Shaping the future of oral cancer diagnosis: advances in salivary proteomics

Figures & data

Figure 1. Steps from biomarker discovery to clinical implementation. The image was created in BioRender.

Figure 2. Main characteristics of saliva as biofluid and types of devices for saliva collection. The image was created with the BioRender.

Figure 3. Proteomics workflow and most used experimental approaches in proteomic studies using saliva as liquid biopsy for oral cancer. This image was created in BioRender.

Table 1. Salivary biomarkers for oral cancer and the main techniques used in each study.

Gene name	Biomarker type	Sample size			Proteomics approaches				IHC	Year
		OSCC	OPMD	Control	Separation of proteins	Identification of proteins	Validation of proteins	Targeted vs Untargeted
LMAN2, PTGR1, RAB13, UQCRC2	Diagnosis	20	Ø	10	SDS-PAGE	LC-MS/MS	Multiplexed bead-based immunoassay	Untargeted	-	2023	[59]
AZGP1, AHSG, KRT6C, BPIFB2	Diagnosis	50	Ø	49	NA	LC-MS/MS and PRM	NA	Untargeted	-	2021	[60]
A2Ml1, CRNN, HBB, Ig ĸ chain V-II region Vĸ167, KNG1, TMPRSS11D	Diagnosis	30	Ø	31	NA	LC-MS/MS	NA	Untargeted	-	2021	[61]
CD44, S100A7, S100P	Diagnosis	30 (2 groups)	15	15	iTRAQ	LC-MS/MS	ELISA	Untargeted	-	2020	[62]
TPM4	Diagnosis	30	Ø	20	2DE	MALDI-TOF	ELISA	Untargeted	+	2019	[63]
A1AT, SERPINA3, AHSG, ANT3, APCS, APOB, APOH, CFB, CFH, CP, FGB, FN1P, HPX, HRG, IVL, KNG1, PLG, S100A12, SERPIND1, VTN	Diagnosis	5	Ø	8 5 non-OSCC subjects	SDS-PAGE	LC-MS/MS	NA	Untargeted	-	2019	[64]
SLC3A2, S100A2, IL1RN	Diagnosis	20	20	20	iTRAQ	LC-MS/MS	ELISA	Untargeted	-	2018	[65]
PRDX-2, ZAG	Diagnosis	92	Ø	83	2-DE	LC-MS/MS	Western blot	Untargeted	-	2018	[66]
C5, SERPIND1, RETN	Diagnosis	20	Ø	17	MS	LC-MS/MS	ELISA	Untargeted	-	2018	[67]
HPA, sAA, K-10, GA-HSA	Diagnosis	2	Ø	2	2-DE	MALDI-TOF-MS	Western blot	Untargeted	-	2013	[68]
THBS2	Diagnosis	49	47	47	SDS-PAGE	LC-MS/MS	Bead-based immunoassay	Untargeted	+	2014	[69]
ANXA1, PRDX2	Diagnosis	22	Ø	20	SDS-PAGE	MALDI-TOF-MS	PSD MALDI-TOF/TOF MS	Untargeted		2012	[70]
CST1	Diagnosis	16	Ø	Ø	SDS-PAGE	SELDI-TOF-MS	NA	Untargeted	-	2009	[71]
TF	Diagnosis	41	Ø	30	2-DE	MALDI-TOF-MS	ELISA and Western blot	Untargeted	-	2010	[72]
24-mer ZNF510 peptide	Diagnosis	47	Ø	30	C8-magnetic beads	MALDI-TOF-MS	ELISA	Untargeted	+	2011	[72,73]
Gene name	Biomarker type	Sample size			Proteomics approaches				IHC	Year
		OSCC	OPMD	Control	Separation of proteins	Identification of proteins	Validation of proteins	Targeted vs Untargeted
STAT3, IKBKB, LY6D	Diagnosis	4	Ø	Ø	FFE and SCE chromatography	LC-MS/MS	Western blot	Untargeted	+	2007	[41]
M2BP, MRP14, CD59, CAT, PFN1	Diagnosis	16	Ø	16	2-DE	LC-MS/MS	ELISA and immunoblotting	Untargeted	-	2008	[42]
S100A8	Diagnosis	100	Ø	35	SDS-PAGE	LC-MS/MS	ELISA and Western blot	Untargeted	+	2014	[74]
