Gastrointestinal Graft-versus-host Disease Research Articles

Amphiregulin, ST2, and REG3α biomarker risk algorithms as predictors of nonrelapse mortality in patients with acute GVHD

AbstractGraft-versus-host disease (GVHD) is a major cause of nonrelapse mortality (NRM) after allogeneic hematopoietic cell transplantation. Algorithms containing either the gastrointestinal (GI) GVHD biomarker amphiregulin (AREG) or a combination of 2 GI GVHD biomarkers (suppressor of tumorigenicity-2 [ST2] + regenerating family member 3 alpha [REG3α]) when measured at GVHD diagnosis are validated predictors of NRM risk but have never been assessed in the same patients using identical statistical methods. We measured the serum concentrations of ST2, REG3α, and AREG by enzyme-linked immunosorbent assay at the time of GVHD diagnosis in 715 patients divided by the date of transplantation into training (2004-2015) and validation (2015-2017) cohorts. The training cohort (n = 341) was used to develop algorithms for predicting the probability of 12-month NRM that contained all possible combinations of 1 to 3 biomarkers and a threshold corresponding to the concordance probability was used to stratify patients for the risk of NRM. Algorithms were compared with each other based on several metrics, including the area under the curve (AUC), proportion of patients correctly classified, sensitivity, and specificity using only the validation cohort (n = 374). All algorithms were strong discriminators of 12-month NRM, whether or not patients were systemically treated (n = 321). An algorithm containing only ST2 + REG3α had the highest AUC (0.757), correctly classified the most patients (75%), and more accurately risk-stratified those who developed Minnesota standard-risk GVHD and for patients who received posttransplant cyclophosphamide-based prophylaxis. An algorithm containing only AREG more accurately risk-stratified patients with Minnesota high-risk GVHD. Combining ST2, REG3α, and AREG into a single algorithm did not improve performance.

High sensitivity of host Helios+/Neuropilin-1+ Treg to pretransplant conditioning hampers development of OX40bright/integrin-β7+ regulatory cells in acute gastrointestinal GvHD.

This study sought to compare the behavior of Treg subsets displaying different coexpression patterns of Neuropilin-1 (Nrp1) and Helios, under the influence of gut stress unrelated to hematopoietic stem cell transplantation, pretransplantation conditioning, and posttransplant gastrointestinal acute graft versus host disease (GI-aGvHD). Host CD4+/CD25hi/Foxp3+ Treg cells, identified by flow cytometry, were isolated from various tissues of mice affected by these stressors. Expression of CD25, CTLA-4, CD39, OX40, integrin-β7, LAG3, TGFβ/LAP, granzyme-A, -B, and interleukin-10 was compared in four Treg subsets displaying Helios or Nrp1 only, both or none. Fluorescence-activated cell sorter-sorted Treg subsets, displaying markers affected in a conditioning- and GI-aGVHD-restricted manner, were further investigated by transcriptome profiling and T-cell suppression assays. We found that conditioning by irradiation greatly diminished the relative frequency of Helios+/Nrp1+ Treg, shifting the balance toward Helios-/Nrp1- Treg in the host. Upregulation of integrin-β7 and OX40 occurred in GI-aGvHD-dependent manner in Helios+/Nrp1+ cells but not in Helios-/Nrp1- Treg. Sorted Treg subsets, confirmed to overexpress Nrp1, Helios, OX40, or integrin-β7, displayed superior immunosuppressive activity and enrichment in activation-related messenger RNA transcripts. Our data suggest that conditioning-induced shrinkage of the Nrp1+/Helios+ Treg subset may contribute to the development of GI-GvHD by impairing gut homing and decreasing the efficiency of Treg-mediated immunosuppression.

Significant impact of antibiotic exposure on GI-GVHD, NRM, and GRFS following allogeneic HCT with non-myeloablative Flu-TBI conditioning

Background Acute gastro-intestinal graft-versus-host disease (GI-GVHD) and non-relapse mortality (NRM) after allogeneic HCT are closely related to loss of microbial diversity and intestinal dominance by single taxa resulting from the use of antibiotics, dietary changes, and mucosal barrier injury. There is a paucity of data on the impact of use of antibiotics in HCT after Flu-TBI-based non-myeloablative (NMA) conditioning where there is absence of mucositis and limited malnutrition. Methods We did a retrospective single-center analysis of patients receiving Flu-TBI-based NMA HCT for a high-grade myeloid malignancy, mostly AML, and MDS, or acute lymphoblastic leukemia (ALL). We analyzed the impact of pre-engraftment antibiotic exposure, prophylactic ciprofloxacin, and or treatment with broad-spectrum cephalosporin/carbapenem, on HCT outcomes, with a focus on the incidence of acute GI-GVHD by day 180 and NRM at 1 year. Results A total of 150 patients were evaluable with a median age of 62 years. Antibiotics were used in 90 patients; 60 prophylactic use only and 30 therapeutic use with or without previous prophylaxis. Antibiotic use resulted in a significant higher incidence of GI-GVHD Stage 1–4; 29% (26/90) versus 5% (3/60) in those not receiving antibiotics (OR 8.1 (95% CI 2.3–28.3), p = 0.001). Use of antibiotics resulted in higher 1-year NRM (19% (17/90) versus 10% (6/60), HR 2.3, p = 0.06), and decreased 2-year GRFS (42% (38/90) versus 55% (33/60), HR 1.7, p = 0.04), but did not impact RFS or OS. Conclusions Use of antibiotics was related to the occurrence of GI-GVHD, NRM, and GRFS in patients receiving truly NMA HCT. Therefore, in the absence of mucositis and low incidence of bacteremia, antibiotics can and should be used restrictively in this setting.

Abstract 2794: Consumption of dietary fiber correlates with a significant increase in a-diversity in the intestinal Microbiome of allo-HCT recipients and with lower GVHD-lethality in pre-clinical models

