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Graft-versus-host disease (GVHD) remains a major limitation of allogeneic hematopoietic stem cell transplantation. Only half of patients with severe acute GVHD respond to first-line treatment with corticosteroids and, for several decades, there was no optimal second-line treatment of patients with corticosteroid-refractory acute GVHD. Ruxolitinib was recently approved for the treatment of corticosteroid-refractory acute GVHD in adult and pediatric patients 12 years and older. Thus, it is important to define the patient population that would now be considered as refractory to ruxolitinib vs ruxolitinib dependent. Here, we propose to define ruxolitinib-refractory acute GVHD as disease that shows: (1) progression of GVHD compared with baseline after at least 5 to 10 days of treatment with ruxolitinib, based either on objective increase in stage/grade, or new organ involvement; (2) lack of improvement in GVHD (partial response or better) compared with baseline after ≥14 days of treatment with ruxolitinib; or (3) loss of response, defined as objective worsening of GVHD determined by increase in stage, grade, or new organ involvement at any time after initial improvement. GVHD manifestations that persist without improvement in patients who had a grade ≥3 treatment-emergent and ruxolitinib-attributed adverse event that did not resolve within 7 days of discontinuing ruxolitinib would serve as a clinical indication for additional treatment. In addition, absence of complete response or very good partial response at day 28 after ruxolitinib could be considered as an eligibility criterion.

Acute graft-versus-host disease (GVHD) is treated with systemic corticosteroid immunosuppression. Clinical response after 1 week of therapy often guides further treatment decisions, but long-term outcomes vary widely among centers, and more accurate predictive tests are urgently needed. We analyzed clinical data and blood samples taken 1 week after systemic treatment of GVHD from 507 patients from 17 centers of the Mount Sinai Acute GVHD International Consortium (MAGIC), dividing them into a test cohort (n = 236) and 2 validation cohorts separated in time (n = 142 and n = 129). Initial response to systemic steroids correlated with response at 4 weeks, 1-year nonrelapse mortality (NRM), and overall survival (OS). A previously validated algorithm of 2 MAGIC biomarkers (ST2 and REG3α) consistently separated steroid-resistant patients into 2 groups with dramatically different NRM and OS (P < .001 for all 3 cohorts). High biomarker probability, resistance to steroids, and GVHD severity (Minnesota risk) were all significant predictors of NRM in multivariate analysis. A direct comparison of receiver operating characteristic curves showed that the area under the curve for biomarker probability (0.82) was significantly greater than that for steroid response (0.68, P = .004) and for Minnesota risk (0.72, P = .005). In conclusion, MAGIC biomarker probabilities generated after 1 week of systemic treatment of GVHD predict long-term outcomes in steroid-resistant GVHD better than clinical criteria and should prove useful in developing better treatment strategies.

Background: Graft-versus-host disease (GVHD) is a dreaded complication after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Such inflammatory diseases are fostered by damage to the intestinal barrier after transplantation. Consequently, the integrity and regeneration of the intestinal barrier is a key factor in the prevention of GVHD. On one side the main driver for regeneration of damaged gut epithelium are intestinal stem cells (ISC), but on the other side these cells are themselves primary targets of donor-derived T cells. One known mechanism of T cell mediated damage to the stem cell compartment is through IFN-γ dependent ISC toxicity. Yet, little is known about how T cells are contributing to the regeneration of damaged tissue after allo-HSCT and GVHD. Methods: To address this, we used preclinical models for allo-HSCT and GVHD including transplantation of recipient mice with escalating doses of Wildtype or IFN-γ-deficient allogeneic T cells and in the presence or absence of the JAK-1/2 inhibitor ruxolitinib. Intestinal regeneration was assessed by RNA-seq, flow cytometry and a newly established ex vivo organoid recovery assay. GVHD outcome was assessed by clinical scoring, histology and survival. Additionally, we established an allogeneic co-culture system of murine or human intestinal organoids with CD4+ conventional T cells or T regs -/+ Ruxolitinib. Effects on organoid growth and cell death were assessed by size measurements and manual counting after passaging. Results: We here demonstrate that recipient mice with increasingly dense intestinal infiltration by allogeneic T cells not only developed more severe GVHD (Fig. 1A), but also showed augmented recovery potential early after allo-HSCT (Fig. 1B). This was associated with intestinal gene signatures related to epithelial regeneration and protection from GVHD. Utilizing ex vivo cultures of intestinal organoids generated from murine allo-HSCT recipients, we found that development of GVHD but also regenerative capacity of ISCs were dependent on interferon (IFN)-γ-producing T cells in the intestine (Fig. 2A-B). Mice with fulminant GVHD and enhanced organoid recovery showed accumulation of intestinal regulatory T cells (Tregs) (Fig. 2C). Ex vivo, T regs nurtured growth of intestinal organoids in an IFN-γ dependent manner (Fig. 2D-E). This effect was diminished in intestinal organoids lacking IFNγR signaling, but was independent of T reg intrinsic IFNγR signaling (Fig. 2E-F). Intriguingly, treatment of murine allo-HSCT recipients with the JAK-1/2 inhibitor ruxolitinib enhanced epithelial organoid regeneration and numbers of intestinal Tregs (Fig. 3A-B). Similarily, growth of human intestinal organoids co-cultured with allogeneic T cells could be augmented by ruxolitinib treatment (Fig. 3C). We thus propose that the level and differentiation of infiltrating intestinal T cells determines both ISC damage and epithelial regeneration during immune-mediated tissue injury, leading to a sensitive equilibrium that can be modulated by therapeutic intervention. We also provide evidence that ruxolitinib improves ISC regeneration via IFNγ-producing Treg cells. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Background: Graft-versus-host disease (GVHD) is a dreaded complication after allogeneic stem-cell transplantation (ASCT). Previously, we and others showed that activation of type I interferon (IFN-I) inducing pathways such as RIG-I/MAVS or cGAS-STING can promote the integrity of the intestinal barrier and limit GVHD (Fischer et al., Sci Transl Med, 2017). However, the signals that drive these protective IFN-I responses are poorly understood. Commensal microbiota can (i) have distant effects on immune responses through modulation of IFN-I signaling (Steed et al, Science, 2017; Swimm et al, Blood, 2018) and (ii) predict mortality in ASCT patients (Peled et al., N Engl J Med, 2020). We hypothesized that microbiota-derived products such as microbial metabolites engage IFN-I signaling in immune and non-immune cells poising them for induction of protective responses. We established a prospective, multi-centric clinical study in patients newly diagnosed with acute leukemia and performed longitudinal stool sampling to track changes in microbial community composition and metabolites expression levels. Submitted as a separate abstract to ASH 2021 (Orberg & Meedt et al.), we show that patients with high metabolite expression going into ASCT are less likely to develop GVHD. In this study, we translate our clinical observations to mouse models of acute GVHD and human and mouse intestinal organoids to uncover the molecular mechanisms via which metabolites protect the intestinal barrier during ASCT. Methods: Stool samples from ASCT patients were obtained in Munich and at Regensburg in accordance to IRB-approved study protocols. Patients were sampled at initial diagnosis (Dx), prior to conditioning and weekly after ASCT up to day 28. We analyzed samples by 16S rRNA sequencing and mass spectrometry to obtain a complete picture of microbiome composition and function. Next, we tested metabolites which we detected in patients (desaminotyrosine [DAT], indole-3-carboxaldehyde [ICA]) as treatment in preclinical models in ASCT and GVHD mouse models. Outcomes were assessed by a novel organoid recovery assay in addition to established read-outs. To obtain a mechanistic understanding of the signaling pathways involved, we stimulated WT or IFN-I-signaling-impaired mouse (incl. STING -/-, MAVS -/-, IFNαR -/-) as well as human intestinal crypt-derived organoids with metabolites. Results: Here, we present a 64-year-old female patient diagnosed with AML who received a 9/10 HLA-matched ASCT. At day 7, i.v. antibiotics were started due to fever and Enterococcus bacteraemia. At day 15, the patient developed skin and GI GVHD (Glucksberg III). At the timepoint initial diagnosis (Dx), i.e. the timepoint when we diagnosed AML but before therapy was initiated, we detected rich alpha diversity (Panel 1a) in the patient's stool. Flavonifractor plautii, a producer of the metabolite DAT, was detectable (Panel 1b). We observed high-level expression of metabolites including short-chain fatty acids, DAT, ICA and secondary bile acids (Panel 1c). Following ASCT, and especially at the early time-points day 0 and day 7, alpha diversity and metabolite expression declined drastically. We confirmed this trend in our multi-centric cohort of ASCT patients by comparing levels of DAT and ICA sampled at admission to the transplantation ward (Conditioning) versus at clinical diagnosis of GVHD: in patients with GVHD, metabolite levels were drastically reduced (Panel 2). Next, we prophylactically administered metabolites in a major mismatch mouse ASCT model. Metabolite-treated mice showed significantly showed better outcomes in our organoid recovery assay, which measures the ability of intestinal stem cells to recover after allogeneic injury (Panel 3). This effect that was abrogated in STING -/- recipients. Metabolite stimulation of mouse small intestinal organoids promoted organoid numbers and size, and required intact STING signaling (Panel 4a). Human colon organoids also responded to DAT and ICA, however the metabolite effect was lost when co-administered with the STING-inhibitor H151. Conclusions: We identify that microbial-derived metabolites detected in patients can engage the STING pathway in humans and mice to confer resistance from immune damage. Thus, prophylactic administration of metabolite cocktails or bacterial consortia that can produce these metabolites may reduce occurrence of GVHD in ASCT patients. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

