Preneoplastic Changes Research Articles

Beyond Metaplasia: Unraveling the Complex Pathogenesis of Autoimmune Atrophic Gastritis and Its Implications for Gastric Cancer Risk.

Autoimmune gastritis (AIG) is a chronic inflammatory condition characterized by immune-mediated destruction of gastric parietal cells, leading to oxyntic atrophy, achlorhydria, and hypergastrinemia. While AIG was historically linked to gastric adenocarcinoma and type I neuroendocrine tumors (NETs), recent evidence suggests the risk of adenocarcinoma in AIG is lower than previously believed, particularly in Helicobacter pylori (H. pylori)-negative patients. The increased cancer risk in AIG is mainly attributed to concurrent or past H. pylori infection. The incidence of gastric adenocarcinoma in AIG ranges from 0.12% to 0.5% per year, with cumulative risks over 10 years reported at 1%-3%. In contrast, type I NETs are more commonly associated with AIG, with an annual incidence of 0.68%-2.8% and cumulative rates as high as 15.3% over five years. Adenomatous polyps, which can progress to malignancy, have been reported in 4.6%-13.6% of AIG patients. This review examines the immune and molecular mechanisms underlying AIG's pathogenesis, positioning it as a model of immune-mediated epithelial injury with limited carcinogenic potential. AIG is associated with reparative metaplastic phenotypes, such as pseudopyloric and complete intestinal metaplasia, which contrast with the more aggressive incomplete intestinal metaplasia observed in H. pylori-induced gastritis. The reduced risk of adenocarcinoma in AIG is attributed to the absence of H. pylori, a T cell-dominated microenvironment, minimal macrophage infiltration, and protective factors such as altered gastric microbiota, epigenetic modifications, increased CD3+ intraepithelial cytotoxic T lymphocytes, and reduced interleukin-33/interleukin-13 signaling. Although AIG is linked to preneoplastic changes, its primary neoplastic risks include the development of type I NETs and adenomatous polyps, which carry a potential for malignant transformation, necessitating long-term surveillance in patients with hypergastrinemia, extensive atrophy, and associated gastric lesions. Challenges persist in distinguishing AIG from other atrophic gastritis types due to limitations in serological and histological markers, but emerging diagnostic tools, such as lymphocyte profiling and molecular assays, promise improved accuracy. This review underscores the importance of tailored surveillance and management strategies to address the distinct neoplastic risks associated with AIG, while advocating for further research into its immune landscape and molecular pathways.

Early detection of cervical tumor cell of origin through Oncogene-induced senescent HPV-positive cells.

Human Papilloma Virus (HPV) is an oncogenic virus and is the most common cause of cervical cancer. HPV has been shown to induce senescence. Cellular senescence is involved in cancer progression and tumorigenesis. Identification and isolation of cells of tumor origin before tumorigeneses is an important step in cancer prevention and treatment. This study aimed to investigate the early cervical atypical senescent cytological preneoplastic change in non-menopausal women. Cervical smears of 121 patients were randomly selected and included in the study which cytopathologically diagnosed as atypical squamous cells of undetermined significance (AS-CUS) in correlation to HPV status, parakeratosis (PK), p16 immunostaining, enlarged Squamous cells nuclei (ES) and inflammatory cells infiltration (ICI). Results revealed that out of the total 121 patients, 32 cases (26%) were positive for high-risk HPV (HR-HPV), 26 cases (22%) were positive for low-risk HPV (LR-HPV) and 63 (52%) were negative for HPV. HPV infections were significantly associated with age groups (p<0.026), PK (p = 0.043), p16 (p = 0.001), ES (p = 0.002) and ICI (p = 0.049). The positive immunostaining expression of p16 was only noticed in two HR-HPV patients. ES cells were found in 9.5% of HPV-negative cases, 27% of LR-HPV cases and 40.5% of HR-HPV cases. High PK cell positivity was seen only in HR-HPV. High ICI scores were seen in 40.6% of HR-HPV patients, 26.9 % of LR-HPV and 17.4 % of negative HPV patients. It was concluded that high PK positivity, high ICI score, positive p16 immunostaining and ES were correlated with HR-HPV in non-menopausal women. These findings could provide potential diagnostic clues for HPV-harboring senescent cells as a strategy for reducing HPV risk of cervical cancer development and identifying the cell of tumor origin, which could be beneficial for improving the utility of senolytic agents and immunotherapy in clinical practice.

