aGVHD Diagnosis Research Articles

Treatment Patterns and Clinical Outcomes of Patients with Moderate to Severe Acute Graft-Versus-Host Disease: A Multicenter Chart Review Study.

Acute graft-versus-host disease (aGVHD) remains a barrier to successful allogeneic hematopoietic stem cell transplantation (HSCT) outcomes. Contemporary comprehensive analyses of real-world clinical outcomes among patients who develop aGVHD post-HSCT are needed to better understand the unmet needs of this patient population. This multicenter, retrospective chart review describes treatment patterns and clinical outcomes among patients (≥18 years old) from Finland, Sweden, and France who developed grades II-IV aGVHD after their first HSCT (January 2016-June 2017). From 13 participating centers, 151 patients were included. The median (Q1, Q3) age at HSCT was 56 (45, 62) years old. One line of aGVHD treatment was received by 47.7%, and the most common first-line treatment was methylprednisolone (alone or in a combination regimen, 74.2%; monotherapy, 25.8%). Among patients treated with methylprednisolone, 79.5% achieved a complete or partial response. The median (Q1, Q3) number of treatment lines was 2.0 (1.0, 3.0). The median (Q1, Q3) time to obtain an aGVHD diagnosis from transplant was 29.5 (21.0, 44.0) days, and 14.5 (7.0, 34.0) days to achieve the best response for 110 evaluable patients. At 6 and 12 months, 53.6% and 49.0%, respectively, achieved a complete response. Chronic GVHD occurred in 37.7% of patients, and aGVHD reoccurred in 26.5%. Following aGVHD diagnosis, mortality rates were 30.0% at 6 months and 37.3% at 12 months. Findings from this study demonstrate a continuing unmet need for new therapies that control aGVHD and improve mortality.

Chronic GVHD after steroid-sensitive, -dependent, and -refractory acute GVHD: incidence and clinical outcomes.

Chronic graft-versus-host disease (cGVHD) is a major limitation to the long-term success of allogeneic hematopoietic cell transplantation (HCT). Our prior study of acute GVHD (aGVHD) defined distinct treatment-response groups based on the response to first-line corticosteroids: steroid-sensitive (SS), steroid-resistant (SR), and steroid-dependent (SD) aGVHDs. We conducted a retrospective, single-institution, cohort study to assess the incidence, risk factors, and clinical outcomes of patients with cGVHD after a previous diagnosis of SS, SD, or SR aGVHD, compared with those with no history of aGVHD. Among 784 consecutive adult and pediatric recipients of HCT for hematologic malignancies between 2008 and 2016, 347 (44%) developed aGVHD, with 13% SS, 12% SD, and 19% SR aGVHD. The 3-year cumulative incidence of cGVHD was 25%. Among those with cGVHD, 39% had no prior aGVHD diagnosis, whereas among those with a prior aGVHD diagnosis, 16% had SS, 24% had SD, and 21% had SR aGVHD. Mild or moderate cGVHD was highest among those with preceding SD aGVHD, whereas severe cGVHD was most frequent among those with previous SR aGVHD. We identified SD and SR aGVHDs as significant independent risk factors for the development of cGVHD after allogeneic HCT, whereas SS aGVHD was not a risk factor. Our study demonstrates that cGVHD after SD aGVHD did not have an intermediate prognosis between SR and SS groups as hypothesized; rather, cGVHD after both SD and SR aGVHD have similar prognoses. Our findings suggest that previous aGVHD response states are important predictors of cGVHD severity and outcomes.

Differential protein expression in endothelial cells exposed to serum from patients with acute graft-vs-host disease, depending on steroid response.

Graft-versus-host disease (GVHD) is a complication of allogeneic haematopoietic cell transplantation. Endothelial injury is crucial as pathophysiological substrate for GVHD. GVHD first-line treatment is high-dose corticosteroids, although some patients are steroid-refractory. Through the present study, we compared the endothelial proteomic profiles in response to serum from steroid-refractory acute GVHD (SR-aGVHD) and steroid-sensitive acute GVHD (SS-aGVHD) patients. Blood samples from SR-aGVHD (n= 4) and SS-aGVHD (n= 8) patients were collected at aGVHD diagnosis. Endothelial cell cultures were exposed (48 h) to patients' serum. Protein extraction and proteomic analysis were performed. Differences were statistically evaluated by multivariate analysis. Forty-four proteins contributed to separate all samples into the two study groups, among which 15 participated significantly (p< 0.05), 10 exhibiting a fold change >1.2. Differentially expressed proteins were mainly associated with oxidative phosphorylation (Cytochrome C oxidase subunit 6B1, CX6B1), inflammation and angiogenesis (Apolipoprotein D, APOD), cell survival (Rapamycin-insensitive companion of mTOR, RICTR), and oxidative stress (Riboflavin kinase, RIFK). This pilot study used a novel approach to distinguish the aGVHD response to steroid treatment. The proteins differentially expressed could constitute potential biomarkers for steroid-treatment response. These findings signify a step forward to identify the mechanisms of response to steroids, of high clinical relevance considering the SR-aGVHD elevated mortality.

Advanced Machine Learning Techniques to Predict Gvhd Occurrence and Severity with High Accuracy

Introduction Graft versus host disease (GVHD) after allo-HSCT is dependent on conditioning regimen, GVHD prophylaxis, HLA matching, as well as recipient-donor discrepancies in sex, age, and CMV status. GVHD is a leading cause of transplant-related mortality with incidence varying between 30% to 70%. Few machine learning (ML) models have been implemented to accurately quantify a patient's risk for aGVHD development and subsequent severity with mixed results, utilizing various ML methodologies of analyzing clinical data, genetic differences, and biomarkers. Comparing across four different ML algorithms, we demonstrate exceptionally high accuracy for prediction of acute GVHD occurrence, severity (Grade I-II vs III-IV), disease relapse, and survival. Materials and Methods Data on 868 adult patients (age 18-76) that received either MA or RIC for any transplant indication between January 2004 and July 2018 at The Ohio State University James Cancer Center was analyzed. Predictive models were developed utilizing four different machine learning algorithms: logistic regression, k-nearest neighbors (KNN), decision tree with hyperparameter tuning, and multilayer perceptron (MLP) neural networks. Models were trained and tuned using K-fold cross validation, and prediction performance was quantified by the receiving operating characteristic area-under-the-curve (ROC AUC) and accuracy. Features included patient demographics (age, gender, KPS/ECOG, race, disease type, disease status, donor type, graft type, sex match, HLA match, conditioning regimens groups, use of (ATG), use of alemtuzumab, GVHD prophylaxis type, date of engraftment, date of aGVHD diagnosis, organ involvement of aGVHD, steroid treatment, specific steroid therapy, date of chronic GVHD diagnosis, relapse date, date of death, and cause of death was collected. The dataset was preprocessed through the standardization of numerical features and encoding of categorical features. This dataset was separated pre-analysis into cohorts - 70% exploratory and 30% validation. Results All 868 patients were included, verified, and data cleaned. The algorithms predicted with high accuracy - >80% for GVHD occurrence and >90% for Grade I-II and Grade III-IV acute GVHD, relapse, and survival. MLP provided the most accurate predictions when compared to decision tree, KNN and logistic regression algorithms. SHapley Additive exPlanations (SHAP) analysis was used to demonstrate the top 20 features contributing to the overall output. SHAP analysis for acute GVHD occurrence is shown for MLP algorithm with age and HLA match as the most critical features. Conclusions We demonstrate the results of utilizing advanced ML algorithms for highly accurate prediction of acute GVHD occurrence, severity (Grade I-II vs III-IV), disease relapse, and survival. The difference between the models' performances may be due to the disproportionate sizes of cohorts in Grade III-IV acute GVHD as opposed to relapse and survival analyses. The risk factors identified as being most influential in affecting outcomes by our ML algorithms are consistent with those historically reported. While the importance of these features are similar to prior models, the ability of the models used to generate highly accurate results is novel contribution. The improved accuracy of our methods compared to previous reports may be due to several factors. MLP algorithm with one or two layers have ample hidden neurons to find patterns to achieve superior and robust classification for GVHD. Such deep learning models have not been used in prior classification. Decision trees also provide higher accuracy compared to KNN and regression due to the deterministic nature of the model which automates feature selection. Our single center data may also present a more homogeneous population with decreased variability of practice and GVHD prophylaxis approaches. Further studies to validate these algorithms in a more recent cohort are planned. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Cell-free DNA levels are increased in acute graft-versus-host disease.