The intestinal microbiome directly effects outcomes in patients undergoing allogeneic stem cell transplantation (ASCT) (Peled et al., N Engl J Med, 2020). Recent developments in microbiome research allow us to look beyond bacterial communities at the composition of the intestinal mycobiome and virome. However, only few studies have addressed these fungal (Zhang et al., Nat Commun, 2021; Legoff et al., Nat Med, 2017) and viral communities (Van Der Velden et al., Biol Blood Marrow Transplant, 2013) in the ASCT setting. Loss of bacterial diversity and domination of single taxa was linked to increased incidence of graft-versus-host-disease (aGvHD) and mortality. However, the functional consequences of intestinal dysbiosis remain poorly understood. Recently, microbial-derived metabolites such as short-chain fatty acids (SCFAs), indoles and secondary bile acids (BA) have been proposed to explain how microbiota exert effects on epithelial and immune responses and shown to protect from GVHD in animal models. To assemble a complete picture of the phylogenetic and metabolic changes which occur as patients undergo ASCT we designed a multiomics approach and identified patterns in bacteriome, mycobiome, virome and metabolome dynamics in relation to clinical factors and GvHD. 353 stool samples from 80 patients undergoing ASCT were obtained at two different transplantation centers (Munich and Regensburg) at defined timepoints before and after transplantation. Changes in metabolite composition were evaluated via mass spectrometry using a targeted metabolomic profiling approach, accompanied by bacterial 16S rDNA-, fungal 18S-ITS- and deep virome sequencing. Results were linked among each other and with clinical metadata such as GvHD, response to steroids, antimicrobial therapy and mortality. Longitudinal profiling of ASCT patients revealed a significant loss of bacterial, fungal and viral diversity following ASCT. At both centers, the effect was most pronounced in the early post-transplant period. Patients that developed GI-GVHD had the lowest diversity scores (Panel 1). GVHD patient samples were dominated by Enteroccocus and Candida, and loss of Caudovirales species, indicative of deep dysbiosis effecting all intestinal communities. ASCT had a strong impact on microbiome function and their ability to produce metabolites. We assayed each sample for production of metabolites with reported protective effects: SCFAs (acetate, butyrate and propionate), indoles (indole-3-carboxylaldehyde, ICA) and BAs (deoxycholic and lithocholic acid). When admitted to the transplantation unit (timepoint "conditioning") most patients had rich metabolite expression profiles. In the early post-transplant period, metabolite levels became progressively more depleted. GVHD patients displayed a nearly complete loss of metabolites (Panel 2a). Importantly, levels of the metabolite ICA at early timepoints could predict the occurrence of GVHD, leading us to consider prophylactic administration in future studies. We performed beta diversity analysis to identify clinical factors that had the highest impact on microbial communities. As expected, we noted that administration of broad-spectrum antibiotics had a profound impact of bacteria, fungi and viruses; but also had a significant negative effect on metabolites levels. Lastly, we report on a case of successful treatment of severe GI-GVHD with fecal microbiota transplant. Response to treatment was characterized by the recipient adopting the FMT donor's rich metabolite expression profile (Panel 2b) . To our knowledge, this is the first multicenter study that combines bacteriome, mycobiome, virome and metabolome analyses a longitudinal fashion in matched patient cohorts. Our results are consistent with previous findings that there are major alterations in gut microbiome and metabolite composition after ASCT. We expand upon those findings by characterizing the time course of phylogenetic alterations and their functional consequences. Those alterations play an important role in GvHD pathogenesis and might serve as potential biomarkers to identify patients at high risk for developing lethal GvHD early on. Our results highlight two strategies for targeted microbiome-based interventions: (1) preventing loss of microbial communities by restrictive use of antibiotics and (2) prophylactic administration of metabolite cocktails. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Introduction Failure to respond to steroid therapy for intestinal acute graft-versus-host disease (aGvHD) is associated with limited further therapeutic options. Fecal microbiotherapy is defined as the perfusion of treated stool from one or several healthy donors via the upper or lower gastrointestinal (GI) route aiming at improving microbial diversity and functionality. Here we report clinical outcomes from a 76-patient cohort with steroid refractory (SR) GI-aGvHD treated with the pooled allogenic fecal microbiotherapeutic MaaT013. Twenty-four patients were treated in the prospective, single-arm, phase IIa, HERACLES study (NCT03359980) while 52 patients were treated in an expanded access program (EAP). Patients and methods For HERACLES, 24 patients with grade III-IV SR-GI-aGvHD were treated with MaaT013 in 26 European sites, as a 2 nd line therapy after SR diagnosis and evaluable for treatment response. In EAP, 52 patients with steroid-dependent or SR-GI-aGvHD (classical n=41, late onset n=3, overlap syndrome n=8) were treated. These patients had previously received and failed 1 to 6 lines (median 3; 40/52 received ruxolitinib) of GvHD systemic treatments. GI-GvHD response was evaluated weekly and 28 days after day (D) 0 (inclusion for HERACLES or 1st dose for EAP). For all patients, GI-overall response rate (ORR) at D28 was defined as the proportion of patients achieving complete response (CR), very good partial response (VGPR) or partial response (PR), compared to D0, without the use of additional systemic therapy. Other endpoints included the best overall response (BOR) achieved at any time, and overall survival (OS). Prepared under GMP, MaaT013 is characterized by a highly consistent richness of 455 ±3% OTUs and an Inverse Simpson index > 20. Treatment comprised 3 MaaT013 doses, each composed of 30 g of feces in 150 mL volume of inoculum (total 90 g of feces from 4 to 8 healthy donors) administered by enema (except for 2 EAP patients by nasogastric tube). All patients received at least 1 MaaT013 dose, 92% (HERACLES) and 87% (EAP) at least 2 doses, and 50% (HERACLES) and 71% (EAP) the full treatment course. In HERACLES, the reasons for not applying the 3 rd dose were death (n=5), physician decision to introduce salvage therapy (n=5), and ICU hospitalization (n=2)). Results In HERACLES, the GI-ORR was 38% including 5 CR, 2 VGPR and 2 PR. In EAP, positive GI-response was achieved in 31/52 patients (60% with 16 CR, 11 VGPR and 4 PR). Considering the GI-BOR, 13/24 (54%) and 35/52 (67%) achieved at least a PR in HERACLES and EAP respectively. In HERACLES, OS was 29% at month (M) 6 and 25% at M12. OS was significantly higher in responding (R) patients (achieving at least PR at D28) compared to non-responding (NR) (44% vs 20% at M6 and 44% vs 13% at M12, logrank p=0.047). In EAP, OS was 48% at M6 and 37% at M12, and significantly higher in R patients compared to NR (71% vs 17% at M6 and 62% vs 6% at M12, logrank p In HERACLES, treatment with MaaT013 was characterized by excellent tolerance: 252 Treatment-Emergent Adverse Events (TEAE) were reported for the 24 patients, the majority being infections (79%) and GI disorders (62%), as expected in GvHD patients. Of these 252 TEAE, only 2% (5 serious events in 2 patients) could not reasonably be excluded from being related to MaaT013 by the investigators. Shotgun sequencing in these 5 TEAE revealed that the causative infectious agents could not be detected in the administered MaaT013. In EAP, the safety profile of MaaT013 was considered satisfactory for all patients. 16S microbiome analyses were performed in the HERACLES population and showed that MaaT013 produced an early increase in α-diversity at genus level with a significant increase in Richness index at all evaluated timepoints (p Conclusion We herein report the treatment of 76 SR-GI-aGvHD patients using a full ecosystem, pooled-donor, high-richness biotherapeutic. The D28 GI-ORR was 38% and 60% in HERACLES and EAP respectively and this clinical benefit positively and significantly impacted OS (44% and 62% M12 in HERACLES and EAP R patients respectively). MaaT013 was shown to be safe and effective in these heavily immunocompromised patients, warranting further exploration of this approach. Figure 1 Figure 1. Disclosures Malard: JAZZ pharmaceuticals: Honoraria; Sanofi: Honoraria; Astellas: Honoraria; Biocodex: Honoraria; Therakos/Mallinckrodt: Honoraria; Janssen: Honoraria. Loschi: CELGENE/BMS: Honoraria; AbbVie: Ended employment in the past 24 months, Honoraria; Gilead: Ended employment in the past 24 months, Honoraria; Novartis: Ended employment in the past 24 months, Honoraria; Servier: Ended employment in the past 24 months, Honoraria; MSD: Honoraria. Cluzeau: Agios: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel, accommodations, expenses, Speakers Bureau; Roche: Consultancy, Honoraria; BMS/Celgene: Consultancy, Honoraria, Speakers Bureau; Jazz Pharma: Consultancy, Honoraria; Abbvie: Consultancy, Honoraria, Speakers Bureau; Amgen: Speakers Bureau; Pfizer: Other: travel, accommodations, expenses; Astellas: Speakers Bureau; Takeda: Other: travel, accommodations, expenses. Huynh: Jazz Pharmaceuticals: Honoraria. Holler: MaaT Pharma: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Vehreschild: SocraTec R&D GmbH: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Ferring: Consultancy, Speakers Bureau; Farmak International Holding GmbH: Consultancy, Honoraria, Speakers Bureau; Bio-Mérieux: Consultancy, Speakers Bureau; Basilea: Consultancy, Speakers Bureau; Arderypharm: Consultancy, Speakers Bureau; Alb Fils Kliniken GmbH: Consultancy, Speakers Bureau; Takeda Pharmaceutical: Research Funding; Seres Therapeutics: Research Funding; Roche: Consultancy, Research Funding, Speakers Bureau; Organobalance: Consultancy, Research Funding, Speakers Bureau; Merck/MSD: Consultancy, Research Funding, Speakers Bureau; MaaT Pharma: Consultancy, Research Funding; Immunic AG: Consultancy, Research Funding, Speakers Bureau; Glycom: Research Funding; Gilead Sciences: Consultancy, Research Funding, Speakers Bureau; Evonik: Research Funding; Da Volterra: Consultancy, Research Funding, Speakers Bureau; Biontech: Research Funding; Astellas Pharma: Consultancy, Research Funding, Speakers Bureau; 3M: Research Funding. Gasc: MaaT Pharma: Current Employment. Plantamura: MaaT Pharma: Current Employment. Mohty: Sanofi: Honoraria, Research Funding; Pfizer: Honoraria; Novartis: Honoraria; Takeda: Honoraria; Jazz: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria; Celgene: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria; Astellas: Honoraria; Amgen: Honoraria; Adaptive Biotechnologies: Honoraria.

To the editor: Allogeneic bone marrow (BM) transplantation (alloBMT) is a unique curative therapy for diverse diseases. However, its use is limited by the development of severe treatment-related complications, most importantly, the occurrence of acute graft-versus-host-disease (aGVHD). Despite the

Late acute (LA) graft-versus-host disease (GVHD) is persistent, recurrent, or new-onset acute GVHD symptoms occurring >100 days after allogeneic hematopoietic cell transplantation (HCT). The aim of this analysis is to describe the onset, course, morbidity, and mortality of and examine angiogenic factors associated with LA GVHD. A prospective cohort of patients (n = 909) was enrolled as part of an observational study within the Chronic GVHD Consortium. Eighty-three patients (11%) developed LA GVHD at a median of 160 (interquartile range, 128-204) days after HCT. Although 51 out of 83 (61%) achieved complete or partial response to initial therapy by 28 days, median failure-free survival was only 7.1 months (95% confidence interval, 3.4-19.1 months), and estimated overall survival (OS) at 2 years was 56%. Given recently described alterations of circulating angiogenic factors in classic acute GVHD, we examined whether alterations in such factors could be identified in LA GVHD. We first tested cases (n = 55) and controls (n = 50) from the Chronic GVHD Consortium and then validated the findings in 37 cases from Mount Sinai Acute GVHD International Consortium. Plasma amphiregulin (AREG; an epidermal growth factor [EGF] receptor ligand) was elevated, and an AREG/EGF ratio at or above the median was associated with inferior OS and increased nonrelapse mortality in both cohorts. Elevation of AREG was detected in classic acute GVHD, but not chronic GVHD. These prospective data characterize the clinical course of LA GVHD and demonstrate alterations in angiogenic factors that make LA GVHD biologically distinct from chronic GVHD.