Pancreatic and Ileal Neuroendocrine Tumors: Metastatic Disease or a Novel MEN Syndrome?

Multiple endocrine neoplasms are a feature of multiple endocrine neoplasia (MEN) syndromes types 1, 2, 4, and 5. However, the ileum is not usually involved in these disorders. We report a series of patients with neuroendocrine tumors (NETs) involving both the pancreas and the ileum. We searched the laboratory information system and personal consultation records of the authors from 2019 to 2023 for patients who had neuroendocrine tumors (NETs) involving both the pancreas and ileum. In a series of 846 patients, we identified 4 patients with pancreatic and ileal NETs, 2 female and 2 male, ages 52 to 75. Two female patients had primary EC cell tumors of the ileum with metastasis to the pancreas that showed expression of CDX2 and serotonin similar to the ileal primary tumors. Two males had primary lesions in the 2 sites with different immunoprofiles; the ileal tumors expressed CDX2 and serotonin and were negative for ARX, whereas the pancreatic tumors expressed ARX, glucagon, and pancreatic polypeptide and were negative for CDX2 and serotonin. In both male patients, the nontumorous pancreas showed preneoplastic changes in the endocrine elements, suggesting germline predisposition to endocrine neoplasia. Testing for known genetic alterations underlying MEN syndromes has not identified a genetic alteration that can be implicated in the development of NETs in both pancreas and ileum. Our series indicates the rare occurrence of NETs in both the pancreas and ileum and emphasizes the importance of using the correct biomarkers to distinguish metastasis from primary neoplasms at the different sites. The rare occurrence of primary ileal and pancreatic NETs may represent a novel MEN syndrome with as yet unknown germline predisposition.

The immunomodulatory impact of naturally derived neem leaf glycoprotein on the initiation progression model of 4NQO induced murine oral carcinogenesis: a preclinical study.

Murine tumor growth restriction by neem leaf glycoprotein (NLGP) was established in various transplanted models of murine sarcoma, melanoma and carcinoma. However, the role of NLGP in the sequential carcinogenic steps has not been explored. Thus, tongue carcinogenesis in Swiss mice was induced by 4-nitroquinoline-1-oxide (4NQO), which has close resemblance to human carcinogenesis process. Interventional role of NLGP in initiation-promotion protocol established during 4NQO mediated tongue carcinogenesis in relation to systemic immune alteration and epithelial-mesenchymal transition (EMT) is investigated. 4NQO was painted on tongue of Swiss mice every third day at a dose of 25µl of 5mg/ml stock solution. After five consecutive treatment with 4NQO (starting Day7), one group of mice was treated with NLGP (s.c., 25µg/mice/week), keeping a group as PBS control. Mice were sacrificed in different time-intervals to harvest tongues and studied using histology, immunohistochemistry, flow-cytometry and RT-PCR on different immune cells and EMT markers (e-cadherin, vimentin) to elucidate their phenotypic and secretory status. Local administration of 4NQO for consecutive 300 days promotes significant alteration in tongue mucosa including erosion in papillae and migration of malignant epithelial cells to the underlying connective tissue stroma with the formation of cell nests (exophytic-hyperkeratosis with mild dysplasia). Therapeutic NLGP treatment delayed pre-neoplastic changes promoting normalization of mucosa by maintaining normal structure. Flow-cytometric evidences suggest that NLGP treatment upregulated CD8+, IFNγ+, granzyme B+, CD11c+ cells in comparison to 4NQO treated mice with a decrease in Ki67+ and CD4+FoxP3+ cells in NLGP treated cohort. RT-PCR demonstrated a marked reduction of MMP9, IL-6, IL-2, CD31 and an upregulation in CCR5 in tongues from 4NQO+NLGP treated mice in comparison to 4NQO treated group. Moreover, 4NQO mediated changes were associated with reduction of e-cadherin and simultaneous up-regulation of vimentin expression in epithelium that was partially reversed by NLGP. Efficacy of NLGP was tested first time in sequential carcinogenesis model and proved effective in delaying the initial progression. NLGP normalizes type 1 immunity including activation of the CD8+T effector functions, reduction of regulatory T cell functions, along with changes in EMT to make the host systemically alert to combat the carcinogenic threat.

