Normal Squamous Epithelium Research Articles

365. METHYLATION BIOMARKERS TO STRATIFY BARRETT’S ESOPHAGUS PATIENTS PROGRESSING AND NON-PROGRESSING TO ESOPHAGEAL ADENOCARCINOMA

Abstract Background Over the last forty years, North America and Europe have witnessed a notable surge in cases of esophageal adenocarcinoma (EAC). This malignancy typically arises from Barrett esophagus (BE), a condition characterized by the replacement of normal squamous epithelium with specialized metaplastic cells. Our study aims to identify specific methylation markers capable of distinguishing between BE patients that will and will not progress to EAC. Methods We conducted differential expression analysis on RNASeq public dataset GSE210647 to distinguish BE progressors from non-progressors. Reads were aligned to hg38 genome using STAR aligner, normalized and analyzed with DESeq2. Significant genes were selected by |log2 fold change| ≥ 0.2 and padj < 0.01. GO, pathway enrichment, and transcription factor (TF) network analyses were performed to uncover biological disparities. Integration with previous methylation data (N.R. Dehkordi et al., 292. Specific methylation markers associated with changes in gene expression distinguish EAC and sub-types of BE patients, ISDE 2023) allowed the identification of overlapping biomarkers, aiding in better discrimination between the two groups. Results Differential expression analysis of GSE210647 identified 2261 significative DEGs between BE progressors and non-progressors. Pathway enrichment analysis in BE progressors revealed significant associations with cellular morphogenesis and reprogramming, alongside the induction of the bile acid pathway, known for its implications in invasiveness and cancer stem cell expansion. TF network analysis identified key regulators such as TBX20, FOXS1, and ZNF454, shedding light on their roles in esophageal carcinogenesis. Among the 505 CpG markers acquired in prior analyses, 127 genomic regions were linked with overexpressed genes either in progressors or in non-progressors BE patients. Leveraging this marker panel, BE samples were stratified into discrete subtypes, aligning either with normal esophageal tissue or EAC, and suggesting a potential stratification by disease progression. Conclusions The integration of expression and methylation analyses allows the identification of key genomic and epigenomic loci able to distinguish BE patients progressing or not-progressing towards EAC. The identified biomarkers are pivotal for personalized risk assessment and for the design of targeted interventions, holding promises for enhanced surveillance protocols, refined early EAC detection approaches, and improved therapeutic strategies aimed at mitigating the risk of EAC development in BE population.

Circular RNA Expression Profile in Cervical Cancer and Construction of the Circular RNA‑MicroRNA‑Messenger RNA Regulatory Network

Introduction. Cervical cancer (CC) remains the most common cancer in women worldwide. However, effective and specific biomarkers for the diagnosis and prognosis of cervical cancer are yet to be found. In recent years, the potential of circular RNAs (circRNAs) as new diagnostic, prognostic and therapeutic tools has received much attention. The current study involved an in-depth bioinformatics research to explore the circRNA-microRNA (miRNA)-messenger RNA (mRNA) regulatory network in order to identify important molecular processes and biological pathways supposedly associated with CC. Materials and methods. The study collected data on the expression of circRNA (GSE102686), miRNA (GSE30656) and mRNA of target genes (GSE9750), based on the Gene Expression Omnibus (GEO) database, in squamous cell carcinoma of the cervix samples and normal squamous epithelium of the cervix, dividing them into study and control groups. Protein-protein interaction (PPI), Gene Ontology (GO) analysis, and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were performed to further understand the function of circRNAs for their target genes. Results. A total of 105 differentially expressed circular RNAs (DECs), 144 differentially expressed microRNAs (DEMs), and 539 differentially expressed target genes (DEGs) were identified for cervical cancer. Concurrently, functional enrichment analysis of GO and KEGG pathways was performed for DEGs. Subsequently, searching databases for circRNA, miRNA and mRNA target genes, as well as PPI network analysis and functional enrichment revealed 3 DECs with significantly high expression levels (hsa_circ_0000745, hsa_circ_0084927 and hsa_circ_0002762), 6 DEMs with reduced expression levels (hsa -miR-145, hsa-miR-876-3p, hsa-miR-1229, hsa-miR-182, hsa-miR-520h and hsa-miR-1252) and 9 key genes such as ANGPT2, COL11A1, MEST, KIF20A, CLN6, FNDC3B, USP18, DLGAP5 and CXCL9, suggesting a potentially significant role in cervical cancer. Conclusion.Understanding the circRNA-miRNA-mRNA regulatory network is of great importance for evaluating the oncogenesis of CC, as well as discoverying new circRNAs as the main regulatory molecules in this network. This is considered to be a new direction in the diagnosis and targeted therapy of cervical cancer.

