Genomic Engineering of Oral Keratinocytes to Establish In Vitro Oral Potentially Malignant Disease Models as a Platform for Treatment Investigation.

Precancerous cells in the oral cavity may appear as oral potentially malignant disorders, but they may also present as dysplasia without visual manifestation in tumor-adjacent tissue. As it is currently not possible to prevent the malignant transformation of these oral precancers, new treatments are urgently awaited. Here, we generated precancer culture models using a previously established method for the generation of oral keratinocyte cultures and incorporated CRISPR/Cas9 editing. The generated cell lines were used to investigate the efficacy of a set of small molecule inhibitors. Tumor-adjacent mucosa and oral leukoplakia biopsies were cultured and genetically characterized. Mutations were introduced in CDKN2A and TP53 using CRISPR/Cas9 and combined with the ectopic activation of telomerase to generate cell lines with prolonged proliferation. The method was tested in normal oral keratinocytes and tumor-adjacent biopsies and subsequently applied to a large set of oral leukoplakia biopsies. Finally, a subset of the immortalized cell lines was used to assess the efficacy of a set of small molecule inhibitors. Culturing and genomic engineering was highly efficient for normal and tumor-adjacent oral keratinocytes, but success rates in oral leukoplakia were remarkably low. Knock-out of CDKN2A in combination with either the activation of telomerase or knock-out of TP53 seemed a prerequisite for immortalization. Prolonged culturing was accompanied by additional genetic aberrations in these cultures. The generated cell lines were more sensitive than normal keratinocytes to small molecule inhibitors of previously identified targets. In conclusion, while very effective for normal keratinocytes and tumor-adjacent biopsies, the success rate of oral leukoplakia cell culturing methods was very low. Genomic engineering enabled the prolonged culturing of OL-derived keratinocytes but was associated with acquired genetic changes. Further studies are required to assess to what extent the immortalized cultures faithfully represent characteristics of the cells in vivo.

The value of regular follow-up of oral leukoplakia for early detection of malignant transformation.

OBJECTIVES: Evaluate whether regular follow-up of oral leukoplakia (OL) resulted in early detection of malignant transformation (MT). METHOD: Two hundred and twenty-two consecutive patients with OL (147 females, 75 males); median follow-up period of 64 months (range: 12-300). Three groups were distinguished: group A (n = 92) follow-up at the hospital; group B (n = 84) follow-up by their dentist; group C (n = 46) lost to follow-up. RESULTS: OLs in group B compared to group A, were smaller in size (<2 cm; p < 0.001), showed more hyperkeratosis (p < 0.001) and less moderate/severe dysplasia (p < 0.001). MT occurred in 45 (20%) patients: 32 (35%) in group A, five (6%) in group B and eight (17%) in group C. There was no significant difference in clinical tumour size between group A (median: 15 mm, range: 1-40) and group B (median: 10 mm, range: 3-25; p = 0.496). Tumour size was smaller for patients in groups A and B (median: 10 mm, range 1-40) compared to group C (median: 33 mm, range: 3-100; p = 0.003). There was a positive correlation between tumour size and interval between the last visit in all patients (p = 0.022). CONCLUSION: Regular follow-up of OL resulted in early detection of MT. If properly selected, follow-up of OL performed by the dentist seems feasible.

The role of differentiated dysplasia in the prediction of malignant transformation of oral leukoplakia.