α1 adrenergic receptors	Diagnosis	26	Ø	26	NA	ELISA	NA	Targeted	-	2021	[75]
LDH, CA125, CRP	Diagnosis	15	20	20	NA	ELISA	NA	Targeted	-	2021	[76]
Cyfra 21–1	Diagnosis	40	Ø	40	NA	ELISA	NA	Targeted	-	2023	[77]
Anti-MMP3, anti-PRDX2, anti-SPARC, anti-HSPA5	Diagnosis	160	Ø	140	NA	Multiplexed bead-based immunoassay	NA	Targeted	-	2022	[78]
MPO, NOX1, NCF2, DNASE1		16	Ø	16	NA	ELISA	NA	Targeted	-	2022	[79]
Exosomal exoAlix	Diagnosis	23	Ø	20	NA	ELISA	Western blotting	Targeted	+	2020	[80]
IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, IFN-g, TNF-α	Diagnosis	14	Ø	14	NA	Bio-Plex ProTM Human Cytokines assay 8-plex	NA	Targeted	-	2021	[81]
KLK5, uPA	Diagnosis	20	80 (4 groups)	20	NA	ELISA	NA	Targeted	+	2021	[82]
B2M	Diagnosis	25	25	25	NA	ELISA	NA	Targeted	-	2020	[83]
MMP1	Diagnosis	9	Ø	5	NA	MALDI-TOF-MS LC-MRM	Luminex multiplex assay	Targeted	-	2020	[45]
IL-6, IL-8, TNF-α	Diagnosis	9	38 (3 groups)	Ø	NA	ELISA	NA	Targeted	+	2020	[84]
CYFRA21–1	Diagnosis	5	30	Ø	NA	ELISA	NA	Targeted	+	2018	[85]
IL-6	Diagnosis	95	Ø	80	NA	ELISA	NA	Targeted	+	2019	[86]
MMP9	Diagnosis	24	20	22 + 22 tobacco users	NA	ELISA	NA	Targeted	+	2019	[87]
Gene name	Biomarker type	Sample size			Proteomics approaches				IHC	Year
		OSCC	OPMD	Control	Separation of proteins	Identification of proteins	Validation of proteins	Targeted vs Untargeted
CTSV, KLK5, ADAM9	Diagnosis	4	8	4	NA	ELISA	Western blotImmunofluorescence	Targeted	-	2018	[88]
IL-1b, IL-6, IL-8, MIP-1b, CCL11, IFN-g, TNF-a	Diagnosis	41	Ø	24	NA	Luminex Bead-based Multiplex Assay	NA	Targeted	-	2018	[89]
EGFR, HGF, sHER2, sIL-6Ra, PECAM-1	Diagnosis	50	Ø	38	NA	Bio-Plex ProTM Human Cancer Biomarker Panel 1, 16-plex angiogenic factors	NA	Targeted	-	2018	[90]
Naa10p, CEA	Diagnosis	112	30	60	NA	ELISA	NA	Targeted	-	2018	[91]
CD44	Diagnosis	20	Ø	20	NA	ELISA	NA	Targeted	-	2018	[92]
MMP9	Diagnosis	30	Ø	30	NA	ELISA	NA	Targeted	-	2017	[92,93]
S100A9, IL-6	Diagnosis	29	Ø	33	NA	Luminex assay and SRM-based assay	ELISA	Targeted	-	2017	[94]
VEGFR-2, IL-6, CRP	Diagnosis	15	32	15	NA	ELISA	NA	Targeted	+	2017	[95]
IL8, IL1B	Diagnosis	60	60	60	NA	ELISA	NA	Targeted	-	2016	[96]
MMP1, KNG1, ANXA2, HSPA5	Diagnosis	131	233 (2 groups)	96	NA	Multiplex LC-MRM-MS	NA	Targeted	-	2016	[97]
BIRC5, CEA	Diagnosis	26	Ø	10	NA	ELISA	NA	Targeted	-	2016	[98]
RARRES2, MMP9	Diagnosis	15	15	15	NA	ELISA	NA	Targeted	-	2017	[86]
C4d	Diagnosis	62	48	Ø	NA	ELISA	NA	Targeted	+	2015	[99]
C1R, LCN2, SLPI, FAM49B, TAGLN2, CFB, C3, C4B, LRG1, SERPINA1	Diagnosis	22 (2 groups)	Ø	8	NA	SRM assay	NA	Targeted	-	2015	[100]
CEA	Diagnosis	27	Ø	26	NA	ELISA	NA	Targeted	-	2016	[100]
Cyfra 21–1	Diagnosis	100	100	100	NA	ELISA	NA	Targeted	-	2015	[101]
IL-10	Diagnosis	22	Ø	23	NA	ELISA	NA	Targeted	+	2015	[102]
ENO1	Diagnosis	3	Ø	3	2-DE	Peptide mass fingerprinting MALDI-TOF-MS	NA	Targeted	+	2015	[103]
