Dysplastic Epithelium Research Articles

Nondysplastic Colon Crypts Intercalated in Tubular Adenomas Support Field Cancerization.

Tubular adenomas of the colon (TA) are neoplastic polyps composed of dysplastic tube-like crypts. Nondysplastic crypts, mostly in asymmetric branching have been previously reported, both beneath and bordering TA. In the present article, intercalated nondysplastic crypts (INDC) amidst dysplastic crypts in TA are showcased. The occurrence of INDC was recorded in 139 TA. Out of the 139 TA, 31% exhibited INDC; of these, 58% were in asymmetric branching (INDCAB), 35% were single intercalated crypts without branching (INDSNB), and 7% were in symmetric branching (INDCSB). Luminal dysplasia occurred in 53% out of the 43 TA: in 37% TA with INDCAB, in 16% TA with INDSNB, but in none of the TA with INDCSB. Thus, INDCAB predominated. The finding of INDC in TA domain contrasts with the infrequency of INDCSB and with the absence of INDCAB in the normal colorectal mucosa. Hence, INDC emerge as integral components in TA. Since only 1 or 2 sections were available per TA, the total number of INDC in the entire TA is likely higher. INDC in TA may be remnants of acquired nondysplastic mucosal cores of abnormal cryptogenesis that were subsequently replaced by top-down growing dysplastic epithelium. The present and previous findings support the concept of field cancerization in the human colorectum.

Human pluripotent stem cell-derived kidney organoids reveal tubular epithelial pathobiology of heterozygous HNF1B-associated dysplastic kidney malformations

Hepatocyte nuclear factor 1B (HNF1B) encodes a transcription factor expressed in developing human kidney epithelia. Heterozygous HNF1B mutations are the commonest monogenic cause of dysplastic kidney malformations (DKMs). To understand their pathobiology, we generated heterozygous HNF1B mutant kidney organoids from CRISPR-Cas9 gene-edited human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) reprogrammed from a family with HNF1B-associated DKMs. Mutant organoids contained enlarged malformed tubules displaying deregulated cell turnover. Numerous genes implicated in Mendelian kidney tubulopathies were downregulated, and mutant tubules resisted the cyclic AMP (cAMP)-mediated dilatation seen in controls. Bulk and single-cell RNA sequencing (scRNA-seq) analyses indicated abnormal Wingless/Integrated (WNT), calcium, and glutamatergic pathways, the latter hitherto unstudied in developing kidneys. Glutamate ionotropic receptor kainate type subunit 3 (GRIK3) was upregulated in malformed mutant nephron tubules and prominent in HNF1B mutant fetal human dysplastic kidney epithelia. These results reveal morphological, molecular, and physiological roles for HNF1B in human kidney tubule differentiation and morphogenesis illuminating the developmental origin of mutant-HNF1B-causing kidney disease.

Assessment of the association of myofibroblasts and structural components of the extracellular matrix with histopathological parameters of actinic cheilitis and lower lip squamous cell carcinoma.

To evaluate the presence of myofibroblasts (MFs) in the development of lip carcinogenesis, through the correlation of clinical, histomorphometric and immunohistochemical parameters, in actinic cheilitis (ACs) and lower lip squamous cell carcinomas (LLSCCs). Samples of ACs, LLSCCs, and control group (CG) were prepared by tissue microarray (TMA) for immunohistochemical TGF-β, α-SMA, and Ki-67 and histochemical hematoxylin and eosin, picrosirius red, and verhoeff van gieson reactions. Clinical and microscopic data were associated using the Mann-Whitney, Kruskal-Wallis/Dunn, and Spearman correlation tests (SPSS, p < 0.05). ACs showed higher number of α-SMA+ MFs when compared to CG (p = 0.034), and these cells were associated with the vertical expansion of solar elastosis (SE) itself (p = 0.027). Areas of SE had lower deposits of collagen (p < 0.001), immunostaining for TGF-β (p < 0.001), and higher density of elastic fibers (p < 0.05) when compared to areas without SE. A positive correlation was observed between high-risk epithelial dysplasia (ED) and the proximity of SE to the dysplastic epithelium (p = 0.027). LLSCCs showed a higher number of α-SMA+ MFs about CG (p = 0.034), as well as a reduction in the deposition of total collagen (p = 0.009) in relation to ACs and CG. There was also a negative correlation between the amount of α-SMA+ cells and the accumulation of total collagen (p = 0.041). Collagen and elastic density loss was higher in larger tumors (p = 0.045) with nodal invasion (p = 0.047). Our findings show the possible role of MFs, collagen fibers, and elastosis areas in the lip carcinogenesis process.

