Year : 2021 | Volume
: 13 | Issue : 5 | Page : 52--56
Salivaomics for oral cancer detection: An insight
V Naga Sirisha Chundru1, Ramadas Madhavan Nirmal2, B Srikanth3, Manasa Bojji1, Namburi Midhun4, B Jaya Lakshmi5,
1 Department of Oral and Maxillofacial Pathology, Malla Reddy Dental College for Women, Hyderabad, Telangana, India
2 Department of Oral and Maxillofacial Pathology, Rajah Muthiah Dental College and Hospital, Chidambaram, Tamil Nadu, India
3 Department of Dental Surgery, MNJ Institute of Oncology and Regional Cancer Centre, Hyderabad, Telangana, India
4 Department of Oral and Maxillofacial Pathology, CKS Theja institute of Dental Sciences, Tirupati, Andhra Pradesh, India
5 Department of Oral Medicine and Radiology, Tirumala Institute of Dental Sciences and Research Centre, Nizamabad, Telangana, India
V Naga Sirisha Chundru
Department of Oral and Maxillofacial Pathology, Malla Reddy Dental College for Women, Hyderabad - 500 055, Telangana
Early detection is very crucial for successful management of oral cancer or any disease as such. Oral squamous cell carcinoma (OSCC) accounts for nearly 90% of malignancy of oral cavity. In the field of cancer research, there is always an ongoing quest for newer methods to lower the morbidity and mortality associated with OSCC. Saliva, a readily available noninvasive biofluid with constant contact with oral cancer lesion, offers an appealing alternative to serum and tissue testing. This review throws light on incorporation of newer technologies for harnessing the saliva to its fullest potential with increased specificity and sensitivity toward identification of cancer-specific molecular signatures for the development of point-of-care applications that could be used at the clinical setting.
|How to cite this article:|
Chundru V N, Nirmal RM, Srikanth B, Bojji M, Midhun N, Lakshmi B J. Salivaomics for oral cancer detection: An insight.J Pharm Bioall Sci 2021;13:52-56
|How to cite this URL:|
Chundru V N, Nirmal RM, Srikanth B, Bojji M, Midhun N, Lakshmi B J. Salivaomics for oral cancer detection: An insight. J Pharm Bioall Sci [serial online] 2021 [cited 2022 Jan 26 ];13:52-56
Available from: https://www.jpbsonline.org/text.asp?2021/13/5/52/317511
Oral cancer is one of the most common cancers in the world accounting for an estimated 354,864 new cases diagnosed annually. India alone accounts for approximately 100,000 incident cases every year. It ranks among the three most common types of cancer in the Indian subcontinent. Oral squamous cell carcinoma (OSCC) accounts for mainly 90% of malignancy of oral cavity. Recent reports revealed that India accounts for 1/3 of the world burden of oral cancer and ranks on the list of the highest incidence oral cancer countries in the world, and Southeast Asian countries collectively account for 80% of OSCC cases globally. The five-year survival rate for OSCC is around 85% if diagnosed early. Mortality is high owing to the initial asymptomatic nature and most patients report when they develop advanced stage symptoms at which stage the disease would have progressed and the survival rate decreases as low as 15%–50%. Thus, early detection is the key to improved survival rate and prognosis for OSCC patients. Carcinogenesis is a multistep process resulting from genetic, epigenetic, and metabolic alteration resulting from exposure to carcinogens. OSCC is usually preceded by precursor lesions oral potentially malignant disorders (OPMDs) such as leukoplakia, erythroplakia, oral lichen planus, and oral submucous fibrosis. Based on the type of lesion, the malignant transformation rate may range from 0% to 20% in 1–30 years. Therefore, thorough clinical examination forms the backbone of oral cancer screening and is especially important in high-risk groups.