ERBB2	Diagnosis	20	20	20	NA	ELISA	NA	Targeted	-	2015	[104]
Gene name	Biomarker type	Sample size			Proteomics approaches				IHC	Year
		OSCC	OPMD	Control	Separation of proteins	Identification of proteins	Validation of proteins	Targeted vs Untargeted
Thiols, BChE	Diagnosis	25	Ø	30	NA	Assay with DTNB/Ellman’s reagent measured spectofotometrically	NA	Targeted	-	2014	[105]
IL-6	Diagnosis	18 OSCC	41 + 21 periodontitis	21	NA	ELISA	NA	Targeted	-	2013	[106]
TSA/TP ratio, NEU1, α2–3 and α2–6 sialoproteins	Diagnosis	100	50	100	NA	TSA and sialidase were estimated by spectrophotometric method Linkage specific biotinylated lectins were used to detect α-2,3 and α-2,6 STs and sialoproteins by ELISA and dot blot respectively	NA	Targeted	-	2014	[107]
anti-p53, anti-survivin, anti-Hsp60, anti-RPLP0, anti-CK8	Diagnosis	131	86 (2 groups)	131	NA	Bio-Plex system and ELISA	NA	Targeted	-	2014	[108]
TFF2, TFF3	Diagnosis	23	Ø	23	NA	ELISA	NA	Targeted	+	2012	[109]
TSA/TP ratio, FUCA1	Diagnosis	100	50	100	NA	Lowry method TSA and a-L-fucosidase assay	NA	Targeted	-	2013	[110]
SPO, SOD, UA	Diagnosis	10	45	30	NA	SPO – enzymatic oxidation reactions SOD – modified Beauchamp method Uric Acid liquicolor Plus assay	NA	Targeted	-	2011	[110]
MMP1, MMP3	Diagnosis	60	15	25	NA	ELISA	NA	Targeted	+	2011	[111]
NNMT	Diagnosis	16	Ø	15	SDS-PAGE	Western blot	NA	Targeted	+	2012	[112]
CCND1, SERPINB5, MMP9, carbonyls	Diagnosis	19	Ø	19	NA	ELISA	NA	Targeted	-	2009	[113]
Gene name	Biomarker type	Sample size			Proteomics approaches				IHC	Year
		OSCC	OPMD	Control	Separation of proteins	Identification of proteins	Validation of proteins	Targeted vs Untargeted
IL1B, IL8, M2BP	Diagnosis	35	Ø	51	NA	ELISA	NA	Targeted	-	2011	[114]
EGFR, Her-2, EGF	Diagnosis	46	Ø	46	NA	ELISA	NA	Targeted	+	2010	[115]
IL-1ª, IL-6, IL-8, VEGFA, TNF-a	Diagnosis	18		56 (4 groups)	NA	ELISA	NA	Targeted	-	2011	[48]
ET-1	Diagnosis	8	Ø	8	NA	ELISA	NA	Targeted	+	2007	[32]
IL-8, IL-1b	Diagnosis	20	Ø	20	NA	Luminex Multianalyte Profiling, ELISA	NA	targeted	-	2008	[43]
IL-6, IL-8	Diagnosis	32	Ø	32	NA	ELISA	NA	Targeted	-	2004	[116]
TNF-a, IL-1ª, IL-6, IL-8	Diagnosis	13	13	13	NA	ELISA	NA	Targeted	-	2005	[54,117]
STAT	Diagnosis	13 OPMD and OSCC 37 – other type		20	NA	HPLC	NA		Targeted	2005	[54]
IL-6, IL-8, TNF-α, HCC-1, PF-4	Diagnosis Prognosis	66	66	25	NA	Multiplex Immunoassay	NA	Targeted	-	2021	[118]
EGFR	Diagnosis Prognosis	63	Ø	60	NA	ELISA	NA	Targeted		2017	[119,120]
VIM	Diagnosis Prognosis	60	47	39	2D-DIGE	LC-MS/MS	ELISA	Untargeted	+	2022	[121]
sMICA	Diagnosis Prognosis	76	Ø	50	NA	ELISA	NA	Targeted	+	2022	[120]
S100A9, FGB, TF, IGHG1, CFL1, TTR	Diagnosis Follow up	8	Ø	8	2-DE	LC-MS/MS	Western blot	Untargeted	-	2008	[41]
AAT, HAP	Diagnosis Follow-up	12	Ø	12	2-DE	MALDI-TOF-MS	ELISA	Untargeted	+	2013	[122]
