Salivary Markers for Oral Cancer Detection
RESEARCH ARTICLE

Salivary Markers for Oral Cancer Detection

Anastasios K. Markopoulos, * Open Modal , 1 Evangelia Z. Michailidou1 Georgios Tzimagiorgis2
Authors Info & Affiliations
The Open Dentistry Journal • 27 Aug 2010 • RESEARCH ARTICLE • DOI: 10.2174/1874210601004010172

Abstract

Oral cancer refers to all malignancies that arise in the oral cavity, lips and pharynx, with 90% of all oral cancers being oral squamous cell carcinoma. Despite the recent treatment advances, oral cancer is reported as having one of the highest mortality ratios amongst other malignancies and this can much be attributed to the late diagnosis of the disease. Saliva has long been tested as a valuable tool for drug monitoring and the diagnosis systemic diseases among which oral cancer. The new emerging technologies in molecular biology have enabled the discovery of new molecular markers (DNA, RNA and protein markers) for oral cancer diagnosis and surveillance which are discussed in the current review.

Keywords: OSCC, DNA, mRNA, miRNA.

INTRODUCTION

Oral cancer refers to all malignancies arising from the lips, the oral cavity, and pharynx [1] and it affects more than 481,000 new patients worldwide. It is the sixth most common cancer in the USA [2]. The 90% of oral cancers are oral squamous cell carcinoma. This cancer, when found early, has an 80 to 90% survival rate. Despite this fact and the great treatment advances, the World Health Organization has reported oral cancer as having one of the highest mortality ratios amongst other malignancies with a death rate at five years from diagnosis at 45% [3]. This high morbidity rate can definitely be attributed to the late diagnosis of the disease [4]. At the moment, a lack in national screening programs together with a lack of definitive and satisfactory biological markers [5-7] for early oral cancer detection has resulted in late stage diagnosis of oral cancer [8]. An increasing number of systemic diseases and conditions, amongst them oral cancer, have been shown to be reflected diagnostically in saliva. Moreover, using saliva as a diagnostic fluid meets the demands for inexpensive, noninvasive, and accessible diagnostic methodology.

Attempts on Early Oral Cancer Detection

The most reliable method for the diagnosis of oral cancer is a tissue biopsy followed by a histopathological evaluation of the tissue specimen [9, 10]. This however takes as granted that a usually asymptomatic lesion will be detected by the patient who will be alerted and will then soon visit a dentist’s or other practitioner’s office [11]. Because oral cancers usually lack early signs, there have been in the past several attempts towards the direction of early oral cancer detection and attention has been drawn to cancer screening programs [12, 13]. Most oral cancer screening programs include the simple visual inspection [9, 14], whereas others attempt the use of toluidine blue [15, 16], brush biopsy (exfoliative cytology) [17, 18], chemiluminesce [19, 20] and fluorescence imaging [21]. The last three screening methods in fact deal with the diagnosis of lesions that have already been detected by the patient, dentist or other clinician but a definitive diagnosis can only be made by a tissue biopsy.

However, according to Kujan et al. [22, 23], “there is not enough evidence to decide whether screening by visual inspection reduces the death rate for oral cancer and also no robust evidence exists to suggest that other methods of screening, toluidine, fluorescence imaging or brush biopsy are either beneficial or harmful”.

Cancer Related Genetic Alterations Identified in Bodily Fluids

In the development of neoplastic disease, progressive genotypic and phenotypic alterations such as the activation of protoncogenes and ongogenes and inactivation of tumor suppressor genes -associated with tumorigenesis- are detected in the affected cells, establishing the model of multistep tumorigenesis [24]. It has been shown that identical mutations can be identified in bodily fluids draining a tumor [7], but, also lately in bodily fluids secreted away from the initial point where a solid tumor is developing [25, 26]. Nucleic acids and proteins related to cancer cells have been detected in plasma/serum [27-29], urine [30, 31], saliva [32, 33], bronchoalveolar lavage fluid [27], cerebrospinal fluid [34] and other bodily fluids. These nucleic acids and proteins have been used as molecular markers for the early diagnosis of the disease [33, 35, 36], recurrence markers [37] survival and metastasis predictors [38, 39] and decide the therapeutic approach [40, 41].

Saliva as a Perfect Diagnostic Medium

Whole saliva is the product of the secretions of the 3 major salivary glands (parotid, submandibular, sublingual) and the numerous minor salivary glands mixed with crevicular fluid, bronchial and nasal secretions, blood constituents from wounds or bleeding gum, bacteria, viruses, fungi, exfoliated epithelial cells and food debris [42, 43]. Saliva has been long proposed and used as a diagnostic medium [44-46] because it is easily accessible and its collection is non-invasive, not time-consuming, inexpensive, requires minimal training and can be used for the mass screening of large population samples [46, 47].

