The role of gut microbiota in acute cellular rejection (ACR) after intestinal transplantation (ITx) remains poorly defined. This study investigated whether ileal microbial dysbiosis precedes ACR and is associated with barrier injury. We conducted a longitudinal and cross-sectional analysis of 16 ITx recipients. Ileal effluents were profiled via 16S ribosomal RNA gene sequencing, short-chain fatty acid quantification, complemented by graft histopathology and assessment of systemic biomarkers of barrier dysfunction. Eight recipients (50.0%) developed biopsy-proven ACR (median 25.5 days post-ITx). Recipients who developed ACR exhibited early and sustained microbial divergence, characterized by Enterobacteriaceae dominance and loss of diversity. Samples collected at a median of 7.0 days before ACR diagnosis revealed a prerejection dysbiotic state marked by reduced microbial diversity, diminished short-chain fatty acid levels, enrichment of Enterobacteriaceae, and depletion of Enterococcaceae and Bacteroidaceae. Functional profiling indicated pre-ACR upregulation of bacterial motility, flagellar assembly, and lipopolysaccharide biosynthesis pathways. These microbial shifts correlated with mucosal injury (mucus thinning, goblet cell loss, and bacterial adherence) and elevated systemic barrier dysfunction biomarkers. Collectively, ileal microbial and metabolic dysbiosis precede clinical ACR and correlate with barrier injury, nominating the ileal microbiota as a predictive biomarker source and therapeutic target in ITx.

The homologous recombination (HR) system repairs DNA double-strand breaks produced by the DNA damage response, which is a complex signaling pathway consisting of the key proteins BRCA1/2 and other DNA repair proteins, such as the ATM, PALB2, BARD1, RAD51, and Fanconi anemia proteins. Mutations and epigenetic alterations in HR-related genes may lead to homologous recombination deficiency (HRD), resulting in genomic instability and contributing to the development of certain solid tumors. The biological significance and molecular mechanism of BRCA1/2 mutation-related HRD are well understood, but the relationships of other HR-related genes and their variant forms with HRD have not been sufficiently studied. These genes exhibit multiple forms of variation, including one or more HR genes, germline or somatic mutations, monoallelic or biallelic variants, and not all variants present HRD. Therefore, HRD is usually defined as HR-related gene variation, but recent studies have shown that defining it as the combined score of loss of heterozygosity, LST and TAI, known as the HRD score, can more accurately assess genomic instability. In patients with HRD, platinum-based therapy and poly ADP-ribose polymerase enzyme inhibitor (PARPi) have been shown to perform well in ovarian, breast, and prostate cancers. For gastrointestinal cancer (GI cancer), HRD has been relatively well studied in pancreatic cancer, but its role in other cancers has rarely been reported. Herein, we review the pathogenesis and predictive value of HRD, including the use of platinum drugs, PARPi, and immunotherapy, in digestive system tumors.

3617 Background: Colorectal cancer (CRC) screening tests based on colonoscopy and fecal DNA testing are limited due to their invasive nature and low compliance rates. Herein, we develop a non-invasive early screening test for CRC based on ColoSeer assay, which combines methylated DNA immunoprecipitation and bisulfite-free sequencing to analyze methylation patterns of circulating tumor DNA (ctDNA). Methods: Tumor and ctDNA methylation patterns in 1,336 subjects (430 healthy controls, 202 colorectal precursor lesions (CPLs), 704 CRCs) undergoing colonoscopy were evaluated using the ColoSeer assay. Results: In the discovery phase, we identified a set of 46 differential methylation markers to screen for CPLs or CRCs from healthy controls. The overall detection sensitivity of ColoSeer was 85.6% for CPL and 97.5% for TMN stage (0-II) CRC, with 92.8% specificity. Conclusions: The ColoSeer assay demonstrated a high degree of accuracy for predicting subjects with CPL or early-stage malignant tumors and has potential as a first-tier health screen for early detection of CRC.

Tissue-resident memory T cells (TRMs) reside in peripheral tissues and provide rapid immune defence against local infection and tumours. Tumour-associated TRMs share common tissue-resident features and formation mechanisms, representing some unique subsets of tumour-infiltrating lymphocytes (TILs). However, differences in the tumour microenvironment(TME) and tumour evolution stage result in TRMs exhibiting temporal and spatial heterogeneity of phenotype and function not only at different stages, before and after treatment, but also between tumours originating from different tissues, primary and metastatic cancer, and tumour and adjacent normal tissue. The infiltration of TRMs is often associated with immunotherapy response and favourable prognosis; however, due to different definitions, it has been shown that some subtypes of TRMs can also have a negative impact. Therefore, it is crucial to precisely characterise the TRM subpopulations that can influence the therapeutic efficacy and clinical prognosis of various solid tumours. Here, we review the spatiotemporal heterogeneity of tumour-associated TRMs, as well as the differences in their impact on clinical outcomes. We also explore the relationship between TRMs and immune checkpoint blockade (ICB) and TIL therapy, providing insights into potential new targets and strategies for immunotherapy.

