Intestinal GVHD Research Articles

Vedolizumab for the prevention of intestinal acute GVHD after allogeneic hematopoietic stem cell transplantation: a randomized phase 3 trial

Acute graft-versus-host disease (aGVHD) of the lower gastrointestinal (GI) tract is a major cause of morbidity and mortality in patients receiving allogeneic hematopoietic stem cell transplantation (allo-HSCT). Vedolizumab is a gut-selective anti-α4β7 integrin monoclonal antibody that reduces gut inflammation by inhibiting migration of GI-homing T lymphocytes. The efficacy and safety of vedolizumab added to standard GVHD prophylaxis (calcineurin inhibitor plus methotrexate/mycophenolate mofetil) was evaluated for prevention of lower-GI aGVHD after unrelated donor allo-HSCT in a randomized, double-blind, placebo-controlled phase 3 trial. Enrollment closed early during the COVID-19 pandemic with 343 patients randomized (n = 174 vedolizumab, n = 169 placebo), and 333 received ≥1 intravenous dose of 300 mg vedolizumab (n = 168) or placebo (n = 165) and underwent allo-HSCT. The primary end point was met; Kaplan–Meier (95% confidence interval) estimated rates of lower-GI aGVHD-free survival by day +180 after allo-HSCT were 85.5% (79.2–90.1) with vedolizumab versus 70.9% (63.2–77.2) with placebo (hazard ratio, 0.45; 95% confidence interval, 0.27–0.73; P < 0.001). For the 5 key secondary efficacy end points analyzed by day +180 after allo-HSCT, rates of lower-GI aGVHD-free and relapse-free survival and grade C–D aGVHD-free survival were significantly higher with vedolizumab versus placebo. No significant treatment differences were found for the other key secondary end points of non-relapse mortality, overall survival and grade B–D aGVHD-free survival, respectively. Incidence of treatment-related serious adverse events analyzed in patients receiving ≥1 dose of study treatment (n = 334) was 6.5% (n = 11 of 169) vedolizumab versus 8.5% (n = 14 of 165) placebo. When added to standard calcineurin inhibitor-based GVHD prevention, lower-GI aGVHD-free survival was significantly higher with vedolizumab versus placebo. ClinicalTrials.gov identifier: NCT03657160.

Mesenchymal stromal/stem cells promote intestinal epithelium regeneration after chemotherapy-induced damage

BackgroundAllogeneic hematopoietic stem cell transplantation (HSCT) is a curative treatment for leukemia and a range of non-malignant disorders. The success of the therapy is hampered by occurrence of acute graft-versus-host disease (aGvHD); an inflammatory response damaging recipient organs, with gut, liver, and skin being the most susceptible. Intestinal GvHD injury is often a life-threatening complication in patients unresponsive to steroid treatment. Allogeneic mesenchymal stromal/stem cell (MSC) infusions are a promising potential treatment for steroid-resistant aGvHD. Data from our institution and others demonstrate rescue of approximately 40–50% of aGvHD patients with MSCs in Phase I, II studies and minor side effects. Although promising, better understanding of MSC mode of action and patient response to MSC-based therapy is essential to improve this lifesaving treatment.MethodsSingle cell human small intestine organoids were embedded in Matrigel, grown for 5 days and treated with busulfan for 48 h. Organoids damaged by treatment with busulfan or control organoids were co-cultured with 5000, 10,000, and 50,000 MSCs for 24 h, 48 h or 7 days and the analyses such as surface area determination, proliferation and apoptosis assessment, RNA sequencing and proteomics were performed.ResultsHere, we developed a 3D co-culture model of human small intestinal organoids and MSCs, which allows to study the regenerative effects of MSCs on intestinal epithelium in a more physiologically relevant setting than existing in vitro systems. Using this model we mimicked chemotherapy-mediated damage of the intestinal epithelium. The treatment with busulfan, the chemotherapeutic commonly used as conditioning regiment before the HSCT, affected pathways regulating epithelial to mesenchymal transition, proliferation, and apoptosis in small intestinal organoids, as shown by transcriptomic and proteomic analysis. The co-culture of busulfan-treated intestinal organoids with MSCs reversed the effects of busulfan on the transcriptome and proteome of intestinal epithelium, which we also confirmed by functional evaluation of proliferation and apoptosis.ConclusionsCollectively, we demonstrate that our in vitro co-culture system is a new valuable tool to facilitate the investigation of the molecular mechanisms behind the therapeutic effects of MSCs on damaged intestinal epithelium. This could benefit further optimization of the use of MSCs in HSCT patients.

Current Developments and Role of Intestinal Ultrasound including the Advent of AI.

Intestinal ultrasound is a non-invasive, safe, and cost-effective technique to study the small and large intestines. In addition to conventional B-mode and color doppler imaging, new US tools have been developed in more recent years that provide auxiliary data on many GI conditions, improving the diagnosis and assessment of relevant outcomes. We have reviewed the more recent literature (from 2010 onwards) on auxiliary tools in bowel ultrasound such as elastography techniques, CEUS, SICUS, and the potential contribution by artificial intelligence (AI) to overcome current intestinal ultrasound limitations. For this scoping review, we performed an extensive literature search on PubMed and EMBASE to identify studies published until December 2023 and investigating the application of elastography techniques, CEUS, SICUS, and AI in the ultrasonographic assessment of the small and large intestines. Multiparametric intestinal ultrasound shows promising capabilities in Crohn's disease, while less is known about the role in ulcerative colitis. Despite some evidence, the CEUS role as a point-of-care examination tool for rare conditions such as intestinal GvHD and ischemic small bowel disease seems promising, possibly avoiding the need to perform further cross-sectional imaging. The use of AI in intestinal ultrasound is still anecdotical and limited to acute appendicitis.