Abstract Background: Graft-versus-host disease (GVHD) is associated with a decrease in intestinal microbiota and beneficial short-chain fatty acids (SCFAs). Dietary fiber can modulate the intestinal microbiome and SCFA production which effect intestinal epithelium health and immunity. Therefore, we hypothesized that a high-fiber diet in recipients of an allogeneic hematopoietic cell transplantation (allo-HCT) could result in an increase of beneficial commensals, concentrations of SCFAs, and decreased risk of GVHD. Methods: We evaluated dietary data and fecal samples of allo-HCT patients receiving unmodified grafts (n=95) at MSKCC (day -10 to day +30 relative to allo-HCT) and correlated microbial α-diversity (16s rRNA, n=568 samples) with fiber intake and acute GVHD in No-GVHD (n=30), Non-LGI-GVHD (GVHD except lower gastrointestinal tract, n=41), and LGI-GVHD patients (lower gastrointestinal tract GVHD, n=24).We tested concentrations of cellulose fiber (0%, 6%, 12% and 40%) in a GVHD mouse model (C57BL/6J into BALB/c) to assess survival, microbial composition (shotgun sequencing n=180; n=30 per group), cecal SCFAs concentrations (GC-MS n=24, n=8 per group), and lastly, T cell subsets (Th1/Th17/Tregs) and colonic epithelial MHC-II expression (flow cytometry n=54, n=18 per group). Results: In allo-HCT patients, fiber intake correlated positively with microbial α-diversity (R=0.3, p=0.04), especially in the LGI-GVHD group (p=0.007). Additionally, patients with high fiber intake had higher microbial α-diversity compared to the low fiber cohort (p<0.0001).GVHD mice receiving a fiber-rich diet (12%), compared to 0% and 6%, had a higher microbial α-diversity (p=0.01); reduced GVHD lethality (p=0.02); and a decrease in the Enterococcus sp. (p=0.004) and Akkermansia sp. (p=0.01) abundance; taxa which are associated with worse overall survival and increased risk of lethal GVHD. In contrast, compared to 12%, the 40% fiber diet resulted in worse survival (p=0.002), lower α-diversity (p=0.05) and a higher abundance of Bifidobacterium sp. (p<0.0001).Cecal butyrate in GVHD mice was higher in response to the 12% vs. the 0% (p=0.01) and 6% (p=0.05) fiber diets. Mice treated with12% fiber displayed higher number of Tregs (CD4+CD25+FOXP3+), than those with 0% (p=0.001) and 6% (p=0.02); and a lower expression of MHC-II in epithelial cells (EpCAM+CD45-) vs. 0% fiber (p=0.02). Conclusions: We demonstrated that a higher fiber intake in allo-HCT patients is associated with an increase in α-diversity; especially in LGI-GVHD patients. In a pre-clinical GVHD-model, optimal fiber consumption (12% cellulose) leads to an increase in α-diversity, a decrease in pathogen relative abundance, increased T-regulatory cells, and a decrease in GVHD lethality. These results suggest that dietary fiber could be utilized to prevent GVHD. Citation Format: Jenny Paredes, Anqi Dai, Ruben J. Faustino Ramos, Peter Adintori, Teng Fei, Chi Nguyen, Harold Elias, Kristen Victor, Romina Ghale, Charlotte Pohl, Ana Gradissimo, Justin Cross, Oriana Miltiadous, Jonathan Peled, Marina Burgos da Silva, Marcel RM van den Brink. Consumption of dietary fiber correlates with a significant increase in a-diversity in the intestinal Microbiome of allo-HCT recipients and with lower GVHD-lethality in pre-clinical models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2794.

Effective treatment of Clostridioides difficile infection improves survival and affects graft-versus-host disease: a multicenter study by the Polish Adult Leukemia Group

Clostridioides difficile infection (CDI) is the most common cause of infectious diarrhea after allogeneic hematopoietic cell transplantation (allo-HCT). The impact of CDI and its treatment on allo-HCT outcomes and graft-versus-host disease (GVHD), including gastrointestinal GVHD (GI-GVHD) is not well established. This multicenter study assessed real-life data on the first-line treatment of CDI and its impact on allo-HCT outcomes. Retrospective and prospective data of patients with CDI after allo-HCT were assessed. We noted statistically significant increase in the incidence of acute GVHD and acute GI-GVHD after CDI (P = 0.005 and P = 0.016, respectively). The first-line treatment for CDI included metronidazole in 34 patients, vancomycin in 64, and combination therapy in 10. Treatment failure was more common with metronidazole than vancomycin (38.2% vs. 6.2%; P < 0.001). The need to administer second-line treatment was associated with the occurrence or exacerbation of GVHD (P < 0.05) and GI-GVHD (P < 0.001) and reduced overall survival (P < 0.05). In the multivariate analysis, the risk of death was associated with acute GVHD presence before CDI (hazard ratio [HR], 3.19; P = 0.009) and the need to switch to second-line treatment (HR, 4.83; P < 0.001). The efficacy of the initial CDI treatment affects survival and occurrence of immune-mediated GI-GVHD after allo-HCT. Therefore, agents with higher efficacy than metronidazole (vancomycin or fidaxomicin) should be administered as the first-line treatment.

Histologic Manifestations of Gastrointestinal Adenovirus Infection After Stem Cell Transplant.

Adenovirus can cause severe disease in hematopoietic stem cell transplant (HSCT) patients. Histopathologic features of this infection in gastrointestinal biopsies and their distinction from graft-versus-host disease (GVHD) have been incompletely studied. We retrospectively identified patients with gastrointestinal adenovirus infection. H&E-stained sections were reviewed and the histologic features were recorded. The extent of immunostaining was determined using a semiquantitative scale and a maximum number of positive cells per high-power field. Information regarding the clinical course and endoscopic findings were obtained from the electronic medical records. The study group included 32 HSCT patients. Most (81%) presented with diarrhea and detectable virus in the serum. Twenty patients had multiorgan involvement in the gastrointestinal tract, mostly in the duodenum (62%) and colon (56%). Characteristic features included apoptotic epithelial cells with nuclear disarray (84%) and tufted aggregates of degenerating epithelial cells (69%), the latter of which was more commonly seen in the study population more than a control group of HSCT patients with GI involvement by GVHD. Viral inclusions were limited to the superficial epithelium in 59% of samples, and the density of viral inclusions within biopsies was variable (grade 1: 40%, grade 2: 38%, and grade 3: 22%). Following therapy, 10 patients (30%) improved and 14 (42%) had progressive disease. Patients with disease progression were often older (64 vs. 36 years, P=0.01) with higher serologic viral loads, prior history of GVHD, multifocal involvement, and increased number and density of immunoreactive nuclei. Adenovirus infection elicits a spectrum of histologic changes that can simulate or occur in combination with gastrointestinal GVHD. Patients with progressive disease are more likely to have high viral loads and more extensive infection of the gastrointestinal tract.

Placenta-Derived Decidua Stromal Cells: A New Frontier in the Therapy of Acute Graft-Versus-Host Disease.

