INTRODUCTION:

Human saliva is a biological fluid of varying diagnostic potential with several advantages for disease diagnosis and prognosis, such as low invasiveness, minimum cost and easy sample collection with minimum discomfort to the patient/subject. Also handling of saliva during the diagnostic procedures is easier than blood as it does not clot and there is no risk of exposure of the laboratory technician to blood borne diseases. Hence processing and analysis of this biological fluid is the most important criteria and if tests could be easily conducted in clinics with saliva, for early detection of diseases, the quality of life for patients would be greatly improved.(1)

Significant proportions of cancers in the initial stages are asymptomatic and are not diagnosed or treated until they reach an advanced stage. Therefore early detection is the most effective means to reduce death from this disease. Currently diagnosis depends on thorough oral examination and [2] histopathological examination by taking a biopsy. Even though a definite diagnosis is based on biopsy, it would be beneficial if it could be done through non invasive techniques like salivary tumour marker analysis.

The diagnosis of oral cancer and/or the malignant potential of an oral lesion is based on various aspects such as (a) etiology‑associated with the use of tobacco, presence of factors such as detection of HPV (b) clinical appearance of the lesion (leukoplakic, erythroplakic, nodular, ulcerative, verrucous) (c) location of the lesion ‑ the high risk sites being floor of the mouth, ventrolateral aspect of the tongue etc., (d) histopathological aspects ‑ presence of epithelial dysplasia and (e) molecular biological aspects of the lesion (3)

CLASSIFICATION OF BIOMARKERS.

S:NO

BASIS

Based on bio molecules:

Based on disease state:

Based on other criteria:

TYPES

(a) DNA biomarkers

(b) RNA biomarkers

(d) Glyco biomarkers.

(a) Prediction biomarkers

(b) Detection biomarkers

(d)Prognosis biomarkers.

(a) Pathological biomarkers

(b) Imaging biomarkers

ROLE OF SALIVARY BIOMARKERS:

Till date, most of the biomarkers have been identified from various body fluids. Among which blood and saliva are the most widely studied body fluids that may contain reliable biomarkers for detecting cancer. It is an informative body fluid containing an array of analytes (Protein, mRNA and DNA) that can be used as biomarkers for translation and clinical applications.[5]

Clinical significance of salivary biomarkers in various malignancies is studied by several investigators. They explored for the presence of salivary proteomics and genomics signatures for breast cancer.[5] The Functions and clinical utility of saliva authors reported Her2/neu as the first salivary biomarker for breast cancer and also documented raised levels of CA15-3 and Her2/neu as well as low levels of p53 in patients with breast cancer. Then elevated salivary levels of CA 125 in patients with ovarian cancer. The salivary leptin was expressed in much higher amount in salivary gland tumors than in healthy parotid tissue. It has been reported that gastric cancer can also be identified at an early stage by using saliva proteome analysis .[5]

Identification of the combination of three mRNA biomarkers (acrosomal vesicle protein 1, ACRV1; DMX like 2, DMXL2 and dolichyl phosphate mannosyl transferase polypeptide 1, catalytic subunit, DPM1) could differentiate pancreatic cancer patients from chronic pancreatitis and healthy individuals.[5]

SALIVARY TRANSCRIPTOMIC BIOMARKERS:

Messenger (m) RNA is the direct precursor of proteins and in general the corresponding levels are correlated in cells and tissue samples [6]. At present, the main strategy to identify salivary transcriptomic biomarkers is through microarray technology. After profiling the transcriptomic biomarkers by microarray, they are validated by quantitative (q) PCR, the gold standard for quantification of nucleic acids.[6].

