A Review on Salivary Genomics and Proteomics ... - Springer Link

Ind J Clin Biochem (Oct-Dec 2011) 26(4):326–334 DOI 10.1007/s12291-011-0149-8

REVIEW ARTICLE

A Review on Salivary Genomics and Proteomics Biomarkers in Oral Cancer Franky D. Shah • Rasheedunnisa Begum • Bhairavi N. Vajaria • Kinjal R. Patel Jayendra B. Patel • Shilin N. Shukla • Prabhudas S. Patel

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Received: 4 July 2011 / Accepted: 4 July 2011 / Published online: 9 August 2011 Ó Association of Clinical Biochemists of India 2011

Abstract Oral cancer has emerged as an alarming public health problem with increasing incidence and mortality rates all over the world. Therefore, the implementation of newer screening and early detection approaches are of utmost importance which could reduce the morbidity and mortality associated with this disease. Sensitive and specific biomarkers for oral cancer are likely to be most effective for screening, diagnosis, staging and follow-up for this dreaded malignancy. Unlike other deep cancers, oral cancer is located in oral cavity. Hence, the direct contact between saliva and oral cancer lesion makes the measurement of tumor markers in saliva an attractive alternative to serum and tissue testing. The DNA, RNA and protein molecules derived from the living cancer cells can be conveniently obtained from saliva. Thus, salivary biomarkers, a non-invasive alternative to serum and tissuebased biomarkers may be an effective modality for early diagnosis, prognostication and monitoring post therapy status. In the current post-genomic era, various technologies provide opportunities for high-throughput approaches to genomics and proteomics; which have been used to evaluate altered expressions of gene and protein targets in saliva of oral cancer patients. The emerging field of F. D. Shah B. N. Vajaria K. R. Patel J. B. Patel P. S. Patel (&) Biochemistry Research Division, The Gujarat Cancer & Research Institute, Asarwa, Ahmedabad 380 016, Gujarat, India e-mail: [email protected] R. Begum Biochemistry Department, M.S. University of Baroda, Vadodara, Gujarat, India S. N. Shukla The Gujarat Cancer & Research Institute, Asarwa, Ahmedabad 380 016, Gujarat, India

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salivary biomarkers has great potentials to prove its clinical significance to combat oral cancer. Hence, we have reviewed importance of several salivary genomics and proteomics biomarkers for oral cancer. Keywords Oral cancer Salivary biomarkers Proteomics Genomics

Oral Cancer: A Leading Malignancy in India Oral cancer is the 15th most prevalent cancer with the age standardized incidence rate of 3.9 per 100,000 population worldwide [1]. This dreaded malignancy stems as the major health concern due to rising trends in younger population. The Indian subcontinent accounts for one-third of the world burden of this malignancy [2]. In India, the age standardized incidence rate of oral cancer is 12.6 per 100,000 population and a sharp increase in the incidence rate of this cancer has been reported in recent years [3]. It is the most common form of cancer and accounts for increasing number of cancer related deaths among men in India [2]. According to the rural and urban registry reports of the Gujarat Cancer and Research Institute, Ahmedabad, the estimated age standardized incidence rate of oral cancer is 24 and 33.3 per 100,000 population, respectively [1]. A recent pooled analysis from the International Head and Neck Cancer Epidemiology consortium based on over 10,000 cases and 15,000 controls support significant role of tobacco and alcohol use in etiology of oral cancer [4]. The high incidence of oral cancer in India has also been linked with habits of tobacco chewing and smoking [5]. The repeated exposures of carcinogenic insults (e.g. tobacco chewing) to oral mucosal cells increase the risk for development of multiple independent premalignant and

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Fig. 1 Stages of oral cancer progression

malignant lesions from the accumulation of genetic alterations of oncogenes and tumor suppressor genes supporting the ‘‘field cancerization’’ theory. In the field cancerization model, multiple oral cancers develop from separate, independent cell clones [6].