MMP1	Diagnosis Follow-up	269	578	313	NA	ELISA	NA	Targeted	-	2020	[123]
IL-1β, IL-8, LGALS3BP	Diagnosis Follow-up	58 OSCC 29 OSCC post-treatment	30	42	NA	ELISA	NA	Targeted	-	2020	[124]
TNF-a, IL-1, IL-6, IL-8	Diagnosis Prognosis	13	13	13	NA	ELISA	NA	Targeted	-	2005	[53]
Gene name	Biomarker type	Sample size			Proteomics approaches				IHC	Year	Ref
		OSCC	OPMD	Control	Separation of proteins	Identification of proteins	Validation of proteins	Targeted vs Untargeted
CFH, FGA, SERPINA1	Diagnosis Prognosis	117	116	115	iTRAQ	MRM-MS	ELISA	Untargeted	-	2019	[125]
Cyfra 21–1	Diagnosis Prognosis	30	Ø	30	NA	ELISA	NA	Targeted	+	2007	[40]
HGF, VEGF, PIGF, MMP-1, MMP-3, MMP-8, MMP-9, MMP-10, MMP-13, TIMP-2	Diagnosis Prognosis	10	Ø	8	NA	Protein chip array	NA	Targeted	+	2022	[126]
IL-4, IL-10, IL-13, and IL-1RA	Diagnosis Prognosis	30	Ø	33	NA	Multiplex immunoassay	NA	Targeted	+	2015	[127]
LDH	Diagnosis Prognosis	25	25	25	NA	BiovisionLactate Dehydrogenase Colorimetric Assay Kit	NA	Targeted	+	2015	[128]
Cyfra 21–1	Diagnosis Prognosis	50	Ø	50	NA	ECLIA	NA	Targeted	+	2016	[129]
MMP9, S100A9, NMMHC II-a, GDI2, PPIA	Prognosis	24	Ø	10	SDS-PAGE	LC-MS/MS	NA	Untargeted	-	2015	[130]
CRP	Prognosis	20	20	20	NA	Immunoturbidometry method	NA	Targeted	-	2016	[131]
CDH1	Prognosis	26	Ø	10	NA	ELISA	NA	Targeted	+	2017	[131,132]
LDH	Prognosis	32	Ø	37	NA	Standard kinetic spectrophotometric method	NA	Targeted	+	2016	[133]
IL-6	Diagnosis Prognosis	25	25	25	NA	ELISA	NA	Targeted	-	2015	[134]
SNCG	Diagnosis Prognosis	79	31	80	NA	ELISA	NA	Targeted	+	2020	[135]
GSS, MA, TNF-α, AFP	Diagnosis Prognosis	10	15	15	NA	ELISA	NA	Targeted	-	2019	[136]
TNF-α	Diagnosis Prognosis	30	30	30	NA	ELISA	NA	Targeted	-	2019	[137]
CA25, TPS	Diagnosis Prognosis	97	36	50	NA	ELISA	NA	Targeted	-	2013	[138]
Gene name	Biomarker type	Sample size			Proteomics approaches				IHC	Year
		OSCC	OPMD	Control	Separation of proteins	Identification of proteins	Validation of proteins	Targeted vs Untargeted
ASAH1, CSTA, ST6GALNAC1, ENDOU, LGALS1, SPINK5, PTX3	Prognosis	9	Ø	3	SDS-PAGE	LC-MS/MS	NA	Untargeted	-	2023	[139]
IL-17ª, IL-17F, TNF-α	Prognosis	71 OSCC/OPSCC			NA	ELISA Luminex Multiplex Assays	NA	Targeted	-	2020	[46]
IL-6	Prognosis	27	Ø	21	NA	Chemiluminescent enzyme immunoassay	NA	Targeted	-	2013	[140]
AKR1B10	Prognosis	86	Ø	35	NA	ELISA	NA	Targeted	-	2018	[141]
IL-6	Prognosis	52	Ø	52	NA	ELISA	NA	Targeted	-	2015	[142]
EGF	Prognosis Response to treatment	10	Ø	12	NA	ELISA	NA	Targeted	-	2004	[3]
SCCA-1, CKM, NME1, retinoic acid-binding protein II, LGALS7, HSAP27, HSP60, HSP71, HSP90, ANXA1	Diagnosis Prognosis Treatment	5	Ø	5	2DE-PAGE	MALDI-TOF-MS	Western blot	Untargeted	-	2014	[143]
HEP2, NHERF-2, MMP2, PGM 1, ACLY, KPCD	Treatment	30	Ø	30	NA	LC-MS/MS	NA	Untargeted	-	2023	[77,144]