Acute graft-versus-host disease (GVHD) is a frequent and potentially life-threatening complication following allogeneic hematopoietic cell transplantation (HCT). Mesenchymal stromal cells (MSCs), rare precursors found in all body tissues, possess immunosuppressive properties and can inhibit alloreactivity both in vitro and in vivo. Two decades ago, we introduced bone marrow-derived (BM) MSCs as a novel therapy for acute GVHD. While some patients responded to BM-MSCs, the response was not universal. Commercially available BM-MSCs are now used for acute GVHD treatment in Canada, Japan, and New Zealand. The fetus is protected from the mother's immune system by the placenta, and our research found that placenta-derived decidua stromal cells (DSCs) offer a stronger immunosuppressive effect than other sources of stromal cells. Safety studies in rabbits, rats, mice, and humans have shown negligible or no side effects from BM-MSCs or DSCs. In a phase I/II trial for severe acute GVHD, we treated 21 patients (median age, 49 years; range 1.6-72 years) with severe biopsy-proven gastrointestinal acute GVHD. The median cell dose of DSCs was 1.2 × 106 (range 0.9-2.9) cells/kg body weight, with a median of 2 (range 1-6) infusions given 1 week apart. The cell viability of DSCs was 93% (range, 69%-100%), and the median cell passage number was 4 (range, 2-4). All patients responded, with a complete response of acute GVHD in 11 patients and partial response in 10 and 1-year survival of 81%. Randomized trials are needed to prove the superiority of DSCs compared to ruxolitinib and/or other novel immunosuppressive therapies.

R-Spondin1 protects gastric stem cells and mitigates gastric GVHD in allogeneic hematopoietic stem cell transplantation

AbstractGraft-versus-host disease (GVHD) is the major obstacle to performing allogeneic hematopoietic cell transplantation (allo-HCT). We and others have shown that intestinal stem cells are targeted in lower gastrointestinal GVHD. A leucine-rich repeat–containing G-protein coupled receptor 5 (Lgr5)–expressing gastric stem cells (GSCs) reside at the base of the gastric glands in mice. After experimental allo-HCT, Lgr5+ GSCs significantly decreased. Parietal cells, which underwent continuous renewal by GSCs, were injured in gastric GVHD, leading to failure of gastric acidification and aerobic bacterial overgrowth in the duodenum. Fate-mapping analysis demonstrated that administration of R-Spondin1 (R-Spo1) that binds to Lgr5 for 6 days in naïve mice significantly increased proliferating epithelial cells derived from Lgr5+ GSCs. R-Spo1 administered on days −3 to −1 and from days +1 to +3 of allo-HCT protected GSCs, leading to amelioration of gastric GVHD and restoration of gastric acidification, and suppression of aerobic bacterial overgrowth in the duodenum. In conclusion, Lgr5+ GSCs were targeted by gastric GVHD, resulting in disruption of the gastric homeostasis, whereas R-Spo1 protected Lgr5+ GSCs from GVHD and maintained homeostasis in the stomach.

Inflammatory Memory Restrains Intestinal Stem Cell (ISC) Regeneration after Allogeneic Stem Cell Transplantation (SCT)

Introduction: Intestinal stem cells (ISCs) are responsible for the remarkable ability to maintain intestinal epithelium homeostasis and regeneration throughout life. Inflammatory damage of ISCs underpins injury caused by graft-versus-host disease (GVHD), inflammatory bowel diseases (IBD) and immune check-point blocker mediated colitis. However, it remains unknown whether the ISCs that survive or tolerate inflammation are fully functional and can return to their full functionality after the resolution of ongoing inflammatory insults. Herein we investigated the consequences of inflammation from GVHD on Lgr5+ISCs in multiple well-defined clinically relevant models of gastro-intestinal acute graft-versus-host disease (GI GVHD). Methods: We utilized single cell RNA (scRNA) sequencing to assess transcriptomics, Assay of transposase-accessible chromatin sequencing (ATAC-seq) to assess epigenomics, and functional metabolomics of ISCs in clinically relevant in vivo models of GVHD after major histocompatibility complex (MHC)-disparate BALB/c→C57BL/6 (B6) and MHC matched minor mismatched C3H.sw→B6 models of allogeneic SCT. Ex vivo intestinal organoids cultures, mitochondrial, and functional biochemical assays were utilized to determine the biological relevance of the changes observed from the ‘omic’ analyses and further validated them in vivo by developing novel Lgr5 +ISC specific succinate dehydrogenase A (SDHA) knock-out mice. Results: We examined the transcriptomes of Lgr5+ISCs with scRNA-seq of the intestinal crypts harvested from MHC-disparate BALB/c→B6 recipients on day +7 after allogeneic SCT. Bioinformatic analyses demonstrated upregulation of interferon and inflammation response genes but significant downregulation of genes involved in mitochondrial function, its complexes including complex II (SDH), ATP metabolic process, OXPHOS, and cytoplasmic translation. To assess the impact of metabolic functional gene changes, we harvested and assessed ISCs in ex vivo organoid cultures from transplanted recipients in the absence of ongoing inflammation. The ISCs harvested from GVHD animals demonstrated significantly reduced regeneration, differentiation and oxygen consumption rates (OCRs) with no change in extracellular acidification rates (ECAR) by Seahorse. FACs analysis confirmed reduction in SDHA component of mitochondrial complex II in Lgr5 +ISCs, demonstrating mechanistic cause for reduction in OXPHOS in ISCs. We next generated and utilized Lgr5 + ISC-specific SDHA KO mice as GVHD recipients and found that demonstrated significantly greater mortality when compared to the WT littermate recipients (P&amp;lt;0.01). Biochemical analyses demonstrated increased levels of succinate, a metabolic intermediary with known epigenetic and inflammatory functions, in ISCs harvested from GVHD animals. We therefore hypothesized that the reduction in ability to form functional organoids ex vivo, in the absence of ongoing inflammation, by the ISCs harvested from GVHD hosts is because of the inflammation epigenetic reprogramming induced by succinate mediated changes in DNA methylation (5-mC) of the ISCs in GVHD recipients. Consistent with the hypothesis greater DNA methylation, with alteration of epigenome by ATAC-seq was observed in ISCs sorted from GVHD recipients. Integrative analyses demonstrated correlation between epigenomic changes and transcriptomic changes. Finally, we analyzed whether the inflammation epigenomic induced changes in the GVHD ISCs with serial ex vivo organoid cultures and in vivo by transfer into secondary hosts. Both ex vivo and in vivo studies demonstrated poor regeneration from GVHD ISCs demonstrating retention of maladaptive memory in ISCs following their exposure to inflammation during GVHD. Conclusions: GI GVHD induced inflammation causes not only quantitative loss of ISCs but also induced qualitative changes in the surviving ISCs. Inflammation induced OXPHOS deficiency in Lgr5 + ISCs leads to accumulation of succinate that reprograms the epigenome and restrains their subsequent regeneration potential.