Oral Cancer: Pathogenesis and Challenges Oral cancer arises through a series of histopathologic stages from benign hyperplasia to dysplasia to carcinoma in situ followed by invasive squamous cell carcinoma (Fig. 1). The malignancy is usually preceded by premalignant lesions like leukoplakia, erythroplakia and oral submucous fibrosis with a transformation rate ranging from 0 to 20% in 1–30 years, according to the type of lesion. In India, oral leukoplakia is considered to be potentially malignant [7]. Globally, the 5 year mortality rate of oral cancer is about 50% and has not changed significantly in recent years despite of the advances in surgery, radiotherapy and chemotherapy. This is attributed mainly to late diagnosis, poor response of tumor to chemotherapy and radiotherapy as well as insufficient biomarkers for early diagnosis and post therapeutic monitoring [8, 9]. The main reason for late stage presentation of the disease is the ignorance of lesions either by patients or clinicians. This is also accounted due to lack of awareness of malignant potentials of small lesions of oral cancer. Health education programs aimed at motivating patients for early diagnosis have also been largely unsuccessful because of incomplete understanding of the disease [10]. Further, detection of an oral cancer at stage I carries a prognosis of 80% survival, while the same lesion at stage III carries a 20% survival. This difference could affect not only the quality of life for

the patients but also the cost of the medical treatments of a stage I versus stage III oral cancer patients. In addition, early detection of cancer would also lead to fewer side effects from cancer treatments such as chemotherapy and radiotherapy and to a better prognosis. Moreover, oral cancer has a very high recurrence rate. Patients who survive a first encounter with this disease have up to a 20 fold increased risk of developing a second cancer [11, 12]. Thus, early identification of recurrence or a second primary tumor remains an important challenge. Therefore, implementation of an early detection scheme would have a positive impact on prognosis of the disease. Microscopic investigations of the progressive cancer are often conducted too late for intervention. It is also impractical to use imaging techniques for cancer screening, since they are time consuming and expensive. These techniques are typically used for confirmation due to their insensitivity for small lesions. Currently, the therapeutic modalities for oral cancer patients are based on traditional stage predicting indices and on histological grading. However, these predictors are subjective and relatively unreliable due to the nature of tumor and its response to therapy [13]. Therefore, scientists have been searching for alternative approaches, which can be helpful in early diagnosis and ultimately improve mortality of oral cancer. Moreover, better understanding of the biological nature of this aggressive disease is also mandatory. There has been ever growing efforts dedicated to the understanding of basic biology of the disease. These efforts have been focused on the identification of biological indicators for early detection of its molecular nature and aggressiveness. Recent advancements in oral cancer research have lead to the development of potentially useful diagnostic tools at clinical and molecular levels for early detection and better

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management of oral cancer. Latest tools developed for early clinical detection of oral cancer include tolonium chloride or toluidine blue dye, Oral brush biopsy kits, ViziLite Plus, salivary diagnostics and several imaging devices such as Velscope and multispectral optical imaging systems [10]. However, oral cancer has remained a great challenge and despite the wide availability of all advances, no noticeable progress has been made in achieving earlier diagnosis of this disease. One way of increasing the range of diagnostic options in the case of primary oral tumors and recurrence is to monitor the level of circulating tumor markers which have adequate sensitivity and specificity. Saliva has emerging role for the investigation of such circulating biomarkers which have relatively better sensitivity and specificity with regards to diagnosis, prognostication and treatment monitoring of the disease.

