Emerging technologies for salivaomics in cancer detection

Abstract Salivary diagnostics has great potential to be used in the early detection and prevention of many cancerous diseases. If implemented with rigour and efficiency, it can result in improving patient survival times and achieving earlier diagnosis of disease. Recently, extraordinary efforts have been taken to develop non‐invasive technologies that can be applied without complicated and expensive procedures. Saliva is a biofluid that has demonstrated excellent properties and can be used as a diagnostic fluid, since many of the biomarkers suggested for cancers can also be found in whole saliva, apart from blood or other body fluids. The currently accepted gold standard methods for biomarker development include chromatography, mass spectometry, gel electrophoresis, microarrays and polymerase chain reaction‐based quantification. However, salivary diagnostics is a flourishing field with the rapid development of novel technologies associated with point‐of‐care diagnostics, RNA sequencing, electrochemical detection and liquid biopsy. Those technologies will help introduce population‐based screening programs, thus enabling early detection, prognosis assessment and disease monitoring. The purpose of this review is to give a comprehensive update on the emerging diagnostic technologies and tools for the early detection of cancerous diseases based on saliva.


Introduction
Saliva is a complex fluid that is composed of water, cells, debris, organic and inorganic molecules that may reflect the physiological state of an individual condition, since many of the componenets of the saliva also play an important role in processes taking part in distal portions of the body [1]. Currently, approximately 40% of markers suggested for diseases such as cancer, cardiovascular disease and stroke can also be found in whole saliva [2]. A biomarker can be defined as a measurable and quantifiable biological parameter that can serve either as an indicator for health, disease status, environmental exposure or pharmacological responses to a therapeutic intervention [3]. Prognostic biomarkers are used as indicators of a benign or a malignant condition, whereas diagnostic biomarkers show the development of a cancer [4].
Siegel et al. reported that 1.7 million Americans are diagnosed with cancer every year and over 500,000 individuals do not survive the disease [5]. Hence, a lot of efforts have been done to advance the field of salivary diagnostic technology, which is likely to revolutionize the way cancerous diseases will be diagnosed in the future [6].
To successfully translate research on salivary biomarkers to the chairside, biomarker studies should follow the principles laid out in the prospective-specimen-collection, retrospective-blinded evaluation (PRoBE design). In this approach, samples are first collected prospectively from a cohort of target population prior to diagnosis. After examination of the patient, individuals with known diagnosis and control subjects will be selected randomly from the cohort and their specimens will be tested in a blinded study design [7]. If applied with rigour and appropriate sampling of patient population, definitive validation of identified biomarkers can result in the Food and Drug Administration regulatory approval.
Despite the acceptance of salivary diagnostics for the detection of cancerous diseases, the absence of a mechanistic rationale in regards to the transmission of biomarkers between the distal tumour and the oral cavity poses a risk that has the potential of undermining saliva's value for the detection of tumour diseases. However, recent studies focused on exosome secretion and biogenesis have attempted to unravel this phenomenon [8][9][10].
Emerging technologies for salivary diagnostics of cancer Point-of-care diagnostics Point-of-care (POC) technologies are newly emerging methods, that when used in conjunction with biomarker identification, have the potential to be used in screening and non-invasive diagnostics in a rapid and convenient fashion [11]. Current diagnostic methods used for the detection of malignant cancers have also significant limitations such as low sensitivity and low specificity. They are time-consuming, invasive, cost-prohibitive, and complex to perform [12,13]. In addition, the long assay time may cause degradation of many important constituents in the patient samples before quantification can be made [14,15]. Therefore, before entering the clinical settings, these POC tests should be appropriately prepared i.e. to prove their validity, reliability, reproducibility and robustness [16].
There are several newly emerging technologies that integrate salivary diagnostics with microfluidics or micro/nanoelectromechanical systems (MEMS/NEMS). Microfluidics consist of manipulation of liquids at the microscale to miniaturize and automate many techniques that may normally require trained personnel with traditional laboratory equipment. MEMS/NEMS devices are composed of mechanical elements, sensors, actuators and electronics on a common silicon substrate developer through microfabrication technology. Those technologies enable users to measure proteins, DNA, transcripts (mRNA), electrolytes and small molecules in saliva [17,18]. Currently developed MEMS/NEMS platforms use a variety of techniques to perform detection, including electrochemical sensing [19], on-chip reverse transcription polymerase chain reaction (RT-PCR) [20], microspherebased optical fibre array [21], high-throughput DNA microarray, surface plasmon resonance optical system [22] or microchip electrophoretic immunoassay [23].
The newly developed POC tests for 'lab-on-a-chip' allows detection of multiple biomarkers, thus facilitating the diagnosis of many human diseases at the same time [36].
In 2003, the University of California at Los Angeles (UCLA) Collaborative Oral Fluid Diagnostic Research Center was established with the major aim of developing the platform for using nanotechnology and microtechnology for detection of salivary proteins and genomic biomarkers. An integrated POC electrochemical multiplexing salivabased platform for oral cancer detection emerged [17,37]. This platform can detect both salivary proteins and nucleic acids as well as measure up to eight different biomarkers in a single test in less than 15 min. under ambient conditions. The salivary test in an Indian cohort of oral cancer saliva samples achieved 90% sensitivity and 90% specificity for both interleukin 8 (IL-8) and IL-8 protein messenger RNA (mRNA) [38]. This method can potentially be used for screening and assessment of the risk for oral cancer, as well as identification of patients that may need a biopsy [37]. Another saliva-based molecular test, OraRisk â human papilloma virus (HPV) with Reflex (Quest Diagnostics, Los Angeles, CA, USA) can determine the presence of HPV types associated with a high risk of developing oral cancer.
Emerging novel POC techniques used specifically for oncogenic mutation detection in clinical practice include: gold nanoparticlebased mutation capture and naked-eye visualization for the detection of single nucleotide polymorphism mutation [39,40] or a combination of magnetic and gold nanoparticle methods to identify KRAS gene mutations [41]. Another method -microfluidic platforms combine genetic analysis with microfluidic systems [40,42], for example, an integrated microfluidic system for JAK2-V617F mutation detection, present in various hematological malignancies [43].  Along with these recent scientific advancements, there is an emerging need to move salivary diagnostics out of the research lab into clinical practice. Point-of-care technologies can provide noninvasive, rapid, easy and accurate measurements directly from saliva for monitoring different medical conditions including cancers [37].