EXOSOMES:

Exosomes are small, right-side out cell-secreted vesicles of about 30–100 nm, derived from fusion of multivesicular bodies to plasma membranes.[7]

More recently, salivary mRNA were localized inside salivary exosomes and these nucleic acids were protected against ribonucleases in saliva ;Moreover, saliva exosomes have been discovered to regulate the cell-cell environment by altering their gene expression allowing us to better understand the molecular basis of oral diseases.[7]

SALIVARY MICRO RNA:

Micro RNAs (miRNAs) are encoded by genes but are not translated into proteins.[8] Hundreds of miRNAs from various organisms have been discovered, and functional assays have established that miRNAs serve important functions in cell growth, differentiation, apoptosis, stress and immune response as well as glucose secretion [8]. Proteins, mRNAs and microRNAs before and after pharmacological interventions may provide important information on drug efficacy and toxicity in the context of therapeutic responsiveness.[8].

CYTOKINES:

The cytokines, which include the interferons, tumour necrosis factor, and the interleukins, are a burgeoning and diverse family of peptide cell regulators. The availability of natural and recombinant cytokines has led to the use as anti-tumour agents and in limiting the myelo suppressive effects of cytotoxic chemotherapy.[9].

In 2004,one group have published that analysis of supernatant of mRNA of IL-8,IL-1beta,dual specificity protein phosphatise-1(DUSP-1)H3 histone family 3A(H3F3A or HA3),S100 calcium binding protein P(S100P), ornithine decarboxylase antizyme-1(OAZ1), spermidine or spermine N1-acetyl tranferase 1(SAT-1), as combination will have a specificity and sensitivity of 91 % in the diagnosis of oral squamous cell carcinoma [10].

SALIVARY BIOMARKERS IN ORAL CANCER:

The prior salivary transcriptomic studies have discovered 7 OSCC-associated salivary RNAs (IL-8, SAT, IL-1B, OAZ1, H3F3A, DUSP, S100P).[11] Report of increased salivary levels of cell cycle regulatory proteins including Cyclin D1 and ki67, glycolytic enzyme lactate dehydrogenase (LDH), matrix metalloproteinase (MMP)-9, as well as reduction in DNA repair enzyme, 8oxoquanine DNA glycosylase (OGG1) and Maspin, a tumor suppressor protein in oral cancer patients.Protein concentrations of both MMP1 and MMP3 were observed to be highly elevated in the saliva of OSCC patients compared to saliva from cancer-free controls [12]. Significantly higher salivary levels of IL-1, IL-6, IL-8 and TNF-a in oral cancer patients as compared to the patients with dysplastic oral lesions and controls[4] After using laser-capture micro dissection, have identified the expression of 2 cellular genes that are uniquely associated with OSCC: interleukin (IL) 6 and IL-8 . 75% positive expression of telomerase in saliva of oral cancer patients suggesting its usefulness as a supportive marker to diagnose oral cancer and also suggested that human telomerase reverse transcriptase (hTERT) analysis may be a potential biomarker for the diagnosis of oral cancer. Presence of p53 autoantibodies in saliva as well as serum of oral cancer patients demonstrated that its detection in saliva can offer a non-invasive method for the detection of a subset of tumors with p53 aberrations

A biomarker that can indicate lymphnode metastasis would be valuable to classify patients with OSCC for optimal treatment. A study have been performed on serum proteomic analysis of OSCC using 2-D gelelectrophoresis and liquid chromatography/tandem mass spectrometry. One of the down-regulated proteins in OSCC was identified as tetranectin, which is a protein encoded by the CLEC3B gene (C-type lectin domain family 3, member B).[13].

NID2 and HOXA9 promoter hypermethylation as biomarkers for prevention and early detection in Oral Cavity Squamous Cell Carcinoma tissues and saliva.[14].

SYSTEMIC TUMOR MARKERS IN SALIVA:

Estrogen Receptor-α:

Several mechanisms have been hypothesized to explain the novel biomarkers of breast cancer in saliva. The most likely mechanism is speculated to be estrogen receptor-alpha (ER-α) dependant for expression. This finding provides a clue to study the mechanism and expression of these proteins of multiple disease states at distant tissues. (15)(16) Estrogen receptors are found to be over expressed in cases of breast cancer. Binding of the estrogen to the estrogen receptor (ER) stimulates the proliferation of mammary cells leading to increased cell division and DNA replication leading to mutations. At the same time, genotoxic wastes are produced during the metabolism. Both these factors are said to be associated with the disruption of cell cycle, apoptosis and DNA repair leading to the formation of tumor. (17) The discovery of this marker for breast cancer in saliva, has therefore offered renewed interest in the potential use of saliva as a diagnostic fluid for other cancers as well.