Abstract 6930: Spatial analysis of multi-ancestral tumors to understand how intercellular signaling impacts tumor invasion

Abstract Colorectal cancer (CRC) persists as a leading cause of cancer-related deaths in the United States. Intratumoral heterogeneity stems from a multi-ancestral origin or clonal divergence and contributes to treatment resistance. Unfortunately, tumors in existing mouse models are largely homogenous, rendering predictions of drug efficacy inaccurate. To investigate whether signaling between distinct neoplastic clones in a tumor and cells in the surrounding microenvironment impacts CRC biology, we performed spatial transcriptomic analysis on samples collected from a transgenic mouse model previously established in the Halberg laboratory. This model (referred to as B6.FCTG3KM) develops three types of tumors: homotypic Adenomatous Polyposis Coli (APC)-deficient tumors that express red fluorescent protein (RFP), homotypic APC-deficient tumors that express constitutively active phosphoinositide 3-kinase (PI3K) and green fluorescent protein (GFP), and heterotypic tumors. Previous characterization of the model clearly demonstrated that heterotypic tumors are significantly more aggressive than either type of homotypic tumor. Analysis was conducted on four serially sectioned slides containing three tumors and adjacent normal tissue obtained from the colon of a male B6.FCTG3KM mouse using the 10x Genomics Visium CytAssist platform with the mouse whole transcriptome probe set. Collected tumors were staged using traditional histopathology techniques by a board-certified pathologist (Matkowskyj). Immunohistochemistry and immunofluorescence were used to assess β-catenin localization and to re-probe for RFP and GFP expression. Sequencing was performed on the Illumina NovaSeqX platform to an average depth of 255,528,294 reads per sample. Data analysis was performed using the 10x Genomics SpaceRanger and Kendziorski laboratory R/SpatialView packages. A fold-change threshold of 1.5 and an adjusted p-value threshold of 0.05 were used when establishing marker genes for each of the 8 clusters identified. One distinct cluster was identified by marker genes including Notum, Wif1, and Dkk2, factors previously implicated in CRC tumorigenesis, and corresponded to the center of each region of surface-level dysplastic epithelia. The remainder of the surface-level dysplastic epithelia corresponded to a second cluster, with marker genes including those coding for TRIM family proteins (Trim5, Trim10, Trim40, Trim12c), and matrix metalloproteinases (Mmp12, Mmp9, Mmp10, Mmp13). A third cluster corresponded to the desmoplastic stroma underlying each neoplasm. Ongoing analyses are underway to identify clonal interactions that alter gene expression and tumor aggressiveness. Citation Format: Mary M. Stangis, Lingxin Cheng, Dawn Albrecht, Santina Snow, Tessa Wirtz, Kristina A. Matkowskyj, Huy Q. Dinh, Christina Kendziorski, Richard B. Halberg. Spatial analysis of multi-ancestral tumors to understand how intercellular signaling impacts tumor invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6930.

Rapid and Label-Free Histopathology of Oral Lesions Using Deep Learning Applied to Optical and Infrared Spectroscopic Imaging Data.

