Introduction

The induction of endoplasmic reticulum unfolded protein responses (UPR^ER) contributes to cancer development and progression. We set out to identify the clinical relevance of signal transducer activating transcription factor 6 (ATF6) and related bacterial genera in colorectal cancer (CRC) patients, and to elucidate the consequence of ATF6 signaling on the murine host.

Aims & Methods

To determine the clinical relevance of ATF6 in CRC, we quantified ATF6 expression in two independent German CRC patient cohorts (n=959; n=256), and a third cohort for validation (n=50), using immunohistochemical staining and artificial intelligence. We used the publicly available The Cancer Genome Atlas (TCGA) Dataset to compare transcription factor activity and microbial profiles. To understand the impact of ATF6 signaling on CRC pathogenesis, we employed our transgenic murine model of intestinal epithelial cell (IEC)-specific activation of the active form of ATF6 (nATF6^IEC) that develops spontaneous and microbiota-dependent colon tumors. We assessed the transcriptional response to ATF6 signaling in colon tissue (bulk mRNA sequencing), and characterized metabolic (untargeted metabolomics) and microbial (16S rRNA sequencing) alterations in murine and CRC tissue. For mechanistic experiments we performed in vivo fatty acid synthase (FASN) inhibition in mice, germ-free mouse colonization, organoid cultures, bioorthogonal non-canonical amino acid tagging (BONCAT) and bacterial growth analysis.

Results

We demonstrate the relevance of ATF6 expression in CRC patients across multiple independent German cohorts by identifying a clear sub-population (19-38% late onset CRC; 11% early onset CRC) of patients that present with high ATF6 expression, underlining the importance of ATF6 in CRC. Transcriptional analysis of nATF6^IEC mice identified bacteria-specific changes in cellular metabolism enriched for fatty acid biosynthesis in IECs. Untargeted metabolomics and isotype-labeling confirmed ATF6-related enrichment of long chain fatty acids (LCFAs) in colonic tissue of CRC patients, nATF6^IEC mice and murine organoid cultures. FASN inhibition and microbiota transfer into germ-free nATF6^IEC mice confirmed the causal involvement of ATF6-induced lipid alterations in tumorigenesis. Multi-omics integration associated tumor-associated LCFAs with tumor-associated bacteria in nATF6^IEC mice. BONCAT analysis reveals the impact of LCFAs on the expansion of specific taxa of the intestinal microbiota. Specifically, the selective expansion of tumor-relevant microbial taxa, including Desulfovibrio fairfieldensis, was mechanistically linked to LCFA exposure. Analyses of the TCGA Dataset identified ATF6 activity to correlate positively with CRC-enriched genera, including Desulfovibrio fairfieldensis, supporting a causal link between ATF6-mediated LCFA synthesis and tumor-relevant changes in the microbiota.

Conclusion

Our study highlights the intricate interplay between UPR^ER, lipid metabolism, and the gut microbiota in CRC. We postulate that chronic ATF6 signaling in the intestinal epithelium promotes the selection of tumor-promoting microbiota by altering lipid metabolism. ATF6 signaling emerges as a key driver that reshapes lipid metabolism and creates a microbiota environment conducive to tumor development, indicating new targetable vulnerabilities in CRC.

Disclosure

no conflict of interest

Introduction

Pancreatic ductal adenocarcinoma (PDAC) is a dismal disease with an average five-year survival rate of approximately 12%¹. The tumor microenvironment (TME) accounts for approximately 40% to 80% of the total tumor mass in PDAC and is composed of cancer-associated fibroblasts and immune cells. PDAC is typically considered a non-immunogenic tumor, showing limited responsiveness to immunotherapy². Nevertheless, the TME plays a crucial role in shaping disease progression and determining patient outcomes.