HER2/neu and CA 15-3:

Appearance of breast cancer changes the set of proteins secreted by the salivary glands and the profile of salivary proteins is different compared to that of the healthy subjects. In a study done to compare the protein expression in benign and malignant breast cancer using isotope tagging, about 49 of the 130 proteins were differentially expressed. Studies also suggest that, additionally, there may be protein profiles that are unique to ductal carcinoma and fibroadenoma cancers. The protein, (c-erbB-2), also known as HER2/neu, is a prognostic breast cancer marker assayed in tissue biopsies from women diagnosed with malignant tumors. An elevated level of this marker indicates aggressiveness and poor prognosis for patient survival. Recent studies suggest that soluble fragments of the c-erbB-2 oncogene may be released from the cell surface and become detectable in patients with carcinoma of the breast. CA 15-3 was set as a standard to measure the diagnostic effectiveness of c-erbB-2 protein. The salivary and serological levels of c-erbB-2 and CA15-3 were significantly higher in the cancer patients, than the salivary and serum levels of healthy controls and benign tumor patients and also it was found that this protein showed high efficiency than the standard CA 15-3 protein. Pilot studies have indicated that the saliva test for this oncogene c-erbB-2 is both sensitive and reliable and is potentially useful not only in initial detection but also of the follow-up screening for breast cancer. (18)

CA125:

CA125 is a widely used tumor marker measured most often in women with cancers of the reproductive tissues including the uterus, fallopian tubes and ovaries. Other cancers that may cause abnormal CA125 levels include cancer of the pancreas, lungs, breast and colon (19).Tumor markers with high sensitivity and high specificity for endometrial cancer are not known at present, although CA-125 is often used in clinical practice. CA-125 values may also be elevated in a number of gynecologic (eg, endometrium, fallopian tube) and nongynecologic (eg, pancreas, breast, colon, lung) cancers. However, the most marked elevations (>1500 U/mL) are generally seen with ovarian cancer. (20) In a study conducted in ovarian cancer patients to compare the CA125 levels in saliva and serum, a linear correlation was observed with respect to the sensitivity of serum and saliva CA125 level. However, there was a drastic difference in the false positive rate of serum CA125 (72.7%) as against salivary CA125 (13.6%). Therefore salivary CA 125 had a better diagnostic value than the serum CA 125.(21)

Prostate specific antigen:

Prostate specific antigen (PSA) is a protein produced by the normal prostate cells. It is normally present in low levels in the serum of all adult men. The normal range is 0 to 4 ng/ml. Rising levels of PSA are associated with the prostate cancer. Also in women the greatest concentrations of this antigen is present in breast milk and amniotic fluid.(22) However, it has been clear that PSA is detectable not only in prostrate tissue but also in cancer tissues away from the prostate and normal tissues as well. Hence, it cannot be entirely relied on PSA for diagnosis of metastatic prostate cancer. PSA is detectable in normal man and often is elevated in benign prostatic hypertrophy, which may limit its value as a screening tool for prostate cancer(23) In an investigation done to determine the free and total PSA levels, and free to total (f/t) ratio in the fasting saliva, in comparison with the serum levels in normal as well as cancer patients, a significant difference between free and total PSA levels in both the saliva and serum samples was noticed, but the salivary f/t ratio was in correlation to that of the normal subjects which indicated that PSA in saliva cannot be efficiently used as an alternate to serum PSA.(24)An increasing number of systemic diseases and conditions have been shown to be reflected diagnostically in saliva. Efforts are also being made to apply salivary proteomics for diseases pecific biomarker discovery, such as lung, gastric and pancreatic cancer. In a study involving detection of salivary biomarkers in pancreatic cancer, revealed eleven mRNA biomarkers with high specificity and sensitivity that differentiated the pancreatic cancer patients from healthy individuals. The altered gene expression in saliva supernatant was discovered using Affymetrix Human Genome U133+2.0 array. The validation for pancreatic cancer detection was done by logistic regression model in combination with three mRNA biomarkers, yielding a high sensitivity and specific method of distinguishing cancer patients from healthy subjects. This is a proof that salivary biomarkers may be a novel diagnostic tool for the non invasive detection of a systemic cancer.( 25) Salivary mRNA and bacterial biomarkers would therefore emerge as highly specific and sensitive tools for lung cancer detection as well. (26)

