Precise analysis of single small extracellular vesicles using flow cytometry.

Methods that enable specific and sensitive quantification of small extracellular vesicles (sEVs) using flow cytometry are still under development. Aggregation or adsorption of antibodies causes sub-nano sized particles or non-specific binding and largely affects the results of flow cytometric analysis of single sEVs. Comparison of control IgG and target-specific IgG is inappropriate because they have different characters. Here, we evaluate four preparation methods for flow cytometry, including ultracentrifugation, density gradient centrifugation, size exclusion chromatography (SEC), and the TIM4-affinity method by using tetraspanin-deficient sEVs. The ultracentrifugation or density gradient centrifugation preparation method has large false-positive rates for tetraspanin staining. Conversely, preparation methods using SEC or the TIM4-affinity method show specific detection of single sEVs, which elucidate the roles of sEV biogenesis regulators in the generation of sEV subpopulations. The methods are also useful for the detection of rare disease-related markers, such as PD-L1. Flow cytometric analysis using SEC or the TIM4-affinity method could accelerate research into sEV biogenesis and the development of sEV-based diagnostics and therapies.

The pattern and spread of invasion can predict late cervical lymph node metastasis in early tongue squamous cell carcinoma.

To determine the predictive indexes of late cervical lymph node metastasis in early tongue squamous cell carcinoma (TSCC). We retrospectively analyzed the cases of 25 patients with stage I/II TSCC who had undergone surgical treatment without elective neck dissection. We evaluated the relationships between clinicopathologic factors and the occurrence of late cervical lymph node metastasis. Of the 25 cases, metastasis to the cervical lymph nodes was observed in nine cases (36.0%). The clinicopathological factors associated with late cervical lymph node metastasis were the mode of invasion (MOI, p = 0.032), depth of invasion (DOI, p = 0.004), and perineural invasion (PNI, p = 0.040). A multivariate analysis revealed that only the DOI was an independent predictor of late cervical lymph node metastasis. The combination of the DOI and MOI or the PNI and MOI was significantly correlated with late cervical lymph node metastasis (p = 0.004 and p = 0.012, respectively). Our findings suggest that combinations of the MOI, DOI, and PNI could be used as an index for predicting late cervical lymph node metastasis in early TSCC.

Tumour: Fibroblast Interactions Promote Invadopodia-Mediated Migration and Invasion in Oral Squamous Cell Carcinoma.

Objectives: In the progression of cancer, interactions between cancer cells and cancer-associated fibroblasts (CAFs) play important roles. Cancer cell invasion is facilitated by filamentous actin (F-actin)-rich membrane protrusions called invadopodia, and the relationship between CAFs and invadopodia has been unclear. We used oral squamous cell carcinoma (OSCC) to investigate CAFs' effects on the formation of invadopodia, and we assessed the expressions of invadopodia markers and CAF markers ex vivo and their relationship with clinical parameters and survival. Materials and Methods: We examined the effect of culture with normal oral fibroblast (NOF)-derived and CAF-derived conditioned medium on the migration and invasion of two OSCC-derived cell lines using Transwells in the absence/presence of Matrigel. Immunoblotting and immunocytochemistry were conducted to assess the expressions of the invadopodia markers tyrosine kinase substrate 5 (Tks5) and membrane type 1 matrix metalloproteinase (MT1-MMP). We also used immunohistochemistry to examine patients with OSCC for an evaluation of the relationship between the CAF marker alpha smooth muscle actin (αSMA) and the expression of Tks5. The patients' survival was also assessed. Results: Compared to the use of culture medium alone, NOF-CM and CAF-CM both significantly increased the OSCC cells' migration and invasion (p < 0.05), and they significantly increased the expressions of both Tks5 and MT1-MMP. After the depletion of Tks5, the OSCC cells' migration and invasion abilities decreased. The expression of Tks5 and that of αSMA were correlated with poor survival, and a high expression of both markers was associated with an especially poor prognosis. Conclusions: These results indicate that the formation of invadopodia is (i) important for OSCC cells' migration and invasion and (ii) regulated by the interaction of OSCC cells and stromal fibroblasts.

Caveolin-1 Expression at Metastatic Lymph Nodes Predicts Unfavorable Outcome in Patients with Oral Squamous Cell Carcinoma.

