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ORIGINAL ARTICLE  
Year : 2017  |  Volume : 60  |  Issue : 4  |  Page : 464-468
Role of salivary biomarkers in early detection of oral squamous cell carcinoma


Consultant Pathologist, SRL Diagnostics, Lucknow, Uttar Pradesh, India

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Date of Web Publication12-Jan-2018
 

   Abstract 

Introduction: Oral cancer is prevalent worldwide and is a common cause of morbidity and mortality. Despite advances in treatment, the survival of patients with oral cancer has not significantly improved over the past several decades owing to late detection and treatment failures. The present study was undertaken with an objective to explore the role of salivary CYFRA 21-1, CA 19-9, lactate dehydrogenase (LDH), total proteins, and amylase as biochemical markers of oral squamous cell carcinoma (OSCC) and premalignant lesions (PML). Materials and Methods: This was a cross-sectional study for diagnostic test evaluation conducted in KGMC Lucknow, between 2010 and 2011. The study population comprised newly diagnosed cases of OSCC (Group I) and PML of oral cavity (Group II) who had not yet received any definitive therapy along with age- and gender-matched healthy controls (Group III). Unstimulated whole saliva was collected from the cases and controls. CYFRA 21-1 and CA19-9 were estimated by ELISA while LDH, total proteins, and amylase were evaluated as per standard kit method. Results: Both OSCC and PML group showed increased salivary CYFRA 21-1, LDH, and total protein concentrations as compared to controls, but the increase in PML was significantly lower as compared to OSCC. A considerable decrease in concentration of amylase was seen in OSCC and PML as compared to control group. Conclusion: The outcome of this study suggests that concurrent analysis of salivary CYFRA 21-1, LDH, total protein, and amylase can be utilized for early detection of oral cancer.

Keywords: Amylase, lactate dehydrogenase, CA 19-9, CYFRA21-1, oral cancer, oral squamous cell carcinoma, premalignant lesions, saliva, total proteins

How to cite this article:
Awasthi N. Role of salivary biomarkers in early detection of oral squamous cell carcinoma. Indian J Pathol Microbiol 2017;60:464-8

How to cite this URL:
Awasthi N. Role of salivary biomarkers in early detection of oral squamous cell carcinoma. Indian J Pathol Microbiol [serial online] 2017 [cited 2018 Jan 19];60:464-8. Available from: http://www.ijpmonline.org/text.asp?2017/60/4/464/222953





   Introduction Top


Oral cancer is the sixth most prevalent cancer worldwide.[1],[2] In the Indian subcontinent, it ranks among the three most common types of cancer.[3] Squamous cell carcinoma (SCC) accounts for nearly 90% cases of malignancy of oral cavity.[1] The history of oral cancer shows that it is preceded in more than 70% of the patients by a recognized premalignant lesion (PML)[4] and intervention at this stage may result in regression of the lesion.

Despite advances in treatment, the prognosis of patients with oral cancer remains dismal with a 5-year survival rate of 62%.[1],[4] The short survival time is attributed largely to late detection and treatment failures. An established tumor marker of oral SCC (OSCC) may help in treatment monitoring apart from early diagnosis. Review of literature suggested that there has been a recent upsurge in studies focusing on the use of saliva as a diagnostic medium[5],[6],[7],[8],[9],[10],[11] and the role of salivary biomarkers in patients with OSCC. Saliva is a readily available fluid that can be collected without much training and used for screening and treatment monitoring purposes if suitable biomarkers are available. CYFRA 21-1, which is the soluble fragment of cytokeratin 19[12] has emerged as a novel marker of SCC. CA19-9, total protein, amylase, and lactate dehydrogenase (LDH) have also been shown to be significantly altered in patients with OSCC as compared to premalignant states and healthy controls.[13],[14],[15]

The present study was undertaken with an objective to compare salivary levels of CYFRA 21-1, CA 19-9, total proteins, amylase, and LDH in patients with OSCC, PML, and age- and gender-matched healthy controls with statistical evaluation of the findings. It also intended to study the various etiological factors associated with oral lesions.


