Unaffected Mucosa Research Articles

Global loss of promoter–enhancer connectivity and rebalancing of gene expression during early colorectal cancer carcinogenesis

Although three-dimensional (3D) genome architecture is crucial for gene regulation, its role in disease remains elusive. We traced the evolution and malignant transformation of colorectal cancer (CRC) by generating high-resolution chromatin conformation maps of 33 colon samples spanning different stages of early neoplastic growth in persons with familial adenomatous polyposis (FAP). Our analysis revealed a substantial progressive loss of genome-wide cis-regulatory connectivity at early malignancy stages, correlating with nonlinear gene regulation effects. Genes with high promoter–enhancer (P–E) connectivity in unaffected mucosa were not linked to elevated baseline expression but tended to be upregulated in advanced stages. Inhibiting highly connected promoters preferentially represses gene expression in CRC cells compared to normal colonic epithelial cells. Our results suggest a two-phase model whereby neoplastic transformation reduces P–E connectivity from a redundant state to a rate-limiting one for transcriptional levels, highlighting the intricate interplay between 3D genome architecture and gene regulation during early CRC progression.

γδ T cells in human colon adenocarcinomas comprise mainly Vδ1, Vδ2, and Vδ3 cells with distinct phenotype and function

Γδ T cell infiltration into tumours usually correlates with improved patient outcome, but both tumour-promoting and tumoricidal effects of γδ T cells have been documented. Human γδ T cells can be divided into functionally distinct subsets based on T cell receptor (TCR) Vδ usage. Still, the contribution of these different subsets to tumour immunity remains elusive. Here, we provide a detailed γδ T cell profiling in colon tumours, using mass and flow cytometry, mRNA quantification, and TCR sequencing. δ chain usage in both the macroscopically unaffected colon mucosa and tumours varied considerably between patients, with substantial fractions of Vδ1, Vδ2, and non-Vδ1 Vδ2 cells. Sequencing of the Vδ complementarity-determining region 3 showed that almost all non-Vδ1 Vδ2 cells used Vδ3 and that tumour-infiltrating γδ clonotypes were unique for every patient. Non-Vδ1Vδ2 cells from colon tumours expressed several activation markers but few NK cell receptors and exhaustion markers. In addition, mRNA analyses showed that non-Vδ1 Vδ2 cells expressed several genes for proteins with tumour-promoting functions, such as neutrophil-recruiting chemokines, Galectin 3, and transforming growth factor-beta induced. In summary, our results show a large variation in γδ T cell subsets between individual tumours, and that Vδ3 cells make up a substantial proportion of γδ T cells in colon tumours. We suggest that individual γδ T cell composition in colon tumours may contribute to the balance between favourable and adverse immune responses, and thereby also patient outcome.

Analysis of the composition of microbiota of the oral mucosa and recurrent aphthae

BACKGROUND: Recurrent aphthous stomatitis is a common disease of the oral mucosa. One of the most substantiated theories of the occurrence of recurrent aphthous stomatitis is the role of immunobacterial mechanisms. AIM: To study the composition of the microbiota of the oral mucosa and recurrent aphthae in children. MATERIALS AND METHODS: Thirty-five children with aphthous stomatitis aged 7–14 years were examined. The patients underwent a microbiological study of the microbiota from the lesion (aphtha) and unaffected oral mucosa. RESULTS: Fifteen types of microorganisms, represented by aerobes and anaerobes, were identified. Streptococcus significantly enriches the microbiota of the oral mucosa, and Streptococcus, E. сoli, and S. epidermidis significantly enrich the microbiota of the aphthae. Additionally, the number of yeast-like cells of the genus Candida in the lesions significantly increased (7.13±2.68 lg CFU/ml). In children, the surface of the aphthae had increased S. mutans (5.47±1.83 lg CFU/ml) compared to the oral mucosa, where the number was 2.35±0.12 lg CFU/ml, with an incidence of 93 and 100%, respectively. S. sanguinis was found equally in both groups (100%), the amount of infestation of which did not differ significantly. S. aureus and Klebsiella spp. were found in 93 and 18% of cases, respectively, only in aphthae, and the number was significantly high (p 0.001) at 5.13±1.68 and 4.13±1.38 lg CFU/ml, respectively, than on oral mucosa. CONCLUSION: A change in the microflora in the oral cavity was established in all children with recurrent aphthous stomatitis — a decrease in the lesions (aphthae) in the representatives of Streptococcus in bacterial communities, Lactobacillus with an increase in the number of yeast-like cells of the genus Candida and the appearance in the biocenosis from the surface of aphthous ulcers S. aureus, E. coli and S. epidermidis. Streptococcus significantly prevails in the oral mucosa, and Streptococcus, E. coli, and S. epidermidis colonize the aphthae.

Similarity Network Analysis of the Adaptive Immune Response in the Proximal Airway.