Oral potentially malignant disorders (OPMDs) are precursors to over 80% of oral cancers. Hematoxylin and eosin (H&E) staining, followed by pathologist interpretation of tissue and cellular morphology, is the current gold standard for diagnosis. However, this method is qualitative, can result in errors during the multi-step diagnostic process, and results may have significant inter-observer variability. Chemical imaging (CI) offers a promising alternative, wherein label-free imaging is used to record both the morphology and the composition of tissue and artificial intelligence (AI) is used to objectively assign histologic information. Here, we employ quantum cascade laser (QCL)-based discrete frequency infrared (DFIR) chemical imaging to record data from oral tissues. In this proof-of-concept study, we focused on achieving tissue segmentation into three classes (connective tissue, dysplastic epithelium, and normal epithelium) using a convolutional neural network (CNN) applied to three bands of label-free DFIR data with paired darkfield visible imaging. Using pathologist-annotated H&E images as the ground truth, we demonstrate results that are 94.5% accurate with the ground truth using combined information from IR and darkfield microscopy in a deep learning framework. This chemical-imaging-based workflow for OPMD classification has the potential to enhance the efficiency and accuracy of clinical oral precancer diagnosis.

Immunophenotypic and Gene Expression Analyses of the Inflammatory Microenvironment in High-Grade Oral Epithelial Dysplasia and Oral Lichen Planus

BackgroundOral lichen planus (OLP) and oral epithelial dysplasia (OED) present diagnostic challenges due to clinical and histologic overlap. This study explores the immune microenvironment in OED, hypothesizing that immune signatures could aid in diagnostic differentiation and predict malignant transformation.MethodsTissue samples from OED and OLP cases were analyzed using immunofluorescence/immunohistochemistry (IF/IHC) for CD4, CD8, CD163/STAT1, and PD-1/PDL-1 expression. RNA-sequencing was performed on the samples, and data was subjected to CIBERSORTx analysis for immune cell composition. Gene Ontology analysis on the immune differentially expressed genes was also conducted.ResultsIn OED, CD8 + T-cells infiltrated dysplastic epithelium, correlating with dysplasia severity. CD4 + lymphocytes increased in the basal layer. STAT1/CD163 + macrophages correlated with CD4 + intraepithelial distribution. PD-1/PDL-1 expression varied. IF/IHC analysis revealed differential immune cell composition between OED and OLP. RNA-sequencing identified upregulated genes associated with cytotoxic response and immunosurveillance in OED. Downregulated genes were linked to signaling, immune cell recruitment, and tumor suppression.ConclusionsThe immune microenvironment distinguishes OED and OLP, suggesting diagnostic potential. Upregulated genes indicate cytotoxic immune response in OED. Downregulation of TRADD, CX3CL1, and ILI24 implies dysregulation in TNFR1 signaling, immune recruitment, and tumor suppression. This study contributes to the foundation for understanding immune interactions in OED and OLP, offering insights into future objective diagnostic avenues.

Activation of the Wnt/β-catenin signaling pathway and CTNNB1 mutations in canine intestinal epithelial proliferative lesions.

Nuclear expression of β-catenin has been reported in canine intestinal epithelial tumors (IETs) and colorectal inflammatory polyps (CIPs) with dysplastic epithelia. However, the role of the Wnt/β-catenin signaling pathway in these lesions remains unclear. To investigate the association between the nuclear β-catenin expression and the activation of the Wnt/β-catenin signaling pathway, immunohistochemistry and mutation analyses were conducted on 64 IETs and 20 CIPs. IETs and CIPs with β-catenin nuclear and/or cytoplasm immunolabeling were classified as β-catenin (+). The immunostaining scores of c-Myc and Cyclin D1 and Ki-67 index were significantly higher in β-catenin (+) cases than in β-catenin (-) cases. Identical APC mutations (p.E154D and p.K155X) were detected in 6/41 β-catenin (+) IETs; all 6 of IETs with APC mutations were Jack Russell Terriers. CTNNB1 mutations were detected in 29/42 β-catenin (+) IETs, 3/11 β-catenin (+) CIPs, and 2/22 β-catenin (-) IETs, most of which were hotspots associated with human colorectal carcinoma. In one Miniature Dachshund diagnosed with a CIP that subsequently developed into an IET, the same CTNNB1 mutation was detected in both lesions. The immunohistochemical results suggest that cell proliferative activity in β-catenin (+) cases may be associated with activation of the Wnt/β-catenin signaling pathway. The mutation analysis results suggest that CTNNB1 mutations may be associated with cytoplasmic β-catenin accumulation in IET and CIP. Furthermore, the dysplastic epithelium in CIP may progress to IET through the activation of the Wnt/β-catenin signaling pathway by the CTNNB1 mutation.

