Salivary cytokines as biomarkers of oral cancer: a systematic review and meta-analysis.

BACKGROUND: Oral cancer (OC) is usually diagnosed at advanced clinical stages due to its asymptomatic nature and absence of pathognomonic signs in its early development phase. Delayed diagnosis is one of the major causes of OC treatment failure and poor prognosis. Development of alternative diagnostic approaches are imperative for improving early detection and therapeutic success rates. Salivary cytokines (SC) have been studied as potential diagnostic biomarkers for OC and may represent a potential tool for improvement of its early detection. METHODS: In this systematic review and meta-analysis we identified SC studied as OC biomarkers by systematically reviewing the PubMed and Cochrane Library databases using the terms: "oral cancer", "cytokine", and "saliva", and also combined with "interleukin" or "interferon". Only case-control studies that measured SC by ELISA from treatment naïve patients were included in the qualitative review. For the meta-analysis were included all comparable studies that provided enough data (sample size, mean and standard deviation or standard error of the mean) for SC levels in OC patients, non-cancer controls and patients with oral potentially malignant disorders (OPMD), including leukoplakia. Comparisons with patients with oral lichen planus (OLP) and gingivitis were included in the qualitative analysis. RESULTS: A total of 28 articles (from 2004 to 2018) were included in the systematic review, describing 10 different SC, being IL-8 and IL-6 the most studied ones. SC levels were consistently higher among OC patients when compared to healthy controls and to patients with OPMD, OLP and gingivitis. Meta-analysis including 23 eligible studies showed that IL-8, IL-6, TNF-α, IL-1ß and IL-10 salivary levels were significantly higher in OC patients compared to controls; and that IL-8, IL-6, TNF-α and IL-1ß salivary levels were also higher in OC patients compared to individuals with OPMD. When compared to healthy controls, OPMD patients showed significantly higher IL-6 and TNF-α salivary levels. CONCLUSIONS: Our analyses showed that the salivary levels of some cytokines are consistently different among OC, OPMD and healthy patients, indicating that these SC may represent potential diagnostic biomarkers for OC and OPMD. Despite of that, SC levels were highly variable among studies, suggesting that further technical improvement and standardization for SC measurement by ELISA is needed in order to successfully translate these biomarkers to the clinical practice.

The neuropeptide calcitonin gene-related peptide links perineural invasion with lymph node metastasis in oral squamous cell carcinoma.

OBJECTIVE: Although perineural invasion (PNI) is well-known to be correlated with and able to predict lymph node metastasis (LNM) in oral squamous cell carcinoma (OSCC), the clinical and molecular correlation between PNI and LNM has not been elucidated, and preoperative biomarkers for LNM prediction in OSCC are urgently needed. MATERIALS AND METHODS: The correlation between PNI and LNM was retrospectively evaluated using a cohort of 218 patients diagnosed with OSCC. Candidate neuropeptides were screened based on TCGA database and verified via immunohistochemistry and Western blot analyses. ELISA was used to detect calcitonin gene-related peptide (CGRP) in patient plasma. In vitro assays were used to explore the effects of CGRP on OSCC cells. RESULTS: OSCC patients with PNI had a higher incidence of LNM (69.86% vs. 26.2%, P < 0.0001, n = 218). CGRP expression was upregulated in the PNI niche and in metastatic lymph nodes, and was correlated with poor overall survival of OSCC patients. Preoperative plasma CGRP levels were higher in OSCC patients (n = 70) compared to healthy donors (n = 60) (48.59 vs. 14.58 pg/ml, P < 0.0001), and were correlated with LNM (P < 0.0001) and PNI (P = 0.0002). Preoperative plasma CGRP levels alone yielded an AUC value of 0.8088 to predict LNM, and CGRP levels combined with preoperative T stage reached an AUC value of 0.8590. CGRP promoted proliferation and migration abilities of OSCC cells, which could be antagonized by either pharmacological or genetic blockade of the CGRP receptor. CONCLUSIONS: The neuropeptide CGRP links PNI and LNM in OSCC, and preoperative plasma CGRP levels can be used to predict LNM in OSCC.

Anticancer activity of Vicenin-2 against 7,12 dimethylbenz[a]anthracene-induced buccal pouch carcinoma in hamsters.

