Sarm1 is not necessary for activation of neuron-intrinsic growth programs yet required for the Schwann cell repair response and peripheral nerve regeneration.

Upon peripheral nervous system (PNS) injury, severed axons undergo rapid SARM1-dependent Wallerian degeneration (WD). In mammals, the role of SARM1 in PNS regeneration, however, is unknown. Here we demonstrate that Sarm1 is not required for axotomy induced activation of neuron-intrinsic growth programs and axonal growth into a nerve crush site. However, in the distal nerve, Sarm1 is necessary for the timely induction of the Schwann cell (SC) repair response, nerve inflammation, myelin clearance, and regeneration of sensory and motor axons. In Sarm1-/- mice, regenerated fibers exhibit reduced axon caliber, defective nerve conduction, and recovery of motor function is delayed. The growth hostile environment of Sarm1-/- distal nerve tissue was demonstrated by grafting of Sarm1-/- nerve into WT recipients. SC lineage tracing in injured WT and Sarm1-/- mice revealed morphological differences. In the Sarm1-/- distal nerve, the appearance of p75NTR+, c-Jun+ SCs is significantly delayed. Ex vivo, p75NTR and c-Jun upregulation in Sarm1-/- nerves can be rescued by pharmacological inhibition of ErbB kinase. Together, our studies show that Sarm1 is not necessary for the activation of neuron intrinsic growth programs but in the distal nerve is required for the orchestration of cellular programs that underlie rapid axon extension.

Salivary microbiome changes distinguish response to chemoradiotherapy in patients with oral cancer.

BACKGROUND: Oral squamous cell carcinoma (SCC) is associated with oral microbial dysbiosis. In this unique study, we compared pre- to post-treatment salivary microbiome in patients with SCC by 16S rRNA gene sequencing and examined how microbiome changes correlated with the expression of an anti-microbial protein. RESULTS: Treatment of SCC was associated with a reduction in overall bacterial richness and diversity. There were significant changes in the microbial community structure, including a decrease in the abundance of Porphyromonaceae and Prevotellaceae and an increase in Lactobacillaceae. There were also significant changes in the microbial community structure before and after treatment with chemoradiotherapy, but not with surgery alone. In patients treated with chemoradiotherapy alone, several bacterial populations were differentially abundant between responders and non-responders before and after therapy. Microbiome changes were associated with a change in the expression of DMBT1, an anti-microbial protein in human saliva. Additionally, we found that salivary DMBT1, which increases after treatment, could serve as a post-treatment salivary biomarker that links to microbial changes. Specifically, post-treatment increases in human salivary DMBT1 correlated with increased abundance of Gemella spp., Pasteurellaceae spp., Lactobacillus spp., and Oribacterium spp. This is the first longitudinal study to investigate treatment-associated changes (chemoradiotherapy and surgery) in the oral microbiome in patients with SCC along with changes in expression of an anti-microbial protein in saliva. CONCLUSIONS: The composition of the oral microbiota may predict treatment responses; salivary DMBT1 may have a role in modulating the oral microbiome in patients with SCC. After completion of treatment, 6 months after diagnosis, patients had a less diverse and less rich oral microbiome. Leptotrichia was a highly prevalent bacteria genus associated with disease. Expression of DMBT1 was higher after treatment and associated with microbiome changes, the most prominent genus being Gemella Video Abstract.

Increased Nerve Density Adversely Affects Outcome in Oral Cancer.

PURPOSE: Perineural invasion (PNI) in oral cavity squamous cell carcinoma (OSCC) is associated with poor survival. Because of the risk of recurrence, patients with PNI receive additional therapies after surgical resection. Mechanistic studies have shown that nerves in the tumor microenvironment promote aggressive tumor growth. Therefore, in this study, we evaluated whether nerve density (ND) influences tumor growth and patient survival. Moreover, we assessed the reliability of artificial intelligence (AI) in evaluating ND. EXPERIMENTAL DESIGN: To investigate whether increased ND in OSCC influences patient outcome, we performed survival analyses. Tissue sections of OSCC from 142 patients were stained with hematoxylin and eosin and IHC stains to detect nerves and tumor. ND within the tumor bulk and in the adjacent 2 mm was quantified; normalized ND (NND; bulk ND/adjacent ND) was calculated. The impact of ND on tumor growth was evaluated in chick chorioallantoic-dorsal root ganglia (CAM-DRG) and murine surgical denervation models. Cancer cells were grafted and tumor size quantified. Automated nerve detection, applying the Halo AI platform, was compared with manual assessment. RESULTS: Disease-specific survival decreased with higher intratumoral ND and NND in tongue SCC. Moreover, NND was associated with worst pattern-of-invasion and PNI. Increasing the number of DRG, in the CAM-DRG model, increased tumor size. Reduction of ND by denervation in a murine model decreased tumor growth. Automated and manual detection of nerves showed high concordance, with an F1 score of 0.977. CONCLUSIONS: High ND enhances tumor growth, and NND is an important prognostic factor that could influence treatment selection for aggressive OSCC. See related commentary by Hondermarck and Jiang, p. 2342.