RNA sequencing
RNA-Sequencing (RNA-Seq) is a newly emerging high-throughput method for performing transcriptome profiling by means of deepsequencing technologies. The transcriptome is composed of transcripts in a cell, and their quantity. It plays a crucial role for unravelling functional elements of the genome, the molecular components of cells, and also the mechanisms of normal development, physiology and pathology. While performing RNA Sequencing, RNA is converted to a library of cDNA fragments. Each molecule is sequenced, with or without amplification, resulting in the large number of reads, that are subsequently aligned either to a reference genome or to a transcriptome [44]. RNA-Seq has many advantages over the currently used DNA microarrays, i.e.: ability to detect transcripts and their isoforms, low background signal, increased dynamic range of expression, measurement of focal changes, splice variants, chimeric gene fusions and applicabilty to each species, etc. [44,45].
Although RNA-Seq of saliva is challenging, because of factors such as the difficulty of performing RNA isolation, stabilitization, RNA library construction, etc. [46][47][48][49], the recent advancements in the field have resulted in the identification of various types of extracellular RNAs (exRNAs) such as: mRNAs and non-coding RNAs (ncRNAs) including microRNAs (miRNAs), piwi-interacting RNAs (piRNAs) and circular RNAs (circRNAs). Specifically, those ncRNAs are emerging regulators of oncogenesis and tumour progression. Because of their small size, they are more stable and less prone to degradation by ribonucleases (RNases) compared to mRNAs [15].
Currently, RNA Sequencing has a wide range of applications in cancer diagnostics. Our group at UCLA is in the process of developing salivary biomarkers for early detection of gastric cancer [50]. This study involves comprehensive RNA-Sequencing performed on 100 randomly selected gastric cancer saliva samples and 100 randomly selected non-gastric cancer matched control subjects. Bioinformatic analysis of RNA-Seq data has revealed various types of exRNAs in cell-free saliva, including 127-418 miRNAs, 32-109 piRNAs and 400 circRNAs, representing the first characterization of circRNAs in extracellular body fluid [50].
Aside from emerging salivary diagnostics, the use of RNA-Seq methodology in cancer diagnostics in other body fluids and tissues is very common [45,51,52]. For example, currently there are known splicing signatures of the three most common types of breast tumours [Triple Negative Breast Cancer (TNBC), non-TNBC and HER2-positive cancers] identified by means of RNA-Seq [51]. Also, alternative breast cancer 1 (BRCA1) transcripts have been detected in a subset of patients with breast cancer and a family history of breast and/or ovarian cancer [53]. Furthermore, diagnosis of acute myeloid leukaemia can be currently made based on the detection of genetic abnormalities such as t(8;21)(q22;q22) translocation or runt-related transcription factor 1 (RUNX1) fusion RUNX1-RUNX1T1 [52].
The recent progress in RNA-Seq technologies have a potential for exRNAs to serve as non-invasive diagnostic indicators of the disease in risk assessement, early diagnostics, prognostics and therapeutics for various diseases, including cancers and infectious diseases [45].

