Survival Of Oral Squamous Cell Carcinoma Patients Research Articles

Streptococcus salivarius-mediated CD8+ T cell stimulation required antigen presentation by macrophages in oral squamous cell carcinoma

It has been shown that the peripheral blood mononuclear cells (PBMCs) from oral squamous cell carcinoma (OSCC) patients presented cytotoxic CD8 T cell response against Streptococcus salivarius (S. salivarius), of which the frequency was positively associated with recurrence-free survival in OSCC patients. To identify the conditions required for regulating S. salivarius-specific CD8 T cell-mediated cytotoxicity, we selectively depleted individual components of the PBMCs, and observed that the depletion of monocytes/macrophages, but not other immune cell subsets, significantly downregulated the S. salivarius-specific CD8 T cell cytotoxicity. Monocyte/macrophage alone was sufficient to reconstitute optimal granzyme B expression from S. salivarius-specific CD8 T cells. Also, both the memory and the naive CD8 T cells reacted to S. salivarius-stimulation, with the memory CD8 T cells presenting significantly higher S. salivarius-reactivity. Using M1- and M2-polarized macrophages from circulating monocytes, we found that M1-polarized macrophages, with significantly higher IL-12 expression and significantly lower IL-10 and MHC class II molecule expression, was more effective at promoting granzyme B responses in CD8 T cells, and required CD80/CD86 costimulating molecules for optimal responses. Interestingly, the tumor-associated macrophages (TAMs) from resected tumors presented characteristics of M2-polarized macrophages with high MHC class II expression and low IL-12 secretion. The frequency of tumor-infiltrating S. salivarius-specific cytotoxic CD8 T cell was inversely correlated with the level of IL-10 secretion and the MHC class II molecule expression in autologous TAMs. Together, we demonstrated that monocyte/macrophages presented essential antigen-presentation and costimulatory roles in CD8 T cell-mediated S. salivarius-specific granzyme B responses, and the polarization of macrophages could influence the potency of CD8 T cell responses.

MiR-182-5p Promotes Growth in Oral Squamous Cell Carcinoma by Inhibiting CAMK2N1

Background/Aims: Emerging evidence suggests that the propagation of oral squamous cell carcinoma (OSCC) is influenced by the abnormal expression of microRNAs (miRNAs). This study aimed to characterize the involvement of miR-182-5p in OSCC by targeting the calcium/ calmodulin-dependent protein kinase II inhibitor CAMK2N1. Methods: miR-182-5p expression was quantified in OSCC tissues and cell lines with reverse transcription polymerase chain reaction (RT-PCR). Cell colony formation, Cell Counting Kit-8 (CCK-8), Ki-67, and nude mouse xenograft assays were used to characterize the role of miR-182-5p in the proliferation of OSCC. A miR-182-5p target gene was identified with western blotting, RT-PCR, and luciferase activity assays. OSCC patient survival based on CAMK2N1 expression was also analyzed. Results: miR-182-5p was up-regulated in in vitro cell lines and in vivo clinical OSCC samples. CCK-8, colony formation, and Ki-67 assays revealed that miR-182-5p promoted the growth and proliferation of OSCC cells. miR-182-5p directly targeted CAMK2N1, as evidenced by luciferase assays and target prediction algorithms. CAMK2N1 operated as a tumor suppressor gene in patients with OSCC. Down-regulating miR-182-5p expression in the CAL-27 cell line restored CAMK2N1-mediated OSCC cell proliferation. miR-182-5p expression inhibited the activation of AKT, ERK1/2, and NF-κB. Mice injected with CAL-27 cells transfected with miR-182-5p-inhibitor demonstrated a significant increase in tumor size and weight and increased CAMK2N1 mRNA and protein expression compared with the miR-negative control group. Conclusion: The miR-182-5p-CAMK2N1 pathway can be potentially targeted to regulate the proliferation of OSCC cells.

Hypoxic volume evaluated by 18F-fluoromisonidazole positron emission tomography (FMISO-PET) may be a prognostic factor in patients with oral squamous cell carcinoma: preliminary analyses

Tumour hypoxia can be detected by 18F-fluoromisonidazole positron emission tomography (FMISO-PET). Few studies have assessed the relationships of new PET parameters, including hypoxic volume (HV), metabolic tumour volume (MTV), and total lesion glycolysis (TLG), with 5-year survival of patients treated surgically for oral squamous cell carcinoma (OSCC). This study evaluated the relationships between these PET parameters and 5-year survival in OSCC patients. Twenty-three patients (age 42–84 years; 15 male, eight female) with OSCC underwent FMISO- and 18F-fluoro-2-deoxyglucose (FDG)-PET computed tomography before surgery. All of them underwent radical surgery and were followed up for more than 5 years. The FDG-PET maximum standardized uptake value (SUVmax), HV, MTV, and TLG were measured. The ability of PET parameters to predict disease-free survival (DFS) and loco-regional recurrence (LR) was evaluated using receiver operating characteristic curve analysis. During the follow-up period, five of the 23 patients (22%) died and six (26%) experienced LR. Although FDG-PET SUVmax was not significantly associated with DFS or LR, HV correlated significantly with both DFS and LR. TLG, but not MTV, was significantly associated with DFS; however neither MTV nor TLG was related significantly to LR. In conclusion, tumour HV may predict outcomes in patients with OSCC.

