Saliva diagnostics: Salivaomics, saliva exosomics, and saliva liquid biopsy

Abstract

Background: Each day, humans produce approximately 0.5 through 1.5 liters of saliva, a biofluid that is rich in biological omic constituents. Our lack of understanding how omic biomarkers migrate from diseased tissue to the saliva has impeded the clinical translation of saliva testing. Although such biomarkers must be conveyed via the vascular and lymphatic systems to the salivary glands, the molecular mechanisms that underlie this transport remain unclear. Although COVID-19 highlighted the need for rapid and reliable testing for infectious diseases, it represents only one of the many health conditions that potentially can be diagnosed using a saliva sample.

Types of studies reviewed: The authors discuss salivaomics, saliva exosomics, and the mechanisms on which saliva diagnostics are based and introduce a novel electrochemical sensing technology that may be exploited for saliva liquid biopsy.

Results: The utility of saliva for screening for lung cancer is under investigation. Saliva testing may be used to stratify patients, monitor treatment response, and detect disease recurrence. The authors also highlight the landscapes of saliva-based SARS-CoV-2 testing and ultrashort cell-free DNA and outline how these fields are likely to evolve in the near future.

Practical implications: Breakthroughs in the study of saliva research, therefore, will facilitate clinical deployment of saliva-based testing.

Keywords: Saliva diagnostics; biomarker; liquid biopsy; saliva exosomics; salivaomics.

Figure 1.

Extracellular RNA (exRNA) Atlas. exRNA profiles are derived from various biofluids, including saliva. Small RNA sequencing assays are used to profile various disease conditions from diverse RNA sources and via different isolation methods. All the RNA-seq data, including saliva exRNAs, were extracted from the results deposited in the exRNA Atlas (https://exrna-atlas.org). Manufacturer information was not available in the database except for the serum and plasma kit.

Figure 2.

Structure and contents of typical salivary exosomes. The exosome is surrounded by a phospholipid bilayer, carrying many cell type–specific cargos. CD: Cluster of differentiation. ctDNA: Circulating tumor DNA. mRNA: Messenger RNA. miRNA: MicroRNA. piRNA: Piwi-interacting RNA. snoRNA: Small nucleolar RNA. Adapted with permission of the publisher from Nonaka and Wong.

Figure 3.

Mechanistic link between cancer and saliva. Cancer-derived exosomes enter the circulation and reach the salivary glands. Exosome uptake at salivary gland acinar cells occurs via endocytosis or membrane fusion. Two types of salivary exosomes are released into the saliva. Cancer-derived exosomes are released through exocytosis, and acinus-derived exosomes are released through fusion of multivesicular bodies with the plasma membrane. Both types of salivary exosomes carry cargos that include cancer-derived products. Adapted with permission of Elsevier from Nonaka and Wong.

Figure 4.

Schematic of electric field–induced release and measurement assay. The cyclic-square wave of the electrical field is applied to release and detect the mutations. Hybridized sequences are measured on the electrochemical sensor with a capture probe precoated in conducting polymer. The horseradish peroxidase (HRP)-labeled reporter probe generates amperometric signals when there is a reaction with tetramethylbenzidine substrate under a −200 mV electrical _field. ctDNA: Circulating tumor DNA. Adapted with permission of the publisher from Nonaka and Wong.

Figure 5.

Liquid biopsy for noninvasive bedside assessment and treatment response monitoring. T790M is a recurrent missense mutation within the tyrosine kinase domain of the epidermal growth factor receptor gene. Exon 19del (Exon 19 deletion) and L858R (amino acid substitution from leucine [L] to arginine [R] at codon 858) are mutations that occur in the epidermal growth factor receptor gene. ctDNA: Circulating tumor DNA.

Figure 6.

Molecular profile of ultrashort cell-free DNA (uscfDNA). A. Broad range cfDNA sequencing reveals a distinct population of ultrashort low-molecular-weight cell-free DNA in plasma. B. Functional peak analysis of plasma fragments reveals an enhanced proportion of regulatory elements in the fragments between 40 base pairs (bp) and 70 bp. mncfDNA: Mononucleosomal cell-free DNA. ncRNA: Noncoding RNA. rRNA: Ribosomal RNA. TTS: Transcription termination site. UTR: Untranslated region. Adapted under a Creative Commons Attribution 4.0 International License from Cheng and colleagues.