Mucocele of the Anorectal Pouch: Mucinous Cystic Neoplasm with Low-Grade Dysplasia Simulating Low- Grade Appendiceal Mucinous Neoplasm (LAMN)

Abstract Introduction/Objective We present the second case of colonic mucocele with dysplasia. To date, only five cases of colonic mucocele have been reported, all in the setting of a pouch or bypass, with only one of them showing dysplasia. The exceptional rarity of the lesion provides unique insight into etiology of mucocele. Methods/Case Report A 44-year-old male with complex surgical history including pull-through procedure for imperforate anus at birth, and later proctectomy with end colostomy due to poor anorectal function, presented with worsening low back pain radiating down to his left leg. An abdominal CT scan revealed a large pelvic cyst, causing compressive neuropathy. Multilocular cystic mass (17.5 cm) was resected, which was partially filled with mucinous material. Mucin was not seen on the cyst surface. On microscopic examination, the cyst was largely denuded however was partially lined by markedly attenuated ano-rectal type mucosa and focally by dysplastic glandular epithelium. Similar to LAMN, pools of acellular mucin dissecting stromal tissues were noted, associated with degenerative changes, calcifications and fibrosis. No invasive carcinoma or high-grade dysplasia was seen. Given the lack of extra- cystic mucin or evidence of overt malignancy, it was reported as mucinous cystic neoplasm with uncertain malignant potential. The patient has been followed for 16 months without evidence of recurrence or peritoneal disease. Results (if a Case Study enter NA) NA Conclusion Extended stasis within a bowel segment appear to contribute to the development of mucoceles. The blind-ended anatomy of the appendix renders it highly susceptible to this phenomenon, explaining the relatively high incidence of LAMN. Conversely, such prolonged stasis rarely occurs elsewhere in colon unless a pouch or bypassed segment is surgically created, particularly placing patients with a complex surgical history at the highest risk. Therefore, close follow-up is indicated in this patient group to screen for mucocele that may progress to develop dysplasia and mucinous neoplasm.

Clinical implications of heat shock protein 70 in oral carcinogenesis and prediction of progression and recurrence in oral squamous cell carcinoma patients: a retrospective clinicopathological study

BackgroundOral cancer is a common cause of death worldwide. The search for novel biomarkers for oral cancer is an ongoing struggle. Prognostic biomarkers are of great importance in diagnosis, and prediction of the cancer outcome. There are several disagreements in oral cancer studies over the role of heat shock proteins as prognostic markers. The current study investigated HSP70 expression in diverse tissues ranging from normal oral mucosa to dysplastic oral epithelium and oral squamous cell carcinoma to determine its role in oral carcinogenesis. Moreover, HSP70 was evaluated concerning different prognostic parameters to determine its capability in predicting cancer progression. Recurrence of tumor was recorded, and patients` disease-free survival was calculated and analyzed considering HSP70 expression to determine the potential utility of HSP70 immuno-expression in predicting recurrence.MethodsA retrospective study was accomplished on 50 cases of OSCC. Biopsies from the cancerous tissue, the free surgical margin, and the normal oral mucosa were used. The grading of dysplastic epithelium and OSCCs followed the criteria of WHO classification (2017). The clinicopathological and follow-up records for each patient were retrieved. Pearson’s Chi-square test, one-way ANOVA, and post hoc tests were used to analyze the variance of HSP70 immuno-expression concerning different parameters. The Kaplan–Meier method was used to compute and visualize disease-free survival, and the log-rank test was used to analyze the data. With Cox regression, univariate and multivariate survival analyses were run. A P-value of 0.05 or less was regarded as statistically significant.ResultsA significant increased expression of HSP70 was observed as the tissue progressed from normal to dysplastic epithelium, and carcinoma (P = 0.000). HSP70 revealed a significant increased expression by progression from mild to severe dysplasia (P = 0.023), and also from well to moderately and poorly differentiated carcinoma (P = 0.000). High HSP70 immuno-expression was significantly associated with progression of OSCC; large-sized tumors (P = 0.002), advanced TNM clinical stages (P = 0.001), positive nodal involvement (P = 0.001), presence of recurrence (P = .008), and reduced DFS (P = 0.014).ConclusionHSP70 has a crucial contribution to oral carcinogenesis, and its immune-expression could potentially be used as predictor of progression and recurrence of OSCC patients.Trial registration: Retrospectively registered.

