Abstract
Background
Oral cancer is the most prominent cancer subtype among all head and neck cancers, the incidence and prevalence of which has been consistently increasing in past years around the globe. At advanced stages, oral cancer imparts significant mortality, morbidity, and mutilation among the patients, and therefore, diagnosis and treatment of the disease at early stages are considered the optimum strategy for the management of the disease. Since molecular changes appear earlier than physical symptoms, several molecular biomarkers are currently being investigated for their role in diagnosing and treating disease. MMP-9 belongs to the family of proteinases that are involved in cytoskeletal degradation, which is a crucial phase of cancer progression.
Objective
In the present study, we analyzed the serum concentration of MMP-9 in oral cancer patients and tried to establish MMP-9 as a predictive biomarker for the progression of oral cancer. We also correlated the clinical, sociodemographic and biochemical parameters with the serum concentration of MMP-9 in oral cancer patients.
Methods
Serum was extracted from the blood sample of 38 oral cancer patients and was analyzed for the concentration of MMP-9 using sandwiched ELISA. Predesigned proforma was used to capture the clinical, sociodemographic and biochemical parameters. Unpaired t-test was used to compare two means, one way ANOVA was used to compare more than two means and Pearson's correlation was used to correlate the variables.
Results
The mean concentration of MMP-9 in patients of oral cancer was 816.9 ± 236.1 ng/mL. The MMP-9 expression level was higher at advanced oral cancer stages than in the early stages. No significant difference in the MMP expression was found in terms of sociodemographic risk factor and tumor site. MMP-9 exhibit significant negative correlation with the HDL and significantly positive correlation with the PTI. Rest of the biochemical parameters does not exhibit significant correlation.
Conclusion
The present study suggests that serum concentration of MMP-9 can be a predictive biomarker for the progression of oral cancer, which needs to be validated by performing further longitudinal cross-sectional studies by taking ample sample size.
Keywords: Matrix metalloproteinases, Biomarker, Oral cancer, Serum, ELISA
Introduction
Oral cancer is the most prominent subtype among all head and neck cancers, following larynx and maxillary cancer. The incidence and prevalence of oral cancer have been increasing consistently in past years around the globe, specifically in low-income countries like India [1]. The patients come to the tertiary care hospital usually when the disease has progressed to advanced stages primarily because oral cancer at early stages is usually misdiagnosed to other diseases or patients waste their time seeking alternative treatment options like allopathic drugs, which usually provide only symptomatic relief [2]. At advanced stages, oral cancer imparts significant mortality, morbidity, and mutilation among the patients [3–10] because patients become physically weak at advance stages of disease [11] and have to undergo a combination of treatment modalities, including surgery, radiotherapy, and chemotherapy [12]. Therefore, diagnosis and treatment of the disease at its early stages are considered the optimum strategy for effective management of the disease. Since molecular changes appear earlier than physical symptoms [13], several molecular biomarkers are currently being investigated for their role in diagnosing and treatment of oral cancer.
Matrix metalloproteinases (MMPs) are the extracellular as well as intracellular peptides that belong to the family of zinc-containing endo proteinases and are able to degrade extracellular matrix components [13]. Till date, 24 different MMPs have been discovered. They are classified according to their location on chromosomes [14] into six groups: collagenases, gelatinases stromelysins, zinc- and calcium-dependent endopeptidases, matrilysins, and membrane-type metalloproteinases [15]. MMPs play important roles in inflammation, cancer wound healing, and many organ developments [16]. MMPs genes are reported to express in the case of tissue remodeling under pathological or physiological conditions [17]. MMPs, including MMP-1, -3, -2, -7, -9, -13, and -14, are reported to involve during cancer cell invasion [18].