Liquid biopsy
Liquid biopsy is a biofluid test (of matrices such as serum, urine, saliva) that detects circulating tumour cells or circulating cell-free tumour DNA (ctDNA) shed into the bloodstream by cancer cells undergoing apoptosis or necrosis. Those tests are much more practical compared to genotyping of tumour tissue, which has significant limitations such as tumour heterogeneity, invasiveness, difficulties with sampling and fact that tumour tissue acquired through a biopsy reflects the condition only at the time of the examination [54][55][56][57]. In addition, tumour-associated mutations detectable in various body fluids provide the information about the early detection, assessment of molecular heterogeneity of general disease, its prognosis, recurrence, monitoring of tumour dynamics and the success or failure of systemic therapies [40,54,57]. Prediction of prognosis in patients with curable cancer disease can already be achieved in several tumours such as breast cancer, melanoma, ovarian or colon cancers [40,57]. Liquid biopsy permits less invasive means of assessing the oncogenic mutation profile of a patient, and can guide the use of targeted molecular therapies resulting in an improvement of clinical outcome in oncological patients [40].

Electromagnetic field-based methods
According to the literature, the electromagnetic field can be a very useful tool in diagnosis and treatment of cancers [68][69][70]. Cormio et al. performed the study aimed to determine the diagnostic accuracy of non-invasive electromagnetic detection of bladder cancer by the tissue-resonance interaction method (TRIM-prob) with its overall sensitivity, specificity, positive and negative predictive values as well as diagnostic accuracy of 97.9%, 89.9%, 86.8%, 98.6%, and 93.6%, respectively. They concluded that TRIM-prob bladder scanning could be used to screen asymptomatic patients at high risk of developing a bladder cancer [70]. Also, surface plasmon resonance can be implemented for cancer diagnosis and photothermal therapy, in which plasmonic gold nanoparticles contribute to evoke strong electromagnetic fields on the particle surface, thus enabling tumour detection [69]. In addition, Zimmerman et al. have reported that intrabuccal administration of 27.12 MHz radiofrequency electromagnetic fields, which are amplitude-modulated at tumourspecific frequencies, results in detecting as well as blocking the growth of tumour cells in a tissue-and tumour-specific fashion in patients with various forms of cancer [68]. Interestingly, there is currently an increased interest in the use of salivary diagnostics in early detection of cancers by means of electromagnetic field, such as lung cancer [71,72].
Electromagnetic phenomena can be highly useful for diagnostic techniques, but if applied improperly, the electromagnetic field may also cause serious harmful effects such as leukaemia [73,74] brain tumour [74,75], breast cancer [74,76,77]. This depends on the intensity of the applied electric and magnetic fields, the time of exposure as well as the nature and the frequency of changes [78,79]. Because of the large variety of these factors, a wide spectrum of different scientific research studies and their applications are currently undertaken to elucidate the usage of electromagnetic phenomena for cancer diagnostics.
One particularly compelling branch where electric and magnetic fields are applied is in the study of exosomal vesicles (EVs), which have a diameter of approximately 30-100 nm. In case of EVs, magnetic beads proved to be very useful in various medical applications including cancer diagnostics [80,81]. Magnetic beads coated with monoclonal antibodies directed against antigens of the specific cells can be easily separated in a magnetic field. They constitute a powerful tool for the isolation of cells, and in particular of exosomes [82]. The schemes of such cell separation in body fluids in vivo and ex vivo are presented in Figures 1 and 2, respectively.