Primary intraosseous carcinoma arising in a dentigerous cyst: A case report

AbstractDentigerous cysts are among the most common cysts of the jaw. They are attached to the crown of an unerupted tooth, most frequently third molars and maxillary canines. They are often asymptomatic and may therefore be diagnosed late. We report a case of a primary intraosseous carcinoma arising from the wall of a dentigerous cyst and describe the expression of typical genes associated with tumour growth and prognosis. The tumour expressed cytokeratins (CK) 5/6, 8, 14 and 19, but not CK7. Ki‐67 and P53 were upregulated in dysplastic epithelium and tumour tissue. Although rare, the possibility for tumour development in the wall of a common cyst should be considered. The case emphasizes the importance of histopathological examination of surgical specimens, even if the clinical diagnosis seems obvious.

Oral Squamous Cell Carcinoma with Comedo Necrosis – A Case Report

Oral squamous cell carcinoma is the common kind of oral malignant neoplasm. Squamous cell carcinomas of the alveolar ridge account for 9% of all oral carcinomas. Oral squamous cell carcinoma is one of the most common cancers in most Asian countries with global distribution. In our country, carcinoma of the oral cavity is one of the five leading sites of cancer in either gender. The dentist should have proper clinical and biological details related to histologic variants. Microscopic documentation of the various histological subtypes of oral squamous cell carcinoma should be emphasised among pathologists to contribute to steady knowledge of the clinical and biological behaviour in the oral cavity and result in more exact treatment protocols. Keywords: Comedo necrosis, Dysplastic epithelium, Oral squamous cell carcinoma

IL-1β transgenic mouse model of inflammation driven esophageal and oral squamous cell carcinoma

Chronic inflammation is integral to the development of esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC), although the latter has not been associated with reflux esophagitis. The L2-IL-1β transgenic mice, expressing human interleukin (IL)-1β in the oral, esophageal and forestomach squamous epithelia feature chronic inflammation and a stepwise development of Barrett’s esophagus-like metaplasia, dysplasia and adenocarcinoma at the squamo-columnar junction. However, the functional consequences of IL-1β-mediated chronic inflammation in the oral and esophageal squamous epithelia remain elusive. We report for the first time that in addition to the previously described Barrett’s esophagus-like metaplasia, the L2-IL-1β mice also develop squamous epithelial dysplasia with progression to squamous cell carcinoma (SCC) in the esophagus and the tongue. L2-IL-1β showed age-dependent progression of squamous dysplasia to SCC with approximately 40% (n = 49) and 23.5% (n = 17) incidence rates for esophageal and tongue invasive SCC respectively, by 12–15 months of age. Interestingly, SCC development and progression in L2-IL-1β was similar in both Germ Free (GF) and Specific Pathogen Free (SPF) conditions. Immunohistochemistry revealed a T cell predominant inflammatory profile with enhanced expression of Ki67, Sox2 and the DNA double-strand break marker, γ-H2AX, in the dysplastic squamous epithelia of L2-IL-1β mice. Pro-inflammatory cytokines, immunomodulatory players, chemoattractants for inflammatory cells (T cells, neutrophils, eosinophils, and macrophages) and oxidative damage marker, iNOS, were significantly increased in the esophageal and tongue tissues of L2-IL-1β mice. Our recent findings have expanded the translational utility of the IL-1β mouse model to aid in further characterization of the key pathways of inflammation driven BE and EAC as well as ESCC and Oral SCC.