Buccal mucosa carcinoma is a significant cause of death in developing nations. Vicenin-2 is a significant bioactive compound found in Ocimum sanctum Linn or Tulsi that possesses several pharmacologic properties. Our focus is to understand the possible impact of Vicenin-2 on 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral carcinogenesis in hamsters. Buccal carcinoma was induced by treatment with carcinogenic DMBA, three times a week for 14 weeks. We determined 100% tumor incidence, abnormal tumor volume, inclined tumor burden, and deduced body weight in DMBA-induced oral squamous cell carcinoma (OSCC) hamsters. The upregulation of cytokine levels (interleukin [IL]-6, IL-1ß, and tumor necrosis factor-alpha [TNF-α]) was observed in DMBA-induced OSCC hamsters. Moreover, dysplastic, hyperplastic, and squamous cell carcinoma was identified in the DMBA-induced OSCC hamsters. The diminished activities of lipid peroxidation and enzymatic/nonenzymatic antioxidants were observed in DMBA-induced hamsters. Furthermore, the high expression of proliferating cell nuclear antigen (PCNA), Cyclin-D1, and Bcl-2, and attenuated Bax expression were observed in DMBA-induced hamsters. Our study results explored that Vicenin-2 (30 mg/kg) treated with DMBA-brushed hamsters averted tumor incidence, improved the antioxidant status, and inhibited lipid peroxidation. Moreover, Vicenin-2 inhibited the immunohistochemical expression of PCNA, Cyclin-D1, and Bcl-2, and significantly restored apoptotic Bax levels. The Vicenin-2 treatment prevents the lesion formation in the oral epithelium of the DMBA-induced hamsters. The Vicenin-2 treatment potentially halts the proinflammatory cytokines (IL-6, IL-1ß, and TNF-α) production in OSCC hamsters. Thus, we proved that Vicenin-2 prevents DMBA-induced buccal carcinogenesis in hamsters via improving antioxidants by modulating apoptotic and cytokines signaling pathways.

Evaluation of synuclein-γ levels by novel monoclonal antibody in saliva and cancer tissues from oral squamous cell carcinoma patients.

The clinical value of synuclein-γ (SNCG) in oral squamous cell carcinoma (OSCC) was evaluated by detecting the expression of SNCG in saliva and tissues and its correlation with clinicopathological parameters (age, gender, ethnicity, degree of differentiation, clinical stage, and lymph node metastasis). Salivary samples were collected from 79 patients with OSCC, 31 patients with oral premalignant lesions (OPMLs), such as oral lichen planus, oral leukoplakia, and erythema, and 80 controls, and levels of SNCG in salivary samples were determined by enzyme-linked immunosorbent assay (ELISA). Tissue expression in formalin-fixed tissue biopsies of 94 cases of OSCC and 30 adjacent normal tissues was analyzed by immunohistochemistry (IHC) using an antibody against SNCG. The results showed that the salivary levels of SNCG in patients with OSCC and OPMLs were significantly higher than those detected in the control group (p<0.001). The immunohistochemical results showed that SNCG was highly expressed in tumor cells of OSCC patients, with low expression in the adjacent normal epithelium (p<0.001, OR=6.074). Salivary SNCG level correlated with differentiation (p=0.022). Besides, the expression of SNCG in OSCC tissues was also significantly associated with differentiation (p<0.001).

Overexpression of KLF17 Predicts a Favorable Prognosis in Patients with Oral Squamous Cell Carcinoma: A Retrospective Study.

BACKGROUND AND OBJECTIVES: Patients with oral squamous cell carcinoma (OSCC), a common malignancy in Asian countries, have a poor prognosis. We investigated the role of Krüppel-like factor 17 (KLF17) and its prognostic significance in OSCC. MATERIALS AND METHODS: KLF17 expression was measured by immunohistochemical staining of specimens from 283 patients with OSCC. We analyzed correlations between KLF17 expression and clinicopathologic features and between KLF17 expression and overall survival. The prognostic value of KLF17 was tested using Kaplan-Meier analysis and Cox proportional hazard models. RESULTS: Among the 283 patients, high KLF17 expression was significantly associated with an early OSCC stage and low T-value (p = 0.033 and p = 0.036, respectively). The five-year survival rates were better in patients with high KLF17 expression than with low expression (66.5% and 49.6%, respectively). The prognostic role of KLF17 was further confirmed through multivariate analysis (hazard ratio 1.506, 95% confidence interval 1.034-2.191, p = 0.033). The prognostic value was more significant in patients with a history of betel quid chewing or with a low T-value. CONCLUSIONS: High KLF17 expression can serve as a marker for a favorable prognosis in patients with OSCC. The prognostic role of KLF17 is more significant in patients with a history of betel quid chewing or a low T-value.

