A CNN-based approach for joint segmentation and quantification of nuclei and NORs in AgNOR-stained images.

BACKGROUND AND OBJECTIVE: Oral cancer is the sixth most common kind of human cancer. Brush cytology for counting Argyrophilic Nucleolar Organizer Regions (AgNORs) can help early mouth cancer detection, lowering patient mortality. However, the manual counting of AgNORs still in use today is time-consuming, labor-intensive, and error-prone. The goal of our work is to address these shortcomings by proposing a convolutional neural network (CNN) based method to automatically segment individual nuclei and AgNORs in microscope slide images and count the number of AgNORs within each nucleus. METHODS: We systematically defined, trained and tested 102 CNNs in the search for a high-performing solution. This included the evaluation of 51 network architectures combining 17 encoders with 3 decoders and 2 loss functions. These CNNs were trained and evaluated on a new AgNOR-stained image dataset of epithelial cells from oral mucosa containing 1,171 images from 48 patients, with ground truth annotated by specialists. The annotations were greatly facilitated by a semi-automatic procedure developed in our project. Overlapping nuclei, which tend to hide AgNORs, thus affecting their true count, were discarded using an automatic solution also developed in our project. Besides the evaluation on the test dataset, the robustness of the best performing model was evaluated against the results produced by a group of human experts on a second dataset. RESULTS: The best performing CNN model on the test dataset consisted of a DenseNet-169 + LinkNet with Focal Loss (DenseNet-169 as encoder and LinkNet as decoder). It obtained a Dice score of 0.90 and intersection over union (IoU) of 0.84. The counting of nuclei and AgNORs achieved precision and recall of 0.94 and 0.90 for nuclei, and 0.82 and 0.74 for AgNORs, respectively. Our solution achieved a performance similar to human experts on a set of 291 images from 6 new patients, obtaining Intraclass Correlation Coefficient (ICC) of 0.91 for nuclei and 0.81 for AgNORs with 95% confidence intervals of [0.89, 0.93] and [0.77, 0.84], respectively, and p-values < 0.001, confirming its statistical significance. Our AgNOR-stained image dataset is the most diverse publicly available AgNOR-stained image dataset in terms of number of patients and the first for oral cells. CONCLUSIONS: CNN-based joint segmentation and quantification of nuclei and NORs in AgNOR-stained images achieves expert-like performance levels, while being orders of magnitude faster than the later. Our solution demonstrated this by showing strong agreement with the results produced by a group of specialists, highlighting its potential to accelerate diagnostic workflows. Our trained model, code, and dataset are available and can stimulate new research in early oral cancer detection.

Continuing education activities improve dentists' self-efficacy to manage oral mucosal lesions and oral cancer.

OBJECTIVES: This cross-sectional study aims to evaluate whether Continuing Education Activities (CEA) influence dentists' behaviour in relation to oral lesions. The secondary aim is to assess the association between dentists' perception of learning adequacy and self-efficacy for oral mucosal lesion management. METHODS: A self-administered online questionnaire was conducted on dentists working at the public health system of Rio Grande do Sul State, Brazil. The questionnaire included questions pertaining to perception of adequacy for oral diagnosis classes upon graduation, participation in oral cancer CEA and self-efficacy in managing oral mucosal lesions. RESULTS: 221 dentists from 91 municipalities answered the questionnaire. Most participants were female (71.5%) with a mean age of 38.3 years. Perception of learning as adequate during undergraduate coursework was associated with self-efficacy to diagnose, biopsy, and treat oral mucosal lesions (P < .05, Chi-squared test). However, 83.3% of dentists considered the time devoted to these topics prior to graduation insufficient. The frequency of oral lesion detection was related to self-efficacy to treat oral lesions and detecting oral cancer (P < .05, Chi-squared test). Among dentists who detected oral lesions frequently, 88.9% had attended CEA, whereas 11.1% of them had never attended these activities. CONCLUSIONS: CEA may improve awareness and efficacy of primary healthcare professional's detection of oral cancer.