Seven-CpG-based prognostic signature coupled with gene expression predicts survival of oral squamous cell carcinoma

BackgroundDNA methylation has started a recent revolution in genomics biology by identifying key biomarkers for multiple cancers, including oral squamous cell carcinoma (OSCC), the most common head and neck squamous cell carcinoma.MethodsA multi-stage screening strategy was used to identify DNA-methylation-based signatures for OSCC prognosis. We used The Cancer Genome Atlas (TCGA) data as training set which were validated in two independent datasets from Gene Expression Omnibus (GEO). The correlation between DNA methylation and corresponding gene expression and the prognostic value of the gene expression were explored as well.ResultsThe seven DNA methylation CpG sites were identified which were significantly associated with OSCC overall survival. Prognostic signature, a weighted linear combination of the seven CpG sites, successfully distinguished the overall survival of OSCC patients and had a moderate predictive ability for survival [training set: hazard ratio (HR) = 3.23, P = 5.52 × 10−10, area under the curve (AUC) = 0.76; validation set 1: HR = 2.79, P = 0.010, AUC = 0.67; validation set 2: HR = 3.69, P = 0.011, AUC = 0.66]. Stratification analysis by human papillomavirus status, clinical stage, age, gender, smoking status, and grade retained statistical significance. Expression of genes corresponding to candidate CpG sites (AJAP1, SHANK2, FOXA2, MT1A, ZNF570, HOXC4, and HOXB4) was also significantly associated with patient’s survival. Signature integrating of DNA methylation, gene expression, and clinical information showed a superior ability for prognostic prediction (AUC = 0.78).ConclusionPrognostic signature integrated of DNA methylation, gene expression, and clinical information provides a better prognostic prediction value for OSCC patients than that with clinical information only.

Overexpression of glutathione peroxidase 1 predicts poor prognosis in oral squamous cell carcinoma.

Intracellular antioxidant enzymes are commonly upregulated in various cancer types and are associated with treatment outcomes. Because the relationship has rarely been examined in oral squamous cell carcinoma (OSCC), we aimed to evaluate the association between the levels of glutathione peroxidase (GPX)1, GPX4, and thioredoxin reductase (TrxR)1 expression and prognosis in patients with OSCC who underwent curative surgical resection. This study included 233 patients who underwent curative surgery for previously untreated OSCC between 2000 and 2012. Tumour GPX1, GPX4, and TrxR1 expression was evaluated by immunohistochemistry and was dichotomised to low and high values according to defined expression levels. The association between GPX1, GPX4, and TrxR1 expression and clinicopathological results was analysed. Univariate and multivariate analyses using the Cox proportional hazards model were conducted to assess the significance of differences in recurrence or survival outcomes between variables. High GPX1, GPX4, and TrxR1 expression was observed in 99 (42.5%), 133 (57.1%), and 46 (19.7%) patients, respectively. GPX1 overexpression was significantly correlated with nodal metastasis, advanced overall stage, depth of invasion of >10mm, high grade and perineural invasion (P<0.05). High GPX4 expression was also related to nodal metastasis, overall advanced stage and high grade (P<0.05). Univariate and multivariate analyses showed that increased GPX1 expression was significantly associated with poor disease-free, cancer-specific and overall survival (all P<0.05), while increased GPX4 or TrxR1 expression was not (all P>0.1). Tumour GPX1 expression is a useful biomarker predictive of recurrence and survival in OSCC patients.

MMP-11 promoted the oral cancer migration and Fak/Src activation.

