Table 1.
Proteomic salivary biomarkers.
| Author | Country | Number of samples | Biomarker category | Biomarker | Saliva collection time | Saliva collection method | Sample processing | Test method | Results |
|---|---|---|---|---|---|---|---|---|---|
| Azeem et al. [28] | India | 96 OSCC patients / 96 tobacco chewers without lesions/96 healthy patients | Protein | PBSA FSA | 9 am-12 pm | Unstimulated whole saliva | Centrifuged at 3000 rpm/15 min. | Spectrophotometric | Salivary PBSA and FSA are significantly raised in tobacco chewers and OSCC patients. |
| Chang et al. [43] | Taiwan | 269 OSCC patients /578 OPMD/313 healthy patients | Protein | MMP1 | — | Unstimulated whole saliva | Centrifuged at 3000 × g/15 min/4°C. Stored at -80°C. |
ELISA | MMP1 levels in saliva distinguish between OSCC patients and healthy controls. MMP1 levels in OSCC patients was strongly correlated with tumor progression. |
| Chi et al. [41] | China | 30 OSCC patients /30 healthy patients | Protein | ANXA2, CA2, CD44, CSTA, DSG3, EGFR, ENO1, FLNA, GANAB, HSPA5, IL-6, ISG15, KRT18, LGALS3BPMMP1, MMP2, MMP3, MYO5A, OASL, PADI1, S100A2, SERPINE1, SPP1, STAT1, TIMP1, TNC, TYMP, ULBP2, WARS, YWHAB | Morning and afternoon | Unstimulated | Centrifuged at 3000 × g/15 min/4 °C. | SISCAPA-MRM | MMP1, PADI1, TNC, CSTA and MMP3 showed significant levels in OSCC patients and a high level of disease discrimination. |
| Cohen et al. [47] | USA | 150 OSCC patients /150 healthy patients | Protein | SolCD44, TP | — | Unstimulated saliva | Transport on ice Storage at -80°C |
Sandwich ELISA | Association of solCD44 with worse progression-free survival data. |
| Csősz et al. [30] | Hungary | 20 OSCC patients/17 healthy patients | Protein | Proteomic profile | Between 9 a.m. and 11 a.m. | Unstimulated saliva | Centrifuged at 4100 × g/15 min/4 °C. Stored -70°C |
LC-MS analysis/ELISA | None of the studied proteins turned to be a potential biomarker in OSCC. |
| Csősz et al. [31] | Hungary | 55 OSCC patients/52 healthy patients | Protein | IL-1α, IL-1β, IL-6, IL-8, TNF-α, VEGF catalase, profilin-1, S100A9, CD59, galectin-3-bindig protein, CD44, thioredoxin and keratin-19 | Between 9 a.m. and 11 a.m. | Unstimulated saliva | Centrifuged at 4.000 × g/15 min/4 °C. Stored at -70°C. |
LC-MS analysis | The validation of IL-6, S100A9 and thioredoxin revealed the potential utility of combination of IL-6 and S100A9. |
| Dikova et al. [40] | Spain | 66 OSCC patients / 33 OHL/33 OPVL/25 healthy patients | Protein | IL-1α, IL-6, IL-8, IP-10, MCP-1, TNF-α, HCC-1, and PF-4 | — | Unstimulated whole saliva | Centrifuged at 3,000 × g/15 min/4 °C. Stored at -80°C. |
ELISA multiplex immunoassay | L-6, IL-8, TNF-α, HCC-1, discriminate between OSCC and healthy patients. IL-6, IL-8, and PF-4 discriminate OSCC from OLK without specifying the type. |
| Ghallab et al. [44] | Egypt | 15 early stage OSCC patients/15 OPMD patients/15 healthy patients | Protein | Chemerin, MMP-9 in serum and saliva | Morning | Unstimulated whole saliva | Centrifuged at 10000 × g/2 min. 2 min/10,000 × g Stored at−80°C. |
ELISA | Serum and salivary levels of chemerin and MMP-9 in patients with OSCC were significantly higher than OPMLs and control group. Patients with OPMLs showed also elevated profiles for serum and salivary chemerin and MMP-9 com- pared to control group. |
| Heawchaiyaphum et al. [48] | Thailand | 92 OSCC patients/83 healthy patients | Protein | HP PRDX-2 ZAG |
— | — | Centrifuged at 1200 rpm /15 min/4°C. Stored at –80°C. |
LC-MS/MS Western blot |
PRDX-2 and ZAG upregulated in OSCCc HP upregulated in OSCC. IGHA2 downregulated in OSCC. |
| Hsiao et al. [49] | Taiwan | 9 OSCC patients / 5 healthy patients | Protein | MMP1 | — | Unstimulated whole saliva | Centrifuged at 3000 × g/15 min/4°C. Sored at-80°C. |
ImmunoMALDI analysis | Elevated MMP1 levels in saliva in 7 of 9 OSCC patients. MMP1 was not detectable in healthy subjects. |
