia. The mean levels were higher in the advanced stages both in OPMDs and OSCC, which made a meaningful conclusion that salivary ET-1 can be a potential diagnostic marker and it can help us to assess the disease progression. However, the levels were not statistically significant amongst different groups which could be attributed to the small sample size.
Table 3 The mean S.D: salivary endothelin-1 level in study groups. Group
Mean
Std. deviation
Std. error
95% confidence interval for mean Lower bound
Upper bound
OSCC SMF Leukoplakia Controls
82.78 65.02 57.76 29.72
5.9 1.8 4.1 14.1
1.54 0.46 1.07 3.65
79.46 64.02 55.46 21.89
86.10 66.03 60.06 37.54
ANOVA Between Groups Within Groups
Sum of Squares 21912.64 3589.01
Mean Square 7304.21 64.08
F 113.969
Sig. < 0.001
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Fig. 1. Mean salivary ET-1 levels in OSCC, SMF, OL and CONTROLS.
Table 4 Pair-wise comparison salivary Endothelin-1 level in study groups. Group
Comparison
Mean Difference
Std. Error
Sig.
OSCC
OSMF Leukoplakia Controls Leukoplakia Controls Controls
17.76 25.02 53.06* 7.26 35.30 28.04
2.92 2.92 2.92 2.92 2.92 2.92
< 0.001 < 0.001 < 0.001 0.016 < 0.001 < 0.001
OSMF Leukoplakia
Table 5 Correlation of salivary ET-1 levels with habit history was done among the study groups. Salivary ET-1 level Leukoplakia
Biserial (rbi)
P-value
Smokeless Type OSMF Smoked Type Gender OSCC Smoked Type Gender
0.41 0.4
0.12 0.1
0.29 0.31 0.18
0.29 0.26 0.51
0.26 0.59 0.36
0.35 0.21 0.18
Hoffman et al in his study in 2012 [10] did not find any significant difference between controls and OSCC patients. They stated that several different factors might be related to that discrepancy, including the sample size. Additionally, other factors might be pointed out to explain the differences between their results and that from literature data, such as the presence of periodontal disease, traumatic lesions, dietary factors and the tumor stage. However, different from the data described by Hoffman et al, we had found statistically significant results between the means of salivary ET-1 levels among the study group. 4.1. Endothelin – 1 and age and gender Few studies have assessed for the salivary or serum ET-1 levels among the different age groups and gender. It has been well described that ET-1 level is vastly implicated in vascular related
Table 6 Correlation of salivary ET-1 with age, clinical and histopathological staging of patient’s with leukoplakia, SMF, OSCC. Salivary ET-1 level Leukoplakia
Spearman’s P
P-value
Histological staging Clinical staging Age groups SMF Histological staging Clinical staging Age Groups OSCC Histological staging Clinical staging Age groups
0.41 0.08 0.38
0.13 0.78 0.16
0.18 0.06 0.48
0.52 0.82 0.06
0.08 0.1 0.16
0.78 0.72 0.57
alterations such as hypertension and ageing. Pritam et al. 2015 [15], found in their study that there was no significant difference among groups of different age (P = 0.6064) and gender (P = 0.1734) in relation to the serum big ET-1 value. In our study, salivary ET-1 levels were elevated in elderly age groups, (P = 0.38, P = 0.16), (r = 0.16, P = 0.57) among leukoplakia and OSCC group respectively and in male gender (rbi = 0.36, P = 0.18), (rbi = 0.36, P = 0.51) among OSCC and SMF groups respectively; but the results could not be generalised because of the small sample size included in the study. Concluding that impact of age and gender on salivary ET-1 levels is statistically insignificant. 4.2. Endothelin-1 and systemic diseases In various studies, it has been shown that ET-1 plays a role in the pathogenesis of acute renal failure after renal ischaemia. Also, ET-1 causes potent vasoconstriction and prolonged elevation of blood pressure in experimental models. It has also been noted that plasma endothelin levels are increased in patients with CHF (congestive heart failure). Increased plasma endothelin levels correlate closely with the degree of haemodynamic and functional impairment in patients. In primary pulmonary hypertension there is proliferation of pulmonary arterial smooth muscle and endothelial injury. It has been observed that, endothelin isopeptides can cause either pulmonary vasodilatation or vasoconstriction. Lam et al. in 2004 [16] showed an increase of salivary IR-ET levels
