|Ahead of print publication
Salivary L-fucose as a biomarker for oral potentially malignant disorders and oral cancer
Mudita Sharma1, Eklavya Sharma2, Vishnudas Prabhu3, Vinitha Ramanath Pai4, Jyothi MP D'souza5, Sindhu Harish6, Maji Jose3
1 Department of Oral Pathology and Microbiology, Geetanjali Dental and Research Institute, Geetanjali University, Udaipur, Rajasthan, India
2 Department of Oral and Maxillofacial Surgery, Geetanjali Dental and Research Institute, Geetanjali University, Udaipur, Rajasthan, India
3 Department of Oral Pathology and Microbiology, Yenepoya Dental College, Yenepoya University, Mangalore, Karnataka, India
4 Department of Biochemistry, Yenepoya Medical College, Yenepoya University, Mangalore, Karnataka, India
5 Department of Biochemistry, Andaman and Nicobar Islands Institute of Medical Sciences, Port Blair, Andaman and Nicobar Islands, India
6 Department of Biochemistry, Kasturba Medical College, Mangalore, Karnataka, India
B-61, Tonk Hospital, Sahkar Marg, Near ICICI Bank, Jaipur - 302 015, Rajasthan
Source of Support: None, Conflict of Interest: None
Background: The objective of this study was to evaluate the serum and salivary L-fucose in oral potentially malignant disorders (OPMDs) and oral cancer (OC) in order to investigate the possibility of using this as biomarker for early diagnosis.
Materials and Methods: The study included 85 participants, who were grouped as control (30), OPMDs patients (25), and OC patients (30). Serum and unstimulated whole saliva were collected from participants of all groups and fucose estimation was done using spectrophotometry. The results were tabulated and analyzed statistically.
Results: The mean serum L-fucose levels in normal, OPMDs, and OC group were 3.49, 19.18, and 35.75 mg/dl, respectively, while the levels of salivary L-fucose were 3.18, 7.02, and 11.66 mg/dl, respectively. A highly significant rise (P < 0.001) in serum and salivary L-fucose was observed in the study participants compared to control.
Conclusions: The present study showed a significant and gradual increase in serum and salivary L-fucose from control to OPMDs to OC. From this study, we suggest that L-fucose can be used as a reliable biomarker and saliva can be used as a diagnostic fluid for screening and early detection of OC.
Keywords: L-fucose, oral cancer, oral potentially malignant disorders, saliva, serum
|How to cite this URL:|
Sharma M, Sharma E, Prabhu V, Pai VR, D'souza JM, Harish S, Jose M. Salivary L-fucose as a biomarker for oral potentially malignant disorders and oral cancer. J Can Res Ther [Epub ahead of print] [cited 2019 Jul 18]. Available from: http://www.cancerjournal.net/preprintarticle.asp?id=257460
| > Introduction|| |
Oral and pharyngeal cancer, grouped together, is the sixth most common cancer in the world. The annual estimated incidence for oral cancer (OC) is around 275,000 worldwide. In the high-risk South Asian countries such as India, Pakistan, Sri Lanka, and Bangladesh, OC is the most common cancer in men and third most common among females. Oral squamous cell carcinomas are often preceded by clinically and histologically evident oral potentially malignant disorders (OPMD). Many times these lesions are diagnosed at an advanced stage necessitating mutilating surgeries as part of the treatment protocol. Therefore, early detection of these lesions is crucial which can reduce the mortality and morbidity rates.
A variety of genetic and molecular signatures of precancerous lesions and oral squamous cell carcinoma (OSCC) have been identified as tumor biomarkers which are biochemical substances elaborated by tumor cells either due to the cause or effect of the malignant process. Tumor markers may be present as intracellular substances in tissues or may be released into the circulation and appear in serum. Glycoproteins and glycolipids, which are important constituents of cell membrane, are reported to be altered in malignant cells and are considered as a hallmark of cancer. Through either increased turnover or secretion and/or shedding from malignant cells, these altered glycoconjugates are released into the circulation and therefore become detectable.