Matrix metalloproteinase-11 (MMP-11) has been observed in most invasive human carcinomas. The current study investigated the association between the clinicopathological characteristics and MMP-11 expression in oral squamous cell carcinoma (OSCC) patients. Immunohistochemistry (IHC) staining was performed to assess MMP-11 expression in 279 patients with OSCC. In addition, the metastatic effects of the MMP-11 overexpression on the OSCC cells were also investigated. We found that MMP-11 expression was present in 118/279 (42.3%) cases and expression of MMP-11 was associated with higher incidence of lymph node metastasis and worse grade of tumor differentiation. Importantly, OSCC patients with strong expression of MMP-11 had a significantly lower survival rate (p=0.010). Furthermore, MMP-11 overexpression in OSCC cells increased in vitro cell migration. Mechanistically, MMP-11 increased the cell motility of OSCC cells through focal adhesion kinase/Src kinase (FAK/Src) pathway. In conclusion, our results revealed that the MMP-11 expression in OSCC samples can predict the progression, especially lymph node metastasis, and the survival of OSCC patients in Taiwan.

MicroRNA-375 Inhibits Growth and Enhances Radiosensitivity in Oral Squamous Cell Carcinoma by Targeting Insulin Like Growth Factor 1 Receptor

Background: MicroRNAs (miRNAs) have emerged as key players in various human biological processes, including tumorigenesis. Here, we investigated the roles of miR-375 in the pathogenesis of oral squamous cell carcinoma (OSCC). Methods: We performed quantitative real-time PCR (qRT-PCR) to detect miR-375 expression in OSCC tissues and corresponding normal oral epithelial tissues and analyze the correlation of miR-375 expression with OSCC metastasis and patient’s survival. Then, the effects of miR-375 expression on proliferation, cell cycle, apoptosis and radiosensitivity in OSCC cells were determined by using MTT, flow cytometry and clonogenic survival assays. A dual-luciferase reporter assay was performed to test whether miR-375 binds to the 3’-untranslated region (3’-UTR) of target mRNA. Results: The expression level of miR-375 in OSCC tissues was significantly lower than that in normal oral epithelial tissues, and low miR-375 expression was correlated with higher incidence of lymph node metastasis and poor survival of OSCC patients. Upregulation of miR-375 significantly inhibits growth, induces cell cycle arrest in G₀/G₁ phase, increases apoptosis and enhances radiosensitivity in OSCC cells. Analysis of luciferase activity demonstrated that miR-375 binds to the 3’-UTR of insulin like growth factor 1 receptor (IGF-1R). Small interfering RNA (shRNA)-mediated IGF-1R knockdown mimics the effects of miR-375 upregulation, while overexpression of IGF-1R partially reverses those effects in OSCC cells. Conclusion: It was obviously demonstrated that miRNA-375 inhibits growth and enhances radiosensitivity in OSCC cells by targeting IGF-1R, suggesting that miR-375 may be a potential therapeutic target for OSCC patients.

Aberrant DNA hypermethylation-silenced SOX21-AS1 gene expression and its clinical importance in oral cancer.

BackgroundLong noncoding RNAs (lncRNAs) are more than 200 nucleotides in length and lack transcriptional ability. The biological function of lncRNAs in oral squamous cell carcinoma (OSCC) remains unclear. The aim of this study was to identify the dysfunction of lncRNA in OSCC.ResultsWe analyzed the transcriptome profiles of human OSCC tissues and paired adjacent normal tissues from two patients through a next-generation sequencing approach. A total of 14 lncRNAs were upregulated (fold change ≥3) and 13 were downregulated (fold change ≤−3) in OSCC tissues compared with the adjacent normal tissues. SOX21-AS1 was subjected to further analysis, revealing that the expression levels of SOX21-AS1 significantly decreased in OSCC compared with the adjacent normal tissue. The promoter activity of SOX21-AS1 was obviously suppressed by in vitro methylation. The DNA methylation status of the SOX21-AS1 promoter was analyzed using combined bisulfite restriction analysis, revealing that the aberrant promoter hypermethylation of SOX21-AS1 was observed frequently in OSCC tissues. The effects of SOX21-AS1 on cell proliferation and invasion were examined through transient transfection. Our data showed that SOX21-AS1 could significantly suppress oral cancer cell growth and invasion. Furthermore, the low expression level of SOX21-AS1 was significantly correlated with an advanced stage (P = 0.047), large tumor size (P = 0.033), and poor disease-specific survival in OSCC patients (P = 0.002).ConclusionsSOX21-AS1 was identified as susceptible dysfunction correlated with promoter hypermethylation in OSCC. Low SOX21-AS1 expression may be an adverse prognostic biomarker for OSCC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-016-0291-5) contains supplementary material, which is available to authorized users.

