3. Modern technologies in salivary diagnostics

## 3.1. Salivary liquid biopsy

recommendations for bioinformatics analysis of salivary RNA-Seq data that differs from other biofluids (blood, urine, etc.) as saliva contains the majority of microbial content, while other physiological fluids are considered to be sterile [48]. Thus, it is recommended to use quite stringent and sensitive criteria, while working with salivary RNA-Seq data to avoid erroneously mapped bacterial reads to the human genome, and to prevent problems with their further annotation to human RNA databases. In addition, the specific sequence of alignment steps and the stringency parameters associated with processing of RNA Sequencing data can

MicroRNAs (miRNAs) are short, single-stranded RNAs that are about 21 nucleotides in length. Their function is to regulate gene expression. Like other types of RNA, miRNAs are transcribed from DNA, but they do not participate in protein translation. They are non- coding RNAs, in which each primary transcript (pri-miRNA) is processed into a pre-miRNA and finally into functional miRNA [49]. Mature miRNA are involved in various biological processes such as cell growth, differentiation, apoptosis, stress and immune response or glucose secretion [50–52]. Studies on miRNA dysregulation in various human diseases have risen rapidly in recent years, including those in cancer, heart disease as well as type II diabetes mellitus and its complications, such as endothelial and vascular smooth muscle cell dysfunction, cardiomyopathy and nephropathy [53–55]. Most importantly, salivary microRNAs (miRNAs) (miR-9, miR-134 and miR-191) can be used as potential biomarkers for head and neck squamous cell carcinoma [56]. The reduction in salivary expression profiles of miR-125a and miR-200a was observed in OSCC patients compared to healthy people [57]. In turn, miR-31 increases in OSCC patients, specifi-

Metabolomics is the study of small molecular metabolites of living tissues, mostly metabolic intermediates such as carbohydrates, lipids, amino acids, nucleic acids, etc. [1]. The major metabolomic technologies include high-performance liquid chromatography-mass spectrometry (HPLC-MS), two-dimensional gas chromatography MS and nuclear magnetic resonance

Salivary metabolites are involved in many biological processes as well as pathogenesis of various diseases such as periodontal diseases, renal diseases, hepatocellular carcinoma and colorectal cancers [59] as well as oral cancer [60]. In case of oral leukoplakia, an upregulation of putrescine, 8-hydroxyadenine and 5,6-dihydrouridine in OSCC can be indicative of increased

Microbiomics include study of bacteria, archaea, protists, fungi and viruses. Microbial profiling (Human Oral Microbe Identification Microarray) of salivary microbiome in early resectable pancreatic cancer revealed that Neisseria elongata and Streptococcus mitis were successfully

grossly increase the final data quality [48].

56 Salivary Glands - New Approaches in Diagnostics and Treatment

cally in saliva, where it rises even more than in plasma [58].

spectroscopy in conjunction with pattern recognition methods [2].

2.3. Micro-RNA-omics

2.4. Metabolomics

2.5. Microbiomics

risk for malignant transformation [61].

Recently, a new trend appeared to reveal emerging role of "liquid biopsy" as identification method of biomarkers in various cancers. Liquid biopsy tests are non-invasive biofluid tests (serum, urine, saliva) that detect circulating tumor cells (CTCs) and fragments of tumor DNA shed into the bloodstream by cells undergoing apoptosis or necrosis [3].

The role of liquid biopsy markers including circulating tumor cells, circulating RNAs (miRNA, lncRNAs and mRNAs), cell-free proteins, peptides and exosomes has been currently investigated as non-invasive cancer biomarkers in different biofluids such as blood, urine, saliva and seminal plasma. Liquid biopsies hold great promise for personalized medicine due to the fact that they enable multiple non-invasive global sampling resulting in longitudinal assessment of the primary and metastatic tumors. Molecular profiling of circulating molecules (proteomic, transcriptomic, genomic, metabolomics, microRNAs) contributed to the successful application of several non-invasive multi-marker tests in the clinic [65].

Nowadays, liquid biopsy enables a variety of clinical and investigational applications such as early detection, assessment of molecular heterogeneity of general disease, monitoring of tumor dynamics (in melanoma, breast, ovarian or colon cancers), identification of genetic determinants for targeted therapy, evaluation of early treatment response, monitoring of minimal residual disease or assessment of resistance evolution in real time [66].