Whole saliva can be collected with or without stimulation. Stimulation can be performed with masticatory movements or by gustatory stimulation (citric acid) [48]. Stimulated saliva however, it can be collected in larger quantities, is a little bit altered in content [49]. Unstimulated saliva can be collected by merely spitting in a test tube or by leaving saliva drool from the lower lip [50] and it is more often used for the diagnosis or follow up of systemic diseases.

Saliva has long been used for the monitoring of drug abuse (drugs and addictive substances) such as cocaine, heroin, amphetamine, barbiturates [51] etc. Moreover salivary testing has largely performed for the diagnosis of HIV-infection [52, 53]. Analysis of salivary parameters such as salivary flow rate, pH, buffer capacity, lactobacillus, and yeast content, presence of IgG, IgM and anti-La autoantibodies and raised protein levels such as that of lactoferrin and cystatin C as has been proposed for the diagnosis of Sjogren’s syndrome [54, 55]. Concerning cancer diagnostics and follow up altered levels of certain mRNA molecules [33, 56] have been detected in saliva in oral cancer patients and of certain proteins in several cancers [25, 26, 57].

Speculations about Possible Mechanisms that Lead to the Presence of Genotypic and Phenotypic Markers in the Saliva

Cell-free nucleic acids and proteins in saliva may derive from serum or can be locally produced [58].

Serum derived nucleic acids and proteins in the saliva may be part of the normal salivary secretion (by the acinar cells) [59] or come there either via intracellular routes such as active transport or passive diffusion [60] from the serum to saliva across cell membranes or extracellular routes such as ultrafiltration through tight junctions [61] or as constituents of the outflowing crevicular fluid.

Cell free nucleic acids and proteins in saliva however can be locally produced by cell necrosis, lysis or apoptosis and trauma and may even be actively released by normal epithelial or cancerous cells. Cell necrosis is a possible mechanism leading to the release of cell free nucleic acids and proteins in the saliva and this idea is also supported by the large amount of DNA in the plasma of patients with cancers in an advanced stage. Moreover, mounting evidence exists concerning the presence of cell-free nucleic acids and proteins in apoptotic bodies [62] which also protect these molecules from degradation [63]. The active release of these molecules in exosomes or microvesicles is another strong possibility [64]. Exosomes or microvesicles are released by living cells. They are membrane vesicles, 40–100-nm in diameter [65], originating from the endoplasmic reticulum and are released when fused with the cell membrane. They contain mRNA [66], miRNA [67] and proteins [68, 69] and are thought to play a role in the cell-free intercellular communication [69-71], Table 1.

Table 1.

Mechanisms that Lead to the Presence of Genotypic and Phenotypic Markers in the Saliva

Cell-free nucleic acids & proteins in saliva Serum derived Normal salivary secretion
Passive diffusion
Active transport
Ultrafiltration through tight junctions
Outflow of crevicular fluid
Locally produced Cell necrosis, lysis
Apoptosis
Trauma
Active release

Salivary Markers for Oral Cancer Detection

Molecular markers for the diagnosis of OSCC can be quested in 3 levels; (I) changes in the cellular DNA, which result in (II) altered mRNA transcripts, leading to (III) altered protein levels (intracellularly, on the cell surface or extracellularly). All these markers are summarized in Table 2.

Table 2.

Molecular Markers for the Diagnosis of Oral Squamous Cell Carcinoma

Changes in the cellular DNA Altered mRNA transcripts Altered protein markersh
Allelic loss on chromosomes 9p Presence of IL8 Elevated levels of defensin-1
Mitochondrial DNA mutations Presence of IL1B Elevated CD44
p53 gene mutations DUSP1 (dual specificity phosphatase 1) Elevated IL-6 andIL-8
Promoter hypermethylation of genes (p16, MGMT, or DAP-K) H3F3A (H3 histone, family 3A) Inhibitors of apoptosis (IAP)
Cyclin D1 gene amplification OAZ1 (ornithine decarboxylase antizyme 1) Squamous cell carcinoma associated antigen (SCC-Ag)
Increase of Ki67 markers S100P (S100 calcium binding protein P) Carcino- embryonic antigen (CEA)
Microsatellite alterations of DNA SAT(spermidine/spermine N1-acetyltransferase) Carcino-antigen (CA19-9)
Presence of HPV CA128
Serum tumor marker (CA125)
Intermediate filament protein (Cyfra 21-1)
Tissue polypeptide specific antigen (TPS)
Reactive nitrogen species (RNS)
8-OHdG DNA damage marker
Lactate dehydrogenase (LDH)
Immunoglobulin (IgG)
s-IgA
Insulin growth factor (IGF)
Metalloproteinases MMP-2 and MMP-11

Changes in the Cellular DNA

Typical changes in the host DNA of dysplastic or cancer cells include point mutations, deletions, translocations, amplifications and methylations, cyclin D1, epidermal growth factor receptor (EGFR), microsatellite instability and HPV presence.