Abstract 1137: Mutation and function of PIK3CA and HRAS in human oral squamous cell carcinoma cells

Abstract Whole-exome sequencing studies showed the mutational landscape of head and neck squamous cell carcinoma including oral cavity. Recent several reports also demonstrated the somatic mutations of oncogenes and tumor suppressor genes in oral squamous cell carcinoma (OSCC), and identified the mutations of HRAS, BRAF, FGFR3, SMAD4, KIT, PTEN, NOTCH1, AKT1, CTNNB1, and PTPN11 to predict disease-free survival of OSCC patients. In this study, we have attempted to elucidate somatic mutations of these genes and their functions in human OSCC cells. We isolated and cultured tumor cells from the resected primary tumor (KT-T), metastatic lymph node (KT-N), and cancerous pleural effusion (KT-M) in the same patient with OSCC. KT-T cells showed epithelial-like shape, whereas both KT-N and KT-M cells indicated the morphological changes to fibroblast-like spindle shape. Genomic DNA was extracted from these cells, and then we performed the mutational analysis by ultra-deep targeted sequencing using Haloplex Cancer Research Panel. All types of KT cells had the mutation of TP53 (R141P, C135S, and E182*) and PIK3CA (H1047R), and an additional active mutation of HRAS (Q61R) was detected in only metastatic KT-N and KT-M cells. Subsequently, we transfected all KT cells with synthetic small interfering RNA (siRNA) specific for PIK3CA (siPIK3CA) and/or HRAS (siHRAS) at the concentration of 10 nM complexed with Lipofectamine RNAiMAX. We confirmed the knockdown effects of these siRNAs in KT cells by quantitative RT-PCR. After transfection of these siRNAs for 72 hours, the growth of KT cells was evaluated by WST-8 assay. Knockdown of PIK3CA or HRAS expression significantly suppressed the growth of KT-T cells only, whereas double knockdown of PIK3CA and HRAS inhibited the growth of KT-N and KT-M cells as well. Next, we investigated the function of mutant HRAS (Q61R) in KT-T cells. Overexpression of mutant HRAS did not change the growth rate and morphology of KT-T cells. Finally, we examined the effects of BEZ-235 (dual PI3K/mTOR inhibitor) and trametinib (MEK1/2 inhibitor) on the growth of KT-T, KT-N, and KT-M cells. BEZ-235 (100 nM) almost completely suppressed the growth of these cells. Trametinib (2 μM) also reduced the growth rate by 16.9% in KT-T, 13.5% in KT-N, and 45.7% in KT-M cells. Furthermore, BEZ-235 combined with trametinib inhibited the cell growth more effectively even at low concentrations. These results suggest that constitutive active mutations of HRAS and PIK3CA support the growth of human OSCC cells and these signaling pathways may be useful therapeutic targets for the patients with OSCC. Citation Format: Hitoshi Akiyama, Koichi Nakashiro, Norihiko Tokuzen, Hiroshi Tanaka, Satoshi Hino, Hiroyuki Hamakawa. Mutation and function of PIK3CA and HRAS in human oral squamous cell carcinoma cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1137.

Insulin-like growth factor-II mRNA binding protein-3 and podoplanin expression are associated with bone invasion and prognosis in oral squamous cell carcinoma

ObjectiveThis study aimed to evaluate the prognostic implications of insulin-like growth factor-II mRNA binding protein-3 (IMP3) and podoplanin (PDPN) as therapeutic targets against oral squamous cell carcinoma (OSCC) with bone invasion. Study designWe elucidated the correlation of IMP3 and PDPN expression with bone invasion in 160 OSCC tissue specimens, and assessed a mouse calvarium xenograft model using an IMP3- and PDPN-depleted OSCC cell line. ResultsThe retrospective analysis revealed that the expression of IMP3 and PDPN is significantly correlated with T stage, lymph node metastasis, and the overall survival of OSCC patients. In addition, the dual expression of IMP3 and PDPN but not the single expression of either IMP3 or PDPN was associated with bone invasion and the number of osteoclasts in patients with OSCC. In support of these findings, IMP3 or PDPN depletion inhibited the invasive capacity of OSCC cells in a three-dimensional culture system, tumorigenesis, and regional bone destruction in a xenograft mouse model. In addition, IMP3 or PDPN depletion inhibited the expression of interleukin (IL)-6 and IL-8 in OSCC cells, and decreased the expression of receptor activator of NF-κB ligand (RANKL) in xenograft tumor tissues of OSCC. ConclusionsThese results suggest that IMP3 and PDPN may have strong influence on the pathogenesis of OSCC, especially in bone invasion, and may serve as novel therapeutic targets with prognostic implications for bone-invasive OSCC.

Aberrant DNA hypomethylation of miR-196b contributes to migration and invasion of oral cancer.