Interactive Visual Exploration of 3D Mass Spectrometry Imaging Data Using Hierarchical Stochastic Neighbor Embedding Reveals Spatiomolecular Structures at Full Data Resolution.

Technological advances in mass spectrometry imaging (MSI) have contributed to growing interest in 3D MSI. However, the large size of 3D MSI data sets has made their efficient analysis and visualization and the identification of informative molecular patterns computationally challenging. Hierarchical stochastic neighbor embedding (HSNE), a nonlinear dimensionality reduction technique that aims at finding hierarchical and multiscale representations of large data sets, is a recent development that enables the analysis of millions of data points, with manageable time and memory complexities. We demonstrate that HSNE can be used to analyze large 3D MSI data sets at full mass spectral and spatial resolution. To benchmark the technique as well as demonstrate its broad applicability, we have analyzed a number of publicly available 3D MSI data sets, recorded from various biological systems and spanning different mass-spectrometry ionization techniques. We demonstrate that HSNE is able to rapidly identify regions of interest within these large high-dimensionality data sets as well as aid the identification of molecular ions that characterize these regions of interest; furthermore, through clearly separating measurement artifacts, the HSNE analysis exhibits a degree of robustness to measurement batch effects, spatially correlated noise, and mass spectral misalignment.

Treponema denticola chymotrypsin-like proteinase may contribute to orodigestive carcinogenesis through immunomodulation.

BACKGROUND: Periodontal pathogens have been linked to oral and gastrointestinal (orodigestive) carcinogenesis. However, the exact mechanisms remain unknown. Treponema denticola (Td) is associated with severe periodontitis, a chronic inflammatory disease leading to tooth loss. The anaerobic spirochete Td is an invasive bacteria due to its major virulence factor chymotrypsin-like proteinase. Here we aimed to investigate the presence of Td chymotrypsin-like proteinase (Td-CTLP) in major orodigestive tumours and to elucidate potential mechanisms for Td to contribute to carcinogenesis. METHODS: The presence of Td-CTLP within orodigestive tumour tissues was examined using immunohistochemistry. Oral, tonsillar, and oesophageal squamous cell carcinomas, alongside gastric, pancreatic, and colon adenocarcinomas were stained with a Td-CTLP-specific antibody. Gingival tissue from periodontitis patients served as positive controls. SDS-PAGE and immunoblot were used to analyse the immumodulatory activity of Td-CTLP in vitro. RESULTS: Td-CTLP was present in majority of orodigestive tumour samples. Td-CTLP was found to convert pro MMP-8 and -9 into their active forms. In addition, Td-CTLP was able to degrade the proteinase inhibitors TIMP-1, TIMP-2, and α-1-antichymotrypsin, as well as complement C1q. CONCLUSIONS: Because of its presence within tumours and regulatory activity on proteins critical for the regulation of tumour microenvironment and inflammation, the Td-CTLP may contribute to orodigestive carcinogenesis.

Differential expression of hsa-miR-221, hsa-miR-21, hsa-miR-135b, and hsa-miR-29c suggests a field effect in oral cancer.

BACKGROUND: The theory of field effect suggests that the tumor-adjacent area, besides histopathologically normal, undergoes genetic and epigenetic changes that can eventually affect epithelial homeostasis, predisposing the patient to cancer development. One of the many molecular changes described in cancer are microRNAs (miRNAs), which regulates the expression of important genes during carcinogenesis. Thus, the aim of this study was to investigate the field effect in oral cancer. METHODS: We investigated the differential expression profile of four miRNAs (hsa-miR-221, hsa-miR-21, hsa-miR-135b, and hsa-miR-29c) in cancerous oral tissue, in tumor-adjacent tissue and and in non-cancerous tissue samples from healthy volunteers. RESULTS: Our results showed significant overexpression profiles of all four studied miRNAs in cancerous oral tissue compared to non-cancerous samples, as well as in tumor-adjacent tissue compared to cancer-free tissue. No significant difference was found when comparing the expression profile of cancerous and tissue-adjacent tissue groups. We found a negative correlation between the expression of hsa-miR-21 expression and STAT3 in oral squamous cell carcinoma. CONCLUSION: These results suggest that the tissue adjacent to cancer cannot be considered a normal tissue because its molecular aspects are significantly altered. Our data corroborates the hypothesis of field cancerization.