OBJECTIVE: Oral leukoplakia is the most common oral potentially malignant disorder. Malignant transformation of oral leukoplakia occurs at an annual rate of 1%-7%. WHO-defined classic epithelial dysplasia is an important predictor of malignant transformation of oral leukoplakia, but we have previously shown in a proof of concept study that prediction improves by incorporation of an architectural pattern of dysplasia, also coined as differentiated dysplasia. We aimed to analyze this finding in a larger cohort of patients. METHOD: For this retrospective study 176 oral leukoplakia patients were included. Biopsies for all patients were assessed for the presence of dysplasia and analyzed for cytokeratin 13 and 17 expression. Moreover, the inter-observer agreement for the diagnosis of differentiated dysplasia was determined. RESULTS: In total, 33 of 176 patients developed oral squamous cell carcinoma during follow-up. Presence of classic epithelial dysplasia increased cancer risk two-fold (HR = 2.18, p = 0.026). Lesions without classic epithelial dysplasia could be further risk-stratified by the presence of differentiated dysplasia (HR = 7.36, p < 0.001). Combined classic epithelial and differentiated dysplasia imparted a seven-fold increased risk of malignant transformation (7.34, p = 0.001). Inter-observer agreement for the diagnosis of dysplasia, including differentiated dysplasia, was moderate (κ = 0.56, p < 0.001). DISCUSSION: This study emphasizes the importance of the recognition of the architectural pattern of differentiated dysplasia as a separate entity for risk prediction of malignant transformation of oral leukoplakia. Presence of any pattern of dysplasia results in accurate prediction of malignant transformation risk of oral leukoplakia.

A narrative review of the role of Eph receptors in head and neck squamous cell carcinoma.

Tyrosine kinase receptors (TKR) coordinate a variety of pathological processes in head and neck squamous cell carcinoma (HNSCC), and eventually play a role in patient outcomes. In this review, the role of Eph receptors in HNSCC progression and the possibility of targeting these receptors are illustrated. All relevant studies were identified through a comprehensive search of four electronic databases, including PubMed, Scopus, web of science, and Embase till August 2022. EphA2 and EphB4, along with ephrin-B2, were the most extensively studied proteins in this family. However, overexpression of EphB4 and its ligand ephrin-B2 were the only proteins that consistently showed association with a poor outcome, indicating that these proteins might serve as valuable prognostic markers in HNSCC. High expression of EphA3 and EphB4 was found to play a crucial role in radioresistance of HNSCC. EphB4 loss, in particular, was observed to induce an immunosuppression phenotypic HNSCC. Currently, ongoing clinical trials are investigating the benefits of EphB4-ephrin-B2 blockade in combination with standard of care treatment in HNSCC. Further efforts are needed to explore the biological role and behavioral complexity of this family of TKR in HNSCC with great attention to avoid heterogeneity of HNSCC subsites.

Significance of immunohistochemistry biomarkers in prediction of malignant transformation of oral lichen planus: A systematic review

Background: Oral lichen planus (OLP) is a chronic inflammatory disorder with increased risk for malignanttransformation. Biomarker validation is a pivotal step in moving newly discovered biomarkers towards clinicalimplementation. We performed a systematic review of studies on biomarkers related to OLP, wherein biomarkershave been described in at least two independent studies. Our aim was to determine whether any of these biomarkers might be promising in predicting the increased risk of malignant transformation of OLP.Material and Methods: We searched the following databases until August 2021: PUBMED, EMBASE, and Web ofScience. Due to high heterogeneity, a qualitative rather than quantitative assessment was conducted. Only proteinsthat consistently showed a significantly high level of expression in neoplastic tissues versus OLP in two or morepublications were considered as promising markers.Results: Initial database researches identified 1671, of which 24 articles were included in the final analysis. Themost frequently reported proteins were p53, Bcl-2 and Ki-67, though there were controversies. PCNA and P21were the only proteins that showed consistent evidence of clinical usefulness as cancer predictors to be consideredas promising markers. Extensive methodological variations in the evaluation of expressions and statistical analyses of the included markers were observed, which hampered comparisons of the results. Conclusions: Multiple levels of heterogeneity with a scarcity of high-quality studies were identified. PCNA and P21were identified as promising predictive markers for evaluating the risk of malignant transformation of OLP, but theyrequire further validation. The focus of future research on validation of predictive biomarkers of OLP should be considered as a high priority because it will accelerate the introduction of newly discovered markers into the clinical setting. (AU)