Saliva: The Mirror of Human Health Saliva is a complex fluid composed of secretions from the salivary glands and gingival crevicular fluid. Ninety percent of saliva is produced by the major salivary glands: the parotid, submandibular and sublingual glands. Approximately, 10% of saliva is produced by minor salivary glands clustered in the oral mucosa (lingual, labial, buccal, palatine, glossopalatine) [14]. Saliva has wide range of functions in normal human physiology (Fig. 2). It plays important role in maintaining oral and dental health. It participates in smooth ingestion and digestion of food; mediate taste sensations. Saliva also exerts a wide range of protective functions on oral tissues and teeth, including facilitating the demineralization and remineralization of teeth, modulating the adhesion of microorganisms to teeth and other oral surfaces and buffering dietary acids [9, 15]. Saliva is an important body fluid for the detection of

Fig. 2 Functions and clinical utility of saliva

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physiological and pathological conditions of human body. It is a complex and dynamic biological fluid containing wide range of compounds. In addition, saliva is a good indicator of the plasma/serum levels of various substances such as hormones and drugs. In the last few years, scientific interest has been raised to salivary analysis not only for various compounds present into saliva (e.g., drugs, pollutants, hormones), but also for its well-documented relation with bacterial, viral and systemic diseases. Therefore, it can be used as a non-invasive method for monitoring plasma concentrations of medicines or other substances and for assessment of the severity of an illness [16]. Due to its diverse biological functions, salivary testing is rapidly growing as a practical and reliable means in clinics and research to recognize early signs of systemic illness and exposure to risk factors. The components of saliva act as a ‘‘mirror of the body’s health’’. Because of the widespread use and growing acceptability of saliva as a diagnostic tool, Various investigators have focused their efforts to establish its clinical usefulness. The data have proved significant utility for researchers, health care professionals and community health program personals to detect and monitor diseases and to improve general health of the public.

Role of Salivary Biomarkers in Cancer 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. Saliva has the advantages that it contains low background of normal material and inhibitory substances as well as fewer complexes than blood [17]. 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 [18]. Saliva has many advantages as a clinical tool over serum and tissues, including simplicity of collection, storing and shipping, cost effectiveness, easy availability of large sample volume for analysis and repeated sampling for monitoring over time. The non-invasive saliva collection techniques dramatically reduce anxiety and discomfort of the patients. Saliva is also easier to handle for diagnostic procedures because no special equipment is needed for saliva sample collection and it does not clot, thus reducing the manipulations which may be required for biochemical analysis [19]. Clinical significance of salivary biomarkers in various malignancies is studied by several investigators. Streckfus et al. [20, 21] explored for the presence of salivary proteomics and genomics signatures for breast cancer. The authors reported Her2/neu as the first salivary biomarker


for breast cancer and also documented raised levels of CA 15-3 and Her2/neu as well as low levels of p53 in patients with breast cancer. Chen et al. [22] described elevated salivary levels of CA 125 in patients with ovarian cancer. Schapher et al. [23] also suggested that 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 [24]. Wong et al. [25] have also identified that combination of three mRNA biomarkers (acrosomal vesicle protein 1, ACRV1; DMX like 2, DMXL2 and dolichyl phosphate mannosyltransferase polypeptide 1, catalytic subunit, DPM1) could differentiate pancreatic cancer patients from chronic pancreatitis and healthy individuals. Thus, the saliva based analysis; a non-invasive alternative to serum analysis can be an effective modality for diagnosis and prognostication of cancer as well as for monitoring post-treatment therapeutic response of the patients. Hence, the development of salivary diagnostic tools is of paramount importance, especially in identification of high risk group, patients with premalignant lesions and patients with previous history of cancer [26].