Allelic loss on chromosomes 9p has been observed in OSCC [72]. Mitochondrial DNA mutations have also been useful targets to detect exfoliated OSCC cells in saliva. They have been identified in 46% of head and neck cancers. The same mitochondrial DNA mutations were detected in 67% of saliva samples from OSCC patients by direct sequencing alone [73]. p53 gene mutations are also present in approximately one-half of head and neck cancers [74, 75]. Using plaque hybridization, Boyle et al. [75] identified tumor specific p53 mutations in 71% saliva samples from patients with head and neck cancer.

Promoter hypermethylation of several genes has been reported in head and neck cancer. Rosas et al. identified aberrant methylation of at least one of three genes (p16, MGMT, or DAP-K) in OSCC. Abnormal promoter hypermethylation was also detected in the matched saliva sample in 65% of OSCC patients [76].

Cyclin D1 gene amplification has been found to be associated with poor prognosis in OSCC [77]. In another study Ki67 markers were increased, while 8-oxoguanine DNA glycosylase, phosphorylated-Src and mammary serine protease inhibitor (Maspin) were found decreased in the saliva of patients with OSCC [78].

Microsatellite alterations of DNA were also observed in the saliva of patients with small cell lung cancer [79]. In the same study it was further demonstrated that 93% of the patients with microsatellite instability in tumor DNA also had similar microsatellite alterations in the corresponding plasma DNA.

The presence of HPV (human papilloma virus) and Epstein Barr virus genomic sequences have been identified as possible DNA molecular markers in detecting OSCC and tumor progression [80, 81].

Altered mRNA Transcripts

RNA for years was thought to quickly degrade in saliva due to the various RNAses that saliva contains [82]. Despite the opposite reports [83], cell-free RNA molecules however, seem to exist in saliva both intact but also fragmented [84]. An intriguing question that remains to be answered is the mechanism by which mRNA in saliva is protected by degradation. A speculation is that salivary mRNA is contained in apoptotic bodies [63, 64] or actively released in exosomes or microvesicles [66, 68,70]. Lately microRNAs, small RNA molecules, 18-24 molecules in length, that seem to regulate transcription were also discovered existing in saliva [85-87].

mRNA detection in saliva has been extensively reported enabling body fluid identification in Forensic Medicine [88, 89]. Moreover mRNA markers in the saliva have been proposed for the diagnosis of primary Sjögren’s syndrome [90] and for the identification of sleep drive both in flies but also in humans [91].

Various mRNA molecules were found up-regulated in the saliva of patients suffering from OSCC by the team of Li et al. [33]. Seven mRNA molecules: transcripts of: 1. IL8 (interleukin 8) playing a role in angiogenesis; replication; calcium-mediated signaling pathway; cell adhesion; chemotaxis ; cell cycle arrest; immune response, 2. IL1B (interleukin 1Β) which takes part in signal transduction; proliferation; inflammation and apoptosis 3. DUSP1 (dual specificity phosphatase 1) with a role in protein modification; signal transduction and oxidative stress, 4. H3F3A (H3 histone, family 3A) having a DNA binding activity, 5. OAZ1 (ornithine decarboxylase antizyme 1) taking part in polyamine biosynthesis 6. S100P (S100 calcium binding protein P) with a role in protein binding and calcium ion binding, and 7. SAT (spermidine/spermine N1-acetyltransferase) which takes part in enzyme and transferase activity- were found significantly elevated in OSCC patients rather than in healthy controls [56].

Altered Protein Markers

Several salivary protein markers for OSCC have been investigated in various studies and have shown relatively moderate sensitivity and specificity values relative to prognosis prediction.

For example, defensins are peptides which possess antimicrobial and cytotoxic properties. They are found in the azurophil granules of polymorphonuclear leukocytes [92, 93]. Elevated levels of salivary defensin-1 were found to be indicative for the presence of OSCC, since higher concentrations of salivary defensin-1 were detected in patients with OSCC compared with healthy controls [94].

In another study soluble CD44 [95] was found to be elevated in the majority of patients with OSCC and distinguished cancer from benign disease with high specificity. Whereas the solCD44 test lacks sensitivity by itself, methylation status of the CD44 gene seems to complement the solCD44 test and provides very high sensitivity and specificity for the detection of OSCC.

St John et al. [32] investigated whether IL-6 and/or IL-8 could serve as informative biomarkers for OSCC in saliva. Interleukin 8 was detected at higher concentrations in saliva, while IL-6 was detected at higher concentrations in serum of patients with OSCC. Thus, they concluded that IL-8 in saliva and IL-6 in serum hold promise as biomarkers for OSCC.

