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Angiogenesis plays an important role in cancer growth and metastasis, and it is considered a therapeutic target to control tumour growth following anti‐angiogenic therapy. However, it is still unclear when tissues initiate angiogenesis during malignant transformation from premalignant condition and whether this premalignant condition could be a therapeutic target of anti‐angiogenic therapy. In this study, we aimed to analyse the onset of angiogenesis by evaluating morphological and functional alterations of microvessels during oral multistep carcinogenesis using a 4‐nitroquinoline 1‐oxide (4NQO)‐induced oral carcinogenesis mouse model. In the study, we initially confirmed that with the use of 4NQO, oral lesions develop in a stepwise manner from normal mucosa through oral epithelial dysplasia (OED) to oral squamous cell carcinoma (OSCC). Evaluation of CD31‐immunostained specimens revealed that microvessel density (MVD) increases in a stepwise manner from OEDs. Histological and functional analyses revealed the structural abnormalities and leakage of blood vessels had already taken place in OED. Then we evaluated the expression profiles of Hif1a and Vegfa along with hypoxic status and found that OED exhibited increased Vegfa expression under hypoxic conditions. Finally, we tested the possibility of OEDs as a target of anti‐angiogenic therapy and found that anti‐VEGFR2 neutralising antibody in OED slowed the disease progression from OED to OSCC. These data indicate that an angiogenic switch occurs at the premalignant stage and morphological, and functional alterations of microvessels already exist in OED. These findings also elucidate the tumour microenvironment, which gradually develops along with carcinogenic processes, and highlight usefulness of the 4NQO‐induced carcinogenesis model in the study of epithelial and stromal components, which will support epithelial carcinogenesis. © 2024 The Author(s). The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

Background: Although cervical lymph node metastasis is an important prognostic factor for oral cancer, occult metastases remain undetected even by diagnostic imaging. We developed a learning model to predict lymph node metastasis in resected specimens of tongue cancer by classifying the level of immunohistochemical (IHC) staining for angiogenesis‐ and lymphangiogenesis‐related proteins using a multilayer perceptron neural network (MNN). Methods: We obtained a dataset of 76 patients with squamous cell carcinoma of the tongue who had undergone primary tumor resection. All 76 specimens were IHC stained for the six types shown above (VEGF‐C, VEGF‐D, NRP1, NRP2, CCR7, and SEMA3E) and 456 slides were prepared. We scored the staining levels visually on all slides. We created virtual slides (4560 images) and the accuracy of the MNN model was verified by comparing it with a hue–saturation (HS) histogram, which quantifies the manually determined visual information. Results: The accuracy of the training model with the MNN was 98.6%, and when the training image was converted to grayscale, the accuracy decreased to 52.9%. This indicates that our MNN adequately evaluates the level of staining rather than the morphological features of the IHC images. Multivariate analysis revealed that CCR7 staining level and T classification were independent factors associated with the presence of cervical lymph node metastasis in both HS histograms and MNN. Conclusion: These results suggest that IHC assessment using MNN may be useful for identifying lymph node metastasis in patients with tongue cancer. [ABSTRACT FROM AUTHOR]

Diagnostic methods of oral squamous cell carcinoma (SCC) using artificial intelligence (AI) and digital-histopathologic images have been developed. However, previous AI training methods have focused on the cellular atypia given by the training of high-magnification images, and little attention has been paid to structural atypia provided by low-power wide fields. Since oral SCC has histopathologic types with bland cytology, both cellular atypia and structural atypia must be considered as histopathologic features. This study aimed to investigate AI ability to judge oral SCC in a novel training method considering cellular and structural atypia and their suitability. We examined digitized histological whole-slide images from 90 randomly selected patients with tongue SCC who attended a dental hospital. Image patches of 1000 × 1000 pixels were cut from whole-slide images at 0.3125-, 1.25-, 5-, and 20-fold magnification, and 90,059 image patches were used for training and evaluation. These image patches were resized into 224 × 224, 384 × 384, 512 × 512, and 768 × 768 pixels, and the differences in input size were analyzed. EfficientNet B0 was utilized as the convolutional neural network model. Gradient-weighted class activation mapping (Grad-CAM) was used to elucidate its validity. The proposed method achieved a peak accuracy of 99.65% with an input size of 512 × 512 pixels. Grad-CAM suggested that AI focused on both cellular and structural atypia of SCC, and tended to focus on the region surrounding the basal layer. Training AI regarding both cellular and structural atypia using various magnification images simultaneously may be suitable for the diagnosis of oral SCC. [ABSTRACT FROM AUTHOR]