MMP-9 belongs to Gelatinases, which are able to digest gelatin with the support of their 3-fibronectin type-II repeats. It can also digest a number of ECM molecules [19]. It is found in chromosome 20q11.2–q13.1 and has a nuclear factor-kB (NF-kB) binding site [20]. It is a potential biomarker for tissue remodeling, as indicated by previous animal models and clinical studies [21]. It acts by releasing bioactive FGF and VEGF sequestered within the ECM. Further release of FGF and VEGF stimulates the other MMPs, which are necessary for angiogenesis [20]. In the present study, we analyzed the serum concentration of MMP-9 in the early and advanced stages of oral cancer in patients and tried to establish MMP-9 as a predictive biomarker for the progression of oral cancer. We also correlated the MMP-9 expression with the sociodemographic determinants, tumor site, and biochemical parameters.
Material and Methods
Study Design and Recruitment of Patients
The present study was a prospective, single-centric, observational, and quantitative study. A total of 38 oral cancer patients have been recruited from the outpatient department of the institute, out of which 13 patients were of early stage (I and II) and 25 patients of advanced stages (III and IV). Patients with metastasis, other comorbidities, and recurrence cancer cases were excluded from the study. Detailed clinical history, sociodemographic determinants, biochemical parameters, and histopathological and radiological findings were noted down on pre-designed proforma.
Blood Collection and Serum Extraction
Two milliliters (mL) of blood was collected in a vacutainer tube by venipuncture. The vein was anchored by holding the patient arm and placing a thumb below the venipuncture site. The patient was asked to form a fist to make veins more prominent. The needle was inserted in the vein swiftly at a 30-degree angle. After collecting 2 mL blood, the needle was withdrawn gently, and a clean gauze was applied with gentle pressure to the site. The blood was transferred from the syringe to the vacutainer tube at a slow and steady pressure [22]. After keeping the blood sample at room temperature for 2 hours, the serum was extracted by centrifuging at 2000 g for 15 min. The serum was collected in microcentrifuge tubes and stored at − 20 °C until analyzed.
Sandwiched ELISA
Sandwiched ELISA Kit (Catalog No: E-EL-H6075, elabscience®, Houston, Texas) was used to analyze the MMP-9 concentration in the serum sample. The sensitivity of the assay was 0.10 ng/mL, and the detection range was 0.16–10 ng/mL. All samples were diluted 100 times, and standards of 10–0.16 ng/mL were prepared using serial dilution (Fig. 1). Each sample, including the standards, were placed in duplicate. The sandwiched ELISA was performed per the manufacturer protocol (Fig. 2). The micro-ELISA plate had a pre-coated MMP-9 antibody. The standards and sample were added to the microwells, which combined pre-coated MMP-9 antibodies. Then, a biotinylated detection antibody and avidin–horseradish peroxidase (HRP) conjugate were added to each microwell and incubated. Unbounded antibodies were washed away using a wash buffer by an ELISA microplate washer (BioTek® Instruments Inc., Winooski, VT, USA). After washing, the substrate solution was added to each microwell. The blue color appears in the microwells; the intensity of which corresponds to the concentration of MMP-9. The enzyme–substrate reaction was terminated by adding a stop solution, and the color turned yellow. The optical density (OD) is measured using a spectrophotometer (BioTek® Instruments Inc., Winooski, VT, USA) at a 450 ± 2 nm wavelength. The OD value is proportional to the concentration of MMP-9. The concentration of MMP-9 in the samples was calculated by comparing the OD of the samples to the standard curve (Fig. 3). The final concentration of MMP-9 was calculated by multiplying the dilution factor of 100 by each value.
Fig. 1.
Preparation of standards of 10–0.16 ng/mL by serial dilution
Fig. 2.
Summary of the standardized protocol of sandwiched ELISA
Fig. 3.
Standard curve of MMP-9 obtained from the standards of 10–0.16 ng/mL
Statistical Analysis
GraphPad Prism 9.0 (GraphPad® Software Inc., San Diego, CA) was used to perform the statistical analysis of data. The data are presented as fraction of the total and percentage. Mean was used as the measure of central tendency, and standard deviation was used as a measure of variability. Unpaired t-test was used to compare two means, and one-way ANOVA was used to compare multiple means. Pearson correlation coefficient (r) was used for correlation analysis. The p-value < 0.05 was taken as significant for all tests.
