Depth of invasion of oral squamous cell carcinoma in Nos2-knockout mice correlated to alterations in systemic inflammatory markers following 4NQO treatment.

BACKGROUND: Peripheral blood analysis is a non-invasive and low-cost technique of prognostic value for several diseases, including oral cancer. Considering the role of inducible nitric oxide synthase in tumor-associated inflammation, this study purposed to evaluate the influence of this enzyme on peripheral blood parameters and systemic inflammatory biomarkers during murine oral carcinogenesis. METHODS: A 50 µg/mL solution of 4-nitroquinoleine-N-oxide was provided to 15 C57BL/6J (Nos2+/+ ) and 16 B6.129P2-Nos2tm1Lau /J (Nos2-/- ) for 16 weeks. Animals were followed for 8 weeks after treatment. Blood samples and tongues were collected for hematological and histopathological analyses. Red blood cells, white blood cells, and platelet cell parameters were analyzed. The neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio, and the systemic immune-inflammation index were also calculated. The depth of invasion of all carcinomas was measured. RESULTS: Differences were found in several blood parameters. The depth of invasion in Nos2-/- was lower than in Nos2+/+ (p = 0.009), and strong correlations were found between depth of invasion and neutrophil count (ρ = -0.68, p = 0.017), lymphocyte count (ρ = 0.72, p = 0.011), neutrophil-to-lymphocyte ratio (ρ = -0.65, p = 0.025), platelet-to-lymphocyte ratio (ρ = -0.73, p = 0.013), and systemic immune-inflammation index (ρ = -0.67, p = 0.037) in Nos2-/- mice. CONCLUSION: Inducible nitric oxide synthase seems to have an important role in OSCC invasion and progression, which might be associated to alterations in immune-inflammatory cell dynamics evidenced by peripheral blood and systemic inflammatory biomarkers.

A retrospective study of 30 basal cell adenomas of the salivary gland in a Brazilian population and literature review.

PURPOSE: The present study aimed to provide the clinicopathological data of Brazilian patients with basal cell adenoma (BCA). METHODS: Records of BCA cases were retrospectively gathered from the Brazilian National Cancer Institute database between 1996 and 2006. All cases were histopathologically reviewed, and the clinicopathological data were collected from the patients' medical files. In addition, an English literature review about this tumor is also presented. RESULTS: Of 1127 salivary gland tumors identified, 30 were BCAs (2.7%). Women were more affected than men (70.0% vs. 30.0%), and the majority (60.0%) were elderly (> 65 years old). The parotid gland was the most frequent location affected (93.3%), followed by the upper lip (3.3%) and submandibular gland (3.3%). Fine-needle aspiration was the main procedure applied to establish a preoperative diagnosis of tumor; however, the results were not always consistent. Histopathologically, the trabecular pattern was the most common type seen (50.0%) among our BCA samples. Most patients underwent superficial or partial parotidectomy. Frey's syndrome was reported only in one case during the follow-up. No recurrence was noted in the present series. The literature review revealed a total of 213 reported cases of BCA in the period investigated. CONCLUSIONS: This is the first case series of BCA reported in Brazil. As occurred in other previously reported series, the clinicopathological data of BCAs are similar and confirm that this type of tumor is rare, develops predominantly in the parotid gland, frequently affects older women, has an indolent behavior, and the affected patients have an excellent prognosis.

The importance of inducible nitric oxide synthase and nitrotyrosine as prognostic markers for oral squamous cell carcinoma.

BACKGROUND: The prognosis of human cancer depends on the deregulations of many molecular patterns. In recent years, a great interest in the intracellular signaling mechanisms related to nitric oxide (NO)-induced carcinogenesis has appeared, as one of the most preeminent prognostic markers for many types of neoplasms. In this study, we identify the levels of iNOS and nitrotyrosine in the sample of normal oral mucosa (NOM), oral leukoplakia (OL), and oral squamous cell carcinoma (OSCC). METHODS: Quantitative polymerase chain reactions (qPCRs) were utilized to detect the NOS2 levels in fresh-frozen tissue samples of NOM (n = 6), OL (n = 20), and OSCC (n = 15). Moreover, the immunohistochemical method was used to examine the levels of iNOS and nitrotyrosine in 85 cases of OSCC (39 cases without metastases and 46 with metastases), 42 cases of OL, and 16 cases of NOM. RESULTS: There are rising tendencies in the iNOS mRNA and protein levels during human oral carcinogenesis. Similar findings were obtained in the nitrotyrosine staining. Furthermore, iNOS and nitrotyrosine immunostaining are associated with several clinical-pathological features of OSCC (site, presence of metastasis, staging, recidivism, and survival). CONCLUSIONS: The NO-signaling pathway plays a vital role in the development and progression of human oral dysplastic and neoplastic diseases. Nitrotyrosine was a significant marker for the discrimination of OSCC prognosis and survival.

