Lectin-like Transcript-1 (LLT1) Expression in Oral Squamous Cell Carcinomas: Prognostic Significance and Relationship with the Tumor Immune Microenvironment.

Lectin-like transcript-1 (LLT1) expression is detected in different cancer types and is involved in immune evasion. The present study investigates the clinical relevance of tumoral and stromal LLT1 expression in oral squamous cell carcinoma (OSCC), and relationships with the immune infiltrate into the tumor immune microenvironment (TIME). Immunohistochemical analysis of LLT1 expression was performed in 124 OSCC specimens, together with PD-L1 expression and the infiltration of CD20+, CD4+, and CD8+ lymphocytes and CD68+ and CD163+-macrophages. Associations with clinicopathological variables, prognosis, and immune cell densities were further assessed. A total of 41 (33%) OSCC samples showed positive LLT1 staining in tumor cells and 55 (44%) positive LLT1 in tumor-infiltrating lymphocytes (TILs). Patients harboring tumor-intrinsic LLT1 expression exhibited poorer survival, suggesting an immunosuppressive role. Conversely, positive LLT1 expression in TILs was significantly associated with better disease-specific survival, and also an immune-active tumor microenvironment highly infiltrated by CD8+ T cells and M1/M2 macrophages. Furthermore, the combination of tumoral and stromal LLT1 was found to distinguish three prognostic categories (favorable, intermediate, and adverse; p = 0.029, Log-rank test). Together, these data demonstrate the prognostic relevance of tumoral and stromal LLT1 expression in OSCC, and its potential application to improve prognosis prediction and patient stratification.

Risk factors associated with fixation-related complications in microsurgical free flap reconstruction of the mandible.

BACKGROUND: Hardware complications (loosening of screws, infection, or exposure of the plate) in mandibular reconstruction with vascularized osseous free flaps impose significant morbidity, and frequently require revision surgery. Purpose of this study was to identify possible contributing factors for hardware complications. METHODS: This is a retrospective cohort study involving case series of patients who underwent microvascular mandible reconstructions between 2000 and 2020. Patient demographics, pathological, clinical, and treatment-related factors were analyzed in univariate and multivariate analyses. RESULTS: Ninety-one patients were enrolled, encompassing 63 reconstructions with fibular free flaps, 26 reconstructions with scapular, and 2 reconstructions with iliac flaps. Rate of hardware complications and plate exposure was 14.3% and 7.7%, respectively, with a median follow-up time for extrusion of 29 months. In univariate analysis, preoperative radiotherapy (odds ratio [OR] = 6.57, p = .01), and secondary mandible reconstruction (OR = 4.3, p = .04) were significant predictors of hardware complications, and plate exposure was most frequently found in secondary reconstruction (37.5%, OR = 11.8, p = .04). Hypertension was the most commonly found comorbidity (24%), and it trended toward significance regarding plate exposure (p = .05). Only secondary mandible reconstruction was associated with osteosynthesis complications (OR = 12.53, p = .01) and plate exposure (OR = 23.86, p = .005) on multivariate analysis, while preoperative radiation therapy did not retain its relevance on plate exposure. CONCLUSION: Secondary mandible reconstructions with vascularized osseous free flaps have a higher risk of osteosynthesis complications than primary reconstructions.

Prognostic implications of preoperative systemic inflammatory markers in oral squamous cell carcinoma, and correlations with the local immune tumor microenvironment.

