The Role of Interferon-γ-Induced Granzyme B in Enhancing Antitumour Immune Responses in Oral Squamous Cell Carcinoma.

Angiogenesis and CD34 Expression as a Predictor of Recurrence in Oral Squamous Cell Carcinoma

The clinicopathological significance of the expression of Granzyme B in oral squamous cell carcinoma

Ultrasound-guided fine-needle aspiration cytology (US-FNAC) is commonly used in the diagnostic work-up of head and neck cancer, but its ability to detect occult lymph node metastases in early-stage oral squamous cell carcinoma (OSCC) with a clinically negative neck remains unclear. A retrospective analysis was performed in 578 patients with early-stage OSCC (cT1-3N0) who underwent US-FNAC prior to surgery. Histopathology, sentinel lymph node biopsy, and follow-up were used as reference standards. Occult nodal metastases were found in 179 patients (31.0%). US-FNAC showed low sensitivity (15.9%) and a negative predictive value of 72.9%, resulting in 149 false-negative cases (25.8%). Specificity (99.5%) and positive predictive value (90.3%) were high, with only 2 false-positive results. In patients with early-stage oral cavity squamous cell carcinoma and a clinically negative neck, US-FNAC demonstrates high specificity but limited sensitivity and negative predictive value. These findings indicate that US-FNAC alone is insufficient to exclude occult nodal metastases and should be regarded as an adjunctive diagnostic tool rather than a stand-alone nodal staging strategy.

The present study aimed to evaluate the correlation of P53 and granzyme B (GB) expression, and also the relationship between P53 expression and GB+ cell density with lymph node metastasis, histologic grade, and inflammation intensity in oral squamous cell carcinoma (OSCC). Immunohistochemical technique with P53 and GB antibodies were performed on stored paraffin blocks from 48 patients with OSCC (with lymph node metastasis n = 24; without lymph node metastasis n = 24). The density of GB expression was quantified both in invasive front (peritumoral ) and within cancer nests (intratumoral ). P53 positivity was seen in 13 (54.16%) cases of the nonmetastatic group and 14 cases (58.3%) in the metastatic group. A significant correlation was seen between P53 immunoexpression and histologic grade (P = 0.047), but there was no significant correlation between P53 expression with lymph node metastasis and inflammation intensity. The density of GB+ cells in the peritumoral zone correlates with a higher intratumoral GB expression (P = 0.001) and was significantly higher in the nonmetastatic group (P = 0.029). No significant correlation between GB and P53 immunoexpression, lymph node metastasis, or inflammation intensity was seen. The present study showed that the presence of a higher density of GB+ cells infiltrating the peritumoral area may have an important role against tumoral cells, prevent lymph node metastasis, and better prognosis in OSCC patients.

BackgroundProgrammed cell death 4 (PDCD4) as a tumor suppressor gene inhibits growth and metastasis of cancer cells, which involved with eIF4A1, the inhibitor of translation initiation. Although the prognosis of early-stage oral squamous cell carcinoma (OSCC) is generally better, but many patients occur recurrence after surgery. Understanding the clinical expression pattern of PDCD4/eIF4A1 signal would provide diagnostic biomarker and target therapy premise for early-stage OSCC patients.MethodsImmunohistochemical analysis was performed on 69 early-stage (T1/2N0M0) OSCC samples to evaluate temporal expression and prognostic value of eIF4A1 and PDCD4 in early-stage OSCC according to cell types and microlocalization. The correlations between PDCD4/eIF4A1 signal and Ki-67, postoperative recurrence and metastasis were determined.ResultsWe found that PDCD4 was presented in tumor cells (TCs) and tumor-infiltrating lymphocytes (TILs) but absent in fibroblast-like cells (FLCs). eIF4A1 was only presented in TCs. PDCD4TCs was negative associated with eIF4A1TCs in tumor center, and patients with low PDCD4TCs or high eIF4A1TCs had poorer differentiation. Moreover, aberrant PDCD4/eIF4A1 signal led to higher Ki-67 level. Interestingly, patients with low expressed PDCD4TILs had better prognosis, indicating the function heterogeneity of PDCD4 in different cell types. Furthermore, low PDCD4 TCs and high eIF4A1TCs predicted higher postoperative recurrence rate and are significant independent risk factors for early-stage OSCC.ConclusionPatients with low PDCD4TCs and high eIF4A1TCs have higher recurrence rate and poor clinical outcome. Of note, PDCD4TILs exerts contradictory function. Thus, PDCD4/eIF4A1 targeting therapeutics should consider the function heterogeneity of PDCD4.

