aGVHD Patients Research Articles

Ruxolitinib-corticosteroid as first-line therapy for newly diagnosed high-risk acute graft versus host disease: study protocol for a multicenter, randomized, phase II controlled trial

BackgroundThe response rate of the first-line therapy with corticosteroid for acute graft versus host disease (aGVHD) is about 50%, and steroid-refractory disease is associated with high mortality. The improved response rate to the first-line therapy of newly diagnosed aGVHD patients would result in therapeutic benefits. Ruxolitinib, a selective Janus kinase (JAK) 1/2 inhibitor, has been approved for the treatment of steroid-refractory acute GVHD. The addition of ruxolitinib to the first-line therapy may improve the efficacy of corticosteroids.MethodsThis investigator-initiated, open-label, multicenter, prospective randomized, and controlled two-arm phase II study compares the efficacy and safety of ruxolitinib combined with 1 mg/kg methylprednisolone versus 2 mg/kg methylprednisolone alone in newly diagnosed aGVHD patients. Patients with intermediate or high-risk aGVHD, as defined by the Minnesota aGVHD high-risk score and biomarker algorithm, are eligible for this study. A total of 198 patients will be randomized at a 1:1 ratio and assigned a GVHD risk (intermediate versus high risk) and disease status before transplantation (complete remission versus no complete remission). The primary endpoint is the overall response rate on day 28, which is defined as an improvement of at least one stage in the severity of aGVHD in one organ without deterioration in any other organ or disappearance of any GVHD signs from all organs without requiring new systemic immunosuppressive treatment. The secondary objectives consist of response time, response duration, overall survival, disease-free survival, non-relapse mortality, failure-free survival, and changes in serum levels of proinflammatory cytokines and GVHD-related biomarkers.DiscussionThis open-label, multicenter, two-arm randomized trial will evaluate whether the addition of ruxolitinib combined with corticosteroid is superior to corticosteroid alone in newly diagnosed high-risk aGVHD.Trial registrationClinicalTrials.gov NCT04061876 (version number: 2019.5.18). Registered on July 16, 2019

Soluble T Cell Immunoglobulin and Mucin Domain-3 (sTIM-3) Predicts Graft-Versus-Host Disease (GVHD) in Iranian Allogeneic Hematopoietic Stem Cell Transplantation

Background: T cell immunoglobulin and mucin domain-3 (TIM-3) is an immune-checkpoint molecule that is upregulated following allogeneic immune responses and could play an important role in the development and pathogenesis of graft-versus-host disease (GVHD). The soluble form of TIM-3 (sTIM-3) is increased following the upregulation of membranous TIM-3. Objectives: The aim of this study was to evaluate the association between plasma level of sTIM-3 and acute GVHD (aGVHD) incidence in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). Methods: Blood samples were collected from 42 allo-HSCT patients and 20 healthy individuals 2 weeks after allo-HSCT. The plasma level of sTIM-3 was measured using enzyme-linked immunosorbent assay (ELISA). The clinical and demographic data of patients were collected from the clinical documents. Data analysis was evaluated using student t-test and one-way ANOVA tests. P-values less than 0.05 were assumed statistically significant. Results: Among 18 (42.8%) patients with aGVHD symptoms, 10 (23.8%) had severe GVHD and 8 (19%) experienced mild GVHD. Plasma sTIM-3 levels at day +14 were significantly higher in patients who developed aGVHD compared to allo-HSCT patients without GVHD and also the healthy control individuals (P-value = 0.015 and < 0.001). Among the aGVHD patients, the sTIM-3 levels in those with severe GVHD were approximately 2.5 times higher than those with mild GVHD (P-value < 0.001). Conclusions: We have identified a high plasma level of sTIM-3 as a valuable biomarker in predicting the development of acute GVHD, especially severe aGVHD in allo-HSCT patients.

Mesenchymal stromal cells plus basiliximab, calcineurin inhibitor as treatment of steroid-resistant acute graft-versus-host disease: a multicenter, randomized, phase 3, open-label trial

BackgroundSteroid-resistant (SR) acute graft-versus-host disease (aGVHD) lacks standard second-line treatment. Mesenchymal stromal cells (MSCs) have potential efficacy in SR aGVHD. We aimed to assess the efficacy and safety of MSCs combined with basiliximab and calcineurin inhibitor as second-line therapy for SR aGVHD.MethodsA randomized phase 3 trial involved 203 SR aGVHD patients at nine centers in China (September 2014–March 2019). Participants were randomized at a 1:1 ratio to receive second-line therapy with (n = 101) or without (n = 102) MSCs. The primary endpoint was the overall response (OR) at day 28. Secondary and safety endpoints included durable OR at day 56, failure-free survival, overall survival (OS), chronic GVHD (cGVHD), infection, hematological toxicity and relapse.ResultsOf 203 patients, 198 (97.5%; mean age, 30.1 years; 40.4% women) completed the study. The OR at day 28 was higher in the MSC group than the control group (82.8% [82 patients] vs. 70.7% [70]; odds ratio, 2.00; 95% confidence interval [CI], 1.01–3.94; P = 0.043). The durable OR at day 56 was also higher in the MSC group (78.8% [78 patients] vs. 64.6% [64]; odds ratio, 2.02; 95% CI, 1.08–3.83; P = 0.027). The median failure-free survival was longer in the MSC group compared with control (11.3 months vs. 6.0 months; hazard ratio (HR) 0.68; 95% CI, 0.48–0.95, P = 0.024). The 2-year cumulative incidence of cGVHD was 39.5% (95% CI, 29.3–49.4%) and 62.7% (51.4–72.1%) in the MSC and control groups (HR 0.55, 95% CI, 0.36–0.84; P = 0.005). Within 180 days after study treatments, the most common grade 3 and 4 adverse events were infections (65 [65.7%] in the MSC group vs. 78 [78.8%] in the control group) and hematological toxicity (37 [37.4%] vs. 53 [53.5%]). The 3-year cumulative incidence of tumor relapse was 10.1% (95% CI, 5.2–17.1) and 13.5% (7.5–21.2%) in the MSC and control groups, respectively (HR 0.75, 95% CI, 0.34–1.67, P = 0.610).ConclusionsMSCs plus second-line treatments increase the efficacy of SR aGVHD, decrease drug toxicity of second-line drugs and cGVHD without increasing relapse, and are well-tolerated. MSCs could be recommended as a second-line treatment option for aGVHD patients.Trial registration clinicaltrials.gov identifier: NCT02241018. Registration date: September 16, 2014, https://clinicaltrials.gov/ct2/show/NCT02241018.