| Ishikawa et al. [50] | Japan | 39 OC patients /31 healthy patients | Protein | Cornulin, Alfa2 macroglobulin-like protein, hemoglobin subunit beta, Ig kapa chain V-II region V kappa 167, Kininogen-1 Transmembrane protease serine 11D |
— | Unstimulated saliva | Centrifuged at 2600 × g/15 min/4°C. | LC-MS/MS | Cornulin lower in patiens with OC. The combination of all the analized biomarkers have a high accuracy for differenciating between patients with oral cancer and control. |
| Kang et al. [33] | China | 20 OSCC patients/20 OSCC patients from OLK/20 OSCC patients from OLP/20 OLK/20 OLP/20 healthy patients | Protein | KLK5 uPA |
— | Stimulated for 10 minutes | Centrifuged at 10000 × g/10 min/4 °C. Stored at -80°C. |
ELISA | The results suggested that the combination of KLK5 and uPA could represent a potential biomarker for determining the malignant transformation of OLK or OLP into OSCC. |
| Khyani et al. [38] | Pakistan | 35 OPMD/35 OSCC patients 35 healthy patients |
Cytokine | IL-6, IL-8 | — | Unstimulated saliva | Centrifuged at 4500 rpm/15 min/4°C. Stored at -20°C. |
ELISA | IL-6 levels not significantly elevated in all groups. IL-8 levels significantly elevated in all groups. |
| Ko et al. [51] | Taiwan | 86 OSCC pateients/35 healthy patients | Protein | AKR1B10 | — | Unstimulated whole saliva | Centrifuged at 1000 × g/10 min/4 °C. Stored at -80°C. |
ELISA | AKR1B10 levels were significantly higher in the patients with OSCC than in the normal controls. Higher salivary AKR1B10 levels were significantly associated with larger tumor size, more advanced clinical stage, and areca quid chewing habit. |
| Lee et al. [39] | Taiwan | 41 OSCC patients/ 24 healthy patients |
Cytokine | EGF, Eotaxin, G-CSF, IFN- g, GRO, IL-10, IL-1α, IL-1β, IL-6, IL-8, IP-10, MIP-1β, TNF-α, VEGF | — | Unstimulated saliva | Centrifuged at 3000 rpm/20 min/4°C. Stored at -80°C. |
Multiplex (Luminex bead-based) | Eotaxin, IL-1β, IL-6, IL-8, MIP-1β, TNF-α: Significant differences between early stage OSCC patients and healthy patients. |
| Lin et al. [52] | Poland | 5 OSCC patients/5 healthy patients | Protein | Global proteomic profile | 10 a.m. to 12 p.m | Unstimulated whole saliva | Centrifuged at 2600 g/20 min/4°C. Stored at -80°C. |
LC-MS/MS | Oral cancer may have varied pathological effects on the saliva proteome. |
| Michailidou et al. [32] | Greece | 34 OSCC patients/20 OLK patients/31 helathy patients | Protein | IL-1B, IL-8, OAZ and SAT | Morning | Unstimulated saliva | Centrifuged at 2600 × g/15 min/4 °C. Stored at -80°C. |
RT-PCR | SAT and IL-8 mRNAs have good discriminatory ability for oral squamous cell carcinoma patients Only 81.3% sensitivity and 73.9% specificity in the diagnosis of oral squamous cell carcinoma at an early stage. |
| Peisker et al. [29] | Germany | 30 OSCC patients/30 healthy controls | Protein | MMP-9 | Between 7 -8 am | Stimulated saliva | Centrifuged at 1000 × g/20 °C/2 min. | ELISA | The elevation of salivary levels of MMP-9 may be a useful adjunctive diagnostic tool for detection of OSCC. |
| Rai et al. [26] | India | 25 OSCC patients /24 healthy patients | Cytokines | IL-6 IL-8 TNF-α IFN-γ GM-CSF |
9.00 am to11.00 am | Unstimulated whole saliva | Centrifuged at 9300 g/5 min. Stored at -20°C. | Bio-Plex pro™ human cytokines assay | The largest amount of S. Anginosus, V. Parvula, P. Endodontalis and P. Anaerobius may help to the development of OSCC via increased expression of proinflamatory cytokines. |
| Seyedmajidi et al. [53] | Iran | 20 OSCC patients/20 healthy patients | Protein | Soluble CD44 in serum and saliva | 10 a.m. to 12 p.m. | Unstimulated whole saliva | Centrifuged at 2000 and 11000 rpm/10 min. Stored at -80°C. |