Background: Different RAS/BRAF allele mutations imply distinct biological properties in various solid tumors. Recently, several studies have focused on the predictive and prognostic roles of various RAS/BRAF allele mutations in colorectal cancer (CRC) but the results remain controversial. Methods: Between March 2017 and September 2022, the patients diagnosed as stages I–IV CRC with detailed medical records including next-generation sequencing (NGS) data and clinicopathological follow-up information available at our center were enrolled. Survival data were estimated using the Kaplan–Meier method, and the difference was tested in a log-rank test. Multivariate tests were carried out using Cox models. Results: A total of 1029 CRC patients were included, and the incidence of RAS/BRAF mutation was 58.4%. The hypermutated cohort was defined as patients with microsatellite instability-H or POLE/D mutation. In the non-hypermutational cohort, only KRAS G13D mutation was associated with a higher incidence and inferior disease-free survival in patients with stage I-III CRC. In the cohort of patients with non-hypermutated metastatic colorectal cancer (mCRC), we assessed the risk of various RAS/BRAF allele mutations and subsequently reclassified patients into four groups based on first-line median progression-free survival: wild type (group 1), low-risk RAS/BRAF mutation (group 2, RAS/BRAF mutations other than KRAS G13D/G12V/G12C or BRAF V600E), high-risk RAS mutation (group 3, KRAS G13D/G12V/G12C), and BRAF V600E mutation (group 4). mCRC patients with high-risk RAS mutation could significantly benefit from intensive triplet chemotherapy (hazard ratio, 2.54; 95% confidence interval, 1.36–5.12; p = 0.0091). Conclusion: In the non-hypermutated CRC cohort, the prognostic risk of various RAS/BRAF allele mutations varied between local and metastatic CRC. KRAS G13D mutation tended to be the only prognostic marker for stages I–III CRC; however, KRAS G13D/G12V/G12C mutations collectively defined a high-risk subgroup of mCRC patients with poor prognosis, who would benefit from intensive triplet chemotherapy.

ObjectControversy remains regarding the safety and efficacy of hemorrhoid ligation and stapled hemorrhoidopexy (SH) in the treatment of hemorrhoids. The study was to explore the operative outcomes of patients underwent multiple thread ligations (MTL) with SH for the management of grade III hemorrhoids.MethodsThis cohort study included patients who underwent MTL (MTL group, 128 cases) or SH (SH group, 141 cases) for grade III hemorrhoids between June 2019 and May 2021. A total of 115 patients in MTL group and 115 patients in SH group were finally included by propensity score matching with a ratio of 1:1. The primary outcome was the recurrence of prolapse within 6 months. Secondary outcomes were operative time, post-operative pain scores, hospital stay, the incidence of complications, Wexner incontinence score, and quality of life of patients with constipation at 6 months post procedure.ResultsMultiple thread ligations and SH resulted in comparable recurrence within 6 months of follow-up, with five and seven cases of recurrence, respectively, (P = 0.352). The two groups had comparable outcomes in terms of post-operative pain, hospital stay, Wexner incontinence scores, and constipation-related quality of life (all P > 0.05). The median operative time was 16 min (15–18 min) in the MTL group versus 25 min (16–33 min) in the SH group (P < 0.01). Univariate analysis showed that the MTL technique had a lower risk of postoperative bleeding than that with the SH technique (P < 0.05).ConclusionThe study indicated that the MTL technique might achieve comparable operative outcomes compared with the SH technique for the management of grade III hemorrhoids, nevertheless, MTL seemed to be associated with less risk of surgical bleeding than SH.