Abstract 2264: Identifying and analyzing independent clonal populations in single Barrett’s esophagus biopsies

Abstract Barrett’s esophagus (BE) is a non-obligate precursor of esophageal adenocarcinoma (EAC), often measuring multiple centimeters in length and characterized by columnar epithelium replacing normal squamous epithelium in the distal esophagus. Early studies in BE suggested that all cells within a patient’s Barrett’s segment are progenies of a single founder cell. However, in recent years this previously accepted model of BE as a monoclonal disease has been retired in favor of a multiclonal model with new evidence suggesting that a patient’s Barrett’s segment can be composed of multiple independent clones with independent founder cells. Nonetheless, single BE samples are still routinely assumed to be monoclonal, leading to independent sets of genomic aberrations erroneously being analyzed as a single set in the case of multiclonal samples. Here, we utilize a previously generated whole-genome sequenced BE dataset and repurpose an established pipeline for detecting subclonal populations to identify BE samples that are comprised of multiple independent clones. Additionally, we present a novel approach for validating multiclonality from sequencing data by phasing pair-end sequencing reads using germline variants. We demonstrate that 20/80 patients have at least one multiclonal sample and that the independent clones contained in these single biopsies can have drastically different genomic profiles. Further, we speculate that these multiclonal samples capture a snapshot of clonal competition and evolution within BE. These findings highlight the importance of accounting for multiclonality when studying BE and provide the first systematic analysis of multiclonal BE samples. Citation Format: Mariya Kazachkova, Phoebe He, Ludmil B. Alexandrov. Identifying and analyzing independent clonal populations in single Barrett’s esophagus biopsies [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 2264.

Inhibition of cytosine 5-hydroxymethylation during progression of cancer precursor lesions in the uterine cervix.

Methylation and hydroxymethylation of cytosine moieties in CpG islands of specific genes are epigenetic processes shown to be involved in the development of cervical (pre)neoplastic lesions. We studied global (hydroxy)methylation during the subsequent steps in the carcinogenic process of the uterine cervix by using immunohistochemical protocols for the detection of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in paraffin-embedded tissues of the normal epithelia and (pre)malignant lesions. This approach allowed obtaining spatially resolved information of (epi)genetic alterations for individual cell populations in morphologically heterogeneous tissue samples. The normal ectocervical squamous epithelium showed a high degree of heterogeneity for both modifications, with a major positivity for 5-mC in the basal and parabasal layers in the ectocervical region, while 5-hmC immunostaining was even more restricted to the cells in the basal layer. Immature squamous metaplasia, characterized by expression of SOX17, surprisingly showed a decrease of 5-hmC in the basal compartments and an increase in the more superficial layers of the epithelium. The normal endocervical glandular epithelium showed a strong immunostaining reactivity for both modifications. At the squamocolumnar junctions, a specific 5-hmC pattern was observed in the squamous epithelium, resembling that of metaplasia, with the typical weak to negative reaction for 5-hmC in the basal cell compartment. The reserve cells underlying the glandular epithelium were also largely negative for 5-hmC but showed immunostaining for 5-mC. While the overall methylation status remained relatively constant, about 20% of the high-grade squamous lesions showed a very low immunostaining reactivity for 5-hmC. The (pre)malignant glandular lesions, including adenocarcinoma in situ (AIS) and adenocarcinoma showed a progressive decrease of hydroxymethylation with advancement of the lesion, resulting in cases with regions that were negative for 5-hmC immunostaining. These data indicate that inhibition of demethylation, which normally follows cytosine hydroxymethylation, is an important epigenetic switch in the development of cervical cancer.

