Oral Stratified Squamous Epithelium Research Articles

Artificial intelligence-based cancer progression prediction of oral premalignant lesions via self-supervised deep learning on histopathology.

90 Background: Premalignant lesions within the oral cavity demonstrate varied degrees of dysplasia. Oral stratified squamous epithelium is sensitive to carcinogenic insults and lesions progress to cancer at variable rates. The current diagnostic standard involves visual and tactile inspection followed by a biopsy. However, thus far, there is clinical ambiguity about which lesions may progress to carcinoma and there are insufficient prognostic tools available to guide clinical decision-making for follow-up. Deep learning is a form of artificial intelligence which can be applied to images. Self-supervised learning (SSL) and attention-based multiple instance learning (aMIL), are deep learning extensions which eliminate the need for pathologist annotations of histology and are more generalizable as the model learns general representations of unlabeled data, compared to traditional tile-based methods. We developed a novel artificial intelligence pipeline to predict progression to oral carcinoma using histological images from oral premalignant lesions. Methods: Digital histopathology images and clinical data cohorts were obtained from the US National Cancer Institute (n=561) and the University of Iowa (n=193) for training, and from the University of Chicago (n=214) for external validation. Patients were defined as progressing if initially non-malignant lesions had documented progression to carcinoma within 12 months of follow-up. Model training and evaluation was done in Python using our publicly available open-source Slideflow platform. We developed a deep learning pipeline utilizing SimCLR as an SSL method for feature extraction paired with an aMIL head for classification. The SimCLR feature generator was trained on only the training cohort, then used to generate features for both training and validation data. The aMIL model was trained on SimCLR-generated features. Trained model performance was evaluated on the external University of Chicago cohort and measured via areas under the receiver operating characteristic curve and precision recall curve (AUROC and AUPRC, respectively). During training and validation, model reliability was interpreted using Class Activation Mapping (Grad-CAM) and generative adversarial network (GAN) methods. Results: On training data, the model achieved average 3-fold cross validation AUROC of 0.86 with AUPRC of 0.83. In the external validation data from UC, the model achieved an AUROC of 0.80 with an AUPRC of 0.79. Deployed as a “rule-in” test for early progression prediction, the model achieved a validation data set specificity of 91% with a sensitivity of 40%. Conclusions: We developed a deep learning model to automatically predict cancer progression from premalignant lesions histopathology using a multi-institutional cohort, with promising external validation performance.

The role of Sonic Hedgehog signaling pathway in the development of oral squamous cell carcinoma

The research of the Sonic Hedgehog signaling pathway activity was carried out on biopsy specimens of 43 patients with oral hyperplasia, squamous intraepithelial neoplasia and oral squamous cell carcinoma. Tissue antigens were determined using a mouse monoclonal antibody to Ki-67 protein and rabbit monoclonal antibody to Gli-1 protein. In squamous intraepithelial neoplasia and oral squamous cell carcinoma specimens Gli-1 was detected in the cytoplasm of cells only. The correlation was found between the proliferative activity of epithelial cells and Gli-1 distribution. Protein Gli-1 can be used for early diagnosis of malignant transformation of oral stratified squamous epithelium.

Liver X receptor reduces proliferation of human oral cancer cells by promoting cholesterol efflux via up-regulation of ABCA1 expression.

Liver X receptors (LXRs) contribute not only to maintain cholesterol homeostasis but also to control cell growth. However, the molecular mechanisms behind the LXR-mediated anti-proliferative effects are largely unknown. Here we show, by immunohistochemistry, that LXRα and LXRβ are differentially distributed in oral stratified squamous epithelia. By immunohistochemical and Western blot analyses, we also reveal that LXRα is abundantly expressed in human oral squamous cell carcinoma (HOSCC) tissues and cell lines. Cell counting, BrdU labeling and cell cycle assay indicated that LXR stimulation led to significant reduction of proliferation in HOSCC cells. Importantly, our study highlights, by using RNA interference, that the ATP-binding cassette transporter A1 (ABCA1)-accelerated cholesterol efflux is critical for the growth inhibitory action of LXRs in HOSCC cells. Moreover, we demonstrate that LXR activation reduces the growth of xenograft tumour of HOSCC cells in mice accompanied by the upregulation of ABCA1 expression and the decline of cholesterol levels in the tumour. These findings strongly suggested that targeting the LXR-regulated cholesterol transport, yielding in lowering intracellular cholesterol levels, could be a promising therapeutic option for certain types of cancers.