Aims & Methods

The aim of this study is to characterize the immunoprofiles of PDAC and analyze their association with tumor stage and patient prognosis.
The study cohort included 443 transcriptomes spanning multiple disease stages (Stage I=35, Stage II/III=244, and Stage IV=164). The contribution of the TME to overall survival (OS) prediction was assessed using four PDAC classifiers: two mono-compartment classifiers based solely on tumoral cell (Bailey³ and Chan-Seng-Yue⁴), and two multi-compartment classifiers (Puleo⁵ and MR-Gradient⁶) that incorporate TME components. The performance of each classifier's Cox regression model was evaluated using the Akaike Information Criterion (AIC). Hierarchical Clustering on Principal Components (HCPC) was applied to inferred immune cell populations (xCell and CIBERSORT) to identify immune clusters (IC). The gene profile of each IC was determined through differential expression analysis. IC assignment in the validation cohorts (TCGA-PAAD and Puleo) was performed using Gene Set Variation Analysis (GSVA) scores.

Results

The multi-compartment classifiers demonstrated superior performance, with the MR-Gradient showing the lowest AIC with hazard ratio (HR) of 0.68 (95% CI, 0.61–0.76; P<0.001), indicating the best model fit. Unsupervised clustering of HCPC revealed four distinct IC. Approximately 18.3% of patients were classified as IC1, characterized by enrichment of B cell and activated dendritic cells populations was associated with the better outcome (HR=0.57; 95% CI, 0.41–0.78; P<0.001), followed by IC2, predominated by CD8⁺ T cells (HR=0.70; 95% CI, 0.53–0.93; P=0.014). In contrast, IC3 and IC4 were defined by an immunosuppressive tumor microenvironment. The frequency of IC1 was highest in Stage I (45.7%) tumors and progressively decreased through Stage IV (17.1%), suggesting a stage-dependent shift in immune composition. These findings were validated in the TCGA-PAAD and Puleo cohorts, where IC1 consistently showed superior outcomes, with HR of 0.37 (95% CI, 0.17–0.78; P=0.009) and 0.43 (95% CI, 0.28–0.66; P<0.001), respectively.

Conclusion

These results validate that incorporating the immunological compartment alongside tumoral cell enhances the prediction of patient outcomes. Moreover, we identified four immunological clusters that span all tumor stages, each defined by distinct inferred immune cell compositions. Notably, patients whose TME was enriched in B cells and activated dendritic cells exhibited the most favorable survival outcomes. Overall, this study provides a framework for stratifying PDAC patients based on the immunological composition of their TME, potentially guiding more personalized therapeutic approaches.

References

1. Siegel, R. L., Giaquinto, A. N. & Jemal, A. Cancer statistics, 2024. CA Cancer J Clin 74, 12–49 (2024).
2. Brouwer, T. P. et al. Local and systemic immune profiles of human pancreatic ductal adenocarcinoma revealed by single-cell mass cytometry. J Immunother Cancer 10, e004638 (2022).
3. Bailey, P. et al. Genomic analyses identify molecular subtypes of pancreatic cancer. Nature 531, 47–52 (2016).
4. Chan-Seng-Yue, M. et al. Transcription phenotypes of pancreatic cancer are driven by genomic events during tumor evolution. Nat Genet 52, 231–240 (2020).
5. Puleo, F. et al. Stratification of Pancreatic Ductal Adenocarcinomas Based on Tumor and Microenvironment Features. Gastroenterology 155, 1999–2013 (2018).
6. Fraunhoffer, N. A. et al. Multi-omics data integration and modeling unravels new mechanisms for pancreatic cancer and improves prognostic prediction. NPJ Precis Oncol 6, (2022).

Introduction

Colon cancer is one of the most common malignant tumors in humans, with colon adenocarcinoma (COAD) being the predominant type. It typically arises from the transformation of colon adenomas. However, the cellular and molecular mechanisms driving the progression from adenoma to adenocarcinoma remain incompletely understood. Investigating this transformation process is crucial for the early intervention and treatment of colon cancer.