MicroRNAs (miRs) are a class of small endogenous non-coding RNAs of ~21–24 nucleotides in length. Previous studies have indicated that miR-196b has either an oncogenic or tumor-suppressive function in various types of cancer. However, the biological role of miR-196b in oral squamous cell carcinoma (OSCC) remains unclear. In the present study, the expression levels of miR-196b were examined in oral cancer tissues and corresponding adjacent normal tissues from 69 OSCC patients using stem-loop reverse transcription-quantitative polymerase chain reaction. The results indicated that miR-196b was significantly overexpressed in OSCC tissues compared with the corresponding adjacent normal tissue samples (64 of 69, 92.7%, P<0.001). Analysis of the methylation status of the miR-196b gene indicated more frequent hypomethylation of the CpG islands located upstream of the miR-196b gene in the OSCC tissues than in the adjacent normal tissues (32 of 69, 46.3%), and the methylation status of miR-196b correlated inversely with its expression levels. Furthermore, the unmethylated status of the miR-196b promoter correlated with poor disease-specific survival in OSCC patients (P=0.035). Functional analysis revealed that ectopic miR-196b expression promoted oral cancer cell migration and invasion abilities, and that silencing of miR-196b could abrogate in vitro migration and invasion of oral cancer cells. Collectively, the present findings indicate that the epigenetic regulation of miR-196b expression plays a crucial role in modulating cell migration and invasion during OSCC progression, and thus may serve as a potential prognosis marker or therapeutic target for OSCC.

Cathepsin B Expression and the Correlation with Clinical Aspects of Oral Squamous Cell Carcinoma.

BackgroundCathepsin B (CTSB), a member of the cathepsin family, is a cysteine protease that is widely distributed in the lysosomes of cells in various tissues. It is overexpressed in several human cancers and may be related to tumorigenesis. The main purpose of this study was to analyze CTSB expression in oral squamous cell carcinoma (OSCC) and its correlation with patient prognosis.Methodology/Principal FindingsTissue microarrays were used to detect CTSB expression in 280 patients and to examine the association between CTSB expression and clinicopathological parameters. In addition, the metastatic effects of the CTSB knockdown on two oral cancer cell lines were investigated by transwell migration assay. Cytoplasmic CTSB expression was detected in 34.6% (97/280) of patients. CTSB expression was correlated with positive lymph node metastasis (p = 0.007) and higher tumor grade (p = 0.008) but not with tumor size and distant metastasis. In addition, multivariate analysis using a Cox proportional hazards model revealed a higher hazard ratio, demonstrating that CTSB expression was an independent unfavorable prognostic factor in buccal mucosa carcinoma patients. Furthermore, the Kaplan–Meier curve revealed that buccal mucosa OSCC patients with positive CTSB expression had significantly shorter overall survival. Moreover, treatment with the CTSB siRNA exerted an inhibitory effect on migration in OC2 and CAL27 oral cancer cells.ConclusionsWe conclude that CTSB expression may be useful for determining OSCC prognosis, particularly for patients with lymph node metastasis, and may function as a biomarker of the survival of OSCC patients in Taiwan.

Pre-treatment neutrophil to lymphocyte ratio predicts the chemoradiotherapy outcome and survival in patients with oral squamous cell carcinoma: a retrospective study.

BackgroundThe Neutrophil to lymphocyte ratio (NLR) has prognostic value in patients with a variety of cancers. However, its prognostic significance in oral squamous cell carcinoma (OSCC) patients has not been fully explored. The purpose of this study was to determine the clinical significance of NLR in patients with OSCC.MethodsOSCC patients who underwent surgery following 5-fluorouracil (5-FU)-based chemoradiotherapy were enrolled in this study. The associations between the NLR status and various clinicopathological features were examined, and the effects of the NLR on the prognosis were evaluated. Analysis of circulating interleukin-6 (IL-6) was carried out and correlation with NLR and C-reactive protein concentration (CRP) was examined.ResultsAn elevated NLR was significantly correlated with advanced T-stage and poor response to chemoradiotherapy. Moreover, a Cox regression analysis based on the disease-free survival (DFS) revealed the NLR status (hazard ratio, 2.013; P = 0.041) and pathological response to chemoradiotherapy (hazard ratio, 0.226; P = 0.001) to be significant prognostic factors in OSCC patients. Furthermore, circulating IL-6 was found to correlate with NLR and CRP.ConclusionThe NLR is a potential biomarker for predicting the clinical response to 5-FU-based chemoradiotherapy and the survival in OSCC patients, and the systemic inflammatory response may be potential target for improving patient’s prognosis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2079-6) contains supplementary material, which is available to authorized users.

Secretome profiling of oral squamous cell carcinoma-associated fibroblasts reveals organization and disassembly of extracellular matrix and collagen metabolic process signatures.

