Saliva liquid biopsy
MMP2, MMP9, and TIMP2 as potential diagnostic biomarkers in oral squamous cell carcinoma. An observational case-control study
DOI:
https://doi.org/10.1590/Keywords:
Squamous cell carcinoma of head and neck, Matrix metalloproteinases, Tissue inhibitor of metalloproteinase-2, SalivaAbstract
Oral squamous cell carcinoma (OSCC) is the most common malignant neoplasm in the oral cavity, characterized by high morbidity and mortality. Various proteins, including matrix metalloproteinases (MMPs), have been investigated as potential biomarkers for diagnosing and prognosing this disease. Objective: To evaluate the utility of MMP-2, MMP-9, and the tissue inhibitor of metalloproteinase-2 (TIMP-2) as biomarkers in diagnosing and prognosing OSCC. Methodology: This retrospective case-control study involved 60 cases and 30 controls from the University Hospital Fundación Santa Fe de Bogotá, part of the Colombian cohort of the InterChange/Headspace study. Saliva samples were collected using a 60-second mouth rinse with 10 mL of sterile saline solution, along with serum and oral tissue samples and clinical and sociodemographic data. MMP-9, MMP-2, and TIMP-2 concentrations in saliva were determined using ELISA, whereas tissue samples were analyzed by immunohistochemistry. MMP activity in saliva was quantified using a generic activity assay. Results: Salivary MMP-9 and MMP-2 concentrations were significantly higher in cases than in controls, whereas no significant differences were observed for TIMP-2. ROC curve analysis showed excellent discriminatory capacity for salivary MMP-9 (AUC=0.946; sensitivity=0.817; specificity=0.867), good performance for MMP-2 (AUC=0.708; sensitivity=0.717; specificity=0.633), and lower discriminatory ability for TIMP-2 (AUC=0.630; sensitivity = 0.617; specificity=0.500). Elevated MMP-9 concentrations were observed in 33% of the serological samples. The immunohistochemistry of MMP-2, MMP-9, and TIMP-2 showed differences between cases and controls. Conclusions: This study highlights salivary MMP-9 and MMP-2, together with MMP activity, as biomarkers of interest for OSCC that may contribute to the understanding of disease-related molecular changes detectable by liquid biopsy. While elevated salivary levels were observed in OSCC cases when compared to controls, these findings should be interpreted as exploratory. Elevated serological MMP-9 levels were observed in a small subset of cases. However, the limited sample size and the absence of a control group in this study preclude diagnostic interpretation. Larger, well-powered, and externally validated studies are required to determine the potential clinical utility of these biomarkers.
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References
1Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, et al. Global Cancer Statistics 2020: GLOBOCAN Estimates of Incidence and Mortality Worldwide for 36 Cancers in 185 Countries. CA A Cancer J Clinicians 2021;71:209-49. doi: 10.3322/caac.21660.
» https://doi.org/10.3322/caac.21660.