Involvement of oral tissues by AL amyloidosis: a literature review and report of eight new cases.

OBJECTIVE: Amyloidosis is a term used to describe a group of diseases in which there is an extracellular deposition of amorphous fibrillar proteins known as amyloid. The aim of this study was to present clinicopathological data from eight oral amyloidosis-affected patients and a deep review of the literature about the disease. MATERIALS AND METHODS: A retrospective study was conducted based on the records of oral amyloidosis-affected patients diagnosed in our institution between 1978 and 2012. The clinicopathological features and immunohistochemical (IHC) staining with anti-kappa and anti-lambda light chain antibodies were carried out and analyzed. RESULTS: Eight patients were diagnosed with the disease; the tongue and women in their sixth decade of life were mostly affected. All lesions demonstrated apple-green birefringence and immunoreactivity for kappa-light chain, and four cases also showed lambda positivity. According to our series, four cases were diagnosed with localized amyloidosis and four with systemic amyloidosis. Prognosis for the systemic ones was gloomy, but good for the localized ones, which was characterized by a slow pattern of deposition without evolution to systemic involvement. CONCLUSIONS: This study reinforces our knowledge about predilections, outcomes, and the importance of making a correct and quick diagnosis of oral amyloidosis and shows the necessity of more studies detailing oral amyloidosis predilection on a global scale. The importance and utility of IHC in the typing of the biochemical nature of amyloid deposits are becoming increasingly necessary for proper management of the patient. Correct classification of the type of amyloid is important for treatment consequences. CLINICAL RELEVANCE: This article highlights the clinicopathological data of patients with amyloidosis affecting oral tissues and compare these new findings with other worldwide descriptions. Because of its rarity, such data are often unfamiliar to most clinicians and pathologists.

Assessment of BRAFV600E and SMOF412E mutations in epithelial odontogenic tumours.

The classification of ameloblastoma in multicystic or unicystic variants is associated with its clinical behaviour. Recently, BRAF and SMO mutations have been reported in ameloblastomas. However, it is not clear if such mutations are shared by the multi- and unicystic variants of ameloblastoma or by odontogenic carcinomas. We assessed BRAFV600E and SMOF412E in multicystic, unicystic and desmoplastic ameloblastomas. In addition, we investigated whether the BRAFV600E mutation occurs in odontogenic carcinomas. A total of 28 formalin-fixed paraffin-embedded samples, comprising 17 ameloblastomas and 11 odontogenic carcinomas, were included. The BRAFV600E mutation was assessed by real-time PCR with a specific TaqMan probe and confirmed by Sanger sequencing. The SMOF412E mutation was assessed by Sanger sequencing. Fourteen out of 17 (82 %) ameloblastomas showed the BRAFV600E mutation, specifically, 5/6 (83 %) unicystic, 7/9 (78 %) multicystic and 2/2 desmoplastic ameloblastomas. BRAFV600E mutation was detected in 4/11 (36 %) malignant tumours, specifically, 3/8 (38 %) ameloblastic carcinomas and 1/1 clear cell odontogenic carcinoma, while the two ghost cell odontogenic carcinomas did not harbour this mutation. The SMOF412E mutation was not detected in ameloblastoma. The BRAFV600E-activating mutation is a common event in ameloblastomas, occurring regardless of site or histological type. This mutation is also detected in odontogenic carcinomas. SMO somatic mutation is a secondary genetic event in the ameloblastoma pathogenesis. Our findings support the possibility for personalised, molecular-targeted therapy for ameloblastomas and odontogenic carcinomas harbouring the BRAFV600E mutation.

Hereditary gingival fibromatosis: clinical and ultrastructural features of a new family.