Salivary Genomics and Proteomics Biomarkers in Oral Cancer Among all the malignancies, oral cancer is one such malignancy where saliva examination for detection can show the greatest benefit because of its direct contact with oral cancer lesions. The most important point for selecting saliva as a diagnostic tool is that it also contains the fallen cells in oral cavity which allow saliva to be the first choice of screening and identification for potentials biomarkers for oral cancer [8]. Several reports on salivary biomarkers in oral cancer have shown significant clinical usefulness for oral cancer as summarized in Table 1. As pointed out in the table, earlier reports have focused on salivary ‘‘tools’’ for measuring changes in specific salivary molecules such as proteins or nucleic acids. The studies have examined genomic and proteomic targets such as DNA aberrations, mRNAs, enzymes, cytokines, growth factors, metalloproteinases, telomerase, cytokeratins etc. in oral cancer [13, 17, 27–31]. The first report of saliva as a diagnostic tool for oral cancer detection was published in 2000 by Liao et al. [32]. The authors claimed that exon 4, codon 63 of the p53 gene was mutated in salivary DNA from five of eight (62.5%) oral cancers patients. In addition, autoantibodies against p53, the aberrantly expressed protein in patients with oral cancer has been identified in both saliva as well as serum [33–35]. TP53 was the only gene with similar incidences of loss of heterozygosity (LOH) and mutations.

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LOH was shown to occur more frequently than gene mutations in oral cancer. El-Naggar et al. [36] found that 49% of the saliva samples of oral cancer patients had LOH in at least one of the 25 markers studied. It is well established that oxidative stress plays an important role in progression of oral cancer. Bahar et al. [37] documented that salivary reactive nitrogen species were significantly higher, while all salivary antioxidants were significantly lower in the oral cancer patients as compared to the controls. This increase in reactive nitrogen species may be the event leading to the consumption and reduction of salivary antioxidants resulting in the oxidative damage to DNA and proteins, and possibly leading to progression of oral cancer. Recently, Shiptzer et al. [13] reported 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, 8-oxoquanine DNA glycosylase (OGG1) and Maspin, a tumor suppressor protein in oral cancer patients. Sato et al. [38] found significantly increased interleukin (IL)-6 levels in saliva of oral cancer patients than controls. Brailo et al. [39] also studied alterations in salivary IL-6 and tumor necrosis factor alpha (TNF-a) in patients with oral leukoplakia. They observed that salivary IL-6 and TNF-a levels were significantly higher in patients with oral leukoplakia as compared to the healthy individuals. IL-6 inactivates p53 tumor suppressor gene by supporting the hypermethylation of its promoter region which results in suppression of apoptosis and uncontrolled cell proliferation. TNFa activates NFjB transcription factor which stimulates cell proliferation and blocks apoptosis and additionally enhances secretion of proinflammatory cytokines. Rhodus et al. [27] reported 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. Considering, the fact that same cytokines were significantly elevated in both oral cancer and oral premalignant lesions, it may have a diagnostic value as the marker of malignant transformation of oral premalignant lesions. In addition, IL-6 also correlated with the recurrence of oral cancer [40]. Zhong et al. [30] found 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. Telomerase is a ribonucleoprotein which aid to elongate repeat sequence at the end of the chromosomes. Telomerase reactivation might be prerequisite for development of malignant cells from the somatic cells by escaping from the proliferative limitations of cellular senescence. Recent research has been directed towards detecting the human papilloma virus (HPV) in

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Table 1 Clinical significance of various salivary biomarkers in oral cancer Biomarkers

Inference

Ref

CycD1, Ki67

All the biomarkers were significantly altered in oral cancer and found to be useful as a supportive tool for diagnosis, prognosis and post-operative monitoring.

[13]

These proangiogenic, proinflammatory cytokines were found to be elevated in whole saliva of oral cancer patients and oral precancers as compared to controls which suggested its utility as surrogate indicators of carcinogenic transformation from oral precancer to oral cancer.

[27]

Telomerase

Increased telomerase activity found in saliva of oral cancer patients suggested that the telomerase in saliva could be useful biomarker for oral cancer.

[30]

p53 Autoantibodies

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. Altered salivary levels of reactive nitrogen species and antioxidants in oral cancer patients suggested the possibility of a direct link between salivary free radicals, antioxidants and oral cancer which may contribute to its diagnosis and treatment.

[33]

LDH, MMP-9 OGG1, Maspin IL-1, IL-6 IL-8, TNF-a

Reactive nitrogen species

[37]

IL-6, TNF-a

Significantly higher levels of salivary IL-6 and TNF-a were observed in patients with oral leukoplakia compared to healthy controls. The alterations in salivary IL-6 and TNF-a might play a significant role in development of oral leukoplakia.

