Tongue Squamous Cell Carcinoma Research Articles

A Multifunctionalized Potyvirus-Derived Nanoparticle That Targets and Internalizes into Cancer Cells

Plant viral nanoparticles (VNPs) are attractive to nanomedicine researchers because of their safety, ease of production, resistance, and straightforward functionalization. In this paper, we developed and successfully purified a VNP derived from turnip mosaic virus (TuMV), a well-known plant pathogen, that exhibits a high affinity for immunoglobulins G (IgG) thanks to its functionalization with the Z domain of staphylococcal Protein A via gene fusion. We selected cetuximab as a model IgG to demonstrate the versatility of this novel TuMV VNP by developing a fluorescent nanoplatform to mark tumoral cells from the Cal33 line of a tongue squamous cell carcinoma. Using confocal microscopy, we observed that fluorescent VNP–cetuximab bound selectively to Cal33 and was internalized, revealing the potential of this nanotool in cancer research.

Prognostic value of radiological T category using conventional MRI in patients with oral tongue cancer: comparison with pathological T category.

This study aimed to compare the radiological tumor (T)-category using multiparametric MRI with the pathological T category in patients with oral tongue squamous cell carcinoma (OTSCC) and to examine which is a better predictor of prognosis. This retrospective study included 110 consecutive patients with surgically resected primary OTSCC who underwent preoperative contrast-enhanced MRI. T categories determined by maximum diameter and depth of invasion were retrospectively assessed based on the pathological specimen and multiparametric MRI. The MRI assessment included the axial and coronal T1-weighted image (T1WI), axial T2-weighted image (T2WI), coronal fat-suppressed T2WI, and axial and coronal fat-suppressed contrast-enhanced T1WI (CET1WI). Axial and coronal CET1WI measurements were divided into two groups: measurements excluding peritumoral enhancement (MEP) and measurements including peritumoral enhancement. The prognostic values for recurrence and disease-specific survival after radiological and pathological T categorization of cases into T1/T2 and T3/T4 groups were compared. The T category of MEP on coronal CET1WI was the most relevant prognostic factor for recurrence [hazard ratio (HR) = 3.30, p = 0.001] and the HR was higher than the HR for pathological assessment (HR = 2.26, p = 0.026). The T category determined by MEP on coronal CET1WI was also the most relevant prognostic factor for disease-specific survival (HR = 3.12, p = 0.03), and the HR was higher than the HR for pathological assessment (HR = 2.02, p = 0.20). The T category determined by MEP on the coronal CET1WI was the best prognostic factor among all radiological and pathological T category measurements.

Relationship between tumor thickness and GATA3 immunoexpression in lip and tongue squamous cell carcinomas.

Lower lip squamous cell carcinomas (LLSCCs) exhibit lower levels of aggressiveness, low relations with metastases and better prognosis when compared with intraoral squamous cell carcinomas. Differently from the oral tongue squamous cell carcinomas (OTSCCs) have a high tendency towards local invasion and lymph nodal dissemination. Our aim was to evaluate tumor thickness in cases of oral squamous cell carcinoma and correlate it with histological grade of malignancy and GATA3 immunoreactivity. Sixty specimens (30 LLSCCs and 30 OTSCCs) were scanned and digitized for the subsequent measurement of tumor thickness, histopathological examination, and quantitative analysis of GATA3 in the parenchyma and stroma of the tumors. Tumor thickness was lower in LLSCC compared to OTSCCs. Immunohistochemical analysis of GATA3 in parenchyma, stroma and both compartments showed higher immunoreactivity in LLSCCs compared to OTSCCs. We observed a negative correlation between tumor thickness and GATA3 expression in parenchyma, stroma, and both compartments. Our results revealed the presence of GATA3 in all cases both in the parenchyma and in the stroma. Higher expression was more related to LLSCCs, which are known to be less aggressive tumors than OTSCCs. A greater tumor thickness was found in OTSCCs, which was correlated with lower expression of GATA3, suggesting that this protein is involved in the inhibition of proliferative, migratory, and invasive capacity.