The most common technologies of liquid biopsy include detection and quantification of ctDNA (circulating tumor DNA) in blood such as Sanger sequencing, pyrosequencing, next generation sequencing, PCR-based technology, high-performance liquid chromatography (HPLC), mutant-enriched liquid chips, amplification refractory mutation system (ARMS), beads, emulsion, amplification and magnetics (BEAMing), pyrophosphorolysis-activated polymerization (PAP) [2, 66] or electric field-induced release and measurement (EFIRM) [67, 68]. The current gold standard methods for detection of ctDNA targets include droplet digital PCR and next-generation sequencing. However, those technologies require extraction of DNA from large volume of biofluid samples. EFIRM can be successfully used for continuous monitoring during treatment. The results are very promising [3].

Circulating tumor DNA (ctDNA) is considered to be stably found in biofluids encapsulated in extracellular vesicles (EVs) and released by cells into the circulation. If the links between distal cancers and the oral cavity will appear to be scientifically proven, it will open a new avenue of clinical utility to effectively, and non-invasively diagnose cancers through saliva. The ctDNA mutant fragments were observed in plasma [69, 70] and saliva samples [71] of head and neck cancer patients.

Acknowledgements

Conflict of interest

Nothing to declare.

Author details

References

19S-24S

Fellowship (Grant Number 17-40-41-KACZ).

Notes/thanks/other declarations

Karolina Elżbieta Kaczor-Urbanowicz

Address all correspondence to: kaczor.karolina@gmail.com

459-472. DOI: 10.1177/1535370216681550

This work was supported by the Public Health Service (PHS) grants from the National Institute of Health (NIH): UH3 TR000923 and R90 DE022734 as well as the 2017 Debbie's Dream Foundation – American Association for Cancer Research (AACR) Gastric Cancer Research

Salivary Diagnostics

59

http://dx.doi.org/10.5772/intechopen.73372

Department of Oral Biology and Medicine, Division of Oral Biology, UCLA Center for Oral/ Head and Neck Oncology Research, UCLA Laboratory of Salivary Diagnostics, UCLA

Department of Orthodontics, University of California at Los Angeles (UCLA), California, USA

[1] Wong DT. Salivaomics. Journal of the American Dental Association. 2012;143(10 Suppl):

[2] Kaczor-Urbanowicz KE, Martin Carreras-Presas C, Aro K, et al. Saliva diagnostics – Current views and directions. Experimantal Biology and Medicine (Maywood). 2017;242:

[3] Aro K, Wei F, Wong DT, Tu M. Saliva liquid biopsy for point-of-care applications. Fron-

[4] Anderson NL, Anderson NG. Proteome and proteomics: New technologies, new con-

tiers in Public Health. 2017;5:77. DOI: 10.3389/fpubh.2017.00077

cepts, and new words. Electrophoresis. 1998;19:1853-1861

The author reports no conflict of interest in relation with the present study.

#### 3.2. Point-of-care technologies

The current knowledge of salivary biomarkers and their role in point-of-care applications highlights the need for development of more advanced technologies. As a consequence, point-of-care diagnostics is definitely approaching reality for salivary research and closely related with its translation into clinical practice [3] as it delivers information of the current status of the disease in a very fast, convenient and non-invasive way. PoCs can be successfully used for early detection and real-time monitoring of the disease [3].

The current PoC technologies are ubiquitous. They comprise microfluidics, micro/nanoelectromechanical systems (MEMS/NEMS), paper-based technology, RNA-sequencing, liquid biopsy, biosensors, fluorescent biosensors, photometric and electrochemical methods, electronic nose and electric field-based methods such as electric field-induced release and measurement (EFIRM) method [3, 68, 72]. Contemporary available PoCs can be delivered in form of small and portable smartphones or "lab-on-chips" [3].

One of such PoC development is the Oral Fluid NanoSensor Test (OFNASET), that is used for multiplex detection of salivary proteomic (thioredoxin and IL-8) and genomic biomarkers (messenger RNA biomarkers, i.e. SAT, ODZ, IL-8, and IL-1b) for oral cancer with 90% sensitivity and 90% specificity for both interleukin 8 (IL-8) and IL-8 protein messenger RNA (mRNA) [67]. In turn, OraRisk human papilloma virus (HPV) test with Reflex (Quest Diagnostics, Los Angeles, CA, USA) can be indicative of HPV infection, high risk factor for development of oral cancer [68]. In addition, electrical controlled magnetic EC Sensor is designed to detect microRNA-200a [73], electrochemical sensor using endonuclease target recycling amplification to capture oral cancer overexpressed 1 (ORAOV1) [74], while wireless mouthguard enzymatic biosensor to detect uric acid [75] or lactic acid [76], potential biomarkers for oral cancer.