Interobserver and intraobserver variability in the radiological assessment of sialolithiasis using cone beam computed tomography

Background: Data regarding the inter- and intraobserver variability in the radiological assessment of sialolithiasis using cone beam computed tomography are missing in the current literature. This study assessed the inter- and intraobserver variability in the radiological assessment of sialolithiasis using cone beam computed tomography (CBCT). Material and Methods: In 107 patients, 130 salivary glands (65 parotid and 65 submandibular) with clinical signs of obstruction were assessed by four independent observers; 2 residents OMFS and 2 experienced OMFS. The observers analyzed the CBCT images and determined the absence or presence of one or more salivary stones in the affected gland. This procedure was repeated after three months. Results: Interobserver agreements showed kappa values of 0.84 for the parotid gland, and 0.93 for the submandibular gland. Intraobserver agreements for the whole group reported kappa values between 0.83 - 0.95. There was no significant difference between residents and experienced OMFS. Conclusions: Due to the good inter- and intraobserver agreement, CBCT appears to be a reproducible imaging modality for detecting salivary stones in patients with signs and symptoms of obstructed parotid and submandibular glands.(AU)

Relationship between volume of submandibular salivary stones in vivo determined with Cone-Beam Computer Tomography and in vitro with micro-Computer Tomography / Relación entre el volumen de cálculos salivales submandibulares in vivo determinado con tomografía computarizada de haz cónico e in vitro con microtomografía computarizada

Background: Successful removal of salivary stones depends on exact pretreatment information of the location, thesize and shape of the stones. This study aimed to compare the volume of submandibular sialoliths determined bypreoperative Cone-Beam Computer Tomography (CBCT) scans with the volume of the removed stones on micro-Computer Tomography (micro-CT) scans.Material and Methods: In this study, using twenty-one submandibular sialoliths, the pretreatment volumes in-vivomeasured on CBCT were compared to the volumes of removed stones determined by micro-CT scans. The volumemeasured on micro-CT scans served as the gold standard. Pre-operative CBCT’s and in-vitro micro-CT’s wereconverted into standard tessellation language models (STL-models) using an image segmentation software pack-age. The CBCT and micro-CT images of the stones were subsequently metrologically assessed and compared toeach other using reverse engineering software.Results: Volumes of submandibular sialoliths determined by CBCT’s correlated significantly with volumes mea-sured on micro-CT’s (Spearman’s coefficient r = 0.916). The interquartile range (IQR) for the volume measuredwith micro-CT was 117.23. The median is 26.41. For the volume measured with CBCT the IQR was 141.3 and themedian 36.61. The average volume on micro-CT is smaller than on CBCT.Conclusions: When using CBCT-scans for the detection of submandibular sialoliths one should realize that in-vivothose stones are actually a fraction smaller than assessed on the preoperative scan. This is important when cut-offvalues of sizes of stones are used in the pretreatment planning of stone removal.(AU)

Enduring rural poverty: Stigma, class practices and social networks in a town in the Groninger Veenkoloniën.

In the Groninger Veenkoloniën, a former peat region in the northeast of the Netherlands, persistent poverty is more prevalent compared to other rural regions in the country. Grounded in participant observations and supplemented by in-depth interviews capturing the social life history of 21 participants, this paper paints a detailed picture of the social networks and class practices of those experiencing persisting poverty in the examined town and surrounding region. In addition, we explore the relations between the rural context and lived experiences of class and poverty. Our findings highlight the complex experience as well as spatial embeddedness of persisting poverty. We find that, although the specific circumstances to which the participants are exposed vary greatly, the repercussions in terms of the characteristics of their social networks and practices are very similar. In general, the social networks of participants are fragmented and small, tightly knit, and characterized by clear power imbalances. The most formative experiences that result in the isolation of networks of poor are found to occur in the home and family situation during childhood years. We argue that poverty and the region's history are intricately interwoven resulting in a socio-spatial stigma which in turn contributes to the persistent and intergenerational character of poverty in the rural context of our study. Due to the long history of stigmatization, dismantling the socio-spatial stigma attached to the Groninger Veenkoloniën will presumably take multiple generations.