   Materials and Methods Top


This was a cross-sectional study for diagnostic test evaluation conducted in KGMC Lucknow, between 2010 and 2011. It included 64 individuals divided into three groups. Group I comprised 30 newly diagnosed cases of OSCC, while group II was constituted by 9 newly diagnosed cases of premalignant oral conditions, patients in both groups had not yet received any definitive therapy at the time of sample collection. Group III included 25 age- and gender-matched healthy controls. Known cases of chronic inflammatory diseases, autoimmune disorders, acquired immune-deficiency syndrome, and patients with the previous history of any head and neck cancer or malignancy at any other site or history of radiation exposure were excluded from the study.

A detailed history was taken with special emphasis on addiction to tobacco, smoking and alcohol, socioeconomic status (according to Kuppuswamy scale), occupation, history of any association with oral premalignant conditions, use of dentures and oral hygiene. A thorough local examination of oral cavity and lymph nodes was done to ascertain the clinical stage of the disease according to the tumor, node, and metastasis classification. Proper clinical evaluation regarding oral hygiene, dental status, and presence of any PML was done.

Saliva samples were collected from histopathologically diagnosed cases of OSCC and precancerous oral lesions presenting at the outpatient department or admitted in the onco-surgery wards at CSMMU, Lucknow before administration of definitive therapy. Unstimulated whole saliva was collected from the study and control group between 10.00 am and 12.00 pm 2 h after the subjects usual breakfast time to ensure that the variability in salivary flow and compositions due to diurnal variations were minimized. Following collection, the saliva sample was centrifuged at 3000 rpm for 15 min to remove squamous cells and debris. The resulting supernatant was used for further biochemical analysis. Saliva samples were stored in deep freezer at −80°C until analyzed. CYFRA 21-1 and CA19-9 were estimated by ELISA method while LDH, total protein, and amylase levels were evaluated according to the standard kit method.

Data so collected were analyzed using SPSS statistical software for Windows version 17, developed by IBM SPSS Software. Following statistical tests were used to test the significance of the differences between the study and control group. Chi-square test was used to analyze the differences between different groups for categorical data. Analysis of variance (ANOVA) and independent samples t-Test was used to compare parametric data of different groups. Independent samples t-Test in case of two groups and ANOVA for more than two groups. The Dunnett's test of pairwise comparison was used to find the significance between two groups which were significant in ANOVA tests. Receiver operator characteristic (ROC) curve analysis was done to evaluate the sensitivity, specificity, and predictive values for diagnostic test. P < 0.05 was considered as statistically significant.


   Results Top


Out of 30 oral cancer patients, 28 (93.3%) were males while 2 (6.7%) were females. Male to female ratio was 14:1. Patients ranged in age from 25 to 70 years with a mean age of 49.6 years. Premalignant oral conditions also showed a male predominance (88.9%). Age of the patients varied from 25 to 40 years with a mean age of 34.2 years. The control group was composed of 22 males (88%) and 3 females (12%), ranging in age from 25 to 68 years with a mean age of 48.1 years. [Table 1] shows age and gender distribution of the study population.
Table 1: Age and gender distribution of study population

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Majority of patients with oral lesions belonged to low social class. Tobacco, both in chewable and smoking form was a strong predisposing factor. It had a synergistic action with concurrent alcohol and proved to be pivotal in the development of OSCC. Association of oral lesions with tobacco chewing (Chi-square, P = 0.0005), smoking (Chi-square, P = 0.0001), and alcohol (Chi-square, P = 0.0005) was statistically significant. Poor oral hygiene was also seen as a contributing factor for the development of oral cavity lesions. All patients with malignant and premalignant conditions in this study presented with poor oral hygiene (Chi-square, P = 0.0005). It showed statistically significant association with tobacco use (Chi-square, P = 0.0005) and low social class (Chi-square, P = 0.002).