Recent immunologic study of the adaptive immune repertoire in the subglottic airway demonstrated high-frequency T cell clones that do not overlap between individuals. However, the anatomic distribution and antigenic target of the T cell repertoire in the proximal airway mucosa remain unresolved. Single-cell RNA sequencing of matched scar and unaffected mucosa from idiopathic subglottic stenosis patients (iSGS, n = 32) was performed and compared with airway mucosa from healthy controls (n = 10). T cell receptor (TCR) sequences were interrogated via similarity network analysis to explore antigenic targets using the published algorithm: Grouping of Lymphocyte Interactions by Paratope Hotspots (GLIPH2). The mucosal T cell repertoire in healthy control airways consisted of highly expressed T cell clones conserved across anatomic subsites (trachea, bronchi, bronchioles, and lung). In iSGS, high-frequency clones were equally represented in both scar and adjacent non-scar tissue. Significant differences in repertoire structure between iSGS scar and unaffected mucosa was observed, driven by unique low-frequency clones. GLIPH2 results suggest low-frequency clones share targets between multiple iSGS patients. Healthy airway mucosa has a highly conserved T cell repertoire across multiple anatomic subsites. Similarly, iSGS patients have highly expressed T cell clones present in both scar and unaffected mucosa. iSGS airway scar possesses an abundance of less highly expanded clones with predicted antigen targets shared between patients. Interrogation of these shared motifs suggests abundant adaptive immunity to viral targets in iSGS airway scar. These results provide insight into disease pathogenesis and illuminate new treatment strategies in iSGS. NA Laryngoscope, 134:3245-3252, 2024.

Localizing Hormone Receptor Expression to Cellular Compartments in Idiopathic Subglottic Stenosis.

Idiopathic subglottic stenosis (iSGS) is an unexplained progressive fibrosis of the upper airway. iSGS almost exclusively affects women; as a result, female hormones (estrogen and progesterone) have been proposed to participate in the pathogenesis of iSGS. Our aim was to localize cell-specific gene expression of estrogen receptors (ESR1 and ESR2) and progesterone receptor (PGR) using an established iSGS single-cell RNA sequencing (scRNAseq) cell atlas. Ex vivo molecular study of airway scar and healthy mucosa from iSGS patients. An established scRNAseq atlas consisting of 25,974 individually sequenced cells from subglottic scar (n = 7) or matched unaffected mucosa (n = 3) in iSGS patients was interrogated for RNA expression of ESR1, ESR2, and PGR. Results were quantified and compared across cell subsets, then visualized using Uniform Manifold Approximation and Projection (UMAP). Confirmatory protein assessment of endocrine receptors in fibroblasts from iSGS patients (n = 5) was performed via flow cytometry. The proximal airway mucosa in iSGS patients demonstrates differential expression of endocrine receptors (ESR1, ESR2, PGR). Within airway scar, endocrine receptors are primarily expressed by fibroblasts, immune cells, and endothelial cells. Fibroblasts show strong ESR1 and PGR expression, while immune cells possess RNA for both ESR1 and ESR2. Endothelial cells predominantly express ESR2. Epithelial cells in unaffected mucosa express all three receptors, which are all reduced in airway scar. scRNAseq data localized endocrine receptor expression to specific cell subsets. These results provide the foundation for future work interrogating how hormone-dependent mechanisms promote, sustain, or participate in iSGS disease pathogenesis. NA; Basic science Laryngoscope, 133:3506-3511, 2023.

P038 Notch pathway in fibrosis: a new anti-fibrotic therapy in Crohn's disease?

Abstract Background Fibrosis represent the main complications related to Crohn's disease (CD). Notch signalling mediate fibrogenic process, including epithelial-mesenchymal transition (EMT) and fibroblast senescence but its role in CD fibrosis is currently unknown. Previous studies have shown a high expression of NOTCH4, NOTCH3, DLL3 and DLL4 in CD fibrotic tissue. DLL4 mainly activates transcription factors involved in EMT, and macrophages could act as a possible source of DLL4 (Edo, et al., 2022. JCC (i200)).The general aim of the present study is to determine the possible potential of Notch pathway as a therapeutic target in intestinal fibrosis associated with CD. Specifically, we pretend: to analyze the localization of NOTCH3/4 receptors in the intestinal tissue of patients with CD complicated; to study the relevance of NOTCH3/4 receptors in the EMT; to study the relevance of Notch pathway in the senescence of intestinal fibroblast. Methods We have analyzed in intestinal samples from CD patients with complicated lesions: the localization of NOTCH3/4 receptors by IH and the protein expression of senescence markers (BCL2 and P53) by WB. We carry out in vitro studies and analyze: the protein expression of HES1 (effector Notch pathway) and EMT markers in DLL4-HT29 treated cells transfected with miNOTCH3 or miNOTCH4; and the protein expression of senescence proteins in HSIF fibroblasts treated with DLL4 or DLL3. Results NOTCH3 was located preferentially in muscular areas -muscularis mucosa, endothelium and muscularis externa- with a striking staining of infiltrated cells in the mucosa of the unaffected area. NOTCH4 is found more specifically in the crypts of the mucosa, as well as in cells of the lamina propria of the unaffected mucosa. The expression of senescence proteins in the tissue showed elevated levels of BCL2 and P53, compared to the unaffected tissue of the same patient. DLL4 increased the protein expression of EMT markers in HT29 cells, and NOTCH4 silencing significantly reverted the expression of these EMT markers. NOTCH3 silencing produced no significant changes after DLL4-HT29 treatment. DLL3, and not DLL4, produced in intestinal fibroblasts a significant increase in the protein expression levels of BCL2, and P53, compared to the vehicle (Figure). Conclusion NOTCH pathway is involved in the regulation of key cellular functions and processes essential for the pathogenesis of intestinal fibrosis in CD patients. DLL4-NOTCH4 interaction triggers transcription factors involved in mesenchymal epithelial transition in colonic epithelial cells, while DLL3 seems to have a more relevant role in activation of senescence in fibroblasts.

