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.

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.

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.

Bone scan index of the jaw: a new approach for evaluating early-stage anti-resorptive agents-related osteonecrosis.

OBJECTIVE: A computer-aided diagnosis of bone scintigraphy using a bone scan index (BSI) has not been applied to a diagnosis of anti-resorptive agents-related osteonecrosis of the jaw (ARONJ). The aim of this study was to validate a diagnostic ability of BSI for early-stage ARONJ. METHODS: A total of 44 cancer patients treated with anti-resorptive drugs were evaluated retrospectively. All patients underwent bone scintigraphy and the tracer uptakes were analyzed by BSI. The software BONENAVI (FUJIFILM RI Pharma; EXINIbone, EXINI Diagnostics) could automatically detect abnormal intensities and calculate each regional BSI (rBSI). Among the rBSIs, the largest one in the jaw was manually selected and defined as maximum BSI of the jaw (BSIJmax). Uptake ratio (UR) between the maximum jaw count-to-average forehead count was also calculated. Screening accuracy of ARONJ based on 2 parameters was compared. Receiver operating characteristic analysis and Fisher's exact test were performed. RESULTS: The BSIJmax was significantly higher in patients who developed ARONJ than in those who did not, 3 months before the diagnosis of stage 2 ARONJ (p < 0.0001 and p = 0.02 in the maxilla and mandible, respectively). Using the cutoff values of 0.09% in the maxilla and 0.06% in the mandible, BSIJmax for predicting stage 2 ARONJ showed sensitivity and specificity of 88 and 96%, respectively, in the maxilla and 64 and 89%, respectively, in the mandible at 3 months before the diagnosis. The BSIJmax >0.09% and BSIJmax >0.06% in the maxilla and mandible, respectively, were much more frequently observed in patients who subsequently developed stage 2 ARONJ 3 months after the bone scintigraphy than in those who did not (p < 0.0001 and odds ratio = 182 in the maxilla and p < 0.005 and odds ratio = 14 in the mandible). The UR showed comparable diagnostic ability. CONCLUSION: The BSIJ provided a new index for evaluating ARONJ. For predicting occurrence of ARONJ, the thresholds of BSIJmax = 0.09 and 0.06% in the maxilla and mandible, respectively, may be used in patients treated with anti-resorptive drugs, and a differential diagnosis including ARONJ is recommended.

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.

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.

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.

Inhibitory effect of neoadjuvant chemotherapy on metastasis of oral squamous cell carcinoma in a mouse model.

The presence or absence of metastasis bears an important influence on the prognosis of head and neck cancer patients. Neoadjuvant chemotherapy has become widely employed as an initial treatment. However, the actual effectiveness of neoadjuvant chemotherapy on metastasis is still unestablished. Therefore, using an orthotopic implantation model in which cervical lymph node metastasis of oral squamous cell carcinoma can be reproduced, we investigated the inhibitory effect of neoadjuvant chemotherapy on metastasis. A highly invasive and metastatic human oral squamous cell carcinoma cell line, OSC-19 cells, was implanted into the tongues of nude mice. After implantation, the mice were divided into four groups: S (surgery), C+S (preoperative chemotherapy+surgery), S+C (surgery+postoperative chemotherapy), and a control (nontreatment) groups. The treatment (tumor resection or chemotherapy) was started 7 days postimplantation. The effects of each treatment on cervical lymph node metastasis were investigated by examining the rate of lymph node metastasis formation at 28 days postimplantation. In the control group, five of the 11 mice died of cachexia before the end of the experiment. However, all mice in the S, C+S, and S+C groups survived until 28 days after implantation. The cervical lymph node metastasis rates were 81.8% in S, 18.1% in C+S, 63.6% in S+C, and 100% in control groups. Thus, metastasis to the cervical lymph node was markedly inhibited by the combination of neoadjuvant chemotherapy and tumor resection. The findings of this study indicate that neoadjuvant chemotherapy is effective for inhibiting metastasis, and that it is necessary to begin chemotherapy as early as possible to achieve an inhibitory effect on metastasis. Considering these effects, if anticancer drugs are used, better therapeutic results can be expected.

Integrin expression levels correlate with invasion, metastasis and prognosis of oral squamous cell carcinoma.

The present study evaluated the relationship between alpha 3, alpha 6A, and beta 1 integrin expression in cancer cells at the invasive front of oral squamous cell carcinoma (OSCC) and survival rates, as well as the clinical and pathological characteristics. Sections of 100 specimens of primary OSCC were immunostained to assess alpha 3, alpha 6A, and beta 1 integrin expression in cancer cells at the invasive front. OSCC patients with higher expression levels of alpha 3, alpha 6A, and beta 1 integrin had significantly better prognosis than those with lower expression levels (median survival at low vs. high expression levels: alpha 3, 37.1 months vs. 55.7 months; alpha 6A , 38.3 months vs. 47.9 months; and beta 1, 26.1 months vs. 46.1 months) (P < 0.05). In addition, beta 1 integrin expression showed the highest correlation with clinical and pathological characteristics. This study concludes that alpha 3, alpha 6A, and beta 1 integrin expression in cancer cells at the invasive front are related to the mode of invasion and prognosis in OSCC.