Impact of peri-transplant RBC transfusion and ABO incompatibility on acute graft-versus-host disease in pediatric hematopoietic stem cell transplant patients

It is well known that stem cell transplantation is curative for many diseases; however, graft versus host disease (GVHD), which is a common posttransplant complication, has still a substantial place among the causes of transplant-related morbidity and mortality.The association between ABO incompatibility and GVHD is controversial. There is also limited available data about the association between blood component transfusions during the peritransplant period and GVHD development in the pediatric setting.Hence, we retrospectively evaluated both the impact of ABO-mismatch and transfusions of RBC and platelets between day -7 pre-transplant and +30 post-transplant to the development of acute GVHD (aGVHD). We analyzed 139 allotransplants in 133 patients who were transplanted by myeloablative conditioning. Fifty-one patients out of 133 (36.7 %) were found to have aGVHD within +100 days post-transplantation. Of them 40 patients had grade I-II and 11 patients had grade III-IV aGVHD. Increased risk of aGVHD is associated with ABO minor mismatch (p: 0.030). Nevertheless, there was no association between ABO mismatch and severity of aGVHD. The median number of RBC transfusions in aGVHD patients was higher than the number of transfusions in patients without aGVHD; however, the difference was not statistically significant (p: 0.11). Platelet transfusion numbers were statistically similar between aGVHD patients and the patients without aGVHD (p: 0.79).In conclusion, major and bi-directional ABO-incompatibility between donors and recipients, and RBC and platelet transfusions between day -7 pretransplant and day +30 post-transplant do not contribute to aGVHD development in children undergoing HSCT by myeloablative conditioning, while ABO minor mismatch is associated with the development of aGVHD.

Ruxolitinib in the management of steroid-resistant/-dependent acute and chronic graft-versus-host disease: results of routine practice in an academic centre.

Graft-versus-host disease (GVHD) is an important complication after allogeneic haematopoietic stem cell transplantation (HSCT). Corticosteroids are the standard first-line treatment. Steroid-resistant/-dependent (SR/D) acute and chronic GVHD (aGVHD, cGVHD) lead to significant morbidity/mortality. The JAK2 inhibitor ruxolitinib has recently been shown in clinical trials to be effective in SR/D aGVHD and cGVHD. We retrospectively analysed the efficacy and safety of ruxolitinib in a cohort of SR/D aGVHD and cGVHD patients treated in a non-trial setting. In the aGVHD cohort, there were 14 men and 12 women, median age at 38 (19-63) years. At day 28 post-ruxolitinib, the overall response rate (ORR) was 86% (complete response, CR, 36%; partial response, PR, 50%). Continued ruxolitinib beyond day 28 resulted in a final CR of 68%. However, 3/15 (20%) of CR patients developed cGVHD. In the cGVHD cohort, there were 16 men and 15 women, median age at 33 (21-64) years. The ORR, CR and PR rates changed with continued ruxolitinib treatment, being 86%, 17% and 69% at 1month; 79%, 38% and 41% at 3months; and 83%, 52% and 31% at 6months. Five patients had overlap GVHD, four of whom achieved CR. Multivariate analysis showed that superior overall survival and failure-free survival were associated with CR at day 28 for aGVHD, and CR at 1year for cGVHD. Ruxolitinib treatment was efficacious for SR/D aGVHD and cGVHD, and continued treatment for at least 6months was needed to maximize benefit.

Letermovir Prophylaxis and Cytomegalovirus Reactivation in Adult Hematopoietic Cell Transplant Recipients with and without Acute Graft Versus Host Disease.

Simple SummaryCytomegalovirus (CMV) infection and graft versus host disease (GVHD) both contribute to increased morbidity and mortality following allogeneic hematopoietic cell transplantation (allo-HCT). Since the development of GVHD can increase a patient’s risk of developing CMV infection post-allo-HCT, the aim of our retrospective study was to assess the effectiveness of letermovir prophylaxis in preventing CMV infection in these patients at high risk for CMV reactivation. Letermovir is an antiviral approved for the prevention of CMV infection. This study demonstrated that patients with GVHD had significantly fewer CMV infections when they received letermovir prophylaxis compared to patients who did not receive letermovir.Cytomegalovirus (CMV) is the most clinically significant infection after allogeneic hematopoietic-cell transplantation (allo-HCT) and is associated with increased mortality. The risk for CMV reactivation increases with graft versus host disease (GVHD). GVHD contributes to significant morbidity and mortality and is treated with immunosuppressive therapies that can further increase CMV infection risk. Prophylaxis with letermovir, an oral antiviral approved to prevent CMV, has been shown to decrease the incidence of CMV infection post-allo-HCT in patients at high risk of CMV reactivation, but there is a lack of data confirming this benefit in patients with GVHD. In this single-center, retrospective study, we assessed the incidence of clinically significant CMV infection (CS-CMVi) in allo-HCT patients who received letermovir prophylaxis (n = 119) and who developed aGVHD compared to a control group (n = 143) who did not receive letermovir. Among aGVHD patients, letermovir prophylaxis decreased CS-CMVi in patients with aGVHD (HR 0.08 [95% CI 0.03–0.27], p < 0.001), reduced non-relapsed mortality (p = 0.04) and improved overall survival (p = 0.04). This data suggests that letermovir prophylaxis improves outcomes by preventing CS-CMVi in patients with aGVHD.