An important role has been attributed to cancer-associated fibroblasts (CAFs) in the tumorigenesis of oral squamous cell carcinoma (OSCC), the most common tumor of the oral cavity. Previous studies demonstrated that CAF-secreted molecules promote the proliferation and invasion of OSCC cells, inducing a more aggressive phenotype. In this study, we searched for differences in the secretome of CAFs and normal oral fibroblasts (NOF) using mass spectrometry-based proteomics and biological network analysis. Comparison of the secretome profiles revealed that upregulated proteins involved mainly in extracellular matrix organization and disassembly and collagen metabolism. Among the upregulated proteins were fibronectin type III domain-containing 1 (FNDC1), serpin peptidase inhibitor type 1 (SERPINE1), and stanniocalcin 2 (STC2), the upregulation of which was validated by quantitative PCR and ELISA in an independent set of CAF cell lines. The transition of transforming growth factor beta 1 (TGF-β1)-mediating NOFs into CAFs was accompanied by significant upregulation of FNDC1, SERPINE1, and STC2, confirming the participation of these proteins in the CAF-derived secretome. Type I collagen, the main constituent of the connective tissue, was also associated with several upregulated biological processes. The immunoexpression of type I collagen N-terminal propeptide (PINP) was significantly correlated in vivo with CAFs in the tumor front and was associated with significantly shortened survival of OSCC patients. Presence of CAFs in the tumor stroma was also an independent prognostic factor for OSCC disease-free survival. These results demonstrate the value of secretome profiling for evaluating the role of CAFs in the tumor microenvironment and identify potential novel therapeutic targets such as FNDC1, SERPINE1, and STC2. Furthermore, type I collagen expression by CAFs, represented by PINP levels, may be a prognostic marker of OSCC outcome.

Detection of cervical lymph node micrometastasis and isolated tumor cells in oral squamous cell carcinoma using immunohistochemistry and serial sectioning.

Introduction:Oral squamous cell carcinoma (OSCC) comprises one of the largest subsets of cancers with a tendency for regional metastasis. Nodal status is a key prognostic indicator in patients with OSCC, particularly with N0 neck. Occult metastasis in the form of micrometastasis (MM) and isolated tumor cells (ITCs), often goes undetected by routine hematoxylin and eosin (H&E) examination using 1–2 sections for analysis. This limitation could be overcome by combining serial sectioning (SS) with immunohistochemistry (IHC) for the detection of MM and ITC. Pan-cytokeratin (pan-CK) (AE1/AE3) is particularly a useful marker to detect these deposits as their presence has resulted in varied interpretations and different applications of the tumor-node-metastasis system.Objectives:The objective of the study was to identify a suitable method for detecting MM and ITC in lymph nodes (LNs) of OSCC by combining SS and IHC and to compare it with conventional H&E staining.Materials and Methods:This laboratory-based, prospective study was conducted on 133 LNs harnessed from ten patients treated with radical neck dissection for primary OSCC. The LNs were subjected to SS at 100 μm intervals. The sections were stained with routine H&E staining, pan-CK and analyzed for MM and ITC according to criteria laid by Hermanek et al.Statistical Analysis:The obtained data were subjected to statistical analysis using Chi-square test.Results:The application of combination of SS and IHC using pan-CK (AE1/AE3) in our study revealed the presence of MM and ITC in 2.25% of the LNs diagnosed as negative on routine H&E examination. The detection of these occult metastatic deposits resulted in upstaging of 33.33% of the patients.Conclusion:In the view of crucial role of occult LN metastasis in prognosis and survival of OSCC patients with N0 neck, diagnostic tools such as IHC staining, particularly with pan-CK (AE1/AE3), combined with SS should be preferred over conventional methods as they result in upstaging, thus sparing the low-risk patients the morbidity of unnecessary treatment.