A group of leading researchers [33, 56, 96-99] using new and sophisticated approaches, such as, Luminex Multianalyte Profiling (xMAP) technology, shotgun proteomics, capillary reversed-phase liquid chromatography with quadruple time-offlight mass spectrometry and matrix-assisted laser desorption/ionization–mass spectrometry (MALDI–MS), has contributed significantly in recent years to the research in saliva for cancer diagnosis. Their studies have shown that saliva contains proteins that may serve as biomarkers for OSCC, since 46 peptides/proteins were found at significantly different levels between the OSCC and control groups. For example Arellano-Garcia et al. [100] using Luminex xMAP technology showed that both IL-8 and IL-1β were expressed at significantly higher levels in OSCC subjects.

Other salivary biomarkers which have been shown to be significantly altered in OSCC patients as compared with healthy controls are inhibitors of apoptosis (IAP) [101], squamous cell carcinoma associated antigen (SCC-Ag) [102-104], carcino- embryonic antigen (CEA) [102, 103], carcino-antigen (CA19-9) [102, 104], CA128 [102, 104], serum tumor marker (CA125) [105], intermediate filament protein (Cyfra 21-1) [106-108], tissue polypeptide specific antigen (TPS) [108, 109], reactive nitrogen species (RNS) and 8-OHdG DNA damage marker [106], lactate dehydrogenase (LDH) and immunoglobulin (IgG) [107], s-IgA [110], insulin growth factor (IGF) [110], metalloproteinases MMP-2 and MMP-11 [110].

CONCLUSIONS

For years saliva is tested as a diagnostic fluid and compared to blood (serum or plasma) in parameters such as sensitivity, specificity and applicability of the method, cost and duration of the procedure, patient compliance [111] etc. Due to the recent advances and emerging technologies in molecular biology new molecular markers (DNA, RNA and protein markers) have been discovered existing in the saliva in measurable quantities [112]. OSCC can be diagnosed with high sensitivity and specificity by merely testing saliva samples from the subjects. This does not of course undermine the value of screening tests by visual examination neither the importance of the tissue biopsy.

Despite the scepticism in the scientific community and the conservatism of the patients, saliva seems to emerge as a valuable tool in cancer diagnostics and mass population screening. In our opinion much attention must be given to the saliva collecting method. An attempt to integrate the simultaneous testing of different salivary molecular markers in order to raise the possibility of an accurate diagnosis by simply using micro- and nano-electrical-mechanical systems biosensors is on the way raising much hope in its future applications [113].

Finally, since the present methods are not ready for immediate clinical use as diagnostic tools, much work is necessary and it can be envisaged that simple, fast, portable and cost-effective clinical diagnostic systems could be available in the near future.