[39]

HPV

Detection of HPV in salivary rinses has potentials for development of molecular screening for HPV-related oral cancer.

[41]

M2BP, MRP14

The data proved that these new targets may lead to a simple clinical tool for the non-invasive diagnosis of oral cancer and suggested that patient-based salivary proteomics is a promising approach to the discovery of biomarkers for oral cancer detection.

[42]

Actin, myosin

Actin and myosin are promising salivary biomarkers for distinguishing premalignant and malignant oral lesions.

[43]

CD59, Profilin 1 Catalase

Transferrin

Salivary transferrin levels in oral cancer patients strongly correlated with the size and stage of the tumor.

[44]

Salivary mRNA

The study developed a method for the multiplex RT-PCR, which made it possible to examine a large number of mRNAs from one droplet of saliva.

[45]

DNA hypermethylation

Methylation array analysis of saliva can produce a set of cancer related genes that are specific and can be used as combined biomarkers for early detection of oral cancer. An assay was developed that could rapidly quantify the promoter hypermethylation of the gene of interest and could potentially be applied into a clinical setting.

[46, 48]

Salivary IL-8 mRNA and protein

EC sensor was developed with multiplexing biomarker detection for salivary diagnostics. IL-8 mRNA and IL-8 protein levels measured by the EC sensors showed significant differences between oral cancer patients and controls.

[49]

saliva, as one of the etiological factor in oral cancer. The incidence of HPV positivity in patients treated for oral cancer is estimated to be more than 45% [9, 41]. The cellular and molecular heterogeneity of oral cancer and the large number of genes potentially involved in oral carcinogenesis emphasize the importance of studying gene expression changes in a global scale by proteomics. The modern high throughput genomic and proteomic approaches have been extensively used to study the altered expressions of genes and proteins in oral cancer. It may be helpful to facilitate the identification of potential biomarkers for oral cancer. With the advances in mass spectrophotometry, there is ongoing development in salivary proteomics for biomarker identification of oral cancer. Indepth analysis of human salivary proteome by Hu et al. [42] revealed several salivary proteins at differential levels between oral cancer patients and matched controls. The

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approach successfully validated five candidate biomarkers including Mac-2 binding protein (M2BP), myeloid related protein 14 (MRP14), CD59, profilin 1 and catalase using immunoassays on independent set of oral cancer patients and matched controls [39]. Using quantitative proteomics approach, de Jong et al. [43] also observed consistently increased levels of actin and myosin in saliva samples from individuals with malignant oral lesions as compared to the premalignant lesions. The authors concluded that actin and myosin are promising salivary biomarkers for distinguishing premalignant and malignant oral lesions. Salivary transferrin is also validated as a biomarker for detection of early stage oral cancer [44]. The tumor-specific DNA in saliva could also be used as biomarker for oral cancer [45]. Methylation array of salivary DNA was supported as an effective biomarker for early detection of oral cancer [46]. Hypermethylation on the promoter of DNA in specific gene