Biochemical composition of salivary stones in relation to stone- and patient-related factors

BACKGROUND: Salivary stones are calcified structures most often found in the main duct of the submandibular or parotid salivary gland. They contain of a core surrounded by laminated layers of organic and inorganic material. MATERIAL AND METHODS: Submandibular and parotid sialoliths (n=155) were collected at the department of Oral and Maxillofacial surgery of a general hospital between February 1982 and September 2012. The weight of the sialo-liths was determined and the consistency was subjectively classified. Subsequently, the biochemical composition of the stones was determined by wet chemical methods or FT-IR spectrometry. Age and gender of the patients were retrieved from their medical records. Data were statistically analyzed using Fisher's exact tests. RESULTS: Sialoliths are mainly composed of inorganic material. Carbonate apatite was identified in 99% of the stones, phosphate in 88%, calcium in 87%, magnesium in 68%, struvite in 44%, oxalate in 38% and carbonate in 35%. Solid salivary stones contain more frequently struvite than stones with a soft consistency (p = 0.05). Larger stones (> 100 mg) contain more frequently carbonate (p = 0.05). Stones from older patients (≥ 38 years) showed an almost significant trend towards more frequent presence of phosphate (p = 0.083). CONCLUSIONS: The biochemical composition of submandibular and parotid sialoliths is related to stone-related factors, probably to age but not to the gender of the patient

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Complications of Sialendoscopy in Patients With Sjögren Syndrome.

PURPOSE: Recent literature suggests that sialendoscopy of the major salivary glands could alleviate symptoms of Sjögren syndrome (SS) and restore salivary function. The aim of this study was to evaluate the authors' experience of sialendoscopy of the salivary glands in patients with SS. MATERIALS AND METHODS: In this retrospective case series study, the surgical data of patients with SS who had undergone sialendoscopy at the VU University Medical Center (Amsterdam, The Netherlands) from November 2014 through April 2015 were used. Outcome measurements were successful entry to the salivary gland and completion of sialendoscopy as planned. Furthermore, pre- and postoperative complications were scored. Descriptive analysis of the data was performed. RESULTS: Surgical data of sialoendoscopic procedures in 26 patients with SS (24 women and 2 men; mean age, 57 yr; range, 27 to 72 yr) were analyzed. Sialendoscopy was successfully performed in 78 of 104 salivary glands (75%; 50 parotid and 28 submandibular glands) in the 26 patients. Sialendoscopy failed in 26 of the 104 sialoendoscopic procedures (25%; 2 parotid and 24 submandibular glands). In 16 salivary glands, the ductal orifice could not be identified. In 7 salivary glands, sialendoscopy could not be performed because of partial or complete stenosis of the salivary duct. In 3 salivary glands, sialendoscopy was not performed because of a ductal perforation. Three patients developed a postoperative infection. CONCLUSION: The overall rate of complications was limited and the sialoendoscopic complications in patients with SS could be regarded as minor. Most complications were seen for sialendoscopy of the submandibular glands in this specific patient category. Careful preoperative selection of patients and salivary glands could contribute to a lower rate of complications and more predictable results.

Fracture of the genial tubercles of the mandible: case report and review of the literature.

Fracture of the genial tubercles is a rare occurrence and can occur as a solitary fracture or an associated fracture of the mandible. A solitary fracture seems to be associated with severe atrophy of the mandible. A report of a case of fractured genial tubercles in an 80-year-old edentulous female patient is described and a review of the literature is presented.

[A woman with a pigmentation of the hard palate]. / Een vrouw met een pigmentatie van het palatum durum.

A bluish flat pigmented lesion of the hard palate of a 51-year-old woman was excised to exclude malignancy, in particular oral malignant melanoma. On histopathological examination, depositions of black pigment were seen accompanied by several foreign body giant cells. Probably due to a childhood trauma, a pencil point had penetrated the hard palate.