We evaluated the clinical and prognostic value of the protein expression of caveolin-1 (CAV1) and p16 at the primary site and metastatic lymph nodes of oral squamous cell carcinoma (OSCC). Primary site specimens from 80 OSCC cases were randomly selected and lymph node specimens from 15 preserved metastatic lymph nodes from among those patients were selected for examination. We evaluated the CAV1 and p16 expression at both the primary site and metastatic lymph nodes, and analyzed the patients' clinicopathological data in relation to CAV1 and p16 expression. Our analysis revealed significant positive correlations between CAV1 expression at the primary site and pathological metastasis, cell differentiation, and mode of invasion (p = 0.019, p = 0.002, p = 0.015, respectively), but p16 expression was not associated with any clinicopathological factors. Patients with high CAV1 expression at the primary sites showed significantly worse prognoses than those with low or negative CAV1 expression (p = 0.002), and multivariate analysis showed that the T classification and CAV1 expression were independent OSCC prognostic factors. CAV1 expression was also present in the metastatic lymph nodes of the OSCC cases with particularly poor differentiation and high invasive grade, and patients with CAV1-positive metastatic lymph nodes showed significantly worse prognoses than those with CAV1-negative metastatic lymph nodes (p = 0.018). CAV1 may activate metastaticity and the invasive capacity of OSCC cells. CAV1 expression, particularly at metastatic lymph nodes, predicts a worse outcome for OSCC, suggesting that CAV1 could be used as a prognostic marker for OSCC.

Focal Adhesion Kinase (FAK) Overexpression and Phosphorylation in Oral Squamous Cell Carcinoma and their Clinicopathological Significance.

Focal adhesion kinase (FAK) is involved in progression of various cancers, and FAK overexpression has been associated with cancer invasion and metastasis. However, the involvement of FAK expression in the clinicopathological malignancy of oral squamous cell carcinoma (OSCC) remains unknown. In addition, there is no consensus regarding the role of p16 expression in OSCC. In this study, the immunohistochemically measured expression of FAK, phosphorylated FAK (FAKpY397) and p16 expressions and their associations with clinicopathological features and 5-year survival rates were examined in surgical samples from 70 patients with primary OSCC. FAK and FAKpY397 were expressed at high levels in 42 cases (60.0%) and 34 cases (48.6%), respectively, and 9 cases (12.9%) were positive for p16. FAK expression was significantly correlated with local recurrence, subsequent metastasis, and the mode of invasion. FAKpY397 expression was significantly correlated with both N classification and the mode of invasion. p16 expression was significantly correlated with clinical stage only. Patients having high expression of FAK, FAKpY397, or both showed significantly worse prognosis, but p16 expression showed no significant relation to prognosis. The results suggested that overexpression and phosphorylation of FAK in OSCC may affect cancer progression, such as local invasion and lymph node metastasis, and thereby contribute to life prognosis.

Detection of sentinel lymph node using contrast-enhanced agent, Sonazoid™, and evaluation of its metastasis with superb microvascular imaging in oral and oropharyngeal cancers: a preliminary clinical study.

BACKGROUND: In sentinel lymph node (SLN) biopsy for head and neck cancers, the radioisotope method has been the gold standard. However, this method has several problems, such as unavoidable radiation exposure and requirements of expensive equipment. AIMS/OBJECTIVES: To overcome these problems, we evaluated the contrast-enhanced ultrasonography (CEUS)-guided SLN-detection method, and predicted the SLN metastatic status using novel ultrasound technology, superb microvascular imaging (SMI). METHODS: Ten patients (6 with oral and 4 with oropharyngeal cancers) without neck lymph node metastasis were enrolled in this study. Ultrasound contrast agent, Sonazoid™, was infiltrated into the mucosa at the primary site to observe the lymphatic ducts and SLNs in the neck field. The detected SLNs were examined for blood flow using SMI to categorize the SLNs metastases-positive or negative. RESULTS: SLNs were successfully detected in 8 out of 10 cases. In 7 out of the 8 cases, in whom SLNs were successfully detected, the metastatic status of SLNs was correctly diagnosed with SMI. CONCLUSIONS AND SIGNIFICANCE: Although more clinical data are needed based on a larger cohort, establishing the CEUS-guided SLN-detection and criteria for the accurate diagnosis of SLN-metastases using SMI would be valuable as an alternative to radioisotope method, in oral and oropharyngeal cancers.