Interferon regulatory factor 4 plays a pivotal role in the development of aGVHD-associated colitis

ABSTRACT Interferon regulatory factor 4 (IRF4) is a master transcription factor that regulates T helper cell (Th) differentiation. It interacts with the Basic leucine zipper transcription factor, ATF-like (BATF), depletion of which in CD4+ T cells abrogates acute graft-versus-host disease (aGVHD)-induced colitis. Here, we investigated the immune-regulatory role of Irf4 in a mouse model of MHC-mismatched bone marrow transplantation. We found that recipients of allogenic Irf4 -/- CD4+ T cells developed less GVHD-related symptoms. Transcriptome analysis of re-isolated donor Irf4 -/- CD4+ T helper (Th) cells, revealed gene expression profiles consistent with loss of effector T helper cell signatures and enrichment of a regulatory T cell (Treg) gene expression signature. In line with these findings, we observed a high expression of the transcription factor BTB and CNC homolog 2; (BACH2) in Irf4 -/- T cells, which is associated with the formation of Treg cells and suppression of Th subset differentiation. We also found an association between BACH2 expression and Treg differentiation in patients with intestinal GVHD. Finally, our results indicate that IRF4 and BACH2 act as counterparts in Th cell polarization and immune homeostasis during GVHD. In conclusion, targeting the BACH2/IRF4-axis could help to develop novel therapeutic approaches against GVHD.

Advantage of First-Line Therapeutic Drug Monitoring-Driven Use of Infliximab for Treating Acute Intestinal and Liver GVHD in Children: A Prospective, Single-Center Study.

The high serum concentrations of TNF-α characterize acute graft-versus-host disease (aGVHD), for which infliximab treatment may be beneficial. In 28 pediatric patients, four doses of 10 mg/kg infliximab every seven days were administered after steroid failure (Standard Group, n = 14) or as a first-line therapy (Early Group, n = 14). Population pharmacokinetic analyses and evaluation of serum cytokines were performed. After two months of treatment, complete response in gastrointestinal and liver aGVHD was achieved in 43% and 100% of patients in the Standard and Early groups, respectively. During follow-up, four patients in the Standard Group (but none in the Early Group) experienced an aGVHD recurrence. Viral infections occurred more frequently in the Standard Group after the fifth dose. Infliximab clearance did not differ between groups or according to treatment outcome for each organ involved in aGVHD, whereas serum levels of cytokines significantly differed. Therefore, present findings show that use of first-line, TDM-driven infliximab to treat aGVHD in children may result in better clinical outcomes and tolerability, with a different pattern of cytokines generated according to the moment of beginning of treatment.

Long-term follow-up of donor-derived CD7 CAR T-cell therapy in patients with T-cell acute lymphoblastic leukemia

BackgroundDonor-derived CD7-directed chimeric antigen receptor (CAR) T cells showed feasibility and early efficacy in patients with refractory or relapsed T-cell acute lymphoblastic leukemia (r/r T-ALL), in a previous phase I trial report, at a median follow-up of 6.3 months. Here we report long-term safety and activity of the therapy after a 2-year follow-up.MethodsParticipants received CD7-directed CAR T cells derived from prior stem cell transplantation (SCT) donors or from HLA-matched new donors after lymphodepletion. The target dose was 1 × 106 (± 30%) CAR T cells per kg of patient weight. The primary endpoint was safety with efficacy secondary. This report focuses on the long-term follow-up and discusses them in the context of previously reported early outcomes.ResultsTwenty participants were enrolled and received infusion with CD7 CAR T cells. After a median follow-up time of 27.0 (range, 24.0–29.3) months, the overall response rate and complete response rate were 95% (19/20 patients) and 85% (17/20 patients), respectively, and 35% (7/20) of patients proceeded to SCT. Six patients experienced disease relapse with a median time-to-relapse of 6 (range, 4.0–10.9) months, and 4 of these 6 patients were found to have lost CD7 expression on tumor cells. Progression-free survival (PFS) and overall survival (OS) rates 24 months after treatment were respectively 36.8% (95% CI, 13.8–59.8%) and 42.3% (95% CI, 18.8–65.8%), with median PFS and OS of respectively 11.0 (95% CI, 6.7–12.5) months and 18.3 (95% CI, 12.5–20.8) months. Previously reported short-term adverse events (< 30 days after treatment) included grade 3–4 cytokine release syndrome (CRS; 10%) and grade 1–2 graft-versus-host disease (GVHD; 60%). Serious adverse events reported > 30 days after treatment included five infections and one grade 4 intestinal GVHD. Despite good CD7 CAR T-cell persistence, non-CAR T and natural killer cells were predominantly CD7-negative and eventually returned to normal levels in about half of the participants.ConclusionsIn this 2-year follow-up analysis, donor-derived CD7 CAR T-cell treatment demonstrated durable efficacy in a subset of patients with r/r T-ALL. Disease relapse was the main cause of treatment failure, and severe infection was a noteworthy late-onset adverse event.Trial registrationChiCTR2000034762.