Legend: Ø, without recruited patients; DNTB, dinitrothiocyanobenzene; ECLIA, electrochemiluminescence immunoassay; FFE, free flow electrophoresis; ELISA, enzyme-linked immunoassay; IHC, immunohistochemistry; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MALDI, matrix assisted laser desorption/ionization; MRM, multiple-reaction monitoring mass spectrometry; NA: non available information; OPSCC, oropharyngeal squamous cell carcinoma; OPMD, oral potentially malignant lesions; OSCC, oral squamous cell carcinoma; PRM, parallel reaction monitoring; SCE chromatography, strong cation exchange chromatography, SDS-PAGE, sodium dodecyl-sulfate polyacrylamide gel electrophoresis; SELDI, surface-enhanced laser desorption/ionization; SRM, selected reaction monitoring; TOF, time of flight mass spectrometry; TSA, trichostatin

Figure 4. Temporal evolution of salivary biomarkers and techniques used in its identification. Vosviewer network of most cited salivary biomarkers and techniques used in proteomics for biomarkers discovery in oral cancer (a). Vosviewer overlay showing the temporal distribution of the most cited salivary biomarkers and techniques used in proteomics for biomarkers discovery in oral cancer (b). Chronological discovery of the most relevant techniques used in studies of proteomics using saliva as source of liquid biopsy for discovery of biomarkers of oral cancer (c).

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