Oral, dental, and craniofacial features in chronic acid sphingomyelinase deficiency.

The aim of this study was to evaluate the oral, dental, and craniofacial features of individuals affected by the chronic forms of acid sphingomyelinase deficiency (ASMD). This study comprised a sample of adult and pediatric patients (n = 8) with chronic ASMD. The individuals underwent oral examinations to evaluate the occurrence of caries, as well as full-mouth periodontal examinations, to assess the occurrence and severity of periodontal diseases. Panoramic and profile radiographs were obtained to analyze dental conditions and craniofacial parameters. Participants also answered questionnaires to identify systemic impairment, parafunctional habits, and bruxism. Dental anomalies of size, shape, and number were found, with agenesis and microdontia being the predominant findings. The average of caries experience was 11.75 (±8.1). Only one patient had periodontal health and all adult individuals had periodontitis at different stages and degrees. Bruxism was found in 87.5% of the sample. The convex profile and maxillary and mandibular retrusion were the most relevant findings in the cephalometric analysis. It is concluded that individuals with chronic ASMD, in addition to several systemic manifestations, present significant modifications in their oral health, from a greater occurrence of dental anomalies, caries, periodontal disease, in addition to skeletal changes.

Cytopathological tests for early detection of oral carcinogenesis.

The carcinogenesis in the oral cavity occurs as a multistep process and is often preceded by potentially malignant lesions. The main risk factors for the development of oral cancer are smoking and alcohol intake. The current challenge is to identify patients at greatest risk for the development of oral cancer using noninvasive and effective methods. The aim of this study is to evaluate the microsatellite mutations in the 9p21 locus, the cell proliferative activity, the pattern of epithelial desquamation, and the nucleus/cytoplasm ratio of exfoliated epithelial cells. Cytopathological samples were collected from 131 individuals divided into four groups: control (n = 26), alcohol-smoking (n = 32), leukoplakia (n = 38), and the oral squamous cell carcinoma group (OSCC, n = 35). From the cytological scraping, a slide was silver impregnated for Ag-stained nucleolar organizer region analysis and another slide was stained using the Papanicolaou technique. The remaining cells were used for DNA extraction, followed by PCR amplification and capillary electrophoresis. The cell proliferation velocity rate was higher in the leukoplakia and OSCC groups compared with the control group (P < 0.05). The leukoplakia group showed increased anucleated scales, whereas the nucleated superficial predominated in the control group and the parabasal cells in the OSCC group (P < 0.05). An increased nucleus/cytoplasm ratio was detected only in the OSCC group (P < 0.05). The 9p21 locus mutation frequency was higher in the alcohol-smoking and leukoplakia groups. 9p21 analysis and Ag-stained nucleolar organizer region methods are promising for the screening and monitoring of individuals at higher risk for the development of oral cancer.

Podoplanin Expression in Odontogenic Keratocysts Associated or not Associated With Nevoid Basal Cell Carcinoma Syndrome.

BACKGROUND: Podoplanin is a transmembrane glycoprotein expressed on various normal or neoplastic cells. Some studies have shown that podoplanin promotes the migration and invasion of tumor cells. This study evaluated a podoplanin expression in Odontogenic Keratocysts (OKs) associated or not associated with Nevoid Basal Cell Carcinoma Syndrome (NBCCS) and in Orthokeratinized Odontogenic Cysts (OOCs). MATERIALS AND METHODS: A total of 50 lesions were obtained in this study, 28 OKs, 18 OKs associated with NBCCS, and 4 OOCs. Immunohistochemical expression of podoplanin in epithelial cells was evaluated using the following score: (a) intensity of immunostaining: (0: absent, 1: weak, 2: moderate, 3: strong, and 4: very strong) and (b) number of positively cells (0: 0%, 1: <25%, 2: 25% to 50%, 3: 50% to 75%, and 4: >75%). The final score was determined by adding the scores of a and b and ranged from 0 to 8 (0: absent, 1 to 4: weak, and 5 to 8: strong). RESULTS: Podoplanin expression was significantly stronger in the basal layer OKs and NBCCS lesions. Further, podoplanin expression was the highest in the suprabasal layer of NBCCS lesions, followed by the suprabasal layers of OK and OOC lesions. CONCLUSIONS: Podoplanin expression is different in lesions of different biological behaviors. Podoplanin seems to play a role in cell proliferation and migration.