REFERENCES

1
The international statistical classification of diseases and related health problems 1 In: World Health Organization; Geneva. 1992.(10 )
2
American Cancer Society. Cancer Facts and Figures 2007. Atlanta: American Cancer Society 2007.
3
Ferlay J, Bray F, Pisani P, Parkin DM. GLOBOCAN 2000, cancer incidence, mortality and prevalence worldwide, Version 10. Lyon: IARC Press 2001.
4
Peacock S, Pogrel A, Schmidt BL. Exploring the reasons for delay in treatment of oral cancer Am Dent Assoc 2008; 139: 1346-52.
5
Schantz SP. Biologic markers, cellular differentiation, and metastatic head and neck cancer Eur Arch Otorhinolaryngol 1993; 250: 424-8.
6
Schantz SP. Carcinogenesis, markers, staging, and prognosis of head and neck cancer Curr Opin Oncol 1993; 5: 483-90.
7
Sidransky D. Emerging molecular markers of cancer Nat Rev Cancer 2002; 3: 210-9.
8
Ellison MD, Campbell BH. Screening for cancer of the head and neck: addressing the problem Surg Oncol Clin N Am 1999; 8: 725-34.
9
Fedele S. Diagnostic aids in the screening of oral cancer Head Neck Oncol 2009; 30: 1-5.
10
Trullenque-Eriksson A, Munoz-Corcuera M, Campo-Trapero J, Cano-Sánchez J, Bascones-Martínez A. Analysis of new diagnostic methods in suspicious lesions of the oral mucosa Med Oral Patol Oral Cir Buccal 2009; 14: E210.
11
Dolan RW, Vaughan CW, Fuleihan N. Symptoms in early head and neck cancer: an inadequate indicator Otolaryngol Head Neck Surg 1998; 118: 463-7.
12
Sankila R, Coll EC. Evaluation and monitoring of screening program. Luxembourg: Office for the Official Publication of the European Communities 2001; pp. 243-54.
13
Warnakulasuriya S, Nanayakkara BG. Reproducibility of an oral cancer and precancer detection program using a primary health care model in Sri Lanka Cancer Detect Prev http://www.jdentaled.org/cgi/external_ref?access_num=1751941&l ink_type=MED 1991; 15: 331-4.
14
Zakzerwska JM, Hindle I, Speight PM. Practical considerations for the establishment of an oral cancer screening programme Commun Dent Health 1993; 10(Suppl 1 ): 79-85.
15
Onofre MA, Sposto MR, Navarro CM. Reliability of toluidine application in the detection of oral epithelial dysplasia and in situ and invasive squamous cell carcinomas Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2001; 91: 535-40.
16
Zhang L, Williams M, Poh CF, et al. Toluidine blue staining identifies high-risk primary oral premalignant lesions with poor outcome Cancer Res 2005; 65: 8017-21.
17
Christian DC. Computer-assisted analysis of oral brush biopsies at an oral cancer screening program J Am Dent Assoc 2002; 133: 357-62.
18
Mehrotra R, Hullmann M, Smeets R, Reichert TE, Driemel O. Oral cytology revisited J Oral Pathol Med 2009; 38: 161-6.
19
Kerr AR, Sirois DA, Epstein JB. Clinical evaluation of chemiluminescent lighting:an adjunct for oral mucosal examinations J Clin Dent 2006; 17: 59-63.
20
Epstein JB, Silverman S Jr, Epstein JD, Lonky SA, Bride MA. Analysis of oral lesion biopsies identified and evaluated by visual examination, chemiluminescence and tolouidine blue Oral Oncol 2008; 44: 538-44.
21
Onizawa K, Saginoya H, Furuya Y, Yoshida H. Fluorescence photography as a diagnostic method for oral cancer Cancer Lett 1996; 108: 61-.
22
Kujan O, Glenny AM, Oliver R, Thakker N, Sloan P. Screening programmes for the early detection and prevention of oral cancer Aust Dent J 2009; 54: 170-2.
23
Omar K, Glenny A, Duxbury J, Thakker N, Sloan P. Evaluation of screening strategies for improving oral cancer mortality: a cohrane systematic review J Dent Educ 2005; 69: 255-65.
24
Farber E. The multistep nature of cancer development Cancer Res 1984; 44: 4217-23.
25
Bigler LR, Streckfus CF, Dubinsky WP. Salivary biomarkers for the detection of malignant tumors that are remote from the oral cavity Clin Lab Med 2009; 29: 71-85.
26
Streckfus C, Bigler L, Dellinger T, Dai X, Kingman A, Thigpen JT. The presence of soluble c-erbB-2 in saliva and serum among women with breast carcinoma: a preliminary study Clin Cancer Res 2000; 6: 2363-70.
27
Schmidt B, Engel E, Carstensen T, et al. Quantification of free RNA in serum and bronchial lavage: a new diagnostic tool in lung cancer detection? Lung Cancer 2005; 48: 145-7.
28
Kopreski MS, Benko FA, Gocke CD. Circulating RNA as a tumor marker: detection of 5T4 mRNA in breast and lung cancer patient serum Ann NY Acad Sci 2001; 945: 172-8.