Results
The mean age of the 38 recruited patients was 51.13 ± 13.38 years, and the mean serum MMP concentration was 816.9 ± 236.1 ng/mL. The number of male patients was higher (78.94%) compared to their counterpart females (21.05%) with a male-to-female sex ratio of 3.75:1. MMP-9 expression was higher in males (826.3 ± 242.5 ng/mL) compared to females (781.5 ± 222.0 ng/mL), but the difference was not significant. All the recruited patients were married, with no unmarried individual. Most of the recruited patients were self-employed (42.10%), and this group also exhibited a higher expression of MMP-9 (850.9 ± 263.1 ng/mL) compared to unemployed (785.6 ± 210.1 ng/mL) and job-employed (798.8 ± 234.5 ng/mL) individuals. A higher proportion of total patients have obtained school-level education (68.42%), while a small proportion represents the individual who was either illiterate (15.78%) or received a graduate (10.52%) and postgraduate level (5.26%) education. Expression of MMP-9 was higher in patients who obtained the school (829.4 ± 256.3 ng/mL) or graduate-level (855.9 ± 227.7 ng/mL) education, compared to the rest of the segment but again the difference is not significant. Most of the patients belonged to low-income families (65.78%), and this segment of the population also represents a higher level of MMP-9 expression (815.9 ± 242.8 ng/mL) compared to high-income families (683.4 ± 285.4 ng/mL). A slightly higher proportion of recruited patients belonged to the rural background (95.78%), and this segment was found to exhibit a higher expression of MMP-9 (852.1 ± 227.9 ng/mL) compared to the urban population (749.1 ± 245.9 ng/mL). In food habits, the proportion of non-vegetarian patients was slightly higher (55.26%) compared to vegetarian ones (44.73%), and the former group shows a higher level of MMP-9 expression (862.4 ± 205.2 ng/mL) compared to the latter one (780.1 ± 257.4 ng/mL). Most patients do not reveal a family history of cancer (94.73%), and MMP-9 concentration was high (680.6 ± 379.8 ng/mL) in those patients. A large segment of recruited patients (68.43%) revealed an addiction history of smoking, tobacco, alcohol, or a combination. MMP-9 expression was higher in patients with a combination of addiction habits (Table 1).
Table 1.
Sociodemographic determinants of the recruited patients along with the serum concentration of MMP-9
| Domain | Variable | Mean ± SD or N (%) | MMP-9 (ng/mL) | P Value |
|---|---|---|---|---|
| Age | Mean age in years | 51.13 ± 13.38 | 816.9 ± 236.1 | |
| Sex | Male | 30 (78.94) | 826.3 ± 242.5 | 0.6399 |
| Female | 8 (21.05) | 781.5 ± 222.0 | ||
| Marital status | Married | 38 (100) | 816.9 ± 236.1 | Nil |
| Unmarried | Nil | |||
| Occupation | Unemployed | 11 (28.94) | 785.6 ± 210.1 | 0.7549 |
| Self-Employed | 16 (42.10) | 850.9 ± 263.1 | ||
| Job-Employed | 11 (28.94) | 798.8 ± 234.5 | ||
| Education | Illiterate | 6 (15.78) | 750.4 ± 211.1 | 0.8784 |
| School level | 26 (68.42) | 829.4 ± 256.3 | ||
| Graduation | 4 (10.52) | 855.9 ± 227.7 | ||
| Postgraduation | 2 (5.26) | 775.7 ± 39.77 | ||
| Monthly Family Income | < 10,000 INR | 25 (65.78) | 815.9 ± 242.8 | 0.2687 |
| 10,000–30,000 INR | 8 (21.05) | 903.5 ± 156.7 | ||