Clinicopathological Significance of the ET Axis in Human Oral Squamous Cell Carcinoma.

The interaction between cancer cells and the surrounding microenvironment in malignant tumor tissue is known to be closely associated with cancer cell invasion and proliferation. Endothelin (ET) present in the microenvironment surrounding tumors has been reported to play a role in cancer cell invasion and proliferation by binding to receptors on the cell membrane of cancer cells. Here, we immunohistologically detected the expression of ET-1 and its receptor ETAR in oral squamous cell carcinoma (OSCC) and evaluated the association between the expression of each as well as their co-expression (ET-axis expression) and clinicopathological factors. A significant difference was observed between the invasion pattern as a parameter of cancer cell malignancy and the expressions of ET-1 and ETAR. The survival rates were significantly lower among the patients who were strongly positive for ET-1 and the ETAR-positive patients compared to negative patients. There was also a significant difference between ET-axis expression and the degree of histological differentiation and mode of invasion, and the survival rate of the positive cases was significantly lower than that of the negative cases. Our findings suggested that ET-axis assessments are important for assessing the malignancy of cancer cells and predicting the prognoses of OSCC patients.

MicroRNA-205-5p suppresses the invasiveness of oral squamous cell carcinoma by inhibiting TIMP­2 expression.

MicroRNAs (miRNAs or miRs) play important roles in carcinogenesis. The miRNA, miR-205-5p, has been reported to suppress the growth of various types of tumor; however, its functional contribution to oral squamous cell carcinoma (OSCC) is not yet clear. Thus, this study was conducted to determine the miRNA expression signatures in OSCC and to investigate the functional role of miR­205­5p in OSCC cells. We measured miR­205­5p expression by RT-qPCR, and examined the function of miR­205­5p by transfecting a miR­205­5p mimic or inhibitor into OSCC cells and measuring cell proliferation, migration and invasiveness. Genes targeted by miR­205­5p were identified using the TargetScan database and verified by western blot analysis, luciferase reporter assay and ELISA. We found that miR­205­5p was significantly downregulated in OSCC cell lines and tissue specimens. Following transfection of miR­205­5p mimic or inhibitor into the cancer cell lines, miR­205­5p overexpression significantly suppressed cancer cell migration and invasion. We further demonstrated that miR­205­5p directly targeted and regulated the tissue inhibitor of metalloproteinases­2 (TIMP­2) gene. The silencing of TIMP­2 suppressed cancer cell invasion and the activation of pro­matrix metalloproteinase­2 (pro­MMP­2). These results suggest that TIMP­2 promotes tumor progression, and that miR­205­5p directly regulates TIMP­2, thereby suppressing pro­MMP­2 activation and inhibiting OSCC cell invasiveness. Our data describing the pathways regulated by miR­205­5p provide new insight into the mechanisms responsible for OSCC development and metastasis.

Regulation of PD-L1 expression in a high-grade invasive human oral squamous cell carcinoma microenvironment.

Blockade of the programmed-death 1 receptor (PD-1)/programmed-death ligand (PD-L1) pathway efficiently reduces tumour growth and improves survival. Durable tumour regression with blockade of the PD-1/PD-L1 checkpoint has been demonstrated in recent clinical studies. Oral squamous cell carcinoma (OSCC) is highly immunosuppressive, and PD-L1 expression has been proposed as a potential mechanism responsible for this phenotype. Despite the fact that anti-PD-1 treatment can produce durable responses, such therapy appears to benefit only a subset of patients. Thus, it is important to understand the mechanisms underlying regulation of PD-L1 expression in the OSCC microenvironment. In this study, we showed that PD-L1 expression in high-grade invasive OSCC cell lines was lower than that in a low-grade invasive OSCC line and found a close correlation between PD-L1 expression and the epithelial-mesenchymal transition (EMT). PD-L1 expression was upregulated in macrophages and dendritic cells (DCs) in high-grade invasive human OSCC tissues or co-cultured with mesenchymal-phenotype OSCC cells in vitro. TLR4-inhibitory peptide successfully suppressed PD-L1 upregulation on macrophages and DCs co-cultured with mesenchymal-phenotype OSCC cells, suggesting that some EMT-induced tumour antigen is critical for PD-L1 induction on tumour-associated macrophages and DCs. Further studies are necessary to explore the impact of EMT on the tumour immune microenvironment and to identify potential biomarkers for selecting patients who might preferentially benefit from PD-1/PD-L1 blockade or immunotherapies more broadly.