The standard visual and tactile examination is easier to perform and requires no additional armamentarium; however, subtle lesions may pass undetected and it is difficult to make a visual distinction between benign, OPMD, and malignant lesions. Although biopsy remains the ultimate gold standard for diagnosis of oral cancer, adjunctive techniques have been developed to aid in making this distinction and to predict malignant transformation. These emergent techniques though promising are often invasive and require high-quality equipment to complete the biopsy, which turns out to be expensive and cumbersome. Moreover, they only prove to be positive if dysplastic changes have occurred. Hence, alternative methods that are more sensitive, noninvasive, cost-effective, and patient-friendly for analysis testing are in great demand.,
Cancer-related Mutations Identified in Body Fluids
Various body fluids such as blood, urine, saliva, cerebrospinal fluid, sweat, tears, peritoneal fluid, vomit, breast milk, semen, and vaginal fluids have unique structural composition specific to certain diseases and conditions. Cancer-related mutations are seen not only in body fluids draining a tumor but also in body fluids secreted away from the initial point where a solid tumor is developing. Cancer-related nucleic acids and proteins identified in body fluids are being used as molecular markers for early diagnosis, recurrence, survival, metastatic indicators, and pharmacological response to therapeutic intervention. The new area of research is early detection of OSCC using salivary diagnostics. Of all biofluids, blood and saliva have been widely studied for reliable biomarkers for cancer detection.
In 1998, the National Institute of Health defined biomarker as a characteristic that is objectively measured and evaluated indicator of normal biologic processes, pathogenic processes, or pharmacological response to therapeutic intervention. Biomarkers are molecular signatures specific to certain disease. The main aim of discovery of biomarkers is to maximize the survivability of cancer through better diagnostics and treatment. Deciphering an individual biomarker signature will aid in the detection, risk assessment, diagnosis, prognosis, and monitoring of disease.
Saliva as Perfect Diagnostic Medium
Human saliva represents whole-body images and is considered “mirror of body's health.” Oral cancer lesion with its constant salivary contact makes the measurement of tumor markers in saliva an attractive alternative to serum and tissue testing. Saliva contains an array of analytes used as biomarkers for translation research and clinical applications. It has a variety of enzymes, hormones, antibodies, antimicrobial constituents, and cytokines. Many of these enter saliva from blood by passing through cells by transcellular, intracellular diffusion, and paracellular routes by extracellular ultrafiltration within salivary glands or through gingival sulcus. Hence, many constituents of blood are also seen in saliva. It is an important indicator of oral and systemic health.,
Rationale Behind the Use of Salivary Biomarkers
Easily accessible, noninvasiveSafe to handle compared to blood, feasibility of greater volume for testing, and repeated sampling for monitoring over timeEasy to ship and store as it does not clotSimplicity of collection with minimal training requirementSelf-collection by patient negates the need for direct interaction with health-care workers and investigators, thus reducing nosocomial infections and less requirement of personal protection equipmentCan be used for mass screening of large population.
Despite several advantages, disadvantages do exist. The concentration of most analytes in saliva is very low (100–1000 fold) as compared to their concentration in blood; however, this may not be a limitation for saliva sampling in oral cancer, as the biomarkers are usually locally released from the tumor site and the availability of highly sensitive technology.,
Method of Collection and Stimulated versus Unstimulated Saliva
Saliva can be collected by either stimulated or unstimulated way. Unstimulated whole saliva (WS) is collected by draining/drool, spitting, suction, or swab. Although these are commonly employed, there is presently no universally accepted method for sample collection. Stimulated saliva is collected by performing masticatory movements or by gustatory stimulation using citric acid, paraffin, or gum base. Irrespective of the method employed, it is vital that subjects rinse the mouth with water before sample collection to avoid any contaminants. Unstimulated saliva is more favorable as stimulated saliva production decreases the concentration of small molecules such as myoglobin, changing the total composition of the analyzed saliva in favor of large molecules. Salivary analysis can also be done for the diagnosis of various conditions such as autoimmune diseases, infections, monitoring of levels of hormones and drugs, forensic evidence, bone turnover marker, and diagnosis of oral diseases with relevance to systemic diseases.,,
Salivaomics for the Detection of Various Cancers and Oral Squamous Cell Carcinoma
Many investigators studied clinical importance of salivary biomarkers in various malignancies., Streckfus et al. reported Her2/neu as the first salivary biomarker for breast cancer and also showed raised levels of CA 15-3 and Her2/neu and low levels of P53 in patients with breast cancer. Various malignancies can also be identified at initial stages using salivary proteomic analysis. Salivary diagnostics aids in identifying high-risk population with OPMD and in those with prior history of malignancy. The first report of saliva as a diagnostic tool for oral cancer detection was published in 2000 by Liao et al. A key development in salivary diagnostics is the advent of omics-based biomarkers. The term salivaomics was coined to reflect the rapid development of translational and clinical tools based on salivary biomarkers. Oral cancer biomarkers can be classified based on biomolecules as DNA markers, RNA markers, or protein biomarkers and based on disease state as diagnostic or prognostic biomarkers. Application of genomics and the knowledge of the sequence of human genome have inspired numerous omics-based disciplines, providing important insight into the pathogenesis of disease, leading to identification of new prognosticators, diagnostic markers, and therapeutic targets. Salivaomics knowledge base provides the first web source dedicated to salivary “omics.” Salivary diagnostics include five major diagnostic alphabets, namely, proteins, messenger RNA (mRNAs), microRNA (miRNAs), metabolic compounds, and microorganisms.