OGC P31 On the role of Cytosponge for Barrett’s surveillance: assessment of viability and safety of this new technology

Abstract Background Barrett’s Oesophagus is a pre-malignant change where the normal stratified squamous epithelium is replaced by intestinal columnar epithelium. Current guidance produced by the British Society of Gastroenterologists describes 'gold-standard' surveillance as regular upper GI endoscopy with quadrantic biopsies for every 2cm length of Barrett’s oesophagus. The Covid pandemic led to significant delays in the Barrett's surveillance; as a result, many patients have fallen outside their maximum recommended surveillance interval. Cytosponge is a novel cytology sampling device which has a potential role in Barrett's surveillance though its use for this condition remains controversial. Methods All patients who were due for Barrett's surveillance in 2022 were identified and screened for suitability for Cytosponge, instead of endoscopy. Patients who were ineligible, or who declined Cytosponge, were sent for endoscopy. The remaining patients underwent a Cytosponge procedure as their primary Barrett's surveillance modality. Early clinical outcomes, patient satisfaction and cytology results were recorded. All patients have been followed up to determine if they have subsequently required endoscopy or had an early diagnosis of oesphago-gastric cancer. Results 193 patients (77%) were suitable for the Cytosponge test. 175 (90%) did not require endoscopy after Cytosponge. 18 patients (10%) had urgent endoscopy after Cytosponge; 9 patients had atypia on cytological analysis, 3 had aberrant p53 and 4 had dysplasia. Three patients had inadequate cytology. 2 of the patients with dysplasia had high-grade dysplasia confirmed on endoscopy; these were successfully treated with radiofrequency ablation. Median follow-up was 14 months (range, 10-22). No patients have been diagnosed with oesophago-gastric cancer so far; 8 patients (4%) were referred later for anaemia or weight loss. None of these patients had oesophago-gastric cancer. Conclusions These data suggest that Cytosponge can be a valuable first-line tool in surveillance of Barrett’s Oesophagus. Cytology results identified specific patients who required urgent endoscopy. No patients have had an early diagnosis of dysplasia or malignancy after a Cytosponge that was normal or showed intestinal metaplasia alone. Our findings suggest that Cytosponge should be integrated as an adjunct in Barrett’s surveillance, with clear benefits in safer and accurate monitoring of the condition without the risks of endoscopy.

Upregulated TC1 and downregulated Chibby were correlated with the aberrant β-catenin expression in laryngeal squamous cell carcinoma.

As an important member of Wnt/β-catenin signaling pathway, the aberrant expression of β-catenin has been implicated in many cancers. Chibby, a β-catenin binding partner, is an antagonist involved in this pathway. In contrast, thyroid cancer 1 (TC1) as an activator of this pathway can relieve the antagonistic activity of Chibby on the β-catenin-mediated transcription and is high expressed in human tumors. The objectives of this study were to examine the expression of TC1, Chibby, and β-catenin and investigate the association among them in laryngeal squamous cell carcinoma (LSCC). The expression of TC1, Chibby, β-catenin, c-Myc, Cyclin D1, and matrix metalloproteinase-7 (MMP-7) were examined by immunohistochemistry in samples from 53 LSCC patients. Compared with normal laryngeal squamous epithelium (NLSE), there were upregulated expression of TC1, downregulated expression of Chibby, and aberrant cytoplasmic expression of β-catenin in the LSCC tissues (P < .001). The high expression of TC1 was correlated with the tumor site, advanced TNM and T stage, lymphovascular invasion, and poor differentiation in LSCC tissues (P < .050). There were correlations between the aberrant expression of β-catenin and the tumor site, advanced TNM and T stage, lymphovascular invasion, perineurial invasion, and poor differentiation in LSCC tissues (P < .050). Upregulated TC1 and downregulated Chibby were correlated with aberrant expression of β-catenin (P < .001), but no correlation between them (P = .076). The percent of abnormal expression of β-catenin in LSCC was 96.00% in TC1+/Chibby-, 73.68% in TC1+/Chibby+, 0.00% in TC1-/Chibby-, and 0.00% in TC1-/Chibby + group (P < .001). High expression of c-Myc, Cyclin D1, and MMP-7 was observed in LSCC tissues (P < .001). There was statistically significant about the expression of Cyclin D1 and MMP-7 among the groups of TC1+/Chibby-, TC1+/Chibby+, TC1-/Chibby-, and TC1-/Chibby + (P < .001), but was not significance about the expression of c-Myc among them (P = .339). No association was found between overall survival and the expression of TC1, Chibby, and β-catenin (P > .05). The upregulated expression of TC1 and downregulated expression of Chibby were correlated with the aberrant expression of β-catenin and the high expression of Cyclin D1 and MMP-7 in LSCC tissues.