Purpose: The aim of this study was to investigate the prognostic significance of preoperative inflammatory markers in peripheral blood of patients with oral squamous cell carcinoma (OSCC), and to establish correlations with the infiltrate of macrophages and lymphocytes in the local immune tumor microenvironment (TME). Materials and Methods: Neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), lymphocyte-monocyte ratio (LMR), and systemic immune-inflammation index (SII) were retrospectively evaluated in a cohort of 348 OSCC patients, and correlated with overall (OS) and disease-specific survival (DSS). Immunohistochemical analysis of tumoral and stromal infiltration of CD8+, CD4+, FOXP3+ and CD20+ lymphocytes and CD68+ and CD163+ macrophages was performed in a subset of 119 OSCC patient samples, and correlations further assessed. Results: NLR, SII, and LMR were significantly associated with a poorer OS in univariate analysis; however, only NLR remained a significant independent predictor in the multivariate analysis (HR = 1.626, p = 0.04). NLR and SII were inversely and significantly correlated with stromal infiltration of CD8+, CD4+, and CD20+ lymphocytes. Moreover, a significant correlation between LMR was also found to significantly associate with stromal infiltration of CD8+, CD4+, and CD20+ lymphocytes, stromal CD68+ and CD163+ macrophages, and also tumoral infiltration of CD4+ and CD20+ lymphocytes. Conclusions: Preoperative NLR, SII, and LMR may serve as valuable systemic markers to predict OSCC patient survival, with NLR emerging as an independent predictor of poor OS. Moreover, strong significant correlations were exclusively observed between systemic inflammatory markers and the local stromal infiltration of lymphocytes in the TME.

Hippo-YAP signaling activation and cross-talk with PI3K in oral cancer: A retrospective cohort study.

OBJECTIVES: This study aimed to investigate the clinical and prognostic relevance of the Hippo-YAP transactivators YAP1 and TAZ in oral squamous cell carcinoma, and their possible relationship with PI3K/mTOR pathway activation. MATERIALS AND METHODS: Immunohistochemical analysis of YAP1, TAZ, PIK3CA (p110α), p-AKT (Ser473), and p-S6 (Ser235) was performed in paraffin-embedded tissue specimens from 165 OSCC patients. Correlations between protein expression and clinical data were further assessed. RESULTS: YAP1 expression was detected in both cytoplasm and nucleus of tumor cells, whereas TAZ expression was only found in the nucleus. Nuclear YAP1 was significantly associated with tumor size (p = 0.03), neck lymph node metastasis (p = 0.02), TNM stage (p = 0.02), and poor differentiation (p = 0.04). Nuclear TAZ was associated with tobacco (p = 0.03) and alcohol consumption (p = 0.04), and poor tumor differentiation (p = 0.04). There was a positive significant correlation between nuclear and cytoplasmic YAP1, nuclear TAZ, p110α expression, and mTORC1 activation p-S6 (S235). Combined expression of nuclear and cytoplasmic YAP1 was prognostic in both univariate and multivariate analyses. Active nuclear YAP1 was significantly and independently associated with poor disease-specific (p = 0.005, HR = 2.520; 95% CI = 1.319-4.816) and overall survival (p = 0.015, HR = 2.126; 95% CI = 1.155-3.916). CONCLUSION: Nuclear YAP1 is an independent predictor of poor survival in oral squamous cell carcinoma.

Imaging of odontogenic keratocysts: a pictorial review.

INTRODUCTION: The aim of the present article was to review and depict the main radiological features of odontogenic keratocysts (OKCs), thus helping the differential diagnoses from other odontogenic cysts and neoplasms. EVIDENCE ACQUISITION: A review of articles published between January 2000 and October 2020 using Medline and the MeSH Term "odontogenic keratocyst" in combination with the following terms "imaging," "radiology," "panoramic radiograph," and "computed tomography," was performed. EVIDENCE SYNTHESIS: Radiographically, OKCs are well-defined unilocular or multilocular radiolucencies bounded by corticated margins. Most lesions are unilocular; instead, multilocular OKCs represent about the 30% of cases, mainly involving the posterior mandible. When, particularly in large lesions, OKCs display a multilocular presentation with adjacent satellite cysts (daughter cysts) a "soap-bubble appearance" can be recognized. DISCUSSION: Panoramic radiograph and CT still play an important role in the diagnosis and treatment planning of OKCs. Unfortunately, it may not be easy to differentiate OKCs from other odontogenic lesions, especially when they are small and unilocular. CONCLUSIONS: Histopathological findings are still necessary to obtain a definitive diagnosis.

Macrophages in Oral Carcinomas: Relationship with Cancer Stem Cell Markers and PD-L1 Expression.

