Malignant transformation of oral leukoplakia is associated with macrophage polarization.

BACKGROUND: Most oral squamous cell carcinomas (OSCC) occur on the basis of oral leukoplakias (OLP). The histologic degree of dysplasia is insufficient for the prediction of OLP malignant transformation. Immunologic parameters are gaining importance for prognostic assessment and therapy of cancer. M2 polarized macrophages were shown to be associated with OSCC progression and inferior prognosis. The current study aims to answer the question if OLP with malignant transformation into OSCC within 5 years differ from OLP without transformation regarding macrophage infiltration and polarization. METHODS: 201 specimens (50 transforming OLP, 53 non-transforming OLP, 49 corresponding OSCC and 49 healthy oral mucosa controls) were processed for immunohistochemistry. Samples were stained for CD68, CD163 and CD11c expression, completely digitalized and computer-assisted cell counting was performed. Epithelial and subepithelial compartments were differentially assessed. Groups were statistically compared using the Mann-Whitney U-test. A cut-off point for the discrimination of transforming and non-transforming OLP was determined and the association between macrophage infiltration and malignant transformation was calculated using the Chi-square test (χ2 test). RESULTS: Macrophage infiltration and M2 polarization in OLP with malignant transformation within 5 years was significantly increased compared to OLP without malignant transformation (p < 0.05). OSCC samples showed the highest macrophage infiltration and strongest M2 polarization (p < 0.05). Additionally, transforming OLP revealed a significant shift of macrophage infiltration towards the epithelial compartment (p < 0.05). χ2 test revealed a significant association of increased macrophage infiltration with malignant transformation (p < 0.05). CONCLUSION: Immunological changes precede malignant transformation of OLP. Increased macrophage infiltration and M2 polarization was associated with the development of oral cancer in OLP. Macrophage infiltration could serve as predictive marker for malignant transformation.

MAGE-A expression in oral and laryngeal leukoplakia predicts malignant transformation.

Leukoplakia is a potential precursor of oral as well as laryngeal squamous cell carcinoma. Risk assessment of malignant transformation based on the grade of dysplasia of leukoplakia often does not lead to reliable results. However, oral squamous cell carcinoma, laryngeal squamous cell carcinoma, and leukoplakia express single or multiple members of the melanoma-associated antigens A (MAGE-A) family, while MAGE-A are absent in healthy mucosal tissue. The present study aimed at determining if there is an association between the expression of MAGE-A in leukoplakia and malignant transformation to oral or laryngeal squamous cell carcinoma. Paraffin-embedded tissues of 205 oral and laryngeal leukoplakia, 90 corresponding tumors, and 40 healthy oral mucosal samples were included in the study. The grade of dysplasia of the leukoplakia samples was determined histopathologically. The leukoplakia samples were divided into lesions that transformed to oral and laryngeal squamous cell carcinoma (n = 91) and lesions that did not (n = 114) during a 5 years follow-up. The expression of MAGE-A3/6 and MAGE-A4 was analyzed by real-time RT-PCR. The expression of MAGE-A 1-4, 6, and 12 was determined by immunohistochemistry. A total of 59.3% of the transforming leukoplakia expressed at least one of the examined antigens as opposed to an expression rate of 3.5% of all non-transforming leukoplakia. There was no MAGE-A expression in healthy oral mucosa. The risk of malignant transformation was statistically significantly associated with MAGE-A expression in immunohistochemistry (p < 0.001) and real-time RT-PCR (MAGE-A3/6, p = 0.001; MAGE-A4, p = 0.002) analyses. There was no significant association between MAGE-A expression and the grade of dysplasia ("low-grade", D0/D1; "high-grade", D2/D3) in immunohistochemistry (p = 0.412) and real-time RT-PCR (MAGE-A3/6, p = 0.667; MAGE-A4, p = 0.756). It seems that the analysis of the MAGE-A expression profile may support the identification of leukoplakia at risk for malignant transformation. Therefore, efforts should be made to establish this analysis as a routine procedure in addition to conventional histopathology.

Pedicled buccal flaps as a backup procedure for intraoral reconstruction.