Comparison of the IHC Markers CD138 and CD43 in Oral Leukoplakia: An Original Research.

In the clinical practice, one of the most common suspicious lesions that may be potentially malignant is oral leukoplakia. Globally, the rate at which it turns malignant varies. This study examines the levels of markers CD138 and 43 in oral leukoplakia. Twenty archival blocks of confirmed epithelial dysplasia were taken from the Department of Oral Pathology. These were processed for the identification of markers CD138 and 43 through Immuno Histo Chemistry (IHC). The blocks were divided equally for both the markers. There was a noticeable difference in staining intensity between dysplastic tissue and nondysplastic epithelium. However, CD138 expression was low or weak in dysplastic epithelium. CD43 expression was negative in all nonhematopoietic tissues. Genes that are cancer associated have been found to have incredibly different impacts in numerous tissues during the multistep process of oral carcinogenesis. In tissues undergoing dysplastic changes, CD138 expression was shown to be decreased, which could point out the malignant changes initiated in the epithelium of the oral tissues.

Image enhanced colonoscopy: updates and prospects-a review.

Colonoscopy has been proven to be a successful approach in both identifying and preventing colorectal cancer. The incorporation of advanced imaging technologies, such as image-enhanced endoscopy (IEE), plays a vital role in real-time diagnosis. The advancements in endoscopic imaging technology have been continuous, from replacing fiber optics with charge-coupled devices to the introduction of chromoendoscopy in the 1970s. Recent technological advancements include "push-button" technologies like autofluorescence imaging (AFI), narrowed-spectrum endoscopy, and confocal laser endomicroscopy (CLE). Dye-based chromoendoscopy (DCE) is falling out of favor due to the longer time required for application and removal of the dye and the difficulty of identifying lesions in certain situations. Narrow band imaging (NBI) is a technology that filters the light used for illumination leading to improved contrast and better visibility of structures on the mucosal surface and has shown a consistently higher adenoma detection rate (ADR) compared to white light endoscopy. CLE has high sensitivity and specificity for polyp detection and characterization, and several classifications have been developed for accurate identification of normal, regenerative, and dysplastic epithelium. Other IEE technologies, such as blue laser imaging (BLI), linked-color imaging (LCI), i-SCAN, and AFI, have also shown promise in improving ADR and characterizing polyps. New technologies, such as Optivista, red dichromatic imaging (RDI), texture and color enhancement imaging (TXI), and computer-aided detection (CAD) using artificial intelligence (AI), are being developed to improve polyp detection and pathology prediction prior to widespread use in clinical practice.

Clinicopathological Features of Oral Leukoplakia Among Snuff Users and Non-Users: An Analytical Study

Oral leukoplakia refers to a white lesion of questionable risk excluding other lesions carrying a risk of conversion into malignancy. Tobacco is regarded as the most common risk factor and may affect the clinicopathological aspect of the said lesion. Objectives: To check the clinicopathological features of oral leukoplakia among snuff users and non-users. Methods: The present analytical study was done on 60 oral leukoplakia cases and was further subdivided into 30 cases of snuff users and 30 non users. Clinicopathological features were assessed in all the cases. Data analysis were done by using SPSS-20. Results: The observed male cases were 43 (71.7 %) and female cases were 17 (28.3%). The ratio was found to be 2.5:1. All the 30 snuff users were males. Among non-users 13/30 (43.3%) were males and 17/30 (56.7%) were females. The relationship was found to be statistically significant with a p-value of &lt;0.01. The mean age among cases who used snuff was 56.97 (SD ± 14.71) while the mean age among non-users was found to be 47.43 (SD ± 13.44). In snuff user’s buccal mucosa was affected in 12/30 (40%) cases whereas in non-user buccal mucosa was also the most common site 18/30 (60%) cases showing a non-significant relationship p-value 0.59. Conclusions: Oral leukoplakia was more prevalent among males with a mean age range of more than fifty years and buccal mucosa and buccal sulcus being the most common sites. Dysplastic epithelium was more common among those cases that used snuff and this showed that chances of malignant transformation are more in such cases.