Expression of syndecan-1 in different grades of oral squamous cell carcinoma: An immunohistochemical study.

Syndecans are a family of transmembrane proteins, belonging to heparin sulphate proteoglycan family and are localized entirely to the epithelial cells with the stratified squamous epithelia. They are involved in cell-cell adhesion and interaction with the extracellular matrix and play a critical role in cell growth, differentiation, cell morphology, and migration. The down regulation of syndecan-1 indicates loss of cellular adhesion and possibility of invasion. The present study is aimed to evaluate the difference in immunohistochemical expression of syndecan-1 in different grades of oral squamous cell carcinoma (OSCC) patients and control group. The present study consists of 42 cases of paraffin-embedded tissue sections of OSCC; 14 well differentiated, 14 moderately differentiated, and 14 poorly differentiated. As a control, 10 paraffin-embedded tissue sections of unaffected oral mucosa were used. The sections were stained for immunohistochemical expression of syndecan -1. The intensity of staining was scored. The immunohistochemistry scores for each sample were obtained by Tissue Quant software. Statistical analysis revealed that there was a significant decrease in intensity of staining between normal and different grades of OSCC. This study shows that as cellular differentiation was lost, syndecan-1 expression was less. This provides an insight and understanding of the pathophysiology of the invasive process of OSCC and helps in establishing the prognostic link.

Intestinal Protein Characterisation of SARS-CoV-2 Entry Molecules ACE2 and TMPRSS2 in Inflammatory Bowel Disease (IBD) and Fatal COVID-19 Infection

—The coronavirus SARS-CoV-2 contributes to morbidity and mortality mainly as a result of immune-pathology in the lungs. Recent data has shown multi-system involvement with widespread viral tropism. Here we present a detailed intestinal protein characterisation of SARS-Cov-2 entry molecules ACE2 and TMPRSS2 in patients with inflammatory bowel disease ([IBD]; ulcerative colitis [UC] and Crohn’s disease [CD]) with age- and sex-matched non-IBD controls, and in those with fatal COVID-19 infection. In our dataset, ACE2 and TMPRSS2 displayed a membrane enterocyte staining in the ileum (due to presence of brush border/microvilli) in contrast to a cytoplasmic pattern in the colon. We also showed a high ACE2/low TMPRSS2 expression pattern in the ileum with a reverse trend in the colon. In UC, colonic ACE2 and TMPRSS2 are cytoplasmic in nature, with significantly higher ACE2 staining intensity compared to non-IBD controls. In inflamed and unaffected IBD mucosa, ileal and colonic enterocyte ACE2 and TMPRSS2 expressions are not modified in the histologic presence of inflammation. We observed immune cells within the lamina propria that expressed ACE2 and TMPRSS2, at higher frequencies in IBD when compared to non-IBD controls. These were identified as plasma cells with multiple myeloma oncogene 1/interferon regulatory factor 4 (MUM1/IRF4) expression. We further analysed the gut histology of six fatal COVID-19 cases, with no difference in colonic and ileal ACE2/TMRPSS2 staining (compared to non-IBD controls) and identified ACE2 + lamina propria plasma cells. Of interest, in this COVID-19 cohort, there was no histologic evidence gut inflammation despite known evidence of viral tropism within the enterocytes. Our data provides evidence for tissue expression of entry molecules ACE2 and TMPRSS2 including a close apposition to plasma cells — both pointing towards a role of the gut in the antecedent immune response to SARS-CoV-2 infection.

Gene expression of cytokinesis regulators PRC1, KIF14 and CIT has no prognostic role in colorectal and pancreatic cancer.

