Clinical Significance of Salivary Biomarkers in Oral Squamous Cell Carcinoma : A Review

ARTICLE INFO Article Type Review Article

Background and Aim:Oral squamous cell carcinoma (OSCC) accounts for approximately 3% of all cancers worldwide, and if diagnosed early, it has a five-year survival rate of around 85%; however, a late diagnosis may decrease the survival rate to 50%.Aberrant expression of several genes is associated with the hallmarks of OSCC including uncontrolled cell proliferation, poor differentiation, invasion, metastasis, and angiogenesis.The potential of molecular biomarkers for the diagnosis, prognosis, or monitoring of the treatment efficacy in OSCC has been extensively explored during the last decades.This study aimed to review the significance of salivary biomarkers in the treatment outcome of OSCC.

Materials and Methods:
The articles in scientific databases including Google Scholar, ScienceDirect, Medline, and PubMed, published between 2004 and 2017, were searched by using relevant keywords including OSCC, biomarkers, diagnosis, prognosis and treatment outcome.Thirty-four articles were reviewed in this study.Results: According to the findings of the reviewed studies, several salivary biomarkers including subcutaneous adipose tissue (SAT), interleukin-8 (IL-8), Cyfra 21-1, 8-hydroxy-2-deoxyguanosine, malondialdehyde, lactate dehydrogenase (LDH), Annexin A8, ErbB2, carcinoembryonic antigen (CEA), C-reactive protein (CRP), and salivary proteomic biomarkers might be used as indicators for the detection of oral cancer and premalignant oral disease (PMOD) and as a potential marker in the prognosis of OSCC.Conclusion: Salivary biomarker analysis seems to be a major advancement in the diagnosis of OSCC, and it is a fast-developing field in scientific research.The results indicate that salivary biomarkers can be useful diagnostic and prognostic tools in OSCC.

Introduction:
Oral squamous cell carcinoma (OSCC) accounts for approximately 3% of all cancers worldwide. (1)OSCC comprises more than 90% of all oral cancers, (2,3) and if diagnosed early, it has a five-year survival rate of about 85%. (4)Nowadays, even with various scientific advancements being explored and discovered every day, the worldwide prognosis of oral cancer has diminished rather than improved.This is a recognized public health care problem with a high mortality and morbidity rate. (5) wide range of molecules is involved in oral carcinogenesis.Aberrant expression of several genes is linked with the hallmarks of OSCC including uncontrolled cell proliferation, defective apoptosis, poor differentiation, epithelial/mesenchymal transition, metastasis, and angiogenesis. (6)The potential of molecular biomarkers for the diagnosis, prognosis, or monitoring of the treatment outcome in OSCC has been vastly explored during the last decades.In the era of personalized medicine, immunohistochemical detection and/or mRNA expression profiling of molecular biomarkers are expected to significantly contribute to the development of new screening tests with a high sensitivity and specificity and tailor-made therapies for OSCC. (7)aliva contains a large number of proteins and peptides, which are used as biomarkers for the detection of different oral and systemic diseases.Saliva is one of the most candid tools for the diagnosis of OSCC because of its direct contact with oral cancerous lesions. (8)Salivary biomarkers have been proven as cost-effective adjuncts in the diagnosis of oral and oropharyngeal carcinoma. (8)In their review on salivary genomics in oral cancer, Shah et al have insisted that saliva is a potential biomarker for OSCC as it contains locally expressed proteins including alpha-amylase, lactoferrins, lysozymes, proline-rich proteins, mucins, histatins, cystatins, and transferrin. (8)Markopoulos et al stated that the molecular diagnosis of OSCC can be done at three levels. (9)lterations in the cellular DNA causes variations in mRNA transcripts and intra-cellular or extracellular protein levels.Salivary biomarkers can be genetic, protein, or metabolomic markers. (10)onsidering the wide range of salivary biomarkers, this study aimed to review the related recent studies in this field to summarize and help in better understanding the clinical significance of salivary biomarkers in the diagnosis of OSCC.

Methods and Materials:
The literature over the past 13 years (2004-2017) were searched through Google Scholar, ScienceDirect, Medline and PubMed databases by using relevant keywords including OSCC, biomarkers, diagnosis, prognosis, and treatment outcome.Finally, 34 articles were reviewed in this study.

Results:
The findings of the reviewed studies indicated that the majority of salivary biomarkers including subcutaneous adipose tissue (SAT), interleukin-8 (IL-8), Cyfra 21-1, 8-hydroxy-2-deoxyguanosine, malondialdehyde, lactate dehydrogenase (LDH), Annexin A8, ErbB2, carcinoembryonic antigen (CEA), C-reactive protein (CRP), and salivary proteomic biomarkers might be used as indicators for the detection of oral cancer and premalignant oral disease (PMOD) and as a potential marker in the prognosis of OSCC.Table 1 demonstrates the summary of the reviewed studies.Salivary levels of immunosuppressive cytokines, IL-4, IL-10, IL-13, and IL-1RA, could prove to be potential biomarkers of OSCC and can be further investigated as markers of early detection and disease progression.Investigation and identification of potential biomarkers in human saliva for the early diagnosis of oral squamous cell carcinoma. (34)14 Wang et al

(30 OSCC patients and 30 apparently healthy individuals)
Ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) in hydrophilic interaction chromatography mode.Salivary metabolite biomarkers for the early diagnosis of OSCC were verified in this study and can be applied as a potential technique of preclinical screening of OSCC.Salivary RNA Signatures in Oral Cancer Detection. (35)

Panta and Venna
Quantitative polymerase chain reaction (qPCR) and microarrays (proven gold standard) followed by qPCR are the principal methods used in salivary RNA analysis.

