Salivary diagnostics holds tremendous potential for the diagnosis, prognosis, and monitoring of cancer.1 The specimen can be obtained simply, inexpensively, and less invasively than blood and thus can be collected without special training.1-3 Saliva is a complex mixture including components such as salivary gland secretions, mucosal transudations, desquamated epithelial cells, expectorated bronchial and nasal secretions, bacteria and bacterial products, viruses, and food debris.1 Due to this complexity, saliva contains a rich variety of potential biomarkers including proteins, enzymes, antibodies, DNA, RNA, and cytokines.2,4
In general, there are three methods for collecting saliva: salivary rinses, whole saliva, and stimulated saliva.2,3 A potential advantage of oral rinse collection methods is that the specimen contains whole saliva; it also has had contact with multiple sites including the oropharynx, oral cavity, and parts of the larynx and hypopharynx, whereas whole saliva or stimulated saliva only permit contact with the anterior oral cavity. Furthermore, concentrations of whole saliva components can vary greatly with hydration level. Since there is more contact with surfaces that contain secreted proteins, it is quite possible that the relative protein composition of the oral rinse would be different and perhaps more favorable than that of whole or stimulated saliva.2
Salivary diagnostics for cancer
Whole saliva contains serum components and therefore may be used for diagnosis, monitoring, and prognostic determination of cancers outside the mouth and throat, as recently reviewed by Malathi et al.1 In their review, they found that cerbB-2 and CA15-3 were differentially expressed in the saliva of women with breast cancer compared to healthy controls. Long noncoding RNA (lncRNA) are associated with lung, breast, and prostate carcinomas. Salivary mRNA markers have been defined for both lung and ovarian cancer. CA 125 is a tumor-associated antigen that is differentially expressed in serum and saliva of patients with oral, breast, and ovarian tumors, and salivary PSA levels correlate with serum PSA levels in patients with prostate cancer and therefore may become a useful marker of this disease.1
In a study involving patients in India, oral cancer screening reduced oral cancer mortality by more than 80 percent in tobacco and/or alcohol users.5 However, this method of screening, visual inspection followed by tissue biopsy, has only 64 percent sensitivity for oral cancer6 and 31 percent specificity for oral dysplasia or cancer.7 Many molecular tests, hypermethylation, RNA, and protein-based panels, are under development, but not validated.8-11 Thus there is a need for something better, and saliva is becoming the biologic sample of choice for head and neck cancer screening.
Work on salivary biomarkers for oral cancer detection has been extensively reviewed by Cheng et al.11 Markers include non-organic compounds; proteins including CD44, IL-8, cyclin D, and many others; DNAs including p53; mRNA including Il-8, OAZ1, and SAT, among others; microRNAs; oxidative stress-related molecules including peroxidase and superoxide dismutase; metabolomics; glycosylation-related molecules; and enzymes such as telemorase.11 Other technologies that use dyes, autofluorescence, or exfoliative cytology as adjuncts to the physical exam are used in clinical practice, but have not improved early detection rates.12,13
Risk assessment in clinical practice
Of the many oral cancer biomarkers studied, only a few are being translated to clinical practice. Among them are CD44, a cell surface transmembrane glycoprotein involved in cell proliferation and migration.14,15 CD44 is also a key tumor initiation marker16 that is over-expressed in the earliest stages of carcinogenesis.17,18 Soluble CD44 (solCD44), released by proteinases, is detectable in body fluids.19,20 Total protein, together with CD44, has also been shown to be an effective tumor marker. Both can be measured with simple, inexpensive assays.21-24
Prior work shows that the combination of solCD44 and protein levels in oral rinses can distinguish head and neck squamous cell carcinoma (HNSCC) cases from controls.22-23 More recent work suggests that sensitivity can reach 88 percent for stage I-III cancer and specificity as high as 95 percent depending on the population studied (unpublished data). Recently, this technology has been converted to a lateral flow test strip point-of-care and a laboratory test, which will be commercially available soon. The inclusion of a cancer stem cell marker allows assessment of risk sufficiently early that reversal of carcinogenesis via behavioral change could be possible even before a lesion is clinically identified.
Separate from CD44 and total protein, another group has performed a validation of the mRNA markers at the University of Michigan, Michigan State University, and the St. Johns Providence Health System in Detroit using technology developed by Dr. Wong at UCLA for the detection of oral cancer. The validation of these biomarkers demonstrated their feasibility in the discrimination of oral squamous cell carcinoma (OSCC) from healthy controls. In the five cohorts studied, the increase in IL-8 and SAT were statistically significant and remained top performers in terms of sensitivity and specificity. However, individual cutoff values for each of these markers and for the combined predictive model need to be further defined in large clinical studies.10 This work has been cited in the development of a risk stratification test recommended for use by the clinician only after suspicious visible lesions are observed and additional testing is warranted. The exact biomarkers used in the risk stratification system are not yet disclosed, to our knowledge.
One saliva-based risk assessment tool for HNSCC currently available is for oral human papillomavirus (HPV) DNA testing. HPV infection is rapidly becoming the most important risk factor for cancers of the oropharynx, with the incidence of HPV+ oropharyngeal cancer rising in the United States.25-26 This HPV is a salivary test that assesses an individual’s risk of developing HPV-related oral cancers by identifying various HPV oncogenic genotypes. However, it is uncertain as to whether the HPV detected by this test shows whether the infection is past or current or even one that will eventually develop into HNSCC, whereas studies show usage of CD44 and total protein able to detect risk of the onset of HNSCC regardless of etiology. As a result, the usefulness of an assay detecting only HPV infection in assessing risk has yet to be determined.27