Relapse Prophylaxis Post-Haploidentical Bone Marrow Transplantation and Cyclophosphamide (Haplo/Cy) By Infusion of Donor-Derived Expanded/Activated Gd T Cells: A Phase I Trial

Background: Effector γδ T cells immediately recognize and kill malignant cells in a broad-based non-MHC restricted manner. Increases in circulating donor-derived γδ T cells during post-bone marrow transplant (BMT) recovery have been significantly associated with improved disease-free survival (DFS). Relapse post Haplo/Cy BMT occurs in approximately 45% of patients. We sought to mitigate relapse in this context by expanding, activating, and infusing donor-derived haploidentical γδ T cells. We now report preliminary clinical and biologic correlative findings from the first cohort of patients who have been treated with ex vivo expanded and activated donor γδ T cells (EAGD). This single-center Phase I clinical trial represents the first systemic infusion of allogeneic EAGD cells in the post-BMT setting Methods: Standard of care reduced-intensity flu/cy/TBI conditioning was followed by an unmanipulated bone marrow graft and 50mg/m2 Cy on days +3 and +4 post-transplant. EAGD were manufactured using the Miltenyi Prodigy bioreactor and cryopreserved. The product was infused intravenously within 5 days of neutrophil engraftment (ANC >500/µL X 3d). Peripheral blood was collected at EAGD infusion and monthly thereafter through day +90, with additional collections every 6 months through 1 year. Biologic parameters included multiparameter flow cytometric immunophenotyping and single cell cytokine analysis of the EAGD graft. Peripheral blood analysis includes leukocyte count and differential, immunophenotyping, and serum Th1/Th2/Th17 cytokine analysis. Primary endpoints include dose-limiting toxicities (DLT) and grade 3-4 adverse events while secondary endpoints include incidence of acute and chronic GvHD, relapse, and overall survival. Results: Three patients have received the first dose level of 1 x 106 EAGD/kg. All three patients remain in morphologic complete remission at 26.5, 24.2, and 12.5 months post-BMT. One patient is receiving ongoing hypomethylating therapy for the occurrence of recipient chimerism. Grade 1-2 toxicities include constipation, CMV reactivation, emesis, fatigue, and hypomagnesaemia. Steroid-responsive cutaneous acute Grade I-II GVHD has been observed in all patients with one patient experiencing Grade II intestinal GvHD. No chronic GVHD, DLTs, treatment-related ≥ grade 3 adverse events, or cytokine release syndrome has occurred. EAGD grafts contained 88.7% - 99.2% Vγ9Vδ2+ γδ T cells with small populations of NK cells and <1.0 x 10 αβ T cells/kg. EAGD principally expressed Granzyme B, MIP1α, MIP1β, and IL-2. Significant peripheral lymphodepletion persisted through the first 100 days post-BMT followed by slow recovery of CD4+, CD8+, γδ T and B cells. NK cells remained within the low normal range throughout. T cells transitioned from a CD45+CD27- effector phenotype to CD45RA CD27 central to effector memory phenotype as recovery progressed. CD3 CD4 CD25 FoxP3 Treg cells remained <3% of circulating T cells. Preliminary serum cytokine and chemokine analysis revealed predominant expression of IFNγ, IL-12p70, IP-10, RANTES and TNFα. Conclusions: Early indications suggest that EAGD transfusion with the initial dose level of 1 x 106 EAGD/kg has manageable toxicity and an appropriate immune recovery profile with 3 of 3 patients alive and progression-free. Clinical Trial Registry: https://clinicaltrials.gov/ct2/show/NCT03533816

Incidence of Common Herpesviruses in Colonic Mucosal Biopsies Following Hematopoietic Stem Cell Transplantation.

Intestinal complications are common after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, only scarce data concern herpesvirus incidence in the colonic mucosa post-HSCT. Our purpose was to assess the frequency and clinical significance of cytomegalovirus (CMV), Epstein-Barr virus (EBV), human herpesvirus type 6 (HHV6), and herpes simplex virus (HSV) in the colonic mucosa post-HSCT. The study group included 119 patients of different ages, mostly with leukemias and lymphomas, subjected to allo-HSCT from haploidentical related (48%) or HLA-compatible donors (52%). In total, 155 forceps biopsies of the colonic mucosa were taken in cases of severe therapy-resistant intestinal syndrome post-HSCT. Most samples were taken from the descending, sigmoid, and transverse colon. Intestinal GVHD or local infections were assessed clinically and by histology. EBV, CMV, HSV, and HHV6 were tested in colonic mucosal lysates with commercial PCR assays. HSV was found in &lt;8% of colonic samples, along with high HHV6 and CMV positivity (up to 62% and 35%, respectively) and a higher EBV incidence at 5-6 months post-HSCT (35%). For CMV and EBV, significant correlations were revealed between their rates of detection in blood and colonic mucosa (r = 0.489 and r = 0.583; p &lt; 0.05). No significant relationships were found between the presence of herpesviruses and most patients' characteristics. EBV positivity in colonic samples was correlated with delayed leukocyte and platelet recovery post-HSCT. Higher EBV frequency in the colonic mucosa was found in deceased patients (56% versus 21%, p = 0.02). The correlations among EBV positivity in the colon, lethality rates and delayed hematopoietic reconstitution suggest some relationship with systemic and local EBV reactivation post-transplant.