29
Hasselmann DO, Rappl G, Rössler M, Ugurel S, Tilgen W, Reinhold U. Detection of tumor-associated circulating mRNA in serum, plasma and blood cells from patients with disseminated malignant melanoma Oncol Rep 2001; 8: 115-8.
30
Bryzgunova OE, Skvortsova TE, Kolesnikova EV, et al. Isolation and comparative study of cell-free nucleic acids from human urine Ann NY Acad Sci 2006; 1075: 334-4.
31
Yoneda K, Iida H, Endo H, et al. Identification of cystatin SN as a novel tumor marker for colorectal cancer Int J Oncol 2009; 35: 33-40.
32
St John M, Li Y, Zhou X, et al. Interleukin 6 and interleukin 8 as potential biomarkers for oral cavity and oropharyngeal squamous cell carcinoma Arch Otolaryngol Head Neck Surg 2004; 130: 929-35.
33
Li Y, St John MA, Zhou X, et al. Salivary transcriptome diagnostics for oral cancer detection Clin Cancer Res 2004; 10: 8442-50.
34
de Bont JM, van Doorn J, Reddingius RE, et al. Various components of the insulin-like growth factor system in tumour tissue, cerebrospinal fluid and peripheral blood of pediatric medulloblastoma and ependymoma patients Int J Cancer 2008; 123: 594-600.
35
Johnson PJ, Lo YM. Plasma nucleic acids in the diagnosis and management of malignant disease Clin Chem 2002; 48: 1186-93.
36
Neves AF, Araújo TG, Biase WK, et al. Combined analysis of multiple mRNA markers by RT-PCR assay for prostate cancer di-agnosis Clin Biochem 2008; 41: 1191-8.
37
Honma H, Kanda T, Ito H, et al. Squamous cell carcinoma-antigen messenger RNA level in peripheral blood predicts recurrence after resection in patients with esophageal squamous cell carcinoma Surgery 2006; 139: 678-85.
38
El-Abd E, El-Tahan R, Fahmy L, et al. Serum metastasin mRNA is an important survival predictor in breast cancer Br J Biomed Sci 2008; 65: 90-4.
39
Voorzanger-Rousselot N, Goehrig D, Journe F, et al. Increased Dickkopf-1 expression in breast cancer bone metastases Br J Cancer 2007; 97: 964-70.
40
Siddiqua A, Chendil D, Rowland R, et al. Increased expression of PSA mRNA during brachytherapy in peripheral blood of patients with prostate cancer Urology 2002; 60: 270-5.
41
Ogawa O, Iinuma M, Sato K, et al. Circulating prostate-specific antigen mRNA during radical prostatectomy in patients with localized prostate cancer: with special reference to neoadjuvant hormonal therapy Urol Res 1999; 27: 291-6.
42
Mandel ID. The functions of saliva J Dent Res 1987; 66: 623-7.
43
Sreebny LM. Salivary flow in health and disease Compend Suppl 1989; 13: S461-9.
44
Kaufman E, Lamster I. The diagnostic applications of saliva: a review Crit Rev Oral Biol Med 2002; 13: 197-212.
45
Streckfus CF, Bigler L. Saliva as a diagnostic fluid Oral Dis 2002; 8: 69-76.
46
Malamud D. Saliva as a diagnostic fluid Br Med J 1992; 8: 207-8.
47
Samaranayake L. Saliva as a diagnostic fluid Int Dent J 2007; 57: 295-9.
48
Fox PC. Salivary enhancement therapies Caries Res 2004; 38: 241-6.
49
da Mata AD, da Silva Marques DN, Silveira JM, et al. Effects of gustatory stimulants of salivary secretion on salivary pH and flow: a randomized controlled trial Oral Dis 2009; 15: 220-8.
50
Navazesh M. Methods for collecting saliva Ann NY Acad Sci 1993; 8: 72-.
51
Bosker WM, Huestis MA. Oral fluid testing for drugs of abuse Clin Chem 2009; 55: 1910-31.
52
Pink R, Simek J, Vondrakova J, et al. Saliva as a diagnostic medium Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 2009; 153: 103-.
53
Roberts KJ, Grusky O, Swanson AN. Outcomes of blood and oral fluid rapid HIV testing: a literature review, 2000-2006 AIDS Patient Care STDS 2007; 21: 621-37.
54
Giusti L, Baldini C, Bazzichi L, Bombardieri S, Lucacchini A. Proteomic diagnosis of Sjögren's syndrome Expert Rev Proteomics 2007; 4: 757-67.
55
Sreebny LM, Zhu WX. The use of whole saliva in the differential diagnosis of Sjögren's syndrome Adv Dent Res 1996; 10: 17-24.
56
Zimmermann BG, Wong DT. Salivary mRNA targets for cancer diagnostics Oral Oncol 2008; 44: 425-9.
57
Di-Xia C, Schwartz P, Fan-Qin L. Salivary and serum CA 125 assays for detecting malignant ovarian tumors Obstet Gynecol 1990; 8: 701-4.
58
Kaufman E, Lamster IB. The diagnostic applications of saliva: a review Crit Rev Oral Biol Med 2002; 13: 197-212.
59
Baum BJ. Principles of saliva secretion Ann NY Acad Sci 1993; 694: 17-23.
60