Acute intestinal graft-versus-host disease (aGVHD) refractory to immunosuppressive treatment is a serious complication after allogenic hematopoietic stem cell transplantation (HSCT). The underlying mechanisms of refractory aGVHD of the gut are not fully understood. Although telomere length (TL) reflects the replicative history of a cell, critically short telomeres have been associated with replicative exhaustion and tissue failure. In this study, we demonstrate that enterocytes of patients with refractory intestinal aGVHD show significantly increased proliferation, which translates into significant and critical telomere attrition following HSCT as compared with unaffected patients undergoing HSCT. Calculated telomere loss in aGVHD patients is 190 bp/wk, thereby massively exceeding physiological steady-state TL shortening rates such as in lymphocytes (∼50 bp/y). Our data support the hypothesis that increased compensatory proliferation following continued tissue damage can result in massive telomere loss in enterocytes of aGVHD patients. The present study introduces aGVHD-triggered increased cellular turnover and telomere loss with subsequent replicative exhaustion as a mechanism for refractory gut GVHD that is compatible with the long-term clinical aspect of the disease and provides a basis for stem cell protective therapies in the treatment of aGVHD.

We analyzed 661 adult patients who underwent single-unit (n = 226) or double-unit (n = 435) unrelated cord blood transplantation (UCBT) following a reduced-intensity conditioning (RIC) consisting of low-dose total body irradiation (TBI), cyclophosphamide, and fludarabine (Cy/Flu/TBI200). Eighty-two patients received rabbit antithymocyte globulin (ATG) as part of the conditioning regimen (ATG group), whereas 579 did not (non-ATG group). Median age at UCBT was 54 years, and diagnoses were acute leukemias (51%), myelodysplastic syndrome/myeloproliferative neoplasm (19%), and lymphoproliferative diseases (30%). Forty-four percent of patients were transplanted with advanced disease. All patients received ≥4 antigens HLA-matched UCBT. Median number of collected total nucleated cells was 4.4 × 10(7)/kg. In the ATG group, on 64 evaluable patients, ATG was discontinued 1 (n = 27), 2 (n = 20), or > 2 days before the graft infusion (n = 17). In multivariate analyses, the use of ATG was associated with decreased incidence of acute graft-versus-host disease (hazard ratio [HR], 0.31; 95% confidence interval [CI], 0.17-0.55; P < .0001), higher incidence of nonrelapse mortality (HR, 1.68; 95% CI, 1.16-2.43; P = .0009), and decreased overall survival (HR, 1.69; 95% CI, 1.19-2.415; P = .003). Collectively, our results suggest that the use of ATG could be detrimental, especially if given too close to graft infusion in adults undergoing UCBT following Cy/Flu/TBI200 regimen.

In this issue of Blood, Kakihana et al report for the first time successful and safe application of related fecal microbiota transplants (FMTs) via nasoduodenal tubes in a small series of patients suffering from steroid-resistant acute intestinal graft-versus-host disease (GVHD). In spite of concomitant cytomegalovirus and Aspergillus infections at the start of treatment, adverse effects were limited and mainly related to the mode of application. Three of 4 patients responded at the end of the observational period of 28 days after first FMT, allowing reduction of concomitant steroid dose by 69%. Four-week response was associated with restoration of commensal microbiota and accompanied by an increase of the effector regulatory T-cell/CD8 T-cell ratio in 3 of 4 patients. In the 3 responding patients, improvement of gut symptoms persisted after the last FMT, with 1 patient being alive 2 years after FMT and 2 patients finally relapsing from underlying disease 106 days and 1 year, respectively, after FMT without recurrence of GVHD.1

Introduction Myeloproliferative Neoplasm (MPN) unclassifiable (MPN-U), is a heterogeneous disease that presents with an MPN-type clinical/ histological phenotype yet fails to meet diagnostic criteria for other MPN entities. Incidence is Methods This registry-based analysis was approved by the Chronic Malignancies Working Party of the EBMT. Patient selection was performed by identifying adult patients who underwent first allo-HCT for MPN-U between 2000-2015, using either Reduced Intensity Conditioning (RIC) or Myeloablative conditioning (MAC) as defined by standard EBMT criteria. Further data collection requests (MED-C) forms were sent to centres to improve data completeness. Statistical analyses were performed with SPSS 22 (SPSS Inc./IBM, Armonk, NY) and R. Overall Survival (OS) was calculated from the date of transplant until death or last observation alive. Cumulative incidence functions were used to estimate Non-Relapse Mortality (NRM) and Relapse Incidence (RI) within a competing risk setting. Results A total of 70 patients, 48 (69%) male and 22 (31%) female, with a confirmed diagnosis of MPN-U were analysed. Median age was 57 years (range (r), 22-70 years). Of these patients, 37 (53%) underwent allo-HCT in the period 2001-2010 and 33 (47%) between 2011-2015. MAC regimens were utilised in 31 (44%) patients while 39 (56%) received RIC. Patients were most frequently transplanted within the first two years from diagnosis, with a median time to allo-HCT of 13 months (r, 3-244 months). Diagnostic karyotype was normal in 36 (51%) and abnormal in 23 (33%) patients, with data missing for 11 (16%) patients. A total of 45 (64%) patients had received prior treatment, 23 (33%) patients were untreated and data was incomplete in 2 (3%) patients. Regarding donor type, 27 (39%) patients had a Matched Sibling Donor (MSD) and 43 (61%) an Unrelated Donor (URD). Most frequent conditioning regimens were TBI-based in the MAC cohort and Fludarabine-Busulphan in the RIC cohort. A trend towards higher rates of delayed/failed engraftment was noted in the RIC compared to MAC cohort (p=0.09). Where successful, median time to neutrophil engraftment in both cohorts was similar (18 days for MAC and 17 for RIC) and both platforms demonstrated similar platelet engraftment times (17.5 days). Incidence of grade II-IV aGVHD at 3 months was higher in the MAC (37%) compared to RIC cohort (16%; p=0.05) and the 12-month cumulative incidence of cGVHD for MAC was 52% (95%CI: 32.4, 71.6) and for RIC 32.1% (95%CI: 14.8, 49.4; p=0.117)). Median follow-up was 87 months (minimum and maximum of censored cases: 31 and 196 months). The median OS estimates at 1, 3 and 5-year were 77%, 55% and 42% (MAC) and 59%, 44% and 41% (RIC), respectively (p=0.33). No significant difference existed in OS rates between those who had pre-transplant therapy versus not. Relapse remained significant: cumulative incidences of relapse at 1,3 and 5-years were 10%, 23% and 27% (MAC) and 28%, 36% and 36% (RIC), respectively (p=0.28). NRM probabilities at 1, 3 and 5-years post allo-HCT were also considerable: 19%, 29%, and 34% (MAC) and 28%, 28% and 28% (RIC), respectively (p=0.84). Main causes of NRM were infection and GVHD. Univariate analysis associated use of an URD with a significantly worse OS and NRM compared with MSD. Moreover, the presence of abnormal karyotype at time of allo-HCT was associated with a trend towards a higher risk of relapse (p=0.06). Conclusions This study highlights the potentially curative role of allo-HCT in MPN-U and provides clinicians with robust engraftment, GVHD and outcome data. Both engraftment and OS rates appear acceptable yet NRM and CIR rates in both settings remain high and need to be addressed. The impact of abnormal karyotype at the time of allo-HCT and a trend towards higher risks of relapse requires further elucidation. Disclosures McLornan: Novartis: Honoraria; Jazz Pharmaceuticals: Honoraria, Speakers Bureau. Robin:Novartis Neovii: Research Funding. Chalandon:Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Incyte Biosciences: Consultancy, Honoraria. Beelen:Medac GmbH Wedel Germany: Consultancy, Honoraria. Kröger:Sanofi-Aventis: Honoraria; Riemser: Research Funding; Novartis: Honoraria, Research Funding; Neovii: Honoraria, Research Funding; Medac: Honoraria; JAZZ: Honoraria; DKMS: Research Funding; Celgene: Honoraria, Research Funding. Milojkovic:Novartis: Honoraria, Speakers Bureau; Incyte: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; BMS: Honoraria, Speakers Bureau. Hernandez Boluda:Incyte: Other: Travel expenses paid.