Colorectal cancer is one of the most common cancers and pancreatic cancer is among the most fatal and difficult to treat. New prognostic biomarkers are urgently needed to improve the treatment of colorectal and pancreatic cancer. Protein regulating cytokinesis 1 (PRC1), kinesin family member 14 (KIF14) and citron Rho-interacting serine/threonine kinase (CIT) serve important roles in cytokinesis, are strongly associated with cancer progression and have prognostic potential. The present study aimed to investigate the prognostic relevance of the PRC1, KIF14 and CIT genes in colorectal and pancreatic cancer. PRC1, KIF14 and CIT transcript expression was assessed by reverse transcription-quantitative PCR in tumors and paired distant unaffected mucosa from 67 patients with colorectal cancer and tumors and paired non-neoplastic control tissues from 48 patients with pancreatic cancer. The extent of transcript dysregulation between tumor and control tissues and between groups of patients divided by main clinical characteristics, namely patients' age and sex, disease stage, localization and grade, was determined. Finally, the associations of transcript levels in tumors with disease-free interval and overall survival time were evaluated. PRC1, KIF14 and CIT transcripts were upregulated in tumors compared with control tissues. PRC1, KIF14 and CIT levels strongly correlated to each other in both colorectal and pancreatic tumor and control tissues after correction for multiple testing. However, no significant associations were found among the transcript levels of PRC1, KIF14 and CIT and disease-free interval or overall survival time. In summary, the present study demonstrated mutual correlation of PRC1, KIF14 and CIT cytokinesis regulators with no clear prognostic value in pancreatic and colorectal cancers. Hence, according to the results of the present study, transcript levels of these genes cannot be clinically exploited as prognostic biomarkers in colorectal or pancreatic cancer patients.

Analysis of the intestinal microbiome in colorectal cancer

Aim. To conduct a comparative analysis of the microbiome of biopsies of a tumor and normal intestinal epithelium of patients diagnosed with colorectal cancer and to identify of functional activities of the obtained bacterial isolates that affect the development of the tumor.
 Methods. The study included 50 patients with malignant neoplasms of the colon: 36 men and 24 women. The mean age of the patients was 64.110.2 years. To analyze the microbiota of the biopsies, DNA samples were obtained from the tissue of the unaffected colon mucosa and tumor of the patients. Bacterial 16S rRNA genes fragments were amplified using bar-coded primer bakt_341f. Metagenomic next-generation sequencing was performed using the MiSeq platform (Illumina, USA). The obtained data were processed by bioinformatic methods using the QIIME package. Recognition of microorganisms depending on the morphotype and gram staining of the microflora was carried out using combination differential media and biochemical tests. Statistical analysis was carried out using Microsoft Excel, Service Pack 2 for Office XP, Statistica 6.0 (StatSoft). A comparative analysis was performed with the Student's t-test and the MannWhitney test in case of unmet conditions of validity. Alpha diversity of bacterial communities was quantified by the Shannon diversity index and the UniFrac distance for beta diversity analysis.
 Results. In patients with colorectal cancer, 5 bacterial phyla were isolated, the predominant of which were Firmicutes and Bacteroidetes, while the content of Actinobacteria was low. In addition, a higher number of representatives of Fusobacteria was observed in the tumor tissue compared to the tissue of a healthy mucosa, at a distance of 5 centimeters proximal to the tumor. The results of this study indicate that the microbiome of a tumor and a healthy mucosa fundamentally differ from each other not only in morphotype and gram staining but also in antagonistic, hemolytic and ribonucleolytic activities.
 Conclusion. Colonization of the tumor by dominant aggressive Gram-negative bacteria leads to significant changes in the tumor microbiome composition compared with normal mucosa, whose representatives are displaced from the damaged epithelium by more aggressive strains.

Mucosa-Associated Microbiota in Barrett's Esophagus, Dysplasia, and Esophageal Adenocarcinoma Differ Similarly Compared With Healthy Controls

INTRODUCTION:Alterations in the composition of the human gut microbiome and its metabolites have been linked to gut epithelial neoplasia. We hypothesized that differences in mucosa-adherent Barrett's microbiota could link to risk factors, providing risk of progression to neoplasia.Methods:Paired biopsies from both diseased and nonaffected esophagus (as well as gastric cardia and gastric juice for comparison) from patients with intestinal metaplasia (n = 10), low grade dysplasia (n = 10), high grade dysplasia (n = 10), esophageal adenocarcinoma (n = 12), and controls (n = 10) were processed for mucosa-associated bacteria and analyzed by 16S ribosomal ribonucleic acid V4 gene DNA sequencing. Taxa composition was tested using a generalized linear model based on the negative binomial distribution and the log link functions of the R Bioconductor package edgeR.Results:The microbe composition of paired samples (disease vs nondisease) comparing normal esophagus with intestinal metaplasia, low grade dysplasia, high grade dysplasia, and adenocarcinoma showed significant decreases in the phylum Planctomycetes and the archaean phylum Crenarchaeota (P < 0.05, false discovery rate corrected) in diseased tissue compared with healthy controls and intrasample controls (gastric juice and unaffected mucosa). Genera Siphonobacter, Balneola, Nitrosopumilus, and Planctomyces were significantly decreased (P < 0.05, false discovery rate corrected), representing <10% of the entire genus community. These changes were unaffected by age, tobacco use, or sex for Crenarcha.DISCUSSSION:There are similar significant changes in bacterial genera in Barrett's esophageal mucosa, dysplasia, and adenocarcinoma compared with controls and intrapatient unaffected esophagus. Further work will establish the biologic plausibility of these specific microbes' contributions to protection from or induction of esophageal epithelial dysplasia.