Intestinal Geographical Pattern of Gut Microbiome and Metabolites Identifies Novel Regulators of Graft-Versus-Host Disease

Introduction: The severity of T cell-mediated gastrointestinal graft-versus-host disease (GI-GVHD) correlates with a decrease in the gut microbiome diversity, loss of obligate anaerobic bacteria and alterations in levels of salutary metabolites like short chain fatty acids (SCFAs). However, it is unknown whether the intestinal anatomic geography of the microbiome and its metabolites impacts GI GVHD severity. Herein, we investigate the pattern of the gut microbiome and metabolites from four distinct anatomical sites of intestines after allogeneic stem cell transplant (allo-SCT) via shotgun metagenomics and untargeted mass-spectrometry. Methods: To determine the composition of the gut microbiome and gut metabolites of mice after allo-SCT, we utilized contemporaneous analyses of shotgun metagenomic sequencing and untargeted mass spectrometry on the gut contents of mice 7 or 21 days after major histocompatibility complex (MHC)-disparate BALB/c→C57BL/6 (B6) allogeneic SCT. We investigated the microbial community composition and gut metabolites from four distinct gut locations (terminal ileum, cecum, transverse colon, descending colon) and performed integrative analysis to assess for novel pathways that may regulate GVHD biology. For shotgun metagenomics analyses, reads were depth normalized with BBnorm v.38.96 and assembled per-sample with MEGAHIT v.1.2.9. Prodigal v.2.6.3 was used to find genes in each bin. KofamScan v.1.3.0 was used to assign KEGG ortholog IDs (KO) to translated genes predicted by Prodigal. Community composition profiles were produced with Kraken2 v.2.1.2 and Bracken v.2.6.1. Functional profiles for each sample were determined with the Kraken community composition profiles and Humann3 v.3.0.0, with a GTDB database built with Struo2. Results: Consistent with previous reports from direct stool analyses, our data demonstrated that after allo-SCT, gut microbial diversity is reduced, relative abundance of obligate anaerobes decreases, and relative abundance of facultative anaerobes increases at all the intestinal anatomic sites, but the difference was maximal in ileo-cecal contents. Furthermore, metabolomic analyses reveal a decrease in previously described metabolites such as, SCFAs, bile acids and indoles at these sites. The distinct patterns of change between control and allogeneic samples depended not only on the gut location probed but also correlated with timepoint after transplant (7 days or 21 days) highlighting the importance of probing different gut locations and timepoints. The largest driver of differences between sample groups was (1) gut location (2) time after transplant (3) transplant type. Integrative analyses demonstrated loss of microbial functional genes related to butyrate processing following allo-SCT complementing previous studies that demonstrated reduction in butyrate levels. Further bioinformatic analyses identified novel microbial functional genes and metabolites that may have biological implications. Analyses revealed that after allo-SCT, phenyllactic acid (PLA) levels increased in the transverse colon and distal colon (fig.1). PLA is an anti-microbial compound and has been shown to have immune modulatory properties. Administration of PLA in drinking water of mice after allo-SCT worsened GVHD severity and increased mortality indicating PLA may play a role in inducing dysbiosis and worsening GVHD severity. Conclusion: Contemporaneous analyses of shotgun metagenomic and untargeted mass spectrometry of gut contents after allo-SCT reveal changes in microbial composition and metabolites consistent with previous data from analyses from stool. However, our analysis demonstrates distinct patterns of key metabolites and microbes in between syngeneic and allogeneic depending on gut location and time after transplant. Furthermore, our analyses have identified novel functional genes and metabolites that are implicated in GVHD severity.

Inb-100: A Pilot Study of Donor Derived, Ex-Vivo Expanded/Activated Gamma-Delta T Cell (EAGD) Infusion Following Haploidentical Hematopoietic Stem Cell Transplantation and Post-Transplant Cyclophosphamide (PTCy)

Background: Gamma-delta (gd) T cells are MHC unrestricted lymphocytes that recognize and lyse malignant cells in allogeneic settings. Although haploidentical transplant with PTCy has reduced the risk of graft-versus-host disease (GvHD); the incidence of relapse remains up to 50% at year 1. Early post-transplant infusion of haploidentical EAGD cells may decrease relapse risk through a graft-versus-leukemia (GvL) effect without severe GvHD. We present updated clinical and correlative data from our Phase I trial using our recommended phase 2 dose (RP2D). Methods: Adults with newly diagnosed or relapsed ALL, CML, AML undergoing first haploidentical transplant with reduced-intensity flu/cy/TBI conditioning received EAGD intravenously within 7 days of neutrophil engraftment. Peripheral blood was collected at EAGD infusion and monthly through day 90, with additional collections every 6 months through 1 year. Primary endpoints include dose-limiting toxicities (DLT), grade (G) 3-4 adverse events including GvHD with secondary endpoints of relapse and overall survival. Biologic parameters included multiparameter flow cytometric immunophenotyping and additional serum cytokine analysis using the Olink ® 48 target panel. Results: 14 subjects were enrolled with 4 treated at Dose Level (DL) 1 of 1 x 10 6 EAGD/kg and 6 subjects treated at the RP2D of DL2 (3 x 10 6 EAGD/kg). Untreated subjects included: One screen failure, a manufacturing failure, one subject who died prior to dosing, and one subject who received an out of study specification product. Treated subjects were 60% male, median age of 68, and primarily AML subjects in CR1. RP2D was defined by an acceptable toxicity profile, no DLT's, prolonged RFS and elevated gd levels. Peripheral lymphodepletion persisted through the first 100 days post-BMT followed by T cell recovery from a CD45+CD27-effector phenotype to central and effector memory phenotype. Notably, DL2 showed significant increases in gd T cell count recovery at days 60, 100 and 180 post-BMT vs. DL1 and historically untreated Haplo/PTCy patients, p = &amp;lt;0.05. NK cells remained within the low normal range and &amp;lt;3% of circulating T cells were Tregs. Preliminary serum cytokine analysis revealed elevated IL-6 and IL-15 in serum post- transplant prior to EAGD infusion, but dropped to normal median levels at day 60, 20-30 days after EAGD infusion. One subject received intermittent hypomethylating therapy for emergence of recipient chimerism. Treatment emergent AE's included transient WBC and ANC decreases (100% each), platelet count decrease and anemia (88.9% each) and maculopapular rash, hypomagnesemia and blood creatine increased (55.6% each). EAGD related toxicities were primarily G1-2, included G1-2 skin acute GvHD and gastrointestinal GvHD, and one case of G3 platelet count decrease. Treatment related serious AE's of G3 nausea and G2 rash were reported. No DLTs, neurotoxicity, cytokine release syndrome or treatment related deaths were reported. Conclusions: Post-BMT infusion of donor-derived, EAGD cells has demonstrated manageable safety with no infusional toxicity or ≥G3 acute GvHD or extensive chronic GvHD. Durable relapse free survival at a maximum of &amp;gt;3 years in subjects with poor risk cytogenetics and adverse clinical risk factors combined with ongoing homeostatic reconstitution of increasing gd T cell effectors post-BMT suggests this therapy may be an effective measure in mitigating relapse after HaploBMT. NCT03533816

Clinical Characteristics, Management, and Allogeneic Hematopoietic Cell Transplantation of Patients with Toll-like Receptor 8 Gain-of-Function Mutations