Eribulin sensitizes oral squamous cell carcinoma cells to cetuximab via induction of mesenchymal-to-epithelial transition.

Inhibition of epidermal growth factor receptor (EGFR) signalling has emerged as a new treatment strategy for oral squamous cell carcinoma (OSCC). Previously, we found that loss of EGFR expression in OSCC was associated with epithelial-mesenchymal transition (EMT), and may have functional implications with regard to resistance to cetuximab, a monoclonal anti-EGFR antibody. Eribulin (a microtubule inhibitor) reportedly renders breast cancer less aggressive, and less likely to metastasise, by triggering mesenchymal­to­epithelial (MET) transition. In the present study we evaluated whether eribulin-induced MET was associated with re-sensitization of resistant OSCC cell lines to cetuximab. In vitro antiproliferative activities were determined in three human OSCC lines (OSC-20, OSC-19 and HOC313) treated with eribulin. These three human OSCC represented different EMT/MET states. Interestingly, HOC313 cells (mesenchymal phenotype) were highly sensitive to eribulin in comparison with other cell lines, and significantly enhanced the anti-proliferative effect of cetuximab in response to the drug. Eribulin also underwent a MET-associated gene switch that resulted in morphological changes and high EGFR expression in HOC313 cells, and abrogated a TGF-ß-induced EMT gene expression signature. Eribulin-dependent sensitization of OSCC to cetuximab is likely due to induction of MET. Combination therapies based on eribulin and cetuximab have potential as a novel treatment regimen in OSCC.

Loss of epidermal growth factor receptor expression in oral squamous cell carcinoma is associated with invasiveness and epithelial-mesenchymal transition.

Inhibition of epidermal growth factor receptor (EGFR) signaling has emerged as a novel therapeutic strategy for the treatment of oral squamous cell carcinoma (OSCC). The EGFR-directed inhibitor cetuximab is currently the only approved targeted therapy for the treatment of OSCC. EGFR status may affect the patient response to cetuximab treatment. In the present study, via analysis of the immunomarker for EGFR, it was revealed that 58.3% of the total cases investigated stained positively for EGFR expression, and furthermore, that invasiveness was inversely correlated with EGFR expression. Expression levels of EGFR were quantified, and the correlation between EGFR expression and cetuximab sensitivity was investigated using three varying grades of invasive human OSCC line. EGFR expression in high-grade invasive cells was significantly downregulated compared with that of low-grade invasive cells. There was no significant antiproliferative effect in the high-grade invasive cells treated with various concentrations of cetuximab. The EMT-associated genes, N-cadherin, vimentin and Snail, were upregulated in the high-grade invasive cells. The low-grade invasive cells exhibited characteristics of typical epithelial cells, including the expression of E-cadherin and absence of the expression of N-cadherin, vimentin and Snail. Transforming growth factor-ß induced low-grade invasive cells to undergo an epithelial-mesenchymal transition (EMT)-associated gene switch, which resulted in low levels of EGFR expression. The results of the present study suggested that loss of EGFR expression in OSCC was associated with EMT, and may have functional implications with regard to tumor invasiveness and the resistance to cetuximab treatment.

Prognostic value of vascular endothelial growth factors A and C in oral squamous cell carcinoma.