Th22 and Tfh Cell Elevation Is Associated with Clinical Response of Photopheresis Therapy in Patients with Steroid-Refractory/ Resistant Graft-Versus-Host Disease (GvHD)

In patients with steroid-refractory/resistant graft-versus-host disease (GvHD), extracorporeal photopheresis (ECP) has been identified as a promising therapeutic strategy due to its safety profile and convincing clinical response rates.While previous studies have reported on the role of Th1, Th2 and Treg cells in ECP therapy, further comprehensive analysis of T helper cells is necessary to provide better understanding of the underlying mechanisms. In this study, we thus investigated the influence of long-term ECP treatment on both Th cells as well as on immune checkpoint molecules and apoptosis. Overall, we investigated 27 patients with GvHD treated by ECP therapy, containing 13 patients with acute GvHD and 14 patients with chronic GvHD. 10/13 (76.9%) of aGvHD and 9/14 (64.3%) of cGvHD patients clinically responded to ECP therapy. Three (23.1%) patients reached CR and seven (53.8%) patients achieved PR under ECP treatment in patients with aGvHD, while in patients with cGvHD CR was achieved in one (7.1%) and PR in eight (57.1%) patients. Stabilization of disease was observed in five (35.7%) patients.On an immunological level, aGvHD, cGvHD and healthy donors (HD) patients showed different profiles of Th populations. In GvHD patients, significantly higher levels for Th2 (aGvHD vs. HD: 36.26% vs. 13.88%, p = 0.014; cGvHD vs. HD: 30.71% vs. 13.88%, p = 0.026), Th17 (aGvHD vs. HD: 19.21% vs. 7.49%, p = 0.038), Th22 (aGvHD vs. HD: 1.22% vs. 0.11%，p = 0.011; cGvHD vs. HD: 0.64% vs. 0.11%, p = 0.012) and GM-CSF + Th cells (aGvHD vs. HD: 1.15% vs. 0.14%, p = 0.022; cGvHD vs. HD: 0.84% vs. 0.14%，p = 0.012) and clearly lower levels for T follicular helper (Tfh) cells including Th1- (aGvHD vs. HD: 0.37% vs. 2.04%, p = 0.002; cGvHD vs. HD: 1.28% vs. 2.04%, p = 0.03) and Th2-like cells (aGvHD vs. HD: 1.17% vs. 4.19%, p = 0.033) were observed in comparison to HDs. This suggests Th cells account for a crucial role in GvHD pathogenesis. ECP therapy was able to accelerate recovery of Tfh cells, including Th1- (Time point 1 vs. Time point 2 in aGvHD: 0.37% vs. 0.97%, p = 0.028), Th2- (T1 vs. T2 in aGvHD: 1.17% vs. 1.35%, p = 0.049; T1 vs. T2 in cGvHD: 2.08% vs. 3.15%, p = 0.048) and Th17-like Tfh cells (T1 vs. T2 in aGvHD: 1.82% vs. 2.70%, p = 0.034), facilitating immune reconstitution after allo-HSCT and thereby alleviating GvHD. Clinical response in aGvHD patients was strongly associated with elevation of Th22 cells by ECP therapy showing 51% upregulation (T1 vs. T2: 0.86% vs. 1.3%，p=0.007). In addition, we also found that the expression of the Fas receptor on effector T cells could be further upregulated with ECP treatment (T1 vs. T2 in cGvHD: 49.65% vs. 56.46%, p = 0.011), which increases the susceptibility of these effectors T cells to Fas-mediated apoptosis. This could lead to the elimination of these overactivated effector T cells and the perseverance of immunological tolerance by triggering activation-induced cell death. In addition, a reduction of Tim-3-expressing effector T cells, which are associated with the severity of GvHD, with ECP therapy (T1 vs. T2 for aGvHD: 18.09% vs. 15.10%, p = 0.044) was discovered.In conclusion, ECP therapy exerts immunomodulatory effects by promoting balanced immune reconstitution and inducing immune tolerance, making it an attractive and promising treatment option for patients with GvHD. DisclosuresSchubert: Gilead: Consultancy. Hegenbart: Alnylam: Honoraria; Akcea: Honoraria; Janssen: Consultancy, Research Funding; Prothena: Research Funding; Pfizer: Consultancy, Honoraria. Schönland: Prothena: Honoraria, Other: Travel grants; Takeda: Honoraria, Other: Travel grants; Pfizer: Honoraria; Janssen: Honoraria, Other: Travel grants, Research Funding; Sanofi: Research Funding. Müller-Tidow: Pfizer: Research Funding; Bioline: Research Funding; Janssen: Consultancy, Research Funding. Dreger: Riemser: Consultancy, Research Funding, Speakers Bureau; Bluebird Bio: Consultancy; Roche: Consultancy, Speakers Bureau; AbbVie: Consultancy, Speakers Bureau; BMS: Consultancy; AstraZeneca: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Janssen: Consultancy; Gilead Sciences: Consultancy, Speakers Bureau. Schmitt: MSD: Membership on an entity's Board of Directors or advisory committees; Hexal: Other: Travel grants, Research Funding; Kite Gilead: Other: Travel grants; TolerogenixX: Current holder of individual stocks in a privately-held company; Bluebird Bio: Other: Travel grants; Novartis: Other: Travel grants, Research Funding; Apogenix: Research Funding. Schmitt: Therakos/Mallinckrodt: Research Funding; TolerogenixX Ltd: Current Employment; Jazz Pharmaceuticals: Other: Travel grant; Hexal: Other: Travel grant.