Tumor-associated macrophages (TAMs) can be polarized into antitumoral M1 and protumoral and immunosuppressive M2 macrophages. This study investigated the clinical relevance of TAM infiltration in oral squamous cell carcinoma (OSCC), evaluating CD68 (M1 and M2 macrophage marker) and CD163 expression (M2 macrophage marker) in the tumor nests and surrounding stroma. Immunohistochemical analysis of both stromal/tumoral CD68+ and CD163+ TAMs was performed in paraffin-embedded tissue specimens from 125 OSCC patients, and correlated with clinical data. Potential relationships with the expression of cancer stem cell (CSC) markers and PD-L1 in the tumors were also assessed. Stromal CD163+ infiltration was significantly associated with the tumor location in the tongue, and stromal and tumoral CD68+ and CD163+-infiltrating TAMs were more abundant in nonsmokers and non-alcohol-drinkers. Strikingly, this study uncovers an inverse relationship between CD68+ and CD163+ TAMs and CSC marker expression (NANOG and SOX2) in OSCC. High infiltration of CD163+ TAMs in both tumor and stroma was strongly and significantly correlated with the absence of NANOG expression. Moreover, infiltration of both CD68+ and CD163+ TAMs was also significantly associated with high tumor expression of PD-L1. Our results suggest that there is a link between TAM infiltration and immune escape in OSCC.

SOX2 Expression Is an Independent Predictor of Oral Cancer Progression.

Potentially malignant oral lesions, mainly leukoplakia, are common. Malignant transformation varies widely, even in the absence of histological features such as dysplasia. Hence, there is a need for novel biomarker-based systems to more accurately predict the risk of cancer progression. The pluripotency transcription factor SOX2 is frequently overexpressed in cancers, including oral squamous cell carcinoma (OSCC), thereby providing a link between malignancy and stemness. This study investigates the clinical relevance of SOX2 protein expression in early stages of oral carcinogenesis as a cancer risk biomarker, and also its impact on prognosis and disease outcome at late stages of OSCC progression. SOX2 expression was evaluated by immunohistochemistry in 55 patients with oral epithelial dysplasia, and in 125 patients with OSCC, and correlated with clinicopathological data and outcomes. Nuclear SOX2 expression was detected in four (7%) cases of oral epithelial dysplasia, using a cut-off of 10% stained nuclei, and in 16 (29%) cases when any positive nuclei was evaluated. Univariate analysis showed that SOX2 expression and histopathological grading were significantly associated with oral cancer risk; and both were found to be significant independent predictors in the multivariate analysis. Nuclear SOX2 expression was also found in 49 (39%) OSCC cases, was more frequent in early tumor stages and N0 cases, and was associated with a better survival. In conclusion, SOX2 expression emerges as an independent predictor of oral cancer risk in patients with oral leukoplakia. These findings underscore the relevant role of SOX2 in early oral tumorigenesis rather than in tumor progression.

The Emerging Role of NANOG as an Early Cancer Risk Biomarker in Patients with Oral Potentially Malignant Disorders.

NANOG, a key regulator of pluripotency and self-renewal in embryonic and adult stem cells, is frequently overexpressed in multiple cancers, including oral squamous cell carcinoma (OSCC). It has been frequently associated with poor outcomes in epithelial cancers, and recently implicated in laryngeal tumorigenesis. On this basis, we investigated the role of NANOG protein expression as an early cancer risk biomarker in oral potentially malignant disorders (OPMD), and the impact on prognosis and disease outcomes in OSCC patients. NANOG expression was evaluated by immunohistochemistry in 55 patients with oral epithelial dysplasia, and 125 OSCC patients. Correlations with clinical and follow-up data were assessed. Nuclear NANOG expression was detected in 2 (3.6%) and cytoplasmic NANOG expression in 9 (16.4%) oral dysplasias. NANOG expression increased with the grade of dysplasia. Cytoplasmic NANOG expression and the histopathological grading were significantly correlated with oral cancer risk, although dysplasia grading was the only significant independent predictor of oral cancer development in multivariate analyses. Cytoplasmic NANOG expression was also detected in 39 (31%) OSCC samples. Positive NANOG expression was significantly associated with tobacco and alcohol consumption, and was more frequent in pN0 tumors, early I-II stages. These data unveil the clinical relevance of NANOG in early stages of OSCC tumorigenesis rather than in advanced neoplastic disease. NANOG expression emerges as an early predictor of oral cancer risk in patients with OPMD.