P017 C86/CD16 macrophages accumulate in the mucosa of B3 patients and could mediate EMT in Crohn’s disease

Abstract Background Macrophages contribute to fibrosis through the release of different mediators and the pattern of secretion may vary according to their phenotype. Methods The aim of the present study is to analyse the pattern of expression of macrophages, of EMT-related genes and cytokines in surgical resections from Crohn’s disease (CD, n = 43) patients which were categorised according to Montreal classification (B2 or B3); unaffected mucosa of patients with ileocecal cancer was used as control (n = 20). mRNA was isolated from intestinal samples and the expression of macrophage, EMT markers and cytokines were analysed by RT-PCR. PBMCS were isolated from healthy donors and treated during 5 days with secretomes, from control, B2 or B3 surgical resections; the mRNA expression of macrophage markers was determined by RT-PCR. U937 cells were differentiated to macrophages and then treated with IFNγ (20 ng/ml) for 4 days, the mRNA expression of macrophages markers were determined by RT-PCR. Results are expressed as mean ± SEM (n ≥ 5). Statistical analysis was performed by ANOVA + Newman–Keuls or t-test. Correlations between data were analysed using Pearson’s correlation coefficient (*p &amp;lt; 0.05). Results The expression of CD16 and CD86 was significantly higher in intestinal samples from B3 CD patients (7.2 ± 1.1 and 7.7 ± 1.3, respectively) than in controls (1.4 ± 0.2 and 2.5 ± 0.4, respectively) or B2 CD patients (4.8 ± 0.9 and 4.5 ± 0.6, respectively). The mRNA expression of CD16 and CD86 were significantly higher in PBMCS treated with B3-secretomes than in those treated with B2- or control secretomes. The expression of CD16 and CD86 significantly correlated with FSP1 (r = 0.74, p = 0.002* and r = 0.66, p = 0.003*, respectively), VIMENTIN (r = 0.60, p = 0.02* and r = 0.82, p = 0.001*, respectively), SNAIL1 (r = 0.61, p &amp;lt; 0.01* r = 0.52, p = 0.04*, respectively), IL4 (r = 0.63, p = 0.01* and r = 0.60, p = 0.02*, respectively) and IFNγ (r = 0.56, p = 0.001* and r = 0.58, p = 0.01*, respectively) in intestinal tissue from the fistulising CD group. U937 cells treated with IFNγ increased significantly the mRNA expression of CD16 (1.94 ± 0.24* vs. vehicle) and CD86 (1.60 ± 0.17* vs. vehicle). Conclusion A macrophage phenotype expressing CD86/CD16 may act as a source of EMT mediators in intestinal tissue from CD patients with a penetrating (B3) behaviour. IFNγ could be responsible for the increase in the number of CD86/CD16 macrophages in the B3 behaviour.

Localization-associated immune phenotypes of clonally expanded tumor-infiltrating T cells and distribution of their target antigens in rectal cancer

ABSTRACTThe degree and type of T cell infiltration influence rectal cancer prognosis regardless of classical tumor staging. We asked whether clonal expansion and tumor infiltration are restricted to selected-phenotype T cells; which clones are accessible in peripheral blood; and what the spatial distribution of their target antigens is.From five rectal cancer patients, we isolated paired tumor-infiltrating T cells (TILs) and T cells from unaffected rectum mucosa (TUM) using 13-parameter FACS single cell index sorting. TCRαβ sequences, cytokine, and transcription factor expression were determined with single cell sequencing. TILs and TUM occupied distinct phenotype compartments and clonal expansion predominantly occurred within CD8+ T cells. Expanded TIL clones identified by paired TCRαβ sequencing and exclusively detectable in the tumor showed characteristic PD-1 and TIM-3 expression. TCRβ repertoire sequencing identified 49 out of 149 expanded TIL clones circulating in peripheral blood and 41 (84%) of these were PD-1− TIM-3−. To determine whether clonal expansion of predominantly tumor-infiltrating T cell clones was driven by antigens uniquely presented in tumor tissue, selected TCRs were reconstructed and incubated with cells isolated from corresponding tumor or unaffected mucosa. The majority of clones exclusively detected in the tumor recognized antigen at both sites.In summary, rectal cancer is infiltrated with expanded distinct-phenotype T cell clones that either i) predominantly infiltrate the tumor, ii) predominantly infiltrate the unaffected mucosa, or iii) overlap between tumor, unaffected mucosa, and peripheral blood. However, the target antigens of predominantly tumor-infiltrating TIL clones do not appear to be restricted to tumor tissue.