[Sialendoscopy for obstructive disorders of the salivary glands]. / Sialendoscopie voor obstructieve speekselklieraandoeningen.

Obstructive disorders of the salivary glands usually manifest as a swelling which is associated with meals ('mealtime syndrome') as well as inflammation of the gland. Obstruction of the salivary ducts of the parotid and submandibular glands is usually caused by a salivary stone, mucous plug or by a stenosis. Until recently, the options for diagnosis and treating these conditions were limited. Ultimately, the decision to remove the salivary gland was often made. Sialendoscopy is a recently developed semi-rigid optical technique that can be used to detect and usually treat obstructions in the salivary ductal system. This new, minimally invasive technique has resulted in the reduction of morbidity related to salivary gland obstruction. In many patients, surgical removal of the salivary gland can be avoided by using this technique.

Traumatic lingual ulceration in a newborn: Riga-Fede disease.

Riga Fede disease is a reactive mucosal disease as a result of repetitive trauma of the tongue by the anterior primary teeth during forward and backward movement. Although the aspect of the lesion might be impressive, its nature is relatively benign. The history and clinical features are most often so typical that there is seldom a need for addititonal histopathological examination. Riga Fede disease can most often be treated with conservative measures only.Beside the presentation of a six-month-old boy with Riga Fede disease, the literature has been reviewed as well. From this review it can be concluded that Riga Fede disease is almost exclusively restricted to the tongue, occurs soon after birth when associated with (neo)natal teeth, has a male predilection, and is in one quarter of the cases associated with neurologic disorders. In the later case, Riga Fede disease develops after the age of 6 months.

The possible premalignant character of oral lichen planus and oral lichenoid lesions: a prospective study.

OBJECTIVES: The possible malignant transformation of oral lichen planus (OLP) is the subject of an ongoing and controversial discussion in the literature. The main criticism of studies on this subject relates to the lack of sufficient data to support the initial diagnosis of OLP in cases that finally developed into squamous cell carcinoma. We describe the possible premalignant character of OLP and oral lichenoid lesions (OLL) of a prospectively followed cohort of patients with detailed documentary data. STUDY DESIGN: A study group of 173 patients, 62 patients diagnosed with OLP and 111 patients with OLL, according to revised, modified World Health Organization diagnostic criteria, was followed up from 6.6 to 72.0 months (mean, 31.9 months). The expected number of patients with oral cancer in the group of patients with OLP and in the group of patients with OLL was estimated by comparing the number of patients, their ages, sex, and the length of follow-up to annual incidence rates of oral cancer for the general Dutch population, to explore the possibility of coincidental carcinomas. The binomial test was used to determine whether the observed number of cases of cancer in the OLP group and the OLL group exceeded the expected numbers. RESULTS: Three of 173 patients (1.7%), 2 men and 1 woman, developed squamous cell carcinomas of the oral mucosa during follow-up. All malignant transformations occurred in the OLL group. The annual malignant transformation rate, based on a mean follow-up of 31.9 months, was calculated as 0.65% per year. A comparison of the expected against actual figures for the development of carcinomas revealed no increase in patients with OLP and a 219-fold increase in patients with OLL, with the latter not statistically significant, but with a P value of .083, suggesting at least a trend. CONCLUSION: Our results give support to the hypothesis that patients with OLL have an increased risk of oral cancer, but this increased risk was not detected in our sample of patients with OLP. Before a final statement with regard to the premalignant character of OLP and OLL can be formulated, the present follow-up study should be prolonged and expanded with a larger number of patients. Until then, we advise that patients with OLP and OLL should undergo biannual follow-up examinations. Follow-up will be particularly important in patients with OLL who have atrophic/erosive/ulcerative lesions.

Cheilitis granulomatosa: overview of 13 patients with long-term follow-up--results of management.