| > 30,000 INR | 5 (13.15) | 683.4 ± 285.4 | ||
| Locality | Rural | 25 (65.78) | 852.1 ± 227.9 | 0.2062 |
| Urban | 13 (34.21) | 749.1 ± 245.9 | ||
| Food habits | Vegetarian | 17 (44.73) | 862.4 ± 205.2 | 0.2918 |
| Non-vegetarian | 21 (55.26) | 780.1 ± 257.4 | ||
| Family history | Yes | 2 (5.26) | 680.6 ± 379.8 | 0.4090 |
| No | 36 (94.73) | 824.5 ± 231.7 | ||
| Addiction history | Smoking | 2 (5.263) | 1049 ± 0.000 | 0.2858 |
| Tobacco | 3 (7.894) | 651.5 ± 428.2 | ||
| Alcohol | 4 (10.52) | 694.4 ± 300.1 | ||
| Smoking + Tobacco | 3 (7.894) | 943.2 ± 164.3 | ||
| Smoking + Alcohol | 7 (18.42) | 904.2 ± 164.9 | ||
| Tobacco + Alcohol | 4 (10.52) | 664.0 ± 278.3 | ||
| Smoking + Tobacco + Alcohol | 3 (7.894) | 920.1 ± 223.6 | ||
| No Addiction History | 12 (31.57) | 803.1 ± 189.9 |
Most patients have a tumor in the tongue region (47.36%) and buccal mucosa (23.68%). Other sites in the oral cavity in which the tumor was reported include the alveolus (7.89%), floor of mouth (7.89%), mandible (7.89%), and palate region (5.26%). High expression of MMP-9 was observed in patients having a tumor in the buccal mucosa (927.8 ± 134.4 ng/mL), mandibles (967.6 ± 141.3 ng/mL), and palate region (828.2 ± 312.5 ng/mL) compared to the rest of the tumor site in the oral cavity. Most of the patients were represented with the advanced stage III (31.57%), and stage IV (34.21%) of the tumor, and only a small fraction of total recruited patients reported with the early stage I (7.89%) and stage II (26.31%). Stage III and stage IV patients have higher mean MMP-9 concentrations of 866.1 ± 184.0 ng/mL and 863.9 ± 236.2 ng/mL, respectively, while stage I and II have lower mean MMP-9 concentrations of 762.9 ± 161.5 ng/mL and 713.0 ± 297.0 ng/mL, respectively (Table 2).
Table 2.
Tumor site and cancer staging of recruited patients of oral cancer along with the serum concentration of MMP-9
| Domain | Variable | N (%) | MMP-9 (ng/mL) Mean ± SD | P Value |
|---|---|---|---|---|
| Tumor site | Buccal mucosa | 9 (23.68) | 927.8 ± 134.4 | 0.4532 |
| Tongue | 18 (47.36) | 758.9 ± 259.1 | ||
| Alveolus | 3 (7.89) | 749.3 ± 99.94 | ||
| Floor of mouth | 3 (7.89) | 741.6 ± 417.7 | ||
| Mandibles | 3 (7.89) | 967.6 ± 141.3 | ||
| Palate | 2 (5.26) | 828.2 ± 312.5 | ||
| Cancer stage | Stage I | 3 (7.89) | 762.9 ± 161.5 | 0.3844 |
| Stage II | 10 (26.31) | 713.0 ± 297.0 | ||
| Stage III | 12 (31.57) | 866.1 ± 184.0 | ||
| Stage IV | 13 (34.21) | 863.9 ± 236.2 |
Among various biochemical parameters, the AST, triglycerides, potassium, creatinine, uric acid, urea, T3, T4, lipase, bilirubin, PT, PTI, INR, and hemoglobin were found to have a positive correlation with the serum concentration of MMP-9. Other biochemical parameters, including the ALT, ALP, cholesterol, LDL, HDL, sodium, calcium, chloride, TSH, amylase, protein, albumin, PTTK, and platelets, were found to have negative correction with the serum concentration of MMP-9. MMP-9 exhibit significant negative correlation with the HDL and significantly positive correlation with the PTI whereas in the case of the rest of the biochemical parameters, the correlation was not significant (Table 3).
Table 3.