such as p16 was found in serum as well as also in saliva [47, 48]. Since mRNA is the direct precursor of protein and as the corresponding levels are correlated in cells and tissue samples, salivary mRNA for dual specificity phosphatase 1 (DUSP1), H3 histone, family 3A (H3F3A), IL-1B, IL-8, ornithine decarboxylase antizyme 1 (OAZ1), spermidine/ spermine N1-acetyltransferase (SAT) and S100 calcium binding protein P (S100P) are documented as the biomarkers of oral cancer [45, 49]. Brinkmann et al. [50] also validated four transcriptome [IL-8, IL-1B, spermidine/ spermine N1-acetyltransferase 1 (SAT1) and S100P] and three protein (IL-1B, IL-8 and M2BP) biomarkers which were significantly elevated in oral cancer. The authors also concluded that these biomarkers are discriminatory and reproducible in different ethnic cohorts. Several studies on polymorphism of several genes like IL-6, IL-8, TNF-a, vascular endothelial growth factor (VEGF), cytochrome P4501A1 (CYP1A1), glutathione-S-transferase T1 (GSTT1) and glutathione-S-transferase M1 (GSTM1) were found to be associated with the development of oral cancer [51–55]. Saliva is a convenient source of genomic DNA that has been proposed to offer non-invasive approach than blood/ tissue based analysis. Thus, it can be envisioned that salivary genomics will eventually be used for genomic screening of oral cancer. Hence, further studies with large cohort size are required to evaluate the role of gene polymorphism in oral cancer development from the fallen cancer cells in saliva.

Recent Data on Salivary Biomarkers from Our Laboratory We have studied several biomarkers associated with cancer susceptibility invasion and metastasis as well as glycosylation from saliva samples obtained from 53 oral cancer patients and 53 controls. Susceptibility Markers in Saliva We evaluated role of CYP1A1, GSTT1 and GSTM1 gene polymorphism from saliva to assess their role as cancer susceptibility markers. The CYP1A1 gene encodes an enzyme with aryl hydrocarbon hydroxylase activity. Formation of aryl epoxides by aryl hydrocarbon hydroxylase is the first step in the metabolism of polycyclic aromatic hydrocarbons from tobacco [53]. GST, a very important Phase II family of enzymes catalyzes the detoxification of a wide variety of active metabolites of tobacco carcinogens. GSTM1 catalyzes the conjugation of glutathione tripeptide to polycyclic aromatic hydrocarbons diol epoxides whereas GSTT1 participates in detoxification of monohalomethanes and reactive diol epoxides [54, 55]. Polymorphisms in the

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genes that code for these enzymes that are potentially involved in either the activation (Phase I) or detoxification (Phase II) of chemical carcinogens in tobacco may alter expression or function, thus increasing or decreasing the activation or detoxification of carcinogenic compounds. The CYP1A1 gene polymorphism (CYP1A1*2A) in our study showed that the prevalence of CYP1A1 homozygous wild (m1/m1) genotype was 56% in cases and 48% in controls. The distribution of CYP1A1 heterozygous (m1/ m2) genotype was 32% in cases and 48% in controls. Whereas, the prevalence of CYP1A1 homozygous variant (m2/m2) genotype was 12% in cases and 8% in controls. GSTM1 analysis from saliva showed that 36% of controls and 36% of patients represent GSTM1 null genotype which is in accordance with our previous blood based studies [56]. Salivary analysis of GSTT1 showed that the frequency of GSTT1 null genotype was 24% in oral cancer patients and 16% in controls. Null GSTT1 genotype was found to be higher in oral cancer patients as compared to the controls which suggested that individuals with null genotype may be more susceptible to develop oral cancer (Unpublished data). Invasion and Metastasis Related Salivary Biomarkers It is well documented that oral cancer has a high degree of local invasiveness and a high rate of metastasis to cervical lymph-nodes. Tissue invasion and metastasis requires extensive remodeling and degradation of extra cellular matrix and basement membrane. Gelatinases; especially, gelatinase A i.e. MMP-2 and gelatinase B i.e. MMP-9 have been shown to play a significant role in invasion and metastasis as they degrade type IV collagen, a major component of basement membrane. Our results indicated that salivary levels of MMP-2 and MMP-9, the invasion and metastasis related markers also play important role in the pathogenesis of oral cancer (Unpublished data). We observed that both active as well as latent forms of salivary MMP-2 and MMP-9 were significantly higher in oral cancer patients as compared to the controls. The receiver operating characteristic (ROC) curve analysis revealed that all forms of MMP-2 and MMP-9 could significantly discriminate between oral cancer patients and controls. The results are in accordance with our previous study on MMP2 and MMP-9 from plasma and tissues obtained from oral cancer patients [57–59]. Salivary Glycoproteins Aberrant glycosylation is the universal feature of cancer. Various glycoconjugates are released into circulation through increased turnover, secretion and/or shedding from malignant cells. Earlier reports from our laboratory