To the editor: Distinct polymorphisms of NOD2/CARD15 (rs2066844 [SNP08], rs2066845 [SNP12], and rs2066847 [SNP13]) have influence on the incidence of Crohn disease (CD), a chronic inflammatory disorder of the gastrointestinal tract. Similar symptoms of CD and GVHD after allogeneic stem cell

Introduction: Sickle cell disease (SCD) is one of the most prevalent monogenic hematological disorders leading to progressive multi-organ damage and reduced life expectancy despite a significant improvement of conventional and supportive care. Allogeneic hematopoietic stem cell transplantation (HSCT) with a matched sibling donor (MSD) represents currently the only curative treatment option, limited by a donor availability Neurotoxicity (NT) contributes significantly to HSCT-associated morbidity and mortality. Calcineurin inhibitor (CNI) related NT after HSCT is mostly caused by cyclosporine A (CsA) and tacrolimus (FK506) with an overall frequency between 4.2% - 28.8%, and severe NT in 4% - 11%, such as posterior reversible encephalopathy syndrome (PRES), seizures, visual disturbance, cortical blindness, aphasia or ataxia. Particularly in hemoglobinopathies (thalassemia, SCD) the reported incidence is around 30%, possibly also due to impaired hepatic metabolization of CsA and FK506 based on iron overload. However, also infusion modalities might impact on NT, since it was more frequently reported after 4-hours bolus injections (10.3%) than continuous infusion (3.3%). However, the excessive NT observed in SCD might be also related to the systemic vasculopathy, increasing with age and pro-inflammatory triggers after allo-HSCT. Methods: A total of 30 pts aged 3 to 31 years with homozygous SCD or HbS 0/+ ß-Thal were treated between 2012 and 2018. Nine pts received a MSD bone marrow (BM) graft, 21 pts received T-haplo-HSCT from a relative (1 second T-haplo-HSCT due to graft rejection) with a α/β CD19 or CD3/CD19 depleted graft. Post-HSCT immunosuppression consisted of either CsA in 6 MSD and 16 T-haplo-HSCT pts or FK506 in 3 MSD and 6 T-haplo-HSCT pts, both mostly in combination with mycophenolate mofetil (MMF). FK506 was administered as a 20-hours continuous infusion, while CsA was given as 4-hours bolus injections. Dosage was adjusted according to usual target levels (CsA: 120ng/ml; FK506: 7ng/ml). Duration of immunosuppression was >6 months in T-haplo-SCT and Results: Overall CNI-related NT was observed in 32.3% of our HSCT pts. Severe NT such as PRES and visual disturbance combined with aphasia was present in 16.1% (Table 1). Overall, NT was more prevalent in MSD (n=5, 55.5%) than in T-haplo-HSCT pts (n=5, 22.7%). The majority of NT events was observed under CsA prophylaxis (8/10; 80%) and in only 22% (2/9) pts with FK506. All 5 cases of severe NT, particularly all PRES, occurred on CsA. Complete recovery of neurologic symptoms was achieved in all pts spontaneously or after switching CsA to FK506 or everolimus (Table 1). Moreover, 60% of pts with NT was >18 years, and 90% >12 years suggesting an increasing risk with age while a third of patients with pre-existing NT experienced no post-HSCT NT. Of note, 4 of 5 pts with severe NT also experienced a mild acute GvHD (Table 1). The overall (OS) and disease-free survival (DFS) with a median follow-up of 17 months in T-haplo-HSCT pts and 22 months in 9 MSD HSCT pts was 90% vs. 100%, respectively. Conclusions: Our data confirm an elevated risk for NT in SCD pts following allo-HSCT. Importantly, the incidence of NT events seems to be related to age (90% of pts with NT were >12years), donor source (MSD vs. T-Haplo; 55.5% vs 22.7%) and CsA rather than FK506 (CSA 80% vs. FK506 20%). All severe NT (particularly all PRES) were observed in pts with CsA prophylaxis, while the continuous infusion of FK506 might have levelled concentration peaks. Nevertheless, the NT observed with CsA post-HSCT could be the consequence of predominantly CsA-related vascular complications inflicting systemically pre-damaged vessels of SCD pts. The mechanism of action could be similar to other systemic endotheliopathies such as sinusoidal obstruction syndrome (VOD/SOS), transplant-associated microangiopathy (TAM) and acute GvHD which was observed in 4/5 pts with severe NT, compared to an overall GvHD rate of 29%. Disclosures Corbacioglu: Gentium: Consultancy, Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria.

Introduction: Increasing evidence suggests that the intestinal microbiota is involved in the development of acute graft-vs.-host disease (GVHD) after allogeneic hematopoietic cell transplantation (allo-HCT). We previously reported in single center studies that Enterococcus a) is associated with GVHD (Holler et al., BBMT 2014) and b) can dominate the post-transplant gut microbiota in up to 50% of allo-HCT patients resulting in a 9-fold increased risk of bacteremia (Taur et al, CID 2012). To further investigate the hypothesis that Enterococcus can trigger the development of GVHD, we studied both allo-HCT patients and pre-clinical mouse transplant models. Methods and Results: Stool samples from 1240 allo-HCT patients at 4 different transplant centers in the U.S., Germany and Japan were collected approximately weekly during inpatient hospitalization. The V4-V5 region of the bacterial 16S rRNA genes from 6718 samples was sequenced at one central site on the Illumina platform. We observed Enterococcus mono-domination (relative abundance > 30%) in post-transplant samples ranging from 20 to 60% of patients at different centers (Fig. A, left). This mono-domination was primarily attributable to E. faecium, and was associated with a significantly increased risk for grade 2-4 acute GVHD (Fig. A, right). In three different mouse models we found a transient bloom of E. faecalis around 7 days after transplant in allo-HCT recipients with GVHD (Fig. B; C57BL/6 -> 129SV model). This bloom did not occur in allo-HCT recipients of a T cell depleted allograft without GVHD. To further investigate this Enterococcus bloom, we treated mice with an experimental E. faecalis-strain on days 4 to 6 after transplant and found significantly increased lethal GVHD. Colonizing germ-free mice with a minimal gut flora also lead to increased lethal GVHD when enterococci were added to the gnotobiotic flora (Fig. C). The allo-HCT recipients with Enterococcus-containing flora had also increased serum IFNg levels. Short chain fatty acids (SCFA) can be protective against GVHD and gut inflammation through maintenance of epithelial homeostasis and increases in anti-inflammatory regulatory T cells in the gut. In BMT mouse models, we found that Enterococcus-dominated allo-HCT recipients with GVHD have significantly less cecal butyrate, a major SCFA. Similarly, Enterococcus domination after allo-HCT also leads to a decrease in fecal SCFAs in patients (Fig. D). Next, we hypothesized that intestinal IgA might have a protective role against this pathogen in mice. 16S sequencing of flow sorted IgA-coated vs. non-coated bacteria from fecal samples of allo-HCT patients and transplanted mice revealed no specific IgA-coating pattern of enterococci both before or after transplant rather excluding the hypothesis that IgA might have a protective role against Enterococci. E. faecalis and E. faecium use the disaccharide lactose as a major carbohydrate source for growth and expansion as observed by analyses of the Enterococcus genome and in vitro growth experiments. In mice, we observed that a lactose-free diet significantly decreases the Enterococcus bloom after transplant in allo-T cell recipients and in first survival experiments attenuates lethal GVHD (Fig. E). Conclusion: Our studies in mouse and man demonstrate that the abundance of Enterococcus in the intestinal flora plays a role in the development of GVHD and the prevention of Enterococcus growth with a lactose-free diet can ameliorate GVHD. Disclosures Peled: Seres Therapeutics: Research Funding.

Introduction. Recurrent disease is the major cause of treatment failure after allogeneic stem cell transplantation (SCT) in patients with AML. Second SCT (SCT2) is a valid treatment option in this setting but outcome is relatively poor. Haplo-identical (haplo) SCT is increasingly used over the last decade due to the introduction of non T-depleted methods. Prior studies have shown similar outcome when using the same or different HLA-matched donor for SCT2. However, there is relatively limited data on the use of haplo-donors. Methods and Results. The study included 556 patients with AML relapsing after a first allogeneic SCT (SCT1) given in CR1 from an HLA-matched sibling (sib, n= 294) or a matched unrelated donor (MUD, n=262) and given SCT2 during the years 2006-2016. The median age at SCT2 was 46 years (20-73). 247 patients were in CR2 (44%) and 309 had active leukemia (55%) at the time of SCT2. The conditioning regimen was myeloablative (MAC, 66%) or reduced-intensity (RIC, 34%) for SCT1, and 41% and 59%, respectively for SCT2. 19% of all patients had acute GVHD grade II-IV and 20% had chronic GVHD after SCT1 and before relapse. Patients were divided into 3 groups based on the donor selected for SCT2; 1) same donor (n=163, sib/sib-112, MUD/MUD-51), 2) different HLA-matched donor (n=305, sib/different sib-44, sib/MUD-93, MUD/ different MUD- 168), 3) haplo-donor (n=88, sib/haplo-45, MUD/haplo-43). All haploSCT were non T-depleted. There were some differences between the 3 groups in the timing of relapse and SCT2. The median time from SCT1 to relapse was similar; 10.6, 12.5 and 9.3 months, respectively (P=0.14). However, the median time from relapse to SCT2 was shorter for the same donor group; 2.8, 3.7 and 3.5 months, respectively (P Conclusions. Second SCT with the same donor or different matched donor is associated with similar outcomes in patients with relapsed AML after a first SCT. However, SCT2 with a haplo-donor is associated with higher NRM and lower LFS, mostly in patients given MAC in SCT1. Prior chronic GVHD after SCT1 is associated with lower relapse rate after SCT2. The role of prior chronic GVHD in donor selection should be further investigated. Disclosures Finke: Medac: Consultancy, Honoraria, Other: travel grants, Research Funding; Neovii: Consultancy, Honoraria, Other: travel grants, Research Funding; Novartis: Consultancy, Honoraria, Other: travel grants, Research Funding; Riemser: Consultancy, Honoraria, Research Funding. Gramatzki:Affimed: Research Funding. Stelljes:Novartis: Honoraria; Amgen: Honoraria; JAZZ: Honoraria; MSD: Consultancy; Pfizer: Consultancy, Honoraria, Research Funding. Stoelzel:Neovii: Speakers Bureau. Mohty:MaaT Pharma: Consultancy, Honoraria.