Surrogate Prognostic Biomarkers in OSCC: The Paradigm of PA28γ Overexpression

Carcinomas of the head and neck represent the sixth most commonly diagnosed type of cancer. Most of head and neck carcinomas are histologically categorized as squamous cell carcinomas (SCC) (Chin et al., 2006). SCC arises from squamous cells lining mucosal surfaces in the nasal cavity, paranasal sinuses, nasopharynx, larynx, trachea, oral cavity, oropharynx, salivary glands, and ears (Argiris et al., 2008). The most common type of head and neck SCC is oral squamous cell carcinoma (OSCC), with an estimated annual incidence of 199 thousands of cases and an estimated mortality of 98 thousands, only in men, as the incidence of OSCC is much higher in men than in women (Bray et al., 2012). The causative risk factors of OSCC include tobacco use and alcohol consumption (Argiris et al., 2008). A wide gamut of molecules is involved in oral carcinogenesis. Aberrant expression of several genes is associated with hallmarks of OSCC, including uncontrolled cell proliferation, defective apoptosis, loss of cell differentiation, epithelial–mesenchymal transition, metastasis, and angiogenesis (da Silva et al., 2011). The potential of such molecules as molecular biomarkers for diagnosis, prognosis or monitoring of treatment efficacy in OSCC has been intensively investigated during the last decades. Immunohistochemical detection and/or mRNA expression profiling of promising molecular biomarkers are expected to significantly contribute to the generation of novel screening tests with high sensitivity and specificity, as well as tailor-made therapies against OSCC, in the era of personalized medicine that has just emerged (Cheng et al., 2014). Besides that, selective targeting of interactions between specific molecules that are key players in cancer-related biochemical pathways could dramatically impede oral carcinogenesis and therefore elongate or ameliorate the survival of OSCC patients. The proteasome activator 28 gamma (PA28γ) encodes the gamma subunit of the 11S regulator of the immunoproteasome, a modified proteasome that processes class I MHC peptides. PA28γ is a multifunctional protein, implicated in the degradation of important cell cycle regulatory proteins in an ATP-independent manner. In fact, PA28γ inhibits apoptosis and promotes cell cycle progression (Rechsteiner and Hill, 2005). A homopolymer form of PA28γ interacts with both MDM2 and p53 proteins and facilitates their physical interaction, which promotes ubiquitination- and MDM2-dependent proteasomal degradation of p53, thus limiting its accumulation and resulting in inhibited apoptosis after DNA damage. Elimination of endogenous PA28γ in human cancer cells has been shown to abolish MDM2-mediated p53 degradation, increase the activity of p53, and enhance apoptosis (Zhang and Zhang, 2008). In the E-Biomedicine, Jing Li and colleagues Li et al., 2015 assess immunohistochemically PA28γ expression as a prognostic biomarker in OSCC. For this purpose, a total of 368 patients from three independent cohorts were included in the current study. Moreover, the authors used an independent cohort of 460 patient specimens obtained between 1992 and 2013 in the TCGA database as an external validation cohort to validate the prognostic value of PA28γ expression. Strong PA28γ expression was shown to predict significantly reduced disease-free and overall survival in OSCC patients. More importantly, the unfavorable prognostic value of PA28γ was independent from other prognostic factors such as smoking history, drinking history, cell differentiation, tumor stage, nodal stage, radiotherapy and chemotherapy. Besides the prognostic significance of PA28γ in OSCC, this study provides evidence for the role of PA28γ in oral tumor growth and metastasis. In more detail, PA28γ silencing in two OSCC cell lines led to a decline in cell viability and colony growth. Moreover, growth of tumor originating from OSCC cells transplanted subcutaneously on the right back of BALB/c nude mice was much slower when PA28γ was silenced in these tumor cells. The authors also showed that PA28γ silencing suppresses tumor angiogenesis and increases tumor cell apoptosis in xenograft models. This study opens the door to assess the clinical value of PA28γ expression as a surrogate prognostic biomarker in larger cohorts of OSCC patients as well as to further investigate the role of PA28γ overexpression in the pathobiology and progression of OSCC. The clinical value of several molecules as putative diagnostic, prognostic, and treatment-response biomarkers in OSCC has been widely investigated during the last decades (Chawla et al., 2015, Ni et al., 2015). The elucidation of the biochemical pathways implicated in oral carcinogenesis, tumor progression, and metastasis, taking also into consideration the heterogeneity of oral tumors, would definitely assist the discovery of novel candidate biomarkers in OSCC. Some molecular biomarkers, including PA28γ, may also have prognostic value in specific OSCC patient subgroups. In conclusion, the dysregulation of PA28γ gene expression in OSCC tissues compared to benign oral tumors, dysplasias, or normal specimens originating from the oral cavity merits further investigation and validation in independent and large cohorts of OSCC patients.

Elevated Serum Gas6 Is a Novel Prognostic Biomarker in Patients with Oral Squamous Cell Carcinoma.