Background & aim:Oral squamous cell carcinoma (OSCC) is a devastating disease with poor prognosis and low survival rates, despite advancements in diagnosis and treatment. Early detection and identification of molecular targets are crucial for improving patient outcomes. This study aims to identify differentially expressed genes (DEGs) and key molecular pathways involved in the OSCC. This study’s findings will contribute to the development of effective targeted therapies, ultimately improving the prognosis and survival rates of OSCC patients.Materials & methods:Three gene expression profiles (GSE37991, GSE30784, and GSE107591) from the GEO database were analyzed for differentially expressed genes using EnrichR. Subsequent downstream analyses of the selected module genes were conducted using various bioinformatics tools including STRING, Cytoscape, GEPIA, cBioPortal, NetworkAnalyst, MirWalk, and a bipartite miRNA-mRNA correlation network.Result:The reanalysis indicated that the Toll-like receptor (TLR) signaling pathway plays a significant role in the development of oral SCC and CXCL8, CCL5, CXCL10, STAT1, IL1B, and TLR2 genes were up-regulated and enriched significantly in the signaling pathways’ interactions in oral SCC. Genetic mutation analysis of hub genes in OSCC revealed that STAT1 have 2.5% mutation rate and 0% for other genes. It was revealed that the development and prediction of OSCC may be affected by hsa-mir-146a-5 and hsa-mir-155-5p.Conclusion:Novel potential biomarkers and signaling pathways associated with OSCC have been identified, which may be important in the transformation of OSCC adenocarcinoma and may serve as therapeutic targets for OSCC.

Oral squamous cell carcinoma (OSCC) constitutes >90% of oral cancers and is the sixth most common malignancy among males worldwide and the fourth leading cause of death due to cancer among males in Taiwan. However, most patients do not receive a diagnosis of OSCC until the late stages, which have a lower survival rate. The use of molecular marker analysis to identify early-stage OSCC would permit optimal timing for treatments and consequently prolong survival. The aim of this study was to identify biomarkers of OSCC using the Illumina GoldenGate Methylation Cancer Panel, which comprised a total of 1,505 CpG sites covering 807 genes. Samples of buccal mucosa resected from 40 OSCC patients and normal tissue samples obtained from 15 patients (normal mucosa from OSCC patients or from patients undergoing surgery unrelated to OSCC) were analyzed. Fms-related tyrosine kinase 4 (FLT4) methylation exhibited a perfect specificity for detecting OSCC, with an area under the receiver operating characteristic curve of 0.91 for both all-stage and early-stage OSCC. Methylation of 7 genes (ASCL1, FGF3, FLT4, GAS7, KDR, TERT, and TFPI2) constitutes the top-20 panels for detecting OSCC. The top-20 panels for detecting early-stage OSCC contain 8 genes: ADCYAP1, EPHA7, FLT4, GSTM2, KDR, MT1A, NPY, and TFPI2. FLT4 RNA expression and methylation level were validated using RT-PCR and a pyrosequencing methylation assay. The median level of FLT4 expression was 2.14-fold for normal relative to OSCC tissue samples (P < 0.0001). Among the 8 pyrosequenced FLT4 CpG sites, methylation level was much higher in the OSCC samples. In conclusion, methylation statuses of selected genes, and especially FLT4, KDR, and TFPI2, might be of great potential as biomarkers for early detection of buccal OSCC.

Expression of multidrug resistance-related genes in oral squamous cell carcinomas.