Fucose is a monosaccharide that is a common component of many N- and O-linked glycans and glycolipids produced by mammalian cells. Fucosylation of glycoproteins (the addition of L-fucose at the terminal end of the oligosaccharide chain) is one of the most important features that mediate several specific biologic functions. It has been documented that tumor cells modulate their surface by increasing fucosylation levels to escape recognition, which contributes to several abnormal characteristics of tumor cells, such as decreased adhesion and uncontrolled tumor growth. Therefore, monitoring serum fucose levels could be a promising approach for the early detection, diagnosis, and prognosis of various cancer types.,,,,,,,,
Estimation of the levels of serum L-fucose in OC patients has shown that it to be elevated significantly which indicates the possibility of this being a reliable biomarker of the disease process.,,,,,, Saliva being a biofluid which is in direct contact with the oral tumor tissue, there is a greater chance that this biomarker is present in saliva than serum. Surprisingly, almost no studies examined L-fucose in the saliva of OPMDs and oral squamous cell carcinoma patients. Therefore, this study was designed to estimate and compare the serum and salivary L-fucose levels in patients with oral precancerous states and carcinomas in order to find the possibility of including saliva as a biofluid, and L-fucose as a marker for early detection of these disease conditions. This is highly relevant in the present era where salivary diagnostics is gaining a lot of significance because of many advantages of saliva over the serum. This monosaccharide in saliva, if established as a reliable biomarker, could be used for widespread screening so that asymptomatic individuals can be detected at an early stage of this disease.
| > Materials and Methods|| |
Individuals visiting the Department of Oral Medicine and Radiology and Department of Oral Surgery of our institution and nearby cancer centers were chosen randomly for the study. Selection of the participants for the study was done, based on the case history and clinical examination. After recording the detailed case history, clinical examination of the participant was carried out. The details of their habits, especially of use of tobacco and smoking, were specifically sought. Once the lesion is clinically diagnosed, biopsy was done to confirm the diagnosis histopathologically. The purpose of the study and the procedure to be carried out were explained to the patients who were selected for the study after histopathological confirmation of diagnosis. The patients who were willing to give written consent only were included in the study. Similarly, age- and sex-matched healthy volunteers without tobacco-related oral habits or oral lesions were included in the control group. Individuals with uncontrolled hypertension, uncontrolled diabetes mellitus, and individuals on medication for any systemic condition were excluded from the study. Thus, the selected individuals were categorized in three groups – 30 participants in the normal control group (NC group), 25 participants in the OPMDs group (OPMD group), and 30 participants in the OSCC group. This study is a case–control study in which comparison of various parameters is done between the control group and two study groups.
The study protocol was approved by the ethical committee of our institution. All information about the patients and their identity was anonymous. Participants were given both verbal and written information about the nature of the study and written consent was obtained. They were allowed to leave the study at any time during the procedures.
Five milliliters of peripheral blood sample was collected from each participant with disposable syringes under aseptic conditions through venipuncture. Serum was separated by centrifugation at 3000 rpm for 15 min. About 1.5–2 ml of unstimulated whole saliva was also collected under resting condition during the hours 10 am–12 pm, 2 h after the participant's usual breakfast time, according to the method of Navazesh. This was to ensure that the variability in salivary flow and composition be minimized due to diurnal variation. The participant was asked to thoroughly rinse their mouth with distilled water to remove any food debris. After 10 min, the participant was directed to spit into a wide-mouth sterile plastic container till the required amount of saliva was collected. The participant was instructed not to spit forcibly to avoid blood contamination. The plastic container was placed in an ice carrier box and transferred to the laboratory. Saliva sample was centrifuged. Serum and saliva samples thus obtained were stored at −80°C until L-fucose analysis was done.