Cell-free DNA (cfDNA) and nucleosomes, consisting of cfDNA and histones, are markers of cell activation and damage. In systemic inflammation these markers predict severity and fatality. However, the role of cfDNA in acute Graft-versus-Host Disease (aGvHD), a major complication of allogeneic hematopoietic stem cell transplantation (HSCT), is unknown. The aim of this study is to investigate the role of cfDNA as a marker of aGvHD. We followed nucleosome levels in 37 allogeneic HSCT patients and an established xenotransplantation mouse model. We determined the origin of cfDNA with a species-specific polymerase chain reaction. In the plasma of aGvHD patients, nucleosome levels significantly increased around the time of aGvHD diagnosis compared to pretransplant, concurrently with a significant increase of known aGvHD markers ST2 and REG3α. In mice, we confirmed that nucleosomes were elevated during clinically detectable aGvHD. We found cfDNA to be mainly of human origin and to a lesser extent of mouse origin, indicating that cfDNA is released by (proliferating) human xeno-reactive PBMC and damaged mouse cells. We show increased cfDNA both in an aGvHD mouse model and in aGvHD patients. We also demonstrate that donor hematopoietic cells and to a lesser degree (damaged) host cells are the cellular source of cfDNA in aGvHD. We propose that nucleosomes and cfDNA might be an additive marker for aGvHD.

Bowel wall thickness is a strong predictor of steroid-refractory acute graft-versus-host disease with gut involvement after allo-HSCT.

Acute graft-versus-host-disease (aGVHD) develops in 10-80% of allo-HSCT patients. More than half of all aGVHD cases are refractory to first-line therapy with steroids. We hypothesized that bowel wall thickness at the time of aGVHD diagnosis could be an early sign of steroid-refractory aGVHD with gut involvement. Our prospective study included 85 patients with hematological malignancies who had undergone allo-HSCT. We used an inexpensive, widespread and simple method of transabdominal ultrasonography to examine bowel wall thickness in patients suspected to have gut aGVHD. Descending colon wall thickness was significantly greater in patients with gut aGVHD later found to be steroid-refractory than in patients with steroid-sensitive gut aGVHD, with AUC-0.73 (95% CI 0.58-0.87, p = 0.013). We showed that bowel wall thickness could predict the steroid-refractoriness of aGVHD. Transabdominal ultrasonography could be used as a marker of steroid-refractory aGVHD with gut involvement after allo-HSCT.

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.

Exposure to Anti-Anaerobic Antimicrobials Around Hematopoietic Cell Transplant (HCT) Is Associated with Poor Outcomes in Pediatric Patients

BackgroundSurvival rates in pediatric hematopoietic cell transplant (HCT) have improved over the last several decades. However, the factors that influence suboptimal outcomes continue to be less well understood. Low diversity of the gut microbiota and dysbiosis are known to be associated with poor outcomes after HCT. Exposure to broad-spectrum antibiotics, particularly those with anti-anaerobic coverage, during the course of HCT can induce both loss of gut microbial diversity and worsen dysbiosis. We explored the relationship of exposure to antibiotics with outcomes after HCT among pediatric patients, specifically with respect to the development of acute graft versus host disease (aGVHD).MethodsWe performed a single institution, retrospective cohort study to evaluate the association of peri-HCT antibiotic exposures with post-HCT outcomes. All patients who underwent their first transplant between 2008-2019 at our institution were included in this study. Patient, disease, HCT, and antibiotic exposure related data were abstracted from patient medical charts. The primary independent variable of interest was exposure to antibiotics for at least 48 hours at any time point from the start of conditioning and up to 100 days after the graft infusion. Antibiotics were further classified based on anti-anaerobic activity. Antibiotic exposure was censored 7 days prior to aGVHD diagnosis. Descriptive statistics are provided. Overall survival (OS) and event-free survival (EFS) curves/functions were estimated by the Kaplan-Meier method, and compared using the log-rank test. Cumulative incidence curve/function of aGVHD (any organ), gastrointestinal aGVHD, relapse and non-relapse mortality (NRM) was estimated by the Kalbafleisch-Prentice method accounting for competing risks and compared by Gray's test. Statistical analyses were performed with SAS version 9.4ResultsA total of 492 patients underwent their first HCT at our center during the study time period. The majority of patients underwent HCT for an underlying malignant disorder (n=408, 83%). A total of 174 patients died (35%); causes of death included GVHD (n=7, 4%), infection (n=31, 17.8%), organ dysfunction (n=34, 19.5%), or relapse (n=102, 58.6%). The cohort was then divided into 2 groups based on exposure to anti-anaerobic antibiotics (n=366, 74%), and no exposure to anti-anaerobic antibiotics (n=126, 26%). Overall survival at 2 years was 59% in the group exposed to any anti-anaerobic antibiotics vs. 82% in the unexposed cohort (p<0.01). EFS at 2 years post- transplant was 61% in the group exposed to any anti-anaerobic antibiotics compared to 77% in the non-exposed group (p<0.01). Non-relapse mortality was higher in those receiving anti-anaerobic antimicrobials at 19 vs. 5%. (p<0.01). There was no significant difference in relapse incidence in the two cohorts ( Twenty-three percent (n=116) of all patients were diagnosed with any grade II-IV aGVHD by day +100 with no difference between the 2 groups (p=0.2). Cumulative incidence of grades II-IV gastrointestinal aGVHD was 5.1% (n=27), with a higher incidence in those patients who did not receive anti-anaerobic antimicrobials (11 vs. 3%, p<0.01).DiscussionIn our cohort, exposure to anti-anaerobic antimicrobials in the peri-transplant setting was associated with increased mortality. The OS and EFS were significantly lower among those exposed to even short periods of anti-anaerobic antimicrobials. No corresponding increase in relapse was seen in those who received anti-anaerobic antibiotics. Infection and organ dysfunction were the leading causes of death in both exposed and unexposed cohorts. The decreased survival does not appear to be driven by aGVHD, as the overall incidence of aGVHD was not significantly different between the two groups. The exact cause-effect relationship between antibiotic exposure and outcomes after HCT in pediatric patients remains to be elucidated. Further studies should focus on the interaction of the nascent immune system, recipient microbiota, and exposure to broad-spectrum antimicrobials, particularly as they relate to the development of GVHD. [Display omitted] DisclosuresSharma: Vertex Pharmaceuticals/CRISPR Therapeutics: Other: Salary support paid to institution; Spotlight Therapeutics: Consultancy; Vindico Medical Education: Honoraria; CRISPR Therapeutics: Other, Research Funding; Medexus Inc: Consultancy; Novartis: Other: Salary support paid to institution.