Aims & Methods

To elucidate the mechanisms driving the adenoma-carcinoma transition in COAD, we conducted single-cell sequencing on samples from adjacent normal tissue, adenomatous tissue, and colon adenocarcinoma tissue. We also integrated single-cell datasets from two GEO databases. Through cell communication analysis, differential gene expression analysis, and pseudotime analysis, we explored the role of the CD8⁺ Tex subset in the adenoma-carcinoma transition. Utilizing GEO transcriptome datasets, we constructed a disease risk prediction model based on nine genes (CEBPB, LEF1, ANXA2, SERPINE2, RUNX2, MLLT6, MAT2A, STAT1, NOTCH1). This model was validated using the TCGA transcriptome dataset and the IMvigor210 immunotherapy cohort. Combining the risk coefficients of the nine genes with survival analysis results, we identified RUNX2 as a potentially significant risk factor. The function of key molecules was further validated using an in vivo AOM/DSS colon cancer model and the HCT116 colon cancer cell line in vitro. Additionally, the interaction between CD8⁺ Tex cells and tumor cells was confirmed through multiplex immunofluorescence.

Results

The study found that the proportion of CD8⁺ Tex cells significantly increased in colon adenoma and carcinoma samples compared to adjacent normal tissue. These cells may promote the transition from adenoma to COAD through multiple mechanisms. Pseudotime analysis indicated that CD8⁺ Tex cells in COAD might differentiate from those in adenomas, and highly malignant COAD epithelial cells may originate from highly malignant adenoma cells. The risk score model based on nine genes identified RUNX2 as a potentially significant risk factor. Both in vivo and in vitro experiments confirmed that RUNX2 promotes the proliferation, invasion, and migration of the HCT116 colon cancer cell line, while the RUNX2 inhibitor CADD522 can suppress the adenoma-to-carcinoma transition. Additionally, TNF-α was found to enhance RUNX2 mRNA and protein expression, thereby promoting the malignant behavior of HCT116 cells and the expression of genes related to the adenoma-to-carcinoma transition.

Conclusion

This study identifies that CD8+ Tex cells may activate the transcription factor RUNX2 in colon cancer cells by binding the ligand TNF-α to the TNFR1 receptor on the membrane of malignant epithelial cells. This activation promotes the transition of malignant epithelial cells in colon adenomas to malignant cancer cells, leading to poor prognosis in COAD. This finding not only elucidates the key cellular and molecular mechanisms of the adenoma-to-carcinoma transition but also provides new potential targets for early intervention and treatment of colon cancer.

Introduction

Early detection of gastric cancer (GC) is crucial for improving patient outcomes, but ongoing surveillance is equally important, given the 16% risk of recurrence or progression after initial treatment. While molecular biomarkers have shown promise for diagnosis, few have been assessed in longitudinal settings.

Aims & Methods

This study aimed to develop and validate a gene expression signature capable of detecting early GC in a screening setting and to explore its utility in predicting metachronous gastric lesions (MGLs) during post-treatment surveillance following curative endoscopic submucosal dissection (ESD). A gene expression signature was developed using RNA-sequencing data from formalin-fixed paraffin-embedded (FFPE) tissue samples, including 23 controls (histologically normal mucosa) and 28 cases (low-/high-grade dysplasia and adenocarcinoma, all submitted to ESD). Gene selection was performed using machine learning within the tidymodels framework. Selected genes were validated by real-time PCR in an expanded cohort (n = 279; 123 controls, 156 cases), which was randomly split into independent discovery and validation sets (60:40 ratio). Logistic regression models were built using gene expression data, age and sex. Predictive performance for MGL development was then assessed in the 156 cases, stratified by MGL occurrence (n = 39 with MGLs, n = 157 without), with at least three years of endoscopic follow-up after curative ESD.

