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I. Introduction
Oral squamous cell carcinoma (OSCC) accounts for 90%-96% incidence of whole head and neck cancers, but there are no sensitive biomarkers for detection of OSCC. Definitive diagnosis has only been possible after examination of removed specimens. This diagnosis has been based on pathological findings such as angiogenesis, proliferation, and metastasis.
Even with radical resection combined with chemotherapy and concurrent radiation therapy, many OSCC cases demonstrate metastasis or recurrence without related or predictive symptoms. Complete eradication, early diagnosis, and metastatic and prognostic factor discovery have been research aims for OSCC management; until now, however, fulfillment of these aims has been elusive.
Like many cancers, early-stage OSCC is difficult to detect. A significant number of patients do not seek clinical care until the OSCC is in an advanced stage. The development and advancement of screening and early diagnosis approaches has been recommended as the most effective strategy for reducing the OSCC-related morbidity and mortality rate1. Biomarkers found in the saliva are an ideal non-invasive diagnostic tool for early diagnosis of cancer2-4. Compared to blood and tissue sampling, saliva sampling is a reliable, non-invasive, convenient, and economical alternative for disease diagnosis and prognosis determination5. Saliva sampling also provides an effective and easily-acquired liquid specimen for large-scale sampling, epidemiologic screening and long-term monitoring6.
Several DNA or RNA sequencing techniques using salivary specimens have been used for karyotyping or submicroscopic chromosomal copy number changes such as microdeletions. These techniques include immunoprecipitation high-performance liquid chromatography, radioimmunoassay, electrophoretic immunoassay, mass spectrometry-based proteomics, microchips or microarrays, microfluidic devices, electrochemical biosensors7,8, fluorescence in situ hybridization and comparative genomic hybridization microarrays. Maxam and Gilbert9 DNA sequencing was first developed in 1976 and is based on nucleobase-specific partial chemical modification of DNA and subsequent cleavage of the DNA backbone at sites adjacent to the modified nucleotides. This sequencing method was advanced by Sanger et al.10 in 1977. Sanger sequencing10, also known as chain termination sequencing, was marketed commercially by Applied Biosystems in 198611 and was the most popular DNA sequencing method until now. These two methods are based on the dideoxy method, the selective incorporation of chain-terminating dideoxynucleotides by DNA polymerase during in vitro DNA replication10,12. These first generation sequencing methods have been improved upon in next-generation sequencing (NGS).(Fig. 1)
NGS is a massive and parallel DNA sequencing technology for large-scale, ultra-high throughput, and automated high-speed genome analyses. NGS is a less expensive method for determining the order of nucleotides in entire genomes or targeted regions of DNA or RNA and has revolutionized the biological sciences. NGS has a wide variety of applications for the study of biological systems at a new level. NGS can be used to sequence entire genomes or specific areas of interest13. Sanger sequencing continues to be useful for smaller-scale, short-read sequencing analysis and for the validation of NGS results14.
The effectiveness of NGS in whole exome sequencing by targeted sequencing of cancer-related genes and in RNA sequencing has been demonstrated. NGS-applied OSCC research has also identified various genetic alterations and detected mutations with low variant allele frequency15. NGS is also becoming an essential method in characterization of salivary gland tumors16. However, NGS has only rarely been applied to the identification of salivary biomarkers of OSCC. We discuss four topics in this review article: definitions and use of NGS, salivary biomarkers and OSCC, current biomarkers using NGS-based technique, and potential candidates as NGS-based salivary biomarkers.
II. Definitions and Use of NGS
In the field of functional genomics, NGS is the most useful method for DNA and RNA analysis. This highly reproducible tool can reveal single nucleotide polymorphisms, related gene variants or spliced transcripts without input of direct DNA or RNA features as in microarray procedures. NGS can also be applied to cDNA molecules for RNA sequencing by reverse transcription from candidate RNA and sequencing-library construction by massive parallel deep sequencing. The most popular NGS methods are summarized in Table 117-31. Illumina Solexa sequencing uses a fluorescence-based Illumina platform to identify 100-150 bp of DNA by emitting a particular fluorescent signal to each chain of nucleic acid. Adaptors can fragment, ligate and anneal longer sequences randomly. Reading is carried out by polymerase chain reaction (PCR) amplification and creation of a unique spot that repeats. The product can be separated into a single strand for final sequencing.(Fig. 1)
The sequencing-by-sequence methods using pyrosequencing is the basis of Roche 454 platform sequencing. The release and incorporation of pyrophosphate into a new strand by polymerase is detected by fluorescence. Multiple reads over 1,000 bp long can be carried out by optical signal detection. One DNA fragment per bead can be annealed, and generic adaptors can add bases to the ends. The product is amplified by PCR as a routine adaptor-specific primers application.