The Potential Roles of Mucosa-Associated Invariant T Cells in the Pathogenesis of Gut Graft-Versus-Host Disease after Hematopoietic Stem Cell Transplantation

Background & Purpose: Gut acute graft-versus-host disease (aGVHD) is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and is associated with high mortality. Mucosal-associated invariant T cells (MAITs) are a group of innate-like T cells enriched in the intestine that can be activated by riboflavin metabolites from various microorganisms. However, little is known about the function or mechanism of action of MAIT in the occurrence of gut aGVHD in the human body.Methods & Cases: In our study, multiparameter flow cytometry (FCM) was used to evaluate the number of MAIT cells (MAITs) and functional cytokines. 16S V34 region amplicon sequencing analysis was used to analyse the intestinal flora of transplant patients. In vitro stimulation and coculture assays were used to study the activation and function of MAITs. The number and distribution of MAITs in intestinal tissues were analysed by immunofluorescence technology. We prospectively studied the enrolled 150 consecutive patients who underwent allo-HSCT in our institute.Results: The number and frequency of MAITs in infused grafts in gut aGVHD patients were lower than those in non-gut aGVHD patients (Figure). Recipients with a high number of MAITs in infused grafts had a higher abundance of intestinal flora in the early post-transplantation period (+14 days). At the onset of gut aGVHD, the number of MAITs decreased in peripheral blood, and the activation marker CD69, chemokines CXCR3 and CXCR4 and transcription factors Rorγt and T-bet tended to increase. Furthermore, when gut aGVHD occurred, the proportion of MAIT17 was higher than that of MAIT1. The abundance of intestinal flora with non-riboflavin metabolic pathways tended to increase in gut aGVHD patients. MAITs secreted more granzyme B, TNF-α and IFN-γ under the stimulation of IL-12/IL-18 (non-TCR signal) and secreted most of the IL-17 under the stimulation of CD3/CD28 (TCR signal). MAITs inhibited the proliferation of CD4+ T cells in vitro.Conclusions: In conclusion, the lower number of MAITs in infused grafts was related to the higher incidence of gut aGVHD, and the number of MAITs in grafts may affect the composition of the intestinal flora of recipients early after transplantation. The flora of the riboflavin metabolism pathway activated MAITs and promoted the secretion of intestinal protective factors to affect the occurrence of gut aGVHD in humans. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Acute GvHD after HLA-Haploidentical Hematopoietic Cell Transplantation with Regulatory and Conventional T Cell Immunotherapy Does Not Adversely Affect Transplantation Outcomes

IntroductionAdoptive immunotherapy with CD4+CD25+FOXP3+ regulatory T cells (Tregs) and conventional T cells (Tcons) has been employed after allogeneic hematopoietic stem cell transplantation (HSCT) to prevent Graft-versus-Host Disease (GvHD) while mantaining Graft-versus-Leukemia effect in the absence of any further immune suppressive GvHD prophylaxis (Di Ianni, Blood 2011; Martelli, Blood 2014). A recent study of age-adapted haploidentical HSCT with Treg/Tcon adoptive immunotherapy (Treg/Tcon haplo-HSCT) resulted in an unprecedented 75% chronic GvHD (cGvHD)/leukemia-free survival (CRFS) in acute myeloid leukemia patients. Acute GvHD (aGvHD) occured in 33% of patients and was the most frequent complication of the procedure (Pierini, Blood Advances 2021). Here, we analyzed clinical and histological characteristics of aGvHD and evaluated its impact on outcomes in patients who received Treg/Tcon haplo-HSCT.MethodsWe retrieved data of patients who underwent Treg/Tcon haplo-HSCT at Perugia University Hospital and who were evaluable for aGvHD from January 2015 until June 2021 (clinicaltrials.gov: NCT03977103). Histological features and lymphoid infiltration by immunohistochemistry were analyzed in gut samples of patients who received diagnostic colonscopy and biopsy for gastrointestinal symptoms.ResultsA total of 105 patients engrafted after Treg/Tcon haplo-HSCT. Mean age at transplant was 48 years (range 18-70). Acute leukemia was the most frequent diagnosis (78/105 myeloid, 23/105 lymphoblastic), 4 patients had myelodisplastic syndrome and 2 multiple myeloma. Twenty-seven (26%) patients had active disease at the time of HSCT. Fourteen (13%) patients received Treg/Tcon haplo-HSCT as a second HSCT procedure. Thirty-one (29%) patients developed grade II-IV aGvHD. Four patients had grade II aGvHD, 23 grade III, and 4 grade IV. Almost all patients (90%) presented gut involvement, while skin was involved in 64% of them and liver in 35%. To explain why gut symptoms such as diarrhea and abdominal pain were more frequently present than symptoms of skin or liver involvement, we evaluated 40 histological samples from 34 patients who received colonoscopy for suspected aGvHD. Twenty-two of them (65%) received a clinical diagnosis of aGvHD and pharmacological immune suppression was promptly started. Clinical diagnosis did not always match histological findings: biopsies were suggestive of aGvHD in 20/22 patients who required treatment, but also in 8/12 patients who did not. We found no difference in median CD3+ cell infiltration among patients who did not need immune suppressive treatments and patients with aGvHD (460 cells/mmq vs 446 cells/mmq). However, FOXP3+ Tregs preferentially infiltrated gut mucosa of patients who resolved clinical symptoms with no need of immune suppressive treatments (120 cells/mmq vs 59 cells/mmq, p = 0.008; FOXP3/CD3 ratio: 0.27 vs 0.18, p = 0.004). This observation suggested Treg defective homing to gut mucosa of aGvHD patients. Indeed, we found infused Tregs expressed lower levels of gut homing receptor α4β7 in comparison with Tcons (p< .001).Despite severe presentation at diagnosis, aGvHD resolved in the majority of patients (71%), and immune suppressive therapy could be completely withdrawn after a relatively short-course (median: 98 days, range 48-404). Sixteen/31 patients (52%) were steroid refractory and required further treatments after first-line (e.g. Ruxolitinib). Only two of them developed moderate/severe cGvHD and one is alive and off-therapy.Indeed, aGvHD diagnosis did not result in higher incidence of non-relapse mortality (cumulative incidence of 29% in aGvHD patients vs 27% in controls; p=non significant, ns, Fig.1A), relapse (13% vs 20%; p=ns, Fig.1B), or cGvHD (6.5% vs 3%; p=ns, Fig.1C). In fact, CRFS of aGvHD patients was similar to CRFS of patients that did not develop aGvHD (Fig. 1D, p=ns, median follow up of live patients: 1246 days).ConclusionsTreg/Tcon haplo-HSCT is followed by low rates of relapse and cGvHD and results in good CRFS in high risk leukemia patients. aGvHD after Treg/Tcon haplo-HSCT preferentially involves the gut where Treg homing is defective. Strategies that enhance Treg homing in the gut (e.g. low-dose IL-2) may help reduce aGvHD after Treg/Tcon haplo-HSCT. In conclusion, despite aGvHD occurred in 29% of patients, it was responsive to treatments and it did not result in disease relapse or cGvHD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Differentiation of a CD4/CD8αβ Double Positive T Cell Population from the CD8 Pool Is Both Predictive and Sufficient to Mediate Graft-Vs-Host Disease