Does radiofrequency ablation of the lower oesophagus allow for clonal expansion of highly mutated neosquamous epithelium?

ObjectiveIn Barrett’s oesophagus (BE), after radiofrequency ablation (RFA), the oesophagus can be repopulated with a stratified ‘neosquamous epithelium’ (NeoSE). While histologically normal, the origin and clonal make-up of this NeoSE...

Promotion of squamous cell carcinoma tumorigenesis by oncogene-mediated THG-1/TSC22D4 phosphorylation.

Carcinoma cells possess high proliferative and invasive potentials and exhibit a resilience against stresses, metabolic disorder, and therapeutic efforts. These properties are mainly acquired by genetic alterations including driver gene mutations. However, the detailed molecular mechanisms have not been fully elucidated. Here, we provide a novel mechanism connecting oncogenic signaling and the tumorigenic properties by a transforming growth factor-β1-stimulated clone 22 (TSC-22) family protein, THG-1 (also called as TSC22D4). THG-1 is localized at the basal layer of normal squamous epithelium and overexpressed in squamous cell carcinomas (SCCs). THG-1 knockdown suppressed SCC cell proliferation, invasiveness, and xenograft tumor formation. In contrast, THG-1 overexpression promoted the EGF-induced proliferation and stratified epithelium formation. Furthermore, THG-1 is phosphorylated by the receptor tyrosine kinase (RTK)-RAS-ERK pathway, which promoted the oncogene-mediated tumorigenesis. Moreover, THG-1 involves in the alternative splicing of CD44 variants, a regulator of invasiveness, stemness, and oxidative stress resistance under the RTK pathway. These findings highlight the pivotal roles of THG-1 as a novel effector of SCC tumorigenesis, and the detection of THG-1 phosphorylation by our established specific antibody could contribute to cancer diagnosis and therapy.

Atrial Flutter After Radiofrequency Ablation for Barrett's Esophagus: A Case Report.

BACKGROUND Barrett's esophagus (BE) is a metaplastic change in the normal esophageal squamous epithelium and is a well-recognized precursor of esophageal adenocarcinoma (EAC). Nowadays, focal radiofrequency ablation is a valid technique for BE treatment by inducing a superficial and focal thermic destruction of metaplastic tissues. According to the literature, the most frequent patient-related adverse events of this procedure are esophageal iatrogenic stenosis, mucosal laceration or perforation of the esophagus, chest pain, and odynophagia/dysphagia. Postoperative heart rhythm abnormalities have been reported very rarely. CASE REPORT A 74-year-old patient with HE was treated by radiofrequency ablation (RFA) with the Barrx™ catheter system. He had 2 symptomatic episodes of atrial flutter in the immediate postoperative period requiring an external electrical cardioversion to induce a return to sinus cardiac rhythm. After atrial flutter ablation, 2 more radiofrequency procedures were performed, without adverse events. A laparoscopic Nissen fundoplication was carried out with complete endoscopic and histologic eradication of BE after 12-month follow-up. To the best of our knowledge, this is the first reported case of atrial flutter after esophageal RFA. Different mechanisms acting on an anatomic predisposing substrate can potentially play a role in starting atrial flutter, and include inflammation, autonomic activation, and myocardial injury. CONCLUSIONS The occurrence of this new type of adverse effect could potentially modify indications and postoperative monitoring of RFA treatment for BE. Endoscopists should know the possibility of this procedural complication in high-risk patients and they should propose alternative techniques or implement close cardiac monitoring in the postoperative period.