Results

RNA-sequencing analysis identified a seven-gene panel with potential for early GC detection, consisting of three upregulated and four downregulated genes in patients with gastric lesions. This signature was successfully validated in the stomach adenocarcinoma cohort of The Cancer Genome Atlas (TCGA-STAD) and Genotype-Tissue Expression (GTEx) datasets, upon filtering stage I cases (n = 81, P < 0.0001). In the discovery set, the model achieved excellent performance in distinguishing lesions from normal tissue (AUC = 0.96, 95% confidence interval (CI): 0.93-0.99; sensitivity = 0.90, 95% CI: 0.79-0.96; specificity = 0.94, 95% CI: 0.85-0.99). One gene retained its expression pattern in patients with MGLs. Although the model was not predictive of MGL development, a new model based on the same signature and trained exclusively on the MGL dataset achieved promising diagnostic performance (AUC = 0.74, 95% CI: 0.59-0.88; sensitivity = 0.85, 95% CI: 0.55-0.98; specificity = 0.72, 95% CI: 0.58-0.84).

Conclusion

This study demonstrates the promise of a gene expression-based model for early GC detection in a Western population. Beyond its diagnostic value, the underlying gene signature also showed potential for predicting MGLs, with good sensitivity and moderate specificity. These findings suggest that this approach could serve a dual purpose in both the detection of gastric lesions and the individualized management of these patients.

Introduction

Prophylactic clip closure is known to avoid delayed adverse events post colorectal endoscopic submucosal dissection (ESD).¹ It may also improve reduce the duration of hospitalization.² However, endoscopic clips have limitation of defect size that can be completely closed due to their limited opening width. Other studies have suggested methods to completely close a mucosal defect using special devices^{3, 4} which have limitations of availability, learning curve, scope removal and reinsertion, and negotiation in narrow spaces. An ideal closure technique has been described as a technique which is technically easy, requiring no special accessories or endoscopes, managed through accessory channel without endoscope removal, possible in narrow space, creating no dead space, and being economical.⁵ We recently developed a Clip with Line Pulley Securing (CLiPS) technique to completely close large mucosal defect which fulfils the criteria for an ideal closure technique.⁶

Aims & Methods

The aim of this study was to report the feasibility and efficacy of CLiPS technique for closure of large mucosal defects following colorectal ESD. This study retrospectively evaluated 53 consecutive patients who underwent closure using CLiPS technique post colorectal ESD. CLiPS technique involves deployment of a clip with nylon line on the edges of the ulcer which are approximated using external counter traction. An endoloop is deployed to fix the line. The principle is supporting the clip line with an endoloop and creating a pulley system by giving external countertraction to the line, to facilitate a strong, stable approximation of edges, thereby making the defect more linear. The line is cut and final closure is completed with conventional clips. The primary outcomes were complete closure rate and adverse events. The secondary outcomes were closure time and defect size.

Results

The lesion characteristics are given in the 'table'. Complete closure was achieved in 94.3%(50/53) of cases. The mean defect size was 48.2­+18.8 mm and median closure time was 14(5-58) minutes. There was one case each of delayed bleeding and perforation which were managed by endoscopic clipping.

Conclusion

CLiPS technique has reasonable outcomes and can be considered to be a safe and effective closure method for large post ESD colorectal defects. Further prospective study may be undertaken to establish wider acceptability of this method.

References

1. Liu M, Zhang Y, Wang Y, et al. Effect of prophylactic closure on adverse events after colorectal endoscopic submucosal dissection: A meta‐analysis. Journal of Gastroenterology and Hepatology. 2020 Nov;35(11):1869-77.
2. Abe S, Saito Y, Tanaka Y, et al. A novel endoscopic hand-suturing technique for defect closure after colorectal endoscopic submucosal dissection: a pilot study. Endoscopy. 2020 Sep;52(09):780-5.
3. Farha J, Ramberan H, Aihara H, et al. A novel through-the-scope helix tack-and-suture device for mucosal defect closure following colorectal endoscopic submucosal dissection: a multicenter study. Endoscopy. 2023 Jun;55(06):571-7.
4. Keihanian T, Zabad N, Khalaf M, et al. Safety and efficacy of a novel suturing device for closure of large defects after endoscopic submucosal dissection (with video). Gastrointestinal Endoscopy. 2023 Sep 1;98(3):381-91.
5. Nomura T, Sugimoto S, Temma T, et al. Suturing techniques with endoscopic clips and special devices after endoscopic resection. Digestive Endoscopy. 2023 Mar;35(3):287-301.
6. Minato Y, Ohata K, Kimoto Y, et al. A modified approach for closing endoscopic submucosal dissection defects using clip with line pulley securing technique and endoloop. VideoGIE. 2024 Mar 27.
   