Biochemical analysis of the sample
L-fucose was estimated according to the method of Winzler. L-fucose is a methylpentose present in glycoproteins. It can be assayed by dissolving ethanol precipitated proteins of serum in alkali, heating with sulfuric acid, and determining the color after the addition of cysteine. The color produced by hexoses under these conditions is corrected by determining absorbance at two wavelengths spectrophotometrically. Five milliliters of 95% ethyl alcohol was added to 0.1 ml of sample (serum/saliva) and mixed. It was centrifuged at 1500 rpm for 15 min, the supernatant was decanted, and the precipitate was suspended in 5 ml of 95% ethyl alcohol. It was recentrifuged for 15 min and the supernatant was completely decanted. The precipitate was dissolved in 1 ml of 0.1N NaOH. About 4.5 ml of ice-cold sulfuric acid-water mixture was added, mixed, and transferred to an already boiling water bath and heated for exactly 3 min. The tubes were then cooled and 0.1 ml of cysteine reagent was added and mixed. After 60 min, the solutions were transferred to appropriate cuvettes and the absorbance was read at 400 nm and 430 nm using 60S ultraviolet-visible spectrophotometer from Thermo Scientific®. Standard L-fucose of varying concentrations was simultaneously run with the test samples to obtain the standard curve. All the chemicals used were of analytical grade and were procured from Hi-Media and Merck companies.
The biochemical values of this study were participated to statistical analysis using SPSS software (version 17.0; SPSS, Inc., Chicago, IL, USA) to specify the statistical correlation between the groups and various parameters. ANOVA test was applied to compare the values in all the groups and Wilcoxon test was applied to compare and correlate serum and salivary L-fucose in all the three groups.
| > Results|| |
Biochemical analysis of serum L-fucose was carried using spectrophotometric method and values were recorded for all the three groups including normal, OPMD, and OSCC groups. The serum L-fucose in normal group ranged from 0.11 to 9.05 mg/dl with mean value of 3.35 mg/dl. The serum L-fucose in precancer group ranged from 11.17 to 29.2 mg/dl with mean value of 19.18 mg/dl. The serum L-fucose in OSCC group ranged from 3.38 to 117.36 mg/dl with mean value of 33.47 mg/dl. The level of serum L-fucose in all the study groups was found to be highly significant when compared to NC group (P < 0.001) [Figure 1]. Comparison of the result between the groups showed a difference in levels, which was found to be highly significant (P < 0.0001) between the normal and OPMD group, normal and OSCC group, and the OPMD and OSCC group [Table 1].
The salivary L-fucose in normal group ranged from 0.38 to 17.40 mg/dl with mean value of 2.94 mg/dl. The salivary L-fucose in precancer group ranged from 0.11 to 18.46 mg/dl with mean value of 7.02 mg/dl. The salivary L-fucose in OSCC group ranged from 0.11 to 30.60 mg/dl with mean value of 11.66 mg/dl. The difference in salivary L-fucose in all the study groups was found to be highly significant when compared to normal (P < 0.001) [Figure 2]. Elevation in levels was found to be highly significant from normal to OPMD group (P < 0.001) and from OPMD to OSCC group [Table 2].
While comparing the serum and salivary L-fucose levels in NC group, only negligible difference was noted which was statistically nonsignificant (P = 0.382). In the OPMD and OC group, the L-fucose levels were less in saliva as compared to serum, but the difference was noteworthy and statistically highly significant (P < 0.005) as shown in [Table 3] and [Figure 3].
| > Discussion|| |
The remodeling of cell surface glycoproteins and glycolipids through modification of oligosaccharide structures is associated with the biological behavior of tumor cells. Fucose is a constituent of oligosaccharides, which are one of the most important factors in the posttranslational modification of proteins and lipids and is notably associated with cancer and inflammation Increased levels of different components of glycoproteins have been associated with different types of malignancies. However, there is a lacuna in the studies using saliva as a diagnostic fluid for OC. Biochemical analysis of saliva is not being evaluated in detail in routine clinical laboratories. This prompted us to evaluate the levels of serum and salivary L-fucose and correlate them.
Serum L-fucose has been reported to be elevated in various malignancies, including breast cancer,,,,, colorectal cancer, cervical cancer, lung cancer, and brain tumors. Significance of serum L-fucose in OC and potentially malignant oral disorders is relatively less investigated.,,,,,, However, few reports available in literature have shown a considerable elevation indicating the value of this biomarker as an indicator of the disease process.