Effectiveness of Autologous Platelet-Rich Plasma and Stromal Vascular Fraction in Autologous Skin Grafting

Introduction: Despite the medical advances, burns remain a serious challenge and require new and promising treatment options, such as biomedical technologies with a high potential for burn wound treatment. However, high cost and complexity of biotechnologies limit their routine use, but autologous tissues, such as stromal vascular fraction (SVF) and autologous platelet-rich plasma (PRP), overcome these shortcomings. Objective: To demonstrate a successful case of using a combination of adipose-derived SVF and autologous PRP during autologous skin grafting in a severely burned patient. Material and methods: We present a successful case of 84-year-old Patient B. treated at Saint Petersburg I. I. Dzhanelidze Research Institute of Emergency Medicine in September-October 2022. He was diagnosed with a 11% (6%) / II-III degree flame burn of the head, neck, trunk, and upper extremities and first-degree inhalation injury. At the first stage, the general condition of the patient was stabilized, and he was prepared for surgery. At the second stage, on day 25 of treatment we performed skin grafting using split-thickness autografts with rare perforations and application of SVF and autologous PRP on a burn wound. We evaluated the treatment effectiveness using planimetry, cytology, and histology. Results: We observed complete engraftment of the autograft with no lysis or rejection during the first dressing change on day 3 after surgery and complete skin restoration on day 11. Cytologic findings over time showed an active decrease in inflammatory cells and an increase in regenerative cells, indicating successful wound healing. Histology demonstrated formation of normal stratified squamous epithelium with areas of hyperkeratosis and young granulation tissue in the superficial layers of the dermis on day 6. Conclusions: Our case demonstrated that the combination of SVF and autologous PRP facilitates split-thickness skin autograft engraftment.

Impact of antigen retrieval protocols on the immunohistochemical detection of epigenetic DNA modifications

This study compares three different pretreatment protocols for the immunohistochemical detection of 5-methylcytosine (5-mC) and 5-hydroxymethylcytosine (5-hmC) in nuclear DNA. The human biological samples analyzed included formalin-fixed and paraffin-embedded (FFPE) normal squamous epithelium, ethanol-fixed cultured cells, and metaphase chromosomes. The antigen retrieval methods included low pH Citrate and high pH Tris–ethylenediaminetetraacetic acid (EDTA) protocols, as well as a method using Pepsin pretreatment combined with HCl for DNA denaturation. A gradual increase in the detection levels of 5-mC and 5-hmC was observed when going from Citrate via Tris/EDTA to Pepsin/HCl retrieval. While the Citrate retrieval protocol was the least efficient for the detection of 5-mC and 5-hmC, it did preserve nuclear morphology and enabled visualization of differences in intra- and internuclear distribution patterns in tissue and cell culture samples by single- and double-fluorescence detection. Quantification of (hydroxy)methylation levels in FFPE material demonstrated a significant heterogeneity and differences in 5-mC and 5-hmC levels within and between nuclei in the different compartments of normal squamous epithelium. It was concluded that immunohistochemical detection of 5-mC and 5-hmC enables the correlation of these DNA modifications with histomorphological features in heterogeneous tissues, but this is influenced by different pretreatment protocols that must be carefully chosen to allow an appropriate interpretation of these epigenetic switches.

LncRNA MALAT1 promotes growth and metastasis of head and neck squamous cell carcinoma by repressing VHL through a non-canonical function of EZH2

Long non-coding RNAs (LncRNAs) are implicated in malignant progression of human cancers. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a well-known lncRNA, has been reported to play crucial roles in multiple malignancies including head and neck squamous cell carcinoma (HNSCC). However, the underlying mechanisms of MALAT1 in HNSCC progression remain to be further investigated. Here, we elucidated that compared with normal squamous epithelium, MALAT1 was notably upregulated in HNSCC tissues, especially in which was poorly differentiated or with lymph nodes metastasis. Moreover, elevated MALAT1 predicted unfavorable prognosis of HNSCC patients. The results of in vitro and in vivo assays showed that targeting MALAT1 could significantly weaken the capacities of proliferation and metastasis in HNSCC. Mechanistically, MALAT1 inhibited von Hippel–Lindau tumor suppressor (VHL) by activating EZH2/STAT3/Akt axis, then promoted the stabilization and activation of β-catenin and NF-κB which could play crucial roles in HNSCC growth and metastasis. In conclusion, our findings reveal a novel mechanism for malignant progression of HNSCC and suggest that MALAT1 might be a promising therapeutic target for HNSCC treatment.

Beige Mucosa Reflecting Active Sites of Eosinophilic Esophagitis.