IntroductionTargetable alterations such as BRAFV600E mutation and NTRK fusion are enriched in microsatellite instability-high (MSI-H) colorectal cancer (CRC). MSI-H with targetable alterations (MSI-H altered) might present unique opportunities for both targeted therapy and immunotherapy. We systematically evaluated the molecular characteristics and immune-related features of MSI-H altered and MSI-H without targetable alterations (MSI-H wt) CRC patients in our study.MethodsAmong 1938 continuously enrolled CRC patients, 126 patients with MSI-H status (6.50%) were included in this retrospective study. Genomic and transcriptomic data were investigated by next-generation sequencing (NGS) and gene expression profiling (GEP), respectively.ResultsBRAFV600E, NTRK1, and FGFR2 mutations were the most frequent targetable alterations in MSI-H CRC patients. The MSI-H altered phenotype was significantly associated with older age (p< 0.001), right side (p=0.024) and females (p= 0.036). No lynch syndrome (LS) patients were identified in MSI-H altered group. The tumor mutational burden (TMB), and tumor neoantigen burden (TNB) of MSI-H altered and wt subgroups were comparable (p<0.05). Subsequently, transcriptomic study analysis further revealed MSI-H altered CRC patients were linked to an immune-active tumor microenvironment with higher levels of Teff IFN-gamma, CYT, and MERCK 18 signatures, and lower levels of the IPRES gene signature, EMT and TGF Beta signatures. In addition, case study supported MSI-H CRC patient harboring targetable alterations might also achieved a long-term disease-free survival benefit from immunotherapy.DiscussionOur study preliminary revealed MSI-H altered as a novel subtype of MSI-H CRC patients with unique molecular signatures and immune-active tumor microenvironment. Given the accessibility of immune checkpoint inhibitors (ICIs) treatment, our results might provide clinical evidence for immunotherapy in MSI-H CRC patients with targetable alterations.

The solute carrier 17 family transports diverse organic anions using two distinct modes of coupling to a source of energy. Transporters that package glutamate and nucleotide into secretory vesicles for regulated release by exocytosis are driven by membrane potential but subject to allosteric regulation by H+ and Cl-. Other solute carrier 17 members including the lysosomal sialic acid exporter couple the flux of organic anion to cotransport of H+. To begin to understand how similar proteins can perform such different functions, we have studied Escherichia coli DgoT, a H+/galactonate cotransporter. A recent structure of DgoT showed many residues contacting D-galactonate, and we now find that they do not tolerate even conservative substitutions. In contrast, the closely related lysosomal H+/sialic acid cotransporter Sialin tolerates similar mutations, consistent with its recognition of diverse substrates with relatively low affinity. We also find that despite coupling to H+, DgoT transports more rapidly but with lower apparent affinity at high pH. Indeed, membrane potential can drive uptake, indicating electrogenic transport and suggesting a H+:galactonate stoichiometry >1. Located in a polar pocket of the N-terminal helical bundle, Asp46 and Glu133 are each required for net flux by DgoT, but the E133Q mutant exhibits robust exchange activity and rescues exchange by D46N, suggesting that these two residues operate in series to translocate protons. E133Q also shifts the pH sensitivity of exchange by DgoT, supporting a central role for the highly conserved TM4 glutamate in H+ coupling by DgoT.

Objective:To examine the outcomes of intestinal autotransplantation (IATx) in patients with locally advanced (LACC) or locally recurrent (LRCC) colon cancer invading the superior mesenteric artery (SMA).Background:SMA involvement in LACC or LRCC is deemed unresectable and is associated with a poor prognosis. Combined extended resections of multiple organs together with SMA, followed by IATx may offer favorable clinical outcomes. However, data on its safety and efficacy are scarce.Methods:This retrospective cohort study included patients undergoing IATx between May 2018 and December 2022 in intestinal transplant programs at 2 university-affiliated hospitals in China. Patients with LACC or LRCC concomitantly with SMA contact of more than 180 degrees were included. Patients with a locoregional peritoneal, pelvic, or distal metastasis were excluded.Results:Ten patients underwent either IATx combined with pancreaticoduodenectomy (n = 8) or IATx alone (n = 2). Eight patients (80%) were males, and the median age was 55 years (range, 32-71 years). The Kaplan-Meier estimates for recurrence-free survival and overall survival at 3 years after IATx were 68% and 80%, respectively. No perioperative deaths occurred. All 10 patients experienced postoperative complications, including Clavien-Dindo grade I (n = 1), grade II (n = 4), grade IIIa (n = 1), grade IIIb (n = 3), and grade IVa (n = 1), which comprised acute venous thromboses, upper gastrointestinal hemorrhage, anastomotic leak, gastropareses, and significant pleural effusions. With an average follow-up of 23.9 months, 8 patients (80%) were currently alive without evidence of disease.Conclusion:Extended resection for LACC or LRCC invading SMA can be performed safely and is associated with prolonged survival.