Introduction Despite substantial progress in the treatment of newly diagnosed AML, 20% to 40% of patients do not achieve remission with conventional induction chemotherapy. In addition, 50% to 70% of patients with CR1 are expected to relapse within 3 years. Currently, allogeneic HSCT is thought to be the most reliable curative option for patients with relapsed and/or refractory (r/r) disease. Compared to HSCT in CR1 allogeneic HSCT beyond CR1 is associated with lower survival rates. So far, different predictive factors influencing survival in r/r AML have been identified. However, pretransplantation variables delineating prognostic subgroups after allogeneic HSCT beyond CR1 are less well known and the total number of patients in trials addressing this issue has been limited. Methods We analyzed the outcome after allogeneic HSCT in non-APL-AML patients transplanted with either subsequent 2nd complete remission (CR2), r/r AML or with persisting cytopenia (PC, including patients with hematologic complete remission with incomplete hematologic recovery and patients with persisting bone marrow hypoplasia) after induction or salvage therapy. Of 3989 patients who enrolled into a multicenter clinical trial (AMLCG1999; NCT00266136) of the German Acute Myeloid leukemia Cooperative Group (AMLCG) we identified 498 subjects aged 17 to 74 years who underwent allogeneic HSCT beyond first CR. Before transplantation, all patients received first line treatment either with 6-Thioguanine, cyararabine, daunorubicin (TAD) and high-dose cytarabine, mitoxantrone (HAM) or HAM/HAM. After induction failure (n=97) or relapse (n=401) patients either received salvage chemotherapy (n=299) or directly underwent allogeneic HSCT (n=199). Overall survival (OS) and relapse free survival (RFS) after allogeneic HSCT were the main endpoints for this analysis. Kaplan-Meier analysis was performed to estimate survival probabilities in subgroups. Log-rank tests were used to compare survival groups. Relative hazard rates and differences in subgroups were calculated using cox regression analysis. Results At a median follow-up of 6.5 years for surviving patients, OS and RFS rates were 32% (95% CI 28-36%) and 30% (95% CI 26-34%), respectively. Subgroup analysis revealed an OS of 49% in 128 patients with allogeneic HSCT in CR2. In comparison, 73 patients who underwent allogeneic HSCT with PC after induction (n = 42) / salvage treatment (n = 31) and 297 patients with refractory disease (RD) exhibited an OS of 39% and 23%, respectively (Figure 1). The corresponding cumulative incidences (CI) at 1/6.5 years for death in remission after allogeneic HSCT were 25%/31% for patients transplanted in CR2, 29%/41% for patients transplanted with PC and 30%/46% for patients transplanted with RD, respectively. The cumulative incidence of relapse (CIR) after 6.5 years was 58% for patients transplanted with RD compared to 34% for patients transplanted in CR2 and 31% for patients with PC. Main prognostic factors for survival outcome were remission status prior to allogeneic HSCT and cytogenetic risk category at diagnosis. By contrast, patient age, donor, graft source, remission status after induction therapy and CMV status had no significant impact on survival outcome. Conclusions Long-term OS rates after allogeneic HSCT for patients with r/r AML are considered to range between 20 and 40%. Our analysis indicates that a substantial fraction of patients who received similar first line treatment within one prospective trial is able to achieve long term survival when transplanted beyond CR1. In addition, remission upon salvage therapy prior to allogeneic HSCT might be a relevant prognostic factor for patients with r/r AML. However, whether remission induction prior to HSCT can provide an overall survival benefit in patients with r/r disease is still elusive and is currently being evaluated in a prospective randomized clinical trial (ETAL3-ASAP; NCT02461537). Disclosures Beelen: Medac: Consultancy, Other: Travel Support. Scheid:Novartis: Honoraria, Research Funding; Janssen: Honoraria; Celgene: Honoraria; Takeda: Honoraria, Research Funding; BMS: Honoraria; Amgen: Honoraria. Lenz:Celgene Corp.: Consultancy, Honoraria, Other: Travel, Accomodations, Expenses, Research Funding, Speakers Bureau; Novartis: Research Funding; Roche: Consultancy, Honoraria, Other: Travel, Accomodations, Expenses, Research Funding; Bayer: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Other: Travel, Accomodations, Expenses, Research Funding, Speakers Bureau; Gilead: Consultancy, Honoraria. Hiddemann:Bayer: Consultancy, Research Funding; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; F. Hoffman-La Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Stelljes:MSD: Consultancy; JAZZ: Honoraria; Amgen: Honoraria; Novartis: Honoraria; Pfizer: Consultancy, Honoraria, Research Funding.

Intestinal microbiota composition is associated with important outcomes after allo-HCT including survival, relapse, GVHD, and infections. These observations have been made almost exclusively by characterizing the microbiota in the first weeks after transplantation, and in single-center studies. We previously reported that intestinal diversity measured peri-neutrophil engraftment is predictive of overall survival in a multicenter cohort. Here, we hypothesized that pre-HCT microbiota configuration may also be an important determinant of post-transplantation outcomes. We report a multicenter analysis conducted at 4 independent international institutions to test this hypothesis. We collected 1922 stool samples ~weekly from 991 unique allo-HCT patients at four international transplant centers: Cohorts 1 and 2 in the US, cohort 3 in Europe, and cohort 4 in Japan. The patients-all adult recipients of allo-HCT-varied in underlying diagnosis, donor-graft sources, conditioning intensity, and GVHD prophylaxis. Samples from all 4 centers were sequenced and analyzed at a central laboratory using the V4-V5 region of 16S rRNA. For patients with multiple samples within the pre-HCT sampling period, which we defined as day -20 to 0, median values were used. On average, patients from all 4 transplantation centers had reduced microbiota diversity pre-HCT, as measured by median α-diversity (inverse Simpson) values that were 1.7-to-2.5-fold lower than those of healthy volunteers. This comparison was made both in volunteers whose samples we sequenced ourselves and in a publicly available dataset (Fig A, p We next asked how similar these pre-HCT communities are across geography. We found that Bray-Curtis distances between institutions were reproducibly much smaller in magnitude than the changes observed over time during transplantation (Fig C, p In order to characterize these clinically relevant low-diversity phenotypes, we defined domination as a microbiota injury in which any operational taxonomic unit comprises >30% of bacterial abundance. The dominating taxa belonged to multiple genera, the most common being Enterococcus, Streptococcus, Lactobacillus, Escherichia, and Klebsiella (Fig E) as annotated by Greengenes and NCBI databases. At all four institutions, the cumulative incidence of intestinal domination by any organism was >50% by day 0 and was >87% by day +28 (Fig F). We performed additional analyses in the largest cohort and found that the low-diversity state is associated with exposure to broad-spectrum antibiotics, conditioning intensity, and low calorie intake. We demonstrate that HCT patients at 4 institutions on 3 continents presented with microbiota configurations that were similar to one another and distinct from those of healthy individuals. Severe microbiota injury as revealed by domination is a common event whose development begins before allograft infusion, and pre-HCT microbiota injury predicts poor overall survival. These observations suggest the pre-HCT period as a window of opportunity to (a) assess microbiota injury as part of comorbidity evaluation, (b) inform selection of antibiotic prophylaxis, gut-decontamination, GVHD-prophylaxis, or conditioning regimens, and (c) intervene with microbiota injury-remediation or prevention strategies. Figure. Figure. Disclosures Peled: Seres Therapeutics: Research Funding. Perales:Merck: Other: Personal fees; Takeda: Other: Personal fees; Abbvie: Other: Personal fees; Novartis: Other: Personal fees; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: Personal fees and Clinical trial support. Jenq:MicrobiomeDx: Consultancy; Ziopharm Oncology: Consultancy; Seres Therapeutics, Inc.: Membership on an entity's Board of Directors or advisory committees; Seres Therapeutics, Inc.: Patents & Royalties.

Background: Sickle cell disease (SCD) is an inherited disorder with an estimate of 300,000 affected newborns per year worldwide. Despite improvements in preventive measures and conventional therapy, substantial morbidity and mortality, resulting in a reduced life expectancy persist. Allogeneic hematopoietic stem cell transplantation (HSCT) with a matched sibling donor (MSD) is currently the curative standard of care. However, matched donor availability is Methods: We report the results of 29 pts with advanced stage SCD transplanted with either a T-cell depleted haploidentical regimen (20 pts, median age 13 years, range 3-31 years) or with bone marrow from a matched family donor (MSD, 9 pts, median age 14, range 9-25 years). Indication for HSCT was advanced stage SCD related complications. Pts with a MSD received a bone marrow (BM) graft. Pts requiring an alternative donor were transplanted with an αß/CD19 (n=5) or CD3/CD19 (n=15) depleted graft from a haploidentical family donor. The conditioning regimen for both groups was identical except that antithymoglobulin (ATG-Neovii®) was given upfront on day -10 to -8 in T-haplo-HSCT and on day -3 to -1 in MSD. Chemotherapy consisted of thiotepa 2 x 5 mg/kg, fludarabine 4 x 40 mg/m2 and treosulfan 3 x 14 g/m2, given between days -10 and -2. Post-HSCT immunosuppression consisted of mofetil mycophenolate and tacrolimus or cyclosporine A for a duration >6 months in T-haplo-HSCT and Results: The conditioning regimen was well tolerated in all pts with no high-grade transplant related toxicity. The overall (OS) and disease-free survival (DFS) with a median follow-up of 17 months in 20 advanced stage mostly adolescent and adult T-haplo-HSCT pts and 22 months in 9 MSD HSCT pts was 90% vs. 100%, respectively. Engraftment was achieved in most pts with stable chimerism over 90%, except for 4 pts with a stable MC in the T-haplo SCT group and 1 patient in the MSD group off immunosuppression. All pts presented a complete BM engraftment of red cell precursors. Only one patient in the T-haplo-HSCT group with a mixed chimerism is still under immunosuppression. The post-HSCT bacterial infectious complications were comparable in both groups and the most common observed viral infections were systemic cytomegaly virus (CMV), adenovirus (ADV), polyomavirus (BKV) and Epstein Barr virus (EBV) reactivations which were successfully treated with intravenous antiviral drugs or virus specific T-cells. Two patients in the T-haplo-HSCT group achieved a DFS off immunosuppression but succumbed to post-HSCT complications. One developed a rotavirus gastroenteritis and an uncontrolled CMV pneumonitis. The other a late graft failure and succumbed to a macrophage activation syndrome. None of our pts developed a Glucksberg Grade III-IV acute GvHD and in the T-haplo SCT group 4 pts (20%) and in the MSD group 2 pts (22%) developed a steroid sensitive mild to moderate cGvHD (fasciitis/oral as well as mild cutaneous GvHD). No extensive cGvHD was observed. To our knowledge this is the largest group of advanced stage SCD pts transplanted with either a T-haplo-HSCT or a MSD approach, according to donor availability, with an almost identical regimen in a direct comparative design. These preliminary results demonstrate increasing evidence for the feasibility, safety and efficacy of a T-haplo-HSCT using CD3/CD19 or αβ/CD19 depleted grafts, in order to offer a curative option to the majority of pts with SCD. A prospective, stratified non-inferiority trial is in preparation (EudraCT number 2018-002652-33) in order to confirm these preliminary results in a large prospective cohort. Disclosures Corbacioglu: Gentium: Consultancy, Honoraria; Jazz Pharmaceuticals: Consultancy, Honoraria.