Predictive value of measuring p53 labeling index at the invasive front of oral squamous cell carcinomas.

Many studies have revealed the frequency of p53 abnormalities in oral cancer. However, it reports only on the relation between clinicopathological findings and p53 expression, and there is no study to examine the relation to the p53 labeling index (p53-LI). The purposes of this study were to examine the correlation between p53 labeling index (p53-LI) at the invasive front of oral squamous cell carcinomas (OSCC) and clinicopathological findings by immunohistochemical staining, and to evaluate clinical significance of measuring p53-LI at the invasive front of OSCC. Sixty-six biopsy specimens of OSCC were randomly selected. Patient age, gender, primary sites, T category, N category, degree of differentiation and mode of cancer invasion were analyzed. p53 expression did not correlate significantly with the clinical findings. However, significant differences were found between p53-LI and the degree of cell differentiation (p < 0.05). The p53-LI of high-grade invasive tumors was significantly larger than that of low-grade invasive tumors (p < 0.05). The overall survival rate (OS) among low-scoring p53-LI cases was 75.5% whereas that for high-scoring p53-LI cases was 40.6%. The disease-free survival rate (DFS) among low-scoring p53-LI cases was 39.5% whereas that for high-scoring p53-LI cases was 76.1%. Patients with low-scoring p53-LI had a significantly worse prognosis than those with among high-scoring p53-LI (p < 0.05). Consequently, the measurement of p53-LI at the invasive front of OSCC is significant as one of the indicators of prognosis.

Correlation of basic fibroblast growth factor expression with the invasion and the prognosis of oral squamous cell carcinoma.

BACKGROUND: The aim of this study was to evaluate the relationship between the expression of basic fibroblast growth factor (bFGF) and fibroblast growth factor receptor-1 (FGFR-1) in cancer cells and fibroblasts at the invasive front of oral squamous cell carcinoma (OSCC), and the pathologic and clinical characteristics. METHODS: Sections of 61 biopsy specimens of primary OSCC were immunostained to assess the expression of bFGF and FGFR-1 in cancer cells and fibroblasts at the invasive front. RESULTS: The bFGF and FGFR-1 expressions in the cancer cells were evident in all specimens, whilst, in fibroblasts, they were detected in 41 (67%) of 61 specimens. These expressions in the fibroblasts occurred notably more often in high-invasive OSCC specimens than low-invasive OSCC specimens. The prevalence of bFGF and FGFR-1 expressions in cases with lymph node metastasis was significantly higher (P < 0.05) than in cases without metastasis. Moreover, these expressions were well correlated with patient prognosis. CONCLUSION: This study concludes that bFGF and FGFR-1 expressions in fibroblasts at the invasive front are linked to the mode of invasion and the prognosis in OSCC.

Influences of angiogenesis and lymphangiogenesis on cancerous invasion in experimentally induced tongue carcinoma.

BACKGROUND: Although it is clear that dissemination via the blood system involves angiogenesis, it is uncertain whether tumors also induce lymphangiogenesis or simply invade existing peritumoral vessels. The purpose of this study was to elucidate changes in tumor blood and lymph vessels in cases involving the invasion of squamous cell carcinoma in the oral cavity, and its significance. Blood and lymph vessels densities in tongue carcinomas induced in hamsters were investigated. METHODS: Tongue cancer was induced by abrading the right margin of the tongue of each hamster with an endodontic barbed broach and subsequently applying 1.0% 9,10-dimenthl-1,2-benzanthracene (DMBA) dissolved in acetone, three times a week, at the same site. Fresh frozen sections were prepared and blood vessels stained blue by perfusion with Coomassie Brilliant Blue and lymph vessels stained brown for 5'-nucleotidase. The effects on the blood vessels and lymph vessels were observed. RESULTS: The results showed that blood and lymph vessel densities were greater in the advanced carcinoma tissues than in normal tissue. These were compared in terms of the mode of cancer invasion. As tumor invasion progressed, the blood vessel density decreased but lymph vessel density tended to be higher in high-degree tumor invasion than in low-degree tumor invasion. The expression of vascular endothelial growth factor-C was seen more frequently as tumor invasion progressed. CONCLUSIONS: The present findings indicated that angiogenesis and lymphangiogenesis are affected by cancerous invasion.

Effects of fibroblast growth inhibitor on proliferation and metastasis of oral squamous cell carcinoma.

Development of a new therapeutic approach to improve the prognosis of high grade invasion of oral squamous cell carcinoma is needed. To elucidate the effect of a fibroblast inhibitor (tranilast), we investigated the proliferation and metastasis of oral squamous cell carcinoma in a mouse model. The effect of tranilast on tumour growth, lymph node metastases, microvessel density, and the proliferating cell nuclear antigen (PCNA) labelling index of oral squamous cell carcinoma implanted into the tongue of nude mice was evaluated. Tumour growth and the incidence of cervical lymph node metastases were significantly suppressed by the administration of tranilast. The amount of fibrous tissue, the microvessel density, and the PCNA labelling index of tumour were also significantly reduced. Administration of a fibroblast inhibitor may well be clinically effective for the treatment of oral squamous cell carcinoma.