Haeckel R, Hanecke P. Application of saliva for drug monitoring: an In vivo model for transmembrane transport Eur J Clin Chem Clin Biochem 1996; 34: 171-91.
61
Aps JK, Martens LC. Review: the physiology of saliva and transfer of drugs into saliva Forensic Sci Int 2005; 150: 119-31.
62
Halicka HD, Bedner E, Darzynkiewicz Z. Segregation of RNA and separate packaging of DNA and RNA in apoptotic bodies during apoptosis Exp Cell Res 2000; 260: 248-56.
63
Hasselmann D, Rappl G, Tilgen W, Reinhold U. Extracellular tyrosinase mRNA within apoptotic bodies is protected from degra-dation in human serum Clin Chem 2001; 47: 1488-9.
64
Ratajczak J, Wysoczynski M, Hayek F, Janowska-Wieczorek A, Ratajczak MZ. Membrane-derived microvesicles: important and underappreciated mediators of cell-to-cell communication Leukemia 2006; 20: 1487-95.
65
Simpson RJ, Lim JW, Moritz RL, Mathivanan S. Exosomes: proteomic insights and diagnostic potential Expert Rev Proteomics 2009; 6: 267-83.
66
García JM, García V, Peña C, et al. Extracellular plasma RNA from colon cancer patients is confined in a vesicle-like structure and is mRNA-enriched RNA 2008; 14: 1424-32.
67
Yuan A, Farber E, Rapoport A, et al. Transfer of microRNAs by embryonic stem cell microvesicles PLoS One 2009; 4: e4722.
68
Skog J, Würdinger T, van Rijn S, et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and pro-vide diagnostic biomarkers Nat Cell Biol 2008; 10: 1470-6.
69
Simpson RJ, Jensen SS, Lim JW, et al. Proteomic profiling of exosomes: current perspectives Proteomics 2008; 8: 4083-99.
70
Al-Nedawi K, Meehan B, Rak J. Microvesicles: messengers and mediators of tumor progression Cell Cycle 2009; 8: 2014-8.
71
Aharon A, Brenner B. Microparticles, thrombosis and cancer Best Pract Res Clin Haematol 2009; 22: 61-9.
72
Nawroz H, van der Riet P, Hruban RH, Koch W, Ruppert JM, Sidransky D. Allelotype of head and neck squamous cell carcinoma Cancer Res 1994; 54: 1152-5.
73
Fliss MS, Usadel H, Caballero OL, et al. Facile detection of mitochondrial DNA mutations in tumors and bodily fluids Science 2000; 287: 2017-9.
74
Liao PH, Chang YC, Huang MF, Tai KW, Chou MY. Mutation of p53 gene codon 63 in saliva as a molecular marker for oral squamous cell carcinomas Oral Oncol 2000; 36: 272-6.
75
Boyle JO, Hakim J, Koch W, et al. The incidence of p53 mutations increases with progression of head and neck cancer Cancer Res 1993; 53: 4477-80.
76
Rosas SL, Koch W, Carvalho MGC, et al. Promoter hypermethylation patterns of p16, O6-methylguanine-DNA-methyltransferase, and death-associated protein kinase in tumors and saliva of head and neck cancer patients Cancer Res 2001; 61: 939-42.
77
Vielba R, Bilbao J, Ispizua A, et al. p53 and cyclin D1 as prognostic factors in squamous cell carcinoma of the larynx Laryngoscope 2003; 113: 167-72.
78
Shpitzer T, Hamzany Y, Bahar G, et al. Salivary analysis of oral cancer biomarkers Br J Cancer 2009; 101: 1194-8.
79
Chen XQ, Stroun M, Magnenat JL, et al. Microsatellite alterations in plasma DNA of small cell lung cancer patients Nat Med 1996; 2: 1033-5.
80
Paz IB, Cook N, Odom-Maryon T, Xie Y, Wilczynski SP. Human papillomavirus (HPV) in head and neck cancer: an association of HPV 16 with squamous cell carcinoma of Waldeyer's tonsillar ring Cancer 1997; 79: 595-604.
81
Shimakage M, Horii K, Tempaku A, Kakudo K, Shirasaka T, Sasagawa T. Association of Epstein-Barr virus with oral cancers Hum Pathol 2002; 33: 608-14.
82
Eichel HJ, Conger N, Chernick WS. Acid and alkaline ribonucleases of human parotid, submaxillary, and whole saliva Arch Biochem Biophys 1964; 107: 197-208.
83
Kumar SV, Hurteau GJ, Spivack SD. Validity of messenger RNA expression analyses of human saliva Clin Cancer Res 2006; 12: 5033-9.
84
Hu Z, Zimmermann BG, Zhou H, et al. Exon-level expression profiling: a comprehensive transcriptome analysis of oral fluids Clin Chem 2008; 54: 824-32.
85
Park NJ, Zhou H, Elashoff D, et al. Salivary microRNA: discovery, characterization, and clinical utility for oral cancer detection Clin Cancer Res 2009; 15: 5473-7.
86
Michael A, Bajracharya SD, Yuen PS, et al. Exosomes from human saliva as a source of microRNA biomarkers Oral Dis 2010; 16: 34-8.
87
Hanson EK, Lubenow H, Ballantyne J. Identification of forensically relevant body fluids using a panel of differentially expressed microRNAs Anal Biochem 2009; 387: 303-14.
88