The 3D's of Neural Phenotypes in Oral Cancer: Distance, Diameter, and Density.

Squamous cell carcinoma of the oral cavity (OSCC) is the most common type of head and neck cancer; survival is poor, and response to treatment varies. Metastasis or recurrence in the regional lymph nodes is associated with poor survival. Consequently, overt or occult spread to the lymph nodes is used to identify patients who will receive adjuvant radiation therapy. Perineural invasion and the diameter of nerves exhibiting perineural invasion have also been suggested to be of prognostic significance. The explosion of interest in cancer neuroscience in the last two decades has led to novel biological insights into interactions between nerves and tumor cells. However, the criteria for defining perineural invasion have lagged behind current knowledge. It is important to re-evaluate the concept of perineural invasion and identify other neural phenotypes in OSCC that can impact treatment selection and prognosis. In addition to perineural invasion, neural phenotypes that are of potential relevance to tumor progression include nerve-tumor distance, nerve diameter, and nerve density. This manuscript discusses the translational significance of recent mechanistic studies on the progression of oral cancer.

Spatial and Transcriptomic Analysis of Perineural Invasion in Oral Cancer.

PURPOSE: Perineural invasion (PNI), a common occurrence in oral squamous cell carcinomas, is associated with poor survival. Consequently, these tumors are treated aggressively. However, diagnostic criteria of PNI vary and its role as an independent predictor of prognosis has not been established. To address these knowledge gaps, we investigated spatial and transcriptomic profiles of PNI-positive and PNI-negative nerves. EXPERIMENTAL DESIGN: Tissue sections from 142 patients were stained with S100 and cytokeratin antibodies. Nerves were identified in two distinct areas: tumor bulk and margin. Nerve diameter and nerve-to-tumor distance were assessed; survival analyses were performed. Spatial transcriptomic analysis of nerves at varying distances from tumor was performed with NanoString GeoMx Digital Spatial Profiler Transcriptomic Atlas. RESULTS: PNI is an independent predictor of poor prognosis among patients with metastasis-free lymph nodes. Patients with close nerve-tumor distance have poor outcomes even if diagnosed as PNI negative using current criteria. Patients with large nerve(s) in the tumor bulk survive poorly, suggesting that even PNI-negative nerves facilitate tumor progression. Diagnostic criteria were supported by spatial transcriptomic analyses of >18,000 genes; nerves in proximity to cancer exhibit stress and growth response changes that diminish with increasing nerve-tumor distance. These findings were validated in vitro and in human tissue. CONCLUSIONS: This is the first study in human cancer with high-throughput gene expression analysis in nerves with striking correlations between transcriptomic profile and clinical outcomes. Our work illuminates nerve-cancer interactions suggesting that cancer-induced injury modulates neuritogenesis, and supports reclassification of PNI based on nerve-tumor distance rather than current subjective criteria.

Phosphorylation of TRIP13 at Y56 induces radiation resistance but sensitizes head and neck cancer to cetuximab.

Radiation therapy, a mainstay of treatment for head and neck cancer, is not always curative due to the development of treatment resistance; additionally, multi-institutional trials have questioned the efficacy of concurrent radiation with cetuximab, the epidermal growth factor receptor (EGFR) inhibitor. We unraveled a mechanism for radiation resistance; that is, radiation induces EGFR, which phosphorylates TRIP13 (thyroid hormone receptor interactor 13) on tyrosine 56. Phosphorylated (phospho-)TRIP13 promotes non-homologous end joining (NHEJ) repair to induce radiation resistance. NHEJ is the main repair pathway for radiation-induced DNA damage. Tumors expressing high TRIP13 do not respond to radiation but are sensitive to cetuximab or cetuximab combined with radiation. Suppression of phosphorylation of TRIP13 at Y56 abrogates these effects. These findings show that EGFR-mediated phosphorylation of TRIP13 at Y56 is a vital mechanism of radiation resistance. Notably, TRIP13-pY56 could be used to predict the response to radiation or cetuximab and could be explored as an actionable target.

Nerve density in cancer: Less is better.

The density of nerves in cancer is emerging as a relevant clinical parameter for patient survival. Nerves in the tumor microenvironment have been associated with poor survival and recurrence, particularly if involved in perineural invasion. However, usually only a few nerves inside a tumor are affected by perineural invasion, while most nerves are not. Mechanistic studies have shown nerve-secreted factors promote tumor growth and invasion thereby making tumors more aggressive. Therefore, the overall number of nerves in the tumor microenvironment should be more representative of the nerve-tumor biological interaction than perineural invasion. This review summarizes the available clinical information about nerve density as a measure of clinical outcome in cancer and explores the mechanisms underlying nerve density in cancer, specifically, neurogenesis, axonogenesis, and neurotropism.