Toll-like receptor 8 gain-of-function (TLR8 GOF) is a recently described inborn error of immunity due to germline or somatic mutations in the TLR8 gene and characterized by severe neutropenia, infections, lymphoproliferation, humoral immune defects, and in some cases, bone marrow failure. Treatment of TLR8 GOF has been challenging to date, and guidance for optimal therapy is lacking. We describe the clinical characteristics and management of 10 patients with TLR8 GOF variants, including the first female patient and further clinical outcome on the 6 patients initially reported with this disease with a focus on the role of allogeneic hematopoietic cell transplantation (HCT). All but one patient were male ranging from 0.5 months to 28 years at age of symptom onset. Eight patients had somatic mutations with variant allele frequencies of 7-26%; two patients had germline mutations. The 9 male patients all had severe neutropenia and varying degrees of anemia and/or thrombocytopenia. Oral ulcers, recurrent infections, hepatosplenomegaly, and hypogammaglobulinemia were common. Immune phenotype was variable, ranging from severe lymphopenia to marked T cell lymphoproliferation. Several patients had an inverted CD4:CD8 T cell ratio and skewing of T cell subsets to the terminal effector memory phenotype. Increased double-negative T cells were also observed. All patients had low class switched memory B cells. Bone marrows were hypo- to hypercellular with marked myeloid hypoplasia, and in most cases, with increased T cells, large granular lymphocytes, and/or lymphoid aggregates. Neutropenia was initially responsive to granulocyte colony stimulating factor (GCSF) +/- steroids in several patients but ultimately become refractory to therapy. Immunomodulatory agents and chemotherapy were unsuccessful. Six patients underwent allogeneic HCT; two patients died at 8 and 18 years without definitive therapy; and one patient was lost to follow-up. The female patient had germline disease and presented with pure red blood cell aplasia at 2 weeks of life and is scheduled to undergo HCT. Transplant data were available for 5 of 6 patients who underwent HCT. Age at transplant was 3 to 21 years. One patient who received a graft from a 7/8 HLA-matched unrelated donor developed severe veno-occlusive disease and secondary graft failure. The patient underwent a second transplant and ultimately died. Another patient received a matched sibling donor HCT while critically ill from Candida lusitaniae and mucormycosis infection with associated multisystem organ failure. The patient engrafted with 100% donor chimerism but died on day +15 post-HCT. The remaining 3 patients are alive and well 8 months to 2 years post-HCT. Two patients received grafts from HLA-matched donors (1 sibling and 1 unrelated) with reduced intensity conditioning and cyclophosphamide, mycophenolate mofetil and vorinostat for graft-versus-host disease (GVHD) prophylaxis. The third patient underwent a haploidentical HCT with myeloablative conditioning and post-transplant cyclophosphamide. Post-HCT complications included acute skin and/or gastrointestinal GVHD that was responsive to therapy in all patients, and severe lung GVHD in one patient. There were no major infectious complications. All three patients had full donor chimerism in myeloid and T cells with complete resolution of disease phenotype at last follow-up. The patient for whom detailed transplant data is not available received a haploidentical HCT. He developed mixed myeloid chimerism for which he received multiple donor lymphocyte infusions and was alive at last contact &amp;gt;3 years post-HCT. TLR8 GOF should be considered in male and female patients with severe, unexplained neutropenia refractory to GCSF, and particular care should be taken when investigating for mutations given the disease-causing variant allele frequencies as low as 7%. Neutropenia and immune dysregulation were refractory to all therapies in our cohort, and TLR8 GOF was fatal in two patients who did not receive definitive therapy. Allogeneic HCT is curative and should be considered without delay for medically suitable patients.

Highly Multiplexed Immunohistochemistry Can Predict Steroid Refractory Gastrointestinal (GI) Acute Gvhd at the Time of Endoscopy

Background: For patients with high-risk hematologic disease, hematopoietic cell transplant (HCT) often represents the only option for cure. However, HCT is fraught with complications, leading to morbidity and mortality. Acute GVHD (aGVHD), specifically steroid refractory (SR) GI aGVHD, is a major cause of early mortality. Two issues with SR GI aGVHD predominate: (1) We do not understand the mechanisms differentiating SR vs steroid sensitive (SS) GI aGVHD; (2) We do not have an accurate test to predict SR vs SS GI aGVHD at the time of endoscopic evaluation. Here, we used state-of-the-art multiplexed immunohistochemistry (IHC) to interrogate immune mediators of GI aGVHD, and to develop a robust assay capable of predicting SR vs SS disease from standard GI biopsies. Methods: We analyzed all HCT patients at Boston Children's Hospital (BCH) from 1/2014-12/2021 (n=551) who received a first lower GI endoscopy for suspected GI aGVHD (n=109). We tracked their SS vs SR status and their Overall Survival (OS) through 2 yr post-HCT. Classic histology was performed and in addition, each sigmoid colon biopsy (formalin-fixed) was analyzed with a series of novel multiplexed IHC panels (interrogating Pan-CK, DAPI, CD3, CD8, CD20, FoxP3, GITR, CD68, Granzymes B and K, CD103, Ki67, ECP, NCR1, BCMA, CD11c, and MPO, with quantitative image analysis performed using HALO Indica Labs Hyperplex module). Density/proportions of multiple cellular infiltrates and their ratios were assessed. Receiver Operator Characteristic (ROC) and K-nearest neighbors (KNN) classification were used to distinguish SS from SR aGVHD. Results: Of 109 biopsies, 60 had histologic evidence of lower GI aGVHD inflammation, 12 had other GI inflammation (eg infections, drug effects) and were not further analyzed, and 15 did not have inflammation and therefore were not diagnosed with histologic aGVHD (termed ‘No Inflammation’ (NI). In an additional 22 biopsies, histology was characterized as “Rare Apoptosis” (RA), a diagnosis for which there is no national consensus about severity or the need for steroid treatment. At BCH, the vast majority of patients with RA on endoscopy were treated as not having aGVHD (with only 3/22 receiving steroids, and all 3 being SS). We tracked OS and IHC characteristics for SS aGVHD, SR aGVHD, NI and RA. Fig 1 shows 2 yr OS, which confirms the inferior OS of SR vs SS aGVHD (SR: 58% vs SS: 93% p &amp;lt;0.0001). Importantly, Fig 1 demonstrates that even without steroids, the OS of RA was equivalent to no GI aGVHD (NI: 89% vs RA: 86% p = 0.74). Multiplexed IHC (performed on 68 biopsies from sequential cases collected from 1/2018-12/2021) was associated with several novel findings ( Fig 2): (1) By IHC, RA had fewer immune infiltrates vs aGVHD and NI: CD3 density: aGVHD (both SS and SR combined): 782 ± 133, NI: 386 ± 37, RA: 165 ± 26, p = 9.5x10 -5; CD8 density: aGVHD: 381 ± 83, NI: 123 ± 24, RA: 42 ± 26, p&amp;lt;0.001; CD4 conventional T cells (CD4 Tconv): aGVHD: 359 ± 47, NI: 237 ± 23, RA: 114 ± 19, p&amp;lt;0.001. Ratios of immune infiltrates also distinguished the 3 subtypes: the ratio of CD8:CD4 Tconv: aGVHD: 0.95 ± 0.14; NI: 0.59 ± 0.10 RA: 0.44 ± 0.29 p = 0.013; CD8:CD68: aGVHD: 0.35 ± 0.03, NI: 0.26 ± 0.04 RA: 0.11 ± 0.02, p&amp;lt;0.001. CD8:Epithelial cells (epi): aGVHD: 0.153 ± 0.04; NI: 0.045 ± 0.01; RA: 0.014 ± 0.003, p&amp;lt;0.001. These results underscore the immunologically benign nature of RA, predicting its superior OS. Importantly, multiplexed IHC could distinguish SR vs SS aGVHD at the time of endoscopy, a finding with major clinical implications. For example, amongst other distinguishing IHC characteristics, the ratio of CD8:CD4Tconv: SR: 1.3 ± 0.22 vs SS: 0.68 ± 0.15 p&amp;lt; 0.01; CD8:epi: SR: 0.220 ± 0.008 vs SS: 0.106 ± 0.042 p = 0.04). A KNN-based classifier identified thresholds with high accuracy (81%), specificity (69%), sensitivity (93%); AUC = 0.852 for SS vs SR aGVHD ( Fig 2). Conclusions: We used state-of-the-art multiplexed IHC to interrogate the immune cells associated with GI aGVHD, with 2 major findings: We find RA associated with superior OS without steroid treatment, and with minimal immune infiltration, confirming its benign nature. We also created a novel multiplexed IHC assay capable of accurately classifying SR vs SS aGVHD from standard-of-care biopsy specimens. If confirmed in a multicenter setting, this would be a major advance, allowing physicians to tailor appropriate therapies to patients at highest risk of developing SR GI aGVHD at the time of initial endoscopic evaluation.