Histopathologic and Preneoplastic Changes in Tubal Ligation Materials.

Background and Objectives: To investigate histopathological changes and serous carcinoma precursors such as secretory cell outgrowths (SCOUTs) and p53 signature in the bilateral tubal ligation (BTL) materials used during cesarean section (S/C). Materials and Methods: In total, 138 patients underwent S/C and tubal sterilization (TS) between October 2020 and May 2021 at Konya City Hospital. Patients' data were obtained from the hospital's system. All data and findings were investigated and statistically evaluated. Results: The mean age was 34.62 years (22-44), the mean gravity was 4.89 (2-15) and the mean parity was 3.46 (1-10). In total, 5.79% SCOUT, 7.24% atypia and 9.42% p53 signatures were observed. Significant correlations were shown between the epithelial cell lineage and age between Ki-67, SCOUT, and gravity; between the Ki-67 results and gravity and parity; and between the p53 score and age. Conclusions: TS is a common, safe, and effective method worldwide. Today, BTL is increasing along with increasing S/C ratios. In addition to the reduced risk of ovarian cancers with ligation alone, precursor lesions such as hyperplasia, SCOUT, p53 signature, and STIL/Serous tubal intraepithelial carcinoma (STIC) are encountered in the ampulla materials obtained. Considering the low rates of re-anastomosis, tubal excision may be recommended instead of ligation in women of relatively higher gravity and age.

Methods to Study Liver Disease Using Zebrafish Larvae.

Liver disease affects millions of people worldwide, and the high morbidity and mortality is attributed in part to the paucity of treatment options. In many cases, liver injury self-resolves due to the remarkable regenerative capacity of the liver, but in cases when regeneration cannot compensate for the injury, inflammation and fibrosis occur, creating a setting forthe emergence of liver cancer. Whole animal models are crucial for deciphering the basic biological underpinnings of liver biology and pathology and, importantly, for developing and testing new treatments for liver disease before it progresses to a terminal state. The cellular components and functions of the zebrafish liver are highly similar to mammals, and zebrafish develop many diseases that are observed in humans, including toxicant-induced liver injury, fatty liver, fibrosis, and cancer. Therefore, the widespread use of zebrafish larvae for studying the mechanisms of these pathologies and for developing potential treatments necessitates the optimization of experimental approaches to assess liver disease in this model. Here, we describe protocols using staining methods, imaging, and gene expression analysis to assess liver injury, fibrosis, and preneoplastic changes in the liver of larval zebrafish.

NOD1 mediates interleukin-18 processing in epithelial cells responding to Helicobacter pylori infection in mice

The interleukin-1 family members, IL-1β and IL-18, are processed into their biologically active forms by multi-protein complexes, known as inflammasomes. Although the inflammasome pathways that mediate IL-1β processing in myeloid cells have been defined, those involved in IL-18 processing, particularly in non-myeloid cells, are still not well understood. Here we report that the host defence molecule NOD1 regulates IL-18 processing in mouse epithelial cells in response to the mucosal pathogen, Helicobacter pylori. Specifically, NOD1 in epithelial cells mediates IL-18 processing and maturation via interactions with caspase-1, instead of the canonical inflammasome pathway involving RIPK2, NF-κB, NLRP3 and ASC. NOD1 activation and IL-18 then help maintain epithelial homoeostasis to mediate protection against pre-neoplastic changes induced by gastric H. pylori infection in vivo. Our findings thus demonstrate a function for NOD1 in epithelial cell production of bioactive IL-18 and protection against H. pylori-induced pathology.

Serum Anti-Thyroglobulin Autoantibodies Are Specific in Predicting the Presence of Papillary-like Nuclear Features and Lymphocytic Infiltrate in the Thyroid Gland.