Expression and Localization of Aqua-glyceroporins AQP3 and AQP9 in Rat Oral Epithelia

Aquaporins (AQPs) are a family of small integral membrane proteins made up of 6 hydrophobic, a-helical, membrane-spanning domains surrounding a highly selective aqueous pore. AQP3, AQP7, and AQP9, termed aqua-glyceroporins, are known to be involved in the transport of water, glycerol, and other small molecules. In this study, we investigated the expression and localization of aqua-glyceroporins in rat oral stratified squamous epithelia of the palate, the buccal mucosa, the inferior aspect of the tongue, and the oral floor by using RT-PCR, immunofluorescence, and immunogold electron microscopy. AQP3 and AQP9 mRNAs were expressed in whole oral epithelium. Immunostaining for AQP3 was recognized in each type of epithelium. The results suggest that AQP3 synthesis begins predominantly in the cytoplasm of the basal cells. During the process of epithelial cell differentiation, AQP3 protein appears to accumulate and be transported to the plasma membrane, from where it is incorporated into the cornified or surface layers. The intracellular localization of AQP3 appears to correlate with the differentiation of keratinocytes, suggesting that it acts as an enhancer of the physiological permeability barrier together with membrane coating granules. The distribution pattern of AQP9 was limited to the marginal areas of the basal and suprabasal layers, which was different from that of AQP3. This difference in distribution between AQP3 and AQP9 suggests that AQP9 in rat oral epithelia acts as a channel by facilitating glycerol uptake from the blood through the endothelial cells of the capillary vessels to the oral stratified squamous epithelium. AQP3 and AQP9 facilitate both transcellular osmotic water flow and glycerol transport as pore-like passive transporters in the keratinocytes of oral epithelia, and may play a key role in not only hydration and the permeability barrier, but also cell proliferation, differentiation, migration, development, and wound healing by generating ATP.

Revisiting a medical case of “stinging” in the human oral cavity caused by ingestion of raw squid (Cephalopoda: Teuthida): new data on the functioning of squid’s spermatophores

Male squid produce intricate spermatophores that, when transferred to the female, undergo the spermatophoric reaction, a complex process of evagination that leads to the attachment of the spermatangium, that is, the everted spermatophore containing the sperm mass. While this process is still not completely understood, the medical literature includes several reports of “oral stinging” (i.e., punctured wounds in the human oral cavity) following consumption of raw male squid, which contains undischarged spermatophores able to inflict such wounds. Here, we revisit a recent medical report of oral stinging by Shiraki et al. (Pathol Int 61:749–751, 2011), providing an in-depth reanalysis of their histological biopsies and revealing vital information on the functioning of squid spermatophores. The morphology of the spermatangia attached within the oral cavity is similar to the condition found in spermatangia naturally attached to female squids. The spermatangia were able to superficially puncture the superficial layers of the oral stratified squamous epithelium, and numerous, minute stellate particles from the squid spermatophore were found adhered to the oral epithelium. These findings corroborate previous hypotheses on the functioning of squid spermatophores, namely that spermatophore attachment generally involves tissue scarification, and that stellate particles play a vital role in the attachment process. Moreover, spermatophore attachment is confirmed to be autonomous (i.e., performed by the spermatophore itself) in another squid species (possibly a loliginid), and the results strongly indicate that the attachment mechanism is not dependent upon a specialized epithelium, nor a mate’s specific chemical stimulus. From the pathological point of view, the best prophylactic measure at present is the removal of the internal organs of the raw squid prior to its consumption.