Raman spectroscopic analysis of the molecular composition of oral cavity squamous cell carcinoma and healthy tongue tissue.

A Raman tissue spectrum is a quantitative representation of the overall molecular composition of that tissue. Raman spectra are often used as tissue fingerprints without further interpretation of the specific information that they contain about the tissue's molecular composition. In this study, we analyzed the differences in molecular composition between oral cavity squamous cell carcinoma (OCSCC) and healthy tissue structures in tongue, based on their Raman spectra. A total of 1087 histopathologically annotated spectra (142 OCSCC, 202 surface squamous epithelium, 61 muscle, 65 adipose tissue, 581 connective tissue, 26 gland, and 10 nerve) were obtained from Raman maps of 44 tongue samples from 21 patients. A characteristic, average spectrum of each tissue structure was fitted with a set of 55 pure-compound reference spectra, to define the best library of fit-spectra. Reference spectra represented proteins, lipids, nucleic acids, carbohydrates, amino acids and other miscellaneous molecules. A non-negative least-squares algorithm was used for fitting. Individual spectra per histopathological annotation were then fitted with this selected library in order to determine the molecular composition per tissue structure. The spectral contribution per chemical class was calculated. The results show that all characteristic tissue-type spectra could be fitted with a low residual of <4.82%. The content of carbohydrates, proteins and amino acids was the strongest discriminator between OCSCC and healthy tissue. The combination of carbohydrates, proteins and amino acids was used for a classification model of 'tumor' versus 'healthy tissue'. Validation of this model on an independent dataset showed a specificity of 93% at a sensitivity of 100%.

An immunohistochemical score to predict the outcome for oral squamous cell carcinoma.

BACKGROUND: Oral cancer is a major public health problem worldwide, with a poor survival. Our aim was to evaluate several protein markers in oral squamous cell carcinomas (OSCC) and analyse their prognostic value on patient's survival. METHODS: We analysed the expression of EGFR, p53, p27, p16, cyclin D1, cyclin A2, COX-2, Ki-67, Bcl-2, VEGFR-1 and VEGFR-2, by immunohistochemistry on 67 primary OSCC. Cancer-specific survival (CSS) analysis was evaluated by the Cox regression model. RESULTS: Markers showed variable expression between 27.9% and 95.2%. In univariate analysis for CSS, we found that four of the tested markers, namely high expression of p53 (P = .001), EGFR (P = .003), cyclin A2 (P = .005) and low expression of p16 (P = .019), along with clinical stage (P < .001), tumour size (P < .001), presence of nodal metastasis (P < .001) and perineural permeation (P = .039) were related to decreased survival. On the basis of these results, we constructed an immunohistochemical score hinging on the possibility that any tumour could express none of these four markers (score 0), one or two markers (score 1) and three or more markers (score 2). In multivariable analysis, this immunohistochemical score revealed an independent prognostic value on cancer-specific survival (P = .001; HR: 3.7: 95%CI 1.7-7.9). Moreover, we confirmed that in early-stage tumours (stage I or II) this score maintained its independent prognostic value (P = .025; HR: 7.9, 95%CI 1.3-49.1) on CSS. CONCLUSION: The expression of the markers p53, p16, EGFR and cyclin A in OSCC, combined to give an immunohistochemical score, may identify high-risk subgroups for decreased survival and to further guide therapeutic decisions.

T cells are involved in the induction of macrophage phenotypes in oral leukoplakia and squamous cell carcinoma-a preliminary report.