Review article
The findings suggest that the detection of lncRNAs in saliva may be used as a noninvasive and rapid diagnostic tool for the diagnosis of oral cancer.Salivary analytes in patients with oral squamous cell carcinoma. (39)11 Fuchs et al 48  Salivary magnesium, calcium, copper, chloride, phosphate, potassium, sodium, total proteins, and amylase were evaluated.Sodium, potassium, and chloride were determined by indirect potentiometry, whereas copper, magnesium and phosphate were determined by atomic absorption spectrophotometry.
In patients with OSCC, increased salivary sodium and chloride might reflect their overall dehydration status due to alcohol consumption rather than a consequence of OSCC itself.Salivary zinc finger protein 510 peptide as a novel biomarker for detection of oral squamous cell carcinoma in early stages. (40)11 Jou et al 77 (47 OSCC patients and 30 healthy donors) A total of 77 salivary samples from both groups were analyzed using MALDI-TOF MS technology.
Identifying 24-mer ZNF510 peptide as OSCC-related salivary biomarkers via proteomic approach proved useful in adjunct diagnosis for early detection rather than specific diagnostic marker for progression of OSCC patients.Tumor and salivary matrix metalloproteinase levels are strong diagnostic markers of oral squamous cell carcinoma.The increase reported in salivary tumor markers may be used as a diagnostic tool, especially when a concurrent analysis for significantly increased markers is done.Salivary transcriptome diagnostics for oral cancer detection. (44)04 Li et al 64 (32 primary T1/T2 OSCC patients and 32 normal subjects) RNA isolation was done from the saliva supernatant, followed by two-round linear amplification with T7 RNA polymerase.
The utility of salivary transcriptome diagnostics is successfully demonstrated in this study for oral cancer detection.

Discussion:
Salivary proteomics is developing through the identification of unique biomarkers for early detection approaches including metabolomics, proteomics, genomics, and bioinformatics. (17)he salivary biomarkers are used because saliva encompasses a wide range of compounds, (45) it is easily accessible, (46) and the sampling process is comfortable for patients. (47)The method is noninvasive and safe with low chances of pathogen transmission in comparison with blood samples. (48)Also, saliva is easy to store (49) and does not coagulate.The systematic analysis of salivary proteomic biomarkers and screening of saliva provides an efficient diagnostic tool to turn salivary diagnostics into clinical reality for oral cancer.Because of cellular and molecular heterogeneity of OSCC, several genes are involved in oral carcinogenesis; therefore, the use of several potential biomarkers renders a more precise diagnosis than the use of any marker alone. (17)In Addition, due to the characteristics of saliva, such as its proximity with the oral cavity and noninvasive sampling, the focus on OSCC has been changed to serum and saliva analysis as an alternative to tissue analysis. (17)Salivary biomarkers of OSCC can be used for cancer detection and monitoring non-cancerous activities. (17)The salivary biomarkers discussed in this study can be used for the detection of altered mRNA transcripts and changed intra-cellular or extra-cellular protein levels, and they can be genetic, protein, or metabolomic biomarkers, (45) The detection of these changes by the use of new technologies such as mass spectrometry, gel electrophoresis, chromatography, microarrays, high performance liquid chromatography (HPLC), polymerase chain reaction (PCR), and enzyme-linked immunosorbent assay (ELISA) is a developing field associated with point-of-care technologies, electrochemical detection, RNA sequencing, and liquid biopsy. (17)urthermore, primary screening by the use of saliva could be an excellent choice considering the advancements in proteomics and genomics.
The extensive sighting of biomarkers and their authentication will change the field of diagnosis for oral cancers and non-cancerous activities.This review provides a comprehensive assessment of the developing diagnostic proteomic tools and biomarkers for the early detection and diagnosis of OSCC by the use of saliva.

Conclusion:
According to the reviewed studies, salivary biomarker analysis seems to be a major development in the diagnosis of OSCC, and it is a fastgrowing field in scientific research.The results show that salivary biomarkers can be a useful diagnostic and prognostic tool in OSCC.
expression of MMP1, MMP3, MMP10, and MMP12 in oral epithelial tissue using Affymetrix U133 2.0 Plus GeneChip arrays, followed by quantitative reverse transcription-PCR (qRT-PCR) for MMP1, and determination of MMP1 and MMP3 concentrations in saliva.