OBJECTIVE: This article describes the diagnosis, clinical and microscopic (histopathology and ultrastructural) features and treatment of a new family with hereditary gingival fibromatosis (HGF) and highlights the importance of this genetic condition. STUDY DESIGN: To characterize the pattern of inheritance and the clinical features, members of a new family with HGF were examined. The pedigree was reliably constructed including the four latest generations of family. Hematoxylin and eosin staining and ultrastructural analysis were performed with the gingival tissue. RESULTS: Examination of the family pedigree revealed that the patient III-2 represent the index patient of this family (initial patient with a mutation), which was transmitted to her daughter through an autosomal dominant mode of inheritance. The affected patients showed a generalized gingival overgrowth. The patient was treated with surgical procedures of gingivectomy and gingivoplasty. The diagnosis was confirmed by histopathology examination that showed a well-structured epithelium with elongated and thin papillae inserted in fibrous connective tissue with increased amount of collagen. The ultrastructural aspects of the tissue show collagen fibrils exhibiting their typically repeating banding pattern with some fibrils displaying loops at their end. Moreover, it was possible to seen in some regions fibrillar component presenting tortuous aspects and loss of the alignment among them. CONCLUSIONS: This HGF frequently resulted in both esthetic and functional problems. The genetic pattern of this Brazilian family suggested a new mutation, which was later transmitted by an autosomal dominant trait.

Hedgehog signaling pathway mediates tongue tumorigenesis in wild-type mice but not in Gal3-deficient mice.

Oral squamous cell carcinoma (OSCC) is one of the most aggressive cancers of the oral cavity and an important cause of death worldwide. Currently, there are limited clinical tools aiding clinicians to establish its early diagnosis, and genetic and epigenetic events leading to the pathogenesis of OSCC remain unsolved. The use of carcinogen-induced knocked out mouse models would help to improve its early detection and also determine the role of proteins such as galectin-3 (Gal3) in this process. Here we used a mouse model of oral carcinogenesis employing two mouse genotypes: wild-type (Gal3+/+) and galectin-3-deficient mice (Gal3-/-) challenged by the carcinogen 4NQO for 16 weeks. After induction, the expression of Wnt1, Wnt3A, Shh and Gli3 proteins in tongue samples was evaluated using an immunohistochemistry approach. All samples of dysplasia and carcinoma were negative for Wnt1. Wnt3A expression was detected in both Gal3+/+ and Gal3-/- mice, at similar levels. Wnt3A expression did not predict tongue tumorigenesis in either genotype. Dysplastic- and carcinoma-expressing Shh was statistically significantly higher in Gal3+/+ mice than Gal3-/- mice (p<0.0001), and was associated with tongue tumorigenesis only in the former. Gli3 expression decreased and increased from dysplasia to carcinoma in Gal3+/+ and Gal3-/- mice, respectively, although the difference was not significant. The results suggest that activated Wnt signaling is present in both mice, and that the Hh signaling pathway might play a role in tongue carcinoma development in Gal3+/+ mice.

OralEpitheliumDB: A Dataset for Oral Epithelial Dysplasia Image Segmentation and Classification.

Early diagnosis of potentially malignant disorders, such as oral epithelial dysplasia, is the most reliable way to prevent oral cancer. Computational algorithms have been used as an auxiliary tool to aid specialists in this process. Usually, experiments are performed on private data, making it difficult to reproduce the results. There are several public datasets of histological images, but studies focused on oral dysplasia images use inaccessible datasets. This prevents the improvement of algorithms aimed at this lesion. This study introduces an annotated public dataset of oral epithelial dysplasia tissue images. The dataset includes 456 images acquired from 30 mouse tongues. The images were categorized among the lesion grades, with nuclear structures manually marked by a trained specialist and validated by a pathologist. Also, experiments were carried out in order to illustrate the potential of the proposed dataset in classification and segmentation processes commonly explored in the literature. Convolutional neural network (CNN) models for semantic and instance segmentation were employed on the images, which were pre-processed with stain normalization methods. Then, the segmented and non-segmented images were classified with CNN architectures and machine learning algorithms. The data obtained through these processes is available in the dataset. The segmentation stage showed the F1-score value of 0.83, obtained with the U-Net model using the ResNet-50 as a backbone. At the classification stage, the most expressive result was achieved with the Random Forest method, with an accuracy value of 94.22%. The results show that the segmentation contributed to the classification results, but studies are needed for the improvement of these stages of automated diagnosis. The original, gold standard, normalized, and segmented images are publicly available and may be used for the improvement of clinical applications of CAD methods on oral epithelial dysplasia tissue images.

Adenoid cystic carcinoma of the salivary gland: a clinicopathological study of 49 cases and of metallothionein expression with regard to tumour behaviour.