2Panarese I, Aquino G, Ronchi A, Longo F, Montella M, Cozzolino I, et al. Oral and Oropharyngeal squamous cell carcinoma: prognostic and predictive parameters in the etiopathogenetic route. Expert Rev Anticancer Ther. 2019;19(2):105-19. doi: 10.1080/14737140.2019.1561288
» https://doi.org/10.1080/14737140.2019.1561288
3Johnson DE, Burtness B, Leemans CR, Lui VWY, Bauman JE, Grandis JR. Head and neck squamous cell carcinoma. Nat Rev Dis Primers 2020;6:92. doi: 10.1038/s41572-020-00224-3
» https://doi.org/10.1038/s41572-020-00224-3
4Chamoli A, Gosavi AS, Shirwadkar UP, Wangdale KV, Behera SK, Kurrey NK, et al. Overview of oral cavity squamous cell carcinoma: Risk factors, mechanisms, and diagnostics. Oral Oncology 2021;121:105451. doi: 10.1016/j.oraloncology.2021.105451
» https://doi.org/10.1016/j.oraloncology.2021.105451
5Liang KH, Lin YY, Chiang SH, Tsai ET, Lo WL, Wang CL, et al. Recent progress of biomarkers in oral cancers. J Chin Med Assoc. 2021;84(11):987-92. doi: 10.1097/JCMA.0000000000000616
» https://doi.org/10.1097/JCMA.0000000000000616
6Wong T-S, Gao W, Li ZH. Matrix metalloproteinase family as molecular biomarkers in oral squamous cell carcinoma. In: Preedy VR, Patel VB, editors. Biomarkers in Cancer, Dordrecht: Springer Netherlands; 2015, p. 771-90. doi: 10.1007/978-94-007-7681-4_10
» https://doi.org/10.1007/978-94-007-7681-4_10
7Nishio K, Motozawa K, Omagari D, Gojoubori T, Ikeda T, Asano M, et al. Comparison of MMP-2 and MMP-9 expression levels between primary and metastatic regions of oral squamous cell carcinoma. J Oral Sci. 2016;58:59-65. doi: 10.2334/josnusd.58.59
» https://doi.org/10.2334/josnusd.58.59
8Cabral-Pacheco GA, Garza-Veloz I, Castruita-De La Rosa C, Ramirez-Acuña JM, Perez-Romero BA, Guerrero-Rodriguez JF, et al. The roles of matrix metalloproteinases and their inhibitors in human diseases. Int J Mol Sci. 2020;21(24):9739. doi: 10.3390/ijms21249739
» https://doi.org/10.3390/ijms21249739
9Stamenkovic I. Extracellular matrix remodelling: the role of matrix metalloproteinases. J Pathol. 2003;200:448-64. doi: 10.1002/path.1400
» https://doi.org/10.1002/path.1400
10Gialeli C, Theocharis AD, Karamanos NK. Roles of matrix metalloproteinases in cancer progression and their pharmacological targeting. FEBS J. 2011;278:16-27. doi: 10.1111/j.1742-4658.2010.07919.x
» https://doi.org/10.1111/j.1742-4658.2010.07919.x
11Rosenthal EL, Matrisian LM. Matrix metalloproteases in head and neck cancer. Head & Neck. 2006;28:639-48. doi: 10.1002/hed.20365
» https://doi.org/10.1002/hed.20365
12Patil R, Mahajan A, Pradeep G, Prakash N, Patil S, Khan S. Expression of matrix metalloproteinase-9 in histological grades of oral squamous cell carcinoma: an immunohistochemical study. J Oral Maxillofac Pathol 2021;25:239. doi: 10.4103/0973-029X.325121
» https://doi.org/10.4103/0973-029X.325121
13Monteiro-Amado F, Castro-Silva II, Lima CJ, Soares FA, Kowalski LP, Granjeiro JM. Immunohistochemical evaluation of MMP-2, MMP-9 and CD31/microvascular density in squamous cell carcinomas of the floor of the mouth. Braz Dent J. 2013;24:3-9. doi: 10.1590/0103-6440201301941
» https://doi.org/10.1590/0103-6440201301941
14Di Zazzo E, Intrieri M, Davinelli S. Liquid biopsy and cancer: an ongoing story. J Clin Med. 2023;12:2690. doi: 10.3390/jcm12072690
» https://doi.org/10.3390/jcm12072690