Buccal mucosa (46.7%) was the most common site of occurrence in the OSCC group whereas in case of PML group tongue (33.3%) was the most preferred location. Lymphadenopathy was present in 33.4% cases of OSCC. Most of the patients belonged to stage I (46.6%, n = 14) followed by stage IVA (26.6%, n = 8), stage II (13.4% (n = 4), stage III (10.0%, n = 3), and stage 0 (3.3%, n = 1).

Histopathologically, most of the cases of OSCC (66.6%) were moderately differentiated followed by well differentiated (30%) and poorly differentiated (3.4%). Out of total 9 cases of PML, 55.5% (n = 5) were leukoplakia followed by oral submucous fibrosis (22.3%, n = 2), lichen planus (11.1%, n = 1), and squamous papilloma (11.1%, n = 1). Squamous papilloma due to its association with human papillomavirus was treated as a potentially malignant condition.[16],[17],[18]

[Table 2] provides the details of levels of salivary biomarkers in various groups of the study population. Mean along with standard deviation (SD) (mean ± 2 SD) and median values are shown for each parameter separately. Mean values for CYFRA 21-1, LDH, and total proteins were considerably higher while for amylase were lower in patients with OSCC and PML as compared to the control group. CA19-9 showed no significant differences between the three groups.
Table 2: Mean and median values of test parameters in study groups

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ROC curve analysis for CYFRA21-1 [Figure 1] showed a sensitivity of 90% and specificity of 97% at a cutoff value of 8.7 ng/ml for malignant lesions. Positive predictive value and negative predictive value observed were 96.4% and 91.7%, respectively. The area under the curve of 0.994 signified that it had a higher chance of correctly identifying oral cancer patients (P = 0.0001).
Figure 1: Receiver operator characteristic curve for CYFRA 21-1

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Correlation between different variables was done using bivariate analysis. CYFRA21-1, LDH, total proteins, and amylase showed statistically significant association with each other. CYFRA21-1, LDH, and total proteins were found to be positively correlated with each other while amylase showed a negative correlation with them. [Table 3] shows association between test variables and clinical parameters.
Table 3: Association between test variables and clinical parameters (χ2)

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   Discussion Top


Oral cancer is prevalent worldwide and is a common cause of morbidity and mortality. Cancers of the oral cavity are surface malignancies whose signs and symptoms can be recognized early. The typical demographic profile of an individual with OSCC, as described in literature is that of a man in the fifth to eighth decade of life, who is a smoker and a drinker.[2],[4],[19],[20] The present study also noted predominance of oral cavity lesions among middle aged to elderly males addicted to tobacco and alcohol. Mean age observed in oral cancer group was 49.6 years as compared to 34.2 years in the premalignant group. This difference of 15 years could be crucial in the entire process of carcinogenesis and potentially malignant lesions detected at earlier stage can be managed more effectively.

This study in accordance with other studies observed a strong association of oral lesions with use of tobacco and alcohol and duration of their use.[1],[2],[3],[4],[5],[6] Association with tobacco and betel quid chewing was seen in 96.7%, with alcohol in 96.7% and smoking in 86.7% cases. This was significantly higher as compared to other studies.[1] Synergistic action of tobacco and alcohol as described by several other studies was decisive in the development of oral malignancy with low social class and poor oral hygiene acting as contributing factors.[3],[4],[19],[21],[22]

Saliva is a complex body fluid composed of secretions from the salivary glands, oral mucosa transudate, and other substances coming from the oropharynx, upper airway, gastrointestinal reflux, gingival sulcus fluid, food deposits, and blood-derived compounds.[6] Although the use of saliva as a diagnostic medium in oral cancer diagnosis is still in its infancy, several authors have published excellent reviews and conducted numerous studies to recommend the use of saliva as a diagnostic medium in various conditions due to its easy availability, noninvasive, cost-effective collection techniques requiring minimal training and time consumption.[5],[6],[7],[8],[9],[10] In the present study too, saliva proved to be an excellent diagnostic medium in case of oral cavity lesions. The biochemical parameters remained stable until analysis when immediately processed and stored at −80°C.