A multicentre study of 268 cases of calcifying odontogenic cysts and a literature review.

OBJECTIVES: To investigate the frequency of calcifying odontogenic cysts (COCs) that have been submitted for microscopic examination from representative geographic regions of Brazil and to compare it with literature data. MATERIALS AND METHODS: A retrospective study was conducted on biopsies obtained from 1953 to 2016 at 10 Brazilian oral and maxillofacial pathology centres. A total of 198,350 biopsy specimens were analysed. Demographic data and histopathological diagnosis were evaluated descriptively and statistically. In addition, a literature review of case series was carried out in four electronic databases. RESULTS: A total of 268 cases of COC were surveyed, representing 0.1% of the oral lesions at the centres studied. Female patients in their second decade of life and the maxilla were more affected. The mean lesion size of symptomatic individuals was larger than that of cases without symptoms (p = 0.026). The literature review showed a higher frequency in Asia and Europe, mainly affecting men in the third decade of life. CONCLUSIONS: COC is a rare lesion. Novel data on the clinicopathological features of 268 cases have been added to the literature. Data regarding gender and age of the Brazilian patients reported herein contrast with findings of case series and retrospective studies reported elsewhere.

Glucose-regulated protein 78 (Grp78) confers chemoresistance to tumor endothelial cells under acidic stress.

OBJECTIVES: This study was designed to investigate the activation of the unfolded protein response (UPR) in tumor associated endothelial cells (TECs) and its association with chemoresistance during acidic pH stress. MATERIALS AND METHODS: Endothelial cells from human oral squamous cell carcinomas (OSCC) were excised by laser capture microdissection (LCM) followed by analysis of UPR markers (Grp78, ATF4 and CHOP) using quantitative PCR. Grp78 expression was also determined by immunostaining. Acidic stress was induced in primary human dermal microvascular endothelial cells (HDMECs) by treatment with conditioned medium (CM) from tumor cells grown under hypoxic conditions or by adjusting medium pH to 6.4 or 7.0 using lactic acid or hydrochloric acid (HCl). HDMEC resistance to the anti-angiogenic drug Sunitinib was assessed with SRB assay. RESULTS: UPR markers, Grp78, ATF4 and CHOP were significantly upregulated in TECs from OSCC compared to HDMECs. HDMECs cultured in acidic CM (pH 6.0-6.4) showed increased expression of the UPR markers. However, severe acidosis led to marked cell death in HDMECs. Alternatively, HDMECs were able to adapt when exposed to chronic acidosis at pH 7.0 for 7 days, with concomittant increase in Grp78 expression. Chronic acidosis also confers drug resistance to HDMECs against Sunitinib. Knockdown of Grp78 using shRNA resensitizes HDMECs to drug treatment. CONCLUSIONS: UPR induction in ECs under acidic pH conditions is related to chemoresistance and may contribute to therapeutic failures in response to chemotherapy. Targeting Grp78, the key component of the UPR pathway, may provide a promising approach to overcome ECs resistance in cancer therapy.

Oral desmoplastic melanoma mimicking inflammatory hyperplasia.

INTRODUCTION: Desmoplastic melanoma (DM) arising in the oral cavity is a rare neoplasm that may be confused with a variety of non-melanocytic benign or malignant lesions. OBJECTIVES: To present a case of DM in the oral mucosa mimicking fibrous inflammatory hyperplasia, discusses the difficulties involved in the diagnosis and offers a literature review on the clinicopathologic and immunohistochemincal aspects of this neoplasm. CASE REPORT: A 62-year-old white male, smoker, was referred with a chief complaint of pain and swelling in the palate. The oral examination revealed multiple brown-to-black patches and a non-pigmented sessile nodule located on the mucosa of the hard palate. The clinical diagnosis of the pigmented lesions was either oral melanosis or melanoma. The nodular lesion was clinically diagnosed as fibrous inflammatory hyperplasia. Incisional biopsy was performed on the pigmented lesion and the microscopic sections revealed a melanotic macule. The nodular lesions histologically revealed an amelanotic desmoplastic melanoma. CONCLUSIONS: Reactive lesions close to a pigmented area should be investigated with great care.