BACKGROUND: Vascular endothelial growth factor (VEGF) family members play a major role in angiogenesis and vascularization. VEGF-A promotes tumor angiogenesis by stimulating the growth of tumor vascular endothelial cells. In addition, VEGF-C has been identified as a potent inducer of lymphangiogenesis in tumor and lymph node metastasis. Previous studies have investigated the association between clinicopathological factors and the expression of VEGF-A and VEGF-C in oral squamous cell carcinoma cancer (OSCC), but the results are contradictory. In this study, we investigated the relationship between VEGF-A and VEGF-C expression and OSCC clinicopathological factors and prognosis. METHODS: Expression of VEGF-A and VEGF-C was evaluated in surgical specimens from 61 patients with OSCC and three human oral cancer cell lines (OSC-19, OSC-20 and HOC313) by immunohistochemical staining and enzyme-linked immunosorbent assay, respectively. We also determined the relationship between the 5-year survival rate and clinicopathological factors, such as TNM classification (Union for International Cancer Control, UICC), lymph node metastasis, recurrence, histological differentiation, location, and mode of invasion. RESULTS: VEGF-A expression correlated significantly with lymph node metastasis. VEGF-C expression was associated with lymph node metastasis, recurrence, and a poorer 5-year survival rate. A multivariate analysis demonstrated that VEGF-C is an independent prognostic factor for patients with OSCC. VEGF-C expression was significantly up-regulated in HOC313 cells compared to OSC-19 and OSC-20 cells. CONCLUSIONS: These results indicate that VEGF-C may be a predictive factor for OSCC outcome, lymph node metastasis, and recurrence. Moreover, VEGF-C may be an important factor in the development of new therapies for OSCC patients.

Loss of claudin-7 is a negative prognostic factor for invasion and metastasis in oral squamous cell carcinoma.

Claudin-7 belongs to the claudin family, which consists of 24 subtypes of essential tight junction (TJ) integral membrane proteins with molecular weights of 20-27 kDa. We investigated the interrelationship between clinicopathological findings and claudin-7 expression in oral squamous cell carcinoma (OSCC). Using immunohistochemical techniques to examine the expression levels of claudin-7 in 67 cases of OSCC, claudin-7 expression was detected in 35 (52.2%) of the 67 cases. We also compared the clinicopathological features of the OSCC cases with claudin-7 expression levels. Moreover, six cell lines with various invasive properties were investigated in vitro to compare mRNA and protein levels of claudin-7 using reverse transcription-polymerase chain reaction (RT-PCR) and the western blotting method. Decreased claudin-7 expression correlated significantly with T-category (p<0.05), lymph node metastasis (p<0.01), and mode of invasion (p<0.001). Patients with positive claudin-7 expression had a significantly better prognosis (p<0.05). Claudin-7 protein and mRNA levels were lower in the HOC313 and TSU cells, which have higher invasive potentials compared with other cell lines. These results suggest that loss of claudin-7 expression is associated closely with invasion and lymph metastasis and is an unfavorable prognostic factor in patients with OSCC.

A hypothesis on the desired postoperative position of the condyle in orthognathic surgery: a review.

It is very important to clarify the relationship between a dentofacial structure and a temporomandibular joint (TMJ) structure in orthognathic surgery. Recently, it was reported that the skeletal and occlusal patterns were associated with the TMJ morphology, including the disk position. In orthognathic surgery, some surgeons state that alterations in the condylar position from surgery can lead to malocclusion associated with the risk of early relapse, and also favor the development of temporomandibular disorders. For these reasons, several positioning devices have been proposed and applied, but now there is no scientific evidence to support the use of condylar positioning devices. There are some reasons why scientific evidence cannot be obtained; however, it also includes the question of whether the preoperative position of the condyle is the desired postoperative position. The purpose of this study was to verify the desired condylar position in orthognathic surgery, based on literature on the postoperative condylar position in orthognathic surgery. From the studies reviewed, it was suggested that the preoperative position of the condyle was not the desired postoperative position in orthognathic surgery.

Changes in temporomandibular joint and ramus after sagittal split ramus osteotomy in mandibular prognathism patients with and without asymmetry.