CYFRA 21-1, first described in the mid-1990s is the soluble fragment of cytokeratin 19. Cytokeratin 19 is an intermediate filament that forms part of the cytoskeleton and is expressed by normal and benign epithelial cells and by various carcinomas.[16] The present study found a significant increase in CYFRA 21-1 level in OSCC compared with PML and healthy controls. Salivary CYFRA 21-1 levels in OSCC were 4.5 times higher while in PML were two-fold higher when compared to healthy controls. Multiple studies conducted to detect the usefulness of CYFRA 21-1 as a biomarker for oral cavity lesions have also reported an increase in its concentration in various premalignant and malignant states.[11],[12],[23],[24],[25] Alkotyfan et al.[16] on the contrary observed no correlation between the CYFRA 21-1 level at the time of initial diagnosis, nor with the clinicopathological parameters. They however found it useful for follow up of the patients.[16] The present study could not find any significant differences in the mean values of CA 19-9 among the three groups, a finding that is in accordance with the study of Nagler et al.[11]

The present study found a significant 3.9 and 2 folds increase in salivary LDH and total proteins in cancer patients while amylase levels were reduced to half as compared to healthy controls. In premalignant conditions LDH and total proteins were raised 2.5 and 1.4 times while amylase levels were decreased to 0.8 times as compared to healthy controls. These findings were consistent with studies of Shetty et al.[13] and Sanjay et al.[14] respectively who have reported increased values of salivary LDH and total proteins in oral cavity lesions. A decrease in salivary amylase concentration has been reported by Hasan and Dawoodin malignant lesions of oral cavity.[15]

CYFRA 21-1 has been described as a fragment of cytokeratin 19 which in turn forms the cytoskeleton, proteins are an inherent constituent of all living cells while LDH is an ubiquitous intracellular enzyme. All the three analytes were found to be increased significantly in the saliva of oral cancer patients and those with PML as compared to healthy controls. A possible explanation for this increase could be the increased cell mass due to uncontrolled cellular proliferation. These cell-free analytes can be locally produced and released in saliva by cell necrosis, lysis or apoptosis or may be actively released by cancerous cells.[26] Reduced salivary amylase concentration in patients with oral lesions has been attributed to low salivary flow rate as a consequence of compromised oral environment and altered concentration of various enzymes, ions, and electrolytes in saliva.[15]

The levels of test variables showed significant association with tumor size, clinical stage, and lymph node status. As shown in [Table 3], Increasing levels were observed for CYFRA 21-1, LDH, and total proteins with increase in tumor size, clinical stage, and presence of lymph nodes. Statistically significant association was seen between levels of CYFRA 21-1 and location of cancer. In case of location over alveolus, palate, or lip the mean value of CYFRA 21-1 was decreased by 41.0% (P = 0.003) when compared to nonalveolus/palate/lip location. Nagler et al. however observed no correlation of CYFRA 21-1 with the above parameters.[23]


   Conclusion Top


OSCC and PML are more prevalent in elderly males as compared to females. Long duration of tobacco, betel quid chewing is a significant risk factor whose carcinogenic effect increases manifold with concurrent addiction to smoking and alcohol. Low socioeconomic status and poor oral hygiene act as contributory factors. Both OSCC and PML group showed increased salivary CYFRA 21-1, LDH, and total protein concentrations as compared to controls, but the increase in PML was significantly lower as compared to OSCC. A considerable decrease in concentration of amylase was seen in OSCC and PML as compared to control group. The outcome of this study suggests that concurrent analysis of salivary CYFRA 21-1, LDH, total protein, and amylase can be utilized for early detection of oral cancer.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Correspondence Address:
Dr. Nidhi Awasthi
521/177, Bhairon Prasad Marg, Bara Chand Ganj, Lucknow, Uttar Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/IJPM.IJPM_140_16

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