The Clinical Observation with Ruxolitinib as Graft-Versus-Host Disease Prophylaxis for Children with Thalassemia after Unrelated or Haploidentical Allo-Hematopoietic Stem Cell Transplantation

To retrospectively analyze the efficacy and safety of ruxolitinib therapy for children with thalassemia after unrelated or haploidentical stem cell transplantation. From March 2020 to March 2021, 22 patients received successfully allogeneic hematopoietic stem cell transplantation in the Zhongshan Hospital of Xiamen University, from +30 to 100 days,those patients received ruxolitinib therapy (2.5 mg, twice daily) and all adverse reactions were observed, include aGVHD, cGVHD, CMV and EBV infection. 22 patients underwent allogeneic stem cell transplantation, 5 patients were diagnosed as aGVHD, 3 patients had grade I－II skin GVHD and 2 patients had grade II intestinal GVHD, those patients were cured. All patients were followed up for more than 21 weeks, 4 cases developed cGVHD, including 3 cases of localized liver GVHD and 1 case of pulmonary GVHD, those were relieved after active treatment. 8 patients had elevated EBV copies (>3×103/ml), and 3 patients had increased CMV copies, the patients recovered after immunosuppressant and antiviral treatment. There was no CMV infection and EBV related post-transplantant lymphoproliferative disorders(PTLD), and no transplant related deaths. Ruxolitinib can effectively reduce the incidence and severity of GVHD without affecting the hematopoietic recovery, and improve the survival status of thalassemia children after transplantation.

Effective Extracorporeal Photopheresis of Patients with Transplantation Induced Acute Intestinal GvHD and Bronchiolitis Obliterans Syndrome.

Background: Patients with steroid-refractory intestinal acute graft-versus-host disease (aGvHD) and bronchiolitis obliterans syndrome (BOS) represent a population with a high need for alternative and effective treatment options. Methods: We report real-life data from 18 patients treated with extracorporeal photopheresis (ECP). This cohort consisted of nine patients with steroid-refractory intestinal aGvHD and nine patients with BOS. Results: We document partial or complete clinical response and reduction of symptoms in half of the patients with intestinal acute GvHD and patients with BOS treated ECP. Responding patients tended to stay on treatment longer. In patients with BOS, stabilization of lung function and forced expiratory volume was observed, whereas, less abdominal pain, less diarrhea, and a reduction of systemic corticosteroids were seen in patients with intestinal acute GvHD. Conclusions: ECP might not only abrogate symptoms but also reduce mortality caused by complications from high-dose steroid treatment. Taken together, ECP offers a serious treatment avenue for patients with steroid-refractory intestinal acute GvHD and BOS.

Updated efficacy and safety report of a phase I trial of donor-derived CD7 CAR T cells for T-cell acute lymphoblastic leukemia.

7023 Background: Results of a phase I trial of donor-derived CD7 chimeric antigen receptor (CAR) T cells for relapsed or refractory T-cell acute lymphoblastic leukemia (r/r T-ALL) (Pan et al. J Clin Oncol 2021;39:3340-3351) have been reported previously, and are updated now. Methods: The target dose is 1 × 106 (±30%) CAR T cells per kg of body weight. Patients with prior stem cell transplantation (SCT) received CAR T cells from prior SCT donors, while patients without SCT history received CAR T cells from new donors who also provided stem cells for transplantation post CAR T therapy. The primary endpoint was safety with efficacy secondary. Survival status were continuously followed up while severe adverse events (SAEs) were recorded until receiving other anti-leukemia therapy. Results: Nineteen (95%) of twenty enrolled patients responded and were followed up with a median time of 15.8 months (range 13-18.3) until Feb, 14th, 2022. Short-term adverse events included grade 3 or higher cytokine release syndrome (10%) and grade 1-2 graft-versus-host disease (GVHD, 60%), which were all reversible. Six late-onset ( &gt; 30 days post-infusion) severe adverse events (SAEs) occurred in 5 responders. Two had been reported previously. Four SAEs were newly observed, including a grade 4 intestinal GVHD at month 11 and a grade 5 pneumonia at month 12.3 in one patient, a grade 5 Pseudomonas Aeruginosa pneumonia at month 8.7 in one patient, and a grade 3 cytomegalovirus (CMV) encephalitis at month 11 which recovered at month 13.3 in another patient. All severe infections occurred in patients with no further therapy, and the total T cells in them reached a median count of 300.03/μL (range 121.46-512.83), which were substantially lower than normal levels despite steadily increasing. The objective response and complete remission rate was 95% and 85% at day 30 post-infusion. Of 19 responders that were followed up, two (11%) withdrew for other therapy at day 55 and 271 respectively. Of 10 (53%) patients who received no further therapy, all had continuously detectable CAR T cells until the last visit, three remained in remission, three had a relapse (one CD7+, and two CD7-), and four died of infection; Seven (37%) patients proceeded to SCT and no CAR T cells were detectable after SCT, and among them two remained in remission, four had a relapse (three CD7+, and one CD7-), and one died of transplant-related mortality. Patients relapsed at a median time of 6 (range 4-10.9) months. The one-year progressive-free survival (PFS) and overall survival (OS) rates were 51.6% (95% CI, 24.7-78.4%) and 72.5% (95% CI, 51.9-93.0%). Conclusions: Donor-derived CD7 CAR T cell therapy showed encouraging activity in treating r/r T-ALL. Relapse emerges as major issues impeding long-term outcomes. CD7-negative relapse was commonly observed under CAR T cell surveillance. Late onset GVHD and infections may occur and should be carefully managed. Clinical trial information: ChiCTR2000034762.