15Hema Shree K, Ramani P, Sherlin H, Sukumaran G, Jeyaraj G, Don KR, et al. Saliva as a diagnostic tool in oral squamous cell carcinoma – a systematic review with meta analysis. Pathol Oncol Res. 2019;25:447-53. doi: 10.1007/s12253-019-00588-2
» https://doi.org/10.1007/s12253-019-00588-2
16Benitha G, Ramani P, Jayaraman S, R A, Ramalingam K, Krishnan M. Evaluation of serum levels of Matrix MetalloProteinase-9 (MMP-9) in oral squamous cell carcinoma and its clinicopathological correlation. Cureus. 2023;15(2):e34954. doi: 10.7759/cureus.34954
» https://doi.org/10.7759/cureus.34954
17Smriti K, Ray M, Chatterjee T, Shenoy R-P, Gadicherla S, Pentapati K-C, et al. Salivary MMP-9 as a biomarker for the diagnosis of oral potentially malignant disorders and oral squamous cell carcinoma. Asian Pac J Cancer Prev. 2020;21:233-8. doi: 10.31557/APJCP.2020.21.1.233
» https://doi.org/10.31557/APJCP.2020.21.1.233
18Zanoni DK, Patel SG, Shah JP. Changes in the 8th Edition of the American Joint Committee on Cancer (AJCC) Staging of Head and Neck Cancer: Rationale and Implications. Curr Oncol Rep. 2019;21:52. doi: 10.1007/s11912-019-0799-x
» https://doi.org/10.1007/s11912-019-0799-x
19Liu X, Su C, Xu J, Zhou D, Yan H, Li W, et al. Immunohistochemical analysis of matrix metalloproteinase-9 predicts papillary thyroid carcinoma prognosis. Oncol Lett. 2019;17(2):2308-16. doi: 10.3892/ol.2018.9850
» https://doi.org/10.3892/ol.2018.9850
20Dalirsani Z, Pakfetrat A, Delavarian Z, Hashemy SI, Mostaan LV, Abdollahnejad M, et al. Comparison of Matrix Metalloproteinases 2 and 9 levels in saliva and serum of patients with head and neck squamous cell carcinoma and healthy subjects. Int J Cancer Manag. 2019;12:E90249. doi: 10.5812/ijcm.90249
» https://doi.org/10.5812/ijcm.90249
21Penttala M, Sorsa T, Thomas JT, et al Grigoriadis A, Sakellari D, Gupta S. Periodontitis home screening with mouth rinse cut-off 20 Ng/mL aMMP-8 Test and mobile application. Diagnostics 2025;15:296. doi: 10.3390/diagnostics15030296.
» https://doi.org/10.3390/diagnostics15030296.
22Leppilahti JM, Tervahartiala T, Kautiainen H, Räisänen I, Ahonen MM, Uitto VJ, et al. Correlations between different protein species of oral rinse MMP-8 and patient-related factors. Clin Exp Dent Res. 2023;9:1021-33. doi: 10.1002/cre2.803
» https://doi.org/10.1002/cre2.803
23Rivera C, Oliveira AK, Costa RA, De Rossi T, Paes Leme AF. Prognostic biomarkers in oral squamous cell carcinoma: a systematic review. Oral Oncology 2017;72:38-47. doi: 10.1016/j.oraloncology.2017.07.003.
» https://doi.org/10.1016/j.oraloncology.2017.07.003.
24Cristaldi M, Mauceri R, Di Fede O, Giuliana G, Campisi G, Panzarella V. Salivary biomarkers for oral squamous cell carcinoma diagnosis and follow-up: current status and perspectives. Front Physiol. 2019;10:1476. doi: 10.3389/fphys.2019.01476
» https://doi.org/10.3389/fphys.2019.01476
25Bastías D, Maturana A, Marín C, Martínez R, Niklander SE. Salivary biomarkers for oral cancer detection: an exploratory systematic review. Int J Mol Sci. 2024;25(5):2634. doi: 10.3390/ijms25052634
» https://doi.org/10.3390/ijms25052634
26Radisky ES. Extracellular proteolysis in cancer: proteases, substrates, and mechanisms in tumor progression and metastasis. J Biol Chem. 2024;300:107347. doi: 10.1016/j.jbc.2024.107347.
» https://doi.org/10.1016/j.jbc.2024.107347.