Potential of Plasmatic Biomarkers to Predict Acute Gvhd Severity: A Validation Study in a Prospective Cohort of 177 Patients

Background: Several biological markers have been reported to predict response to treatment and mortality in patients with acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplant. aGVHD is a major concern since it significantly impacts outcome, especially in patients with steroid refractory aGVHD, leading to a high mortality rate (>50%). The present study was designed to validate a prognostic score based on 6 plasmatic biomarkers: Elafin, IL2Ra, TNFRI, HGF, IL8, and REG3a (Levine et al. Blood 2012).Methods: A prospective study was conducted between 2013 and 2017 in our institution in patients presenting with aGVHD after transplantation for hematological disease. One blood sample was taken at diagnosis of aGVHD and clinical data were prospectively collected. No response at day 30 after aGVHD onset was defined as non-complete response or requirement of a second line therapy. Association of biomarkers with day-30 response and overall survival was analyzed by logistic and Cox models, respectively. Discriminative ability of the models was quantified by the c index. The biomarkers were measured by a multiplex Luminex based assay (R&D systems) except one which was measured by ELISA (REG3a).Patient characteristics: Median age at inclusion was 45 years, 63 (36 %) patients were women; hematological disease prior transplant was acute myeloid leukemia or myelodysplastic syndrome in 36 (20%), lymphoma in 31 (18%), acute lymphoid leukemia in 32 (18%) and myeloproliferative neoplasms in 24 (14%) patients. A reduced intensity conditioning regimen was used in 117 (66%) and the donor was an HLA-matched sibling donor in 51 (29%) patients, matched unrelated in 82 (46%) and HLA-mismatched in 44 (25%). Sources of stem cells were G-CSF mobilized peripheral blood stem cells in 138 (78%), bone marrow in 23 (13%) and unrelated cord blood in 16 (9%) patients. aGVHD occurred at a median of 22 days after transplant (range 6 - 77) and was graded I, II and III at time of aGVHD diagnosis for 12%, 57% and 31% of patients respectively. At inclusion, aGVHD involved skin (n=143), gut (n=138) and/or liver (n=29). At day 30 after treatment initiation, 123 (69%) patients had a complete response. The follow-up was greater than 6 months at time of analysis for all living patients. Forty patients were dead 180 days after the aGVHD episode.Results: Plasmatic biomarkers were measured at time of corticosteroids initiation at aGVHD onset in 177 patients. Predictive potential was assessed on response at day 30 and mortality at 6 months. The score was significantly associated with day-30 response and 6-month mortality, as already reported. In a multivariable analysis, after adjustment for aGVHD grade at initiation of treatment, age, donor type (related or unrelated), source of stem cells, albumin level and fever, the score was still predictive for day-30 response and 6-month mortality, along with aGVHD grade (table 1). None of the other parameters were associated with day-30 response while age and donor type were associated with 6-months mortality. The same multivariate analysis was further conducted with ST2, another strong prognostic biomarker previously described (Vander Lugt et al NEJM 2013). After adjustment for the other variables, ST2 was associated with 6-month mortality only (table 1).Finally, we used the c-index to estimate the discriminative ability of the score alone, ST2 alone or the combination of score and ST2. The score had the highest value to discriminate response at day-30 (c-index=0.68) as compared to ST2 (c-index=0.60) and the combination score+ST2 (c-index=0.66). The combination score+ST2 had the highest value to predict 6-month mortality (c-index=0.68) as compared to the score (c-index=0.64) and ST2 (c-index=0.59) alone.Conclusion: The present study shows the potential of a score based on 6 biomarkers (Elafin, IL2Ra, TNFRI, HGF, IL8, and REG3a) and of ST2, to predict severity of aGVHD, after adjustment for other variables. However, when tested for discriminative value by the c-index, our results support the need for improved biomarker studies and prospective trials designed to re-enforce immunosuppressive therapy in patients with unfavorable biomarkers, while patients with favorable biomarkers could possibly benefit from less intensive aGVHD therapy. This study corroborates the ability of a score based on 6 biomarkers to predict severity of aGVHD in an independent cohort of patients. [Display omitted] DisclosuresPeffault De Latour:Alexion Pharmaceuticals, Inc.: Consultancy, Honoraria, Research Funding; Amgen: Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding. Socié:Alexion Pharmaceuticals, Inc.: Consultancy.

Tyrosine supplement ameliorates murine aGVHD by modulation of gut microbiome and metabolome.

BackgroundMicrobial communities and their metabolic components in the gut are of vital importance for immune homeostasis and have an influence on the susceptibility of the host to a number of immune-mediated diseases like acute graft-versus-host disease (aGVHD) after allogeneic hematopoietic stem cell transplantation (allo-HSCT). However, little is known about the functional connections between microbiome and metabolome in aGVHD due to the complexity of the gastrointestinal environment.MethodInitially, gut microbiota and fecal metabolic phenotype in aGVHD murine models were unleashed by performing 16S ribosomal DNA gene sequencing and ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS)-based metabolomics.FindingsThe group with aGVHD experienced a significant drop in Lachnospiraceae_unclassified but an increase in the relative abundance of Clostridium XI, Clostridium XIVa and Enterococcus. Meanwhile, a lower content of tyrosine was observed in the gut of aGVHD mice. The correlation analysis revealed that tyrosine-related metabolites were inversely correlated with Clostridium XIVa, besides, Blautia and Enterococcus also displayed the negative tendency in aGVHD condition. Apart from exploring the importance and function of tyrosine, different tyrosine diets were offered to mice during transplantation. Additional tyrosine supplements can improve overall survival, ameliorate symptoms at the early stage of aGVHD and change the structure and composition of gut microbiota and fecal metabolic phenotype. In addition, aGVHD mice deprived from tyrosine displayed worse manifestations than the vehicle diet group.InterpretationThe results demonstrated the roles and mechanisms of gut microbiota, indispensable metabolites and tyrosine in the progression of aGVHD, which can be an underlying biomarker for aGVHD diagnosis and treatment.FundingThis research was funded by the International Cooperation and Exchange Program (81520108002), the National Key R&D Program of China, Stem Cell and Translation Research (2018YFA0109300), National Natural Science Foundation of China (81670169, 81670148, 81870080 and 91949115) and Natural Science Foundation of Zhejiang Province (LQ19H080006).