Biochemical profile of the recruited patients and their correlation with the serum concentration of MMP-9
| Domain | Variable | Mean ± SD, N | Pearson correlation (r) | P value |
|---|---|---|---|---|
| Liver function | ALT (U/L) | 33.95 ± 18.99, 22 | − 0.1656 | 0.4615 |
| AST (U/L) | 40.73 ± 41.66, 22 | 0.01829 | 0.9356 | |
| ALP (U/L) | 114.3 ± 52.74, 21 | − 0.2255 | 0.3258 | |
| Lipid profile | Cholesterol (mg/dL) | 217.9 ± 41.43, 13 | − 0.0233 | 0.9398 |
| Triglycerides (mg/dL) | 229.8 ± 110.7, 12 | 0.3094 | 0.3278 | |
| LDL (mg/dL) | 140.0 ± 40.35, 12 | − 0.1417 | 0.6604 | |
| HDL (mg/dL) | 43.41 ± 7.94, 13 | − 0.5825 | *0.0367 | |
| Blood glucose | FBS (mmol/L) | 115.9 ± 0.00, 1 | ||
| Electrolytes | Sodium (mg/dL) | 139.9 ± 4.11, 23 | − 0.1017 | 0.6443 |
| Calcium (mg/dL) | 9.28 ± 0.72, 14 | − 0.09351 | 0.7505 | |
| Potassium (mg/dL) | 4.52 ± 0.41, 22 | 0.04599 | 0.839 | |
| Creatinine (mg/dL) | 0.90 ± 0.24, 31 | 0.2356 | 0.202 | |
| Uric acid (mg/dL) | 4.96 ± 1.36, 25 | 0.1705 | 0.4152 | |
| Phosphate (mg/dL) | 3.59 ± 0.33, 2 | |||
| Urea (mg/dL) | 28.10 ± 14.13, 31 | 0.348 | 0.0551 | |
| Chloride (mg/dL) | 102.3 ± 4.10, 23 | − 0.01135 | 0.959 | |
| Thyroid function | T3 (mIU/mL) | 30.71 ± 59.16, 9 | 0.2306 | 0.5505 |
| T4 (mIU/mL) | 7.89 ± 1.97, 9 | 0.4629 | 0.2096 | |
| TSH (mIU/mL) | 3.75 ± 2.56, 10 | − 0.5505 | 0.0992 | |
| Enzymes | Lipase (U/L) | 106.2 ± 49.92, 3 | 0.8015 | 0.408 |
| Amylase (U/L) | 89.67 ± 54.78, 3 | − 0.6297 | 0.5664 | |
| Proteins | Protein (mg/dL) | 7.09 ± 0.63, 21 | − 0.04114 | 0.8595 |
| Albumin (mg/dL) | 4.21 ± 0.83, 22 | − 0.1412 | 0.5309 | |
| Bilirubin (mg/dL) | 0.56 ± 0.40, 24 | 0.3676 | 0.0772 | |
| Prothrombin | PT | 19.49 ± 22.22, 18 | 0.2805 | 0.2596 |
| PTI | 83.16 ± 26.68, 12 | 0.7896 | *0.0023 | |
| INR | 1.08 ± 0.11, 17 | 0.1114 | 0.6702 | |
| PTTK | 29.08 ± 4.78, 10 | − 0.1865 | 0.6059 | |
| Hemogram | Hemoglobin (g/dL) | 13.36 ± 2.06, 28 | 0.2837 | 0.1435 |
| Platelets (109/L) | 232.1 ± 103.2, 26 | − 0.1918 | 0.3479 |
Discussion
Previous studies have reported a significantly high expression of MMP-9 in patients with head and neck cancer compared to the healthy control in the various body fluids and tissue, including saliva [23–27], biopsy tissue [28–31], plasma [32], and in serum [27, 33] (Table 4). In an recent study, patients with head and neck cancer were reported to have a significantly higher concentration of serum MMP-9 (range between 39 and 1547 ng/mL with a mean concentration of 417 ng/mL) compared the healthy individuals (range between 30 and 537 ng/mL with a mean concentration of 189 ng/mL) [34]. In another study, serum levels of MMP-9 in head and neck cancer patients were reported to be 78.77 ± 27.51 ng/mL, which was substantially higher than that of the healthy control at 39.89 ± 20.54 ng/mL [27]. In the present study, the mean serum concentration of MMP-9 in oral cancer patients was 816.9 ± 236.1 ng/mL, which supports the previous data.