Rational Modification of Human Gut Microbiome and Metabolites By Dietary Resistant Starch in Allogeneic Hematopoietic Stem Cell Transplantation: A Feasibility Study

Allogeneic hematopoietic stem cell transplantation (allo-HCT) is limited by acute graft versus host disease (GVHD). GVHD is the principal cause of non-relapse mortality (NRM) and a major cause of morbidity after allo-HCT. Recent published experimental data demonstrated that altering the microbiome-metabolite axis regulates acute GVHD severity. Specifically, short chain fatty acid (SCFA) butyrate was significantly decreased in the intestinal epithelial cells (IECs) of mice experiencing GVHD, while restoring butyrate levels by increasing intestinal butyrate-producing bacteria, reduced experimental acute GI GVHD severity and mortality. Prebiotics usually refer to indigestible carbohydrates that are metabolized by the intestinal microbiota to produce microbial metabolites, such as SCFAs, that serve as nutrients for IECs. Administration of defined quantities of resistant potato starch (RPS), as a prebiotic, to normal healthy human volunteers promoted increase in butyrogenic bacteria and increased intestinal levels of SCFA butyrate to a greater extent than other commercially available resistant starch preparations tested. These data form the rationale for this study and led to prospectively study the clinical feasibility and impact of the administration of RPS as a prebiotic dietary intervention on the intestinal microbiota and its dependent metabolites in allo-HCT recipients. Adults undergoing human leukocyte antigen-matched, related-donor myeloablative allo-HCT were recruited and received RPS orally daily from day -7 to day 100 after allo-HCT. Stool samples were collected in the OMNIgene-Gut® (DNA Genotek) collection kit. Fecal samples were subjected to 16S rRNA gene sequencing to determine microbiota composition and quantification of fecal SCFAs was performed by liquid chromatography. Blood specimens were collected using standardized protocols. The effect of RPS on plasma SCFAs and &amp;gt; 200 other metabolites was longitudinally assessed using targeted and global metabolomic analyses. Mass spectrometry was utilized for quantification of plasma metabolites. The primary objective was to assess the feasibilty and test the effect of RPS on the structure of the patients' intestinal microbiome. Metrics for the feasibility of this dietary intervention were prespecified by setting a target for ≥ 60 % of patients to adhere to ≥ 70% of scheduled doses. We hypothesized that RPS would be feasible and increase stool butyrate levels as a byproduct of microbial metabolism. Key secondary objectives were to longitudinally evaluate plasma metabolites in recipients of RPS compared to historical controls as well as assess tolerability of RPS in allo-HCT recipients. Ten subjects were enrolled. The primary endpoint was met. Feasibility exceeded the preset goal of ≥ 70% adherence to scheduled dosages in ≥ 60 % of patients as 8 of the 10 patients (80%) received ≥ 70 % of scheduled doses. Intestinal butyrate levels were significantly higher while participants were on RPS as compared to when they were not on RPS (p &amp;lt; 0.0001) (fig. 1A). We observed longitudinal changes in plasma metabolites post allo-HCT compared to baseline independent of whether allo-HCT recipients received RPS (p &amp;lt; 0.0001) (fig. 1B). In RPS recipients, the dominant plasma metabolites were, however, much more stable across timepoints when compared to historic controls suggesting a greater equilibrium in their production and consumption (fig. 1C). The median age of participants was 57 years (range 52-62 years). All subjects received standard GVHD prophylaxis with tacrolimus and methotrexate as well as standard antibiotic prophylaxis with levaquin, and standard neutropenic fever treatment with IV cefepime (90%) or IV vancomycin, No adverse effects/toxicities attributed to RPS were observed and longitudinal specimens were collected successfully (table 1). This study showed that a dietary intervention using RPS in allo-HCT recipients is feasible, and was able to rationally alter the salutary intestinal microbial metabolite SCFA butyrate, despite the utilization of several HCT related medications, including antibiotics. These data demonstrate translation of fundamental discoveries in mouse allo-HCT studies, to testing RPS in healthy humans, to showing that RPS is a feasible microbiome-modifying intervention in allo-HCT recipients, and that a prebiotic strategy can now be applied and tested to mitigate acute GVHD in allo-HCT.