PD-L1 Expression in Tumor Cells Is an Independent Unfavorable Prognostic Factor in Oral Squamous Cell Carcinoma.

BACKGROUND: The immune checkpoint PD-1 and its ligand PD-L1 are involved in the induction of immunological tolerance of solid tumors including oral squamous cell carcinoma (OSCC). The aim of the study was to establish the clinical and prognostic significance of PD-L1 in OSCC. METHODS: Tissue microarrays of 125 resected OSCC were stained with two different commercially available PD-L1 antibodies (clones E1L3N and 22C3), alongside PD-1 immunostaining. RESULTS: PD-L1 expression in more than 10% of tumor cells was associated with poorer survival, and established as a clinically relevant cut-off point. This relevant PD-L1 expression was detected in 10% to 15% OSCC specimens depending on the anti-PD-L1 antibody, and showed an inverse correlation with tobacco and alcohol consumption. We consistently found that PD-L1 expression was associated with tumor recurrence and lower disease-specific survival. Multivariate analysis further revealed that neck node metastasis (HR 2.304; P = 0.009) and tumor PD-L1 expression (HR 2.571; P = 0.01) were significant independent factors for poor prognosis. CONCLUSIONS: PD-L1 expression in more than 10% of tumor cells was a significant and independent factor of poor prognosis in OSCC. IMPACT: PD-L1 expression in more than 10% of tumor cells was consistently established as a clinically relevant cut-off point by using two different antibodies. Remarkably, PD-L1 expression emerges as an independent poor prognosis marker in patients with OSCC.

Distinct Expression and Clinical Significance of Zinc Finger AN-1-Type Containing 4 in Oral Squamous Cell Carcinomas.

Zinc finger AN1-type containing 4 (ZFAND4) has emerged as a promising prognostic marker and predictor of metastasis for patients with oral squamous cell carcinoma (OSCC). However, further validation is fundamental before clinical implementation. Hence, this study evaluated the expression pattern of ZFAND4 protein expression by immunohistochemistry using an independent cohort of 125 patients with OSCC, and correlations with the clinicopathologic parameters and disease outcome. Remarkably, ZFAND4 expression, while negligible in normal epithelium, exhibited two distinct expression patterns in tumors that did not overlap. A gross granular staining was characteristic of the undifferentiated cells at the invasive front of tumors, whereas the most differentiated cells located at the center of the tumor nests showed diffuse non-granular staining. ZFAND4 staining was higher in undifferentiated than in differentiated areas of tumors. High ZFAND4 expression in differentiated cells was significantly associated to well-differentiated (p = 0.04) and non-recurrent tumors (p = 0.04), whereas ZFAND4 expression in undifferentiated cells correlated with tumor location (p = 0.005). No correlations between the ZFAND4 expression and patient survival were found. These data question the clinical relevance of ZFAND4 expression as a prognostic biomarker in OSCC, and also reveal distinct ZFAND4 expression patterns depending on the differentiation areas of tumors that should be evaluated separately.

Dysregulation of Mir-196b in Head and Neck Cancers Leads to Pleiotropic Effects in the Tumor Cells and Surrounding Stromal Fibroblasts.

The miR-196 family members have been found dysregulated in different cancers. Therefore, they have been proposed as promising biomarkers and therapeutic targets. This study is the first to investigate the role of miR-196b in the development and progression of head and neck squamous cell carcinomas (HNSCC), and also the impact on the surrounding tumor microenvironment. Increased miR-196b levels were detected in 95% of primary tumors and precancerous lesions, although no significant differences were observed between non-progressing versus progressing dysplasias. Furthermore, increased levels of both miR-196a and miR-196b were successfully detected in saliva samples from HNSCC patients. The functional consequences of altered miR-196 expression were investigated in both HNSCC cell lines and cancer-associated fibroblasts (CAFs) by transfection with specific pre-miR precursors. Results showed that both miR-196a and miR-196b elicit cell-specific responses in target genes and downstream regulatory pathways, and have a distinctive impact on cell proliferation, migration and invasion. These data reveal the early occurrence and prevalence of miR-196b dysregulation in HNSCC tumorigenesis, suggesting its utility for early diagnosis and/or disease surveillance and also as a non-invasive biomarker in saliva. The pleiotropic effects of miR-196a/b in HNSCC cell subpopulations and surrounding CAFs may complicate a possible therapeutic application.