Presence of parasite DNA in clinically unaffected nasal mucosa during cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis

ObjectivesWe aimed to detect Leishmania DNA carriage in nasal mucosa of individuals with cutaneous leishmaniasis (CL) caused by Leishmania (Viannia) braziliensis. MethodsA cross-sectional study was performed in all individuals with CL without nasal lesions (n = 153) attended within 2 years in an endemic area of L. (Viannia) braziliensis in Bahia (Brazil). An otorhinolaryngologist assessed the clinical status of the nasal mucosa by anterior rhinoscopy and endoscopic examinations. Swab samples were collected for parasite DNA detection by PCR from all individuals before standard treatment for leishmaniasis. A second evaluation 3 months after treatment was performed to assess clinical outcomes. ResultsParasite DNA was detected in 7.8% (12/153) of clinically healthy nasal mucosa of individuals with CL. Interestingly, DNA was more frequently identified in individuals with more skin lesions (median 1.5, interquartile range (IQR) 1–3.5 versus 1.0, IQR 1–1.5; p 0.044), or larger injuries (median 2.7, IQR 2–3.8 versus 1.6, IQR 1–2.5; p 0.013). Additionally, the disease of those individuals with positive PCR evolved more frequently to unusual forms of leishmaniasis (recidiva cutis and disseminated) (45.5% (5/11) versus 11.5% (14/122); p 0.009), and required more cycles of treatment to reach clinical cure (median 2, IQR 1–4 versus 1, IQR 1–2; p 0.05). ConclusionThese findings suggest an early parasite tropism to nasal mucosa in L. (Viannia) braziliensis infection and a clinical phenotype of CL cases associated with parasite DNA in nasal mucosa. Future studies should evaluate whether PCR of nasal swab samples could serve as a prognostic tool for individuals at risk of mucocutaneous leishmaniasis.

The ASAMET trial: a randomized, phase II, double-blind, placebo-controlled, multicenter, 2\u2009\xd7\u20092 factorial biomarker study of tertiary prevention with low-dose aspirin and metformin in stage I-III colorectal cancer patients

BackgroundEpidemiological studies and cardiovascular prevention trials have shown that low-dose aspirin can reduce colorectal cancer (CRC) incidence and mortality, including inhibition of distant metastases. Metformin has also been associated with decreased colon adenoma recurrence in clinical trials and lower CRC incidence and mortality in epidemiological studies in diabetics. While both drugs have been tested as single agents, their combination has not been tested in cancer prevention trials.Methods/designThis is a randomized, placebo-controlled, double-blind, 2 × 2 biomarker trial of aspirin and metformin to test the activity of either agent alone and the potential synergism of their combination on a set of surrogate biomarkers of colorectal carcinogenesis. After surgery, 160 patients with stage I-III CRC are randomly assigned in a four-arm trial to either aspirin (100 mg day), metformin (850 mg bis in die), their combination, or placebo for one year. The primary endpoint biomarker is the change of IHC expression of nuclear factor kappa-B (NFκB) in the unaffected mucosa of proximal and distal colon obtained by multiple biopsies in two paired colonoscopies one year apart. Additional biomarkers will include: 1) the measurement of circulating IL-6, CRP and VEGF; 2) the IHC expression of tissue pS6K, p53, beta-catenin, PI3K; 3) the associations of genetic markers with treatment response as assessed by next generation sequencing of primary tumors; 4) the genomic profile of candidate genes, pathways, and overall genomic patterns in tissue biopsies by genome wide gene expression arrays; and 5) the evaluation of adenoma occurrence at 1 year.DiscussionA favorable biomarker modulation by aspirin and metformin may provide important clues for a subsequent phase III adjuvant trial aimed at preventing second primary cancer, delaying recurrence and improving prognosis in patients with CRC.Trial registrationEudraCT Number: 2015–004824-77; ClinicalTrial.gov Identifier: NCT03047837. Registered on February 1, 2017.

Abstract 4679: Rectal cancer-infiltrating T cells show clonal expansion associated with spatially restricted tolerogenic phenotypes