AIMS: Adenoid cystic carcinoma (AdCC) of the salivary glands shows heterogeneous behaviour, with metastasis as a key indicator of poor prognosis. Metallothionein (MT) expression has been associated with poor prognosis of diverse neoplasms. We evaluated prognostic factors for AdCC and the role played by MT, focusing on metastatic behaviour. METHODS AND RESULTS: We reviewed the files of the Brazilian National Institute of Cancer between 1997 and 2004, obtaining 49 cases. Fourteen tumours had metastasized during follow-up. Among these, we identified cases presenting with metastasis at patient admission as showing the poorest survival rates. MT immunostaining of the tumours was performed (using the E9 antibody), and evaluated for the parameters of proportion, intensity and distribution in tumour cells. Extent and intensity of staining, and Quickscore (a combined measure of extent and intensity), were higher in metastatic than non-metastatic tumours (for Quickscore, P = 0.044), with highest values found for cases of early metastasis. Most cases showing weak staining, and all with a predominantly cytoplasm-restricted staining pattern, were non-metastatic. Metastatic tumours of solid type received higher scores than solid non-metastatic (Intensity, P = 0.0239; Quickscore, P = 0.0481). CONCLUSIONS: Our results demonstrated metastasis to be the most significant indicator of poor prognosis and deterioration for AdCC. Consistent patterns of MT expression were observed to correlate with metastatic behaviour, indicating that MT may potentially serve as a prognostic marker for AdCC.

Detection of cytokeratins in ghost cells of calcifying cystic odontogenic tumor indicates an altered keratinization and hair follicle differentiation for their development.

Calcifying cystic odontogenic tumors (CCOTs) are benign cystic lesions of odontogenic origin characterized by an ameloblastoma-like epithelium and the presence of a group of cells named ghost cells. The pattern of cytokeratin (Ck) expression on these lesions remains unclear and needs to be clarified. To this end, the expression of Ck6, Ck13, Ck14, Ck18, and Ck19 in the epithelium lining of 7 cases of CCOTs was evaluated by immunohistochemistry. For this, the epithelium lining was divided into 3 distinct regions: basal layer, suprabasal layer, and the compartment composed of ghost cells. In this study, 6 cases (85.7%) were classified as type 1 and 1 (14.3%) as type 4. All cases were negative for Ck13 and Ck18, despite the epithelial layer, as well as in the ghost cells. Ck6 was only positive in the ghost cells. Positivity for Ck14 and Ck19 was found in the basal and suprabasal layers, including the ghost cells. The results showing positivity for Ck14 and Ck19 in all of the analyzed cases reinforce CCOT as being of odontogenic origin, and the restricted expression of Ck6 in the ghost cells may be indicative that these cells suffer an altered differentiation into hair follicles in CCOTs.

Histopathological diagnosis in pediatric stomatology: A 43-year retrospective study of 1,480 cases from a Brazilian institution.

OBJECTIVES: the aim of this study was to analyze the prevalence of histopathological diagnoses in oral biopsied tissues obtained from a Brazilian pediatric population. METHODS: an analytical, cross-sectional retrospective study was performed with biopsy files of patients ≤14 years of age from a Brazilian oral pathology laboratory over a 43-year period. Data included sex, age, location, and diagnoses. The prevalence was calculated by means of relative frequency. Associations between sex, age groups and diagnoses were verified with Pearson's chi-square test. RESULTS: from 19,456 oral biopsies, 1480 (7.6%) were obtained from patients aged ≤14 years. Most children were 10-14 years of age (60.1%) and females (55.1%), with an overall M:F of 1:1.2. Children aged 0-9 years and males had a higher frequency of lesions of the oral mucosa, whilst the 10-14 year age group showed a higher frequency of cysts, odontogenic tumors, and salivary gland lesions. The latter was also significantly higher in females. Samples consisted mostly of soft tissue lesions (53%) obtained from the lower lip (30.7%). Intraosseous lesions showed a slight predilection for the mandible (21.2%). Salivary gland lesions (28.8%) was the most common diagnostic category, followed by reactive lesions (18.8%), and cysts (16.1%). Mucocele (33.5%), dentigerous cyst (6.7%), and fibrous hyperplasia (5.9%) were the top three histopathological diagnoses. Malignant lesions affected only 0.9% of this population. CONCLUSION: our results were similar to other retrospective studies. Due to the low frequency of oral biopsies in children, data on the prevalence of oral pathology in this population might aid in the clinical and histopathologic diagnoses.

Absence of galectin-3 does not affect the development of experimental tongue carcinomas in mice.