Saliva is a repository of protein biomarkers even though the proteomic content was found to be only 30% of that of blood. More than 2300 minor proteins or peptides are present in saliva defining it as the first salivary biomarker alphabet. In 2008, 1166 salivary proteins have been identified in a National Institute of Dental and Craniofacial Research (NIDCR)-funded project that sought to catalogue and annotate the human salivary proteome. This project was an important step for saliva to be clinically useful in disease diagnosis and health monitoring. A consortium funded by NIDCR cataloged the proteins in saliva of major human salivary glands and found 1100 nonredundant proteins. The salivary proteome helps in identifying biomarkers for both local and distant diseases. Analysis of salivary proteome may unravel morbidity signatures in the preliminary stage and monitor disease progression. For early detection of cancer, salivary protein biomarkers are analyzed either individually or as a panel. Salivary protein markers such as interleukin 8 (IL-8), IL-6, IL-1β, matrix metalloproteinase (MMP2, MMP9), transforming growth factor-1, Ki-67, cyclin-D1, Cyfra-21.1, transferrin, α-amylase, tumor necrosis factor alpha (TNF-α), and catalase have been detected in OSCC by various studies. Cytokines are intercellular signaling proteins that mediate normal growth, cellular proliferation, tissue repair, and angiogenesis. They are involved in immune response against infection and inflammation. Proangiogenic, proinflammatory cytokines such as IL-1, IL-6, IL-8, and TNF-α are elevated in WS of oral cancer and oral precancers compared to controls, suggesting their utility as surrogate indicators of carcinogenic transformation from oral precancer to oral cancer. IL-8 levels have been reported to be significantly higher in saliva of OSCC patients compared to severe periodontal disease. Recent advancements in proteomic technologies such as Luminex multianalyte profiling, shotgun proteomics, capillary reversed-phase liquid chromatography with quadruple time-of-flight mass spectrometry (MS), and matrix-assisted laser desorption/ionization-MS (MALDI-MS) have contributed enormously to salivary diagnostics allowing detection of very low abundance molecules in salivary proteome. Oral fluid nanosensor test is an automated system that enables simultaneous quick detection of multiple salivary proteins and nucleic acids for the determination of various diseases. This point-of-care (POC) lab-on-a--chip works on electrochemical detection. Electric field-induced release and measurement (EFIRM) sets an exciting precedent for future POC salivary diagnostics. EFIRM enables polymerase chain reaction (PCR) free rapid biodetection of oncogenic targets with minimal volume of saliva., Even though the proteomic constituents are considered as the first salivary diagnostic biomarker alphabet, the genomic targets are considered as highly discriminatory and informative.