The purpose of this study was to examine the changes in the temporomandibular joint (TMJ) and ramus after sagittal split ramus osteotomy (SSRO) with and without Le Fort I osteotomy. The subjects consisted of 87 Japanese patients diagnosed with mandibular prognathism with and without asymmetry. They were divided into 2 groups (42 symmetric patients and 45 asymmetric patients). The TMJ disc tissue was assessed by magnetic resonance imaging (MRI) and the TMJ space, condylar and ramus angle were assessed by computed tomography (CT) preoperatively and postoperatively. Medial joint space on the deviation side in the asymmetry group was significantly larger than that in the symmetry group (P = 0.0043), and coronal ramus angle on the non-deviation side in the asymmetry group was significantly larger than that in the symmetry group preoperatively (P = 0.0240). The horizontal condylar angle on the deviation side in the asymmetry group was significantly larger than that in the symmetry group (P = 0.0302), posterior joint space on the non-deviation side in the symmetry group was significantly larger than that in the asymmetry group postoperatively (P = 0.00391). The postoperative anterior joint space was significantly larger than the preoperative value on both sides in both groups (the deviation side in the symmetry group: P = 0.0016, the non-deviation side in the symmetry group: P < 0.0001, the deviation side in the asymmetry group: P = 0.0040, the non-deviation side in the asymmetry group: P = 0.0024). The preoperative disc position could was not changed in either group. These results suggest that significant expansion of anterior joint space could occur on the deviation side and non-deviation side in the asymmetry group as well as on both sides in the symmetry group, although disc position did not change in either group.

Expression of urokinase-type plasminogen activator/urokinase-type plasminogen activator receptor and maspin in oral squamous cell carcinoma: Association with mode of invasion and clinicopathological factors.

It is well documented that the binding of urokinase-type plasminogen activator (uPA) to its receptor (uPAR), which has been implicated in cancer invasion and metastasis, is regulated by several inhibitors such as maspin. In this study, we investigated the interrelationship between clinicopathologic findings and expression of uPA, uPAR and maspin in oral squamous cell carcinoma (OSCC) to elucidate the participation of maspin in the uPA/uPAR system in the malignant behavior of OSCC. Using immunohistochemical techniques to examine the expression levels of uPA, uPAR and maspin in 54 cases of OSCC, we also compared the clinicopathologic features of OSCC with the expression levels of each. Moreover, we examined the expression of uPA, uPAR and maspin in six cell lines derived from OSCC using reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. uPA and uPAR showed a positive correlation with the mode of cancer invasion; conversely maspin showed a negative correlation with the mode of invasion. Multivariate analysis revealed that only two factors (N-category and uPA+/uPAR+/maspin- expression pattern) were significant and independent variables with relative risks of 3.84 and 2.52, respectively. In particular, tumors exhibiting an expression pattern of uPA+/uPAR+/maspin- were highly malignant and were associated with the worst survival rate (5-year overall survival rate, 29.4%), while tumors with an expression pattern, uPA-/uPAR-/Μaspin+, showed the most favorable survival rate (5-year overall survival rate, 77.8%). In vitro, lower expression of maspin was also noted in the cell lines derived from grade 4D OSCC, which exhibited a stronger invasive potential than the cells lines derived from the other grades of OSCC, while uPA and uPAR demonstrated an expression trend opposite to maspin. These results indicate that uPA, uPAR and maspin expression patterns may be useful markers for evaluating the clinical course or prognosis of OSCC patients.

Expression form of p53 and PCNA at the invasive front in oral squamous cell carcinoma: correlation with clinicopathological features and prognosis.

BACKGROUND: Abnormalities in cell-cycle-controlling genes are important in the malignant transformation and proliferation of tumors. Among these genes, the tumor suppressor gene p53 is the most notable, and its mutations provide an indicator of tumor progression and prognosis. Proliferating cell nuclear antigen (PCNA) is a highly conserved nuclear protein that is expressed during cell replication and DNA repair. This study examined the expression of p53 and PCNA at the invasive front of oral squamous cell carcinomas (OSCC) by immunohistochemical staining, and investigated the relationship of these proteins to clinicopathological findings and prognosis. METHODS: Fifty-nine biopsy cases of OSCC were examined by immunohistochemical staining. Clinicopathological data were gathered and patient survival was analyzed. RESULTS: The p53 labeling index (p53-LI) and PCNA labeling index (PCNA-LI) were examined at the invasive front of the tumors. A high p53-LI (p53+) was observed in 17 of the 59 cases (28.8%) and a high PCNA-LI (PCNA+) was observed in 28 of the 59 cases (47.5%). Among the modes of cancer invasion, many of the p53+/PCNA+ cases could be confirmed as highly invasive cancer (P < 0.05). In addition, the p53+/PCNA+ cases showed a high risk of tumor recurrence compared with the other expression forms, and patients with p53+/PCNA+ had a worse prognosis than those with the other expression forms. High labeling indices of p53 and PCNA are associated with poor prognosis in patients with OSCC. CONCLUSION: We suggest that it is important to investigate the expression of p53 and PCNA at the invasive front of OSCC.