Cancer Cell Membrane-Coated Nanoparticles for Treatment of Oral Tongue Squamous Cell Carcinoma by Combining Photothermal/Photodynamic Therapy and Autophagy Inhibition

Cancer Cell Membrane-Coated Nanoparticles for Treatment of Oral Tongue Squamous Cell Carcinoma by Combining Photothermal/Photodynamic Therapy and Autophagy Inhibition

Abstract LB175: Discovery of HP567, a highly potent, brain-penetrating, and H1047R-selective PI3Kα inhibitor for the treatment of tumors bearing H1047R mutation

Abstract The phosphoinositide 3 kinase (PI3K)-protein kinase B (PKB/AKT) is one of the most altered pathways in human cancer and its hyperactivation promotes tumor growth and survival. H1047R increases kinase activity and is the most common PI3Kα mutation with a mutation rate of 7.8% in all cancers, and 21%, 5% and 3% in breast cancer, endometrium, and ovarian cancer, respectively. Alpelisib is an FDA approved PI3Kα inhibitor targeting both wild-type (WT) and mutant PI3Kα, yet its clinical benefit is limited by the toxicities including hyperglycemia and rash (which are considered on target effects of WT- PI3Kα inhibition). Here, we report an allosteric and brain-penetrating PI3Kα inhibitor, HP567 that is 14-fold selective for PI3Kα H1047R over PI3Kα WT and is 80-fold selective over other PI3K isoforms. HP567 strongly inhibits proliferation and p-AKT (S473) in H1047R mutant cell lines with an average IC50 of 138 nM and 8 nM, respectively, and has no activity against PI3Kα WT cell lines. HP567 strongly inhibits growth of tumors bearing H1047R in multiple CDX models including breast cancers (MDA-MB-453, T47D, and CAL-148), ovarian cancer (SK-OV-3), tongue squamous cell carcinoma (CAL-33), and a brain-metastatic breast cancer (MDA-MB-453), without causing hyperglycemia or increases in plasma insulin levels. In conclusion, HP567 is a novel, potent, and brain-penetrating PI3Kα H1047R selective inhibitor. The pre-clinical data supports the clinical development of HP567 for the treatment of tumors bearing PI3Kα H1047R mutations. Structure of HP567 will not be disclosed. Citation Format: Jing Li, Lei Fan, Chengcheng Liu, Yao Luo, Xiaohui Chu, Hua Yu, Fei Wang, Xinghai Li. Discovery of HP567, a highly potent, brain-penetrating, and H1047R-selective PI3Kα inhibitor for the treatment of tumors bearing H1047R mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB175.

Early growth response 1 regulates dual‑specificity protein phosphatase 1 and inhibits cell migration and invasion of tongue squamous cell carcinoma.

Oral squamous cell carcinoma (OSCC) is one of the most common malignant tumors in the head and neck, and among the OSCCs, tongue squamous cell carcinoma (TSCC) is one of the most common types. Although therapy strategies have recently advanced, the prognosis of TSCC has not substantially improved. Metastasis is one of the main causes of patient mortality in TSCC; therefore, it is necessary to elucidate the mechanism by which TSCC metastasis is regulated. In the present study, the early growth response 1 (Egr-1) expression in TSCC was analyzed based on GEO datasets and the effect of Egr-1 in TSCC tumor cell migration and invasion was measured using Transwell assay. By overexpressing dual-specificity protein phosphatase 1 (DUSP1) in cells with Egr-1 knockdown using lentivirus infection, the role of DUSP1 in Egr-1-regulated TSCC cell migration and invasion was determined. By using luciferase and ChIP assays, the mechanism behind how DUSP1 is regulated by Egr-1 was detected. In the present study, it was demonstrated that Egr-1 was downregulated in TSCC and the knockdown of Egr-1 increased TSCC cell migration and invasion. The expression of Egr-1 was also correlated with DUSP1. The overexpression of DUSP1 in Egr-1 knockdown cells, reduced the level of cell migration and invasion. Furthermore, it was demonstrated that knockdown of Egr-1 inhibited the promoter activity of DUSP1 and the site through which Egr-1 regulates DUSP1 transcription was identified. In conclusion, the present study demonstrated that Egr-1 regulates TSCC cell migration and invasion through modulating DUSP1, suggesting the potential of Egr-1 and DUSP1 as therapy targets for TSCC.

SOX9 promotes stemness in the CAL27 cell line of tongue squamous cell carcinoma.