Phosphorylated ribosomal protein S6 correlation with p21 expression and inverse association with tumor size in oral squamous cell carcinoma.

BACKGROUND: The purpose of this study was to investigate the clinical relevance of phosphorylated ribosomal protein S6 (p-S6), a surrogate marker of mammalian target of rapamycin (mTOR) activation, and p21 in a series of 125 patients with oral squamous cell carcinomas (OSCCs). METHODS: Immunohistochemical analysis was performed to ascertain the phosphorylation status of p-S6 at Ser235/236 and Ser240/244, p21, and p53 protein expression. RESULTS: Expression of phosphorylated S6 protein on either serine 235/236 or serine 240/244 was detected in 83% and 88% tumors, respectively, and both of them were inversely and significantly correlated with the tumor size and local infiltration. Positive p21 expression was found in 91.5% of the cases, and was inversely correlated with tumor size. In OSCC, p21 expression correlates with p-S6 levels, a surrogate marker of mTOR activation, independently of p53 status. CONCLUSION: Expression of both p21 and p-S6 was found to inversely associate with tumor size but not survival outcomes in patients with OSCC.

Searching for proprioceptors in human facial muscles.

The human craniofacial muscles innervated by the facial nerve typically lack muscle spindles. However these muscles have proprioception that participates in the coordination of facial movements. A functional substitution of facial proprioceptors by cutaneous mechanoreceptors has been proposed but at present this alternative has not been demonstrated. Here we have investigated whether other kinds of sensory structures are present in two human facial muscles (zygomatic major and buccal). Human checks were removed from Spanish cadavers, and processed for immunohistochemical detection of nerve fibers (neurofilament proteins and S100 protein) and two putative mechanoproteins (acid-sensing ion channel 2 and transient receptor potential vanilloid 4) associated with mechanosensing. Nerves of different calibers were found in the connective septa and within the muscle itself. In all the muscles analysed, capsular corpuscle-like structures resembling elongated or round Ruffini-like corpuscles were observed. Moreover the axon profiles within these structures displayed immunoreactivity for both putative mechanoproteins. The present results demonstrate the presence of sensory structures in facial muscles that can substitute for typical muscle spindles as the source of facial proprioception.

The prognostic role of claudins -1 and -4 in oral squamous cell carcinoma.

Claudin dysregulation has been described in various tumor types; however, its clinical relevance is poorly understood. We present a study in which we assessed the expression of claudin 1 (CLDN1) and CLDN4 in oral squamous cell carcinoma (OSCC), as well as their prognostic relevance. Immunohistochemical analysis of CLDN1 and CLDN4 expression was carried out on tissue sections from 65 OSCCs. The presence of CLDN1 in the invasive front of tumor islands was associated with neck node metastasis, and the expression of CLDN4 was associated with higher histological grade, and tumor recurrence. Membranous staining for CLDN4 in tumor cells, and weak intensity of CLDN4 immunoexpression were predictive for poorer survival. In a multivariate analysis for disease recurrence, CLDN1 immunostaining was statistically significant. Specifically, CDLN1 expression in the tumor invasive front was associated with tumor recurrence. Our results indicate that CLDN4 expression is correlated with poor prognosis, and CLDN1 expression may be an indicator of recurrence of OSCC.

E-cadherin and ß-catenin expression in well-differentiated and moderately-differentiated oral squamous cell carcinoma: relations with clinical variables.

The aim of this study was to establish the expression and localisation of E-cadherin and ß-catenin in oral squamous cell carcinomas (SCC) so that we could correlate the findings with prognostically-relevant clinicopathological variables. E-cadherin and ß-catenin expression in normal oral mucosa and in oral squamous cell carcinomas were examined immunohistochemically, and their association with clinicopathological factors and prognosis were then analysed in 69 patients who had been operated on for oral SCC. E-cadherin expression was found in all 69 cases: in 11 cases (16%) it was weak; in 21 (30%) moderate, and in 37 (54%) high. ß-Catenin expression was found in 64 cases (93%): in 18 cases (26%) cell-membrane expression was weak; in 26 (38%) it was moderate; in 19 (28%) it was high, and in one case (1%) there was cytoplasmic staining. No nuclear staining was detected. E-cadherin was significantly associated with histological grade (p=0.002) and alcohol consumption (p=0.05), and ß-catenin was significantly associated with nodal stage (p=0.02), TNM stage (p=0.009), and E-cadherin expression (p=0.01). However, none of them were independent prognostic factors in the disease-specific survival analysis. E-cadherin is closely linked to ß-catenin expression in oral SCC and to tumour differentiation. Alcohol consumption could increase the aggressiveness of SCC, leading to reduced expression of E-cadherin. ß-catenin could be an early marker for the identification of occult metastases in patients with oral SCC.