Opsoclonus-Myoclonus Syndrome and Acquired Von Willebrand Syndrome As Manifestations of Graft-Versus-Host Disease

Background: Opsoclonus-myoclonus syndrome (OMS), known as dancing eye syndrome, is a rare neurological disorder characterised by a triad of opsoclonus (rapid, involuntary eye movements), myoclonus (uncontrolled muscle twitches) and ataxia. The syndrome is usually seen in children, often associated with neuroblastoma, and is extremely rare in adults. The pathogenesis is thought to be immune in nature and aetiology is variable although most cases are associated with solid tumors or after an infection. Treatment involves a combination of managing the underlying condition and immunosuppression. Acquired von Willebrand disease is a rare acquired bleeding disorder with a range of different aetiologies including lymphoproliferative, myeloproliferative, cardiovascular and autoimmune disorders. The pathogenic mechanism, in most cases is due to increased plasma clearance of von Willebrand factor (vWF) caused by such mechanisms as antibodies, cell adsorption, shear stress or increased proteolysis. Therapeutic approaches focus on prevention of bleeding through supportive treatment, and management of the underlying cause. To the best of our knowledge, this is the first case of opsoclonus-myoclonus syndrome with concurrent acquired von Willebrand disease as manifestations of graft versus host disease. Case: Here we present the case of a 47-year-old man presenting with opsoclonus myoclonus syndrome and concurrent acquired von Willebrand disease as manifestations of graft versus host disease. The patient had a background of relapsed myeloid sarcoma after haploidentical allogeneic haematopoietic stem cell transplant with subsequent chemotherapy to achieve full remission. He received donor lymphocyte infusion as consolidation therapy to enhance the graft-versus leukaemia effect. Six weeks following donor T cell infusion of 1 x 10^6 CD3 cells/kg, he presented with initial features suggestive of graft versus host disease (GVHD) of the digestive tract and subsequently developed neurological manifestations in the form of rapid, repetitive, involuntary and multidirectional eye movements; tremors and ataxia. Further tests prior to planned invasive testing revealed an additional new diagnosis of acquired von Willebrand disease, requiring supportive von Willebrand factor concentrate infusion. Multiple extensive investigations including cerebral MRI imaging, cerebrospinal fluid analysis and paraneoplastic antibodies were all negative for associated malignancy, infection, other autoimmune disorder or disease relapse. Skin, bone marrow examination and cross-sectional imaging confirmed complete remission with complete donor chimerism in all blood cell lineages. The patient commenced steroid immunosuppression for treatment of gastrointestinal GVHD with complete resolution of all symptoms and clinical parameters. Conclusion: We highlight a rare case of central nervous system involvement with graft versus host disease following cellular therapy, manifesting as the rare opsoclonus-myoclonus syndrome with associated acquired von Willebrand disease. The proposed pathological mechanism is the activation of host tissue antigen-specific T and B cells, resulting in autoantibody and cytokine-mediated effects upon neuronal tissue and coagulation proteins. Use of glucocorticoids in this case to suppress auto-antibody formation, block transcription of pro-inflammatory genes and induce lymphocyte depletion was rapidly effective and provided durable remission of symptoms, without recurrence on cessation of immunosuppression. This case highlights the need for close clinical and biochemical monitoring of patients during the risk period following allogeneic cellular therapy, for serious allo-immune manifestations not fitting classical descriptions of GVHD.

Budesonide Added to Post-Transplantation Cyclophosphamide, Tacrolimus and Mycophenolate Mofetil for Prevention of Graft-Versus-Host Disease

Background: Topically active corticosteroids have been shown to effectively treat inflammatory bowel disease with a systemic steroid-sparing effect. However, previous studies have not demonstrated their efficacy in preventing graft-versus-host disease (GVHD) when combined with the standard GVHD prophylaxis regimen consisting of calcineurin inhibitors and methotrexate. We hypothesized that the addition of orally administered ileal-controlled-release budesonide to the post-transplant cyclophosphamide (PTCy)-based regimen could effectively prevent severe acute gastrointestinal GVHD. In this study, we present outcomes of the first 43 consecutively treated patients (pts) with the PTCy-tacrolimus (T), mycophenolate mofetil (M) and budesonide (B) (PTCy-TMB) as a GVHD prophylaxis regimen after allogeneic stem cell transplantation (alloSCT). Methods: Pts with hematologic malignancies received their first alloSCT at our institution between 05/2020-04/2023. Thirty one pts received conditioning with fludarabine (160 mg/m 2) + melphalan (100-140 mg/m 2) + TBI (2-4Gy) or thiotepa 5 mg/kg. Twelve pts received fludarabine (150mg/m 2) + cyclophosphamide (29mg/kg) and TBI 4Gy ± ATG (4.5 mg/kg). GVHD prophylaxis consisted of Cy (50 mg/kg on D+3 and +4), T (trough goal 6-10 ng/mL), M (15mg/kg TID) and budesonide (3 mg TID) starting on D+5 and continued until D+90 followed by a weekly taper if no acute GVHD (aGVHD) occurred. T was continued for at least 6 months followed by a taper, unless otherwise indicated. Results: The median age was 50 years (range 22-73), 39 pts had hematologic malignancies including AML (N=14), ALL (N=11) and NHL (N=8). Seven pts (16%) classified as high/very-high DRI. The median of HCT-CI was 4 (range 0-7). Donors were haploidentical (N=32), MSD (N=8), MUD (N=1), MMSD (1) and MMUD (N=1). PBSCs were used in 28 pts (65%). Thirty pts (70%) received RIC regimens. Median follow-up was 407 days (range 91-996 days). At day 100, the cumulative incidence (CI) of grade 2 aGVHD was 7.0% (95%CI 1.8-17.2), while none developed grade 3-4 aGVHD (Figure 1). One patient had severe chronic GVHD (bronchiolitis obliterans) with CI at 2 years of 3.2% (95%CI 0-14.0). The CI of any systemic steroid use at D+100 and D+180 was 30.2% (95%CI 17.4-44.1) and 44.8% (95%CI 28.9-59.5), respectively. Among the pts who received treatment for aGVHD, the median cumulative systemic steroid exposure (in prednisone equivalents) was 1,318 mg (IQR 623-3,775) at D+100 and 1,325 mg (IQR 525-1,640) at D+180, with a median treatment duration of 40 days (IQR 25-110). At 1 year, the OS, PFS, GRFS, relapse and NRM were 79.6% (95%CI 61.4-89.9), 69.8% (95%CI 51.6-82.3), 66.6% (95%CI 48.4-79.8), 15.3% (95%CI 6.2-28.2) and 14.9% (95%CI 5.3-29.1), respectively. The 1-year CI of newly diagnosed/worsening hyperglycemia and Cushing syndrome was 18.2% (95%CI 7.9-31.9) and 5.0% (95%CI 0.9-14.2), respectively. 3 pts (7%) required insulin treatment. The median absolute lymphocyte count (ALC) at day 30, 90 and 180 was 200/mcL, 300/mcL and 500/mcL, respectively, with no differences in the median ALC at D+180 among pts with and without GVHD. Median cell numbers of CD3 +, CD3 +/CD4 +, CD3 +/CD8 +, CD19 + and CD56 + at 1, 3, 6 months post-transplant were D+30 - 63/mcL, 18/mcL, 34/mcL, 1/mcL, 87/mcL, D+90 - 111/mcL, 64/mcL, 34/mcL, 9/mcL, 216/mcL and D+180 - 269/mcL , 78/mcL , 126/mcL , 7/mcL, 189/mcL, respectively. After 10/2022 pts were tested routinely for Reg3α and ST2 (baseline and retested if developed any signs/symptoms of aGVHD). Among 18 tested pts at baseline, the median number (range) of Reg3α and ST2 at baseline was 28 ng/mL (16-90) and 23,308 pg/mL (4,678-56,226), respectively. Three low-risk pts developed grade 1 aGVHD (N=2) and grade 2 aGVHD (N=1). Fourteen pts with GI sign/symptoms after transplant showed median number (range) of Reg3α and ST2 of 78 ng/mL (16-837) and 34,904 pg/mL (13,329-160,000), respectively. Only one pt with high-risk aGVHD algorithm developed grade 2 aGVHD (Figure 1). Conclusions: The addition of budesonide to PTCy-based GVHD prophylaxis regimen (PTCy-TMB) is feasible and appears to be effective in preventing severe GI aGVHD, without compromising transplant outcomes and immunologic reconstitution.