(1) Background: Previous studies have reported a correlation between serum anti-Thyroglobulin-antibodies (TgAb) and papillary thyroid carcinoma. The aim of our study was to evaluate whether serum TgAb and anti-thyroid-peroxidase antibody (TPO) positivity was also related to pre-neoplastic histological changes such as papillary-like nuclear features (PLNF) and with the presence of lymphocytic infiltrate (LI) in thyroid surgical specimens. (2) Methods: The study was retrospectively carried out on 70 consecutively recruited patients who underwent thyroidectomy for benign process and whose TgAb and TPOAb values were retrieved from clinical records. Histological sections of thyroid surgical samples were revised, looking for PLNF and lymphocytic infiltrate. HBME1 expression was assessed by immunohistochemistry. (3) Results: Our results showed a significant association between TgAb, PLNF, and lymphocytic infiltrate. The presence of TgAb was highly specific, but less sensitive, in predicting the presence of PLNF (sensitivity = 0.6, specificity = 0.9; positive predictive value (PPV) = 0.88; negative predictive value (NPV) = 0.63). TgAb positivity showed a good association with the presence of lymphocytic infiltrate (sensitivity = 0.62, specificity = 0.9; PPV = 0.88 and NPV = 0.68). HBME1 immunoreactivity was observed in the colloid of follicles showing PLNF and/or closely associated with LI. (4) Conclusions: The presence of PLNF and LI is associated with serum TgAb positivity. The presence of TgAb and of LI could be triggered by an altered thyroglobulin contained in the HBME1-positive colloid, and could be a first defense mechanism against PLNF that probably represent early dysplastic changes in thyrocytes.

125 Effect of Multi-Walled Carbon Nanotube Exposure on wild type and p53+/- Rat Lungs

Abstract Carbon nanotubes (CNTs) are nanomaterials that can be inhaled by employees during production or handling. One CNT, Mitsui-7 (MWNT-7), has been classified by the International Agency for Research on Cancer as “possibly carcinogenic to humans”. In addition, there is a proposal coming from the risk assessment committee of ECHA to classify all fibres with dimension’s compatible with the “Fibre Paradigm” (FP), as carcinogenic. However, there is a lack of clarity regarding the fibres that do not fill the criteria of the FP. In this study, we compared Mitsui-7, compatible with the FP criteria, to a CNT, NM-403, too short and thin to fill this principle. We exposed Sprague Dawley female rats deficient for p53 (GMO), or wild type (WT), by intratracheal instillation to these two CNTs. The animals (GMO and WT) were exposed once a week for 4 consecutive weeks to 0.25 mg/rat of CNT and were sacrificed 3 days and 8 months after the last instillation. Exposure to both CNTs induced acute lung inflammation, which persisted after 8 months in the lungs of rats only exposed to NM-403. On the top of a persistent inflammation, NM-403 also stimulated hyperplasic changes in the rat lungs, which can be considered as a precursor stage for neoplasia. However, no adenoma nor carcinoma was detected. The hyperplasia was significantly more pronounced in the GMO rats. The results suggest that CNT not filling the criteria of the FP can cause persistent inflammation and pre-neoplastic changes. Their safety should be addressed with caution.

Frequency of Preneoplastic and Neoplastic Lesions in Cholecystectomy Specimen from a Tertiary Care Hospital in Uttar Pradesh, India: A Cross-sectional Study

Introduction: Cholelithiasis is known to cause a spectrum of changes, including inflammation in the gallbladder wall, cholesterolosis, atrophy, metaplasia, dysplasia, polyps, and eventually cancer. Gallbladder Carcinoma (GBC) is a rare and highly fatal malignancy. Aim: To determine the frequency of preneoplastic and neoplastic lesions, along with demographic and clinical aspects of different gallbladder lesions. Materials and Methods: This cross-sectional study was performed on 788 patients who underwent elective cholecystectomy for gallbladder disease at Hind Medical College, Sitapur, Uttar Pradesh, India, from August 2017 to October 2021. All patients underwent a history and physical examination, followed by routine laboratory tests and other investigations, including full abdominal ultrasound. Histopathologic evaluation of the gallbladder specimen was also performed after surgical resection. Categorical variables were presented as numbers and percentages (%), while continuous variables were presented as mean ± Standard Deviation (SD). Results: A total of 788 cholecystectomy specimens were analysed. The mean age of the patients was 42.49±1.39 years with female predominance 640 (82.05%). The majority of cases were between 40-49 269 (34.13%) and 60-69 399 (50.63) years of age. Preneoplastic changes were found in 764 (96.96%) cases, while neoplastic pathology was evident in 24 (3.04%) cholecystectomy specimens. The most commonly observed preneoplastic change was chronic cholecystitis in 532 (67.54%) cases. Other lesions associated with chronic cholecystitis were cholesterolosis in 76 cases (9.64%), xanthogranulomatous cholecystitis in 34 cases (4.31%), follicular cholecystitis in 30 cases (3.80%), and GBC in 24 cases (3.04%). Conclusion: This study observed that chronic cholecystitis and cholesterolosis, followed by xanthogranulomatous cholecystitis, were associated with metaplastic changes in gallbladder pathologies. It is believed that metaplasia-dysplasia could be linked to GBC. Therefore, routine microscopic examination is required for all cholecystectomies. However, further studies on gallbladder carcinogenesis are needed.