BACKGROUND: Cheilitis granulomatosa, often regarded as a subtype of orofacial granulomatosis, is characterized by recurrent or persistent swelling of one or both lips. Classically, a non-necrotizing granulomatous inflammation is seen at histologic examination. Although a relationship has been proposed between Melkersson-Rosenthal syndrome (and the monosymptomatic form, cheilitis granulomatosa) and Crohn's disease on the basis of the orofacial swelling and similar histology, several studies of Melkersson-Rosenthal syndrome have not found an association with Crohn's disease. METHODS: The clinical features, histopathology, association with Crohn's disease, and results of nonsurgical and surgical therapy in 13 patients with cheilitis granulomatosa were investigated in a retrospective case study with a mean follow-up period of 8.2 years. RESULTS: There was a low chance of developing Crohn's disease. Most patients in this study responded to nonsurgical treatment modalities. Patients with deterioration of lip swelling usually responded to intralesional injections with triamcinolone or to short courses of systemic glucocorticoids. Nonsteroidal systemic modalities, such as clofazimine, hydroxychloroquine, or sulfasalazine, were alternatives to glucocorticoid regimens, thus avoiding the long-term side effects of corticosteroids. Surgical intervention should only be performed in severely disfiguring cases. CONCLUSIONS: The management of cheilitis granulomatosa remains a challenge. As this study revealed a low chance of developing Crohn's disease, it does not seem justified to inform patients with cheilitis granulomatosa of the possibility that they might develop Crohn's disease. Patients with a negative history of gastrointestinal complaints should not be exposed to routine investigations of the gastrointestinal tract.

Queilitis granulomatosa: revisión de 13pacientes con seguimiento a largo plazo.Resultados del tratamiento / Cheilitis granulomatosa: overview of 13 patients with long-term follow-up-results of management

Antecedentes: La queilitis granulomatosa, que generalmente se considera como un subtipo de la granulomatosis orofacial, se caracteriza por inflamación persistente o recurrente de uno o ambos labios. Típicamente, en la exploración histológica se observa una inflamación granulomatosa no necrótica. Aunque se ha propuesto que existe una relación entre el síndrome Melkersson-Rosenthal (y la forma monosintomática, queilitis granulomatosa) y la enfermedad de Crohn, en función de la inflamación orofacial y la histología similar, en varios estudios del síndrome Melkersson-Rosenthal no se ha encontrado ninguna asociación con la enfermedad de Crohn. Métodos: Se investigaron las manifestaciones clínicas, la histopatología, la asociación con la enfermedad de Crohn y los resultados del tratamiento no quirúrgico y quirúrgico en 13 pacientes con queilitis granulomatosa en un estudio de casos retrospectivo con un seguimiento medio de 8,2 años. Resultados: Hubo pocas posibilidades de desarrollar la enfermedad de Crohn. La mayoría de los pacientes de este estudio respondieron a modalidades de tratamiento no quirúrgicas. Los pacientes con deterioro de la inflamación de los labios, generalmente, respondieron a inyecciones intralesionales con triamcinolona o a ciclos cortos de glucocorticoides sistémicos. Los tratamientos sistémicos no esteroideos, como clofacimina, hidroxicloroquina o sulfasalacina, fueron alternativas a los tratamientos con glucocorticoides, evitando así los efectos secundarios de los corticosteroides a largo plazo. Sólo debería realizarse una intervención quirúrgica en algunos casos de pacientes muy desfigurados. Conclusiones: El tratamiento de la queilitis granulomatosa sigue siendo un reto. Puesto que este estudio demostró que la posibilidad de desarrollar enfermedad de Crohn era muy baja, no está justificado informar a los pacientes con queilitis granulomatosa de que tienen la posibilidad de desarrollar la enfermedad de Crohn. Los pacientes sin antecedentes de problemas gastrointestinales no deberían someterse a investigaciones sistemáticas del tracto gastrointestinal (AU)