27Quintero-Fabián S, Arreola R, Becerril-Villanueva E, Torres-Romero JC, Arana-Argáez V, Lara-Riegos J, et al. Role of Matrix Metalloproteinases in angiogenesis and cancer. Front Oncol. 2019;9:1370. doi: 10.3389/fonc.2019.01370
» https://doi.org/10.3389/fonc.2019.01370
28Gordon GM, Ledee DR, Feuer WJ, Fini ME. Cytokines and signaling pathways regulating matrix metalloproteinase-9 (MMP-9) expression in corneal epithelial cells. J Cell Physiol. 2009;221:402-11. doi: 10.1002/jcp.21869
» https://doi.org/10.1002/jcp.21869
29Ramos-DeSimone N, Hahn-Dantona E, Sipley J, Nagase H, French DL, Quigley JP. Activation of matrix metalloproteinase-9 (MMP-9) via a converging plasmin/stromelysin-1 cascade enhances tumor cell invasion. J Biol Chem. 1999;274(19):13066-76. doi: 10.1074/jbc.274.19.13066
» https://doi.org/10.1074/jbc.274.19.13066
30Rashid ZA, Bardaweel SK. Novel Matrix Metalloproteinase-9 (MMP-9) inhibitors in cancer treatment. Int J Mol Sci. 2023;24:12133. doi: 10.3390/ijms241512133
» https://doi.org/10.3390/ijms241512133
31Li S, Pritchard DM, Yu LG. Regulation and function of matrix metalloproteinase-13 in cancer progression and metastasis. Cancers. 2022;14:3263. doi: 10.3390/cancers14133263
» https://doi.org/10.3390/cancers14133263
32Hua H, Li M, Luo T, Yin Y, Jiang Y. Matrix metalloproteinases in tumorigenesis: an evolving paradigm. Cell Mol Life Sci. 2011;68:3853-68. doi: 10.1007/s00018-011-0763-x
» https://doi.org/10.1007/s00018-011-0763-x
33Cheng Y, Chen J, Shi Y, Fang X, Tang Z. MAPK signaling pathway in oral squamous cell carcinoma: biological function and targeted therapy. Cancers 2022;14:4625. doi: 10.3390/cancers14194625
» https://doi.org/10.3390/cancers14194625
34He Y, Sun MM, Zhang GG, Yang J, Chen KS, Xu WW, et al. Targeting PI3K/Akt signal transduction for cancer therapy. Sig Transduct Target Ther. 2021;6:425. doi: 10.1038/s41392-021-00828-5
» https://doi.org/10.1038/s41392-021-00828-5
35Liu F, Wu Q, Dong Z, Liu K. Integrins in cancer: emerging mechanisms and therapeutic opportunities. Pharmacol Ther. 2023;247:108458. doi: 10.1016/j.pharmthera.2023.108458
» https://doi.org/10.1016/j.pharmthera.2023.108458
36Niland S, Riscanevo AX, Eble JA. Matrix metalloproteinases shape the tumor microenvironment in cancer progression. Int J Mol Sci. 2021;23:146. doi: 10.3390/ijms23010146
» https://doi.org/10.3390/ijms23010146
37Kessenbrock K, Plaks V, Werb Z. Matrix metalloproteinases: regulators of the tumor microenvironment. Cell. 2010;141:52-67. doi: 10.1016/j.cell.2010.03.015
» https://doi.org/10.1016/j.cell.2010.03.015
38Itoh Y. Membrane-type matrix metalloproteinases: Their functions and regulations. Matrix Biol. 2015l;44-46:207-23. doi: 10.1016/j. matbio.2015.03.004
» https://doi.org/10.1016/j. matbio.2015.03.004
39McKaig BC, McWilliams D, Watson SA, Mahida YR. Expression and regulation of tissue inhibitor of metalloproteinase-1 and matrix metalloproteinases by intestinal myofibroblasts in inflammatory bowel disease. Am J Pathol. 2003;162(4):1355-60. doi: 10.1016/S0002-9440(10)63931-4