Extracellular vesicles from gastric epithelial GES-1 cells infected with Helicobacter pylori promote changes in recipient cells associated with malignancy.

Chronic Helicobacter pylori (H. pylori) infection is considered the main risk factor for the development of gastric cancer. Pathophysiological changes in the gastric mucosa initiated by this bacterium can persist even after pharmacological eradication and are likely attributable also to changes induced in non-infected cells as a consequence of intercellular communication via extracellular vesicles (EVs). To better understand what such changes might entail, we isolated EVs from immortalized normal gastric GES-1 cells infected (EVHp+) or not with H. pylori (EVHp-) by ultracentrifugation and characterized them. Infection of GES-1 cells with H. pylori significantly increased the release of EVs and slightly decreased the EV mean size. Incubation with EVHp+ for 24 h decreased the viability of GES-1 cells, but increased the levels of IL-23 in GES-1 cells, as well as the migration of GES-1 and gastric cancer AGS cells. Furthermore, incubation of GES-1 and AGS cells with EVHp+, but not with EVHp-, promoted cell invasion and trans-endothelial migration in vitro. Moreover, stimulation of endothelial EA.hy926 cells for 16 h with EVHp+ promoted the formation of linked networks. Finally, analysis by mass spectrometry identified proteins uniquely present and others enriched in EVHp+ compared to EVHp-, several of which are known targets of hypoxia induced factor-1α (HIF-1α) that may promote the acquisition of traits important for the genesis/progression of gastric pre-neoplastic changes associated with H. pylori infection. In conclusion, the harmful effects of H. pylori infection associated with the development of gastric malignancies may spread via EVs to non-infected areas in the early and later stages of gastric carcinogenesis.

Simulated galactic cosmic radiation (GCR)-induced expression of Spp1 coincide with mammary ductal cell proliferation and preneoplastic changes in ApcMin/+ mouse

Female astronauts inevitably exposed to galactic cosmic radiation (GCR) are considered at a greater risk for mammary cancer development. The purpose of this study is to assess the status of mammary cancer-associated preneoplasia markers after GCR and γ-ray irradiation using a mouse model of human mammary cancer. Female ApcMin/+ mice were irradiated to 50 cGy of either γ-ray (137Cs) or full-spectrum simulated galactic cosmic radiation (GCR) (33-beam), and at 110 – 120 days post-irradiation mice were euthanized, and normal-appearing mammary tissues were analyzed for histological and molecular markers of preneoplasia. Whole-mount staining, hematoxylin and eosin-based histological assessment, and Cyclin D1 immunohistochemistry (IHC) were performed to analyze ductal outgrowth and cell proliferation. Additionally, mRNA expression of known mammary preneoplasia markers (Muc1, Exo1, Foxm1, Depdc1a, Nusap1, Spp1, and Rrm2) was analyzed using qPCR, and their respective protein expression was validated using immunohistochemistry. A significant increase in ductal outgrowth and cell proliferation in mammary tissues of GCR-irradiated mice was noted which indicates a higher risk of mammary cancer, relative to γ-rays. Increased mRNA and protein expression of Spp1 was observed in the GCR group, relative to γ-rays. This study demonstrates the plausibility of Spp1 as a preneoplasia marker in the early detection of mammary cancer after space radiation exposure.