BACKGROUND: Intraoral soft tissue deficiency and impaired wound beds are common problems after cleft and tumour surgery or after dental trauma. Frequently, limited defects are overtreated with extensive microvascular reconstruction procedures, but pedicled flaps remain useful, as they are simple to harvest, and they provide a reliable outcome. The buccal flap, first described in the 1970s, has been used for palatine lengthening in cleft patients over decades. In the following, we present an expanded indication in cases of palatal fistula, complex vestibulum, exposed bone in orthognathic surgery, and osteoradionecrosis. METHODS: We conducted a retrospective chart review and report on all buccal flaps harvested in our department within the last 3 years with a follow-up period of at least half a year after flap surgery. Patients of all age groups and treatment indications in which a buccal flap was used were implicated in the evaluation. RESULTS: Sixteen buccal flaps were performed in 10 patients. The median age at the time of surgery was 42 years, reaching from 12 up to 66 years. Fourteen buccal flaps were used for upper jaw or palatal coverage; two buccal flaps were used in the mandible. In terms of complications (four flaps; 25%), there were two partial flap failures, one wound dehiscence and one wound dehiscence. There were no failures of the remaining mucosal flap islands after pedicle dissection. CONCLUSION: The buccal flap is a reliable and straightforward approach to challenging intraoral wound beds with soft tissue deficiency. We thoroughly discuss the additional indications for buccal flap surgery, describe the harvest technique, and provide strategies to prevent intra- and postoperative complications.

Beyond PD-L1-Identification of Further Potential Therapeutic Targets in Oral Cancer.

BACKGROUND: The involvement of immune cell infiltration and immune regulation in the progression of oral squamous cell carcinoma (OSCC) is shown. Anti-PD-1 therapy is approved for the treatment of advanced OSCC cases, but not all patients respond to immune checkpoint inhibitors. Hence, further targets for therapeutic approaches are needed. The number of identified cellular receptors with immune checkpoint function is constantly increasing. This study aimed to perform a comparative analysis of a large number of immune checkpoints in OSCC in order to identify possible targets for therapeutic application. MATERIALS AND METHODS: A NanoString mRNA analysis was performed to assess the expression levels of 21 immune regulatory checkpoint molecules in OSCC tissue (n = 98) and healthy oral mucosa (NOM; n = 41). The expression rates were compared between the two groups, and their association with prognostic parameters was determined. Additionally, relevant correlations between the expression levels of different checkpoints were examined. RESULTS: In OSCC tissue, significantly increased expression of CD115, CD163, CD68, CD86, CD96, GITRL, CD28 and PD-L1 was detected. Additionally, a marginally significant increase in CD8 expression was observed. BTLA and PD-1 levels were substantially increased, but the differential expression was not statistically significant. The expression of CD137L was significantly downregulated in OSCC compared to NOM. Correlations between immune checkpoint expression levels were demonstrated, and some occurred specifically in OSCC tissue. CONCLUSIONS: The upregulation of inhibitory receptors and ligands and the downregulation of activators could contribute to reduced effector T-cell function and could induce local immunosuppression in OSCC. Increased expression of activating actors of the immune system could be explained by the increased infiltration of myeloid cells and T-cells in OSCC tissue. The analysis contributes to the understanding of immune escape in OSCC and reveals potential targets for oral cancer immunotherapy.

PD1 expression and correlation with its ligands in oral cancer specimens and peripheral blood.

OBJECTIVES: This study analyzed the expression of the PD1 receptor in tumor tissue and peripheral blood of oral squamous cell carcinoma (OSCC) patients, and correlated it with the PD1 ligands PD-L1 and PD-L2. The currently low response rates of checkpoint inhibitor treatment in OSCC could be increased by a better understanding of immune checkpoint biology. Despite evidence in the literature for upregulation of PD1 checkpoint ligands in OSCC tissue, there has been no correlation analysis of the PD1 receptor with its ligands in tissue specimens and peripheral blood of OSCC patients. MATERIALS AND METHODS: An RT-qPCR analysis of PD1 mRNA expression was performed in oral cancer specimens, healthy mucosa, and corresponding blood samples. A cut-off point (COP) was determined and a chi-square (χ2) test was carried out. PD1 expression was correlated with previously reported PD-L1 and PD-L2 expression values using the Spearman test. RESULTS: Tissue and blood specimens of 48 OSCC patients and 26 healthy individuals were analyzed. PD1 expression in OSCC specimens was significantly increased (p = 0.006) compared with healthy oral mucosa. PD1 overexpression in tissue samples showed a significant association with the presence of malignancy (p = 0.006). PD1 expression in tissue samples showed a significant positive correlation (p < 0.001) with the ligands PD-L1 and PD-L2. In contrast, there was no correlation between PD1 and its ligands in blood samples. However, there was a significant positive correlation (p < 0.001) between the ligands PD-L1 and PD-L2, both in tissue and blood samples. CONCLUSIONS: Increased PD1 expression might be a manifestation of T-cell exhaustion in OSCC specimens, leading to immune tolerance. PD-L1/PD-L2-PD1 interaction may be a major mediator of local immunosuppression in OSCC, requiring advanced multimodal treatment protocols.