Acute graft-vs-host disease (aGVHD) and cancer relapse remain the primary complications following an allogeneic hematopoietic stem cell transplantation (allo-HSCT) for malignant blood disorders. While post-transplant cyclophosphamide combined with standard GVHD prophylaxis has greatly reduced the overall prevalence and severity of aGVHD, relapse rates remain a concern. There is thus a need to identify the specific human T cell populations mediating GVHD vs GVL activity as a means to develop targeted therapeutics capable of controlling aGVHD without inhibiting GVL activity. In this study, we identify a novel human T cell population that develops after transplant that is predictive and sufficient for GVHD pathology.To determine the role of human T cell populations in aGVHD, we performed xenogeneic transplantation studies using primary human graft tissue from a variety of sources (peripheral blood, G-CSF mobilized peripheral blood, bone marrow and umbilical cord blood) in addition to collecting primary human aGVHD blood samples from our clinic. Using the LD50 dose of human graft tissue, we identified a novel mature CD4 +/CD8αβ + double positive (DP) T cell population that only developed after transplantation. The development of this population was further confirmed in aGVHD patients from our clinic. The presence of DP T cells, irrespective of graft source, was also predictive of lethal GVHD in as early as one week after xenogeneic transplantation.To identify the origin of DP T cells, we transplanted isolated human CD4 or CD8 T cells into mice which showed that DP T cells only arise from the CD8 pool. Furthermore, re-transplantation of flow-sorted CD8 T cells from GVHD mice did not reveal a 2nd wave of DP T cell differentiation. This data, in addition to their highly proliferative state, suggests that DP T cells represent highly activated CD8 T cell clones. The ability of these CD8-derived DP T cells to gain CD4 expression coincides with their co-expression of both RUNX3 and THPOK, the master transcription factors of the CD8 and CD4 lineages respectively, that classically repress each other. Intracellular cytokine staining also revealed that DP T cells are the primary activated T cell population in xenogeneic GVHD, secreting both modulatory and cytotoxic cytokines (e.g. IFNγ, IL-17A, IL-22, perforin and granzyme). Ex vivo re-stimulation or re-transplantation of flow-sorted DP T cells showed that this T cell population is capable of dividing and expanding independent of CD4 and CD8 single positive T cells with the majority of the isolated DP T cells retaining their co-expression of CD4 and CD8.Finally, transplantation of either isolated human peripheral blood CD4 or CD8 T cell populations were capable of causing lethal GVHD. Conversely, re-transplantation of flow-sorted DP, CD8 or CD4 T cells from GVHD mice revealed that DP and CD4 T cells are sufficient to mediate GVHD pathology but re-transplanted CD8 T cell are not. This correlates with the absence of DP T cell differentiation in that re-transplanted CD8 population. The differentiation of DP T cells from chronically activated CD8 T cells represents a novel mechanism of GVHD pathology not previously described. The presence of DP T cells in other chronic inflammatory human diseases also suggests a broader pathology mediated by DP T cells. Further understanding of DP T cell differentiation and pathology may lead to targeted prophylaxis and/or treatment regimens for aGVHD and other human chronic inflammatory diseases. [Display omitted] DisclosuresCapitini: Nektar Therapeutics: Honoraria; Novartis: Honoraria.

Antigenic Modulation of CD6 By Itolizumab Is a New Mechanism for Effector T Cell Inhibition

IntroductionAcute Graft-Versus-Host Disease (aGVHD) is a serious complication of hematopoietic stem cell transplantation (HSCT) that is primarily driven by alloreactive T cells. CD6 is a costimulatory receptor primarily expressed on T cells that promotes synapse formation, T cell activation, and migration into tissues by engaging with its ligand, activated leukocyte cell adhesion molecule (ALCAM). CD6 is expressed on reconstituting T cells soon after HSCT (Rambaldi et al., 2019) and early studies have shown that ex-vivo depletion of CD6 + donor cells prior to HSCT decreases the incidence of aGVHD (Soiffer et al., 1992; Soiffer et al., 1998). The reduced levels of aGVHD were attributed to an increased prevalence of CD6 - T cells that were less alloreactive (Rasmussen et al., 1994). Therefore, modulating activity of the CD6-ALCAM pathway may ameliorate aGVHD. Itolizumab is a humanized anti-CD6 monoclonal antibody that was previously described to block the engagement of ALCAM, thereby inhibiting T cell activity and trafficking. Here, we elucidate a second and highly novel mechanism in which antibody-mediated loss of CD6 from the surface of T cells results in CD6 low T cells that are hyporesponsive to T cell stimulation.MethodsTo assess the molecular mechanisms for itolizumab-induced loss of cell surface CD6, peripheral blood mononuclear cells (PBMCs) were exposed to different conditions including treatment with selected protease inhibitors, a membrane inhibitor, or Fc receptor blocking antibodies in the presence of itolizumab. Furthermore, B cells, NK cells, and monocytes were isolated from PBMCs and mixed with T cells in combination with itolizumab to evaluate cell contact requirements for loss. Following treatment, cell surface levels of CD6 protein were assessed by flow cytometry using a non-competing anti-CD6 detection antibody while analysis of full length CD6 was detected by western blot from total protein lysate. The soluble form of CD6 (sCD6) was analyzed by electrochemiluminescence and immunoprecipitation from supernatant of treated cells.ResultsFollowing treatment with itolizumab, the levels of cell surface CD6 was reduced as assessed by flow cytometry and western blot. Concurrent with a loss of cell surface CD6, levels of sCD6 in the cell supernatant increased, suggesting CD6 is primarily cleaved from the cell surface rather than internalized. Characterization of the cleaved sCD6 product by western blot revealed a 30KDa product. Itolizumab-induced changes in both levels of cell surface CD6 and sCD6 was abrogated by both 4-benzenesulfonyl fluoride hydrochloride (AEBSF), a serine protease inhibitor and cytochalasin D, an inhibitor of actin polymerization that prevents movement within the cell membrane and blocks endocytic trafficking. This suggests that a membrane-bound serine protease is responsible for cleavage.Itolizumab-induced CD6 cleavage did not occur with T cells alone and required the presence of other immune cells including monocytes and NK cells, but not B cells (Figure 1). In addition, when monocytes and T cells were separated by a membrane, cleavage of CD6 was not observed in the presence of itolizumab, indicating that cell-to-cell contact is required. Furthermore, blocking antibodies indicated that functional Fc receptors, especially FcγRIA, were required, suggesting that binding of itolizumab to both an Fc receptor on monocytes and CD6 on T cells predicated the cleavage event. The resulting CD6 low T cells were hyporesponsive to T cell stimulation, even in the absence of itolizumab, as indicated by reduced expression of PD-1, CD71 and CD25 as well as inflammatory cytokines (Figure 2). Inhibition was observed for both naïve and effector/memory T cell subsets.ConclusionsOur results reveal a novel mechanism of antigenic modulation by itolizumab in which CD6 is cleaved from the T cell surface and released in a soluble form. Cleavage of CD6 occurs via a membrane-bound serine protease and appears dependent upon the engagement of itolizumab with Fc receptor(s) present on monocytes and NK cells. The loss of cell surface CD6 results in T cells with reduced responses to TCR-mediated stimulation. As such, treatment of aGVHD patients with itolizumab may reduce the alloreactivity of donor T cells, ameliorating disease symptoms and improving the clinical outcomes in these patients. [Display omitted] DisclosuresChu: Equillium: Current Employment. Marrocco: Equillium, Inc.: Current Employment. Tiet: Equillium, Inc.: Current Employment. Ampudia: Equillium, Inc.: Current Employment. Connelly: Equillium: Current Employment, Divested equity in a private or publicly-traded company in the past 24 months, Membership on an entity's Board of Directors or advisory committees. Ng: Equillium: Current Employment, Current equity holder in publicly-traded company, Divested equity in a private or publicly-traded company in the past 24 months.