Recurrence of premalignant oral cavity and oropharynx lesions after pulsed diode laser treatment

PurposeLeukoplakia is common with a 1 % incidence in the population and may harbor preneoplastic changes. Diode lasers provide both precision and coagulation for excision of superficial lesions in clinical and operative settings with little damage to deeper tissue. We aim to determine the rate of oral and oropharyngeal hyperkeratosis and dysplasia recurrence after treatment with diode laser. Materials and methodsPatients with oral or oropharyngeal hyperkeratotic or mild dysplastic lesions treated with pulsed diode laser between 2013 and 2020 at a tertiary academic institution were analyzed. The main outcome measure was recurrence of hyperkeratosis and dysplasia after treatment. ResultsFourteen patients received diode laser treatment for hyperkeratotic or mild dysplastic lesions of the oral cavity or oropharynx. Demographic features included 9 (64.3 %) females and mean age of 66.6 years. In these 14 patients, 18 distinct lesions were identified. Eleven (61.1 %) lesions were located on the oral tongue, 4 (22.2 %) on the buccal mucosa, 2 (11.1 %) on the hard palate, and 1 (5.6 %) on the soft palate. Average time from lesion clinical diagnosis to the first diode laser treatment was 8.3 months with an average number of 1.4 treatments per lesion. Three (16.7 %) lesions experienced recurrence after the most recent treatment. None of the lesions underwent malignant transformation. None of the patients experienced bleeding, tethering, or dysarthria after treatment. One patient developed pyogenic granuloma and reported chronic tongue pain. ConclusionsPulsed diode laser treatment of leukoplakia was well tolerated with low complication rates and reasonable control of precancerous lesions.

Abstract 2199: Molecular dissection of a novel, highly proliferative lineage in a model of Helicobacter pylori-driven gastric metaplasia and dysplasia

Abstract At least 80% of gastric cancer cases are attributed to stomach infection with the bacterium Helicobacter pylori (Hp), which causes lifelong chronic inflammation. In some individuals, this inflammation can cause gastric atrophy, metaplasia (conversion of one normal cell type to another), dysplasia (presence of abnormal cells) and finally cancer, but the specific mechanism(s) through which Hp triggers this cascade are not well understood. Similar preneoplastic changes are recapitulated in a mouse model through tamoxifen-induced expression of active KRAS in the chief cells of the stomach (KRAS+ mice). We found that Hp infection of KRAS+ mice exacerbated disease: compared to Hp-KRAS+ mice, Hp+KRAS+ mice had an altered trajectory of metaplasia and accelerated dysplasia. In accordance with the hypothesis that Hp causes cancer through eliciting chronic inflammation, Hp+KRAS+ mice had severe inflammation marked by a ten-fold increase in T cells compared to Hp-KRAS+ mice. To understand gene expression changes in gastric cells in the context of metaplasia with and without active Hp infection, we performed single cell RNA-sequencing (scRNA-seq) on eight mouse stomachs (+/- Hp, +/- KRAS) using the 10x Chromium platform. Cluster analyses followed by UMAP (Uniform Manifold Approximation and Projection) visualization allowed us to identify 25 clusters, including epithelial and immune cell types. Hp+KRAS+ mice had a striking expansion of a population of metaplastic cells (most similar to surface mucus-producing “pit” cells), which comprised ~40% of epithelial cells in these mice. Metaplastic pit cells expressed markers of intestinal metaplasia and gastric cancer, and their abundance in the stomach was correlated with helper T cell abundance. Analysis of RNA velocity suggested that metaplastic pit cells arose from a progenitor cell type, rather than arising from pit cells directly. As well, a 10x Visium spatial gene expression experiment revealed the spatial organization of metaplastic pit cells near the lumen of the gastric epithelium. Finally, antibiotic eradication of Hp prevented metaplastic pit cell development and other disease phenotypes. Taken together, these studies provide new understanding of how Hp infection and inflammation cause gastric preneoplastic progression. Citation Format: Valerie Phoebe O'Brien, William C. Young, Greg Finak, Raphael Gottardo, Nina R. Salama. Molecular dissection of a novel, highly proliferative lineage in a model of Helicobacter pylori-driven gastric metaplasia and dysplasia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2199.

Three dimensional reconstruction of the mouse cerebellum in Hedgehog-driven medulloblastoma models to identify Norrin-dependent effects on preneoplasia

Spontaneous mouse models of medulloblastoma (MB) offer a tractable system to study malignant progression in the brain. Mouse Sonic Hedgehog (Shh)-MB tumours first appear at postnatal stages as preneoplastic changes on the surface of the cerebellum, the external granule layer (EGL). Here we compared traditional histology and 3DISCO tissue clearing in combination with light sheet fluorescence microscopy (LSFM) to identify and quantify preneoplastic changes induced by disrupting stromal Norrin/Frizzled 4 (Fzd4) signalling, a potent tumour inhibitory signal in two mouse models of spontaneous Shh-MB. We show that 3DISCO-LSFM is as accurate as traditional histology for detecting Norrin/Fzd4-associated changes in PNL formation in Ptch+/− mice and EGL hyperplasia in Neurod2-SmoA1+/− mice. Moreover, we show that the anti-tumour effect of Norrin/Fzd4 signalling is restricted to the posterior region of the cerebellum and is characterized by defective neural progenitor migration away from the EGL. In conclusion, 3DISCO-LSFM is a valid way to monitor tumour initiation events in mouse MB models and reveals an unanticipated regional restriction of stromal signalling in constraining tumour initiation.