Non-Invasive Diagnosis of Pediatric Intestinal Graft-Versus-Host Disease: A Case Series

Intestinal graft-versus-host disease (I-GvHD) represents a life-threatening complication in allogeneic stem cell transplantation (SCT). Unfortunately, non-invasive validated diagnostic tools to diagnose I-GvHD, evaluate treatment response, and guide the duration of immunosuppression are still lacking. We employed standard ultrasound and power Doppler to diagnose and follow up on pediatric intestinal GvHD. We herein report on three patients, prospectively evaluated among 24 pediatric patients referred to our center for allogeneic SCT. These three patients presented abdominal pain and diarrhea within the first 200 days after transplantation. In the reported cases, we performed small- and large-intestine ultrasound (US) at clinical onset of lower-intestinal symptoms and, when intestinal GvHD was confirmed, at GvHD flares, if any, and at follow-up. US constantly (3/3 patients) revealed increased bowel wall thickening (BWT) with different bowel segments’ involvement from patient to patient. Further, a moderate or strong increased Doppler signaling was seen in 2 out of 3 patients, according to clinical GVHD staging (e.g., the more the increase, the more the staging). Standard sonography corroborated GvHD diagnosis in all patients considered and was able to detect GvHD progression or complete normalization of findings, thus simplifying ensuing clinical decisions. Our report highlights the need to design clinical trials for the validation of non-invasive radiologic tools for diagnosis and follow-up of GvHD, especially in pediatric patients.

72 - The Amphiregulin/EGFR Axis Regulates Donor T Cell Expansion and Contributes to Intestinal Gvhd Pathology after Allogeneic Bone Marrow Transplantation

72 - The Amphiregulin/EGFR Axis Regulates Donor T Cell Expansion and Contributes to Intestinal Gvhd Pathology after Allogeneic Bone Marrow Transplantation

18F-FDG-PET-MRI for the assessment of acute intestinal graft-versus-host-disease (GvHD)

BackgroundGraft versus host disease (GvHD) is a frequent complication of allogeneic stem cell transplantation (alloSCT), significantly increasing mortality. Previous imaging studies focused on the assessment of intestinal GvHD with contrast-enhanced MRI/CT or 18F-FDG-PET imaging alone. The objective of this retrospective study was to elucidate the diagnostic value of a combined 18F-FDG-PET-MRI protocol in patients with acute intestinal GvHD.MethodsBetween 2/2015 and 8/2019, 21 patients with acute intestinal GvHD underwent 18F-FDG-PET-MRI. PET, MRI and PET-MRI datasets were independently reviewed. Readers assessed the number of affected segments of the lower gastrointestinal tract and the reliability of the diagnosis on a 5-point Likert scale and quantitative PET (SUVmax, SUVpeak, metabolic volume (MV)) and MRI parameter (wall thickness), were correlated to clinical staging of acute intestinal GvHD.ResultsThe detection rate for acute intestinal GvHD was 56.8% for PET, 61.4% for MRI and 100% for PET-MRI. PET-MRI (median Likert-scale value: 5; range: 4–5) offers a significantly higher reliability of the diagnosis compared to PET (median: 4; range: 2–5; p = 0.01) and MRI alone (median: 4; range: 3–5; p = 0.03). The number of affected segments in PET-MRI (rs = 0.677; p < 0.001) and the MV (rs = 0.703; p < 0.001) correlated significantly with the clinical stage. SUVmax (rs = 0.345; p = 0.14), SUVpeak (rs = 0.276; p = 0.24) and wall thickening (rs = 0.174; p = 0.17) did not show a significant correlation to clinical stage.Conclusion18F-FDG-PET-MRI allows for highly reliable assessment of acute intestinal GvHD and adds information indicating clinical severity.

Phase II Trial of Natalizumab (Tysabri®) with Corticosteroids As Initial Treatment of Gastrointestinal Acute Graft Versus Host Disease