Mesenchymal Stromal Cells Plus Anti-CD25 Antibody and Calcineurin Inhibitors for Steroid-Resistant Acute Graft-Versus-Host Disease: A Multicenter, Randomized, Phase 3 Trial

INTRODUCTION Steroid-resistant (SR) acute graft-versus-host disease (aGVHD) lacks standard second-line treatment. Mesenchymal stromal cells (MSCs) have potential efficacy in SR aGVHD. To assess efficacy and safety of MSCs combined with anti-CD25 antibody and calcineurin inhibitors as second-line therapy for SR aGVHD.METHORDS A randomized phase 3 trial involved 203 SR aGVHD patients at 10 centers in China (September 2014-March 2019). Final follow-up was on June 30, 2020. Participants were randomized in a 1:1 ratio to receive second-line therapy (anti-CD25 antibody combined with calcineurin inhibitors) with MSCs (n=101) or without MSCs (n=102). The primary endpoint was overall response (OR) at day 28 with a predefined threshold of -20%. Secondary and safety endpoints included durable OR at day 56, failure-free survival, overall survival (OS), chronic GVHD (cGVHD), infection, haematological toxicity and relapse.RESULTS Of 203 patients, 198 (97.5%; mean age, 30.1years; 40.4% women) completed the study. OR at day 28 was higher in the MSCs group than in the control group (82.8% [82 patients] vs 70.7% [70]; odds ratio, 2.00; 95% confidence interval [CI], 1.01-3.94; P=.043). Durable OR at day 56 was also higher in the MSCs group (78.8% [78 patients] vs. 64.6% [64]; odds ratio, 2.02; 95% CI, 1.08-3.83; P=.027). The median failure-free survival was longer in the MSCs group compared with control group (11.3 months vs. 6.0 month; hazard ratio (HR) 0.68; 95% CI, 0.48-0.95, P=.024). The 2-year cumulative incidence of cGVHD was 39.5% (95% CI, 29.3%-49.4%) and 62.7% (51.4%-72.1%) in the MSCs and control groups (HR 0·55, 95% CI, 0·36-0·84; P=0·005). Within 180 days after study treatments, the most common grade 3 and 4 adverse events were infections (65 [65.7%] in the MSCs group vs 78 [78.8%] in the control group), haematological toxicity (37 [37.4%] vs 53 [53.5%]).CONCLLUSIONS MSCs plus second-line treatments may increase the efficacy of SR aGVHD, decrease drug toxicity of second-line therapy and cGVHD development, and are well tolerated. DisclosuresNo relevant conflicts of interest to declare.

The Potential Roles of Mucosa-Associated Invariant T Cells in the Pathogenesis of Gut Graft-Versus-Host Disease After Hematopoietic Stem Cell Transplantation.

Gut acute graft-versus-host disease (aGVHD) is a serious complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and is associated with high mortality. Mucosa-associated invariant T (MAIT) cells are a group of innate-like T cells enriched in the intestine that can be activated by riboflavin metabolites from various microorganisms. However, little is known about the function or mechanism of action of MAIT cells in the occurrence of gut aGVHD in humans. In our study, multiparameter flow cytometry (FCM) was used to evaluate the number of MAIT cells and functional cytokines. 16S V34 region amplicon sequencing analysis was used to analyze the intestinal flora of transplant patients. In vitro stimulation and coculture assays were used to study the activation and function of MAIT cells. The number and distribution of MAIT cells in intestinal tissues were analyzed by immunofluorescence technology. Our study showed that the number and frequency of MAIT cells in infused grafts in gut aGVHD patients were lower than those in no-gut aGVHD patients. Recipients with a high number of MAITs in infused grafts had a higher abundance of intestinal flora in the early posttransplantation period (+14 days). At the onset of gut aGVHD, the number of MAIT cells decreased in peripheral blood, and the activation marker CD69, chemokine receptors CXCR3 and CXCR4, and transcription factors Rorγt and T-bet tended to increase. Furthermore, when gut aGVHD occurred, the proportion of MAIT17 was higher than that of MAIT1. The abundance of intestinal flora with non-riboflavin metabolic pathways tended to increase in gut aGVHD patients. MAIT cells secreted more granzyme B, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ under the interleukin (IL)-12/IL-18 stimulation [non-T-cell receptor (TCR) signal] and secreted most of the IL-17 under the cluster of differentiation (CD)3/CD28 stimulation (TCR signal). MAIT cells inhibited the proliferation of CD4+ T cells in vitro. In conclusion, the lower number of MAIT cells in infused grafts was related to the higher incidence of gut aGVHD, and the number of MAIT cells in grafts may affect the composition of the intestinal flora of recipients early after transplantation. The flora of the riboflavin metabolism pathway activated MAIT cells and promoted the expression of intestinal protective factors to affect the occurrence of gut aGVHD in humans.