Incidence of Pancreatic Intraepithelial Neoplasia in an Autopsy Series.

Pancreatic intraepithelial neoplasia (PanIN) is the currently preferred designation for putative preneoplastic changes in the pancreas. There are few data for the incidence of PanIN in the general population. Our goal was to determine the incidence of PanIN in a large group of pancreases obtained at autopsy. Slides stained with hematoxylin and eosin were scanned to count PanIN. We found multiple PanINs in most pancreases and at least 1 in 86.4% of 154 pancreases when multiple slides (8-12) from each were examined. The average age at autopsy was 62 years, and 90% of the patients were 40 years or older. Several questions were raised by our observations. Should a minimum size be defined for classification as PanIN? Do PanINs occur in lesions that apparently arise from acinar to ductal metaplasia? Does squamous metaplasia in PanIN have any special significance, and do purely squamous lesions have preneoplastic significance? We conclude that the incidence of PanIN is higher than previously reported.

Differential Expression of Long Noncoding RNA HOTAIR in Intestinal Metaplasia and Gastric Cancer.

INTRODUCTION:High expression of HOTAIR promotes tumor growth and carries a dismal prognosis for the patient. We investigated the prognostic value of HOTAIR expression in gastric cancer (GC) and systematically delineate the expression in relation to Helicobacter pylori infection and preneoplastic changes.METHODS:HOTAIR expression was analyzed in surgical paired tissue samples of patients with GC and biopsy samples from patients with atrophic gastritis and/or intestinal metaplasia (AG ± -IM), chronic nonatrophic gastritis, and controls. The cancer genome atlas (TCGA) data were used for validation. HOTAIR expression was evaluated in sera and ascites of patients with GC. Quantitative HOTAIR expression analysis was performed using quantitative polymerase chain reaction, and LINE-1 methylation was assessed by bisulfite pyrosequencing.RESULTS:HOTAIR was more frequently detected in tumor tissues compared with adjacent gastric mucosa (65.4% vs 8.6%). HOTAIR expression was associated with depth of tumor invasion and tumor location and with shorter overall survival in patients with diffuse-type GC as confirmed in the TCGA cohort. HOTAIR was not detectable in controls but was found in 2.2% of patients with chronic nonatrophic gastritis and 18.3% of patients with AG ± IM, which was further associated with IM, grade of IM, and H. pylori positivity.DISCUSSION:HOTAIR expression was associated with GC and preneoplastic changes of stomach mucosa. Although HOTAIR expression was strongly linked to IM, HOTAIR expression was only associated with worse prognosis in Lauren diffuse and not intestinal type of GC. Further studies are needed to evaluate the value of HOTAIR as diagnostic and predictive biomarker in IM and translational therapeutic relevance of HOTAIR in diffuse-type GC.

Detection of hepatocarcinogens by combination of liver micronucleus assay and histopathological examination in 2-week or 4-week repeated dose studies