Early-stage oral squamous cell carcinoma (OSCC) treatment is based on anatomic location, clinical TNM staging, and histological grade. It is a heterogeneous disease group. Classification of patients with OSCC by immunohistochemical analysis of established oncoproteins and evaluate disease course was our primary objective. Characterization of stage I OSCC patients in Southwest Finland was our secondary objective. Immunohistochemical analysis of tumor specimens and retrospective analysis of patient data of the patient treated in Turku University Hospital for T1N0M0 OSCC during the years 2000-2004. Paraffin-embedded tumor specimens from 35 OSCC patients were collected and analyzed for HIF-1α, CD44, p16, Ki67, and podoplanin by immunohistochemistry and correlated with clinical findings. Tumoral CD44 and HIF1-α expression levels, in combination, predicted 5-year disease-free survival. Reduced expression of CD44 and elevated expression of HIF1-α is associated with the lowest probability of disease-free survival compared to the population as a whole (P < .001 in Kaplan-Meier analysis). Patients with grade I tumors demonstrated improved disease-specific survival compared to those with grade II tumors (P = .027). No association was seen between p16 expression, Ki67 labeling index, or podoplanin expression and prognosis in our 35 specimens. HIF-1α and CD44 immunohistochemical detection could potentially serve as a prognostic tool in therapy selection for early-stage OSCC. 2b.

The search for molecular markers to improve diagnosis, individualize treatment and predict behavior of tumors has been the focus of several studies. This study aimed to analyze homeobox gene expression profile in oral squamous cell carcinoma (OSCC) as well as to investigate whether some of these genes are relevant molecular markers of prognosis and/or tumor aggressiveness. Homeobox gene expression levels were assessed by microarrays and qRT-PCR in OSCC tissues and adjacent non-cancerous matched tissues (margin), as well as in OSCC cell lines. Analysis of microarray data revealed the expression of 147 homeobox genes, including one set of six at least 2-fold up-regulated, and another set of 34 at least 2-fold down-regulated homeobox genes in OSCC. After qRT-PCR assays, the three most up-regulated homeobox genes (HOXA5, HOXD10 and HOXD11) revealed higher and statistically significant expression levels in OSCC samples when compared to margins. Patients presenting lower expression of HOXA5 had poorer prognosis compared to those with higher expression (P=0.03). Additionally, the status of HOXA5, HOXD10 and HOXD11 expression levels in OSCC cell lines also showed a significant up-regulation when compared to normal oral keratinocytes. Results confirm the presence of three significantly upregulated (>4-fold) homeobox genes (HOXA5, HOXD10 and HOXD11) in OSCC that may play a significant role in the pathogenesis of these tumors. Moreover, since lower levels of HOXA5 predict poor prognosis, this gene may be a novel candidate for development of therapeutic strategies in OSCC.

Simple SummaryIn early-stage (cT1-2N0) oral cancer, occult lymph node metastases are present in 20–30% of patients. Accordingly, accurate staging of the clinically negative cervical nodal basin is warranted in these patients. Sentinel lymph node biopsy has proven to reliably stage the clinically negative cervical nodal basin in early-stage oral cancer. However, due to the limited resolution of conventional sentinel lymph node imaging, occult lymph node metastasis may be missed in particular circumstances. Therefore, technical developments are necessary to bring the diagnostic accuracy of sentinel lymph node biopsy, in early-stage oral cancer, to a higher level. This review evaluates novel sentinel lymph node imaging techniques for early-stage oral cancer, such as MR lymphography, CT lymphography, PET lymphoscintigraphy and contrast-enhanced lymphosonography. Their reported diagnostic accuracy is described and their relative merits, disadvantages and potential applications are outlined.Sentinel lymph node biopsy (SLNB) is a diagnostic staging procedure that aims to identify the first draining lymph node(s) from the primary tumor, the sentinel lymph nodes (SLN), as their histopathological status reflects the histopathological status of the rest of the nodal basin. The routine SLNB procedure consists of peritumoral injections with a technetium-99m [99mTc]-labelled radiotracer followed by lymphoscintigraphy and SPECT-CT imaging. Based on these imaging results, the identified SLNs are marked for surgical extirpation and are subjected to histopathological assessment. The routine SLNB procedure has proven to reliably stage the clinically negative neck in early-stage oral squamous cell carcinoma (OSCC). However, an infamous limitation arises in situations where SLNs are located in close vicinity of the tracer injection site. In these cases, the hotspot of the injection site can hide adjacent SLNs and hamper the discrimination between tracer injection site and SLNs (shine-through phenomenon). Therefore, technical developments are needed to bring the diagnostic accuracy of SLNB for early-stage OSCC to a higher level. This review evaluates novel SLNB imaging techniques for early-stage OSCC: MR lymphography, CT lymphography, PET lymphoscintigraphy and contrast-enhanced lymphosonography. Furthermore, their reported diagnostic accuracy is described and their relative merits, disadvantages and potential applications are outlined.