In oral squamous cell carcinoma, high FAK expression is correlated with low P53 expression.

Focal adhesion kinase (FAK) and p53 have been associated with metastatic activity and a poor prognosis in oral squamous cell carcinoma (OSCC). Recently, a feedback mechanism in which FAK regulates p53 has been proposed. The present study aims to determine the role of p53 in FAK regulation in these tumors. FAK and p53 expression was examined by immunohistochemistry in normal oral mucosa and in 67 oral squamous cell carcinomas. p16(INK4a) was also studied in view of its association with human papillomavirus infection. The association between FAK and p53 was subsequently analyzed. FAK expression in OSCCs was heterogeneous: 22 (33 %) cases showed weak expression, 16 (24 %) showed moderate expression, and 22 (33 %) cases showed high expression. Regarding p53, 31 of 67 (46 %) available tumor specimens showed negative staining, and 36 of 67 (54 %) showed positive nuclear staining for p53. FAK expression was inversely correlated with p53 expression (Fisher's exact test, p = 0.005). There was no association between p16(INK4a) and p53 or FAK expression. In conclusion, our results support the hypothesis that FAK activity might be involved in the down-regulation of p53 expression in OSCC.

Retrospective analysis of 314 orbital fractures.

OBJECTIVE: The aim of this study was to retrospectively review the orbital fractures treated at our institution, especially epidemiologic aspects, treatment options, and postoperative sequelae. STUDY DESIGN: Three hundred fourteen patients with orbital fractures treated at the Department of Oral and Maxillofacial Surgery of the Central University Hospital in Asturias (Spain) between 2000 and 2006 were retrospectively reviewed. The patients were evaluated by age, gender, etiology, diagnostic tools, fracture pattern, treatment, and complications. RESULTS: The most common causes of injury were motor vehicle accidents (29.6%), followed by falls (27.4%). Men in the sixth decade were most affected. One hundred forty-four patients (46%) underwent internal fixation with titanium miniplates, and 17 (5.4%) required orbit floor implants. The most frequent sequelae were infraorbital nerve hypoesthesia (24.5%), enophthalmos (3.8%), and diplopia (2.2%). CONCLUSIONS: In our area of 1 million inhabitants, falls are the second cause of orbital fractures, which can be attributed to the large aged population. Postoperative complications cannot be definitely evaluated until a few months after the surgery.

[Superficial angiomyxoma of the parotid region and review of the literature]. / Angiomixoma superficial de la región parotídea y revisión de la literatura.

Superficial angiomyxoma (SA) is a rare benign cutaneous neoplasm first described by Allen et al in 1988. To the best of our knowledge, we report the first case of superficial angiomyxoma located in the parotid region. We also stress the importance of distinguishing this entity from other lesions that may be involved in this location such as cutaneous neoplasms, parotid tumours or cysts. We emphasise the need to rule out the Carney complex, which has been associated with these tumours.

Posttraumatic frontal mucocele.

Mucoceles of the frontal sinus are expansive cysts filled of the mucus secreted by goblet cells interspersed in the ciliated mucosa. The aim of this article was to present a case of a frontal mucocele that was developed in a 31-year-old man 19 years after having had a frontal sinus fracture. This is a rare entity. Frontal sinus fractures must be treated by a multidisciplinary team to avoid all possible sequelae. An adequate primary management of frontal sinus fractures is essential to prevent complications. This may include conservative attitude, reduction and fixation with miniplates, obliteration, cranialization, or grafting. Removal of any rest of epithelium is mandatory in this sense.