Extracorporeal photopheresis in the treatment of acute graft-versus-host disease: A multicenter experience

Background and objectivesExtracorporeal photopheresis (ECP) is one of the second-line treatment strategies in steroid-refractory acute graft-versus-host disease (aGvHD). We aimed to share our multicenter experience using ECP in our steroid-refractory aGvHD patients. Materials and methodsA retrospective observational series of 75 aGvHD patients from 4 transplant centers were analyzed. All ECP procedures were performed with the off-line system. All patients received ECP as second-line therapy. Results74.7 % of aGvHD patients were grade 3 or 4. The overall response rate was 42.7 % (32/75) in aGvHD including 17 complete responses (22.7 %). Median follow-up was 6 months (range, 1–68). Median overall survival (OS) was 5 months for non-responders and 68 months for responders (p < 0.001). Twenty-seven (36 %) patients are alive, and 48 (64 %) patients have died. ConclusionsEarly initiated ECP could be an effective treatment alternative in patients with steroid-refractory aGvHD.

IFNG-AS1 and MAF4 long non-coding RNAs are upregulated in acute leukemia patients who underwent bone marrow transplantation

Purpose of the studyAcute graft versus host disease (aGVHD) is an immune-mediated reaction that results in impaired immune and body function after allogeneic hematopoietic stem cell transplantation (allo-HSCT). lncRNAs have been discovered as particular T cell regulators, and alloreactive T cells have been known as a critical factor in aGVHD. As a result, we investigated the importance of lnc-MAF4 and IFNG-AS1 expression levels in aGVHD patients versus non-aGVHD patients. Material and MethodsThis research included 38 patients with hematological disorders who were undergoing primary allo-HSCT. Human identical siblings or unrelated donors were used to collect stem cell. Samples were taken within days 0, 7, 14, 28, and 52±8 after transplantation. The expression of lncRNA levels was measured using the QRT-PCR technique. And the data were analyzed using GraphPad Prism 6 ResultsOur data revealed that LncRNA MAF4 and INFG-AS1 expression levels in aGVHD were not significantly different compared to the non-GVHD group immediately after transplantation, nor at day 7 or 14. However, the aGVHD group showed an overt up-regulation of the two lncRNAs on samples taken at day 28 and 52±8 compared to non-GVHD patients. DiscussionSince the intracellular pathway of these lncRNAs shows a direct relationship with the IFNγ cytokine production resulting in differentiation to TH1 cells and inhibition of differentiation to TH2 cells, they can be, therefore, considered as suitable molecular candidates for the prediction of aGVHD in patients receiving HSCT.

Circulating miR-455-3p, miR-5787, and miR-548a-3p as potential noninvasive biomarkers in the diagnosis of acute graft-versus-host disease: a validation study.

Currently, acute graft-versus-host disease (aGVHD) diagnosis is based on clinical features and pathological findings. Until now, there is no non-invasive diagnostic test for aGVHD. MicroRNAs may act as promising predictive, diagnostic, or prognostic biomarkers for aGVHD. The purpose of the current study was to validate circulating microRNAs as diagnostic biomarkers to assist clinicians in promptly diagnosing aGVHD, so that treatment can be initiated earlier. In the present study, we evaluated six microRNAs (miR-455-3p, miR-5787, miR-6729-5p, miR-6776-5p, miR-548a-3p, and miR-6732-5p) selected from miRNA array data in 40 aGVHD patients compared to 40 non-GVHD patients with RT-qPCR. Target genes of differentially expressed microRNAs (DEMs) were predicted using Targetscan, miRanda, miRDB, miRWalk, PICTAR5, miRmap, DIANA, and miRTarBase algorithms, and their functions were analyzed using EnrichNet, Metascape, and DIANA-miRPath databases. The expressions of plasma miR-455-3p and miR-5787 were significantly downregulated, whereas miR-548a-3p was significantly upregulated in aGVHD patients compared to non-GVHD patients. Moreover, DEMs showed potentially high diagnostic accuracy for aGVHD. In silico analysis of DEMs provided valuable information on the role of DEMs in GVHD, immune regulation, and inflammatory response. Our study suggested that miR-455-3p, miR-5787, and miR-548a-3p could be used as potential noninvasive biomarkers in the diagnosis of aGVHD in addition to possible therapeutic targets in aGVHD.