We evaluated a 30-year-old woman with a 7-year history of eosinophilic esophagitis (EoE) with an initial complaint of moderate dysphagia. She gradually recovered after receiving proton pump inhibitors and oral budesonide. Only proton pump inhibitors were continued as a maintenance therapy until she developed very mild dysphasia 2 months earlier. Although endoscopy using white light imaging revealed characteristic findings of EoE, such as furrows and rings, with unclear images (Figure A), narrow-band imaging (NBI) revealed beige mucosa regions more clearly (Figure B). Magnified endoscopy using NBI revealed dot-shaped intrapapillary capillary loops and the absence of subepithelial vessels in the beige mucosa (Figure C). Target biopsied beige mucosa specimens revealed numerous intraepithelial eosinophils with basal layer hyperplasia and dilated intercellular spaces, reflecting active sites of EoE, whereas in nonbeige mucosa, normal squamous epithelium with few eosinophils was observed (Figure D). A recent study revealed that the loss or thinning of the superficial differentiated cell layer due to EoE activity allows 415-nm light to contact the hemoglobin component, resulting in a beige color change under NBI (Ayaki M, et al. J Gastroenterol Hepatol. 2022). The presence of beige mucosa under NBI in EoE patients, especially in cases without prominent EoE characteristic findings in white light imaging, indicates a suitable biopsied site for correct EoE diagnosis along with its histological activity.

Evaluation of Sirtuin1 Overexpression by Immunohistochemistry in Cervical Intraepithelial Lesions and Invasive Squamous Cell Carcinoma.

Cervical cancer is one of the most common genital cancers in the woman with approximately half a million new cases per year. Development of invasive squamous cell carcinoma (SCC) is the result of persistent and frequent human papilloma virus infection in premalignant lesions of cervix. Thereby identification of biomarkers that could predict progression of dysplastic mucosa to invasive cancer is of great clinical significance. Overexpression of SIRT1 has been reported to induce tumorogenesis in several organs. We evaluated SIRT1 expression in normal squamous epithelium of cervix, low-grade and high-grade cervical intraepithelial lesions and invasive SCC. A total of 104 cases were selected including 34 low-grade cervical intraepithelial lesions (CINs), 37 high-grade CINs, and 35 cases of invasive SCC. The normal cervical epithelium showed negative or weak SIRT1 positivity only in basal layers. SIRT1 cytoplasmic expression was found in 13 of 34 (38.2%) of low-grade CINs, 31 of 37 (83.8%) of high-grade CINs and all 35 (100%) cases of invasive SCC. Expression between 2 groups of CIN was statistically significant ( P =0.001). Thus, SIRT1 appears to be a promising biomarker for predicting the progression of CIN to invasive SCC.

S2047 Is It Anal or Rectal? A Rare Case of Squamous Cell Carcinoma

Introduction: Primary rectal squamous cell carcinomas (SCC) are extremely rare and difficult to distinguish from anal cancers. The majority of rectal SCC are secondary to anal SCC extension and detected at an advanced stage. Although rectal and anal cancers are anatomically close, they are distinct entities with risk factors, different histologic features, patterns of spread, staging systems, and treatment pathways. We present a rare case of a middle-aged man with AIDS who presented with bloody diarrhea, eventually found to have a primary rectal SCC. Case Description/Methods: A 59-year-old-male with a history of AIDS presented with bloody diarrhea and fevers. He denied abdominal or rectal pain. Physical exam only revealed mild tenderness of the abdomen. Labs were unremarkable. Gastrointestinal PCR revealed shigella, enteroaggregative E. Coli, enterotoxigenic E. coli, and enteropathogenic E. coli infection. Computed tomography of the abdomen and pelvis revealed pancolitis and left irregular rectal wall thickening, concerning for rectal carcinoma. He was started on IV ciprofloxacin and metronidazole with improvement in symptoms. Colonoscopy revealed a non-obstructing mass in the rectum. Pathology demonstrated high-grade anal intraepithelial lesion (AIN-3) and focally invasive squamous cell carcinoma, with acute and chronically inflamed rectal mucosa. Given the incongruence between colonoscopy and pathology, it was uncertain whether a primary lesion of the rectum with spread to anus existed or if 2 separate pathologies were present. Repeat colonoscopy to define the anatomic location of the mass revealed a severely ulcerated, friable, rectal lesion, discontinuous from an area of anal inflammation, separated by normal mucosa. Repeat pathology revealed invasive and in situ SCC of the rectum. The patient was referred for chemoradiation with 5-fluorouracil and mitomycin (Figure). Discussion: Primary rectal SCC accounts for 0.01% of all colo-rectal carcinomas. Risk is increased in patients with a history of Human Papilloma virus infection. The diagnosis of primary rectal SCC requires 3 criteria: (i) exclusion of metastasis, (ii) no squamous-lined fistulous tract involving the affected rectum, and (iii) exclusion of anal SCC with proximal extension (absence of continuity between the tumor and the normal anal squamous epithelium). Our patient ultimately fulfilled all these criteria. Primary rectal SCC should be considered a differential diagnosis in patients with HIV/AIDS, presenting with bloody diarrhea and a rectal mass.Figure 1.: CT of the abdomen and pelvis (A) revealing an irregular, asymmetric rectal wall thickening centered laterally on the left, highly concerning for rectal carcinoma, in addition to a focal left-sided outpouching from the rectum, suggesting a thick-walled diverticulum. Colonoscopy showing a rectal mass with ulcerated and friable mucosa (B&D). Area of anal inflammation (yellow circle, C) discontinuous from rectal mass with an adjacent small area of normal epithelium.