BACKGROUND: Galectin-3 is a lectin that presents pivotal roles in tumor biology and there are no studies evaluating their expression in dysplasias and carcinomas developed from tongue carcinogenesis models. AIMS: To investigate the role of galectin-3 in the development of tongue carcinomas using a mouse model of oral carcinogenesis. METHODS: Galectin-3-deficient (gal3(-/-)) and wild-type (gal3(+/+)) mice were challenged with 4-nitroquinoline-1-oxide in drinking water for 16weeks and killed at different times. Tongues were removed and the number of dysplasias and carcinomas was counted. An immunohistochemical study for galectin-3 was performed only in the tongue from gal3(+/+) mice. RESULTS: In both groups, a reduction of dysplasias and an increase of carcinomas from week 16 to week 32 (p>0.05) were observed. A predominance of high cytoplasmic and nuclear galectin-3 expression was observed in carcinomas (64.7%) and dysplasias (55.5%), respectively (p>0.05). The perilesional areas always presented a statistical cytoplasmic and nuclear galectin-3 overexpression. CONCLUSIONS: Absence of galectin-3 did not directly affect the process of carcinogenesis and a cytoplasm shift of galectin-3 seems to be associated with development of tongue carcinomas.

Vascular endothelial growth factor and thymidine phosphorylase expression in salivary gland tumors with distinct metastatic behavior.

BACKGROUND: Metastasis of salivary gland tumors has a negative impact on survival. Angiogenesis and its factors are potential markers for predicting metastasis in different malignant tumors, but this is not the case for salivary gland tumors. METHODS: Salivary gland tumors of distinct biologic behavior were analyzed according to the semiquantitative immunoexpression of vascular endothelial growth factor (VEGF) and thymidine phosphorylase (TP). RESULTS: Vascular endothelial growth factor expression was predominantly weak in benign tumors. Weak TP expression was observed in 100% cases of benign tumors and in 74.3% of primary malignant tumors. High VEGF and TP expression levels were significantly associated with primary malignant tumors but not with primary non-metastasizing and primary metastasizing malignant tumors or with subtypes of malignant tumors. CONCLUSIONS: Vascular endothelial growth factor and TP expression levels discriminate benign and malignant tumors but cannot predict metastasis from non-metastasizing tumors.

The role of the histones H3K9ac, H3K9me3, HP1γ, and H3K36me3 in oral squamous cell carcinoma loco-regional metastasis and relapse.

Molecular markers with unequivocal significance in predicting cervical lymph node metastasis of oral squamous cell carcinoma (OSCC) has not yet been identified. Histones are DNA-binding proteins that can regulate gene expression, and some studies have shown that such proteins are implicated with tumor development and progression. This study aimed to investigate the expression of some histone modifications in OSCC and their roles in cervical lymph node metastasis. To address this goal, H3K9ac, H3K9me3, HP1γ, and H3K36me3 expression levels were investigated immunohistochemically in a retrospective metastatic and non-metastatic OSCC samples. We analyzed the association between these markers with clinical-pathological data and survival rates. Hyperacetylation of H3K9ac was associated with cervical lymph node metastasis and local relapse. High expression levels of H3K9m3 were related to age and symptomatology. Furthermore, it was also found a statistically significant association between high HP1γ-expressing tumors and tumor size. However, no markers were associated with reduced overall survival rate. Our results suggest that covalent histone modifications contribute to OSCC behavior, and H3K9ac may play a critical role in OSCC-derived cervical lymph node metastasis.

Computational normalization of H&E-stained histological images: Progress, challenges and future potential.

Different types of cancer can be diagnosed with the analysis of histological samples stained with hematoxylin-eosin (H&E). Through this stain, it is possible to identify the architecture of tissue components and analyze cellular morphological aspects that are essential for cancer diagnosis. However, preparation and digitization of histological samples can lead to color variations that influence the performance of segmentation and classification algorithms in histological image analysis systems. Among the determinant factors of these color variations are different staining time, concentration and pH of the solutions, and the use of different digitization systems. This has motivated the development of normalization algorithms of histological images for their color adjustments. These methods are designed to guarantee that biological samples are not altered and artifacts are not introduced in the images, thus compromising the lesions diagnosis. In this context, normalization techniques are proposed to minimize color variations in histological images, and they are topics covered by important studies in the literature. In this proposal, it is presented a detailed study of the state of art of computational normalization of H&E-stained histological images, highlighting the main contributions and limitations of correlated works. Besides, the evaluation of normalization methods published in the literature are depicted and possible directions for new methods are described.

Color normalization of faded H&E-stained histological images using spectral matching.

