Introduction

Undifferentiated carcinoma (UC) of the pancreas is a rare and aggressive variant of pancreatic cancer. UCs are often concurrent with adenocarcinoma and harbor common genetic features, and pathogenic mutations of KRAS and TP53, and chromosomal polyploidy may associate with the development of UC. However, due to its rarity, the tumorigenesis of UC still needs to be fully elucidated. We aimed to clarify the molecular mechanisms of UC arising from intraductal neoplasms of the pancreas.

Aims & Methods

We performed molecular tracing on 2 cases of UCs derived from intraductal papillary mucinous neoplasms (IPMNs). Multi-regional targeted sequencing using a gene panel targeting pancreatic cancer and IPMN-related 37 genes, copy number variation analysis for KRAS and GNAS using droplet digital PCR, and fluorescence in-situ hybridization (FISH) of KRAS/CEN 12 and MYC/CEN 8 were performed. Tumor phenotypes were evaluated by immunohistochemistry.

Results

Case 1 was an intestinal-type IPMN with UC composed of pleomorphic cells expressing vimentin, ZEB1, and Snail and with mucinous carcinoma. The histological transition was observed each from IPMN. Case 2 was a pancreatobiliary-type IPMN; when it recurred in the remnant pancreas, IPMN was associated with intra-ductal invasive growth of UC composed of spindle cells expressing vimentin and ZEB1 and with an extra-ductal invasion of moderately differentiated tubular adenocarcinoma. Identical somatic mutations; KRAS Q61_E62delinsRK, GNAS R201S, TP53 F113V, and RNF43 S321* in case 1, and KRAS Q61H and TP53 R175H in case 2, and copy number variations; CDKN2A deletion and GNAS and MYC amplification in case 1 and amplifications of KRAS, GNAS, MYC, ATM, and ERBB2 and deletions of CDKN2A and SMAD4 in case 2, were detected in both IPMNs and invasive carcinomas. FISH showed marked polyploidy in the KRAS locus (copy number average 5.1 and 6.1, max 26 and 15, respectively) and amplification of MYC locus (copy number average 6.3 and 5.3, respectively) in UC components in both cases. IPMN components showed intermediate polyploidy of KRAS (copy number average 2.2 and 4.3, max 6 and 7, respectively) and amplification of MYC (copy number average 7.8 and 4.9, respectively). Protein expression of MYC was upregulated in IPMN components, concordant with MYC amplification, while relatively down-regulated in UC components. Interestingly, IPMN revealing papillary nodule showed wild-type allele dominant KRAS imbalance and nuclear accumulation of β-catenin in case 1 and amplifications of GATA6 in case 2.

Conclusion

Increased KRAS burden due to marked polyploidy and mutant-allele specific imbalance, TP53 pathogenic mutation, and MYC amplification were the molecular alterations related to UC derived from IPMNs. Moreover, intermediate polyploidy of KRAS and amplification of MYC had already begun subsequent to TP53 mutation in intraductal components. On the other hand, WNT activation and amplification of GATA6 may be related to intraductal dominant growth and de-selection along with the invasion. This is the first report tracing the molecular alterations associated with tumorigenesis of UC from pre-invasive intraductal neoplasm.

Introduction

There is growing awareness of the importance of histological outcomes in ulcerative colitis (UC). However, direct comparisons of histological endpoints are lacking. Thus, we aimed to conduct a network meta-analysis (NMA) to compare the effectiveness of various biologics and small molecules in achieving histological endpoints in UC.

Aims & Methods

We searched four online databases to identify all randomized controlled trials (RCTs) investigating advanced therapies for moderate-to-severe UC that reported histological outcomes. The analysis focused on the proportion of patients achieving histological improvement, histological remission, and histo-endoscopic improvement during induction and maintenance therapy. Using a random-effects frequentist model, we conducted a NMA, presenting the results as odds ratios (OR) with 95% confidence intervals. To evaluate the relative effectiveness of each treatment, we calculated the P-score, which ranges from 0 to 1. We also assessed the risk of bias and evidence certainty using established scoring systems.

Results

Twenty-four RCT were included, comprising 15 different therapies, and enrolling over 8800 patients. Of these trials, 19 provided data on induction therapy, while 10 assessed histological outcomes during maintenance treatment. Outcome definitions were similar among studies. We found that etrasimod 2 mg/day ranked highest in achieving histologic response (P-score 0.98) and remission (P-score 0.90) following induction, while guselkumab ranked first for histoendoscopic improvement. In maintenance trials, upadacitinib 30 mg/day ranked highest in achieving histologic and histo-endoscopic outcomes (P-score 0.88 for both endpoints). Etrasimod 2 mg/day ranked second for histologic remission (P-score 0.70) and histo-endoscopic improvement (P-score 0.73), while mirikizumab 200 mg/month ranked second for histologic response.