Decrease in T-Lymphocyte Mitochondrial DNA Copy Number Is Associated with Acute Graft Versus Host Disease

Introduction Mitochondria play an important role in T-lymphocyte activation and initiation of the inflammatory response, two pathways that are important in the pathogenesis of acute graft-versus-host disease (aGvHD). We evaluated whether change in mitochondrial DNA (mtDNA) copy number in CD3+ T-lymphocytes measured at two time-points pre and post aGvHD could be used as a biomarker for aGvHD diagnosis that correlates with clinical and/or biopsy proven aGvHD. Methods 99 patients who underwent an allogeneic hematopoietic cell transplant (allo-HCT) between March 2012 and December 2016, were diagnosed with aGvHD and had samples available before and after aGvHD diagnosis were selected for this analysis. We measured mtDNA copy number in CD3+ T-lymphocytes at two time points using real time quantitative polymerase chain reaction (PCR); once prior to diagnosis of aGvHD (median (Interquartile range): 11 (5 – 22) days prior to aGvHD diagnosis) and once after aGvHD diagnosis (median: 23 (12-34) days after aGvHD diagnosis) in 99 patients. 16% (n=16) of the patients had grade I aGvHD and 84% (n=83) had grade II-IV aGvHD. We used linear mixed models with random subject and batch effects to evaluate the change in mtDNA copy number before to after aGvHD diagnosis within each patient. We also used Fine and Gray regression models to evaluate whether reduction in mtDNA copy number after aGvHD diagnosis was associated with one year non relapse mortality (NRM). Results The median age was 58 (interquartile range 38 to 65). 35% patients underwent a transplant from a HLA-identical sibling donor, 19% matched unrelated donor, 2% mismatched related donor, 14% single and 30% double umbilical cord blood. Conditioning was myeloablative in 36% and reduced intensity in 64%. aGvHD was diagnosed at a median of 42 (range 14-180) days from HCT. 16 (16%) of the patients were diagnosed with grade 1, 38% with grade II, 34% grade III and 11% grade IV aGvHD. Overall, geometric mean mtDNA copy number decreased 14% after aGvHD diagnosis (95% CI: 3% to 26%; p=0.01). MtDNA copy number decreased from pre-aGvHD to post-aGvHD in all grades of aGvHD though a decrease was seen in a larger proportion of patients with grade IV aGvHD (9/11; 82%) vs. patients with grade I aGvHD (8/16; 50%). There were 25 patients who experienced NRM within the first year of transplant. Exploratory analysis showed that mortality was lower among patients in whom mtDNA copy number increased post-aGvHD diagnosis (6/38; 16%) as compared to those in whom mtDNA copy number decreased post-aGvHD diagnosis (19/61; 31%) (p=0.12). Conclusion This study shows that mtDNA copy number was more likely to decrease after diagnosis of aGvHD. Thus, change in mtDNA copy number among CD3+ T lymphocytes may be a useful biomarker to identify individuals with aGvHD. The prognostic significance of reduced mtDNA copy number on NRM needs to be evaluated in larger studies.

Disease Progression, Treatment Patterns, Hospitalization, and Clinical Outcomes of Patients with Acute Graft-Versus-Host Disease (aGVHD) after Allogeneic Hematopoietic Stem Cell Transplant (HSCT): A Multicenter Chart Review

Introduction aGVHD is a significant cause of morbidity and mortality following allogeneic HSCT. Objectives To describe the clinical course, treatments, hospitalizations, and clinical outcomes of patients who developed aGVHD after HSCT. Methods A multicenter retrospective chart review was conducted at 11 large US academic and community transplant centers to identify patients with first allo HSCT from 1/1/2014–6/30/2016 who subsequently developed grade II–IV aGVHD. Changes in aGVHD grade or organs involved, aGVHD recurrence, inpatient resource utilization, all-cause mortality, and 12-month overall and nonrelapse mortality (NRM) based on Kaplan-Meier estimates were reported. Results Data were collected for 475 patients (mean age, 52 years; 57% male). Median (interquartile range [IQR]) time from transplant to aGVHD diagnosis was 32 (22–48) days. At time of diagnosis, most (63%) patients had grade II disease (Figure 1), and 33% had ≥2 organs involved. Among all patients, 29% had an increase in their aGVHD grade, with a median time of 19 days from diagnosis to max grade. At max aGVHD grade, 46% had ≥2 organs involved (Figure 2). Overall, an increase in grade, new organ involvement, and progression by either organ or grade was experienced by 29%, 27%, and 40% of patients, respectively. Among patients with grade II–IV at diagnosis (n=431), 73% (314) received systemic corticosteroids (CS) as initial therapy, and median (IQR) time from diagnosis to initiation of systemic CS was 0 (0–2) days. Median starting dose was 1.0 mg/kg (79 mg/day) for prednisone and 2.1 mg/kg (190 mg/day) for methylprednisolone. During follow-up (median [IQR] 524 [165–954] days from aGVHD diagnosis to death/last visit), 23% of patients had CS dose increases, and 44% were unable to taper below 10 mg/day. Upon tapering CS, aGVHD recurred in 199 of 475 (42%) and was managed in 51% by increasing CS doses. 89 patients received additional systemic therapy for GVHD; 42% increased CS dose before additional therapy. Overall, 55% of patients required hospital readmission(s) within 100 days post-HSCT (mean length of stay, 27.5 days). Relapse of the underlying malignancy was reported in 110 (23%) patients. The 12-month overall mortality and NRM rates were 35% and 26%, respectively. Overall, 53% of patients died at a median (IQR) of 213 (77–430) days from aGVHD diagnosis (Figure 3). Conclusion 40% of patients with aGVHD experienced progression of GVHD despite systemic treatment; 27% with grade II progressed to higher grade. Approximately half of all patients required readmissions for inpatient stays of almost a month. Half of those patients died within 7 months of aGVHD onset. Although patients with grade III–IV aGVHD had the worst outcomes, patients with grade II at onset also had high mortality rates (51%). These data indicate the need for effective and tolerable treatments administered early in the disease course, even for grade II aGVHD.