BACKGROUND: The prognosis of human malignancies has been shown to depend on immunological parameters, such as macrophage polarization (M1 and M2). In this study, we identify the phenotype of macrophages, and investigate an involvement of infiltrated T cells that participate in the polarization of macrophages, in oral leukoplakia (OLK), and oral squamous cell carcinoma (OSCC). METHODS: Immunohistochemical method was used to examine the number of CD68+ , CD163+ (M2), iNOS+ (M1) macrophages, and CD4+ , CD8+ , CCR4+ (Th2), CCR5+ (Th1) cells in 102 cases of OSCC: without metastases-OSCC M(-) (n = 54), and with metastases-OSCC M(+) (n = 48), 23 cases of OLK, and 18 control cases. RESULTS: The mean number of CD68+ , CD163+ , iNOS+ , CD4+ , CCR4+ , CCR5+ cells was significantly increased in OSCC M(+) group compared with OLK, OSCC M(-) and control group. We found positive correlations between the number of CD4+ T cells and CD163+ and iNOS+ macrophages as well as CCR4+ and CCR5+ cells in both OSCC groups. The mean number of CD8+ cells was significantly increased in OSCC M(-) and OLK compared with OSCC M(+) and control group. In OSCC M(+) and OSCC M(-) groups, a negative correlation between the number of CD8+ cells and CD163+ and iNOS+ macrophages was found. CONCLUSIONS: The number and co-localization of lymphocytes and macrophages in OLK and OSCC may indicate that infiltrating cells influence the early and subsequent stage of oral carcinogenesis.

Cancer-associated fibroblasts promote bone invasion in oral squamous cell carcinoma.

BACKGROUND: The molecular mechanisms involved in the invasion of bone by oral squamous cell carcinomas (OSCC) are poorly understood, and little is known about the role of cancer-associated fibroblasts (CAF), the presence of which confers a poor prognosis. METHODS: Clinicopathological data from 277 OSCC cases involving bone resections were reviewed, and 32 cases thoroughly analysed histologically. Immunohistochemistry was used to examine αSMA, RANKL and OPG. Western blotting and qPCR were used to assess myofibroblast (CAF-like) differentiation, RANKL and OPG expression in vitro, and RANKL secretion was analysed by ELISA. Osteoclastogenesis was examined using TRAP staining, multinucleation and pit forming assays. RESULTS: Fibrous stroma intervened between tumour and bone in the majority of cases, with no direct contact between cancer cells and bone. RANKL and OPG, two proteins key to regulating bone resorption, were expressed in tumour cells as well as fibrous stroma adjacent to bone and αSMA-positive myofibroblastic CAF were consistently seen infiltrating into bone ahead of tumour cells. Human primary osteoblasts cultured with conditioned media from human OSCC-derived cells and human primary CAF showed a significant increase in RANKL and a decline in OPG mRNA expression. RANKL secretion was significantly increased in primary oral fibroblasts induced to differentiate into a CAF-like phenotype by transforming growth factor-ß1 (TGF-ß1) treatment and in primary CAF. Indirect co-culture of murine macrophages with conditioned media from CAF (experimentally derived and isolated from OSCCs) resulted in a marked increase in osteoclastogenesis (in excess of that provoked by cancer cells) determined by tartrate-resistant acid phosphatase activity, multinucleation and resorption pit formation. CONCLUSIONS: This study is the first to describe a functional role for CAFs in bone invasion and turnover, identifying a novel potential therapeutic target and diagnostic indicator in this difficult to treat bone invasive malignancy.

Expression of hypoxia-induced factor-1 alpha in early-stage and in metastatic oral squamous cell carcinoma.

To investigate hypoxia-induced factor-1 alpha expression in distinct oral squamous cell carcinoma subtypes and topographies and correlate with clinicopathological data. Hypoxia-induced factor-1 alpha expression was assessed by immunohistochemistry in 93 cases of OSCC. Clinical and histopathological data were reviewed from medical records. Hypoxia-induced factor-1 alpha status was distinct according to tumor location, subtype and topography affect. In superficial oral squamous cell carcinomas, most tumor cells overexpressed hypoxia-induced factor-1 alpha, whereas hypoxia-induced factor-1 alpha was restricted to the intratumoral region in conventional squamous cell carcinomas. All basaloid squamous cell carcinomas exhibited downregulation of hypoxia-induced factor-1 alpha. Interestingly, metastatic lymph nodes (91.7%, p = 0.001) and the intratumoral regions of corresponding primary tumors (58.3%, p = 0.142) showed hypoxia-induced factor-1 alpha-positive tumor cells. Overall survival was poor in patients with metastatic lymph nodes. Hypoxia-induced factor-1 alpha has distinct expression patterns in different oral squamous cell carcinoma subtypes and topographies, suggesting that low oxygen tension promotes the growth pattern of superficial and conventional squamous cell carcinoma, but not basaloid squamous cell carcinoma. Indeed, a hypoxic environment may facilitate regional metastasis, making it a useful diagnostic and prognostic marker in primary tumors.