Acute graft-versus-host disease (GVHD) remains the major obstacle to a more favorable therapeutic outcome of allogeneic hematopoietic stem cell transplantation (HSCT). GVHD is characterized by tissue damage in gut, liver, and skin, caused by donor T cells that are critical for antitumor and antimicrobial immunity after HSCT. One obstacle in combating GVHD used to be the lack of understanding the molecular mechanisms that are involved in the initiation phase of this syndrome. Recent research has demonstrated that interactions between microbial-associated molecules (pathogen-associated molecular patterns [PAMPs]) and innate immune receptors (pathogen recognition receptors [PRRs]), such as NOD-like receptors (NLRs) and Toll-like receptors (TLRs), control adaptive immune responses in inflammatory disorders. Polymorphisms of the genes encoding NOD2 and TLR4 are associated with a higher incidence of GVHD in HSC transplant recipients. Interestingly, NOD2 regulates GVHD through its inhibitory effect on antigen-presenting cell (APC) function. These insights identify important mechanisms regarding the induction of GVHD through the interplay of microbial molecules and innate immunity, thus opening a new area for future therapeutic approaches. This review covers current knowledge of the role of PAMPs and PRRs in the control of adaptive immune responses during inflammatory diseases, particularly GVHD.

Introduction The composition of the intestinal microbiota is associated with important outcomes after allo-HSCT including survival, relapse, GVHD, and infections. The microbial composition changes drastically after allo-HSCT, with precipitous drops in α-diversity and domination by single organisms. Low diversity is particularly associated with poor overall survival from transplant-related mortality, including death after GVHD. These observations were all made in single-center cohorts. Here we compare-for the first time-the kinetics of microbiota diversity in allo-HSCT patients from three independent international institutions. We also explore the role of nutrition as well as antibiotics in a single-center pilot study and the recovery from microbiota injury after discharge. Methods We collected stool samples approximately weekly from adult allo-HSCT inpatients and approximately monthly post-discharge. Samples from all three centers were sequenced at MSKCC using the V4-V5 region of 16S rRNA. In the nutrition cohort, patients self-documented oral intake at each meal; data were confirmed in focused patient interviews by a registered dietician three times weekly and nutritional compositions were obtained from the hospital kitchen database. Oral swish samples were collected prior to allo-HSCT and during post-transplant neutropenia. Results 5,823 samples from 1,118 patients were compared: 5,508 samples from 947 MSKCC patients, 108 samples from 40 Duke patients, and 152 samples from 109 Regensburg, Germany patients. The patients-all adult recipients of allo-HSCT -varied in underlying diagnosis, donor-graft sources, conditioning intensity, and GVHD prophylaxis. We mapped the microbiota composition of each sample in 2-D space using t-Distributed Stochastic Neighbor Embedding (t-SNE). Samples from all three centers spread throughout t-SNE space, revealing no transplant-center-specific effect (Fig 1A). Enterococcus domination, a low-diversity state we previously associated with subsequent enterococcal bacteremia was observed in all three centers (Fig 1A). Diversity decreased comparably after allo-HSCT transplant at all centers (Fig 1B), validating our prior findings in this larger, international multicenter cohort. In a separate cohort of MSKCC patients, we explored the relative contributions of antibiotics and nutritional intake using a multivariate linear regression model of α-diversity dynamics. Exposure to piperacillin-tazobactam (pip-tazo), a drug with broad-spectrum antibacterial activity, and calorie intake were significant predictors (p Conclusion We determine that striking microbiota injuries after allo-HSCT occur generally across geographic regions. Nutritional perturbations and antibiotic exposure are both associated with microbiota injury, and recovery from the injured state occurs over several months post-discharge. Importantly, diversity at the time of neutrophil engraftment is associated reproducibly with overall survival. Disclosures Peled: Seres: Research Funding. Sung: Merck: Research Funding; Novartis: Research Funding; Cellective: Research Funding. Jenq: Seres: Research Funding. van den Brink: Jazz Pharmaceuticals: Consultancy; Seres: Research Funding; PureTech Health: Consultancy; Therakos Institute: Other: Speaking engagement.

Introduction: Amphiregulin (AREG) is an epidermal growth factor receptor (EGFR) ligand that can restore integrity to damaged intestinal mucosa in murine models of acute graft-versus-host disease (aGVHD). We previously described AREG as a circulating biomarker of late-onset aGVHD, but its relevance combined with clinical risk factors has not yet been tested in a large cohort of patients with aGVHD occurring prior to day 100 post-transplant. We therefore tested samples from two aGVHD first-line treatment trials, Blood and Marrow Transplant Clinical Trials Network (BMT CTN) 0302 and 0802, and identified a clinically relevant threshold level of circulating AREG at aGVHD onset. We then investigated whether incorporating AREG into the refined Minnesota Risk Score could further risk-stratify patients. Patients and Methods: Blood samples were obtained at the onset of systemic aGVHD treatment within BMT CTN 0302 (serum) and BMT CTN 0802 (plasma). All patients with response data and samples for analysis from both trials were included (N=251). We determined the association of AREG with clinical outcomes, including risk stratification by the refined Minnesota criteria, day 28 complete/partial response (CR/PR) to first-line therapy, 2-year overall survival (OS) and 6-month and 2-year non-relapse mortality (NRM). We investigated the effect of AREG on clinical endpoints per a doubling in the value of AREG, defined a clinically relevant threshold level in the AREG values using two-fold cross-validation, and confirmed the clinical relevance of this cut-point in independent samples (N=92) from pooled from drawn from the Chronic GVHD Consortium and the Mount Sinai Acute GVHD International Consortium (MAGIC). Results: In patients enrolled in BMT CTN 0302/0802, AREG levels were 1.7-fold higher in patients with Minnesota high-risk (HR) compared to standard-risk (SR) aGVHD (HR median 53.4 vs SR 31 pg/ml, p Conclusion: AREG is elevated in patients with poor aGVHD outcomes and adds to the accuracy of risk stratification when combined with the refined Minnesota Risk Score. AREG ≥33 pg/mL at aGVHD onset is associated with lower day 28 CR/PR and higher mortality in samples from 4 multicenter cohorts. The mechanism of elevated circulating AREG in severe aGVHD is not yet known, although we hypothesize the degree of AREG elevation reflects the intensity of immune-mediated tissue injury resulting in AREG release. With accumulating evidence of altered EGFR ligands in aGVHD, further investigation into epithelial repair pathways involving AREG may lead to new adjunctive therapies to overcome poor steroid response in high-risk aGVHD. Disclosures Holtan: Incyte: Other: One-time advisory board member. Khera: Novartis: Consultancy. Lee: Mallinckrodt: Honoraria; Amgen: Other: One-time advisory board member; Bristol-Myers-Squibb: Other: One-time advisory board member; Kadmon: Other: One-time advisory board member. Chen: Immudex: Research Funding. Arora: Takeda Oncology: Consultancy. Flowers: Pharmacyclics: Consultancy. Cutler: Pfizer: Consultancy; Kite: Consultancy; Incyte: Consultancy; Bristol-Myers Squibb: Consultancy; Pharmacyclics: Consultancy; Astellas: Consultancy. Jagasia: Janssen: Consultancy, Research Funding; Therakos: Consultancy, Research Funding; Mallinckrodt: Consultancy. Hexner: Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding. Levine: Novartis Pharmaceuticals Corporation: Membership on an entity's Board of Directors or advisory committees. MacMillan: Magenta Therapeutics: Research Funding.

Urinary 3-IS levels predict outcome after ASCT and are associated with antibiotics and NOD2/CARD15 variants.Publisher’s Note: There is an Inside Blood Commentary on this article in this issue.Indole, which is produced from l-tryptophan by commensal bacteria expressing tryptophanase, not only is an important intercellular signal in microbial communities, but also modulates mucosal barrier function and expression of pro- and anti-inflammatory genes by intestinal epithelial cells. Here, we hypothesized that decreased urinary excretion of 3-indoxyl sulfate (3-IS), the major conjugate of indole found in humans, may be a marker of gut microbiota disruption and increased risk of developing gastrointestinal (GI) graft-versus-host-disease. Using liquid chromatography/tandem mass spectrometry, 3-IS was determined in urine specimens collected weekly within the first 28 days after allogeneic stem cell transplantation (ASCT) in 131 patients. Low 3-IS levels within the first 10 days after ASCT were associated with significantly higher transplant-related mortality (P = .017) and worse overall survival (P = .05) 1 year after ASCT. Least absolute shrinkage and selection operator regression models trained on log-normalized counts of 763 operational taxonomic units derived from next-generation sequencing of the hypervariable V3 region of the 16S ribosomal RNA gene showed members of the families of Lachnospiraceae and Ruminococcaceae of the class of Clostridia to be associated with high urinary 3-IS levels, whereas members of the class of Bacilli were associated with low 3-IS levels. Risk factors of early suppression of 3-IS levels were the type of GI decontamination (P = .01), early onset of antibiotic treatment (P = .001), and recipient NOD2/CARD15 genotype (P = .04). In conclusion, our findings underscore the relevance of microbiota-derived indole and metabolites thereof in mucosal integrity and protection from inflammation.

Single nucleotide polymorphisms (SNPs) of the NOD2/CARD15 gene resulting in a diminished nuclear factor-κB (NF-κB) response to bacterial cell wall products have been associated with an increased incidence of Crohn disease. To assess a possible contribution of NOD2/CARD15 mutations to graft-versus-host disease (GvHD) and complications following allogeneic stem cell transplantation, we retrospectively typed DNA from donor/recipient pairs in 169 consecutive patients receiving transplants from related or unrelated donors. Mutated alleles were observed in 21% of patients and in 14% of donors. Cumulative incidence of 1-year, transplant-related mortality rose from 20% in donor/recipient pairs without mutated SNPs to 49% in pairs with recipient mutations (P = .03) and 59% in pairs with donor mutations (P < .005), and was highest in 12 pairs with mutated alleles in both donor and recipients (83%; P < .001). Similar associations were observed for severe overall and severe gastrointestinal GvHD. The impact of NOD2/CARD15 mutations was more prominent for HLA-identical sibling transplantations but was also observed in unrelated donor transplantation. Mutations proved to be independent risk factors for transplant-related mortality. Our findings indicate a major role of monocyte/macrophage dysfunction in the pathophysiology of GvHD and strongly suggest a future risk assessment or even donor selection through NOD2/CARD15 typing.