Tongue squamous cell carcinoma (TSCC) is a prevalent form of oral malignancy, with increasing incidence. Unfortunately, the 5-year survival rate for patients has not exceeded 50%. Studies have shown that sex-determining region Y box 9 (SOX9) correlates with malignancy and tumor stemness in a variety of tumors. To investigate the role of SOX9 in TSCC stemness, we analyzed its influence on various aspects of tumor biology, including cell proliferation, migration, invasion, sphere and clone formation, and drug resistance in TSCC. Our data suggest a close association between SOX9 expression and both the stemness phenotype and drug resistance in TSCC. Immunohistochemical experiments revealed a progressive increase of SOX9 expression in normal oral mucosa, paracancerous tissues, and tongue squamous carcinoma tissues. Furthermore, the expression of SOX9 was closely linked to the TNM stage, but not to lymph node metastasis or tumor diameter. SOX9 is a crucial gene in TSCC responsible for promoting the stemness function of cancer stem cells. Developing drugs that target SOX9 is extremely important in clinical settings.

Melatonin inhibits tongue squamous cell carcinoma: Interplay of ER stress-induced apoptosis and autophagy with cell migration

Tongue squamous cell carcinoma (TSCC) occupies a high proportion of oral squamous cell carcinoma. TSCC features high lymph node metastasis rates and chemotherapy resistance with a poor prognosis. Therefore, an effective therapy strategy is needed to improve patient prognosis. Melatonin (MT) is a natural indole compound shown to have anti-tumor effects in several cancers. This study focused on the role and mechanism of MT in TSCC cells. The results of the study suggest that MT could inhibit cell proliferation in CRL-1623 cells. Western blot analysis showed the down-regulate of cyclin B1 and the up-regulate P21 protein by MT. MT was also shown to down-regulate the expression of Zeb1, Wnt5A/B, and β-catenin protein and up-regulate E-cadherin to inhibit the migration of CRL-1623 cells. MT also promoted the expression of ATF4, ATF6, Bip, BAP31 and CHOP in CRL-1623 cells leading to endoplasmic reticulum stress, and induced autophagy and apoptosis in CRL-1623 cells. Western blots showed that MT could promote the expression of Bax, LC3, and Beclin1 proteins and inhibit the expression of p62. We screened differentially expressed long non-coding RNAs (lncRNAs) in MT-treated cells and found that the expression of MALAT1 and H19 decreased. Moreover, MT inhibited tumor growth in nude mice inoculated with CRL-1623 cells. These results suggest that MT could induce autophagy, promote apoptosis, and provide a potential natural compound for the treatment of TSCC.

CD44 positive cancer stem cells express invasion and metastasis markers: Analysis of three tongue squamous cell carcinoma cell lines

CD44 positive cancer stem cells express invasion and metastasis markers: Analysis of three tongue squamous cell carcinoma cell lines

Evaluation of the Anti-Cancer Effect of Camel Milk on Tongue Squamous Cell Carcinoma Cell Line Using Flow Cytometric Analysis, Nuclear Morphometric Assay, and Western Blotting Technique: An In-Vitro Study

Evaluation of the Anti-Cancer Effect of Camel Milk on Tongue Squamous Cell Carcinoma Cell Line Using Flow Cytometric Analysis, Nuclear Morphometric Assay, and Western Blotting Technique: An In-Vitro Study

MicroRNA expression as a prognostic biomarker of tongue squamous cell carcinoma (TSCC): a systematic review and meta-analysis

BackgroundRecent studies have indicated that microRNA (miRNA) expression in tumour tissues has prognostic significance in Tongue squamous cell carcinoma (TSCC) patients. This study explored the possible prognostic value of miRNAs for TSCC based on published research.MethodsA comprehensive literature search of multiple databases was conducted according to predefined eligibility criteria. Data were extracted from the included studies by two researchers, and HR results were determined based on Kaplan‒Meier curves according to the Tierney method. The Newcastle‒Ottawa Scale (NOS) and GRADE (Grading of Recommendations Assessment, Development, and Evaluation) pro-GDT were applied to assess the quality of all studies. Publication bias was estimated by funnel plot, Egger’s rank correlation test and sensitivity analysis.ResultsEleven studies (891patients) were included, of which 6 reported up-regulated miRNAs and 7 mentioned down-regulated miRNAs. The pooled hazard ratio (HR) from the prognostic indicator overall survival (OS) was 1.34 (1.25–1.44), p < 0.00001, indicating a significant difference in miRNA expression between TSCC patients with better or worse prognosis.ConclusionMiRNAs may have high prognostic value and could be used as prognostic biomarkers of TSCC.