Importance of the PD-1/PD-L1 Axis for Malignant Transformation and Risk Assessment of Oral Leukoplakia.

BACKGROUND: The programmed cell death ligand 1/programmed cell death receptor 1 (PD-L1/PD-1) Immune Checkpoint is an important modulator of the immune response. Overexpression of the receptor and its ligands is involved in immunosuppression and the failure of an immune response against tumor cells. PD-1/PD-L1 overexpression in oral squamous cell carcinoma (OSCC) compared to healthy oral mucosa (NOM) has already been demonstrated. However, little is known about its expression in oral precancerous lesions like oral leukoplakia (OLP). The aim of the study was to investigate whether an increased expression of PD-1/PD-L1 already exists in OLP and whether it is associated with malignant transformation. MATERIAL AND METHODS: PD-1 and PD-L1 expression was immunohistologically analyzed separately in the epithelium (E) and the subepithelium (S) of OLP that had undergone malignant transformation within 5 years (T-OLP), in OLP without malignant transformation (N-OLP), in corresponding OSCC and in NOM. Additionally, RT-qPCR analysis for PD-L1 expression was done in the entire tissues. Additionally, the association between overexpression and malignant transformation, dysplasia and inflammation were examined. RESULTS: Compared to N-OLP, there were increased levels of PD-1 protein in the epithelial and subepithelial layers of T-OLP (pE = 0.001; pS = 0.005). There was no significant difference in PD-L1 mRNA expression between T-OLP and N-OLP (p = 0.128), but the fold-change increase between these groups was significant (Relative Quantification (RQ) = 3.1). In contrast to N-OLP, the PD-L1 protein levels were significantly increased in the epithelial layers of T-OLP (p = 0.007), but not in its subepithelial layers (p = 0.25). Importantly, increased PD-L1 levels were significantly associated to malignant transformation within 5 years. CONCLUSION: Increased levels of PD-1 and PD-L1 are related to malignant transformation in OLP and may represent a promising prognostic indicator to determine the risk of malignant progression of OLP. Increased PD-L1 levels might establish an immunosuppressive microenvironment, which could favor immune escape and thereby contribute to malignant transformation. Hence, checkpoint inhibitors could counteract tumor development in OLP and may serve as efficient therapeutic strategy in patients with high-risk precancerous lesions.

The altered expression levels of miR-186, miR-494 and miR-3651 in OSCC tissue vary from those of the whole blood of OSCC patients.

BACKGROUND: Altered expressions of miR-186, miR-494 and miR-3651 in whole blood of OSCC patients have already been demonstrated. However, nothing is known about their expression in tumor tissues. This study aimed at evaluating differences in miRNA expression in OSCC tissues compared to healthy oral mucosa and at checking if the altered expression is reflected in corresponding blood. METHODS: In 53 OSCC and 40 healthy mucosal tissues the expression of miR-186, -494 and -3651 was determined by RT-qPCR and expression levels were compared between the groups. The altered expression in tissues was compared with that determined for corresponding blood. MiR-3651 target genes were identified using TargetScan software. RESULTS: Differential expression of miR-186 and miR-494 could not be observed in tumor tissues compared to normal mucosa (pmiR-186= 0.13; pmiR-494= 0.35). Contrary to the detected upregulation of the miR-3651 in the blood of OSCC patients, a significant downregulation was observed in OSCC tissues. A significant correlation between underexpression and diagnosis was ascertained (p= 0.0001). 1710 possible target genes of miR-3651 were identified. CONCLUSIONS: Decreased expression of miR-3651 in OSCC tissues may be a diagnostic biomarker. The opposite change in expression level in the blood might indicate different functions of this miRNA in circulation and tissue.

Prognostic significance of PD-L2 expression in patients with oral squamous cell carcinoma-A comparison to the PD-L1 expression profile.