Background: Graft-vs.-host disease (GVHD) is caused by allorecognition of host antigens by tissue-specific donor lymphocytes. We have previously demonstrated that α4ß7 integrin is upregulated on both naïve and memory T cell subsets in patients who subsequently developed intestinal acute GVHD (Chen, BMT 2013). This integrin interacts with endothelial cell adhesion molecules and mediates homing of lymphocytes to the gastrointestinal (GI) tract. Natalizumab (Tysabri®, Biogen Inc.) is a selective adhesion molecule inhibitor, acting directly against the α4 subunit of α4 integrins. Given the poor outcomes of GI GVHD when treated with corticosteroids alone, we initiated a phase II study to determine the safety and efficacy of natalizumab in combination with corticosteroids for the initial treatment of acute GI GVHD.Methods: Subjects with new-onset, acute GVHD of the lower GI tract were eligible to participate. Other organ involvement was permitted but not required. Biopsy confirmation of GI GVHD and exclusion of infectious causes of diarrhea were required. Natalizumab was given at a dose of 300 mg IV, with a second dose permitted 4 weeks later in the event of an incomplete response. An initial corticosteroid dose of 2 mg/kg of methylprednisolone was suggested, but dosing and dose reductions were at the discretion of the treating physician. Steroids could be initiated up to 7 days prior to the initiation of natalizumab while biopsy and infectious disease specimens were being analyzed. The primary endpoint was GVHD-free survival rate 56 days from enrollment. Secondary endpoints included response rates at day 28 and 56, steroid dose at days 28, 56, 100, 180 and 365, incidence of systemic infections at day 56 and 180, and overall survival.Results: 18 subjects were enrolled and received at least one dose of natalizumab. Subjects had a median age of 63.5 years, and 12 were male. Fifteen had a non-myeloablative transplant, 12 had a matched unrelated donor, and all had received PBSCs. All subjects had newly diagnosed acute GI GVHD at time of enrollment; 4 had concomitant cutaneous and 1 had concomitant hepatic involvement. Stage 1, 2, 3, and 4 GI involvement was noted in 7, 4, 4, and 3 subjects respectively, corresponding to overall grade II GVHD in 7 and grade III in 11 subjects. Sixteen patients had data for response assessment. The day 56 GVHD-free survival rate was 37.5%. The overall response rate (PR+CR) for GI GVHD at day 28 and day 56 was 75% and 62.5% respectively. All surviving subjects except one achieved steroid dose reduction by day 56, with a median reduction of 61.6%. Three patients experienced treatment-related toxicity possibly related to natalizumab; one subject had grade 2 hepatic toxicity, one had grade 4 encephalopathy, and one developed hepatic failure. Three subjects have developed chronic GVHD. Four subjects had documented infections with bacteria (2), yeast (1), and viral pathogens (1- polyomavirus). Six subjects had measurable JC viremia prior to initiation of natalizumab and 8 subjects had measurable JC viremia at day 56, although only 1 subject had a viral load greater than 1000 copies/ml. No subject has developed JC-associated disease. Seven subjects have died. Causes of death include GVHD (2), disease relapse (2), and organ failure (3). With a median follow-up of 180 days, the 6 month overall survival was 52% (95% CI 25-74%).Conclusion: Natalizumab in combination with corticosteroids as initial treatment of acute gastrointestinal GVHD is feasible and well tolerated. These early results suggest a higher rate of response at day 56 than is typically observed with steroids alone. Inhibition of T-cell homing to the GI tract may improve GI GVHD response and potentially mitigate progression of acute GVHD. DisclosuresKoreth:Kadmon Corp: Membership on an entity's Board of Directors or advisory committees; Amgen Inc.: Consultancy; Prometheus Labs: Research Funding; Takeda Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Millennium Pharmaceuticals: Research Funding. Armand:Bristol-Myers Squibb: Consultancy, Research Funding; Tensha: Research Funding; Affimed: Research Funding; Sequenta/Adaptive: Research Funding; Merck & Co., Inc.: Consultancy, Research Funding; Roche: Research Funding; Genmab: Consultancy; Pfizer: Consultancy, Research Funding; Infinity: Consultancy; Otsuka: Research Funding; Sigma Tau: Research Funding. Antin:Gentium SpA/Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Cutler:Pfizer: Consultancy; Pharmacyclics: Consultancy; Kite: Consultancy; Bristol-Myers Squibb: Consultancy; Incyte: Consultancy; Astellas: Consultancy.

The Multicentre, Double-Blinded, Placebo-Controlled Clinical-Trial (Pre-GvHD) for Prediction and Pre-Emptive Treatment of Acute GvHD

Allogeneic stem cell transplantation (HSCT) is a curative treatment for adult patients with hematologic malignancies, but is limited by severe, life-threatening complications such as acute graft-versus-host disease (aGvHD). We have developed a proteomic urine pattern “aGvHD_MS17”, consisting of 17 differentially excreted peptides, capable to predict aGvHD grade II or more (1, 2). In 2008, a multicenter, randomized, placebo-controlled, double blind clinical trial (Pre-GvHD) was initiated testing aGvHD_MS17 for prediction of aGvHD and to initiate pre-emptive therapy using prednisolone 2-2.5mg/kg.Patients and Methods:Eleven German transplant-centres contributed 267 patients. Urine was collected weekly from day +7 to +35 and on days +50 and +80 (all +/-3 days) frozen, shipped to Hannover and analyzed using capillary electrophoresis coupled on-line to mass spectrometry (CE-MS) within 72h as described (1). aGvHD_MS17 was considered positive, when the dimensionless classification factor (CF) was +0.1 or more. Eight patients were excluded from analyses, either due to no medication (n=5) or protocol violations (n=3) and 92 were randomized according to the positivity of aGvHD-MS17 to receive either prednisolone (2-2.5mg/kg, n=44) or placebo (n=48) for 5 days followed by a taper for 19 days, if no aGvHD occurred. The remaining 167 patients formed the observation group according to pattern negativity.About half of the patients had acute leukemia (placebo group: n=24/48 (50%), prednisolone group: n=21/44 (50%); observation group: n= 91/167 (54%)) and were in complete remission/chronic phase (CR/CP) (placebo n=23/48 (47%), prednisolone n=27/44 (61%) and observation n=68/167 (41%). The majority was transplanted from matched donors (placebo: n=42/48 (87%); prednisolone: n=37/44 (84%); observation: 146/167 (87%), using reduced intensity conditioning regimens (RIC; 64%), and a calcineurin-inhibitor based GvHD-prophylaxis with MTX or MMF).Results:Prospective and blinded evaluation of aGvHD_MS17 revealed that the first analysis time point (day +7; range: 2-17) most accurately predicted aGvHD grade II or more with a sensitivity of 87% and a specificity of 81% prior to clinical signs with a CF of +0.1 (2). Patients with samples positive for aGvHD_MS17 in the early analyses time points had a 21-fold higher risk to develop aGvHD grade II or more ((p<0.0001), Figure 1). By day +28 the predictive value of aGvHD_MS17 was lost. Confounding factors were conditioning with RIC-protocols and early death after HSCT. Patients with one sample positive for aGvHD_MS17 had a 3-fold higher risk to die, 35% of those died prior to day +500, compared to only 10% of the patients with negative samples. Analysis of pre-emptive therapy using prednisolone revealed that the incidence and severity of acute GvHD was not significantly different between the placebo and prednisolone arm suggesting that prednisolone 2-2.5mg/kg was insufficient to prevent aGvHD in this setting. Further analyses of aGvHD according to organ manifestation are ongoing. The occurrence of aGvHD after a positive proteomic pattern test in the placebo group of this trial was lower than in the previous pilot study. Possible reasons include different patient populations (pilot study: 18% of patients transplanted in relapse; current trial: only 4% transplanted in relapse, increasing the risk to develop aGvHD), different intestinal decontamination and GvHD prophylaxis protocols. The frequency of adverse and serious adverse events was not higher in the prednisolone arm than the placebo arm. No specific safety risk of the pre-emptive therapy with prednisolone was identified.Conclusions:Taken together our results indicate that pre-emptive treatment of imminent aGvHD based on proteomic-pattern-diagnostic with prednisolone 2-2.5mg/kg appears to be safe, but did not influence severity or incidence of aGvHD grade II or more. The prospective evaluation of aGvHD_MS17 confirms the highly reproducible results in the early analysis time points (day +7 to +21) for prediction of aGvHD (day +7; range 2-17). Patients with aGvHD_MS17 positive samples have a 21-fold risk to develop severe GvHD (grade II-IV). Moreover, patients with one sample positive for aGvHD_MS17have a 3-fold increased risk of death by day +500 after HSCT.(1) Blood 2007 :109(12):5511-9.(2) Leukemia, 2014: , 28(4):842-52 [Display omitted] DisclosuresJochen:Mosaiques GmbH: Employment. Schmid:Jazz: Membership on an entity's Board of Directors or advisory committees; Incyte: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau; MoilMed: Membership on an entity's Board of Directors or advisory committees; Celgene: Research Funding, Speakers Bureau. Bethge:Miltenyi Biotech GmbH: Consultancy, Honoraria, Research Funding; Neovii GmbH: Honoraria, Research Funding. Raad:Mosaiques GmbH: Employment. Durban:Mosaiques GmbH: Employment.