S2706 Orofacial Granulomatosis in Crohn's Disease

Introduction: Extra-intestinal manifestations (EIMs) of inflammatory bowel disease (IBD) are rare complications that affect roughly 6-25% of IBD patients. Orofacial granulomatosis (OFG) is a rare manifestation that does not have reliable epidemiologic data and is characterized by orofacial swelling with non-caseating granulomas seen on pathology. Case Description/Methods: A 23-year-old male with a history of Crohn’s colitis presented with loose stools and hematochezia for 7 months and 1 year of left sided lip swelling. He was diagnosed with Crohn’s disease at age 15 and was maintained on oral mesalamine and 6-mercaptopurine (6-MP). At age 16, the patient was evaluated for supraglottic edema. Urgent tonsillectomy was performed, with pathology revealing necrotizing and non-caseating granulomas. He recovered without complication. He achieved endoscopic remission at age 18 and his medications were discontinued. Unfortunately, he was lost to follow-up. During this subsequent presentation, his vitals were within normal limits. Exam was notable for left-sided lip and cheek swelling without mass or lesion. Bloodwork was notable for an ESR of 19 and CRP of 7.5. Lip biopsy showed normal squamous epithelium and non-caseating granulomas, consistent with OFG. Patient was treated for a Crohn’s flare with oral prednisone as a bridge to 6-MP. His OFG was treated with intralesional corticosteroids without relief and has on-going outpatient follow up. His current treatment plan includes further cycles of steroid injections before considering alternative therapies. His most recent colonoscopy showed endoscopic and histologic remission of his luminal Crohn’s while on infliximab. (Figure) Discussion: OFG is frequently seen as a manifestation of a systemic condition such as IBD. OFG is diagnosed by biopsy and clinical suspicion. OFG specific treatments include topical steroids and calcineurin inhibitors. Recent data suggests that a cinnamon/benzoate free diet may provide modest benefit to patients. Adequate treatment of underlying IBD is equally important. This patient has the classic symptoms and histopathologic findings of OFG and likely had undiagnosed OFG for many years given his tonsillar pathology years prior. Despite treatment, this patient has persistent and refractory symptoms. This case highlights a classic presentation of a rare IBD extra-intestinal manifestation and demonstrates how EIMs can occur even with well controlled IBD.Figure 1.: Orofacial Granulomatosis: Lip and Cheek Swelling.

MO43-1 Aspirin and nonsteroidal anti-inflammatory drugs (NSAIDs) and associated risk of Barrett's esophagus: A systematic review

Barrett's esophagus (BE) is a condition characterized by conversion of normal squamous epithelium into metaplastic columnar epithelium. Individuals with BE are predisposed to esophageal adenocarcinoma, a cancer with high morbidity and mortality. Aspirin and other NSAIDs have been suggested to protect against esophageal adenocarcinoma. The aim of this study is to evaluate the risk of developing Barrett's esophagus with aspirin or other NSAIDs intake.

Unique Glycoform-Dependent Monoclonal Antibodies for Mouse Mucin 21.