Clinical Case Summary

Introduction: Intra-abdominal sepsis from colonic perforation is a surgical emergency with high mortality. While diverticulitis is a common cause in older adults, its presence in young, healthy individuals suggests additional etiologies. Parasitic infestations such as Ascaris lumbricoides, though rare in developed regions, can complicate outcomes.
Case presentation: A 35-year-old previously healthy male presented with 3 days of severe abdominal pain and vomiting. CT scan revealed pneumoperitoneum. Emergency laparotomy showed perforated sigmoid diverticulitis with fecal peritonitis. A Hartmann’s procedure was performed with an open abdomen due to gross contamination. Despite surgery, the patient developed persistent sepsis, requiring seven relook laparotomies. During the fourth, live Ascaris worms were seen emerging from the bowel during ileostomy creation, prompting Albendazole treatment. Histopathology confirmed diverticulitis with perforation. Recovery was complicated by multidrug-resistant infections, anemia, malnutrition, and thrombotic events, prolonging ICU stay.
Discussion: This case highlights how unrecognized parasitic infestation can worsen intra-abdominal sepsis. Ascaris may contribute to mucosal damage, obstruction, and inflammation, exacerbating outcomes. Early hypoalbuminemia might have reflected chronic enteric loss or parasite burden.
Conclusion: In endemic areas, chronic helminthic infections should be considered in young patients with unusual presentations or poor postoperative progress. Timely recognition may impact treatment and recovery.

References

Figure 1: CT Abdomen with Contrast showing Moderate Pneumoperitoneum
Figure 2: Worm coming out of iliostomy
Figure 3: acsaris
Figure 4 : CT Chest showing bilateral moderate pleural effusion more on the right side with underlying lung consolidation collapse.
Figure 5 : US Neck showing Left internal jugular vein central line with echogenic thrombus not completely occluding the lumen.

Introduction

Acute appendicitis is a widely encountered emergency across the globe, showcasing the resilience of modern medicine, as it is traditionally addressed through the transformative procedure of appendectomy. Endoscopic Retrograde Appendicitis Therapy (ERAT) has emerged as a minimally invasive alternative; however recurrence is a worry.

Aims & Methods

This multi-center retrospective study aimed to evaluate the long-term safety, efficacy, and recurrence-related risk factors of ERAT to guide clinical decision-making. A total of 435 patients who underwent ERAT for acute appendicitis between October 2017 and October 2024 at three Chinese medical centers were included. Appendicitis recurrence was defined as imaging-confirmed symptoms returning within one-month post-ERAT. Univariate and multivariate regression analysis identified independent recurrence risk variables.

Results

All ERAT procedures were technically successful and achieved a clinical success rate of 92.4%. Over a median follow-up period of 37 months, the overall recurrence rate was 16.1%, with the majority of recurrences (13%) observed within the first year. Independent risk factors for recurrence included recurrent appendicitis (HR=1.916), appendiceal lumen distortion (HR=7.996), absence of stent placement (HR=0.375, protective), and operator inexperience (HR=2.318). Stent implantation effectively minimizes recurrence risk, especially in complicated incidents. ERAT facilitated overseeing appendicolith elimination, which assisted in occurring appendicolith-associated appendicitis.

Conclusion

ERAT is deemed safe and effective for acute appendicitis, with the highest recurrence risk occurring within the first year. Effective stent placement and skilled operators are crucial for reducing recurrence in complex cases. The aforementioned findings put forward significant insights for enhancing ERAT and advancing therapeutic outcomes.