Intensified Conditioning Is Associated with Loss of Stool Bacterial Diversity and Pronounced Gastrointestinal Graft-Versus-Host Disease in Allogeneic Hematopoitic Stem Cell Transplantation

AbstractIn allogeneic hematopoitic stem cell transplantation (allo-HSCT), intestinal microbiota and acute graft-versus-host disease (aGVHD) were impacted by broad-spectrum antibiotics. Whereas besides the influence of antibiotics, other factors may influence intestinal microbiota and aGVHD but were undetermined, and the correlations between microbiota and immune homeostasis were still poorly understood. We performed a prospective study group (n =57) whose antibiotics was balanced, including III-IV intestinal acute GVHD (n = 12), Ӏ-II intestinal aGVHD (n = 14) and non-aGVHD (n = 31) groups, retrospective analysis of the intestinal microbiota among the groups by 16S rRNA gene sequencing at engraftment time post-transplantation. Meanwhile, we detected T cell related cytokines. And then T lymphocyte subsets were detected at day 28 following allo-HSCT. Samples of intestinal biopsies from the above III-IV aGVHD patients were stained by immunofluorescence histology. Our results demonstrated that intensive conditioning was likely to be associated with lower microbiota diversity compared with standard conditioning (61.1% vs. 25.6%, P = 0.017). Abundance of Lachnospiraceae, Ruminococcaceae and Clostridiaceae was lower in III-IV aGVHD than in non-aGVHD (P = 0.001, 0.016 and 0.028, respectively). Then, loss of microbiota diversity was associated with the subsequent aGVHD after engraftment. Whereas the III-IV aGVHD patients with low diversity accompanied by depletion of RORγt Tregs. Furthermore, our findings suggested that microbiota diversity correlated with the levels of IL-17A (r = -0.341, P = 0.010) and the amounts of Th1, Th17 cells negatively (r = -0.329 and -0.265; P= 0.012 and 0.046, respectively). In conclusion, loss of microbial diversity and development of aGVHD may begin with the intensive conditioning besides the administration of antibiotics during allo-HSCT, and microbiota diversity might correlate with immune homeostasis at early stage.Keywords: conditioning, intestinal microbiota, immune homeostasis, acute graft-versus-host disease, allogeneic hematopoietic stem cell transplantation DisclosuresNo relevant conflicts of interest to declare.

Mucosal Foxp3+ Regulatory T Cells Show Significant Numerical Differences in Patients with Intestinal Graft-Versus-Host Disease Compared to Infectious Diseases of the Gut.

Objectives: The clinical diagnosis of intestinal GvHD is often complicated, even if bioptical material is present. So far, it is difficult to separate between GvHD and infectious complications, since distinguishing immunological features are not established. In a multicenter study, we quantified Foxp3+ immunoregulatory T cells (Treg) and effector T cells in the intestinal mucosa of patients with acute or chronic GvHD, and infectious diseases of the gut.Methods: 95 paraffin-embedded tissue samples (44 with acute GvHD, 18 with chronic GvHD and 33 without histological GvHD) derived from intestinal biopsies from 49 patients undergoing allogeneic stem cell transplantation, and 12 inflammatory controls (6 patients with CMV-colitis and 6 with diverticulitis) were analysed by double immunoenzymatic labeling. Serial sections were stained for CD4, CD8 and co-stained for CD3/Foxp3 and CD25/Foxp3. In addition, lamina propria lymphocytes were isolated from intestinal biopsies by collagenase digestion and further characterized by flow-cytometry in comparison to peripheral blood lymphocytes in 24 patients with suspected GvHD after allogeneic stem cell transplantation and healthy controls.Results: The number of CD3+ T cells was extensively increased in intestinal GvHD, CMV colitis and diverticulitis. Though the number of CD8+ cells was similar, we saw striking differences in the frequency of Foxp3+ Treg in relation to CD8+ effector T cells. The number of Foxp3+ T cells per 100 CD8+ T cells was significantly lower in GvHD samples (1.7 ± 0.2) than in CMV colitis (27.4 ± 2.5) or diverticulitis (50.0 ± 4.4). Within the allogeneic transplanted patients, samples without histological signs of GvHD had also significantly elevated numbers of Foxp3+/100 CD8+ T cells (14.1 ± 2.0) compared to acute (1.7 ± 0.2) and chronic GvHD (2.9 ± 0.8) and healthy controls (2.0 ± 0.3).Conclusion: In contrast to infectious diseases of the gut, GvHD is characterized by a considerable decrease of mucosal Treg cells. Together with recent experimental data, our results give new insights in the pathophysiology of intestinal GvHD and can be helpful for the precise diagnosis of intestinal complications after allogeneic stem cell transplantation.

