Identification of Proteomic Biomarkers in Proliferative Verrucous Leukoplakia through Liquid Chromatography With Tandem Mass Spectrometry.

Proliferative verrucous leukoplakia (PVL) is an oral potentially malignant disorder associated with high risk of malignant transformation. Currently, there is no treatment available, and restrictive follow-up of patients is crucial for a better prognosis. Oral leukoplakia (OL) shares some clinical and microscopic features with PVL but exhibits different clinical manifestations and a lower rate of malignant transformation. This study aimed to investigate the proteomic profile of PVL in tissue and saliva samples to identify potential diagnostic biomarkers with therapeutic implications. Tissue and saliva samples obtained from patients with PVL were compared with those from patients with oral OL and controls. Label-free liquid chromatography with tandem mass spectrometry was employed, followed by qualitative and quantitative analyses, to identify differentially expressed proteins. Potential biomarkers were identified and further validated using immunohistochemistry. Staining intensity scan analyses were performed on tissue samples from patients with PVL, patients with OL, and controls from Brazil, Spain, and Finland. The study revealed differences in the immune system, cell cycle, DNA regulation, apoptosis pathways, and the whole proteome of PVL samples. In addition, liquid chromatography with tandem mass spectrometry analyses showed that calreticulin (CALR), receptor of activated protein C kinase 1 (RACK1), and 14-3-3 Tau-protein (YWHAQ) were highly expressed in PVL samples. Immunohistochemistry validation confirmed increased CARL expression in PVL compared with OL. Conversely, RACK1 and YWHA were highly expressed in oral potentially malignant disorder compared to the control group. Furthermore, significant differences in CALR and RACK1 expression were observed in the OL group when comparing samples with and without oral epithelial dysplasia, unlike the PVL. This research provides insights into the molecular mechanisms underlying these conditions and highlights potential targets for future diagnostic and therapeutic approaches.

DNA Methylation by Bisulfite Next-Generation Sequencing for MLH1 and MGMT in Oral Squamous Cell Carcinomas and Potentially Malignant Disorders: An Integrative Analysis towards Field Cancerization.

Background and Objectives: MGMT methylation is a well-described biomarker in several solid tumors and MLH1 seems to occur in the initial stages of oral carcinogenesis. The aims of this study were to evaluate MHL1 and MGMT methylation levels in oral squamous cell carcinoma (OSCC) and oral potentially malignant disorders (OPMDs), and to integrate this information with The Cancer Genome Atlas (TCGA) database. Materials and Methods: To determine the percentage of gene methylation in MLH1 and MGMT, pyrosequencing analysis was conducted. Samples were divided as follows: (1) patients diagnosed with OSCC (N = 16); (2) patients with OPDM who developed OSCC in the same location (N = 47); and (3) patients with OPDM who developed OSCC in a different location (N = 22). As a validation cohort in this study, data from The Cancer Genomic Atlas (TCGA) database, particularly regarding Head and Neck Squamous Cell Carcinoma, was used. Results: Overall MLH1 methylation levels of 8.6 ± 11.5% and 8.1 ± 9.2% for MGMT were obtained. With regard to MHL1, the OSCC presented the highest degree of methylation with 9.3 ± 7.3% (95%CI 5.1-13.6), and with regards to MGMT, the simultaneous malignancy group presented the highest degree of methylation with 10 ± 13.5% (95%CI 6-10), although no significant differences were found between the groups (p = 0.934 and p = 0.515, respectively). The estimated survival was higher for MGMT methylated cases (19.1 months, 95%CI 19.1-19.1) than for unmethylated cases (9.4 months, 95%CI 6-12.8), but not statistically significant. Conclusions: Our results did not show a correlation between MGMT and MLH1 methylation and any clinicopathological feature or survival in our institutional cohort. MLH1 methylation was present mainly in OSCC, whilst MGMT in OPMD represented a modest contribution to field cancerization, with an overall consistency with the TCGA database.

Efficacy of different sentinel lymph node biopsy protocols in oral squamous cell carcinoma: Systematic review and meta-analysis.