ObjectiveThis study explored the level and clinical significance of serum Gas6 in patients with oral squamous cell carcinoma (OSCC).MethodsA total of 128 OSCC patients and 145 normal controls were selected. Enzyme-linked immunosorbent assay was used to detect Gas6 concentration in sera from the OSCC patients and controls. The correlations of serum Gas6 concentration and clinicopathological characteristics of OSCC patients were assessed, and the prognostic significance of serum Gas6 was evaluated with a Kaplan–Meier curve and log-rank test.ResultsThe results showed that serum Gas6 concentration was significantly higher in OSCC patients than in controls (P < 0.05). OSCC patients with late TNM stage (III, IV) had a relatively high serum Gas6 concentration compared with those with early stage (I, II) (P < 0.01) and patients with poorly differentiated tumors had a higher level of serum Gas6 than those with well-differentiated tumors (P < 0.01). Multivariate logistic regression analysis demonstrated that high serum Gas6 was an independent risk factor for lymph nodal metastases in OSCC patients (OR = 2.79, 95% CI: 1.72–4.48). For predicting OSCC development, ROC curve analysis showed a sensitivity of 0.63 with a specificity of 0.92 (AUC = 0.79, 95% CI: 0.74–0.85). Cox analysis revealed that high serum Gas6 was an independent biomarker for predicting poor overall survival in OSCC patients (HR = 2.07, 95% CI: 1.79–3.62). In addition, we found that Gas6 expression was increased in OSCC tissues and it may significantly decrease E-cadherin expression, and increase P-cadherin and N-cadherin expression, in OSCC cells. Further, Gas6 could promote the migratory and invasive ability of OSCC cells in vitro.ConclusionTaken together, these results suggest that Gas6 increases the metastatic capacity of OSCC cells and serum Gas6 could be a candidate biomarker for diagnostic and prognostic use in OSCC patients.

Carbonic anhydrase IX overexpression regulates the migration and progression in oral squamous cell carcinoma.

Carbonic anhydrase IX (CAIX) is reportedly overexpressed in several types of carcinomas and is generally considered a marker of malignancy. The current study investigated the association between membrane expression of CAIX and the clinicopathological characteristics in oral squamous cell carcinoma (OSCC) patients. The study used immunohistochemistry to examine CAIX expression in 271 OSCC specimens by tissue microarray (TMA) and assessed the effect of CAIX overexpression and knockdown on migration of oral cancer cells in vitro. We found that CAIX expression was associated with more advanced clinical stages (p = 0.030) and positive lymph node metastasis (p = 0.026). Importantly, CAIX expression was correlated with a poorer patient prognosis in a univariate survival analysis (p = 0.025). Moreover, CAIX suppression by small interfering RNA (siRNA) significantly reduced cellular migration in OECM-1 oral cancer cell. In conclusion, our study showed that the expression of CAIX in OSCC samples can predict the progression of OSCC and survival of OSCC patients in Taiwan.

CMTM3 inhibits cell growth and migration and predicts favorable survival in oral squamous cell carcinoma.

Downregulation of CKLF-like MARVEL transmembrane domain-containing member 3 (CMTM3) has been reported in a number of human tumors. However, the role of CMTM3 in oral squamous cell carcinoma (OSCC) remains largely unknown. In this study, we showed that the expression of CMTM3 was significantly reduced in OSCC cell lines and primary tumor specimens (P < 0.001). Methylation-specific PCR showed hypermethylation in CMTM3 promoter in a significant proportion of tumor tissues (61 %). The expression of CMTM3 was associated with T stage, lymph node metastasis, tumor node metastasis (TNM) stage, and recurrence of OSCC patients (P < 0.05, n = 201). More importantly, CMTM3 expression was associated with the prognosis of OSCC patients (P < 0.001) and was an independent prognostic factor (hazard ratio = 0.593, 95 % confidence interval, 0.272-1.292; P = 0.039). Overexpression of CMTM3 inhibited the growth and migration of OSCC cells. In vivo experiments also showed that the growth of OSCC xenografts in nude mice was significantly inhibited by CMTM3 overexpression. These findings indicate that downregulation of CMTM3 due to promoter hypermethylation contributed to the proliferation and migration of OSCC cells and suggest that CMTM3 is an independent prognostic factor for the evaluation of the survival of OSCC patients.

Expression of myeloid zinc finger 1 and the correlation to clinical aspects of oral squamous cell carcinoma.

The myeloid zinc finger 1 (MZF1) is a zinc finger transcription factor which regulates myeloid differentiation and oncogenesis. However, little information is available concerning the MZF1 expression in oral squamous cell carcinoma (OSCC) and its correlation with patients' prognosis. We detected the expression of MZF1 in 274 patients with OSCC using tissue microarrays (TMAs) and evaluated the associations between nuclear MZF1 expression and the clinical parameters of OSCC patients. We found that nuclear MZF1 expression was present in 190/274 (69.3 %) cases, and loss of nuclear expression of MZF1 was associated with more advanced clinical stages (p = 0.011) and larger tumor size (p = 0.002), but not associated with positive lymph node metastasis and distal metastasis. Importantly, tongue squamous cell carcinomas (SCC) patients with negative nuclear MZF1 expression had significantly worse overall survival rates (log-rank test, p = 0.028). In conclusion, our results revealed that the loss of nuclear expression of MZF1 in OSCC samples can predict the progression of OSCC and the survival of OSCC patients in Taiwan.