Apart from proteins, saliva also contains nucleic acids considered as second diagnostic alphabet. mRNA being a direct precursor of proteins, its corresponding levels are correlated in cells and tissue samples. Compared to DNA, RNA is more labile and is susceptible to degradation by RNases. Against the earlier hypothesis that human mRNA cannot survive extracellularly in saliva, recent studies showed that RNAs in saliva exist as stable molecules and are protected from degradation by RNases by harboring in exosomes. Exosomes are membrane-bound organelles originating from endoplasmic reticulum, whose size ranges from 30 to 100 nm. They are abundantly filled with mRNA and miRNA and aid in shuttling RNA from tumor site into saliva to target sites. Exosomes regulate cell-to-cell environment by altering their gene expression facilitating to understand molecular basis of oral diseases. RNAs in saliva are derived either from serum or produced locally. Several researchers have shown that the levels of mRNA reflect physiological state and disease process. Li et al. found significant upregulation of seven mRNA transcripts such as dual specificity phosphatase 1, H3 histone family 3A, ornithine decarboxylase antizime 1, spermidine/spermine N1 acetyl transferase, S100 calcium binding protein P (S100P), IL 8, and IL 1β in the saliva of OSCC patients. They used microarrays followed by quantitative PCR (qPCR), gold standard for quantification of nucleic acids. Multiplex RT-PCR-based preamplification allows quantification of >50 targets from one reaction, enabling a small volume of preamplification product to be used for further qPCR measurement, thereby reducing the cost and time involved.
These constitute third salivary diagnostic alphabet. They are short RNA transcripts that range from 19 to 20 nucleotides, discovered in the early 90s in a transparent nematode. More than 1000 miRNAs have been profiled so far. Compared to mRNAs, their expression increases to a significant proportion, i.e., 10–100-fold in oral cancers. They are important regulators of mRNA and protein expression and assumed to regulate the expression of almost 1/3 of all human transcripts. They function as either oncogenes or tumor suppressors based on their target transcripts. Their dysregulation effects cell growth, apoptosis, differentiation, motility, and immunity. miRNAs have been observed in OSCC to be epigenetically regulated by DNA methylation. They can accurately differentiate even poorly differentiated carcinomas, suggesting them as potential biomarkers. Most studies showed significant change in three miRNA molecules such as miR-125A, miR-200A, and miR-31.,,
Metabolomics is a measure of all intracellular metabolites and is a potent tool for understanding cellular functions. Metabolomics helps in understanding metabolic dynamics associated with disease process and drug exposure. Metabolomics complement data obtained from genomics and transcriptomics facilitating study of individual variation in a disease state or during therapeutic intervention. Sugimoto et al. in their study on salivary metabolomics found that twenty-eight metabolites including pyrroline, choline, and valine were found to be discriminatory between healthy controls and OSCC patients. Their study showed that cancer-specific signatures are embedded in saliva metabolites. Sridharan et al. found significant upregulation and downregulation of various salivary metabolites in OSCC and oral leukoplakia compared to controls.
Methylomics and Microbiomics
DNA methylation induces cells to maintain or alter unique characteristics by controlling and modulating gene expression. Hypermethylated genes cause alterations in proliferation, DNA repair, apoptosis, cell-to-cell adhesion, and angiogenesis, suggesting them as potential biomarkers for oral cancer. A salivary study on OSCC patients and normal controls showed homeobox protein HOXA9 and nidogen2 as methylated genes in OSCC patients. This promoter hypermethylation of genes in saliva may serve as potential biomarkers for the early detection of OSCC. Establishing disease-specific microbiological signatures could lead to development of simple tests targeting discriminatory microbes capable of identifying particular pathologies. Capnocytophaga gingivalis, prevotella melaninogenica, and streptococcus mitis can be used as diagnostic markers to distinguish OSCC from healthy controls. Studies have shown increased candidal carriage in the saliva of OSCC compared to controls. Human papilloma virus is also associated with oropharyngeal SCC.
OSCC is multifactorial with heterogenic pathogenesis, so a panel of markers could yield highly discriminatory nucleic acids, proteins, metabolomes, etc., Incorporation of newer technologies for simultaneous testing of different salivary biomarkers using micro- and nanoelectrical mechanical system biosensors could yield results with high sensitivity and specificity. Standardization of methods of saliva collection and extensive validation of biomarkers in large patient cohorts of different ethnicities and combining multiple markers from different “omics” is very much essential before any biomarker candidates can be tailored for clinical use.
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