Disease Progression, Hospital Readmissions, and Clinical Outcomes of Patients with Steroid-Refractory Acute Graft-Versus-Host Disease: A Multicenter Chart Review

Background: Acute graft-versus-host disease (aGVHD) post allogeneic hematopoietic stem cell transplantation (HCT) has a significant impact on morbidity and mortality. The standard first-line therapy for grade II-IV aGVHD is systemic corticosteroids (CS), which provide effective control in 40%-60% of patients. Patients may be unable to taper steroids due to new organ involvement or aGVHD flares. The objective of this retrospective multicenter study was to describe the clinical course, outcomes, and hospital readmissions for patients with aGVHD who were refractory to or dependent on systemic CS. Methods: A multicenter retrospective chart review was conducted at 11 large US academic and community transplant centers to identify patients who had their first HCT between Jan 1, 2014, and June 30, 2016, and subsequently developed grade II-IV aGVHD (defined by International Bone Marrow Transplant Registry Severity Index). Patients who participated in a GVHD prophylaxis trial or used Janus kinase inhibitors were excluded. Steroid dependence (SD)/steroid refractory (SR) was defined as the use of additional systemic anti-GVHD therapy, inability to taper high-dose steroids (≥1 mg/kg) by ≥25%, or tapering of CS dose by ≥25% but not &lt;10 mg/day. Clinical outcomes included changes in aGVHD grade or organs involved, aGVHD reoccurrence, and all-cause mortality. Results: Patient (N=168) mean age was 55 years, and 64% were male. The stem cell source was peripheral blood for 74% of patients. Median time from transplant to aGVHD diagnosis was 30 days (interquartile range [IQR]: 21-49). At the time of aGVHD diagnosis, most patients had grade I or II (65%; Figure 1) disease: 39% had skin involvement only; 38% had lower gastrointestinal (GI) involvement; and 37% had ≥2 organs involved (Figure 2). At the time of maximum aGVHD grade, most patients (66%) had grade III-IV disease: 25% had skin involvement only; 54% of patients had lower GI involvement; and 54% had ≥2 organs involved. Between the time of diagnosis and maximum aGVHD grade, 54% of patients had new organ involvement or an increase in aGVHD grade. Median time from diagnosis to maximum grade was 6.0 days. Of the 146 patients with grade II-IV aGVHD at diagnosis, 82% (119/146) were given systemic CS as first-line therapy, and 49% (72/146) initiated systemic CS on the day of diagnosis. Among the 119 patients, the average starting daily dose was 77 mg (0.9 mg/kg) for prednisone and 166 mg (1.8 mg/kg) for methylprednisolone. During follow-up (median 194 days from aGVHD diagnosis to death/last visit, IQR: 58-720), 36% of patients had an increase in steroid dose, and 88% were unable to taper below 10 mg/day. aGVHD recurred in 42% of patients (70/168) and was managed by increasing the CS dose in 79% of these patients. Over half of patients (53%; 89/168) received additional systemic anti-GVHD therapy; of those, 41% had an increase in CS dose before receiving additional therapy. Median time from CS initiation to additional therapy was 21 days. Frequently used additional therapies included sirolimus (19%), polyclonal antibodies (antithymocyte globulin/antilymphocyte globulin; 18%), mycophenolate mofetil (17%), tocilizumab (16%), extracorporeal photopheresis, and etanercept (both 13%). 26% (23/89) used ≥2 additional therapies. Overall, 57% of patients (95/168) required hospital readmission(s); 24% had ≥2 readmissions within 100 days post-HCT, with a mean inpatient length of stay of 50 days. Half (51%) of patients experienced an infection within the first 100 days post-HCT. Relapse of the underlying malignancy was reported in 35 patients (21%). Overall, 70% of patients (118/168) died at a median of 118 days (IQR: 62.0-234.1) from aGVHD diagnosis; 82% of patients with aGVHD progression died at a median of 116.0 days (IQR: 49-223), and 80% of patients with maximum grade III-IV aGVHD died at a median of 80 days (IQR: 42-216). Additionally, 86% of patients with lower GI aGVHD died at a median of 74.0 days (IQR: 44-174) (Figure 3). Conclusions: The majority of patients with aGVHD had severe and rapidly progressing disease. These findings indicate that clinical outcomes were poor among patients with SR and SD, with a mortality rate of 70% within 4 months of aGVHD diagnosis. Most patients required hospital readmission with an extended length of stay. The rapidly worsening clinical course further emphasizes the need for effective and tolerable therapies that prevent or reverse disease progression. Disclosures Yu: Incyte: Employment, Equity Ownership. Hanna:Asclepius Analytics: Employment. Paranagama:Incyte: Employment, Equity Ownership. Tang:Asclepius Analytics: Employment. Naim:Incyte: Employment, Equity Ownership. Galvin:Incyte: Consultancy.

Fecal Microbiota Transplantation As a Treatment of Severe Steroid-Resistant Acute and Chronic Graft Versus Host Disease. Spectrum of Responses and Complications

INTRODUCTION Only half of the patients with gut predominant acute graft-versus-host disease (aGvHD) respond to first-line treatment (corticosteroids). No consensus has yet been reached on treatment in the second and subsequent lines. There are several case reports supporting the role of fecal microbiota transplantation (FMT) in the treatment of steroid-resistant GvHD. Here we present the results of FMT in one of the largest cohorts of patients with acute (a) and chronic (c) GvHD, mostly with gut manifestation. MATERIAL AND METHODS 12 patients aged 26-67 years underwent a total of 15 FMTs to decolonize the gastrointestinal tract (GI) from antibiotic-resistant bacteria (ARB). In all of them steroid resistant GvHD (2 patients with cGvHD and 10 with aGvHD) coexisted. All patients with aGvHD had GI tract involvement (median grade: 3), 7 had skin (median grade: 3) and 5 had liver (median grade: 1) GvHD symptoms. Patients with cGvHD presented progressive involvement of the skin, lungs, pericardium and eyes. FMT was performed as two infusions by nasoduodenal tube of a 100g/200 ml solution of feces, on following days. OUTCOMES The median time from aGvHD diagnosis to the first FMT treatment was 46.5 days (8-489 days). Most patients were still on steroids during FMT (median dose 0.5 mg metyloprednisolone/kg of body weight). As a result, in 10/12 patients (83%) ARB decolonization was observed. Overall response rate (ORR) in the case of aGvHD reached 58% (7/12 performed FMTs), including CR in 4/12 (33%) cases. Median duration of response to relapse or death was 168 days (10-1080 days). The majority of patients who responded to FMT (5/6, 83%) underwent the procedure in a better general condition (ECOG≤2). Severe general condition (ECOG&gt; 2) seemed to be related with the presence of serious complications after FMT. Both patients with cGvHD achieved stabilization or improvement of organ disease, which may be related with FMT procedure (there is no data on use FMT in cGvHD). COMPLICATIONS Among the complications of FMT with a possible cause-effect relationship there were: 1 death in close relation with FMT (respiratory failure in the course of pneumonia), 3 septic episodes, 2 subileus episodes and one case of diarrhea. In the latter case, the workup revealed norovirus infection originating from the fecal material of the donor (who was asymptomatic). CONCLUSIONS The described results indicate that FMT may be an effective treatment option for severe GvH disease. Patients undergoing the procedure should be qualified early enough to avoid life-threatening complications and maximize the likelihood of responding to treatment. Figure Disclosures Jedrzejczak: Amgen: Consultancy, Other: travel support for hematology meetings (ASH, EBMT, EHA) ; Celgene: Other: travel support for hematology meetings (ASH, EBMT, EHA) ; Takeda: Consultancy; Novartis: Research Funding; Roche: Other: travel support for hematology meetings (ASH, EBMT, EHA) . Basak:Teva: Honoraria; Celgene: Honoraria.