A phase 1 diagnostic study was performed to evaluate a novel technology for clinical proteom research based on capillary electrophoresis and mass spectrometry. Urine from 40 patients after hematopoietic stem cell transplantation (HSCT; 35 allogeneic, 5 autologous) and 5 patients with sepsis was collected for a period of 100 days and analyzed. More than 1000 different polypeptides could be detected in individual samples. Polypeptide patterns excreted in the urine of patients were significantly different from those of healthy volunteers. No significant differences were detected comparing different conditioning regimens. The aim of this study was to identify polypeptide patterns functioning as early indicators of graft-versus-host disease (GVHD). Eighteen patients developed GVHD after allogeneic HSCT. Sixteen differentially excreted polypeptides formed a pattern of early GVHD markers, allowing discrimination of GVHD from patients without complications with 82% specificity and 100% sensitivity, cross-validated. Inclusion of 13 sepsis-specific polypeptides allowed us to distinguish sepsis from GVHD with a specificity of 97% and a sensitivity of 100%. Sequencing 2 prominent GVHD-indicative polypeptides led to the identification of a peptide from leukotriene A4 hydrolase and a peptide from serum albumin. The data reveal that capillary electrophoresis and mass spectrometry allow identification of biomarkers for a variety of diseases or related complications. (Blood. 2004;104:340-349)

Graft-versus-host disease (GVHD), the primary cause of non-relapse mortality (NRM) following allogeneic hematopoietic stem cell transplantation, does not always respond to treatment with high dose systemic corticosteroids. We have recently shown that a combination of three biomarkers (TNFR1, ST2, and REG3α) measured at onset of GVHD can predict day 28 response to treatment and 6-month NRM (Levine, Lancet Haem, 2015). Our goal in the current study was to determine if the same biomarker-based Ann Arbor GVHD algorithm can alsopredict treatment response andmortality whenapplied after one week of systemic corticosteroid treatment. The study population consisted of 378 patients (pts) with acute GVHD from 11 centers in the Mount Sinai Acute GVHD International Consortium. All pts were treated with systemic steroids and provided a plasma or serum sample obtained after one week of treatment (±3 days). The median starting dose of systemic steroids for Grade II-IV GVHD was 2.0 mg/kg/day and for Grade I was 1.0 mg/kg/day, after which treatment varied. Patients were divided into test (n=236) and validation (n=142) cohorts. We applied the Ann Arbor GVHD algorithm to concentrations of TNFR1, ST2, and REG3α measured after one week of treatment to generate a predicted probability of 6-month NRM, which we term the treatment score (TS). We employed unsupervised k-medoidclustering to partition TS values from the test cohort into two groups (high and low). This unbiased approach identified a high score group made up of 25% of pts (n=58) in the test cohort. We observed that the day 28 response rate (complete, CR + partial, PR) was significantly lower in pts with high scores compared to low scores in the test cohort (24% vs 65%, p Approximately half of the pts in each cohort (test: 48%; validation: 44%) responded (CR+PR) to the first week of steroids and these ptshad significantly lower 6-month NRM than non-responders (NR) (test: 17% vs 36%, p=0.0002; validation: 13% vs 36%, p=0.0014). Yet the TS continued to stratify mortality risk independently of clinical response. In the test cohort, pts with a high score comprised 16% of all early responders and experienced more than twice the NRM of early responders with a low score (33% vs 13%, p=0.022) (Fig 2A). Conversely, test cohort pts who did not respond by day 7, but had a low score, fared much better than non-responders with a high score (NRM 21% vs 68%, p In conclusion, a treatment score based on three GVHD biomarkers measured after one week of steroids stratifies pts into two groups with distinct risks for treatment failure and 6-month NRM. It is particularly noteworthy that the TS identifies two subsets of pts with steroid refractory (SR) GVHD who have highly different outcomes (Fig 2B/D). The much larger group, approximately two thirds of all SR pts, may not need the same degree of treatment escalation as is traditional for clinical non-response, and thus overtreatment might be avoided. Because the TSis measured at a common decision making time point, it may prove useful to guide risk-adapted therapy. Disclosures Mielke: Novartis: Consultancy; MSD: Consultancy, Other: Travel grants; Celgene: Other: Travel grants, Speakers Bureau; Gilead: Other: Travel grants; JAZZ Pharma: Speakers Bureau. Kroeger:Novartis: Honoraria, Research Funding. Chen:Incyte Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding. Jagasia:Therakos: Consultancy. Kitko:Therakos: Honoraria, Speakers Bureau. Ferrara:Viracor: Patents & Royalties: GVHD biomarker patent. Levine:Viracor: Patents & Royalties: GVHD biomarker patent.

No laboratory test can predict non-relapse mortality (NRM) after hematopoietic cellular transplantation (HCT) prior to the onset graft-versus-host disease (GVHD). Recently, we have shown that a signature of three GVHD plasma biomarkers (TNFR1, ST2, and REG3α) can predict response to GVHD therapy and NRM at the onset of clinical GVHD (Levine, Lancet Haem, 2015). Our goal in the current study was to identify a blood biomarker signature that could predict lethal GVHD and six-month NRM well in advance of the onset of GVHD symptoms. Patient samples on day +7 after HCT were obtained from 1,287 patients from 11 HCT centers in the Mount Sinai Acute GVHD International Consortium (MAGIC). Samples from two large centers (n = 929) were combined and randomly assigned to a training set (n = 620) and test set (n = 309). 358 patients from nine others centers constituted an independent validation set. The overall cumulative incidences of 6-month NRM were 11%, 12%, and 13% for the training, test, and validation sets respectively. The incidence of lethal GVHD, defined as death without preceding relapse while under steroid treatment for acute GVHD, were 18%, 24%, and 14% in the same groups, respectively. The median day of GVHD onset was 28 days in the training set and 29 days in the test and validation sets. We measured four GVHD related biomarkers [ST2, REG3α, TNFR1, and IL2Rα] in all samples and used the training set alone to develop competing risks regression models that used all 13 possible combinations of one to four biomarkers to predict 6-month NRM. The best algorithm, which we rigorously confirmed through Monte Carlo cross-validation of 75 different combinations of training sets, included ST2 and REG3α. No combination of one, three, or four biomarkers was superior to the combination of these two biomarkers. The day 7 algorithm identified high risk (HR) and low risk (LR) groups with 6-month NRMs of 28% and 7%, respectively (p In conclusion, we have developed a blood biomarker algorithm that predicts the development of lethal GVHD seven days after HCT, which performed successfully in large multicenter validation sets. The GVH reaction is already in progress by day +7, even though clinical symptoms may not occur until days or weeks later. We speculate that the blood biomarker concentrations at this early time point reflect subclinical GI pathology, a notion that is reinforced by the fact that ST2 and REG3α, the two biomarkers in the algorithm, are closely associated with GI GVHD. The algorithm identified HR and LR strata in several patient groups with different overall risk for lethal GVHD (donor, HLA match, conditioning regimen intensity, age). This day +7 algorithm should prove useful in clinical BMT research by identifying patients at high risk for lethal GVHD who might benefit from aggressive preemptive treatment strategies. Disclosures Chen: Novartis: Research Funding; Incyte Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Jagasia:Therakos: Consultancy. Kitko:Therakos: Honoraria, Speakers Bureau. Kroeger:Novartis: Honoraria, Research Funding. Levine:Viracor: Patents & Royalties: GVHD biomarkers patent. Ferrara:Viracor: Patents & Royalties: GVHD biomarkers patent.

The influence of interferon- (IFN) pretreatment on the outcome after allogeneic bone marrow transplantation (BMT) in chronic myelogenous leukemia (CML) is controversial. One goal of the German randomized CML Studies I and II, which compare IFN ± chemotherapy versus chemotherapy alone, was the analysis of whether treatment with IFN as compared to chemotherapy had an influence on the outcome after BMT. One hundred ninety-seven (23%) of 856 Ph/bcr-abl–positive CML patients were transplanted. One hundred fifty-two patients transplanted in first chronic phase were analyzed: 86 had received IFN, 46 hydroxyurea, and 20 busulfan. Forty-eight patients (32%) had received transplants from unrelated donors. Median observation time after BMT was 4.7 (0.7 to 13.5) years. IFN and chemotherapy cohorts were compared with regard to transplantation risks, duration of treatments, interval from discontinuation of pretransplant treatment to BMT, conditioning therapy, graft-versus-host disease prophylaxis and risk profiles at diagnosis and transplantation, and IFN cohorts also with regard to performance and resistance to IFN. Outcome of patients receiving related or unrelated transplants pretreated with IFN, hydroxyurea, or busulfan was not significantly different. Five-year survival after transplantation was 58% for all patients (57% for IFN, 60% for hydroxyurea and busulfan patients). The outcome within the IFN group was not different by duration of prior IFN therapy more or less than 5 months, 1 year, or 2 years. In contrast, a different impact was observed in IFN-pretreated patients depending on the time of discontinuation of IFN before transplantation. Five-year survival was 46% for the 50 patients who received IFN within the last 90 days before BMT and 71% for the 36 patients who did not (P = .0057). Total IFN dosage had no impact on survival after BMT. We conclude that outcome after BMT is not compromised by pretreatment with IFN if it is discontinued at least 3 months before transplantation. Clear candidates for early transplantation should not be pretreated with IFN.

Acute graft-versus-host disease (GVHD) of the gastrointestinal (GI) tract is an often lethal complication of allogeneic hematopoietic cell transplant. Clinical severity correlates with outcomes, but histopathologic grading is primarily used to confirm the clinical diagnosis. One barrier to using histopathologic grading to predict clinical outcomes is inter-grader variability among transplant centers. Recent experimental models have shown that the loss of Paneth cells, which are located in the small intestine and help regulate the GI microbiome by secreting antimicrobial peptides, correlates with clinical GVHD severity. Because Paneth cells are easy to identify and quantify by light microscopy, we evaluated the mean number of Paneth cells per high-powered field (hpf) in 116 duodenal biopsies obtained at diagnosis of GI GVHD at 2 different centers with their clinical outcomes. Paneth cell counts were reproducible between centers (r(2) = 0.81; P.0001). Lower numbers of Paneth cells at diagnosis correlated with clinically more severe GI GVHD (P.0001) and less likelihood of response to GVHD treatment (P.0001). A threshold of 4 Paneth cells per hpf stratified patients according to nonrelapse mortality (28% vs 56%; P = .004). We conclude that the enumeration of duodenal Paneth cells is a readily available index of disease severity that provides important information regarding GVHD prognosis.