» https://doi.org/10.1016/S0002-9440(10)63931-4
40Braakhuis BJ, Leemans CR, Brakenhoff RH. Using tissue adjacent to carcinoma as a normal control: an obvious but questionable practice. J Pathol. 2004;203:620-1. doi: 10.1002/path.1549
» https://doi.org/10.1002/path.1549
41De Vicente JC, Fresno MF, Villalain L, Vega JA, Hernández Vallejo G. Expression and clinical significance of matrix metalloproteinase-2 and matrix metalloproteinase-9 in oral squamous cell carcinoma. Oral Oncology. 2005;41:283-93. doi: 10.1016/j.oraloncology.2004.08.013
» https://doi.org/10.1016/j.oraloncology.2004.08.013
42Jose D, Mane DR. Correlation of matrix metalloproteinase-9 expression with morphometric analysis of mucosal vasculature in oral squamous cell carcinoma, oral epithelial dysplasia, and normal oral mucosa. Int J Health Sci (Qassim) 2018;12:36-43.
43Zhao Y, Shen M, Wu L, Yang H, Yao Y, Yang Q, et al. Stromal cells in the tumor microenvironment: accomplices of tumor progression? Cell Death Dis. 2023;14:1-24. doi: 10.1038/s41419-023-06110-6
» https://doi.org/10.1038/s41419-023-06110-6
44Fan HX, Li HX, Chen D, Gao ZX, Zheng JH. Changes in the expression of MMP-2, MMP-9, and ColIV in stromal cells in oral squamous tongue cell carcinoma: relationships and prognostic implications. J Exp Clin Cancer Res. 2012;31:90. doi: 10.1186/1756-9966-31-90
» https://doi.org/10.1186/1756-9966-31-90
45Nath D, Shivasekar M, Vinodhini VM. Smoking induces the circulating levels of matrix metalloproteinase-9 and its association with cardiovascular risk in young smokers. Medeni Med J. 2022;37:306-12. doi: 10.4274/MMJ.galenos.2022.45057
» https://doi.org/10.4274/MMJ.galenos.2022.45057
46Sivaraman SreekanthK. Effect of smoking on metalloproteinases (MMPs) activity in patients with Acute Myocardial Infarction (AMI). J Clin Diagn Res. 2014;8(2):27-30. doi: 10.7860/JCDR/2014/7052.3998
» https://doi.org/10.7860/JCDR/2014/7052.3998
47Koken T, Gursoy F, Kahraman A. Long-term alcohol consumption increases pro-matrix metalloproteinase-9 levels via oxidative stress. J Med Toxicol. 2010;6:126-30. doi: 10.1007/s13181-010-0081-y
» https://doi.org/10.1007/s13181-010-0081-y
48Wang Z, Zhang C, Zhang X, et al. Assessing the impact of long-term storage on the quality and integrity of biological specimens in a reproductive biobank. Bioeng Transl Med. 2024 Jun 26;9(6):e10692. doi: 10.1002/btm2.10692
» https://doi.org/10.1002/btm2.10692
49Amadeu JK, Lemes AL, Schussel JL, Amenábar JM.. Effect of storage time and temperature on salivary total antioxidant capacity, total oxidant status, and oxidant stress index. Acta Stomatol Croat. 2019;53:119-24. doi: 10.15644/asc53/2/3
» https://doi.org/10.15644/asc53/2/3
50Boynes SG, Sofiyeva N, Saw T, Nieto V, Palomo L. Assessment of salivary matrix metalloproteinase (MMP8) and activated salivary matrix metalloproteinase (aMMP8) in periodontitis patients: a systematic review and meta-analysis. Front Oral Health. 2025;6:1444399. doi: 10.3389/froh.2025.1444399
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