In this study mean serum L-fucose in normal, OPMD, and OSCC groups was 3.32, 19.18, and 33.47 mg/dl, respectively. The rise of serum L-fucose from normal to OPMD and OSCC was found to be highly significant (P < 0.0005) on statistical analysis. All the previous studies reported on OPMD and OSCC patients have shown a significant rise of serum L-fucose compared to the control group which is consistent with our findings.,,,,,, The variation in values noted in different studies may be due to difference in the methodology followed for the estimation. This observation of elevated serum L-fucose indicates that fucose from the altered tissue is released into the serum, probably due to increasing turnover and release of cells into the circulation.
A gradual and consistent increase in salivary L-fucose levels was noted in the OPMDs and OC patients as compared to the normal healthy individuals with a mean value of 2.94, 7.02, and 11.66 mg/dl, respectively. The elevation of levels between groups was highly significant (P < 0.0005). To the best of our knowledge, there is no published data on salivary L-fucose in OC or potentially malignant disorders. The elevation of L-fucose in saliva reinforces the fact that neoplastically altered cells overexpress fucosylated glycans on their surface and are continuously shed into the saliva which is constantly bathing the tumor tissue.
In this study, the comparison between serum and salivary L-fucose in different groups was done. In normal group, the mean serum and salivary L-fucose was 3.32 and 2.94 mg/dl, respectively. In OPMD group, the mean serum and salivary L-fucose was 19.18 and 7.02 mg/dl, respectively, and in OSCC group, it was 33.47 and 11.66 mg/dl, respectively. It is interesting to note that the salivary and serum fucose levels were fairly equal (P = 0.382). However, contrary to what was expected, both in OPMD and OSCC groups, salivary fucose level was much lesser than in serum with highly significant difference (P < 0.0005). The probable reason could be degradation of salivary fucose by bacterial action. Supporting evidence for this fact is the results obtained in investigations carried out by Leach and Critchley which has shown that sugars associated with the salivary glycoproteins (3 hexoses, 2 hexosamines, and fucose) are spontaneously lost from saliva by bacterial action, and this loss may also be prevented by either heat or antibiotics. Large number of bacteria accumulated in the oral cavity in the form of dental plaque are capable of metabolizing carbohydrate components of the glycoproteins and this is also confirmed by observation that dental plaque is practically devoid of both sialic acid and fucose (<0.002%), the two essential sugar constituents of salivary glycoproteins. The great difference observed in OPMD and OC patients compared to control group could be due to their poor oral hygiene that would have led to heavy bacterial load leading to faster degradation rate of fucose.
Although the actual values of L-fucose are less in saliva than serum, they are present in detectable amount in saliva and significant rise in precancer and OC. Thus, from the results of our study, we suggest that L-fucose can be used as a biomarker for screening purposes for OC. There are compelling reasons to use saliva as a diagnostic fluid to monitor health and disease. Saliva-based diagnostics are less invasive, less expensive, and present less risk to both the patient and the health-care provider than current methodologies.
Although utmost care was taken in all steps of the study process, including collection, transportation, and storage, to reduce the degradation of highly labile salivary glycoproteins, the results show that total bacterial enzymatic action could not be controlled. Therefore, it would be ideal to use saliva which is directly collected from the salivary ducts which are not contaminated by plaque bacteria for fucose estimation. Further research is recommended to reconfirm our findings with more samples and advanced techniques available for the estimation of L-fucose. This study throws light on the promising future of saliva to be used as a diagnostic medium and fucose as marker for OPMD and OC. Salivary cancer diagnostics is in its infancy and there is a necessity for additional research and more efforts to overcome the multitude of barriers.
We would like to acknowledge the financial support received by the Indian Council of Medical Research for this research project, cooperation of the participants included, and the technical staff involved in the study.
Financial support and sponsorship
The study was financially supported by the Indian Council for Medical Research.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3]
[Table 1], [Table 2], [Table 3]