The principle of measuring the direct hydrogen ion release from individual bases, not fluorescence, is the basic principle of Ion torrent and proton platform sequencing. Direct measurement of the emission of hydrogen ions by polymerase during the incorporation of deoxynucleoside triphosphate to a growing DNA strand. This is executed by uniform 200 to 400 bp fragmentation, and adapters are added to effect amplification by emulsion PCR.
III. Salivary Biomarkers and Oral Cancer
Saliva sampling is rapidly expanding; and arrays of analytes including proteins, messenger RNA (mRNA), and DNA in saliva have been studied for their potential use as biomarkers for OSCC screening programs32,33. OSCC is a malignancy in which salivary diagnosis has the greatest potential because the OSCC environment contains saliva. One of the useful aspects of saliva is its containment of exfoliated tissues or cells from the oral cavity. This aspect suggests the existence of potential salivary biomarkers for OSCC33. A number of candidate salivary biomarkers for oral cancer, including genomic and proteomic biomarkers for OSCC, have been reported and are summarized in Table 2.
Proteomic studies using human saliva have been targeting the biological activities of various peptides and proteins in normal individuals and in those with various pathologies. Proteomic targets such as CD59, catalase, M2BP, and MRP14 were suggested from a shotgun proteome analysis for oral cancer detection34. Li et al.35 reported the presence of more than 3000 RNA species, mostly mRNAs; and reports of Park et al.36 and Patel et al.37 support the role of microRNAs (miRNAs) in oral cancer progression and various cancers.
Several salivary biomarkers such as DAPK, TIMP3, p16, and MGMT are potential screening markers for OSCC38, and changes in DNA methylation patterns could be a useful screening tool for predicting the rate and the likelihood of malignant transformation. The methylation of DNA in saliva has been suggested as an effective biomarker for early detection of OSCC39. For example, hypermethylation of the DNA promoter of certain genes, such as p16, has been found in saliva as well as serum40,41. Promoter hypermethylation has been suggested to be an early event during OSCC genesis. Any change of transcription factor or receptor functions or other factors in tumorigenesis might be involved in loss of cell cycle control. This loss of control was reported by Liao et al.42; this group suggested the first salivary biomarker, genomic p53. Other cell cycle regulatory proteins such as mammary serine protease inhibitor and cyclin D1 were also increased in OSCC saliva43.
Due to the basic precursor characteristics of mRNA for protein expression, other potential salivary biomarkers display increased mRNA production. These include IL-8, IL-1β, S100P, and spermidine/spermineN1-acetyltransferase44. The circulating levels of CD44 are reported to be related to head and neck cancer metastasis, and CD44 is a potential salivary protein biomarker45. Actin and myosin are also regarded as promising salivary biomarkers for premalignant differentiation and malignant oral lesions33 IL-6 is another premalignant differentiation biomarker found especially in the saliva of oral leukoplakia patients. IL-6 inactivated the p53 tumor suppressor gene by hypermethylation of its promoter region resulting in uncontrolled cell proliferation and suppression of programmed cell death33.
Researchers have also directed their research toward detection of human papilloma virus (HPV) and Epstein–Barr virus (EBV) in saliva; these viruses are etiological factors in cancer. The incidence for HPV positivity has been reported to be more than 45% in patients treated for oral cancer33.
IV. Current Cancer Biomarkers Using NGS-based Technique
Currently, several authors have reported data from the search for biomarkers using a NGS-based technique36,46-69.(Tables 3, 4) Most of these studies started from Gene Expression Omnibus datasets (https://www.ncbi.nlm.nih.gov/gds) in combination with differentially expressed genes, the protein-protein interaction network, gene ontology and the Kyoto Encyclopedia of Genes and Genomes pathway. Shanmugam et al.70 have reported a customized NGS analysis of unique coding regions of seven mutated genes from OSCC saliva, and similar data from whole-exome sequencing were also discovered in tumors from The Cancer Genome Atlas71, the International Cancer Genome Consortium gingiva-buccal cohort69, and MD Anderson Cancer Center OSCC cohort72.
From this review, NGS-based salivary biomarkers in OSCC could be categorized primarily as genomic (Table 3) and transcriptome markers.(Table 4) Within these main categories, we could present promoter hypermethylation of p16 and three miRNAs as the main candidate salivary biomarkers. Especially, miRNAs, endogenous, non-coding, single-stranded RNA molecules 22 nucleotides long, have tumor-controlling characteristics including tumor-suppression.