Conclusion

Our findings underscore the importance of small molecules in treating moderate-to-severe UC, particularly etrasimod and upadacitinib. Nonetheless, additional head-to-head RCTs are imperative to better inform clinical practice and to address unanswered questions.

Introduction

Ulcerative colitis (UC) is a chronic, non-specific inflammatory disease of the bowel of unknown etiology. This disease is characterised by high difficulty in cure, high recurrence and cancer rates, which seriously endangers the lives and health of our population. Taraxasterol (TA), a major constituent of the traditional Chinese medicine dandelion, has been reported in several studies to have good anti-inflammatory and antioxidant activities as well as anti-tumour effects.

Aims & Methods

However, the molecular targets and mechanisms of action of TA against UC have not been fully elucidated.The Swiss Target Prediction, PharmMapper databases were used to obtain relevant targets for the drug TA. Targets for UC were obtained from GeneCards, OMIM, PharmGkb, DrugBank databases. Targets of TA anti-UC identified by intersection of drug target and disease target. The relevant targets of TA anti-UC were uploaded to the String database and the resulting data were imported into Cytoscape 3.9.1 software to construct a protein-protein interaction (PPI) network of drug-disease targets. At the same time, the top 10 hub targets were screened using CytoHubba software. Gene ontology (GO) and Kyoto Encyclopedia of Genomes (KEGG) pathway enrichment analyses were performed on TA anti-UC targets using R software, and molecular docking was performed by Autodock Vina 1.1.2. The GEO dataset was used to analyze the differential mRNA expression of the TA anti-UC top 10 hub gene in UC patients and the normal population. Lipopolysaccharide (LPS)-induced intestinal epithelial cells (NCM460) were used to construct an in vitro cellular inflammation model and a Dextran Sulfate Sodium Salt (DSS) induced acute mouse colitis model. The pivotal targets and key signaling pathways were also validated by RT-qPCR and Western blotting.

Results

A total of 129 relevant targets of TA against UC were obtained, of which the top 10 hub targets were GRB2, PTPN11, SRC, PIK3R1, LCK, EGFR, MAPK1, IGF1, ESR1 and JAK2. The PI3K-Akt pathway was significantly associated with TA treatment of UC as identified by KEGG enrichment analysis. GEO dataset analysis demonstrated that EGFR and GRB2 were upregulated in UC patients, while SRC, PIK3R1 and JAK2 were down-regulated. In vitro experiments revealed that TA treatment reversed the expression of EGFR, GRB2, PIK3R1 and JAK2 differential genes. Furthermore, TA was able to up-regulate the mRNA expression of pro-inflammatory factor IL-10 and decrease the mRNA levels of anti-inflammatory factors IL-6, IL-1β and TNF-α in LPS-induced NCM460 cells. In vivo assay confirmed that TA significantly alleviated DSS-induced colonic inflammation in mice, including reversal of weight loss and shortening of colon length, reduction of DAI score in mice, and improvement of intestinal barrier disruption. In vivo and in vitro experiments have confirmed that TA also inhibits the activation of the PI3K/Akt pathway, thus exerting an anti-inflammatory effect.

Conclusion

TA may play a therapeutic role in UC by inhibiting the PI3K-Akt pathway. EGFR, GRB2, PIK3R1 and JAK2 may be the most relevant potential therapeutic targets for TA in the treatment of UC.

Introduction

Aloe barbadensis Mill. (Aloe) with its complex polysaccharide composition has been suggested to have prebiotic properties, however no human intervention trials have previously been undertaken to study this effect.

Aims & Methods

With the aim of designing prebiotic strategies for human gut health promotion, this randomized, placebo-controlled study evaluated the effects of increasing doses of chemically characterized Aloe extract on gastrointestinal function and overall gastrointestinal microenvironment, including fecal microbiota and metabolite composition in healthy adults.
Fecal samples were collected from healthy subjects (56 females and 21 males, age 19 - 69) at baseline, and week 3 and week 6 after intervention with placebo or 0.5 g, 1.0 g or 3.0g of Aloe extract/day. Gastrointestinal symptoms were assessed using validated questionnaires and stool consistency was evaluated using the Bristol Stool Form Scale. A food questionnaire was used to evaluate dietary quality, adapted using an index created by the Swedish National Food Agency. Fecal microbiota was evaluated by GA-map^TM Dysbiosis Test, while H-1 nuclear magnetic resonance (NMR) spectroscopy was used for metabolomic profiling. Multivariate Orthogonal Projections to Latent Structures Discriminatory Analysis (OPLS-DA) was applied to fecal microbiota and metabolite data sets. Statistical analyses were carried out using non-parametric tests.