Expression and associations of TRAF1, BMI-1, ALDH1, and Lin28B in oral squamous cell carcinoma.

Tumor necrosis factor receptor-associated factor 1, an adaptor protein of tumor necrosis factor 2, is involved in classical nuclear factor (NF)-κB activation and lymphocyte recruitment. However, less is known about the expression and association of tumor necrosis factor receptor-associated factor 1 with cancer stem cell markers in oral squamous cell carcinoma. This study aimed to investigate the expression of tumor necrosis factor receptor-associated factor 1 and stem cell characteristic markers (lin28 homolog B, B cell-specific Moloney murine leukemia virus integration site 1, and aldehyde dehydrogenase 1) in oral squamous cell carcinoma and analyze their relations. Paraffin-embedded tissues of 78 oral squamous cell carcinomas, 39 normal oral mucosa, and 12 oral dysplasia tissues were employed in tissue microarrays, and the expression of tumor necrosis factor receptor-associated factor 1, B cell-specific Moloney murine leukemia virus integration site 1, aldehyde dehydrogenase 1, and lin28 homolog B was measured by immunohistostaining and digital pathological analysis. The expression of tumor necrosis factor receptor-associated factor 1 was higher in the oral squamous cell carcinoma group as compared with the expression in the oral mucosa (p < 0.01) and oral dysplasia (p < 0.001) groups. In addition, the expression of tumor necrosis factor receptor-associated factor 1 was associated with those of B cell-specific Moloney murine leukemia virus integration site 1, aldehyde dehydrogenase 1, and lin28 homolog B (p = 0.032, r2 = 0.109; p < 0.0001, r2 = 0.64; and p < 0.001, r2 = 0.16) in oral squamous cell carcinoma. The patient survival rate was lower in the highly expressed tumor necrosis factor receptor-associated factor 1 group, although the difference was not significant. The clustering analysis showed that tumor necrosis factor receptor-associated factor 1 was most related to aldehyde dehydrogenase 1. These findings suggest that tumor necrosis factor receptor-associated factor 1 has potential direct/indirect regulations with the cancer stem cell markers in oral squamous cell carcinoma, which may help in further analysis of the cancer stem cell characteristics.

A panel of microRNAs can be used to determine oral squamous cell carcinoma.

BACKGROUND: Subjective histopathology is currently used to diagnose oral squamous cell carcinoma (OSCC). We tested if abundances of a panel of microRNA could be an objective OSCC indicator. METHOD: Literature review enabled identification of 10 microRNAs associated with oral and head and neck malignancies. We extracted RNA from formalin-fixed paraffin-embedded biopsies; 20 each with OSCC, dysplasia, or histologically normal epithelium (HNE) and 10 with oral lichen planus (OLP). Relative abundances of microRNAs in HNE and OSCC were determined using reverse transcription and then real-time PCR with global mean normalization. MicroRNAs differentially expressed (test microRNA, T-miR) and non-differentially expressed (normalization microRNA, N-miR) were identified. The raw microRNA Cq data were incorporated in a developed algorithm that output a T-miR expression value (T-miREV) score. Raw Cq data from HNE, OSCC, dysplasia, and OLP samples were then used to test the algorithm scoring and OSCC classification. RESULTS: Four test and normalization microRNAs were identified. Algorithm output of T-mirEV >1 or <-1 indicated high and low OSCC probability score, respectively, and gave 88.9% sensitivity, 100% specificity, and 93.5% accuracy. Grouping high and intermediate T-mirEV scores (T-miREV ≥-1) resulted in sensitivity of 90%, specificity of 65%, and accuracy of 77.5% in OSCC classification. All 20 dysplasias and eight of 10 OLP had T-miREV ≥-1 indicating intermediate to high probability of malignant changes. CONCLUSION: A microRNA panel combined with our algorithm can identify tissue with probable oncogenic changes. IMPACT: The developed algorithm serves as a baseline for prospective trials, which may result in potential clinical utility.