Mucin 21(Muc21)/epiglycanin is expressed on apical surfaces of squamous epithelia and has potentially protective roles, which are thought to be associated with its unique glycoforms, whereas its aberrant glycosylation is implicated in the malignant behaviors of some carcinomas. Despite the importance of glycoforms, we lack tools to detect specific glycoforms of mouse Muc21. In this study, we generated two monoclonal antibodies (mAbs) that recognize different glycoforms of Muc21. We used membrane lysates of Muc21-expressing TA3-Ha cells or Chinese hamster ovary (CHO)-K1 cells transfected with Muc21 as antigens. Specificity testing, utilizing Muc21 glycosylation variant cells, showed that mAb 1A4-1 recognized Muc21 carrying glycans terminated with galactose residues, whereas mAb 18A11 recognized Muc21 carrying sialylated glycans. mAb 1A4-1 stained a majority of mouse mammary carcinoma TA3-Ha cells in vitro and in engrafted tumors in mice, whereas mAb 18A11 recognized only a subpopulation of these. mAb 1A4-1 was useful in immunohistochemically detecting Muc21 in normal squamous epithelia. In conclusion, these mAbs recognize distinct Muc21 epitopes formed by combinations of peptide portions and O-glycans.

Novel DNA Methylation Biomarker Panel for Detection of Esophageal Adenocarcinoma and High-Grade Dysplasia.

Current endoscopy-based screening and surveillance programs have not been proven effective at decreasing esophageal adenocarcinoma (EAC) mortality, creating an unmet need for effective molecular tests for early detection of this highly lethal cancer. We conducted a genome-wide methylation screen to identify novel methylation markers that distinguish EAC and high-grade dysplasia (HGD) from normal squamous epithelium (SQ) or nondysplastic Barrett's esophagus (NDBE). DNA methylation profiling of samples from SQ, NDBE, HGD, and EAC was performed using HM450 methylation arrays (Illumina) and reduced-representation bisulfate sequencing. Ultrasensitive methylation-specific droplet digital PCR and next-generation sequencing (NGS)-based bisulfite-sequencing assays were developed to detect the methylation level of candidate CpGs in independent esophageal biopsy and endoscopic brushing samples. Five candidate methylation markers were significantly hypermethylated in HGD/EAC samples compared with SQ or NDBE (P < 0.01) in both esophageal biopsy and endoscopic brushing samples. In an independent set of brushing samples used to construct biomarker panels, a four-marker panel (model 1) demonstrated sensitivity of 85.0% and 90.8% for HGD and EACs respectively, with 84.2% and 97.9% specificity for NDBE and SQ respectively. In a validation set of brushing samples, the panel achieved sensitivity of 80% and 82.5% for HGD and EAC respectively, at specificity of 67.6% and 96.3% for NDBE and SQ samples. A novel DNA methylation marker panel differentiates HGD/EAC from SQ/NDBE. DNA-methylation-based molecular assays hold promise for the detection of HGD/EAC using esophageal brushing samples.

Expression of LATS1 in head and neck squamous cell carcinoma and its effect on tumor cell proliferation and invasion.

Nowadays, the incidence and mortality of head and neck tumors are gradually increasing. Head and neck malignant tumors (such as laryngeal cancer, hypopharyngeal cancer, oral cancer, nasopharyngeal cancer, oropharyngeal cancer, and other head and neck malignancies) are more common among systemic tumors. The most common pathological head and neck tumor type is squamous cell carcinoma, accounting for about 90%. In this study, immunohistochemical methods were used to collect the normal squamous epithelial tissues of the head and neck, atypical hyperplasia tissues, and head and neck squamous cell carcinomas on a tissue chip for detection. The recombinant LATS1 overexpression plasmid was prepared and transferred into B88 cells. Western blotting, MTT, and Transwell chamber methods were used to detect the effects of LATS1 proliferation, migration, and B88 cell overexpression. The experimental results showed that in head and neck squamous cell carcinoma, the expression of LATS1 protein decreased from 59.3% to 11.3%. At the same time, this protein inhibited the proliferation, migration, and invasion of head and neck squamous epithelial cells and also inhibited epithelium- Interstitial transformation exerts its tumor suppressor effect, indicating that LATS1 may play a tumor suppressor effect as a tumor suppressor gene. An in-depth study of the role and mechanism of LATS1 protein in the occurrence of head and neck squamous cell carcinoma may provide new opportunities for the treatment of head and neck squamous cell carcinoma in the future.