Enhanced laser-induced fluorescence and Raman spectroscopy with gold nanoparticles for the diagnosis of oral squamous cell carcinoma

BackgroundThe incidence, mortality, and recurrence rates of oral cancer are high worldwide. It is a common and aggressive type of tumor. Owing to the challenges associated with early illness diagnosis, squamous cell carcinoma, a kind that is prevalent of oral cancer, has an unacceptably high fatality rate. The management of the condition and the prevention of cancer, on the other hand, depend greatly on early detection. Therefore, alternative methods for the treatment and early diagnosis are essential for oral cancer. The detection of tongue squamous cell carcinoma is aided by coupled surface plasmon resonance, which can occur in gold nanoparticles (AuNPs). Compared to the currently utilized imaging contrast chemicals, AuNPs are more biocompatible and capable of targeting specific surface molecules. In the current study, AuNPs were synthesized in one step via citrate reduction and applied to tongue samples of a Caucasian man's Homo sapiens (Squamous cell carcinoma from ATCC cell-lines) in order to improve early detection using and laser-induced fluorescence and Raman spectroscopy.ResultsUV–visible spectroscopy, Zeta potential, TEM, and FTIR spectroscopic technique were used to characterize the synthesized nanoparticles. The synthesized AuNPs measured 13 ± 3 nm with uniform size distribution and high stability. Results demonstrate the significance of AuNPs in improving the identification of tongue squamous cell carcinoma.ConclusionObtained results revealed that the use of AuNPs modifies the emitted spectra in the two employed spectroscopic techniques and provides more significant receiver operating characteristic curve parameters, hence a higher detection rate of cancer.

Depth of invasion as an independent prognostic factor in early-stage oral cavity squamous cell carcinoma

PurposeTo determine the significance of depth of invasion as a predictor of recurrence and mortality in tongue and non-tongue early-stage oral cavity squamous cell carcinoma patients treated with surgery and no postoperative radiotherapy. Materials and methods344 patients with oral cavity squamous cell carcinoma from 2005 to 2022 at a tertiary academic medical center were reviewed. Patients were included if they had newly diagnosed, previously untreated T1-T2N0 disease treated with surgery alone that was observed within a follow-up of 5 years. For each patient, anatomic site of oral cavity squamous cell carcinoma was categorized as either tongue or non-tongue. Cox proportional hazards regression analyses were performed to determine the association of depth of invasion with recurrence and mortality, with anatomic site, smoking status, and age at biopsy as covariates. Model assumptions were tested by statistical and graphical evaluation using Schoenfeld residuals. ResultsOf 108 patients with T1-T2N0 disease, 78 (72.2 %) had tongue disease, and 30 (27.8 %) had non-tongue disease. Median follow-up was 18.2 months (range, 0.01–58.2 months). In the Cox proportional hazards models, with adjustment for anatomic site and other covariates, depth of invasion positively predicted recurrence (HR 1.16, 95 % CI: 1.01–1.32, p = 0.034) and death (HR 1.42, 95 % CI: 1.11–1.83, p = 0.006). ConclusionsDepth of invasion is an independent predictor of recurrence and death across early-stage tongue and non-tongue squamous cell carcinoma. Therefore, depth of invasion may indicate a need for more aggressive treatment than surgery alone, such as postoperative radiotherapy, even in the absence of other adverse features on pathology within the early-stage population.

Progressive loss of upper limb function as a presenting feature of glenohumoral metastasis from a primary tongue malignancy

Skeletal distant metastasis occurs rarely in head and neck squamous cell carcinoma. Palliative chemo or radio-therapy, immunotherapy or surgical treatment are indicated depending on the site, number of bony metastases and performance score of the patient; however with guarded prognosis. Due to the high mortality of patients with distant metastasis, early detection by imaging and identification of biomarkers can aid in prognostication and treatment planning.This paper describes a rare occurrence of shoulder skeletal metastasis in two patients who underwent initial definitive surgery followed by adjuvant therapy for lateral tongue squamous cell carcinoma.