Squamous cell carcinoma subverts adjacent histologically normal epithelium to promote lateral invasion.

Recurrent and new tumors, attributed in part to lateral invasion, are frequent in squamous cell carcinomas and lead to poor survival. We identified a mechanism by which cancer subverts adjacent histologically normal epithelium to enable small clusters of cancer cells to burrow undetected under adjacent histologically normal epithelium. We show that suppression of DMBT1 within cancer promotes aggressive invasion and metastasis in vivo and is associated with metastasis in patients. Cancer cells via TGFß1 and TNFα also suppress DMBT1 in adjacent histologically normal epithelium, thereby subverting it to promote invasion of a small population of tumor cells. The sufficiency of DMBT1 in this process is demonstrated by significantly higher satellite tumor nests in Dmbt1-/- compared with wild-type mice. Moreover, in patients, invasion of small tumor nests under adjacent histologically normal epithelium is associated with increased risk for recurrence and shorter disease-free survival. This study demonstrates a crucial role of adjacent histologically normal epithelium in invasion and its important role in the tumor microenvironment and opens new possibilities for therapeutic strategies that reduce tumor recurrence.

The Chick Chorioallantoic Membrane In Vivo Model to Assess Perineural Invasion in Head and Neck Cancer.

Perineural invasion is a phenotype in which cancer surrounds or invades the nerves. It is associated with poor clinical outcome for head and neck squamous cell carcinoma and other cancers. Mechanistic studies have shown that the molecular crosstalk between nerves and tumor cells occurs prior to physical interaction. There are only a few in vivo models to study perineural invasion, especially to investigate early progression, before physical nerve-tumor interactions occur. The chick chorioallantoic membrane model has been used to study cancer invasion, because the basement membrane of the chorionic epithelium mimics that of human epithelial tissue. Here we repurposed the chick chorioallantoic membrane model to investigate perineural invasion, grafting rat dorsal root ganglia and human head and neck squamous cell carcinoma cells onto the chorionic epithelium. We have demonstrated how this model can be useful to evaluate the ability of cancer cells to invade neural tissue in vivo.

Redefining Perineural Invasion: Integration of Biology With Clinical Outcome.

A diagnosis of perineural invasion (PNI), defined as cancer within or surrounding at least 33% of the nerve, leads to selection of aggressive treatment in squamous cell carcinoma (SCC). Recent mechanistic studies show that cancer and nerves interact prior to physical contact. The purpose of this study was to explore cancer-nerve interactions relative to clinical outcome. Biopsy specimens from 71 patients with oral cavity SCC were stained with hematoxylin and eosin and immunohistochemical (IHC; cytokeratin, S100, GAP43, Tuj1) stains. Using current criteria, PNI detection was increased with IHC. Overall survival (OS) tended to be poor for patients with PNI (P = .098). OS was significantly lower for patients with minimum tumor-nerve distance smaller than 5 µm (P = .011). The estimated relative death rate decreased as the nerve-tumor distance increased; there was a gradual drop off in death rate from distance equal to zero that stabilized around 500 µm. In PNI-negative patients, nerve diameter was significantly related to OS (HR 2.88, 95%CI[1.11,7.49]). Among PNI-negative nerves, larger nerve-tumor distance and smaller nerve diameter were significantly related to better OS, even when adjusting for T-stage and age (HR 0.82, 95% CI[0.72,0.92]; HR 1.27, 95% CI[1.00,1.62], respectively). GAP43, a marker for neuronal outgrowth, stained less than Tuj1 in nerves at greater distances from tumor (OR 0.76, 95% CI[0.73,0.79]); more GAP43 staining was associated with PNI. Findings from a small group of patients suggest that nerve parameters other than presence of PNI can influence outcome and that current criteria of PNI need to be re-evaluated to integrate recent biological discoveries.

An Uncommon Occurrence of Three-Fourth Molars Concomitant to Hypodontia in a Nonsyndromic Patient.

Bilaterally mandibular and maxillary fourth molars, concomitant to hypodontia, are uncommon. These dental anomalies, called hypohyperdontia, are rare and etiology is not well defined. Several complications may occur as a consequence of supernumerary teeth, such as teeth impaction, resorption of adjacent teeth, and development of cysts and tumors. The authors present an uncommon occurrence of three-fourth molars concomitant to hypodontia and dentigerous cyst associated with impacted mandibular second and third molars in a nonsyndromic patient. The authors attempt to discuss the occurrence of bilaterally fourth molars and reinforce the importance of clinical approach, considering risks and benefits for the patient.