The sentinel node biopsy (SNB) is highly protocolized in other cancers, however, this is not the case for oral squamous cell carcinoma patients, hence our objective was to evaluate the different protocols published. A specific study protocol was designed and subsequently registered on PROSPERO (Ref. CRD42021279217). Twenty-three articles were included in the meta-analysis. The grouped sensitivity of the SNB was 82% (95% CI: 0.74-0.88), and the grouped specificity was 100% (95% CI: 0.99-1.00). The use of other radiotracers other than pre-operative lopamidol showed higher values of sensitivity of 82.80% (95% CI: 76.90%-87.50%; p < 0.001). The use of the blue dye stain showed higher sensitivity values of 85.60% (95% CI: 71.90%-93.20%), compared to sensitivity values of 77.50% when it was not used (95% CI: 69.10%-84.20%) (p < 0.001). Diagnostic rates are variable and they were significantly better when 99mTc was used in all its variations and accompanied by the blue dye staining.

Immunohistochemical Characterization of Bcl-2 in Oral Potentially Malignant Disorders.

Bcl-2 is a group of apoptotic proteins that play a key role in cellular homeostasis. Overexpression of Bcl-2 has been associated with the poor prognosis of oral squamous cell carcinoma (OSCC). The aim of this study is to analyze the immunohistochemical expression of Bcl-2 in healthy oral mucosa, different oral potentially malignant disorders and OSCC, and to determine its diagnostic value. A retrospective observational study was carried out in the Oral Medicine Unit of the University of Santiago de Compostela. All the clinicopathologic data were collected and paraffin-embedded blocks were available to perform the immunohistochemistry study with Bcl-2. We studied 18 fibromas, 15 OSCC, 29 oral leukoplakia lesions (OL), 59 oral lichen planus (OLP) cases, and 16 healthy controls. OL with epithelial dysplasia (31.2%) showed the highest expression of Bcl-2 and OLP (1.9%) showed the lowest expression of Bcl-2 (P=0.025). Receiver operating characteristics curves showed that the detection of Bcl-2 enables discrimination between OL and OLPs (sensitivity: 58.6%, specificity of 99.32%). Bcl-2 negative expression in the OLP diagnosis obtained an odds ratio of 13.750 (95% confidence interval: 3.354-56.369; P<0.0001) and the positive expression in the OL 4.468 (95% confidence interval: 1.889-10.565; P=0.001). Bcl-2 could be used as a diagnostic biomarker to study their malignant transformation.

Immunoexpression of Apoptosis and Cell-cycle Arrest Markers in Oral Lichen Planus.

The expression pattern of a panel of 5 molecular markers (p53, cyclin D1, Ki-67, BCL-2, and BAX) was studied in samples from patients with oral lichen planus (OLP) and normal oral mucosa (NOM) of healthy controls to investigate the implications of cell cycle and apoptosis in OLP. The 59 OLP and 16 NOM biopsies were stained by an inmunoperoxidase technique for p53, cyclin D1, Ki-67, BCL-2, and BAX and assessed microscopically for semiquantitative analysis. Positivity for BCL-2 and Ki-67 was significantly more frequent in NOM than in OLP (P<0.05). p53 levels were upregulated in atrophic/erosive clinical presentations when compared with reticular presentations and in cases with discontinued inflammatory infiltrate. Multivariate analysis through logistic regression showed that BCL-2 in OLP versus NOM was the only significantly altered marker in the present cohort (adjusted odds ratio=12.42; 95% confidence interval: 2.5-61.65; P=0.002). The cell patterns in OLP and NOM are distinct according to the present molecular markers panel. The presence of BCL-2 altered expression may be related to various molecular pathways that connect/link this condition to other autoimmune disorders and also may be involved in complex roles that evoke malignant transformation of OLP.

Identification of Prognosis Associated microRNAs in HNSCC Subtypes Based on TCGA Dataset.