Leishmaniasis with oral mucosa involvement.

INTRODUCTION: The term leishmaniasis comprises a group of diseases caused by different protozoan species of the genus Leishmania. There are three main clinical forms of leishmaniasis: visceral, cutaneous and mucocutaneous. Exclusive involvement of the mucosa is very rare. OBJECTIVES: To present a case of mucocutaneous leishmaniasis in an elderly patient, discuss the clinical presentation, diagnostic process and treatment emphasizing the distinctions from other granulomatous lesions. CASE REPORT: A 71-year-old male presenting with a symptomatic lesion on the hard and soft palate, which had developed over a period of 6 months was evaluated. The oral exam revealed a lesion with multiple ulcerated nodules on the hard and soft palate extending to the oropharynx. The diagnostic hypothesis was chronic infectious disease (paracoccidioidomycose, tuberculosis and leishmaniasis) or squamous cell carcinoma. Histopathological, histochemical and immunohistochemical analysis were performed. A chest x-ray revealed a normal pulmonary pattern. The Montenegro skin test was positive. The definitive diagnosis was leishmaniasis with exclusive oral manifestation and the patient was treated with liposomal amphotericin. CONCLUSIONS: Localized oral mucosa leishmaniasis is an uncommon event in an immunocompetent patient. Dentists play an important role in the diagnosis of oral leishmaniasis, which has systemic repercussions.

Oral ulceration with bonesequestration: case report / Ulceração bucal com sequestro ósseo: relato de caso

This report describes the case of a 38-year-old male patient that presented bilateral ulcerations with bone necrosis in the mandibular lingual area at the level of the mylohyoid ridge for 30 days. The patient reported discrete pain and eventually bleeding. Occlusal radiograph showed bilateral bone exostoses in the lingual mandibular cortical bone. Oral ulceration with bone sequestration diagnosis was based on clinical examinationand on oclusal radiograph. The patient was treated with antibiotics, 0.12% chlorhexidine mouth washes and removal of bone sequestrations. At clinical follow-up at 60 days, it was observed that the region was completely healed. The patient had bruxism, which was diagnosed based on the examination of wear on the surfaces of anterior incisors and on family reports. For this reason, he was referred to a clinical dentist for treatment with an occlusal splint. This is a rare lesion and the recognition of its characteristics is important for an accurate diagnosis and adequate clinical management.

Este estudo relata o caso de um paciente do gênero masculino, 38 anos de idade, que apresentava úlceras bilaterais com necrose óssea na região lingual da mandíbula no nível da linha miloioidea há trinta dias. O paciente relatava dor discreta e sangramento eventual.A radiografia oclusal mostrava exostoses ósseas bilaterais na cortical óssea lingual da mandíbula. O diagnóstico de úlcera com sequestro ósseo baseou-se no exameclínico e na radiografia oclusal. O paciente foi tratado com antiobióticos, bochechos com clorexidina a 0,12%e remoção dos sequestros ósseos. Após sessenta dias, na consulta de proservação, observou-se que a região estava completamente cicatrizada. O paciente apresentava bruxismo, o qual foi diagnosticado pela observação de facetas de desgaste nos incisivos inferiores e por meio de entrevista com os familiares. Em razão disso,o paciente foi encaminhado para um clínico geral para que fosse realizado um tratamento com placa miorrelaxante. Essa lesão é rara e o reconhecimento de suas características é importante a fim de que se realize o diagnóstico preciso e o tratamento apropriado.

NF1 diagnosis criteria and associated sarcomatous tumor review of the literature and case report.