Healthcare Costs in Patients with Lethal Acute Graft-Versus-Host Disease (aGVHD) Post Allogeneic Hematopoietic Stem Cell Transplant (allo-HSCT) in the United States

Introduction Acute GVHD is a common complication and cause of death after allo-HSCT. Previous analysis showed that patients with aGVHD incurred significant healthcare costs (ie, average $33,675 monthly costs) over the first year post transplant in the US (Yu et al. Biol Blood Marrow Transplant. 2018; 24(3):S189–S190). Objective To estimate healthcare utilization and costs incurred among patients with aGVHD who died within 100 days or 1 year of aGVHD diagnosis in the United States. Method Administrative claims from the Optum Research Database were used to identify allo-HSCT patients from 01/01/10 to 08/31/16. Patients with aGVHD had ≥2 outpatient or ≥1 inpatient claims with diagnosis of aGVHD (International Classification of Disease [ICD]-9 279.51 or ICD-10 D89.810) within study period or until onset of cGVHD. Patients were included if ≥12 years old, enrolled in commercial or Medicare Advantage plan ≥6 months preceding and ≥360 days post-HSCT, or less if death occurred. Death was determined based on Social Security Death Master File data available for a portion of the patients in the database. Average per patient monthly all-cause healthcare costs while surviving were reported for patients with aGVHD who died within 100 days post-aGVHD diagnosis for the 100-day analysis, or died within 360 days for the 360-day analysis. Result Included in the analysis were 26 and 54 patients who died within 100 and 360-days after aGVHD diagnosis, respectively. Mean age was 57 years for both groups; and 46% and 44% were female, respectively. Patients with aGVHD who died within 100-days of aGVHD diagnosis had a median of 35 inpatient days (interquartile range [IQR]: 22-54) during median 44 day survival time from aGVHD diagnosis, and incurred $114,720 median (IQR: $52,744–$198,815) per-patient per-month costs for all-cause inpatient care while alive, accounting for 82% of median per-patient per-month all-cause total costs ($139,948; IQR: $91,793–$205,317). Patients who died within 1 year of aGVHD diagnosis had a median of 46 all-cause inpatient days (IQR 29-72) over a median 108-day survival time from aGVHD diagnosis and incurred $69,622 median (IQR: $26,823–$139,599) per-patient per-month costs for all-cause inpatient care while alive, accounting for 76% of per-patient per-month total costs (median $91,717; IQR: $43,893–$158,890). Conclusion Patients with aGVHD who survived less than 1 year post-diagnosis of aGVHD incurred substantial inpatient healthcare resource use and costs, which were mostly in the first 100 days of acute GVHD diagnosis. The economic burden associated with this high risk complication could be limited through early and effective therapies, resulting in fewer deaths and reduced in-patient care use.

Outcomes of Infliximab in Management of Steroid-Refractory Acute Graft Versus Host Disease

Corticosteroids are considered the standard first-line treatment for moderate/severe acute graft versus host disease (aGVHD), yet there is no consensus on the optimal management of patients that are steroid-refractory (SR). Infliximab is a chimeric monoclonal antibody with anti-TNF-α activity that has previously demonstrated efficacy in SR-aGVHD. We performed a retrospective review of 59 patients at Indiana University who underwent allogeneic stem cell transplantation and developed SR grade III-IV aGVHD from January 2007 to August 2018. Infliximab was administered 10 mg/kg weekly intravenously. Response criteria was in accordance with Martin et al (BBMT 2009; 12:777-784). Median age of treated patients was 49.5 years (range, 34.5-60) and median time from transplant to diagnosis of aGVHD was 106 days (range, 42-185) Patients received a median of 12 days (range, 8-20) of systemic steroids prior to infliximab. At the start of infliximab, 52 (88%) patients were on non-steroidal immunosupressants and 39 (66%) on non-absorbable steroids concurrently with systemic steroids. Patients received a median of 3 doses of infliximab with 37 (62.7%) patients receiving There was no significant difference between responders (n=16) and non-responders (n=43) in baseline demographic, transplant-related characteristics, initial GVHD prophylaxis regimen, number of non-steroidal systemic immunosuppressants at time of aGVHD diagnosis, or use of non-absorbable steroids. There was no difference in time from diagnosis of aGVHD to start of infliximab (median 16 vs. 11 days, P=0.06). Responders were less likely to have liver aGVHD involvement vs. non-responders (18% vs. 53%, P=0.016) and had slower tapering of systemic steroids, both at median time to 50% steroid dose (18 vs. 12 days, P=0.02) and 25% steroid dose (36 vs. 24 days, P=0.01). Responders had lower incidence of bacterial infection (44% vs. 74%, P=0.04), but not viral infections (56% vs. 47%, P=0.57). Responders had a longer overall survival (median 20.3 vs. 2.8 months, P=0.01). Our single center report of 59 patients with SR-aGVHD treated with infliximab is among the largest reported. The outcomes of response to SR-aGVHD are poor and there is an unmet need for better agents. Additional studies with greater patient numbers are needed to identify factors associated with improved response to infliximab.

Mortality, length of stay and costs associated with acute graft-versus-host disease during hospitalization for allogeneic hematopoietic stem cell transplantation

Objective: Acute graft-versus-host disease (aGVHD) is a common and life-threatening complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). The extent to which aGVHD increases inpatient costs associated with allo-HSCT has not been thoroughly evaluated. In this analysis, mortality, hospital length of stay (LOS) and costs associated with aGVHD during allo-HSCT admissions are evaluated.Methods: This is a retrospective analysis of discharge records from the National Inpatient Sample database for patients receiving allo-HSCT between 1 January 2009 and 31 December 2013. Allo-HSCT discharges with an aGVHD diagnosis were included in the aGVHD group and those without any graft-versus-host disease (GVHD) diagnosis comprised the non-GVHD group. Mortality, LOS and costs were compared between the two groups, as well as within subgroups, including age (<18 vs. ≥18 years) and survival status (alive vs. deceased) at discharge.Results: Overall, mortality (16.2% vs. 5.3%; p < .01), median hospital LOS (42.0 vs. 26.0 days; p < .01) and median total costs ($173,144 vs. $98,982; p < .01) were significantly increased in patients with aGVHD versus those without GVHD during hospitalizations for allo-HSCT, irrespective of age group. Patients with aGVHD who were <18 years of age had a lower mortality rate but greater hospital LOS and total costs versus patients aged ≥18 years. Patients who died during allo-HSCT hospitalization had longer LOS and incurred greater costs than those who survived in both the aGVHD and non-GVHD groups.Conclusion: Occurrence of aGVHD during allo-HSCT admissions resulted in a tripling of the mortality rate and a near doubling of hospital LOS and total costs. In addition, death during allo-HSCT hospitalizations was associated with greater healthcare utilization and costs. Effectively mitigating aGVHD may improve survival and substantially reduce hospital LOS and costs for allo-HSCT.