Alpha fetoprotein:

Alpha fetoprotein (AFP) is a protein that has been encoded by the AFP gene. AFP is normally produced during fetal and neonatal development, by the liver, yolk sac and in small concentrations by the gastrointestinal tract. By the second year of life, AFP concentrations decrease rapidly and thereafter, normally, only trace amounts are detected in serum. Normal adults have serum AFP concentrations of less than 10ng/ml. AFP has also been demonstrated in various tumours such as hepatoma, hepatoblastoma, acute and chronic liver cirrhosis and so on. However, it has been implicated that salivary glands are not involved in the synthesis of these proteins. (27) In a preliminary investigation carried to detect the presence of AFP in the human saliva in patients with hepatocellular carcinoma, significant increase in the levels of AFP was observed with respect to the normal subjects. Also a significant correlation was noticed between saliva and serum levels of AFP. It is highly probable that the salivary AFP appears in saliva from plasma by passive seepage. AFP is a useful marker in hepatocellular carcinoma and germ cell tumors associated with extreme elevations >500ng/ml. Hence salivary AFP detection may prove to be a promising technique for detection of hepatocellular carcinoma with saliva as the diagnostic fluid.(28)

CA 19-9:

CA 19-9 is not sensitive or specific enough to be used as a screening marker for cancer, and it is not of diagnostic value to any specific type of cancer. Yet, it is mainly used as a general tumor marker. CA 19-9 can only be used as a tumor marker if the cancer is producing elevated amounts of it. Since CA 19-9 is elevated in about 65% of those with bile duct (hepatobiliary) cancer, it may be used to help evaluate and monitor people with this type of cancer. CA 19-9 might be a valuable new diagnostic tool in the preoperative differentiation between malignant and benign parotid tumors but further investigation in a larger number of patients is required to confirm its use as an efficient biomarker to detect specific tumors. (29)

Immunoglobulins:

Immunoglobulins are not classic tumor markers but are antibodies/blood proteins which are normally synthesized by the immune system cells to fight against the foreign substances/antigens. There is an elevated level of immunoglobulin in blood in certain bone marrow cancers such as multiple myeloma and Waldenstrom macroglobulinemia. Therefore the presence of high levels of specific monoclonal immunoglobulins is a sign of patients with myeloma or macroglobulinemia. These paraproteins are usually complete antibody molecules but may be isolated light chains or, rarely, heavy chains. They may be lambda or kappa light chains and of any immunoglobulin subtype. The levels of these immunoglobulins can be followed to see the prognosis of the related tumor, i.e., the amount of paraprotein serves as the index for tumor volume which can be used to know the response to the treatment. (30) A study in OSCC patients to detect the levels of IgA, IgG and haptoglobulin reported the significant increase of these markers in untreated OSCC patients compared to the control group. Also a significant increase in IgG and IgA was noticed in the OSCC treated patients compared to the level of salivary haptoglobulin. The IgG and IgA levels were also found to be increased in patients with precancerous lesions. The possible appearance of these immunoglobulins in saliva may be because of direct transudation from the blood or as a local defense mechanism against the tumor development. These salivary parameters evaluated from OSCC patients indicated the presence of IgG and secretory IgA which may prove as a significant prognostic value in early detection of OSCC. (31) Research studies have also reported the presence of β (2)-microglobulin in the saliva of patients with malignant tumors in the case of head and neck tumors. The malignant tumor cells are likely to excrete β (2)-microglobulin in higher amounts resulting in the infiltration of these proteins into saliva making this a possible method of diagnosing tumors.(32)