Hypermethylation of the p16 promoter is a useful serum biomarker for the early detection of alimentary tract cancer, especially gastric cancer. Zammit et al.73 reported on the etiology of OSCC using NGS while focusing on smoking and HPV factors in a prospective observational study. This group showed that the most frequent mutations were found in TP53 and CDKN2A in the salivary specimen.(Table 3) Fadhil et al.74 reported five miRNAs using NGS data in saliva of 12 HNSCC patients and 12 healthy controls. Among these, miR-let-7a-5p and miR-3928 were suggested to be NGS-based salivary biomarkers for early diagnosis of HNSCC when compared with other miRNAs such as miR-7703, miR-345-5p, and miR-1470.(Table 4) A recent study also showed the potential benefits of NGS for mRNA expression profiling by using miR143-3p to detect chronic periodontitis75.
Lu et al.76 reported that miR-31-5p may be an independent OSCC biomarker by showing its tumor growth inhibition capacity in oral cancer patient-derived xenograft models. Wang et al.77 showed that mature miR-125b could control metabolism and immunity of cancer cells by regulating NF-κB or p53 signaling pathways through translation inhibition of 3′ untranslated regions of target mRNAs. Korpal and Kang78 also showed that miR-200 family members inhibit epithelial-mesenchymal transition (EMT) and metastasis. This occurs through a miRNA-mediated regulatory pathway by direct targeting of transcriptional repressors of E-cadherin, ZEB1, and ZEB2. The validity of this report was strengthened by Kabzinski et al.79 who showed that DNA methylation of the miR-200c promoter in an epithelial originated tumor may occur during EMT. Majewska et al.80 discovered a potentially targetable novel anaplastic lymphoma kinase fusion in an intraductal carcinoma of a minor salivary gland by NGS analysis.
Compared with known genomic salivary biomarkers, such as p53, FAT1, CASP8, PIK3CA, HRAS, NOTCH1, and CDKN2A, NGS-based genomics consider mutation of p53 and promoter hypermethylation of DAPK, TIMP3, p16, and MGMT, cyclone D1 and mammary serine protease inhibitors. Also, up-regulated oncogenic tissue miRNAs and down-regulated tissue suppressive marker miRNAs, including miR 31, miR 125, and miR 3928, are recommended as subjects for further research70,74,76-79,81.(Table 5)
V. Discussion
This review article aimed to summarize current applications of NGS in cancer research and to propose potential genomic and proteomic saliva biomarkers for NGS-based study in OSCC screening and diagnosis programs. For the establishment of a standardized research and designed protocol for the detection of trace protein or nucleic acids biomarkers from saliva samples, efficient and stable collection, processing and preservation methods should first be confirmed. For the collection of saliva samples, confirmation of the patient’s mouth cleanness is essential. This can be accomplished by rinsing the mouth with water to remove substances and by ensuring that the patient avoids any eating or drinking for at least 30 minutes prior to sampling. Approximately 2.5 mL of saliva in the buffered solution is recommended. Preparation should include addition of 2.5 mL of DNA stabilization buffer into a 10- or 15-mL conical tube. Excessive saliva might to be degraded due to insufficient DNA stabilization buffer, and insufficient saliva collection may not provide adequate results. The cap needs to be replaced, mixing needs to occur by inversion not shaking, and the specimen needs to be stored at room temperature for immediate use or at 4°C for future use.
NGS-based research has been used to identify factors in metastasis of lung, prostate, ovarian, and bile duct cancer. NGS-based evaluation of candidate biomarkers in saliva could also be routinely used as a simple, non-invasive test in patients with OSCC. NGS has also been used as a valuable tool for detecting novel biomarkers in periodontal disease75. More recently, NGS-based research has led the ability to differentiate patients with primary Sjögren’s syndrome from those with non-Sjögren’s sicca82.
In various medical and non-medical institutions, prevention of unnecessary medical expenses may be possible by the quick and easy diagnosis of oral cancer in healthy or suspect patients. There would be no additional cost to the public health system, and provision of information on the potential of benefits to patients with OSCC by using NGS based salivary biomarkers can be effected. Since both proteins and genes can be applied as biomarkers, the accuracy of diagnosis can be increased. New biomarkers can be added quickly for expansion of a preventive platform for a variety of medical and dental diseases. Since both proteins and genes can be applied as biomarkers, the accuracy of diagnosis can be increased, and new biomarkers can be added quickly, so that it can be expanded to a preventive platform through early diagnosis of various diseases as well as various medical and dental treatments.
In conclusion, selection of potential OSCC biomarkers through NGS-based protein studies from epithelial tissue cells in collected saliva will be possible. These non-invasive methods could be a useful tool for the improvement of an immune biosensor for ensuring general public health and for diagnosis of OSCC before its metastasis or progressive infiltration to adjacent tissues. Based on NGS analysis, we will be able to verify the biomarkers of OSCC in saliva and to effect early diagnosis of oral cancer based on this.
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