EBV-Related Post-Transplant Lymphoproliferative Disease in Allogeneic Hematopoietic Cell Transplantation: Single-Center Experience and Considerations Regarding CNS Involvement

EBV-related post-transplant lymphoproliferative disease (EBV-PTLD) is a serious complication of allo-HCT, for which antithymocyte globulin (ATG) administration has been identified as the most important predisposing factor. Although the development of EBV-PTLD used to be detrimental to patients, use of rituximab has largely changed both the incidence and the outcome of this complication, either administered as preemptive treatment based on peripheral blood (PB) EBV titles, or for the management of the disease itself.We determined the incidence of EBV-PTLD through chart review of 797 consecutive allo-HCT recipients transplanted in our center (7/1990-7/2018) and evaluated factors potentially influencing EBV-PTLD occurrence and outcome.Among 797 allo-HCT recipients (n=465 sibling, n=277 MUD, n=47 haploidentical, n=2 twin and n=6 cord blood), 14 (1.7%) patients developed EBV-PTLD. The diagnosis was confirmed by biopsy in 9/12 cases (7 monomorphic, 1 polymorphic and 1 HL-like PTLD), 1 case developed sole EBV encephalitis, 1 case developed CNS lymphoma, and the remaining 3 cases were clinically diagnosed on the basis of generalized lymphadenopathy coupled with high PB EBV title. The patients suffered from ALL (n=6), AML (n=2), CML (n=2), severe aplastic anemia (n=2), MDS (n=1) and plasmacytic leukemia (n=1). Notably, 6/14 patients had high tumor burden at the time of transplantation [refractory AML (n=1), relapsed refractory ALL (n=2), CML blastic crisis (n=2), refractory MDS RAEB II (n=1)]. EBV-PTLD incidence was significantly higher in MUD versus sibling allo-HCT (3.4% versus 0.6%, p=0.006), in haploidentical versus sibling allo-HCT (6.4% versus 0.6%, p<0.001), and in haploidentical versus conventional allo-HCT (6.4% versus 1.5%, p=0.01). Among the haploidentical allo-HCTs complicated with EBV-PTLD, 2 were T-cell depleted with add-back infusions of transduced donor lymphocytes, and 1 was T-cell replete. These results possibly reflect the standard use of ATG as acute GVHD prophylaxis in MUD and haploidentical allo-HCT. Indeed, 12/14 (85.7%) patients who developed EBV-PTLD had received ATG as part of their conditioning regimen, at doses ranging from 5-10mg/kg (median 5mg/kg), and 1 additional patient had received ATG 20mg/kg as 2nd-line treatment for hyperacute GVHD. The conditioning intensity was not significantly associated with EBV-PTLD development [classic myeloablative (n=8), reduced-toxicity (n=3), reduced-intensity (n=3)]. EBV-PTLD occurred early in the post-transplant period (median: 73, range 41-603 days). In 7/14 cases, it was preceded by the onset of aGVHD (median interval from aGVHD diagnosis: 58 days), in one case it coincided with aGVHD diagnosis +45 days from allo-HCT, and in one case it developed soon (+55 days) after induction of GVHD through DLIs. At EBV-PTLD diagnosis, the median EBV title in PB was 78,400 copies/ml (range 84-2,860,000). Of note, one patient developed sole CNS EBV-PTLD with extremely low PB EBV viral load. This patient had received preemptive rituximab 500mg 7 months prior to CNS EBV-PTLD development, on the basis of elevated PB EBV title (70,900 copies/ml). Since then, PB EBV title was always tested below <500 copies/ml by conventional PCR, and was practically negative at the time of EBV-PTLD diagnosis (84 copies/ml). Ten patients were treated with intravenous rituximab: in six patients the disease resolved, but 3/4 cases with CNS involvement succumbed. The fourth patient with CNS involvement is currently under treatment with high-dose MTX and intravenous rituximab and has achieved partial remission. The remaining four patients were unsuccessfully treated with combinations of bleomycin/vindesine/IFNa/immunoglobulins/DLIs.In conclusion, EBV-PTLD is an early complication of alloHCT, associated mainly with defects in T cell immunity. Selective ATG administration to MUD and haploidentical allo-HCT may explain the higher incidence of EBV-PTLD as compared to sibling allo-HCT, yet we identified higher incidence among haploidentical alloHCTs that cannot be attributed to a particular lymphodepletion scheme. Prompt administration of intravenous rituximab is very effective, except for CNS involvement. Importantly, PB EBV monitoring may be misleading in CNS EBV-PTLD, particularly following preemptive intravenous rituximab administration. Taking into account the dismal outcome, intrathecal rituximab should be considered. DisclosuresVardi:Gilead: Research Funding; Janssen: Honoraria. Gavriilaki:European Hematology Association: Research Funding.

Repeated Courses of Orally Administered Fecal Microbiota Transplantation for the Treatment of Steroid Resistant and Steroid Dependent Intestinal Acute Graft Vs. Host Disease: A Pilot Study (NCT 03214289)