Mediator of DNA damage checkpoint protein 1 (MDC1) as a prognostic marker for patients with oral squamous cell carcinoma.

BACKGROUND: The mediator of DNA damage checkpoint protein 1 (MDC1) is involved in the regulation of cell cycle checkpoints and recruitment of several repair proteins to the site of DNA double-stranded breaks (DSBs). This study aimed to correlate the expression of MDC1 protein with clinicopathological parameters and to evaluate its prognostic significance in patients with oral squamous cell carcinoma (OSCC). METHODS: MDC1 protein expression was evaluated immunohistochemically from untreated 100 patients with OSCC using modified H-score method. The association of MDC1 immunostaining was evaluated with clinicopathological parameters and disease outcome using univariate and multivariate survival analysis for relapse-free survival (RFS) and overall survival (OS). RESULTS: Incidence of nuclear and cytoplasmic expression of MDC1 protein was 85% & 92%, respectively. Strong nuclear MDC1 protein expression was found to be significantly correlated with lymph node metastasis (P = 0.032). For RFS, Kaplan-Meier survival analysis demonstrated that presence of metastatic lymph node (P = 0.001), lymphatic permeation (P = 0.020), and nuclear MDC1 (P = 0.005) remained significant risk predictors. In multivariate survival analysis, nuclear MDC1 (P = 0.027) entered at step 2 after presence of metastatic lymph node (P = 0.002) at step 1 for predicting reduced RFS. In relation to treatment, OSCC patients exhibiting weak expression of nuclear MDC1 protein were benefited significantly when treated with surgery followed by radiation therapy (P = 0.001). CONCLUSION: Thus, this study showed that MDC1 protein expression could be used as a prognostic marker in predicting relapse-free survival in patients with OSCC. OSCC patients expressing weak MDC1 protein could be benefited by adjuvant radiotherapy instead chemo-radiotherapy.

Evaluation of the p-AKT, p-JNK and FoxO3a function in oral epithelial dysplasia.

OBJECTIVES: To evaluate the expression of p-AKT, p-JNK, FoxO3a, and Ki-67 in samples of oral squamous cell carcinoma (OSCC) and oral epithelial dysplasias (OEDs) to understand their possible involvement in the malignant transformation process of oral lesions. MATERIALS AND METHODS: Tissue samples of 20 cases of OSCCs, 20 OEDs, and normal oral mucosa were subjected to immunohistochemistry reactions for anti-p-AKT, anti-p-JNK, anti-FoxO3a, and anti-Ki-67 antibodies. It was analyzed using quantitative (number of immunostained cells) and qualitative (immunostaining intensity) parameters in different cell immunostaining sublocations. RESULTS: Nuclear p-AKT was observed significantly greater immunostaining in OSCC (21.2 ± 19.0) than in dysplasias (7.9 ± 8.1) and controls (1.8 ± 4.7) (P = 0.002). Immunostaining of strong nuclear p-JNK was greater in controls (48.3 ± 13.7) than in OEDs (11.0 ± 10.3) and OSCCs (1.1 ± 1.3) (P < 0.001). Strong nuclear immunostaining of FoxO3a proved to be absent in OSCCs (0.0 ± 0.1) with little staining on dysplasias (3.2 ± 5.4) and increased expression in controls (13.5 ± 4.8) (P < 0.001). Immunostaining of strong nuclear Ki-67 was grater in OSCCs (48.1 ± 49.6) than in OED (11.8 ± 10.6) and controls (1.9 ± 2.0) (P < 0.001). CONCLUSIONS: Malignant process of OEDs in this research may involve the same mechanisms of established malignant lesions.

SATB1 promotes tumor metastasis and invasiveness in oral squamous cell carcinoma.