Oral Squamous Cell Carcinoma-Derived Cell-Free DNA Modulates Stemness and Migration of Oral Squamous Cell Carcinoma Cell Line by Inducing M2 Macrophage Polarization

To investigate the effect of oral squamous cell carcinoma (OSCC)-derived cell-free DNA (cfDNA) on the polarization of macrophages and the regulatory effect of polarized macrophages on the stemness and migration of OSCC cells. A total of 30 OSCC tissue samples, 10 dysplastic oral tissue samples, and 10 normal oral tissue samples were collected. The status of all tissue samples was confirmed by pathology analysis. Immunohistochemical (IHC) staining and immunofluorescence (IF) staining were performed to examine the cell count and location of M2 macrophages in different types of oral tissue samples. The conditioned medium (CM) of OSCC cell line CAL-27 from the human tongue was collected and the cfDNA was concentrated and isolated for identification. The macrophages were treated by cfDNA and their morphological characteristics were observed under microscope. The expression levels of polarization-related indicators were determined by RT-qPCR. CAL-27 cell line was treated with macrophage CM induced by cfDNA and the expression levels of stemness-related genes were determined by RT-qPCR. Scratch-wound assay was conducted to verify that the migration ability of CAL-27 was modulated by macrophages induced by cfDNA. There were more M2 macrophages in the deep connective tissue of dysplastic oral epithelium and the stroma of OSCC compared with those in the normal oral tissues ( P<0.05). OSCC cell line CAL-27 could secret cfDNA of 10000-15000 bp in length. cfDNA secreted by CAL-27 could induced in macrophages significantly higher expression of M2-macrophage-related genes ( P<0.05). cfDNA-treated macrophages induced significantly increased expression of stemness-related genes in CAL-27 cell line ( P<0.05) and promoted the migration ability of CAL-27 cell line ( P<0.05). OSCC-derived cfDNA promotes stemness and migration of OSCC cell line by inducing M2 macrophage polarization.

Partial decellularization eliminates immunogenicity in tracheal allografts.

There is currently no suitable autologous tissue to bridge large tracheal defects. As a result, no standard of care exists for long-segment tracheal reconstruction. Tissue engineering has the potential to create a scaffold from allografts or xenografts that can support neotissue regeneration identical to the native trachea. Recent advances in tissue engineering have led to the idea of partial decellularization that allows for the creation of tracheal scaffolds that supports tracheal epithelial formation while preserving mechanical properties. However, the ability of partial decellularization to eliminate graft immunogenicity remains unknown, and understanding the immunogenic properties of partially decellularized tracheal grafts (PDTG) is a critical step toward clinical translation. Here, we determined that tracheal allograft immunogenicity results in epithelial cell sloughing and replacement with dysplastic columnar epithelium and that partial decellularization creates grafts that are able to support an epithelium without histologic signs of rejection. Moreover, allograft implantation elicits CD8+ T-cell infiltration, a mediator of rejection, while PDTG did not. Hence, we establish that partial decellularization eliminates allograft immunogenicity while creating a scaffold for implantation that can support spatially appropriate airway regeneration.