Biomarkers for Clinical Response of Extracorporeal Photopheresis

Biomarkers for Clinical Response of Extracorporeal Photopheresis

Similar Outcomes of Early Failure Steroid Dependent Acute GvHD and Upfront Steroid Refractory Acute GvHD: Implications on Clinical Trials

BackgroundSecond-line therapy for acute GvHD (aGvHD) remains an unmet need. Clinical trials for second-line therapy typically enroll patients with steroid refractory (SR) aGvHD, defined as worsening after 3 days or no improvement after 7-10 days of corticosteroid based therapy. Approximately 60% patients respond by day 28 of initial corticosteroid based therapy. A significant proportion of these patients lose their response during corticosteroid taper, and are thus steroid dependent (SD). We hypothesized that patients who have recurrent or progressive aGvHD prior to reaching 50% of the initial corticosteroid dose (early failure, SD-EF) will have an outcome similar to aGvHD that is upfront SR (USR) at initial presentation, and thus should be enrolled on clinical trials for SR aGvHD without re-challenging with corticosteroids to prove refractoriness.MethodsThis was a retrospective, single-center study of allo-HCT recipients between 2005 and 2013. Records of all patients with a diagnosis of aGvHD were reviewed. Patients were included if they were on a calcineurin inhibitor and received systemic corticosteroids for clinical or histologically proven aGvHD and were USR or SD. Patients were excluded if they had progression or relapse of their underlying disease before aGvHD or if they received >1 allo-HCT. USR aGvHD was defined as worsening of aGvHD within the first 3 days or no improvement after 10 days of starting ≥1 mg/kg of prednisone-equivalent. SD aGvHD was defined as initial response to corticosteroids with recurrence or worsening on tapering. The SD group was subdivided into early failure (SD-EF) and late failure (SD-LF) if they recurred at ≥50% of the starting corticosteroid dose and <50% of the starting corticosteroid dose, respectively. The primary outcomes were overall survival (OS) and non-relapse mortality (NRM) at 1 y and 2 y after initiation of corticosteroids for aGvHD. Secondary outcomes were cause of death and use of second line therapy. Cause of death due to GvHD included patients who died from infection while on therapy for active GvHD.ResultsOf 596adult patients who received allo-HCT, 465 developed aGvHD (78%). Patients were excluded for: recurrent or progressive disease prior to aGvHD onset (6), receiving >1 allo-HCT (6), and not being on a calcineurin inhibitor (4). Systemic corticosteroids were used in 345 (74%). Incidence of USR and SD aGvHD among those treated with corticosteroids was 13% (43/345) and 29% (100/345). SD-EF and SD-LF accounted for 45% and 55% of all SD aGvHD. Table 1 outlines baseline characteristics. Median follow up of the cohort was 1.7 y (range, 0.03 to 12.2) and OS was 1.8 y (95% CI, 1.2 to 3.1). Second line therapy was required in 65%, 42%, and 27% (p=0.001) of USR, SD-EF and SD-LF patients, respectively.Primary endpoints were OS and NRM at 1 y and 2 y. OS at 1 y and 2 y for USR vs SD were 47% vs 68% (p=0.002) and 44% vs 51% (p=0.077), respectively. NRM at 1 y and 2 y for USR vs SD groups were 40% vs 20% (p=0.007) and 42% vs 28% (p=0.04). As hypothesized, outcomes for USR and SD-EF were similar: OS at 1 y and 2 y in USR vs SD-EF were 47% vs 60% (p=0.08) and 44% vs 42% (p=0.49), respectively; NRM at 1 y and 2 y for USR vs SD-EF were 40% vs 29% (p=0.19) and 42% vs 40% (p=0.53). GvHD leading to NRM was similar in USR and SD-EF (63% vs 44%, p=0.24).The OS was similar for SD-EF vs SD-LF at 1 y (60% vs 75%, p=0.1) and 2 y (42% vs 58%, p=0.09). NRM was higher for SD-EF compared to SD-LF at both 1 y (29% vs 13%, p=0.04) and 2 y (40% vs 18%, p=0.02), primarily due to a higher rate of GvHD related deaths (SD-EF vs SD-LF 44% vs 16%, p=0.03). This was in contrast to the SD-LF group, where relapse accounted for a higher proportion of death compared with 53% of deaths due to relapse for SD-LF vs 28% for SD-EF (p=0.03)ConclusionsForty-five percent of patients with SD aGvHD have SD-EF. Our data suggests that patients with USR and SD-EF remain at a high risk of death from complications of GvHD, and both groups should be considered for second line therapy of aGvHD. The current approach of requiring SD aGvHD patients to fulfill SR aGvHD criteria prior to enrolling on a study of second-line therapy increases corticosteroid exposure and related morbidity, without clear benefit of long-term outcome. As interventions with safer adverse effect profiles are developed, it is imperative that the eligibility criteria of second-line therapy be modernized to include SD-EF aGvHD, to allow this high-risk group to derive the benefit of experimental therapeutics. [Display omitted] DisclosuresSavani:Jazz Pharmaceuticals: Speakers Bureau. Byrne:Pfizer: Membership on an entity's Board of Directors or advisory committees; Karyopharm: Research Funding; Concert Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Jagasia:Therakos: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Mallinckrodt: Consultancy.

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.

The significance of focally enhanced gastritis in haematopoietic stem cell transplant recipients.

The histological diagnosis of acute gastric graft-versus-host-disease (aGVHD) in patients with a history of haematopoietic stem cell transplant (HSCT) is based on the presence of epithelial cell apoptosis and karyorrhectic debris. There is, however, limited information on the histological findings in patients who develop symptoms several months after transplant. Focally enhanced gastritis (FEG), defined by the presence of focal periglandular lymphohistiocytic inflammation with neutrophilic or lymphocytic intra-epithelial infiltration of gastric glands, has been described in patients with inflammatory bowel disease and in HSCT patients. The pattern closely resembles the focal periductal inflammation and lymphocytic exocytosis seen in chronic GVHD of the salivary gland. We sought to evaluate the significance of FEG in HSCT patients. Gastric biopsies from 151 HSCT patients who underwent endoscopies for GVHD-like symptoms were identified. Time from transplant to biopsy, presence of extra-gastric GVHD, medications and outcome were noted. Thirty-five biopsies showed FEG and 21 showed aGVHD; the remainder were either normal or showed non-specific changes. Twenty-one (60%) FEG patients had concurrent histologically proven extra-gastric GVHD. The time to biopsy in FEG patients was significantly longer than in aGVHD patients (162 versus 57days, P<0.01). Prior or subsequent gastric biopsies of 14 patients in the FEG cohort were also evaluated and, of these, six showed aGVHD while one showed persistent FEG. These findings suggest that FEG probably represents a form of late-occurring GVHD. This histological pattern should not be overlooked when identified in gastric biopsies from HSCT patients.