Electric field induced release and measurement method
The electric field induced release and measurement method (EFIRM) is a newly developed technology at UCLA. EFIRM is an electrochemical-based technique that measures the oxidation and reduction rates of a chemical reaction to perform quantification of a target biomolecule [28,38]. This is similar to the principles used for traditional glucose metres, which measure the oxidation and reduction rates of glucose oxidase reacting with glucose.
In case of EFIRM, a capture probe that is complementary to a ctDNA target is designed and then immobilized on the surface of a  gold electrode by encapsulating it in a conducting polymer matrix [83]. After the immobilization of the capture probe on the surface of the electrode, the clinical specimen (i.e. saliva or plasma) is placed on the surface of the electrode and a cyclic square wave (CSW) is applied. This CSW is designed to specifically lyse the exosomal structure that encapsulates the ctDNA sequence and aid in the DNA hybridization process [28] (Fig. 3). Following the incubation of the target sequence to the capture probe, a detector probe that is also complementary to the ctDNA is hybridized. This detector probe has fluorescein isothiocyanate (FITC) located at its terminal end, that is then complexed to an anti-FITC antibody with horseradish peroxidase (HRP) (Fig. 4).
Finally, the quantification of the amount of the target DNA is performed by adding 3,3 0 ,5,5 0 -Tetramethylbenzidine (TMB) and hydrogen peroxide readout substrate mix to the surface of the electrode. Reactions between the HRP enzyme, TMB and hydrogen peroxide will occur, and electronic circuits will be interfaced with the electrode to measure the magnitude of the reaction. If there is a large amount of ctDNA present, then, there will be a high electric current, but if no ctDNA is present, no significant amounts of electrochemical current will be measured.
The EFIRM technique was first deployed in 2009 for the examination of salivary biomarkers for oral cancer detection in a collaborative project between the UCLA School of Dentistry and UCLA School of Engineering [38]. More recent work on EFIRM with this platform on detecting ctDNA EGFR mutations in saliva has demonstrated near perfect clinical sensitivity and specificity for detection of lung cancer [38,56,84]. In a blinded pilot study, 40 patient saliva samples were   analysed using EFIRM and compared to tissue-based oncogenic analysis. Characterizing the performance of EFIRM (area-under-the-curve of 0.94 and 0.96 was achieved for detecting exon-19 deletion and the L858R mutations, respectively). A comparison of saliva with plasma samples showed R values of 0.98 and 0.99 for the exon-19 deletion and L858R mutation, respectively [40]. Another clinical application of the method that is currently investigated is the detection of oncogenic KRAS gene mutations in patients diagnosed with pancreatic cancer [40].

Conclusions
A wide variety of emerging saliva-based technologies have already demonstrated their credibility in the early detection of many cancerous diseases. It is evident that the trend will develop towards constant improvement of POC diagnostic tools, NGS methods, advanced PCR-and electromagnetic field-based technologies as well as liquid biopsy. Further research studies will reveal which method will be the most suitable to be applied in a clinical practice.