Conhecimento dos alunos de Odontologia na identificação do câncer oral / Knowledge of dental students on the identification of oral cancer

Objetivo: Este estudo buscou avaliar o conhecimento e as percepções dos estudantes de Odontologia sobre o câncer oral, nas quatro faculdades de Odontologia do estado do Espírito Santo. Material e Métodos: Um questionário objetivo composto por 16 perguntas foi aplicado presencialmente aos alunos matriculados nos dois últimos períodos de Odontologia das respectivas instituições mediante assinatura do termo de consentimento livre e esclarecido. Resultados: Os dados obtidos revelam que, dos 196 participantes voluntários, 94,9% acreditam ser responsáveis pelo diagnóstico e prevenção do câncer oral, 67,3% consideram seu grau de conhecimento sobre o assunto insuficiente ou mediano e 53,6% não reconhecem, com precisão, os principais fatores de risco para este câncer. Conclusão: Esses achados reiteram a importância de instruir e capacitar o cirurgião-dentista, desde a sua formação, sobre o tema câncer oral, a fim de que estes sejam profissionais aptos a intervir, diagnosticar e prevenir essa enfermidade.

Objective: This study assessed the knowledge and perceptions of dental students about oral cancer in the four dental schools in the state of Espirito Santo. Material and Methods: A 16-question questionnaire was given to the participants after agreeing to informed consent. All participants were students enrolled in the last two semesters of dental school. Results: The results showed that among 196 participants, 94.9% believed they were responsible for the diagnosis and prevention of oral cancer, 67.3% considered their level of knowledge on the subject insufficient or moderately sufficient and 53.6% did not recognize with precision the main risk factors for oral cancer. Conclusion: These findings reaffirm the importance of proper instruction in diagnosing oral cancer in dental school in order for graduates to be able to properly diagnose and prevent the disease.

Hyperplastic dental follicle: a case report and literature review.

Hyperplastic dental follicle is an odontogenic hamartomatous lesion associated with delayed or tooth eruption failure in young patients. The occurrence of this pericoronal dental lesion may be single or multiple and it seems to be more frequent than literature has reported. We present a literature review focusing on the etiopathogenesis and clinicopathological features of this hamartomatous lesion in young patients. In addition, we reported a case of hyperplastic dental follicle causing delayed tooth eruption of 14-year-old male patient. Microscopic analyses based on routine staining and immunohistochemistry were used to discuss the cells found in pericoronal follicle. This paper reinforces the importance of association between clinical history and radiographic features with microscopic pericoronal follicle examination for diagnosis of this hamartomatous lesion.

Comparison of radiographic measurements obtained with conventional and indirect digital imaging during endodontic treatment.

The aims of this study were to evaluate the quality of indirect digitized radiographic images taken during endodontic procedures and to compare the measurements recorded with this technique to those obtained from conventional radiographs. Two-hundred conventional periapical radiographs taken at the undergraduate Endodontics Clinic of the Dental School of Bauru were digitized. The conventional and indirect digitized images were compared by three examiners as to the quality and accuracy of the measurements recorded during endodontic treatment, in canal length determination, gutta-percha adaptation, lateral condensation and final obturation. The conventional radiographs were observed on a film viewer, surrounded by a dark card, and measured with magnifying glass and a millimeter ruler; the indirect digitized images were evaluated on the Digora for Windows software, with free utilization of the bright/contrast tool. Unlike the conventional radiographic images, all indirect digitized images were considered as having a high quality. The distance between the filling material and the root apex was 0.117 mm larger, on average, for the Digora system (p<0.01). The measurements achieved by the investigated radiographic methods were clinically similar and they are thus equivalent. Changes in brightness and contrast of the images using Digora software improved the diagnosis.

Comparison of radiographic measurements obtained with conventional an indirect digital imaging during endontic treatment

The aims of this study were to evaluate the quality of indirect digitized radiographic images taken during endodontic procedures and to compare the measurements recorded with this technique to those obtained from conventional radiographs. Two-hundred conventional periapical radiographs taken at the undergraduate Endodontics Clinic of the Dental School of Bauru were digitized. The conventional and indirect digitized images were compared by three examiners as to the quality and accuracy of the measurements recorded during endodontic treatment, in canal length determination, gutta-percha adaptation, lateral condensation and final obturation. The conventional radiographs were observed on a film viewer, surrounded by a dark card, and measured with magnifying glass and a millimeter ruler; the indirect digitized images were evaluated on the Digora® for Windows software, with free utilization of the bright/contrast tool. Unlike the conventional radiographic images, all indirect digitized images were considered as having a high quality. The distance between the filling material and the root apex was 0.117 mm larger, on average, for the Digora® system (p<0.01). The measurements achieved by the investigated radiographic methods were clinically similar and they are thus equivalent. Changes in brightness and contrast of the images using Digora®software improved the diagnosis.