Narrow Band Imaging: A New Diagnostic Approach to Visualize Angiogenesis in Superficial Neoplasia

Although numerous gastrointestinal endoscopes pass through the oropharynx and the hypopharynx, it is extremely difficult to detect an early cancer in these sites during routine endoscopic examination. Most patients with cancer in these sites are usually diagnosed in advanced stages. If effective screening methods can detect an earlier stage, such as carcinoma in situ, it would obviously be of great benefit. Narrow band imaging is an innovative optical technology that can clearly visualize the microvascular structure of the organ surface. Herein, we demonstrate that narrow band imaging combined with magnifying endoscopy can identify a carcinoma in situ in oropharyngeal and hypopharyngeal mucosal lesions. Scattered irregular foci of microvascular proliferation projecting to the dysplastic squamous epithelium are the typical features. These results indicate that an approach to visualize angiogenesis or morphologic changes of microvessels in the superficial neoplasm can be a new diagnostic method not only for the head and neck region but also for other sites in the gastrointestinal tract.

P73 expression for human buccal epithelial dysplasia and squamous cell carcinoma: does it correlate with nodal status of carcinoma and is there a relationship with malignant change of epithelial dysplasia?

TP73, a p53 homologue gene, shares similar structural sequences with p53. The aim of this study was to investigate the p73 expression for human buccal epithelial dysplasia (ED) and squamous cell carcinoma (SCC). Seventy-five samples of human buccal ED, including mild, moderate, and severe ED (25 samples in each category), were analyzed for p73 protein expression by means of immunohistochemistry. Twenty-five samples of human buccal SCCs were analyzed for p73 mRNA expression with reverse transcription-polymerase chain reaction (RT-PCR) and were also analyzed for protein expression with immunohistochemical analysis. By use of immunohistochemical analysis, nuclear staining of p73 protein was detected in a subset of normal mucosa, buccal ED, and SCC specimens. p73 nuclear staining was noted for the basal cells of normal buccal mucosa. For buccal lesions deriving from mild, moderate, and severe ED, p73 protein was observed in basal and parabasal layers and in more superficial cell layers corresponding to the spinous layer. For well-differentiated carcinomas, p73 immunoreactivity was chiefly observed among the less-differentiated cells in the periphery of carcinomatous clusters, whereas moderately differentiated carcinomas revealed homogeneous staining, involving nearly all of the tumor cells. On RT-PCR, the expression of p73 mRNA from buccal SCC was noted to be compatible with the findings of immunohistochemical analysis. An electrophoretic band with a 180-bp PCR product corresponding to p73 mRNA has been observed. The expression of p73 seemed to be significantly elevated for specimens of buccal ED (protein level) and SCC (protein and mRNA levels) compared with the analogous expression for normal control tissue (Fisher's exact test, p <.001). Also, p73 expression (protein and mRNA levels) correlated significantly with cervical lymph node metastasis for cases of buccal SCC (Fisher's exact test, p <.001). Eight cases of ED (protein level) showing p73 positivity have undergone malignant transformation to develop SCCs in 2 years follow-up, but no statistical significance was established (Fisher's exact test, p >.05). The data suggest that p73 expression may be (1) associated with the differentiation of oral stratified squamous epithelium, (2) an early event in human oral carcinogenesis, and (3) associated with the nodal status of patients with oral carcinoma and a possible indicator for malignant change of oral ED.