OBJECTIVE: Our aim is to evaluate the expression of SATB1 in human oral squamous cell carcinomas (OSCC) and its role in the invasiveness and metastasis of OSCC. SUBJECTS AND METHODS: A human OSCC tissue microarray was used to evaluate the expression pattern of SATB1. SATB1 mRNA knockdown was performed in human OSCC cell lines SCC25 and Cal27 to assess the function of SATB1 in the invasiveness and metastasis of OSCC. RESULTS: SATB1 is highly expressed in human OSCC determined by immunohistochemistry, and its nuclear/cytoplasmic ratio of histoscore is significantly correlated with patients' prognosis. Reduced cell motility, invasiveness, expression of epithelial to mesenchymal transition (EMT) markers (N-cadherin and ß-catenin), and elevated expression of epithelial markers were observed in SATB1-knockdown cells in in vitro studies. Depletion of SATB1 also restored a cobblestone-like morphology in TGF-ß1-treated cells. CONCLUSIONS: These findings suggest SATB1 may play an important role in OSCC invasiveness and metastasis.

Is There Evidence for the Presence and Relevance of the PD-1/PD-L1 Pathway in Oral Squamous Cell Carcinoma? Hints From an Immunohistochemical Study.

PURPOSE: To examine oral squamous cell carcinoma (OSCC) specimens for programmed death ligand-1 (PD-L1) expression and presence of programmed death-1 (PD-1)-positive tumor-infiltrating lymphocytes (TILs) and to determine possible clinicopathologic implications. It was hypothesized that PD-L1 expression and PD-1-positive TIL presence in OSCC would have no clinical relevance. MATERIALS AND METHODS: The authors implemented a retrospective cohort study design. The study cohort was chosen in compliance with predefined inclusion criteria. Demographic, clinical, and histopathologic data were gathered. Tissue microarrays were obtained from paraffin-embedded OSCC specimens and analyzed immunohistochemically for PD-L1 expression and PD-1-positive TIL infiltration. PD-L1 positivity of OSCC specimens served as the predictor variable and neck node metastasis served as the primary outcome variable. Descriptive and inferential statistics were computed and the significance level was set at a P value less than or equal to .05. RESULTS: The study sample was composed of 88 patients (48 men, 40 women; mean age, 61.34 yr). Marked PD-L1 expression was detected in 29% of OSCC specimens (26 of 88) and 83% of specimens (73 of 88) exhibited a high rate of PD-1-positive TIL infiltration. PD-L1 positivity of OSCC samples was significantly associated with the anatomic origin of OSCC (P = .039), presence of cervical metastasis (P = .039), and high PD-L1-positive TIL infiltration (P = .033). CONCLUSION: A considerable proportion of OSCCs exhibited marked PD-L1 expression. This could be associated with clinical parameters. PD-L1 expression in OSCC might differ depending on its anatomic origin. PD-1-positive TILs could be detected in most OSCC specimens. These findings might indicate a potential role for the PD-1 and PD-L1 pathway in OSCC.

Detection of Glutathione in Oral Squamous Cell Carcinoma Cells With a Fluorescent Probe During the Course of Oxidative Stress and Apoptosis.

PURPOSE: To detect glutathione (GSH) in oral squamous carcinoma cells (OSCCs) with a GSH selective fluorescent probe during the course of oxidative stress and apoptosis. MATERIALS AND METHODS: A novel GSH probe was applied to assess GSH in human tongue squamous cell carcinoma cells (cal-27). The cellular GSH and reactive oxygen species (ROS) levels were assessed with a GSH probe and DCF-DA (2,7-dichlorofluorescin diacetate) probe. The mitochondrial GSH and ROS levels were assessed with a GSH probe, DCF-DA probe, and Mitotracker Red CM-H2XRos probe (Invitrogen, Carlsbad, CA). To further study whether oxidative stress would induce apoptosis of OSCCs, we then applied a GSH probe and annexin V-fluorescein isothiocyanate probe to assess cellular GSH levels and eversion of phosphatidylserine, and the cellular GSH levels and mitochondrial membrane potential (ΔΨm) were assessed with a GSH probe and JC-1 probe during the course of oxidative stress and apoptosis induced by hydrogen peroxide and ethacrynic acid. The fluorescence was observed under laser confocal fluorescence microscopy. RESULTS: The intensity of fluorescence that represented intracellular alteration of GSH levels, cellular ROS formation, mitochondrial ROS formation, and apoptosis occurrence, respectively, could be visualized under laser confocal fluorescence microscopy. CONCLUSIONS: The GSH selective fluorescent probe can evaluate cellular GSH levels sensitively during the course of oxidative stress and apoptosis of OSCCs induced by exogenous hydrogen peroxide, which could be enhanced by depletion of mitochondrial GSH.