Table 4.
Summary of various findings related to MMP-9 expression in head and neck cancer with reference to sample used in the study and methods of analysis
| Finding related to MMP-9 expression | Sample | Method | Authors |
|---|---|---|---|
| Elevated expression of MMP-9 compared to healthy controls | Saliva | ELISA | Peisker et al. [23], Shpitzer et al. [24], Shpitzer et al. [25], Smriti et al. [26], Dalirsani et al. [27] |
| Tissue | IHC | Kato et al. [29], Ikebe et al. [30] | |
| RT-PCR | Pornchai et al. [28] | ||
| Plasma | ELISA | Singh et al. [32] | |
| Serum | ELISA | Dalirsani et al. [27], Lee et al. [33] | |
| Significantly high expression of MMP-9 found at advanced stages of cancer compared to local stages | Tissue | IHC | Atla et al. [35] |
| Plasma | ELISA | Lin et al. [36] | |
| Serum | ELISA | Riedel et al. [34], Lotfi et al. [37] | |
| High level of MMP-9 expression is positively correlated with the regional lymph node metastasis or distant metastasis | Tissue | IHC | Katayama et al. [38], Kurahara et al. [39], Zhou et al. [40], Fan et al. [41] |
| Serum | ELISA | Lotfi et al. [37] | |
| MMP-9 level significantly decreased after tumor surgery | Saliva | ELISA | Shin et al. [43] |
| Serum | ELISA | Stanciu et al. [44] | |
| MMP-9 expression positively correlated with dysplasia | Tissue | IHC | Champatyray et al. [31] |
| MMP-9 expression is not correlated with tumor stage or recurrence rate | Tissue | IHC | de Vicente et al. [45] |
| MMP-9 expression is not correlated with the tumor size and metastasis | Tissue | IHC | Guttman et al. [42] |
Significantly high expression of MMP-9 was observed in the advanced stage of oral cancer compared to early stages in several studies [34–36]. Serum levels of MMP-9 in oral cancer at the T1 stage were reported to be 1119.80 ± 307.71 ng/mL, which was found to increase at the T2 stage to 1308.30 ± 372.92 ng/mL and was highest in the T3 stage to 1483.75 ± 134.85 ng/mL [37]. In the present study also, the mean serum concentration was found to increase with the advanced cancer stage. Patients with cancer stage I and stage II have a mean MMP-9 concentration of 762.9 ± 161.5 ng/mL and 713.0 ± 297.0 ng/mL, respectively, whereas the patients with stage III and stage IV cancer have a mean MMP concentration of 866.1 ± 184.0 ng/mL and 863.9 ± 236.2 ng/mL, respectively.
Several studies correlated the MMP-9 expression with metastasis found that the elevated level of MMP-9 expression is positively correlated with regional lymph node metastasis or distant metastasis in oral cancer [38–41]. However, contradictory to those studies, a study conducted by Guttman et al. found that the high MMP-9 expression in tongue cancer patients is not correlated with neck metastasis [42]. MMP-9 expression has also been found to be decreased significantly after surgery in oral cancer patients [43]. The mean serum level of MMP-9 in preoperative oral cancer cases was reported to be 59.10 ± 13.61 ng/mL, whereas that in healthy controls was significantly lower at 24.83 ± 17.33 ng/mL [33]. In a cohort study in oral cancer patients, the serum concentration of MMP-9 at the preoperative stage ranged from 914 to 1964 ng/L (mean, 1619 ng/mL) and at the postoperative stage, was in the range from 877 to 2001 ng/mL (mean, 1402 ng/mL) [44]. However, in a particular study by de Vicente et al., no correlation between the MMP-9 expression and recurrence rate was found in oral cancer patients [45]. In present study, however, we did not conducted the follow up of patients to analyze the MMP-9 concentration at postoperative stages or over long term survival.