AIM: This paper reports the case of a sarcomatous tumor (probably a pleomorphic sarcoma) in a patient with Neurofibromatosis type 1 (NF1) and discusses the association between these two diseases. BACKGROUND: NF1 is an autosomal dominant tumor predisposition syndrome. Malignant transformation is observed in patients with this disease, usually in the form of malignant peripheral nerve sheath tumors (MPNSTs). REPORT: A 72-year-old female patient with diagnosis of NF1 presented with left mandibular region enlarged for about 6 months, intra-oral examination revealed a mass growth on the floor of the lower left posterior area of the oral cavity measuring 5 cm in its greatest diameter and covered by ulcerated mucosa. SUMMARY: Few cases of association of neurofibromatosis with other types of soft tissue sarcomas have been reported. Undifferentiated pleomorphic sarcomas are the most common of these tumors. We intend to call attention to the importance of patient follow-up and counseling.

Odontogenic cysts: analysis of 680 cases in Brazil.

The purpose of this study was to evaluate the prevalence of 680 odontogenic cysts diagnosed in Porto Alegre, RS, Brazil, and to compare results with findings in the literature. Data of odontogenic cysts diagnosed from 1985 to 2005 were collected from the files of the Oral Pathology Laboratory of Pontifícia Universidade Católica do Rio Grande do Sul, Porto Alegre, RS, Brazil, and entered in a standardized form for later comparisons. The most prevalent odontogenic cysts were radicular (72.50%), dentigerous (22.20%) and residual (4.26%) cysts. The mandible of white patients was the anatomic site and ethnic group most frequently affected by this disease. Four of the six types of cysts were more frequent in the second and fourth decades of life, and no significant differences were found between sexes in the diagnosis of odontogenic cysts. In conclusion, the prevalence of odontogenic cysts was similar to that reported in the literature, which shows that inflammatory cysts are the most frequent.

Quantification of silver-staining nucleolar organizer region in epithelial cells of tongue of mice after exposure to, or intake of, alcohol.

The purpose of the current study was to evaluate the effect of alcohol on the proliferative activity of epithelial cells in the lingual mucosa of mice by means of silver-staining nucleolar organizer region (AgNOR) count and area measurements. Forty-eight CF1 mice were separated into three groups. The test groups were submitted to topical exposure to, or intake of, 40% (volume/volume) ethyl alcohol. Biopsy specimens were collected from the middle third of the dorsal tongue at 0, 6, and 12 months, and samples were stained according to the AgNOR technique. Mean number and mean area of AgNOR per nucleus were calculated for 50 basal layer cells and 50 intermediate layer cells. Increases in mean number and mean area of AgNOR per nucleus in intermediate cells were observed at 12 months in the alcohol intake group (P < .05). Results showed that intake of 40% alcohol increased epithelial cell proliferation in the dorsal surface of lingual mucosa.

Proliferating cell nuclear antigen expression on tongue of mice after intake of, or topical exposure to, alcohol.

The purpose of the current study was to evaluate the effects of alcohol on cellular proliferation. Sixty mice were separated into three groups of 20 mice in each. The first group, exposed to alcohol continuously, ingested 40% [volume/volume (vol./vol.)] alcohol instead of water during the experiment. For the second group, exposed to alcohol topically, alcohol was applied to the dorsum of the tongue twice a week. The third group served as the control group. We used the proliferating cell nuclear antigen (PCNA) immunohistochemical expression technique to perform quantitative measurements of cellular proliferation in the basal and intermediate layers of the epithelial tissue of the tongue. Cell proliferation was quantified at three different time points: just before the beginning of the experiment and at 6 and 12 months. Results were compared for mice in each group and for the three groups. At 12 months, we observed an increase in cellular proliferation in the intermediate layer of the epithelium of mice in the group that consumed alcohol (P=.01). Results for topical alcohol-exposed and control groups did not show significant differences in cellular proliferation at any time point during the study. We concluded that the effects of alcohol on cellular proliferation may be caused by continuous intake of alcohol and occur throughout life.