BACKGROUND:Limited data have been used to evaluate the efficacy of immunotherapy in metastatic colorectal cancer (mCRC). Furthermore, potential markers that can be used to identify responding patients and to further improve efficacy have not been fully explored. METHODS AND RESULTS:In our study, we included a total of 97 patients with mCRC, who each received programmed death-1 (PD-1) inhibitor-based combination therapy at our center. All 12 hypermutated patients benefited from immunotherapy, with median progression-free survival (mPFS) reaching 28.3 months, regardless of liver metastasis. The objective response rate (ORR) of non-hypermutated patients was 16.5% (14/85), with an mPFS of 4.0 months. For non-hypermutated patients, multivariate analysis revealed that the combination of liver metastasis and baseline lesion number significantly stratified response and survival. The lesion-based analysis indicated that the lymph node was the most responsive, followed by the peritoneum and lung, with liver metastasis being the least responsive. None of the patients (0/7) with negative programmed ligand-1 (PD-L1) expression responded, and positive PD-L1 expression may serve as a biomarker (mPFS 5.7 vs. 2.2 months, p = 0.002) that can be used to further guide treatment in non-hypermutated mCRC with liver metastasis (CRLMs). CONCLUSION:Patients with hypermutated mCRC benefited significantly from immunotherapy, whereas the non-hypermutated cohort with liver metastasis and numerous lesions showed less benefit. The lesion sites reflected varying levels of efficacy, among which PD-L1 potentially cooperated to guide the immunotherapy of CRLMs.

Abstract Background Circulating soluble programmed death ligand 1 (sPD-L1) can negatively regulate T-cell function and serve as a prognostic or predictive marker in a variety of cancers. However, rare studies have evaluated the potential roles of sPD-L1, and no study has estimated its predictive value for the efficacy of immune treatment in colorectal cancer (CRC). Methods Plasma samples from 192 CRC patients were used to estimate correlations between clinicopathological features and sPD-L1, secreted PD-L1 (secPD-L1) and exosomal PD-L1 (exoPD-L1). Baseline and posttreatment sPD-L1 levels were also investigated in 55 patients with metastatic CRC (mCRC) treated with chemotherapy ± targeted therapy and 40 patients with proficient mismatch repair (pMMR) mCRC treated with combination immunotherapy. Both sPD-L1 and secPD-L1 were quantified by enzyme-linked immunosorbent assay, while exoPD-L1 was analyzed using flow cytometry. Results secPD-L1 was the major component and positively correlated with sPD-L1 in CRC, while exoPD-L1 was almost undetectable. Higher levels of sPD-L1 were detected in patients with distant metastasis, especially those with distant lymph node metastasis and tissue combined positive score (CPS) instead of tumor proportion score (TPS). Chemotherapy or targeted therapy did not significantly impact sPD-L1 concentration. Progressive disease on combination immunotherapy was associated with an increase in sPD-L1 level, whereas no significant change was observed in patients with durable clinical benefit. Conclusion sPD-L1 mainly consisted of secPD-L1, and its level was higher in patients with distant metastasis, especially distant lymph node metastasis and positive CPS. sPD-L1 is a potential dynamic marker to identify rapid progression on combination immunotherapy and avoid ineffective treatment for pMMR CRC.

Background: LDITx is not routinely performed because of surgical risks to the donor and the potential inferior physiologic performance of the segmental graft. However, data on the effectiveness of LDITx are scarce. This study aimed to examine outcomes of living-donor intestinal transplant (LDITx) recipients. Methods: This retrospective cohort study included patients undergoing LDITx between May 1999 and December 2022 in intestinal transplant programs in two university-affiliated hospitals in China. Results: Actuarial survival rates were 82%, 75.7%, 68.9% for patient and 76.3%, 66.6%, 63% for graft at 1, 3, and 5 years, respectively. Recipients with > 3/6 HLA-matched grafts had superior patient and graft survival rates than those with ≤ 3/6 HLA-matched grafts (P < 0.05). There were 12 deaths among the recipients, with infection being the leading cause (41.7%), followed by rejection (33.3%), surgical complications (16.7%), and others (8.3%). There were 16 graft losses among the recipients, with acute cellular rejection being the predominant cause (37.5%), followed by infection (25%), technical failure (12.5%), chronic rejection (12.5%), and others (12.5%). With an average follow-up of 3.8 (range, 0.7-24) years, the rates of acute and chronic rejection were 33% and 4%, and the rate of CMV disease and PTLD were 5% and 2.5%, respectively. Of the 46 patients, 34 (73.9%) are currently alive and have achieved enteral autonomy. None of the donors experienced life-threatening complications or mortality. Six (15%) of 46 donors experienced minor operative complications. Conclusions: LDITx is a valuable treatment option for patients with end-stage intestinal failure. Living-donor ileal resection is associated with minimal short- and long-term morbidity and remains an attractive alternative for potential recipients when suitable deceased donors are unavailable.