Prospective longitudinal evaluation of microbiome diversity in patients with hematological malignancy undergoing allogeneic hematopoietic stem cell transplantation (HSCT).

7005 Background: Studies suggest that decreased microbial diversity due to chemotherapeutic and antibiotic exposure may be associated with acute graft vs host disease (aGvHD) and mortality in patients undergoing allogeneic HSCT. In addition, disruption of the microbiome by antibiotics may lead to intestinal domination by pro-inflammatory bacteria, resulting in increased risk of aGvHD. This relationship has been described in settings with prophylactic antibiotic use, a standard of care in most transplant centers. Here we assessed how the microbiome and GvHD outcomes differ when prophylactic use of antibiotics is avoided. Methods: We collaborated on an observational study (COLLECT) to evaluate changes in microbial diversity over time in subjects undergoing allogeneic HSCT. According to protocol at the University Hospital of Cologne, antibiotics were administered only as empiric treatment for febrile neutropenia or as targeted treatment. Stool was collected weekly from 65 subjects at baseline (pre-HSCT) to day 28 with additional time points taken at day 56, day 90, day 365, and upon diagnosis of intestinal GvHD (GvHD-day 0 and GvHD-day 7). Patients were monitored for incidence of GvHD, including acute GvHD of the liver, intestine, and skin. Microbiome 16SV4 profiles were generated from 381 stool samples. Linear effects models were developed to evaluate the association between Shannon diversity, intestinal domination, and the incidence of intestinal GvHD and mortality. Results: Of the 65 subjects, 28 subjects (42%) went on to develop intestinal GvHD, and 16 subjects (25%) did not survive to day 365. A decline in Shannon diversity was observed during the neutropenic period following HSCT. Subjects who went on to develop intestinal GvHD had significantly lower Shannon diversity at the time of stem cell engraftment (p &lt; 0.0468). Furthermore, lower diversity was observed throughout the study period in subjects experiencing intestinal GvHD. We developed a linear model evaluating the association between mortality and Shannon diversity and found a significant relationship at days 28 and 90 post HSCT (p &lt; 0.0001 and 0.0121, resp). Intestinal domination by Enterobacteriaceae or Enterococcus was significantly associated with the incidence of intestinal GvHD (p &lt; 0.0082) or mortality (p &lt; 0.001), respectively. Conclusions: Data from this observational study (COLLECT) suggests decreases in microbial diversity over time occur in subjects undergoing allogeneic HSCT despite the lack of prophylactic antibiotics. Investigation of whether administration of microbiome therapeutic drugs prior to transplant and/or at the time of engraftment can reduce morbidity and mortality in this high-risk patient population is warranted. Clinical trial information: NCT03148197.

Exploration of the relationship between intestinal flora changes and gut acute graft-versus-host disease after hematopoietic stem cell transplantation.

BackgroundAcute graft-versus-host disease (aGVHD) is a life-threatening factor for post-hematopoietic stem cell transplantation (HSCT) patients. To investigate the relationship between intestinal flora changes and gut aGVHD after HSCT, we performed this cross-sectional study.MethodsWe selected children from our medical center from July 2016 to January 2017. Fifty-six samples from 42 patients and 6 samples from normal children met the study criteria and were analyzed. Fecal 16S RNA sequencing was completed before transplantation or on days 7, 28 or 100 post-transplantation. The intestinal infection and GVHD clinical data were retrospectively analyzed, and the survival risk factors were analyzed. Correlation analysis was performed with the feces bioinformatic data.ResultsThe GVHD group alpha diversity was the lowest, which was significantly different than that of the non-diarrhea group (P value=0.032). A richer posttransplantation relative abundance of Moraxellaceae was conducive to survival, while that of Enterococcaceae and Alphaproteobacteria was not. Similarly, a rich relative abundance of Proteobacteria, Gammaproteobacteria and Odoribacteraceae in the intestinal flora before HSCT contributed to patient death thereafter. Regarding diarrhea, the GVHD group exhibited a richer Pasteurellales and Pasteurellaceae relative abundances, which showed strong correlations with diarrhea severity. Peptostreptococcaceae, Bifidobacteriales and Bifidobacteriaceae were richer in relative abundance in the intestinal infection group and correlated with pretransplant characteristics.ConclusionsThe gut microbiota diversity was lowest when gut aGVHD occurred, which was consistent with the clinically higher mortality rate and greater treatment difficulty. Pasteurellaceae played an important role in gut aGVHD and diarrhea severity. Bifidobacteriaceae led to infectious diarrhea after HSCT. Specific bacteria were biomarkers for survival: Moraxellaceae, Enterococcaceae and Alphaproteobacteria from the intestinal flora after HSCT and Proteobacteria, Gammaproteobacteria and Odoribacteraceae before HSCT.