Objectives: This study sought to identify tumor characteristics that associate with regional lymph node metastases in squamous cell carcinomas originating in the upper gingiva. Materials and methods: Data from 113 patients from Osaka University Dental Hospital were included. We measured each primary tumor's width, length, depth, and the extent of bone invasion. Additionally, tumor signal intensity for T1 and T2-weighted images as well as the center of the tumor's location and T classification was assessed, and a histopathological analysis was performed. Results: Tumor signal intensity was not found to be a significant prognostic factor. However, bucco-lingual width, histopathological classification as well as the tumor's location were significantly different between metastatic and non-metastatic groups in both univariate and multivariate analysis. Superior–inferior depth and T classification were significant only in the univariate (and not the multivariate) analysis. Conclusions: Bucco-lingual width, histopathological grading as well as the tumor's location are likely to be important predictors for the occurrence of LN metastasis in upper gingival carcinoma patients and should be considered when managing care for these patients. [ABSTRACT FROM AUTHOR]

Objective: Although class III beta‐tubulin (TUBB3) is not expressed in normal epithelium, its expression in cancers of some organs has been reported. Herein, we investigated the expression pattern and expression levels of TUBB3 in oral squamous cell carcinoma (SCC), and assessed whether TUBB3 immunostaining could improve the diagnostic accuracy of oral scraping liquid‐based cytology (LBC). Methods: Paraffin sections of biopsies from 107 patients with primary SCC and 30 patients with squamous papilloma of the tongue or gingiva were immunostained for TUBB3. In addition, 15 LBC samples obtained from the study participants with SCC were immunostained for TUBB3. Seven LBC samples were false‐negative. The TUBB3 expression level in each sample was evaluated and classified as 3+, 2+, 1+, or 0. Results: TUBB3 expression was confirmed in 91.6% of paraffin‐embedded SCC specimens. Clear and diffuse positivity (2+ or above) was observed in 77.6% of the total cases. In the well‐differentiated type, tumour cells in the middle layer of the parenchyma specifically expressed TUBB3. In almost LBC samples, cancerous intermediate cells showed immunopositivity similar to that of paraffin samples, even if cellular atypia was not clear in Papanicolaou staining. Conclusions: TUBB3 immunostaining is useful for diagnosing oral SCC in scraping LBC, especially when samples consist of intermediate cells with little morphological change. Moreover, TUBB3 immunostaining could improve the diagnostic accuracy of oral scraping LBC by reducing false‐negatives. [ABSTRACT FROM AUTHOR]

Background: Previous reports concerning the prevalence of human papillomavirus (HPV) in oral squamous cell carcinoma (OSCC) have observed varied results. The aim of this study was to evaluate the prevalence of HPV in oral premalignant lesions (OPL) and OSCC. For accurate HPV detection in oral lesions, comparative analysis was performed on cervical lesions as positive controls. Methods: Fifty-seven cases with OPL and 50 with OSCC were selected. Twenty-nine control cases were selected from cervical lesions. The HPV infection rate was analysed by consensus polymerase chain reaction (PCR) using the My09/My11 and Gp5+/Gp6+ primers, and genotyping detection was employed using a PCR-based micro-array. Immunohistochemical staining for p16 was performed. Results: Twenty-eight (96.6%) cases of cervical lesions were positive for HPV by consensus PCR and 24 cases (82.8%) were positive by genotyping. The total HPV-positive rate in cervical lesions was 96.6%. HPV-DNA was detected in nine cases (15.8%) of OPL and six cases (12.0%) of OSCC by consensus PCR. Six cases (10.5%) of OPL and three cases (6.0%) of OSCC were positive by genotyping. The total HPV-positive rate in oral lesions was 22.4% (26.3% of OPL and 18.0% of OSCC). In cervical lesions, immunohistochemistry of p16 identified 27 cases (93.1%) as positive. Fifteen cases (26.3%) of OPL and eight cases (16.0%) of OSCC were positive for p16. Conclusions: The HPV infection and p16-positive rates in oral lesions are lower than previously reported. This suggests that HPV may not play a major role in oral lesions although its involvement cannot completely be ruled out. [ABSTRACT FROM AUTHOR]