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documented elevated serum levels of total sialic acid (TSA), lipid bound sialic acid (LSA), and TSA to total protein ratio in oral cancer and also suggested that alterations in sialic acid levels have significant clinical usefulness for oral cancer patients [60–63]. Our recent data on salivary glycoproteins including TSA and a-L-fucosidase showed that the changes in these biomarkers play an important role in oral carcinogenesis. It was observed that salivary a-L-fucosidase levels were significantly higher in oral cancer patients as compared to the controls. Also, the TSA/total protein ratio was found to be significantly increased in oral cancer patients as compared to the controls. ROC curve analysis revealed that a-L-fucosidase levels have significant efficacy to discriminate between oral cancer patients and controls (Unpublished data). Electrochemical Sensor for Multiplex Salivary Biomarker Detection In a collaborative research project with University of California at Los Angeles (UCLA), Los Angeles, California, USA, we developed an electrochemical (EC) sensor based on the simultaneous detection of multiple salivary biomarkers for oral cancer. We have found that multiplex assay of both IL-8 mRNA and IL-8 protein levels measured by the EC sensors have significant differences between oral cancer patients and controls. Under the multiplexing mode, the limit of detection of salivary IL-8 mRNA reaches to 3.9 fM and 7.4 pg/ml for salivary IL-8 protein. The ROC analysis revealed that the EC sensor yields as high as 90% sensitivity and specificity for both IL8 mRNA and IL-8 protein. These results were very close to the data measured by traditional assays (enzyme-linked immunosorbent assay, ELISA and polymerase chain reaction, PCR) using same saliva samples. The area under curve (AUC) value was 0.90 for IL-8 mRNA and 0.91 for IL-8 protein. While the combined IL-8 mRNA and protein showed better AUC (0.93) compared with single biomarker [49]. Therefore, combination of multiple biomarkers exhibited improved accuracy instead of single biomarker for oral cancer detection. Promising results from these data suggested that in-depth analysis of these salivary biomarkers have a great potentials to be clinically useful noninvasive biomarkers for screening, diagnosis and treatment monitoring of oral cancer patients.

Concluding Remarks Currently, the saliva research field is rapidly evolving and advancing due to the use of novel approaches including metabolomics, genomics, proteomics and bioinformatics. Implication of saliva as a diagnostic tool for various

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diseases has proved that saliva contains much more clinical information apart from its functional value. Due to its proximity to oral cavity and non-invasive collection procedure, salivary screening can be the best choice as primary screening test for oral cancer. The systematic analysis of salivary genomics and proteomics biomarkers facilitates the identification of sensitive and specific parameters for oral cancer that may aid in effective screening to identify patients with high risk and also help in designing better treatment modalities thus improving survival of oral cancer patients. Collectively, the promising field of salivary genomics and proteomics biomarker analysis may strengthen and transform the field of oral cancer diagnosis which will enable clinicians to monitor patients’ saliva for diagnosis and prognostication of oral cancer. It will thus advance the clinical efforts to overcome the severity of the disease. However, there may be cultural and behavioral perceptions against using saliva; these barriers will need to be overcome with time. Further, enormous efforts from researchers and clinicians are essential to turn salivary diagnostics into clinical and commercial reality to combat oral cancer. Acknowledgment Our ongoing work on salivary biomarkers is partially funded by research project grants from Indian Council of Medical Research (ICMR, Grant no.: 5/13/95/2003-NCD III) and Gujarat Cancer Society, Ahmedabad (Grant no.: RE/28/BRD 1/09). The authors are also thankful to The Gujarat Cancer and Research Institute for administrative support and allowing to use the clinical material.

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