Background: Steroid-resistant (SR) intestinal acute graft versus host disease (aGVHD) is a devastating complication of allogeneic hematopoietic stem cell transplantation. Preliminary reports suggest that fecal microbiota transplantation (FMT) administered through a nasogastric tube or colonoscopy may be an effective treatment. We report the results of a single-arm pilot study (NCT 03214289) using FMT in capsules to treat SR or steroid dependent (SD) intestinal aGVHD.Methods: The primary outcome was the occurrence of severe adverse events (SAEs) at 28 days post last FMT course. Secondary outcomes included GVHD response. Complete response (CR) was defined as resolution of gastrointestinal symptoms or reduction of steroid dose to 5 mg of prednisone. Partial response was defined as a decrease in severity of GVHD by at least one stage or a ≥40% reduction in steroid dose. Patients were eligible if they had SR or SD gut aGVHD without active infection or neutropenia. Per-protocol, participants received a course of 30 frozen capsules of fecal matter over two consecutive days. FMT courses could be repeated from the same or a different donor, at the treating physician's discretion. Capsules are produced from healthy unrelated donors who underwent vigorous screening. They are taken orally and are flavorless and odor-free. To characterize the impact of the FMT on the gut microbiota, stool samples of recipients were serially collected and underwent 16s rRNA sequencing.Results: To date, we have enrolled 7 patients with intestinal aGVHD (6 SR, 1 SD) (Table). The median dose of methylprednisolone (MP) was 1 mg/kg (interquartile range [IQR] 0.8-1.3 mg/kg). FMT was administered at a median of 39 days (IQR 21-58 days) from aGVHD diagnosis. A total of 15 courses of FMT were given. Patients received a range of 1-3 FMT courses (median 2). The capsules were well tolerated. Patient #1 developed Enterococcus Faecium bacteremia 2 days following the second FMT. To track the source of bacteremia, we performed targeted metagenomic sequencing. The enterococcus strain from the blood culture was identified in the recipient's pre-FMT stool sample but not in the FMT inoculum (i.e., capsule), confirming that the bacteremia was not an FMT complication. Similarly, patient #6 developed Pseudomonas aeruginosa bacteremia 3 days after the 2nd FMT. 16s rRNA sequencing of the donor capsule failed to demonstrate Pseudomonas taxa. No other SAEs suspected to be related to the FMT were observed. Two patients achieved a CR with complete resolution of GVHD symptoms. Patient #6 had a partial improvement following the 1st FMT, with a reduction of MP from 2 mg/kg to 1.3 mg/kg. Three days after the 2nd FMT, she developed fatal pseudomonas bacteremia, not related to the FMT as detailed above. At last follow-up (median 61 days, IQR 40-99), 3/7 patients were alive. Three patients died from consequences of active GVHD, while one patients who responded to FMT and was free of GVHD, succumbed to an invasive Aspergillus infection of the brain.16s rRNA sequencing of stool samples revealed bacterial domination (i.e., occupation of at least 40% of the microbiota by a single predominating taxon) of Escherichia(E) coli in four patients before FMT, with a major reduction following therapy. FMT was associated with the introduction of new bacteria and an increase in bacterial diversity in the recipient's stool (Figure).Conclusions: We demonstrate for the first time the utility of fecal microbiota transplantation in orally administered capsules for the treatment of severe intestinal acute GVHD. The capsules were well tolerated and safe. Metagenomic sequencing proved that a bacterial infection following FMT was not related to the procedure. Sequencing of the stool sample revealed bacterial domination with E.coli in 4/7 patients prior to the first FMT. Following FMT, bacterial diversity increased. Finally, 2/7 patients attained a complete response following therapy, suggesting a potential role of FMT in patient management.Figure. (A) Heatmap of operational taxonomics units (OTU). Each column marks a sequenced stool sample at a specific time point and rows individual taxas. The color code indicates relative abundance. Dotted lines represent an FMT course. Before FMT all patients, aside from patient #6, had markedly reduced diversity, with enrichment of OTUs following treatment. (B). Change of bacterial diversity, measured by the Shanon diversity index before and after FMTs. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

T Regulatory Cell Receptor Repertoire Focusing and Clonal Expansion Indicates Control of Acute GvHD after Donor Lymphocyte Infusion

Background: T effector cells (Teff) within the stem cell graft in allogeneic hematopoietic stem cell transplantation (HSCT) can elicit disabling acute graft-versus-host disease (aGvHD) contributing to transplant-related mortality. Teff as donor lymphocyte infusion (DLI) are a therapeutic option to re-induce complete remission (CR) after leukemia relapse. Usually DLIs are given in dose escalating regimens until CR is achieved or first signs of aGvHD develop. To monitor the DLI induced allo-immune response and the efficacy of aGvHD treatment is a clinical challenge, since no established biomarkers are available.T regulatory cells (Tregs) are thought to play an important role in balancing immune responses and studies have shown effects of adoptive Treg transfer as a therapeutic option in GvHD. T cell receptor (TCR) sequencing of T cell subsets, such as Tregs, after DLI might allow the identification of clones inducing control of GvHD. Here we report data of 29 DLI patients with a median follow-up of >1 year allowing us to thoroughly analyze differences of the TCR repertoire in patients with or without aGvHD.Aims: We aimed to analyze Treg TCR diversity and clonality changes over time as a potential biomarker for development and treatment response of aGvHD following DLI.Patients and Methods: The study cohort consisted of 29 leukemia patients after HSCT who received DLI treatment for recurrence of disease, molecular relapse or high risk phenotype. Blood samples were taken before and after DLI. A median of 4 (range 2-6) blood samples per patient were available for TCR-sequencing. The last sample was taken at a median of 123 (27-530) days post DLI. After generation of PBMCs CD4+CD127-CD25+ Tregs were FACS-sorted for cDNA-based CDR3-region amplification of the TCR-β chain. CDR3 amplicons were sequenced on the Illumina MiSeq platform and annotated using the IMGT.org database. Further bioinformatic analyses were based on VDJtools and the tcR R-package.Results: In 18/29 patients we observed clinical symptoms of aGvHD, with blood samples available of the acute onset in 12/18 cases. Treg frequencies and absolute numbers did not differ between aGvHD and noGvHD samples. Treg TCR diversity, assessed via inverse Simpson's diversity index (1/D) increased on average by 322% at the first occurrence of aGvHD compared to the previous sample (Figure 1A, B). Stratifying for aGvHD severity (total grade 1-2 vs. 3-4) did not reveal any group differences. However, stratifying by organ involvement (skin vs. GI/liver) showed a more pronounced increase of 1/D in patients with aGvHD of the GI tract and/or the liver. Moreover, in 11 subjects blood samples were available a median of 7 (3-14) days prior to aGvHD diagnosis. Already at this preclinical time point we detected an increased 1/D of +361% (Figure 1A, B). Again, aGvHD organ involvement significantly affected the result, with GI/liver involvement mainly driving this effect (+567% vs. +1% 1/D). In contrast, patients who did not develop aGvHD at any time after DLI showed on average a slight decrease of -8% 1/D compared to the previous time point. Next, we analyzed all aGvHD patients with at least one sample available after initiation of treatment (local and/or systemic steroids). Control or remission of aGvHD symptoms was accompanied by decreased 1/D of on average -58% compared to the previous samples (Figure 2). In 9/11 patients we saw a focusing of the Treg TCR repertoire; in the other two (Figure 2, red lines) no or only partial clinical response to systemic steroids was reported.Conclusion: Our data describe detailed changes in the Treg compartment on the TCR level following development and treatment of aGvHD. Currently, aGvHD diagnosis following DLI treatment relies solely on clinical symptoms, deciding whether further dose increments of DLI can be administered. As our data suggest, Treg TCR sequencing may support transplant specialists with (1) detection of patients at risk for aGvHD, even prior to clinical symptoms, (2) identification of patients eligible for further dose increments (compare Figure 1B), and (3) assessment of aGvHD treatment with guidance whether higher dosage of steroids and/or alternative immunosuppressive treatment might be required (compare Figure 2, red lines). Future prospective studies are needed to replicate these findings in a large cohort, potentially enabling identification of specific Treg TCR clones controlling the allo-immune response in aGvHD. DisclosuresGanser:Novartis: Membership on an entity's Board of Directors or advisory committees. Koenecke:BMS: Consultancy; abbvie: Consultancy; Amgen: Consultancy; Roche: Consultancy.