Background and Objectives: Head and Neck Squamous Cell Carcinoma (HNSCC) includes cancers from the oral cavity, larynx, and oropharynx and is the sixth-most common cancer worldwide. MicroRNAs are small non-coding RNAs for which altered expression has been demonstrated in pathological processes, such as cancer. The objective of our study was to evaluate the different expression profile in HNSCC subtypes and the prognostic value that one or several miRNAs may have. Materials and Methods: Data from The Cancer Genome Atlas Program-Head and Neck Squamous Cell Carcinoma (TCGA-HNSCC) patients were collected. Differential expression analysis was conducted by edge R-powered TCGAbiolinks R package specific function. Enrichment analysis was developed with Diana Tool miRPath 3.0. Kaplan-Meier survival estimators were used, followed by log-rank tests to compute significance. Results: A total of 127 miRNAs were identified with differential expression level in HNSCC; 48 of them were site-specific and, surprisingly, only miR-383 showed a similar deregulation in all locations studied (tonsil, mouth, floor of mouth, cheek mucosa, lip, tongue, and base of tongue). The most probable affected pathways based on miRNAs interaction levels were protein processing in endoplasmic reticulum, proteoglycans in cancer (p < 0.01), Hippo signaling pathway (p < 0.01), and Transforming growth factor-beta (TGF-beta) signaling pathway (p < 0.01). The survival analysis highlighted 38 differentially expressed miRNAs as prognostic biomarkers. The miRNAs with a greater association between poor prognosis and altered expression (p < 0.001) were miR-137, miR-125b-2, miR-26c, and miR-1304. Conclusions: In this study we have determined miR-137, miR-125b-2, miR-26c, and miR-1304 as novel powerful prognosis biomarkers. Furthermore, we have depicted the miRNAs expression patterns in tumor patients compared with normal subjects using the TCGA-HNSCC cohort.

Comprehensive Genomic Review of TCGA Head and Neck Squamous Cell Carcinomas (HNSCC).

The aim of this present study was to comprehensively describe somatic DNA alterations and transcriptional alterations in the last extension of the HNSCC subsets in TCGA, encompassing a total of 528 tumours. In order to achieve this goal, transcriptional analysis, functional enrichment assays, survival analysis, somatic copy number alteration analysis and somatic alteration analysis were carried out. A total of 3491 deregulated genes were found in HNSCC patients, and the functional analysis carried out determined that tissue development and cell differentiation were the most relevant signalling pathways in upregulated and downregulated genes, respectively. Somatic copy number alteration analysis showed a "top five" altered HNSCC genes: CDKN2A (deleted in 32.03% of patients), CDKN2B (deleted in 28.34% of patients), PPFIA1 (amplified in 26.02% of patients), FADD (amplified in 25.63% of patients) and ANO1 (amplified in 25.44% of patients). Somatic mutations analysis revealed TP53 mutation in 72% of the tumour samples followed by TTN (39%), FAT1 (23%) and MUC16 (19%). Another interesting result is the mutual exclusivity pattern that was discovered between the TP53 and PIK3CA mutations, and the co-occurrence of CDKN2A with the TP53 and FAT1 alterations. On analysis to relate differential expression genes and somatic copy number alterations, some genes were overexpressed and amplified, for example, FOXL2, but other deleted genes also showed overexpression, such as CDKN2A. Survival analysis revealed that overexpression of some oncogenes, such as EGFR, CDK6 or CDK4 were associated with poorer prognosis tumours. These new findings help us to develop new therapies and programs for the prevention of HNSCC.

A novel in silico reverse-transcriptomics-based identification and blood-based validation of a panel of sub-type specific biomarkers in lung cancer.

Lung cancer accounts for the highest number of cancer-related deaths worldwide. Early diagnosis significantly increases the disease-free survival rate and a large amount of effort has been expended in screening trials and the development of early molecular diagnostics. However, a gold standard diagnostic strategy is not yet available. Here, based on miRNA expression profile in lung cancer and using a novel in silico reverse-transcriptomics approach, followed by analysis of the interactome; we have identified potential transcription factor (TF) markers that would facilitate diagnosis of subtype specific lung cancer. A subset of seven TF markers has been used in a microarray screen and was then validated by blood-based qPCR using stage-II and IV non-small cell lung carcinomas (NSCLC). Our results suggest that overexpression of HMGA1, E2F6, IRF1, and TFDP1 and downregulation or no expression of SUV39H1, RBL1, and HNRPD in blood is suitable for diagnosis of lung adenocarcinoma and squamous cell carcinoma sub-types of NSCLC. Here, E2F6 was, for the first time, found to be upregulated in NSCLC blood samples. The miRNA-TF-miRNA interaction based molecular mechanisms of these seven markers in NSCLC revealed that HMGA1 and TFDP1 play vital roles in lung cancer tumorigenesis. The strategy developed in this work is applicable to any other cancer or disease and can assist in the identification of potential biomarkers.