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in patients with gastritis, gastric and duodenal ulcers, which may contribute to the formation of peptic ulcer diseases in humans. Hoffman et al. in 2011 [10] separated their patients according to their sex and hypertension history; there was no significant association with the ET-1 levels. Cheng et al. in 2011 [13] analysed the systemic diseases of the participants in each group; cardiovascular diseases, bone and joint diseases, gastrointestinal diseases, hypothyroidism and diabetes. Due to the small sample size of each group, they did not analyse the statistical correlation between the salivary ET-1 level and the systemic diseases of participants. Looking at the association between hypertension and salivary ET-1 levels; our study did not include cases with a history of any systemic diseases, which can contribute to the limitation of the study. 4.3. Endothelin-1 and habit history Lam et al. 2004 [16] assessed the levels of ET-1 in patients with a history with smoking. In their study, the smokers show significantly higher salivary levels of IR-ET than those of the non-smokers. Very few studies have analysed for the habit history and its correlation with ET-1 levels in saliva of the participants. In our study, within the study group, there was a positive correlation (rbi = 0.41, P = 0.12) of salivary ET-1 level with Smokeless form of tobacco in the leukoplakia group. None of the cases in this group had a history of consuming smoked form of tobacco, therefore we couldn’t comment on its association with salivary ET-1. 4.4. Endothelin -1 and periodontitis Yamamoto et al. in 2003 [17] found that the expression of ET-1 in gingival cells was strongly induced by the P. gingivalis infection. However, Pradeep et al. in 2008 [18] conducted a study where it was found that the gingival crevicular fluid samples in all the groups tested negative for the endothelin-1 molecule. These results were not in accordance with those of a previous study by Yamamoto et al. in 2003 [19]. So to conclude as mentioned by Hoffman et al. [10, the levels of ET-1 in periodontitis might affect its mean levels in the result data. However, our study did not assess for the periodontal status of the patients for evaluation of ET-1 levels, which accounts for a drawback. 4.5. Endothelin- 1 and oral fibroblasts Hinsley et al. in 2012 [20] stated that the significance of the tumour stroma in carcinogenesis and progression of disease is increasingly recognised, and represents an attractive target for emerging therapeutic approaches. The predominant cell type in the stroma, fibroblasts, frequently undergo a number of changes in response to signals released by malignant epithelial cells including increased proliferation, migration and acquisition of a contractile, myofibroblastic phenotype. In OSCC, the presence of myofibroblasts in the tumour stroma was recently identified as the strongest negative prognostic indicator, and myofibroblasts were shown to promote cancer cell invasion. They showed that ET-1 stimulation of fibroblasts increases their migration and confers on them a more contractile phenotype. This is associated with an increased ability to stimulate oral cancer cell invasion but is not concomitant with the acquisition of markers of myofibroblastic transdifferentiation, suggesting ET-1may synergise with other factors to generate the reactive, myofibroblast rich stroma associated with aggressive oral malignancies. Lieu et al in 2016 [21] found that the proteome analysis in saliva of normal buccal mucosa, SMF and OSCC revealed a number of potential biomarker and they seem to have large prognostic value for patient survival, which suggest that SMF and OSCC biomarkers
are potential targets for therapeutical intervention. Therefore, ours was the first study to hypothesize that ET-1 levels can be expressed in SMF patients and the mean levels of salivary ET-1 were significantly high when compared to that of normal controls. The limitation of the present study is that, lesser number of samples was being considered for analysis. Although significant levels have been found in assessing salivary ET-1 levels in OPMD and OSCC, the findings of the study cannot be generalised owing to smaller sample size. Studies on larger populations to validate the results of the present pilot study are therefore necessary. 5. Conclusions and future directions In the present study, salivary ET-1 levels were highly expressed in patients with OSCC, SMF and OL as compared to normal healthy controls. Also, the salivary levels of ET-1 were highly expressed in initial clinical stages of OSCC, implicating the role of ET-1 as a growth factor and tumour initiation factor. It was found that the levels of ET-1 were highly expressed in all stages of SMF, which may indicate its role in oral fibroblast proliferation and disease progression. Therefore, our results demonstrate potential utility of salivary analysis for ET-1 levels to monitor patients at risk for OSCC. This study provides the basis for a larger prospective study to determine the critical levels of salivary ET-1 necessary to diagnose and monitor OPMD’s and its potential to undergo malignant transformation. More research in the direction of including other oral potentially malignant disorders will help us to estimate the levels of salivary ET-1, thereby reducing the incidence of OPMDs and consequently preventing oral carcinogenesis. Disclosure of interest The authors declare that they have no competing interest.
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