Abstract T cell infiltration correlates with prognosis and outcome in colorectal cancer regardless of the tumor stage. However, data on clonal expansion, phenotypes, functions, and spatial distribution of tumor-infiltrating T cells (TILs) are limited. We hypothesized that subsets of clonally expanded rectal cancer-associated T cells are specifically recruited into the tumor and show characteristic phenotypes and functions. Paired TILs and T cells from adjacent unaffected mucosa were isolated from five treatment-naïve rectal cancer patients. Although T cell immune phenotypes were heterogeneous, the frequencies of CD38+, PD-1+, and TIM-3+ cells were significantly higher in CD8+ TILs when compared to T cells from unaffected mucosa (p &amp;lt; 0.05). To track clonal expansion and phenotypes at the single cell level, we combined 13-parameter FACS single cell index sorting with next generation T cell receptor (TCR) and phenotype sequencing. Paired TCRαβ sequences were obtained from 1237 TILs (on average 412 TILs per patient) and 1257 T cells infiltrating the unaffected mucosa (on average 419 T cells per patient) from three selected patients. Clonal T cell expansion was not restricted to TILs and occurred predominantly in CD8+ T cells (p &amp;lt; 0.05). Expanded TIL clones were more frequently TIM-3+, PD-1+, and CD38+ (p &amp;lt; 0.05) and occupied characteristic phenotype compartments when compared to T cells from unaffected mucosa with t-stochastic neighbor embedding (t-SNE) visualization. FOXP3 expression was enriched in non-expanded TILs (p &amp;lt; 0.05). Only 11 out of 179 TIL clones were also detectable in unaffected mucosa of the same patients and these overlapping clones were exclusively PD-1- TIM-3-. In conclusion, rectal cancer is infiltrated by oligoclonal T cells. Selected clones were exclusively detected in TILs suggesting their expansion and recruitment driven by tumor-associated cues. Tolerance was induced i) in an antigen-specific manner by the expression of immune checkpoint molecules predominantly on clonally expanded TILs, and ii) in an antigen-independent manner by infiltrating polyclonal FOXP3+ regulatory T cells. Citation Format: Livius Penter, Kerstin Dietze, Felix Aigner, Lars Bullinger, Thomas Blankenstein, Leo Hansmann. Rectal cancer-infiltrating T cells show clonal expansion associated with spatially restricted tolerogenic phenotypes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4679.

Genetic and epigenetic alterations in the tumour, tumour margins, and normal buccal mucosa of patients with oral cancer

Despite adequate surgical resection, oral squamous cell carcinoma (OSCC) shows a high rate of recurrence and metastasis, which could be explained by the presence of molecular alterations in seemingly normal tumour margins and the entire oral mucosa. The aims of this study were (1) to assess the presence of gene amplification (c-Myc and HER2) and promoter methylation (p14 and p16) in the tumours, tumour margins, and unaffected oral mucosa of 40 OSCC patients, and (2) to evaluate the possibility of using these alterations as prognostic markers. c-Myc and HER2 genes were quantified by means of real-time PCR (qPCR), and p14 and p16 methylation status was determined by methylation-specific PCR (MSP PCR). All tissues examined exhibited molecular alterations in various proportions. Tumour tissues, as expected, showed the highest prevalence of alterations, while oral mucosa showed the lowest. Multiple alterations (co-alterations) in tumours and tumour margins were significantly more frequent than in unaffected oral mucosa (P<0.001 and P=0.027, respectively). HER2 amplification in margin tissue (P<0.001) and swabs (P=0.013), as well as the existence of three co-alterations in margins (P=0.001) and macroscopically unaffected oral mucosa (P<0.001) were correlated with shorter disease-specific survival.

Colorectal cancer-infiltrating T lymphocytes display a distinct chemokine receptor expression profile

BackgroundT lymphocytes exert important homeostatic functions in the healthy intestinal mucosa, whereas in case of colorectal cancer (CRC), infiltration of T lymphocytes into the tumor is crucial for an effective anti-tumor immune response. In both situations, the recruitment mechanisms of T lymphocytes into the tissues are essential for the immunological functions deciding the outcome. The recruitment of T lymphocytes is largely dependent on their expression of various chemokine receptors. The aim of this study was to identify potential chemokine receptors involved in the recruitment of T lymphocytes to normal human colonic mucosa and to CRC tissue, respectively, by examining the expression of 16 different chemokine receptors on T lymphocytes isolated from these tissues.MethodsTissues were collected from patients undergoing bowel resection for CRC. Lymphocytes were isolated through enzymatic tissue degradation of CRC tissue and nearby located unaffected mucosa, respectively. The expression of a broad panel of chemokine receptors on the freshly isolated T lymphocytes was examined by flow cytometry.ResultsIn the normal colonic mucosa, the frequencies of cells expressing CCR2, CCR4, CXCR3, and CXCR6 differed significantly between CD4+ and CD8+ T lymphocytes, suggesting that the molecular mechanisms mediating T lymphocyte recruitment to the gut differ between CD4+ and CD8+ T lymphocytes. In CRC, the frequencies of cells expressing CCR2 and CXCR5 were significantly lower in both the CD4+ and CD8+ T lymphocyte populations compared to unaffected colonic mucosa, and the frequency of CCR9+ cytotoxic T lymphocytes was significantly decreased in CRC tissue.ConclusionsWith regard to the normal gut mucosa, the results suggest that the molecular mechanisms mediating T lymphocyte recruitment differ between CD4+ and CD8+ T lymphocytes, which are important for understanding gut homeostasis. Importantly, T lymphocytes from CRC compared to normal colonic tissue displayed a distinct chemokine receptor expression profile, suggesting that mechanisms for recruitment of T lymphocytes to CRC tissue are skewed compared to normal colonic mucosa. Understanding these mechanisms could help in developing new strategies in cancer immunotherapy and to optimize already available alternatives such as immune checkpoint inhibitors.