BackgroundCurrently, revisions to the ICH S1 guidance on rodent carcinogenicity testing are being proposed. Application of this approach would reduce the use of animals in accordance with the 3Rs principles (reduce/refine/replace). The method would also shift resources to focus on more scientific mechanism-based carcinogenicity assessments and promote safe and ethical development of new small molecule pharmaceuticals. In the revised draft, findings such as cellular hypertrophy, diffuse and/or focal cellular hyperplasia, persistent tissue injury and/or chronic inflammation, preneoplastic changes, and tumors are listed as histopathology findings of particular interest for identifying carcinogenic potential. In order to predict hepatocarcinogenicity of test chemicals based on the results from 2- or 4-week repeated dose studies, we retrospectively reanalyzed the results of a previous collaborative study on the liver micronucleus assay. We focused on liver micronucleus induction in combination with histopathological changes including hypertrophy, proliferation of oval cells or bile duct epithelial cells, tissue injuries, regenerative changes, and inflammatory changes as the early responses of hepatocarcinogenesis. For these early responses, A total of 20 carcinogens, including 14 genotoxic hepatocarcinogens (Group A) and 6 non-liver-targeted genotoxic carcinogens (Group B) were evaluated.ResultsIn the Group A chemicals, 5 chemicals (NPYR, MDA, NDPA, 2,6-DNT, and NMOR) showed all of the 6 early responses in hepatocarcinogenesis. Five chemicals (DMN, 2,4-DNT, QUN, 2-AAF, and TAA) showed 4 responses, and 4 chemicals (DAB, 2-NP, MCT, and Sudan I) showed 3 responses. All chemicals exhibited at least 3 early responses.Contrarily, in the Group B chemicals (6 chemicals), 3 of the 6 early responses were observed in 1 chemical (MNNG). No more than two responses were observed in 3 chemicals (MMC, MMS, and KA), and no responses were observed in 2 chemicals (CP and KBrO3).ConclusionEvaluation of liver micronucleus induction in combination with histopathological examination is useful for detecting hepatocarcinogens. This assay takes much less time than routine long-term carcinogenicity studies.

Morphological Changes in Major Salivary Glands in Mice Treated With a Choline and Methionine Deficient Diet.

It has been shown that the methionine-choline deficient (MCD) diet induces hepatocarcinogenesis, but not in extrahepatic organs, such as the testis, and pancreas, although may increase chemical-induced carcinogenesis in the colon, mammary gland, esophagus, and pancreas. Accumulating evidence suggests that salivary glands are very susceptible to stress conditions, such as radiation, hyperglycemia, and exposure to xenobiotics in vivo. This study aimed to analyze the histological changes on the major salivary glands (parotid, submandibular, and sublingual) after MCD diet administration. Male Swiss mice were submitted to ad libitum access to the control (AIN-76) or MCD diet for 28 days. The rebound group received the MCD diet for 24 days and the control diet for 10 days. Using the AxioImager A2 microscope, the hematoxylin-eosin (HE) stained specimens (4 mm) were evaluated for tissue degeneration, nuclear hyperchromatism and atrophy. In the parotid gland from the MCD group, tissue degeneration, pyknosis, apoptosis and atrophy were observed, which remained in the rebound group, associated with hyperchromatism. In the submandibular gland from both MCD and rebound groups, severe tissue disorganization was associated with cell pleomorphism, hyperchromatic cells, apoptosis, increased eosinophilia, and inflammatory infiltrate. Finally, in the sublingual gland, there were no histological alterations in the experimental groups compared to the control. MCD can induce pre-neoplastic changes in the mouse parotid and submandibular glands, which are not reversed by a change in the diet.

Clinical Significance of Endometrial Cells in Pap Smear of Women Aged 40 Years and Older.

To investigate the histopathological follow-up results in women diagnosed with endometrial cells in the Papanicolaou (Pap) test. Between January 2013 to December 2018, women with endometrial cells on the Pap test were searched from the hospital electronic database. The patients with endometrial cells on the Pap test who underwent further histopathological evaluation and who were followed-up for at least 1 year were enrolled in the study, while those who had a Pap test result other than endometrial cells, were lost during follow-up, or had missing data were excluded. Out of 91,142 Pap smears, 121 (0.1%) cytologically had endometrial cells, and of those 65 cases were eligible for final analysis. The mean age of patients with premalignant/malignant lesions (57.7 ± 2.9) was higher than those with benign lesions (50.1 ± 0.7), with 77% of them in the postmenopausal period. Gynecologic premalignant/malignant lesions were detected in 9 (17.7%) patients including 2 (3.1%) endometrial hyperplasias and 7 (10.8%) endometrial cancers. The menopausal status (p=0.010) and being 50 years and older (p=0.002) were significantly associated with pre-neoplastic or neoplastic changes in patients with endometrial cells. The presence of endometrial cells in Pap tests may be a harbinger of endometrial pathologies, especially at the age of 50 years and over. The menopausal status is another possible determinant in detecting endometrial carcinoma. Further investigation may be suggested in women aged ≥50 years and postmenopausal in the event of endometrial cell detection.