BACKGROUND: Despite the observed association of increased PD-L1 expression in peripheral blood of oral squamous cell carcinoma (OSCC) patients with histomorphologic parameters, the role of the PD1 ligands-PD-L1 and PD-L2-is insufficiently understood. Aim of the study was to investigate whether the alterations of PD-L1 and PD-L2 expression in blood are associated with survival and could serve as immune monitoring parameter. Moreover, it should be analyzed if PD-L2 is differentially expressed in tissue and blood samples of OSCC patients compared to healthy controls and if there is an association of PD-L2 expression with histomorphologic and prognostic tumor parameters. METHODS: PD-L2 mRNA expression was analyzed in tumors and healthy oral mucosa specimens and in corresponding peripheral blood samples of 48 OSCC patients and 26 healthy controls using RT-qPCR. A cutoff point (COP) was determined and a chi-square test (χ2 test) was carried out. Survival analysis of PD-L2 and previously reported PD-L1 expression data was performed using Kaplan-Meier analysis (Log-rank test). RESULTS: PD-L2 expression in tissue samples was significantly (P < 0.001) higher in OSCC patients compared to healthy controls. A significant association of PD-L2 expression above the COP (positive) with malignancy was ascertained (P < 0.001). A significant (P = 0.01) association of previously reported PD-L1 expression rates in peripheral blood with survival could be shown. CONCLUSION: Peripheral blood PD-L1 expression might be a prognostic marker for OSCC patients and a possible parameter to monitor immune dysfunction in malign diseases. In the peripheral blood, PD-L1 might be more relevant for immune tolerance than PD-L2. Local PD-L2 expression in tissue samples might be useful as a diagnostic parameter for malignancy and could contribute to the immunosuppressive local microenvironment in OSCC.

Prognostic significance of altered miRNA expression in whole blood of OSCC patients.

Currently, there is a lack of blood markers for the detection of recurrent oral squamous cell carcinoma (OSCC). The present study aimed to investigate whether the aberrant expression of single microRNAs (miRNAs) in whole blood of patients could serve as a biomarker for persistent or recurrent OSCC. Whole blood of 2 groups of formerly treated OSCC patients was investigated by RT-qPCR for their circulating miRNA profiles. The R-OC group included patients with recurrence of OSCC (n=21) and the NR-OC group included patients without recurrence (n=21). Fold-changes and significance of the differences in miRNA expression levels between the groups were determined. A cut-off point (COP) for the discrimination between the R-OC and NR-OC groups was calculated and the significance between over/under expression of the miRNAs and the recurrence of malignancy was determined. Significant differences in the miRNA expression in whole blood of the R-OC and NR-OC groups were found. The levels of miR-3651 and miR-494 were significantly increased and the level of miR-186 was significantly decreased in whole blood of the R-OC patients (pmiR-3651=0.001, pmiR-494=0.003 and pmiR-186=0.001). By the determination of the COP, increased or decreased expression of the markers was significantly correlated to the recurrence of the disease. Altered expression of miR-494, miR-3651 and miR-186 appears to be associated with the recurrence of OSCC. The present study may form the basis for establishing a blood test as a minimally invasive method for the detection of the recurrence of OSCC.

PD-L1 expression in tumor tissue and peripheral blood of patients with oral squamous cell carcinoma.

BACKGROUND: Immune checkpoints like programmed cell death-1 (PD-1) and its ligand PD-L1 are involved in immune escape mechanisms of solid tumors including oral squamous cell carcinoma (OSCC). Inhibitors of the pathway are successfully used for treating especially advanced disease. However, the physiological relevance of PD-1/PD-L1-signaling in OSCC is insufficiently understood. The aim of the study was to analyze if PD-L1 expression in tumor tissue and peripheral blood samples of OSCC patients is associated with histomorphological tumor parameters and if PD-L1 expression in patients is different from controls. RESULTS: OSCC tumor specimens showed a significantly higher PD-L1 expression than oral mucosa controls (p < 0.001; upregulation more than 3-fold). Cross-tabulation revealed an association of increased expression of PD-L1 mRNA in tissue specimens with malignancy (p < 0.001).OSCC cases with higher tumor grade and cases with lymph node metastases (N+) were significantly (p < 0.05) associated with increased PD-L1 expression in peripheral blood. Cross-tabulation revealed an significant association with lymph node metastases (N+) (p ≤ 0.002). MATERIALS AND METHODS: PD-L1 mRNA expression was analyzed in tumor specimens and corresponding samples of healthy oral mucosa and peripheral blood of 45 OSCC patients and 36 healthy control persons using RT-qPCR analysis. A Mann-Whitney U-test, a cut-off point analysis and a Chi-square test were carried out. CONCLUSIONS: PD-L1 expression in OSCC could contribute to the immunosuppressive local tumor microenvironment. Increased malignant behavior (higher tumor grade, positive nodal status) might be associated with PD-L1 mediated systemic immune tolerance. Thus, PD-L1 expression in peripheral blood might be an indicator of the existence of metastatic disease (N+) in OSCC.