Digital spatial profiling of intraductal papillary mucinous neoplasms: Toward a molecular framework for risk stratification

The histopathologic heterogeneity of intraductal papillary mucinous neoplasms (IPMN) complicates the prediction of pancreatic ductal adenocarcinoma (PDAC) risk. Intratumoral regions of pancreaticobiliary (PB), intestinal (INT), and gastric foveolar (GF) epithelium may occur with either low-grade dysplasia (LGD) or high-grade dysplasia (HGD). We used digital spatial RNA profiling of dysplastic epithelium (83 regions) from surgically resected IPMN tissues (12 patients) to differentiate subtypes and predict genes associated with malignancy. The expression patterns of PB and GF lesions diverged from INT, suggesting that PB and GF arise from a common lineage. Transcriptional dysregulation within PB lesions mirrored that of PDAC, whereas INT and GF foci did not. Tumor necrosis factor/nuclear factor κB (TNF-NFκB) and cell cycle (cycling S and cycling G2-M) programs occurred with relative prominence in PB and INT subtypes, respectively. Together, this study delineates markers of high-risk IPMN and insights into malignant progression.

Importance of P16ink4a marker in oral neoplasms

We have increase in number of oral lesions at ASRAMS, as it is a referral centre and there by we have determined to study the prevalence of these lesions and its association with P16ink4a marker.The purpose of this study is to evaluate the role of P16ink4a in oral epithelial dysplasias and oral squamous cell carcinoma by immunohistochemistry. In clinically significant patients, correlation between histopathology findings and its association with P16ink4a marker has been studied in Department of Pathology, ASRAMS. A retrospective study done from July 2017 to July 2019.The study was carried out after approval of the Institutional Ethics Committee. The biological material received was resection specimen and punch biopsies of the lesion which was fixed in 10% formalin for 6hrs later processed, followed by paraffin embedding and hematoxylin &amp; eosin staining of the sections. Histopathological classification of lesions was done according to the criteria proposed by WHO for oral cavity lesions.Immunohistochemical analysis with P16ink4a was performed on the serial sections. Development of reactions was performed with DAB (Diamino benzidine). The panel used was clone E6H4, requiring no dilution followed by epitope retrieval solution. All data were tabulated in a Windows Excel spreadsheet (Microsoft Excel 2011) and statistically analyzed using software version. The expression of P16ink4a in various lesions was tabulated.The study included 29 cases of oral lesions, most of them belonged to age group of 40 to 60 yrs. Male preponderance is noted. Most of the cases in our study were located on tongue. Histopathologically well differentiated squamous cell carcinomas were more in number. The immunoexpression of p16 stain was present at nuclear and cytoplasmic level mainly found at the basal dysplastic epithelium. P16 had a weak intensity at level of tumor proper and also at invasion front.P16ink4a immunohistochemical marker can be used as a surrogate biomarker for HPV detection of oral epithelial dysplasias and oral squamous cell carcinomas. P16 is useful marker in identifying dysplastic lesions and decrease of its immunoexpression is a predictive factor notified for neoplastic transformation.

Impact of CD34-dependent Micro Vessel Density on Periapical Odontogenic Cysts.

Odontogenic cysts belong to a type of lesions with endodontic origin that in some cases mimic even aggressive odontogenic tumors sharing with them similar radiographic features. Periapical cysts (PCs) belong to the inflammatory odontogenic cysts sub-category and rarely squamous cell carcinoma arises from their hyperplastic/ dysplastic epithelia. This study aimed to explore the impact of cluster differentiation 34 (CD34) protein expression combined with micro vessel density (MVD) on PCs. Forty-eight (n=48) archival, formalin-fixed, and paraffin-embedded PC tissue specimens were included in the study. Immunohistochemistry (IHC) was performed in the corresponding tissue sections using an anti- CD34 antibody. CD34 expression levels and also MVD in the examined cases were measured by implementing a digital image analysis protocol. CD34 over-expression (moderate to high staining intensity levels) were detected in 29/48 (60.4%) cases, whereas the rest of them (19/48-39.6%) were characterized by low levels of expression. Extended MVD was identified in 26/48 (50.1%) cases correlated with CD34 over-expression, epithelial hyperplasia (p-value=0.001), and marginally with inflammatory infiltration level in the examined lesions (p-value=0.056). CD34 over-expression combined with increased MVD is associated with a neoplastic-like (hyperplastic) phenotype in PCs as a result of increased neo-angiogenic activity. These histopathological characteristics rarely form an eligible substrate for squamous cell carcinoma onset in untended cases.