Bacterial and Bacteriophage Consortia Are Associated with Protective Intestinal Immuno-Modulatory Metabolites in Allogeneic Stem Cell Transplantation Patients

The human microbiome is a predictor of clinical outcome in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-SCT). Besides bacteria, fungi and viruses as well as intestinal microbiota-derived metabolites are involved. However, it is still unclear how dynamic shifts in these three kingdoms contribute to the production of intestinal metabolites, how metabolites are impacted by graft-versus-host disease (GvHD) or antibiotics and whether they are associated with clinical outcome. We report the two-year follow-up of a prospective, observational, longitudinal cohort of allo-SCT patients (n=78) at two German transplantation centers that combined three-kingdom (bacteria, fungi, viruses) analysis of intestinal microbial communities with targeted metabolomics ( Figure 1). Using Multi-omics factor analysis (MOFA), we uncovered a functional microbiome signature of bacteria from the Lachnospiraceae and Oscillospiraceae families and their associated bacteriophages, which correlated with the production of immuno-modulatory metabolites (IMMs) including short-chain fatty acids (SCFAs), branched-chain fatty acids (BCFA), metabolites associated with induction of type-I IFN signaling (IIMs) and immuno-modulatory secondary bile acids ( Figure 2). We established an Immuno-modulatory Metabolite Risk Index (IMM-RI) consisting of five index IMMs, which was associated with improved survival, less transplant-related mortality and reduced relapse rate. Onset of gastrointestinal GvHD and exposure to antibiotics significantly impacted intestinal levels of protective IMMs. Using whole shotgun metagenomic sequencing, we observed that in IMM-RI low-risk patients, sustained production of protective IMMs was associated with a high abundance of microbial SCFA biosynthesis pathways, specifically butyric acid via butyryl-CoA:acetate CoA-transferase (BCoAT). Through genome assembly from viral metagenomic sequencing data, we detected two distinct bacteriophages which encoded BCoAT as an auxiliary metabolic gene. They were more abundant in IMM-RI low-risk patients and positively correlated with intestinal butyric acid levels, suggesting that these bacteriophages may modulate bacterial metabolite biosynthesis. Our study identifies a specific microbiome signature associated with protective IMMs that could improve fecal microbiota transplantation (FMT) donor selection and provides a rationale for the development of engineered metabolite-producing consortia and defined metabolite combination drugs as novel microbiome-based therapies for cancer patients.

Spatial transcriptomics reveals distinct tissue niches linked with steroid responsiveness in acute gastrointestinal GVHD

AbstractSevere acute graft-versus-host disease (aGVHD) is associated with significant mortality and morbidity, especially in steroid-resistant (SR) cases. Spatial transcriptomic technology can elucidate tissue-based interactions in vivo and possibly identify predictors of treatment response. Tissue sections from 32 treatment-naïve patients with biopsy-confirmed lower gastrointestinal (GI) aGVHD were obtained. The GeoMx digital spatial profiler was used to capture transcriptome profiles of >18 000 genes from different foci of immune infiltrates, colonic epithelium, and vascular endothelium. Each tissue compartment sampled showed 2 distinct clusters that were analyzed for differential expression and spatially resolved correlation of gene signatures. Classic cell-mediated immunity signatures, normal differentiated epithelial cells, and inflamed vasculature dominated foci sampled from steroid-sensitive cases. In contrast, a neutrophil predominant noncanonical inflammation with regenerative epithelial cells and some indication of angiogenic endothelial response was overrepresented in areas from SR cases. Evaluation of potential prognostic biomarkers identified ubiquitin specific peptidase 17–like (USP17L) family of genes as being differentially expressed in immune cells from patients with worsened survival. In summary, we demonstrate distinct tissue niches with unique gene expression signatures within lower GI tissue from patients with aGVHD and provide evidence of a potential prognostic biomarker.

Stability assessment and content determination test of oral liquid forms of beclomethasone dipropionate prepared in hospital pharmacy

ObjectivesOral beclomethasone dipropionate (BDP) is known for its use as a therapeutic medicine for gastrointestinal graft-versus-host disease (GI-GVHD). Despite growing demand for oral BDP formulation, no commercial forms have yet...

Impact of the histologic grade of acute gastrointestinal graft-versus-host disease on outcomes in pediatric patients treated with allogeneic hematopoietic stem cell transplantation.

Acute gastrointestinal graft-versus-host disease (GVHD) is a common life-threatening complication after hematopoietic stem cell transplantation (HCT). We aimed to investigate outcomes according to the clinical, endoscopic, and histologic severity of gastrointestinal GVHD in pediatric patients treated with allogeneic HCT. This retrospective cohort study included pediatric patients who underwent sufficient endoscopic and histopathologic evaluation for clinically suspected acute gastrointestinal GVHD between 2010 and 2020. Fifty-one patients were included (male proportion, 68.6% [35/51]; median age at HCT, 6.4 years). When the patients were classified according to the histologic severity of gastrointestinal GVHD, the severe group had an earlier onset of GVHD symptoms and a higher proportion of patients with severe clinical gastrointestinal GVHD than the mild-to-moderate and "absent" groups. In Cox proportional hazards regression analysis, the groups with more severe clinical and histologic gastrointestinal GVHD showed a higher risk of non-relapse mortality (NRM). The 5-year overall survival (OS) rates were 58.3 and 36.4% in the mild-to-moderate and histologic gastrointestinal GVHD groups, respectively (p = 0.0384). Patients with higher clinical and histologic grades of gastrointestinal GVHD showed higher cumulative incidence of NRM. Our results demonstrated that histologic severity of gastrointestinal GVHD is a relevant factor affecting OS and NRM, and patients with mild-to-moderate or severe histologic gastrointestinal GVHD have worse outcomes than patients without histologic GVHD. These findings support the importance of assessing the histologic grade in the diagnostic evaluation of patients with clinical gastrointestinal GVHD.