Histological samples stained with hematoxylin-eosin (H&E) are commonly used by pathologists in cancer diagnoses. However, the preparation, digitization, and storage of tissue samples can lead to color variations that produce poor performance when using histological image processing techniques. Thus, normalization methods have been proposed to adjust the color of the image. This can be achieved through the use of a spectral matching technique, where it is first necessary to estimate the H&E representation and the stain concentration in the image pixels by means of the RGB model. This study presents an estimation method for H&E stain representation for the normalization of faded histological samples. This application has been explored only to a limited extent in the literature, but has the capacity to expand the use of faded samples. To achieve this, the normalized images must have a coherent color representation of the H&E stain with no introduction of noise, which was realized by applying the methodology described in this proposal. The estimation method presented here aims to normalize histological samples with different degrees of fading using a combination of fuzzy theory and the Cuckoo search algorithm, and dictionary learning with an initialization method for optimization. In visual and quantitative comparisons of estimates of H&E stain representation from the literature, our proposed method achieved very good results, with a high feature similarity between the original and normalized images.

Unsupervised method for normalization of hematoxylin-eosin stain in histological images.

Histological images stained with hematoxylin-eosin are widely used by pathologists for cancer diagnosis. However, these images can have color variations that highly influence the histological image processing techniques. To deal with this potential limitation, normalization methods are useful for color correction. In this paper, a histological image color normalization is presented by considering the biological and hematoxylin-eosin properties. To this end, the stain representation of a reference image was applied in place of the original images representation, allowing the preservation of histological structures. This proposal was evaluated on histological images with great variations of contrast, and both visual and quantitative analyzes yielded promising results.

Warthin's tumor at the Brazilian National Cancer Institute: additional evidence of homogeneous sex prevalence and association with other neoplasms.

PURPOSE OF THE STUDY: To report the largest Brazilian series of Warthin's tumor (WT). PROCEDURES: The medical files of 76 patients with WT treated in the Brazilian National Cancer Institute from 1996 to 2006 were reviewed. RESULTS: The male:female ratio was 2:1, with a predominance of white, old, and smoking patients. However, there were more women among the nonsmokers. One fifth of the patients presented with synchronous multiple lesions. Parotid lesions were prevalent, but there were patients with lesions in cervical lymph nodes and in the inferior lip. Most cases were treated by superficial parotidectomy, without recurrences. One fourth of the patients also developed other primary neoplasms. CONCLUSIONS: The observed data do not differ from those in the international literature. Multiplicity and the development of other neoplastic diseases require close clinical management of patients with WT. MESSAGE: The previously unreported observation of female prevalence among nonsmoking people with WT should be confirmed and explored in future studies.

Pathologic significance of AKT, mTOR, and GSK3ß proteins in oral squamous cell carcinoma-affected patients.

Phosphatidylinositol-3-kinases are kinases that lead to AKT phosphorylation and thus mTOR and GSK3ß activation. These proteins are linked to tumorigenesis, but their roles in driving cervical lymph node (CLN) metastasis of oral squamous cell carcinoma (OSCC) cells are unknown. This study aimed to investigate the role of AKT, mTOR, and GSK3ß proteins in the occurrence of CLN metastasis in OSCC patients. Ninety and 18 paraffin-embedded OSCC and oral mucosa samples were included, respectively. We divided our OSCC patients into non-metastasizing (PNM) and metastasizing (PM) groups, and the expression of total AKT, pAKT1Thr308, pAKTSer473, GSK3ß, pGSK3ßSer9, and pmTORSer2448 was analyzed by immunohistochemistry. The mean expression of GSK3ß, pGSK3ßSer9, total AKT, and pmTOR2448 was always higher in the OSCC tissues than that in the controls. A positive correlation was also found among these proteins. Total AKT, pmTORSer2448, and pGSK3ßSer9 expression was significantly higher in the PNM and PM groups than that in the control group. However, only GSK3ß expression was significantly higher in the PM group compared with the PNM group. High expression levels of GSK3ß and pGSK3ßSer9 were significantly associated with CLN metastasis, but only GSK3ß remained an independent predictor of CLN metastasis. pGSK3ßSer9 and CLN metastasis were associated with a poor prognosis, but only the latter remained an independent prognostic parameter. Kaplan-Meier survival curves showed that pGSK3ßSer9 and CLN metastasis were significantly related to reduced survival rates. These results suggest that AKT and mTOR proteins are involved in OSCC biology and that GSK3ß itself may drive CLN metastatic spread of OSCC cells.