Peri-implant defect regeneration in the diabetic pig: A preclinical study.

OBJECTIVES: The study aims to establish a peri-implant dehiscence-type bone defect in a diabetic animal model of human bone repair and to quantify the influence of diabetes on peri-implant bone regeneration. MATERIAL AND METHODS: Experimental diabetes was induced in three domestic pigs by streptozotocin. Three animals served as healthy controls. After 12 months four standardized peri-implant dehiscence bone defects were surgically created in the ramus mandibulae. The animals were sacrificed after 90 days. Samples were histologically analyzed to quantify new bone height (NBH), bone-to-implant-contact (BIC), area of newly formed bone (NFB), bone-density (BD), and bone mineralization (BM) in the prepared defect (-D) and in a local control region (-L). RESULTS: After 90 days, diabetic animals revealed a significantly lower BIC (p = 0.037) and BD (p = 0.041) in the defect area (-D). NBH and BM-D differences within the groups were not significant (p > 0.05). Significant more NFB was measured in the healthy control group (p = 0.046). In the region of local bone BIC-L was significant less in the diabetic group (p = 0.028). In the local control region BD-L and BM-L was lower in the diabetic group compared to the healthy control animals (p > 0.05). CONCLUSION: Histological evidence indicates impaired peri-implant defect regeneration in a diabetic animal model.

Alterations in miRNA expression patterns in whole blood of OSCC patients.

BACKGROUND/AIM: Aberrant microRNA (miRNA) expression in blood of cancer patients is a common finding. The present study aimed at evaluating the differences in miRNA expression in whole blood samples of oral squamous cell carcinoma (OSCC) patients compared to healthy controls. MATERIALS AND METHODS: Microarray based miRNA profiling was performed on whole blood samples of 20 OSCC patients and 20 healthy volunteers and the differences in expression patterns between the two groups were evaluated. The results were validated by Reverse Transcription quantitative-Polymerase Chain Reaction (RT-qPCR) in 50 OSCC patients and 35 volunteers. RESULTS: 21 miRNAs were identified to be significantly differentially expressed in whole blood of OSCC patients compared to healthy controls. The impact of miR-186 (p=0.002), miR-494 (p=0.001) and miR-3651 (p=0.0001) could be validated by RT-qPCR. CONCLUSION: The aberrant expressions of miR-186, miR-494 and miR-3651 in whole blood of OSCC patients may serve as the basis for establishing minimally-invasive screening methods for OSCC based on miRNAs as biomarkers.

miR-186, miR-3651 and miR-494: potential biomarkers for oral squamous cell carcinoma extracted from whole blood.

microRNAs (miRNAs) are aberrantly expressed in the whole blood of patients suffering from different types of cancer. Collection of whole blood samples is a minimally invasive procedure. To date, little is known concerning the altered miRNA expression in patients suffering from oral squamous cell carcinoma (OSCC). The present study aimed to evaluate the difference in miRNA expression in whole blood samples in OSCC patients as compared to healthy volunteers who served as controls. In 20 blood samples from patients and healthy volunteers, the expression patterns of 1,205 human miRNAs were examined by miRNA microarray in order to identify those with the most pronounced differential expression. The results were verified by quantitative RT-PCR (RT-qPCR) for miR-186, miR-3651 and miR-494 using 57 samples of patients and 33 samples of healthy volunteers. Receiver operating characteristic (ROC) curves and the highest Youden index were calculated in order to assess cut-off points (COPs) that allowed the distinguishing of blood samples of OSCC patients from those of healthy volunteers. Significantly different expression rates were found for miR-186 (p=0.01), miR-3651 (p=0.0001) and miR-494 (p=0.004) between the OSCC patients and healthy controls. In the OSCC patients, there was a 2-fold upregulation for miR-494 and miR-3651 and a 2-fold downregulation for miR-186. Based on the determined COPs, significant correlations between miR-3651 overexpression and lymph node status (p=0.04), tumor grade (p=0.02) and clinical stage (p=0.04) were indicated. Aberrant expression levels of miR-186, miR-494 and miR-3651 in whole blood samples of OSCC patients may provide the possibility to establish a minimally invasive screening method for OSCC.