Effects of dietary lipids on cell proliferation of murine oral mucosa

BackgroundThe lack of certain essential polyunsaturated fatty acids (PUFAs) induces perturbation in cell proliferation, apoptosis and dedifferentiation that could be linked to an increased protumorigenic trend. Contrarily, n-3 essential fatty acids (EFAs) arrest cell proliferation in several tumor models. According to the concept of field cancerization, multiple patches of abnormal epithelial proliferation may coexist in the vicinity of oropharyngeal neoplasms.The purpose of the present study is to determine whether certain dietary PUFAs differentially modulate the patterns of cell proliferation and apoptosis at non-tumoral sites of the oral mucosa in mice bearing DMBA induced salivary tumors.After weaning, BALB/c mice were assigned to four diets: Control (C), Corn Oil (CO), Fish (FO) and Olein (O). Two weeks later, DMBA was injected into the submandibular area. The animals were sacrificed between 94 and 184 days at 4–6 PM. Fixed samples of lip, tongue and palate were stained using H-E and a silver technique. A quantification of AgNORs in the basal (BS) and suprabasal stratum (SBS) of the covering squamous epithelia as well as of mitosis and apoptosis was performed.ResultsAnalysis of Variance showed greater proliferation in tongue than in palate or lip. According to the diet, a significant difference was found in the Fish Oil, in which palate exhibited fewer AgNOR particles than that of the control group, both for BS and SBS (p < 0.05 and 0.152, respectively), indicating a reduced cell proliferation.ConclusionsThese results corroborate and reaffirm that the patterns of cell proliferation, apoptosis and differentiation of the oral stratified squamous epithelium may be differentially modulated by dietary lipids, and arrested by n-3 fatty acids, as shown in several other cell populations.

Immunohistochemical detection of Fas antigen in oral epithelia.

A monospecific and high titer polyclonal antibody, designated as Fas D, raised against synthetic polypeptides selected from a part of the human Fas antigen (aa 104-114), was used to identify the Fas antigen in human oral epithelia in normal and pathological states. The human gingival proteins had been extracted and analysed by an ABC (avidin-biotin complex) immunoblotting technique. The antibody interacted with a single band in gingival proteins with an estimated molecular weight of 35,000, which is in good agreement with that calculated from amino acid sequences of the human Fas antigen. Using an indirect immunohistochemical method, the antibody localized on the stratum spinosum and the basal part of the stratum corneum of normal human gingiva. Specimens obtained from patients with odontogenic keratocysts, leukoplakia, lichen planus, and squamous cell carcinoma were also stained with the antibody. The pattern of the Fas antigen distribution in oral stratified squamous epithelia was, with some overlapping, characteristic for each disease.

Epstein‐Barr virus receptors but not viral DMA are present in normal and malignant oral epithelium

The presence and distribution of Epstein-Barr Virus receptors (EBVR's) on a range of normal (n = 18), dysplastic (n = 10) and malignant (n = 20) oral mucosa were studied by immunocytochemical methods using the monoclonal antibodies (MAb's) HB5 and B2. EBVR's were demonstrated as membrane staining of the spinous layers of normal non- and parakeratinized epithelium, indicating that EBVR's are differentiation-linked. This distribution was retained in dysplastic epithelium. Tissue from oral squamous cell carcinomas (SCC's) showed variable reactivity of only a few cells scattered randomly within the samples. Furthermore, a sensitive in situ hybridization (ISH) technique was used to determine if Epstein-Barr virus (EBV) was present in normal (n = 15) and oral squamous cell carcinoma tissue (n = 20). No EBV DNA was demonstrated within either normal or malignant epithelium, suggesting that the virus does not persist in normal oral stratified squamous epithelium nor is there any evidence for a role in oral carcinogenesis.

Ultrastructure of oral squamous-cell carcinoma

Fresh surgical specimens of sixteen cases of oral squamous-cell carcinoma were processed for electron microscopic study. All cases were histologically graded as moderately differentiated carcinoma. As compared to normal oral stratified squamous epithelium, some unusual ultrastructural features were present in carcinoma. These features were spherical or ovoid nuclear bodies composed of concentrically arranged filaments and granules, clustered ribosomes, many lysosomal bodies, cell residues in other cells, absence and multilayering of basal lamina, pseudopodal cytoplasmic projections, microfilaments in peripheral cytoplasm, clusters of swirled tonofilaments, intracytoplasmic desmosomes, and a small amount of glycogen. These features are interpreted as being related to hyperactivity, phagocytosis, locomotion, and differentiation of cancer cells.

Field cancerization in oral stratified squamous epithelium; clinical implications of multicentric origin.

Field cancerization in oral stratified squamous epithelium; clinical implications of multicentric origin.