ASAMET: A randomized, 2x2 biomarker prevention trial of low-dose aspirin and metformin in colorectal cancer.

TPS1591 Background: Epidemiological studies and cardiovascular prevention trials have shown that low-dose aspirin (ASA) can inhibit colorectal cancer (CRC) incidence and mortality, including inhibition of distant metastases. Metformin (MET) has also been associated with decreased CRC incidence and mortality in meta-analyses of epidemiological studies in diabetics and has been shown to decrease by 40% colorectal adenoma recurrence in a randomized trial. Recent studies have shown that ASA is an inhibitor of mTOR/S6K1 and an activator of AMPK, targeting regulators of intracellular energy homeostasis and metabolism, and that the combination of ASA and MET, another AMPK activator and S6K1 inhibitor, has a striking additive effect on AMPK activation and mTOR inhibition, with increased autophagy and decreased cell growth in CRC cell lines. While both drugs are being tested as single agents, their combination has not been tested in clinical trials. Methods: This is a randomized, placebo-controlled, double-blind, 2x2 biomarker trial of ASA and MET to test the activity of either agent alone and the potential synergism of their combination on a set of surrogate biomarkers of colorectal carcinogenesis. After surgery, 160 patients with stage I-III CRC will randomly be assigned in a 4-arm trial to either ASA, 100mg qd, MET 850mg bid, their combination, or placebo for 1 year. The primary endpoint is the change, defined as the difference between pre- and post-treatment expression, of NFκBin the unaffected mucosa of proximal and distal colon obtained by multiple biopsies in paired colonoscopies one year apart. Additional biomarkers will include: 1) the genomic profile of candidate genes, pathways, and overall genomic patterns in tissue biopsies by genome-wide gene expression arrays; 2) the IHC expression of tissue pS6K, p53, beta-catenin, PI3K; 3) the associations of mutations and SNPs with treatment response by NGS of primary tumors; 4) the measurement of circulating IL-6, CRP and VEGF, and 5) plasma and colonic MET concentrations and their correlation with biomarker profiles. A favorable biomarker modulation may provide important clues for a subsequent phase III adjuvant trial. Clinical trial information: EudraCT Number: 2015-004824-77.

P761 Gene expression differences between Crohn's disease aphthous ulcers and healthy Peyer's patches highlights potential treatment strategies

Background: The earliest macroscopic lesion in Crohn's disease (CD) is the aphthous ulcer, which overlies Peyer's patches and lymphoid follicles. Our aim was to characterize differences in gene expression of aphthous ulcers and Peyer's patches. Methods: Biopsies were obtained from the terminal ileum of 12 patients (6 with CD, 6 healthy controls). Aphthous ulcers and adjacent unaffected mucosa were obtained from CD patients, and Peyer's patches and adjacent mucosa from the controls. All patients were medication-free, except one. RNA was extracted using Qiagen kits. NextSeq 500 libraries were constructed using NextSeq 500/550 High output kits (Illumina) in a 150 bp paired-end format. Transcripts were assessed for quality using FASTQC, trimmed using Trimmomatic, and aligned to the human reference genome using subread mapper. Fragment counts were obtained using featureCount, and expression values normalized using the trimmed mean of M-values normalization method (TMM). Differential gene expression analyses were performed using generalized linear models in edgeR. Cell-specific gene expression was determined using ImSig. Results: We obtained 36 million tags per sample, 87% were retained for downstream processing, and 93% of these mapped to the human genome. 685 genes were significantly differentially expressed between aphthous ulcers and Peyer's patches, all were upregulated in aphthous ulcers. Differential gene expression analysis revealed 34 pathways that were upregulated in aphthous ulcers relative to Peyer's patches. Receptors for the constant region of immunoglobulin (Ig) G were represented in 13 of these pathways. Expression of the high affinity FCGR1A (CD64), and low affinity FCGR3A (CD16), FCGR2A and FCGR2C (CD32), was significantly upregulated in aphthous ulcers (p=1.14E-12), but not the inhibitory FCGR2B (CD32B). Other pathways that were highly upregulated in aphthous ulcers included those involved in responding to bacteria, leukocyte chemotaxis, inflammatory response, and creation of C2 and C4 activators. ImSig, which is capable of using transcriptome data to indicate cell types and their activation state, revealed that core marker genes for plasma cells were overrepresented in aphthous ulcers relative to Peyer's patches and unaffected/normal mucosa. Conclusions: The most significantly upregulated Fc gamma receptors in aphthous ulcers are the major receptors expressed on monocytes/macrophages, which are involved in regulating innate and adaptive immune responses, as well as plasma cell survival (FCGR2B), making them an attractive target for immunotherapy.