ELISA | There was no statistically significant difference in serum and saliva solCD44 level between the patient and control groups. There was no significant correlation between the solCD44 level in each patient and control group in serum. |
| Shan et al. [46] | China | 20 OSCC patients/20 OPMD patients/20 healthy patients | Protein | SLC3A2 S100A2 IL1RN |
Morning | Unstimulated whole saliva | Centrifuged at 2600 × g/15 min/4°C. Stored at -80°C. |
iTRAQ y ELISA | SLC3A2 and S100A2 levels were significantly increased in the OSCC group compared to healthy controls and OPMD. IL1RN levels were significantly decreased in the OSCC group compared to healthy controls and OPMD. |
| Singh et al. [25] | India | 31 OSCC TNM I-II and 27 27 TNM III-IV patients/30 OPMD patients/29 post treatment patients/ 42 healthy patients |
Protein | IL-1β IL-8 LGALS3BP |
Morning | Unstimulated | Centrifuged at 1000 × g/20 min/2-8°C. Stored at -80°C. |
ELISA | IL-1β and IL-8 were significantly elevated in all stages OSCC patients. LGALS3BP was elevated in early OSCC patients. |
| Sivadasan et al. [22] | India | 15 dysplasticOLK/15 LP 15 OSCC N0 15 OSCC N+ patients/15 healthy patients | Protein | 93 proteins | Morning | Unstimulated | Centrifuged at 2000 rpm/4°C/10 min and centrifuged at 14000 rpm. Storage at -80°C. |
LC-MS and ELISA | CD44, S1000A7 and S100P levels significantly altered in patients with leukoplakia with dysplasia and OSCC. |
| Wang et al. [54] | China | 79 OSCC patients/31 OPMD patients 80 healthy patients |
Protein | SNCG | — | Unstimulated | Centrifuged at 1000 × g/2 min/room temperature. Stored at -80°C. |
ELISA | SNCG expression is related with medium and low degrees of cell differentiation in OSCC. |
| Yu et al. [55] | Taiwan | 103 low risk OPMD/130 high riskOPMD/ 131 OSCC patients/ 96 healthy patients |
Protein | ANXA2 HSPA5 KNG1 MMP1 |
— | Unstimulated whole saliva | Centrifuged at 3000 × g/15 min/4°C. Stored at -80°C. |
LC-MRM-MS | The four-protein panel could be used to diagnose OSCC with an 80% specificity. |
| Zanotti et al. [45] | Italy | 63 OSCC patients/ 60 healthy patients |
Protein | EGFR | — | Unstimulated | Centrifuged 13000 rpm/3 min. Storage at -80°C. | Sandwich ELISA | Higher EGFR concentration in saliva in OSCC patients. Higher EGFR levels in patients with higher T category. Worse prognosis in patients with higher EGFR levels. |
| Zheng et al. [27] | China | 112 OSCC patients 30 OPML 60 healthy subjects |
Protein | Naa10p CEA |
9.00 am to 11.00 am | Unstimulated whole saliva | Saliva: Centrifuged at 800 × g/2 min/room temperature. Blood: Centrifuged at 1200 × g/10 min/room temperature. Stored at -80 °C. |
ELISA | Combined diagnosis of Naa10p and CEA significantly increased both sensitivity and specificity. Combined diagnosis with Naa10p and CEA used like salivary biomarkers resulted more sensitive than serum. |
FSA: free sialic acid; PBSA: salivary protein-bound sialic acid; OHL: oral homogeneous leukoplakia; OPVL: oral proliferative verrucous leukoplakia; OPMD: oral potentially malignant disorder; OLK: oral leukoplakia; OLP: oral lichen planus; OC: oral cancer; OSCC: oral squamous cell carcinoma; ELISA: enzyme-linked immunosorbent assay; LC-MRM-MS: liquid chromatography-multiple reaction monitoring-mass spectrometry; SISCAPA-MRM: stable isotope standards and capture by anti-peptide antibodies coupled with multiple reaction monitoring-mass spectrometry; RT-PCR: real-time polymerase chain reaction; iTRAQ: isobaric tags for relative and absolute quantitation, SNCG: synuclein-gamma; EGFR: epidermal growth factor receptor; CEA: carcinoembryonic antigen; KLK5: kallikrein 5; Upa: urokinase-type plasminogen activator; MMP: metalloproteinase; TNF-α: tumour necrosis factor-α: IL: interleukine; KNG1: kininogen 1, HSPA5: heat shock 70 kDa protein 5; ANXA2: annexin 2; S1OOA2: S100 calcium-binding protein A2; IFN-γ: interferon-γ; GM-CSF: granulocyte macrophage colony-stimulating factor;.