Prognostic Factors for Locoregional Control in Early-Stage Oral Tongue Squamous Cell Carcinoma Treated with Partial Glossectomy and Elective Neck Dissection

Prognostic Factors for Locoregional Control in Early-Stage Oral Tongue Squamous Cell Carcinoma Treated with Partial Glossectomy and Elective Neck Dissection

Sentinel Lymph Node Biopsy (SLNB) for Early-Stage Head and Neck Squamous-Cell Carcinoma of the Tongue: Twenty Years of Experience at I.N.T. "G.Pascale".

Oral tongue squamous-cell carcinoma (OTSCC) is the most prevalent malignancy in the head and neck region. Lymphatic spread, particularly to cervical lymph nodes, significantly impacts 5-year survival rates, emphasizing the criticality of precise staging. Metastatic cervical lymph nodes can decrease survival rates by 50%. Yet, elective neck dissection (END) in T1-2 cN0 patients proves to be an overtreatment in around 80% of cases. To address this, sentinel lymph node biopsy (SLNB) was introduced, aiming to minimize postoperative morbidity. This study, conducted at the ENT and Maxillofacial Surgery department of the Istituto Nazionale Tumori in Naples, explores SLNB's efficacy in early-stage oral tongue squamous-cell carcinoma (OTSCC). From January 2020 to January 2022, 122 T1/T2 cN0 HNSCC patients were enrolled. Radioactive tracers and lymphoscintigraphy identified sentinel lymph nodes, aided by a gamma probe during surgery. Results revealed 24.6% SLN biopsy positivity, with 169 SLNs resected and a 21.9% positivity ratio. The study suggests SLNB's reliability for T1-2 cN0 OTSCC patient staging and early micrometastasis detection.

[The analysis of unconventional lymph node metastasis in tongue squamous cell carcinoma].

Objective: To analyze the characteristics of cervical lymph node metastasis in tongue squamous cell carcinoma (TSCC). Methods: A retrospective study was conducted. A total of 329 patients with TSCC who underwent en bloc resection of primary tumor and neck dissection in the Second Xiangya Hospital of Central South University from June 2010 to March 2018 were included. There were 283 males and 46 females, aged from 26 to 80 years. All patients underwent the modified neck dissection. The main difference between the modified neck dissection and the traditional neck dissection lay in the managements of unconventional lymph nodes. The lymphatic adipose tissues adjacent to the superior thyroid artery, the base of facial artery and the branches of external carotid artery were thoroughly dissected. The primary tumor as well as lingual artery, tissues along the lingual artery and lymph nodes in the mouth floor were resected. χ2 test was used for comparison of count data, and linear regression model was used for multivariate analysis. Results: Cervical lymph node metastases were found in 136 patients (41.3%). Among 142 patients (T1-2cN0) with supraomohyoid neck dissection, 22 patients had pathologically occult lymph node metastases (15.5%), with a 5-year overall survival rate of 90.2%, which was similar to the 5-year overall survival rate of 92.1% in 120 patients without lymph node metastasis (χ2=0.156, P=0.693). Multivariate linear regression analysis showed that T stage, clinical stage and unconventional lymph node metastasis were important factors for cervical lymph node metastasis in tongue cancer patients (P<0.05). Unconventional lymph node metastases occurred in 30 patients (9.1%), including the metastases of lymph nodes in the floor of mouth (3.0%), the lingual artery (2.4%), the base of the external maxillary artery (2.1%), the superior thyroid artery (0.9%), and the external carotid artery (0.6%). There were significant differences in the unconventional lymph node metastasis rates between patients with negative and positive conventional lymph node metastases [4.9%(10/203) vs. 15.9%(20/126), χ2=11.242, P=0.001] and also between patients with depth of invasion ≤5 mm, 5 mm <depth of invasion ≤10 mm or depth of invasion >10 mm [3.1%(2/64) vs. 5.7%(6/106) vs. 13.8%(22/159), χ2=7.907, P=0.005]. Conclusion: Supraomohyoid neck dissection can achieve reliable control efficacy in patients with cN0 tongue cancer. All patients with lymph node dissection should undergo unconventional lymph node dissection. Unconventional lymph node dissection is strongly recommended for patients with conventional lymph node metastasis.