Juusola J, Ballantyne J. Multiplex mRNA profiling for the identification of body fluids Forensic Sci Int 2005; 152: 1-12.
89
Juusola J, Ballantyne J. Messenger RNA profiling: a prototype method to supplant conventional methods for body fluid identifica-tion Forensic Sci Int 2003; 135: 85-96.
90
Hu S, Wang J, Meijer J, et al. Salivary proteomic and genomic biomarkers for primary Sjögren's syndrome Arthritis Rheum 2007; 56: 3588-600.
91
Seugnet L, Boero J, Gottschalk L, Duntley S, Shaw P. Identification of a biomarker for sleep drive in flies and humans Proc Natl Acad Sci USA 2006; 103: 19913-8.
92
Lichtenstein A, Ganz T, Selsted ME, Lehrer RI. In vitro tumor cell cytolysis mediated by peptide defensins of human and rabbit granulocytes Blood 1986; 68: 1407-0.
93
Lehrer RI, Ganz T, Selsted ME. Defensins: endogenous antibiotic peptides of animal cells Cell 1991; 64: 229-30.
94
Mizukawa N, Sugiyama K, Fukunaga J, et al. Defensin-1, a peptide detected in the saliva of oral squamous cell carcinoma patients Anticancer Res 1998; 18: 4645-9.
95
Franzmann EJ, Reategui EP, Pedroso F, et al. Soluble CD44 is a potential marker for the early detection of head and neck cancer Cancer Epidemiol Biomarkers Prev 2007; 16: 1348-55.
96
Hu S, Arellano M, Boontheung P, et al. Salivary proteomics for oral cancer biomarker discovery Clin Cancer Re 2008; 14: 6246-52.
97
Tan W, Sabet L, Li Y, et al. Optical protein sensor for detecting cancer markers in saliva Biosens Bioelectron 2008; 24: 266-71.
98
Hu S, Yen Y, Ann D, Wong DT. Implications of salivary proteomics in drug discovery and development: a focus on cancer drug discovery Drug Discov Today 2007; 12: 911-6.
99
Yang CY, Brooks E, Li Y, et al. Detection of picomolar levels of interleukin-8 in human saliva by SPR Lab Chip 2005; 5: 1017-23.
100
Arellano-Garcia ME, Hu S, Wang J, et al. Multiplexed immunobead-based assay for detection of oral cancer protein biomarkers in saliva Oral Dis 2008; 14: 705-12.
101
Kurokawa H, Tsuru S, Okada M, Nakamura T, Kajiyama M. Evaluation of tumor markers in patients with squamous cell carcinoma in the oral cavity Int J Oral Maxillofac Surg 1993; 22: 35-8.
102
Hoffmann J, Munz A, Krimmel M, Alfter G. Intraoperative and postoperative kinetics of serum tumor markers in patients with oral carcinoma J Oral Maxillofac Surg 1998; 56: 1390-3.
103
Krimmel M, Hoffmann J, Krimmel C, Cornelius CP, Schwenzer N. Relevance of SCC-Ag, CEA, CA 19 and CA 125 for diagnosis and follow-up in oral cancer J Craniomaxillofac Surg 1998; 26: 243-8.
104
Nagler RM, Braun J, Daitzman M, Laufer D. Spiral CT angiography - analternative vascular evaluation technique for head and neck microvascular reconstruction: a preliminary experience Plast Reconstr Surg 1997; 100: 1697-703.
105
Kurokawa H, Yamashita Y, Tokudome S, Kajiyama M. Combination assay for tumor markers in oral squamous cell carcinoma J Oral Maxillofac Surg 1997; 55: 964-6.
106
Nagler RM, Barak M, Ben-Aryeh H, Peled M, Filatov M, Laufer D. Early diagnostic and treatment monitoring role of Cyfra 21-1 and TPS in oral squamous cell carcinoma Cancer 1999; 35: 1018-25.
107
Yen TC, Lin WY, Kao CH, Cheng KY, Wang SJ. A study of a new tumour marker, CYFRA 21-1, in squamous cell carcinoma of the head and neck, and comparison with squamous cell carcinoma antigen Clin Otolaryngol 1998; 23: 82-6.
108
Nagler R, Bahar G, Shpitzer T, Feinmesser R. Concomitant analysis of salivary tumor markers: a new diagnostic tool for oral cancer Clin Cancer Res 2006; 12: 3979-84.
109
Bahar G, Feinmesser R, Shpitzer T, Popovtzer A, Nagler RM. Salivary analysis in oral cancer patients DNA and protein oxidation, reactive nitrogen species and antioxidant profile Cancer 2007; 109: 54-9.
110
Shpitzer T, Bahar G, Feinmesser R, Nagler RM. A comprehensive salivary analysis for oral cancer diagnosis Cancer Res Clin Oncol 2007; 133: 613-7.
111
Pesce MA, Spitalnik SL. Saliva and the clinical pathology laboratory Ann N Y Acad Sci 2007; 1098: 192-9.
112
Segal A, Wong DT. Salivary diagnostics: enhancing disease detection and making medicine better Eur J Dent Educ 2008; 12: 22-9.
113
Wong DT. Towards a simple, saliva-based test for the detection of oral cancer. ‘Oral fluid (saliva), which is the mirror of the body, is a perfect medium to be explored for health and disease surveillance’ Expert Rev Mol Diagn 2006; 6: 267-72.