Epigenetic loss of mucosa-associated lymphoid tissue 1 expression in patients with oral carcinomas.

Mucosa-associated lymphoid tissue 1 (MALT1), which is located in a genomic region that encodes unknown tumor suppressor gene(s), activates nuclear factor-kappaB in lymphocyte lineages. However, its expression and role in the pathology of malignant tumors of epithelial origin is not known. In the present study, we examined MALT1 expression and its implications for the pathology of oral carcinomas. Immunostaining localized MALT1 in the nucleus of normal oral epithelial cells, but the expression was absent in 45.0% of carcinomas (49 of 109 cases) especially at the invasive front. The loss of expression was correlated with tumor recurrence (P = 0.007) and poor patient survival (P < 0.001), and it was an independent prognostic determinant (P < 0.001). MALT1-negative carcinomas exhibited microsatellite instability at the MALT1 locus and a specific cytosine methylation positioned at -256 from the gene, and the expression was recovered by demethylation treatment. In contrast to lymphocyte lineages, carcinoma cells showed MALT1 located at the nucleus independent of its domain structures, and its loss of expression induced the epithelial-mesenchymal transition. These results show that MALT1 is expressed in the nucleus of oral epithelial cells and that its expression is epigenetically inactivated during tumor progression, suggesting that the detection of MALT1 expression is a useful predictive and prognostic determinant in the clinical management of oral carcinomas.

Loss of maspin is a negative prognostic factor for invasion and metastasis in oral squamous cell carcinoma.

OBJECTIVE: Maspin, a 42-kDa protein, belongs to the serpin family of protease inhibitors and is known to have tumor-suppressor function. In this study, we investigated the interrelationship between clinicopathologic findings and maspin expression in oral squamous cell carcinoma (OSCC). METHODS: Using immunohistochemical techniques to examine the expression levels of maspin in OSCC, maspin expression in OSCC was detected in 46 (64.8%) of 71 cases. We also compared the clonicopathologic features of OSCC cases with maspin expression levels. Moreover, we examined expression of maspin in eight cell lines derived from OSCC using reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting. RESULTS: There was a significant correlation between decreased maspin expression and T-category (P < 0.01), lymph metastasis (P < 0.0001), and mode of invasion (P < 0.0001). Patients with positive maspin expression had a significantly better prognosis (P < 0.001). Lower expression of maspin was also seen in cell lines derived from grade 4D, which shows stronger invasive potential than other grades of OSCC. CONCLUSION: Maspin may be a useful marker to identify the potential for progression in OSCC.

Significance of stromal desmoplasia and myofibroblast appearance at the invasive front in squamous cell carcinoma of the oral cavity.

BACKGROUND: Tumor invasion involves complex interactions between tumor and stromal cells. We examined the extent of connective tissue in the tumor stroma and whether myofibroblasts play a role in assisting cancer invasion and metastasis. METHODS: Biopsy materials from 84 patients with oral squamous cell carcinoma (SCC) were used. We compared data from intrastromal collagen fibers using Azan staining, immunohistochemical identification of myofibroblasts by cytoskeletal markers, alpha-smooth muscle actin, vimentin, desmin, and clinicopathological parameters. Clinical outcome was compared by 5-year survival rate. RESULTS: There were high levels of stromal collagen fibers in invasive tumors. Myofibroblast appearance increased with increasing tumor invasiveness. Lymph node metastasis occurred more frequently in the myofibroblast-positive group, and the survival rate was significantly poorer in this group. CONCLUSIONS: Fibrous stroma in SCC appeared to have a desmoplastic response. However, an independent invasive mechanism may regulate the stroma, with tumor desmoplasia occurring in highly developed, invasive tumors.