RUVBL1 accelerates tongue squamous cell carcinoma by mediating CRaf/MEK/ERK pathway

RAF/MEK/ERK pathway is frequently activated in tumor. Therefore, this study will investigate the function of RUVBL1 (RAF-binding protein) in tongue squamous cell carcinoma (TSCC). Bioinformatics was performed to identify differentially expressed mRNAs (DE-mRNAs) in TCGA-oral squamous cell carcinoma, GSE13601, and GSE34105 datasets. A total of 672 shared DE-mRNAs were identified in three datasets, and they are regulating metastasis and angiogenesis. Patients with RUVBL1 low expression had high overall survival. Overexpressing RUVBL1 enhanced the viability, wound healing percentage, invasion, sphere formation, angiogenesis, and resistance to cisplatin and 5-fluorouracil in CAL-27 and SCC-4 cells, and the opposite results were obtained by knocking down RUVBL1. Moreover, overexpression of RUVBL1 bolstered tumor growth invivo. Strikingly, RUVBL1 diminished the phosphorylation of CRAF Ser259, which led to activation of the MEK/ERK pathway. In conclusion, RUVBL1 contributes to the malignant biological behavior of TSCC via activating the CRAF/MEK/ERK pathway. This provides molecular mechanisms and perspectives for targeted therapy of the CRAF/MEK/ERK pathway.

SCUBE3 exerts a tumor-promoting effect in tongue squamous cell carcinoma by promoting CEBPA binding to the CCL2 promoter.

Tongue squamous cell carcinoma (TSCC) is the main pathological subtype of oral cancer, and the current therapeutic effect is far from satisfactory. The signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) has been shown to be a tumor-promoting factor in several malignancies. However, little is known about the role of SCUBE3 in TSCC. In this study, we identified that SCUBE3 was highly expressed in TSCC. Clinically, high expression of SCUBE3 was positively associated with tumor stage and T stage of TSCC. Functionally, SCUBE3 silence remarkably restrained cell proliferation, migration and invasion, induced apoptosis as well as cell cycle arrest in G2 phase, and weakened the tumorigenicity of TSCC cells in vivo. Mechanistically, SCUBE3 promoted the direct binding of CCAAT enhancer binding protein alpha (CEBPA) to C-C motif chemokine ligand 2 (CCL2) promoter in TSCC cells. Interestingly, CCL2 overexpression partially reversed the inhibitory effect of SCUBE3 deficiency on TSCC cell viability and migration. Moreover, signal transducer and activator of transcription 3 (STAT3) signaling contributed to CCL2-mediated phenotypes in TSCC cells. Implications: our data revealed a tumor-promoting role for SCUBE3 in TSCC via the CEBPA/ CCL2/STAT3 axis, which provided new insight into novel potential therapeutic target for TSCC.

Novel DNA methylation biomarkers for early diagnosis of oral tongue squamous cell carcinoma (OTSCC).

Oral tongue squamous cell carcinoma (OTSCC) is the most common malignancy type among males across the world. However, analysis of molecular markers could be useful in detecting the early-stage OTSCC, which would allow optimal clinical treatments and prolong the survival rate of patients consequently. The study has the objective of detecting the role of salivary biomarkers based on gene promoter hypermethylation. Sample data from 45 OTSCC and normal groups were analyzed to exhibit the methylation levels of salivary biomarkers (TRH, FHIT, MGMT, p16, and RASSF1A). The specificity and sensitivity analysis of methylation biomarkers was conducted in addition to the receiver operating characteristic (ROC) curve for both early-stage and advanced OTSCC stages. Quantitative data findings showed the perfect sensitivity and specificity for TRH, MGMT, p16, and RASSF1A with 100%, and > 90%, respectively. In addition, the results indicated an inefficient area under curves (> 0.7) for these biomarkers to detect the OTSCC. There were no significant differences observed between TRH and FHIT and p16 and MGMT based on the Wilcoxon signed-rank test. The methylation statuses of genes TRH, RASSF1A, p16, and MGMT might become utilized as predictive biomarkers for clinical application in early diagnosis of OTSCC and noninvasive oral cancer screening.