The objective of this study was to explore the prognostic value of in situ memory T cells and patterns of invasion (POI) in early stage oral squamous cell carcinoma (OSCC). One hundred fifty-seven patients with early stage (cT1T2N0) OSCC were identified from a pre-existing database of patients with oral cancer and were classified by POI according to hematoxylin-eosin staining. We examined the impact of the immunohistochemical expression of CD45RO in OSCC. Overall survival (OS) curves were calculated using the Kaplan-Meier method. Margin status was significantly associated with local recurrence in early stage OSCC (p= 0.000), and depth of invasion was also associated with regional recurrence (2 mm: p= 0.034; 4 mm: p= 0.015). The expression of CD45RO (p= 0.021) and POI (p= 0.027) individually was associated with OS, and patients in the group with combination score tended to have worse OS (p= 0.012). Combined evaluation of POI and CD45RO might prove to be a useful indicator for high-risk patients with occult metastases from early stage OSCC.

Gene expression signatures that can discriminate oral leukoplakia subtypes and squamous cell carcinoma

Background: M6A is the most widespread RNA internal modification that contributes to the transcription of RNA-mediated genes. Increasing evidence suggest that m6A has vital role in cancer initiation and progression. However, little is known about its roles in oral squamous cell carcinoma(OSCC). Therefore, this study aimed to explore the prognostic relevance of m6A related genes in OSCC through comprehensive bioinformatics analysis. Materials: Transcriptome expression profile were downloaded from The Cancer Genome Atlas (TCGA) following extracting m6A related genes and long non-coding RNA (lncRNA) based on expression level. The differentially expressed genes between OSCC samples and control samples was analyzed using R software. Furthermore, cluster analysis and principal component analysis (PCA) were used to analyze m6A related genes, and a risk model was constructed on basis of m6A related genes. In addition, co-expression network was established according to m6A related genes and differentially expressed lncRNA (DElncRNA) expression profile and crucial m6A genes were selected based on univariate, multivariate cox regression and survival analysis. Then, CCK-8, colony formation assays, transwell assay and scratch wound healing were performed to explore the biological effects of identified m6A-related genes in OSCC. Furthermore, Western blot assay was conducted to detect the EMT-associated genes expression level. Findings: A total of 13 m6A related genes were extracted and 8 genes were differentially expressed in OSCC. Subsequently, 418 DElncRNA were selected. M6A-Based Clustering Shows 2 Subtypes of OSCC with different clinical outcome.Then, a risk model including HNRNPC, METTL14, YTHDF2, ALKBH5 genes was established on basis of m6A expression profile, which can indicate the survival time and differentiation grade and may be an independent prognostic biomarkers. Furthermore, a co-expression network including 7 m6A related genes and 21 DElncRNA was constructed based on DElncRNA and m6A related genes and HNRNPC was identified as an independent prognostic biomarker in OSCC. Functional studies revealed that HNRNPC knockdown inhibited cell proliferation, migration and invasion of OSCC cells in vitro. Conversely, HNRNPC overexpression showed the opposite biological effects. Moreover, we identified that HNRNPC regulated epithelial-mesenchymal transition in OSCC. Interpretation: This study revealed that m6A may play a significant role in OSCC initiation and progression through integrated bioinformatics analysis. And co-expression network may be helpful for revealing potential correlation of lncRNA and m6A in OSCC. In addition, a risk model was established to predict the prognosis of OSCC.At last, HNRNPC was proved to promote OSCC carcinogenisis and be an independent prognostic biomarker of OSCC, suggesting that it may be a new therapeutic target of OSCC. Funding Statement:This study was supported by the National Natural Science Foundation of China (81472536); the Science and Technology Planning Project of Guangdong Province (No. 2017A020215181 and No. 2014A020212440); Project of Educational Commission of Guangdong Province of China (2018KTSCX026); the Scientific Research Planning Project of Southern Medical University (CX2018N016, PY2018N031) and the Presidential Foundation of the Nanfang Hospital (2014027, 2019Z030). Declaration of Interests: All authors declare no conflicts of interest. Ethics Approval Statement: The study protocol was approved by the Ethics Committees of Nanfang Hospital of Guangdong Province (NO: NFEC-2018-027).

Noninvasive genomic profiling of somatic mutations in oral cavity cancers