ß Glucuronidase:

ß glucuronidase is an acid hydrolase that deconjugates glucuronides, however little is known about human β glucuronidase.(33) The salivary ß glucuronidase is said to have application in the detection of head and neck tumors. The subsequent changes in the enzyme level can be used as a diagnostic index to detect the high risk group for malignancy and also to determine the prognosis during the course of the disease.(34) The assessment of cytokines / hormones in saliva has gained wide acceptance but little is known about its presence in saliva. Salivary peptide hormones such as EGF and transforming growth factor α (TGF-α) are likely to be involved in the promotion of cell proliferation. There has been evidence proving the role of salivary cytokines such as IL-8 and leptin in tumorigenesis in the oral cavity and salivary glands.(35) The expression of the cytokine leptin in saliva was found to be increasing significantly compared to the healthy parotid tissues in the salivary gland tumors. This cytokine was known to be produced directly by the salivary gland tumors and not imported from the blood. The assessment of these salivary hormones provides new ways for the detection of promising tumor markers.(36) A study aimed at the early detection of OSCC of the tongue using quantitative ELISA revealed five cytokines that were elevated in the treatment group when compared to the healthy subjects, which correlated with the decreased survival rate. IL-1α, IL-6, IL-8, vascular endothelial growth factor A (VEGF-A) and TNF-α were the five cytokines that could serve as potential biomarkers in screening and early detection of cancer.(37)

Kallikreins:

Kallikreins are a group of 15 secreted serine proteases possessing numerous physiological roles which are encoded by the genes Tissue kallikrein (KLK1) and Kallikrein related peptidase (KLK215). The aberrant expression of these kallikreins using immunological assays makes them useful as a tool for screening and diagnosing malignancy.(38)Kallikreins are gaining increased attention as they play a role of biomarkers in screening, diagnosis, and prognosis and monitoring of various cancers such as breast, lung, ovarian and prostate. Recent studies have proved the involvement of kallikreins with the establishment and progression of malignancy. These secretor products from the salivary vesicles of parotid gland are known to function in the proteolytic cascade pathway leading to the cleavage of extracellular matrix components and also in the processing of peptide growth factors such as EGF which facilitates cancer cell invasiveness and metastasis.(39) Human kallikrein 6 (hK6), also known as protease M, is known to express in salivary gland tumors. However, further studies are required to assess whether it can be used as a specific biomarker in salivary gland tumors.(40) Assessment of the expression of hK14 in normal salivary glands and tumors including pleomorphic adenoma, adenoid cystic carcinoma and mucoepidermoid carcinoma, clearly show significantly increased levels of the protein in pleomorphic adenoma and adenoid cystic carcinoma than normal salivary glands and mucoepidermoid carcinoma which is suggestive of its use as a potential biomarker in differential diagnosis of the salivary gland tumors.(41) Picogram quantities of Human glandular kallikrein 2 (hK2) have been detected in saliva. hK2 is a prostate secreted protease known to activate the zymogen form of PSA suggesting its function in a combined form. hK2 functions as an activator molecule and PSA as an effector molecule in amniotic fluid, colostrums and seminal plasma but not in saliva. Only low concentrations of hK2 were detected in saliva(42)

CONCLUSION:

With an attentive approach towards building novel techniques for the detection and validation of salivary markers for oral as well as systemic disease, saliva would serve as a very good diagnostic tool to improve the quality of life of the cancer patients. This review suggests that pursuing of saliva as a tool to detect the different types of cancer can pave the way for improved outcome of future non invasive investigation in the same field giving the patient a chance for a better quality of life.

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Received on 13.05.2016 Modified on 28.05.2016

Research J. Pharm. and Tech. 2016; 9(7):1007-1012.

DOI: 10.5958/0974-360X.2016.00190.6