Results

All participants completed the study and demographic parameters such as age, sex, diet quality and BMI did not differ between the study groups at baseline. Intervention with increasing doses of Aloe was safe and well tolerated and did not cause gastrointestinal symptoms, as assessed using the gastrointestinal symptoms rating scale. Stool consistency changed after supplementation with Aloe, (n= 57; 3.9 (3.2 – 4.4) vs 3.6 (2.8 – 4.2), P = 0.005), but not for placebo (n= 20; 3.9 (3.2 – 4.5) vs. 3.7 (3.2 – 4.3), P = 0.82), comparing baseline vs. end of intervention, without difference between the groups (P = 0.33).
No alterations were seen in the fecal microbiota profiles before vs. after intervention, in either of the groups, indicative of a stable overall gut microbiota. Furthermore, the fecal metabolite profiles remained unaltered after intervention with Aloe. However, Aloe modulated the fecal metabolite profile in healthy subjects with BMI ≥25 (n = 24; R2 = 0.87, Q2 = 0.51), but not healthy subjects with BMI ≤24 (n = 32; R2 = 0.44, Q2 = 0.12), when comparing baseline vs. end of intervention by OPLS-DA. Moreover, Aloe intervention showed potential to reduce the difference seen in the fecal metabolite profiles between subjects with BMI ≤24 and those with BMI ≥25 from R2 = 0.57 and Q2 = 0.30 at baseline to R2 = 0.38 and Q2 = -0.11 at end of intervention. No such tendencies were seen in the smaller placebo group (BMI ≤24, n = 12 vs. BMI ≥25, n =6).

Conclusion

Supplementation with increasing doses of Aloe extract is safe and well tolerated in healthy adults. Overall, our findings support safety of Aloe extract, and show that supplementing diet with Aloe extract has the potential to modulate intestinal microbiota function, especially in healthy adults with a BMI ≥25, with an altered intestinal microenvironment.

Disclosure

Presenting Author - Bani Ahluwalia is employed by Calmino Group AB, while carrying out Post-doctoral research at the University of Gothenburg.

Introduction

Most immune-mediated disorders display a degree of heritability, with specific genetic variants linked to disease susceptibility. Variants in Endoplasmic Reticulum Aminopeptidase 2 (ERAP2) have been associated with numerous inflammatory conditions, including Inflammatory Bowel Disease (IBD). As several species (rodents included) lack the ERAP2 gene, functional studies have been relatively limited. Several immunological functions of ERAP2 beyond peptide trimming have been proposed, yet most of these findings have been reported in immune cell populations. As the epithelial involvement in IBD pathogenesis is getting increasingly recognized, we set out to explore the impact of epithelial ERAP2 presence during inflammatory encounter using human-derived colonoids as a model system. To investigate the effect of genotype on ERAP2 expression in different disease states, patient material from a IBD cohort was used to correlate genotype to ERAP2 expression in inflamed and noninflamed colonic biopsies.

Aims & Methods

Two single nucleotide polymorphisms (SNPs) linked to ERAP2 expression (rs2910686/rs2248374) were analyzed in a cohort of IBD patients. Genotypes for both SNPs were correlated with colonic ERAP2 expression levels in noninflamed and inflamed biopsies. Based on rs2910686 genotype, ERAP2-proficient (n=4) and ERAP2-deficient (n=4) colon organoids were established to study differential expression between proficient and deficient donors after proinflammatory stimulation.

Results

Conditioning on genotype reveals upregulation of ERAP2 in the inflamed colon of IBD patients. ERAP2 expression proficiency correlates with rs2910686 genotype. IFNɣ induces colonic epithelial ERAP2 expression. Western blot confirms that mechanisms independent of the frequently studied SNP rs2248374 can cause ERAP2-deficiency. A total of 586 genes involved in various molecular mechanisms are differentially expressed between ERAP2-proficient and -deficient colonoids upon proinflammatory stimulation, including AOC1, CPE, ANPEP and MEP1A (catalytic activity), TNFSF9, MDK, GDF15, ILR6A, LGALS3 and FLNA (regulator activity), SLC40A1 and SLC5A1 (transmembrane transporter activity), FGL2, HMCN2, and MUC17 (extracellular matrix structural constituents).

Conclusion

Our findings demonstrate that ERAP2 expression is significantly upregulated in the inflamed colon of IBD patients, and expression levels correlates with rs2910686 genotype. Although rs2248374 is commonly used to assess ERAP2 expression proficiency, our findings confirm that mechanisms independent of rs2248374 can lead to ERAP2-deficiency. We further demonstrate that stratifying organoid donors based on genotype is a useful way of exploring the impact of genetic variants. Our data support that ERAP2 play a multifaceted role in modulating the inflammatory response in epithelial cells.