There has been a debate about whether demographic factors or the disease condition influence the expression of molecular markers. It is incorrect to associate MMP-9 with demographic factors or disease conditions alone. MMP-9 expression at any time is the sum of the disease condition and demographic determinants. In the present study, we analyze the MMP-9 expression per disease condition and demographic factors. Gender differences tend to influence the MMP-9 expression as there is a huge difference between male and female hormonal profiles, thus the underlying physiology. Patients from the upper economic class tend to have enriched healthcare access more than low economic class. Education level influences lifestyle and hygienic conditions. All these variables tend to influence the MMP-9 expression in oral cancer patients.
Previously, Shin et al. reported that the MMP-9 expression in oral squamous cell carcinoma is influenced by the addiction history, education level, physical activity, periodontitis, diabetes and hypercholesterolemia, obesity, and hypertension [43]. Patil et al. analyzed the MMP-9 expression in oral squamous cell carcinoma tissue samples using immunohistochemistry and found higher MMP-9 expression in females compared to males; also, MMP-9 expression was found to increase with advancing age [46]. Dai et al. analyzed MMP-9 expression in oral squamous cell carcinoma and reported elevated expression of MMP-9 in females compared to males [47].
The present study revealed the serum concentration of MMP-9 in oral cancer patients and highlighted its correlation with the various sociodemographic, clinical, and biochemical parameters. The present study provided important information to support the existing studies to establish the MMP-9 as a biomarker of clinical significance for oral cancer. As the present study was a pilot study, sample size is limited, which suggests that future studies could take a large sample. A further study that involved the simultaneous analysis of MMP-9 concentration in all body fluids, including the serum, plasma, saliva, and tissue extract, could help rule out the contradictions in the literature. Apart from body fluids, it would be more interesting to simultaneously analyze the MMP-9 expression at the mRNA level. MMP-9 can occur in an active and inactive form in the cancer microenvironment; it would be of great application to analyze and compare the active and inactive concentrations of MMP-9 using advanced techniques like fluorogenic substrate assay. A study establishing the MMP-9 concentration in various subtypes of head and neck cancer could also be of great importance. We have reported the MMP-9 serum concentration only before the surgery, and a further follow-up study that analyzes the MMP-9 expression in oral cancer patients over long-term survival could be of great importance. A more elaborated study that could correlate the MMP-9 expression and treatment relapse rate will also be very significant.
Conclusion
The serum concentration of MMP-9 was found to be high in advanced stages of oral cancer compared to early stages, which suggests that MMP-9 has the potential to be a predictive biomarker for the progression of oral cancer. The MMP-9 expression level was also found to be strongly correlated with certain sociodemographic risk factors, including addiction habits, which suggests that the disease progression could be halted by arresting the addiction habits. Since most of the biochemical parameters were not found to exhibit a significant correlation with MMP-9, further research is required to search for their potential correlation with the MMP-9 expression. Further longitudinal, cross-sectional, multi-institutional randomized studies, which undertake a large sample size analyzing the expression of MMP-9 over long-term survival in oral cancer patients, could help establish the MMP-9 as a marker of clinical relevance to check the progression of oral cancer.
Acknowledgements
Authors highly acknowledged to our colleagues for reviewing the present work. Authors are also acknowledged to doc navigator© New Chandigarh, India for providing the proofreading services for this manuscript.
Author Contributions
JB and NP conceptualized the work and helped with the patient recruitment; MS and AKY helped in the molecular analysis; JS, KY, and AKG performed the experiments; JS and AKG compiled the raw data, generated the results, and drafted the manuscript.
Funding
The work in present article was funded by the intramural research grant (Letter No. 71/2-Edu-16/605) and institute research fellowship (Letter No. A30011/Ph.D./1Trg/July2019) provided by the host institute.
Data Availability
This manuscript has no associated data to be deposit. All data generated or analyzed during this study are included in this article.
Code Availability
No Code has been used in this article.
Declarations
Conflict of interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Ethical Approval
Present study has been approved by the institute ethics committee (Letter No. INT/IEC/2021/SPL-427 with Ref No. NK/6993/MS/782).
Consent to Participate
Participant information sheet was provided to all participants, and written consent was taken from every participant.
Consent for Publication
Authorship consent form has been signed by all the authors.
Footnotes
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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