Contribution of host-related cytokine release in the course of pretransplant conditioning to early tissue damage and induction of acute graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT) has been shown in experimental models. We performed a clinical phase I/II trial applying a monoclonal antibody neutralizing human tumor necrosis alpha (TNF alpha) during pretransplant conditioning as additional prophylaxis in high-risk patients admitted to allogeneic BMT; TNF alpha serum levels and clinical courses in 21 patients receiving anti-TNF alpha prophylaxis were compared with data from 22 historical controls. Absence of significant release of TNF alpha in the period of busulphan (BUS) treatment, but significant induction of TNF alpha by total body irradiation (TBI) and cyclophosphamide (CY) conditioning were correlated with significantly earlier onset of acute GVHD in patients receiving TBI/CY regimens as compared with BUS/CY-treated patients. Prophylactic application of monoclonal anti-TNF alpha seemed to postpone onset of acute GVHD from day 15 to day 25 (P < .05) after TBI/CY and from day 33 to day 53 after BUS/CY (P < .10) conditioning. Application of monoclonal anti-TNF alpha in low and intermediate doses was safe and not associated with an increased incidence of infectious or hematologic complications. Thus, our data provide indirect and direct evidence for involvement of conditioning-related cytokine release in induction of early acute GVHD in the clinical setting and support further investigation of this novel approach in randomized trials.

The lower gastrointestinal tract (LGI) and liver are the GVHD target organs most associated with treatment failure and nonrelapse mortality. We recently identified regenerating islet-derived 3-α (REG3α) as a plasma biomarker of LGI GVHD. We compared REG3α with 2 previously reported GI and liver GVHD diagnostic biomarkers, hepatocyte growth factor (HGF) and cytokeratin fragment 18, in 954 hematopoietic cell transplantation patients. All 3 biomarkers were significantly elevated in LGI GVHD compared with non-GVHD diarrhea; REG3α discerned LGI GVHD from non-GVHD diarrhea better than HGF and cytokeratin fragment 18. Although all 3 biomarkers predicted nonresponse to therapy at day 28 in LGI GVHD patients, only REG3α and HGF concentrations predicted 1-year nonrelapse mortality (P = .01 and P = .02, respectively). Liver GVHD without GI involvement at GVHD onset and non-GVHD liver complications were uncommon; all 3 biomarkers were elevated in liver GVHD, but did not distinguish GVHD from other causes of hyperbilirubinemia.

Clinical GVHD staging varies between centers and is not agreed upon by independent reviewers (Weisdorf, BBMT 2003). These inconsistencies help explain why promising GVHD treatments from single center studies have not reproduced in multicenter trials. To address this issue, our international GVHD research consortium has developed guidance that has been refined through consensus following case discussions, resulting in uniform and reproducible GVHD clinical symptom reporting. We record all raw target organ symptom data and apply staging based upon this data (Table 1). Only areas of erythema, bullae and desquamation are quantified because other skin changes are not typical of active GVHD rash. Upper GI symptoms must meet thresholds to diagnose GVHD: anorexia with associated weight loss, nausea and/or 2+ vomiting episodes/day lasting 3+ days. For lower GI GVHD we collect stool volumes excluding formed/mostly-formed stools. Unquantified episodes are counted at 200 ml per episode (3 ml/kg for children We classify biopsy report interpretation into four standardized categories: Positive (unequivocal presence of GVHD), Equivocal (findings suggestive of GVHD, but the pathologist cannot confirm the diagnosis), Non-diagnostic (no abnormalities or subtle findings insufficient to determine etiology), and Non-GVHD etiology (definitive evidence of another diagnosis without concomitant features suggestive of GVHD). We then combine the biopsy interpretation with clinical decision making to assign an overall confidence level to the diagnosis of GVHD in each target organ (Table 2): Confirmed (positive biopsy, regardless of clinician treatment decision), Probable (GVHD diagnosis is sufficiently favored that treatment is given without a positive biopsy), Possible (symptoms present without a positive biopsy; not treated as GVHD), Negative (no symptoms or symptoms are present but a GVHD diagnosis is not entertained and a non-GVHD etiology is identified). Uniform clinical data collection is essential for future GVHD trials and requires application of clear guidance. While still subject to refinement, these guidelines, in use for 2 years by an international research consortium, are designed to be clear, easy to apply, and for clinical trial use. Table 1. GVHD Target Organ Staging Stage Skin (active erythema only) Liver (bilirubin) Upper GI Lower GI (stool output/day) 0 No active (erythematous) GVHD rash < 2 mg/dl No or intermittent nausea, vomiting or anorexia Adult: < 500 ml/day or 50% BSA 6.1-15 mg/dl - Adult: >1500 ml/day or >7 episodes/day Child: > 30 ml/kg/day or >10 episodes/day 4 Generalized rash (>50% BSA) and bullous formation or desquamation > 5% BSA >15 mg/dl - Severe abdominal pain with or without ileus, or grossly bloody stool (volume independent) Table 2. Confidence Levels Pathologic evidence Clinician assessment Treatment for acute GVHD Comments Confirmed Unequivocal evidence of GVHD GVHD is the etiology for symptoms Not required GVHD is clearly present even if other etiologies co-exist simultaneously Probable Not required (includes equivocal and non-diagnostic biopsies) GVHD most likely etiology for symptoms Yes GVHD is most likely present but other etiologies may also explain the symptoms and there insufficient evidence to make a confirmed diagnosis Possible GVHD in differential diagnosis (but no treatment is being provided) No GVHD may be present, but other etiologies are favored Negative Unequivocal evidence of a diagnosis other than GVHD (e.g., drug rash) GVHD is not considered as an explanation for the symptoms No and the symptoms resolve without GVHD treatment A "negative" biopsy (e.g., normal skin) is not unequivocal evidence of a diagnosis other than GVHD Disclosures Devine: Genzyme: Research Funding. Chen:Bayer: Consultancy, Research Funding. Porter:Novartis: Patents & Royalties, Research Funding. Levine:Novartis: Consultancy.

Introduction: With many treatment options for chronic myeloid leukemia (CML), endpoints like health-related quality of life (HRQoL) move into focus and might be essential for deciding on treatment strategies. We sought to evaluate HRQoL in CML patients who had been registered in four consecutive studies of the German CML study group. Methods: The EORTC QLQ-C30 questionnaire was used to assess HRQoL of CML patients. Functional scales and global health status were calculated in accordance with Aaronson (1993) and Fayers (2001). With scales ranging from 0 to 100, 8 points are regarded as a minimally important difference (Efficace et al., 2013). Baseline data of responders (R) and non-responders (NR) were compared. Associations between two variables were assessed by the Fisher or Mann-Whitney tests, as appropriate. The global health status and the functioning scores were compared between groups with the van Elteren test, if the groups were stratified for another variable. Furthermore, results of the global health status and the functioning scores in our sample were standardized in accordance with the age (18-29, 30-39, 40-49, 50-59, 60-69, ≥70 years) and sex distribution of the 2448 participants of the German HRQoL outcome study reported by Hinz et al. 2014. The outcome of our sample was then compared with the outcome of these 2448 patients representing QoL of the German population in general. Comparison was performed using a t test. Results: A questionnaire was sent to 1634 patients. During January to April 2011, 858 questionnaires (53%) were sent back. Compared to NR, R were older (median age: 55 vs. 58, p=0.0426); years since diagnosis (median 6.5 vs. 7.4) and the percentage that had been transplanted were lower (24%vs.18%). No differences were observed regarding sex, Euro score, or time after allogeneic hematopoietic stem cell transplantation (HSCT). When answering the questionnaire, 517 (60%) patients received imatinib 400mg (IM400) and 102 (12%) were off therapy after HSCT. Less than 10% of patients received imatinib 800mg, imatinib+AraC or interferon alpha, nilotinib, or dasatinib. Time since diagnosis was ≤3 years in 156 (18%), >3 and ≤7 years in 309 (36%), and >7 years in 393 (46%) of the patients. Women (352, 41%) perceived a significant reduction in global health status (mean: 62.7, p Compared to the German population, the 858 CML patients had significantly lower scores for global health status (mean: 67.9, p To evaluate HRQoL in patients with long standing disease, 100 patients with diagnosis >7 years off therapy after HSCT and 203 patients receiving IM400 were analyzed. Adjusted for age group and sex, CML patients receiving IM400 for more than 7 years had lower scores for global health status (mean: 63.8, p With respect to all six HRQoL scores, significantly lower scores than from the German population were also observed for the CML patients being seven years without treatment after HSCT: global health status (mean: 69.2, p Conclusions: In this cross-sectional study, women showed an impaired global health status, role, emotional, and physical functioning compared to males. Considering all 858 CML patients, the HRQoL was significantly impaired in all scales when compared to the German population. The same results were observed for the subgroups of patients either receiving IM400 for at least 7 years or being off therapy 7 years after HSCT. Reduced HRQoL remains an issue for all patients after long-term TKI treatment or after HSCT. These data may serve as a basis to evaluate HRQoL in stopping studies in CML. Disclosures Saussele: BMS: Honoraria, Other: Travel grant, Research Funding; Novartis Pharma: Honoraria, Other: Travel grant, Research Funding; ARIAD: Honoraria; Pfizer: Honoraria, Other: Travel grant. Kremers:Novartis: Honoraria; Bristol Myers Squibb: Other: Travel costs, supporting educational meeting; Novartis: Other: supporting educational meeting. Hochhaus:Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding. Müller:BMS: Honoraria, Other: Consulting or Advisory Role, Research Funding; Novartis: Honoraria, Other: CONSULTING OR ADVISORY ROLE, Research Funding; ARIAD Pharmaceuticals Inc.: Honoraria, Other: Consulting & Advisory Role, Research Funding. Hehlmann:Novartis Pharma: Research Funding; BMS: Consultancy. Pfirrmann:BMS: Consultancy, Honoraria; Novartis Pharma: Consultancy, Honoraria.

Induction of indoleamine 2,3-dioxygenase (IDO), the rate-limiting enzyme in tryptophan degradation along the kynurenine pathway, acts as a potent immunoregulatory loop. To address its role in human allogeneic stem cell transplantation, we measured major tryptophan metabolites, such as quinolinic acid and kynurenine, in serial urine specimens from 51 patients by liquid chromatography-tandem mass spectrometry. Samples were collected between admission and day 90 after transplantation, and metabolite levels were correlated with early clinical events and outcome. In selected patients, IDO gene expression was assessed by quantitative RT-PCR in intestinal biopsies. Surviving patients had significantly lower metabolite levels on days 28, 42, and 90, respectively, compared with patients dying of GVHD and associated complications (n = 10). Kynurenine levels were directly correlated with severity and clinical course of GVHD: Mean urinary quinolinic acid levels were 4.5 ± 0.3 μmol/mmol creatinine in the absence of acute GVHD, 8.0 ± 1.1 μmol/mmol creatinine for GVHD grade 1 or 2, and 13.5 ± 2.7 μmol/mmol creatinine for GVHD grade 3 or 4 (P < .001), respectively. GVHD-dependent induction of IDO was further suggested by increased expression of IDO mRNA in intestinal biopsies from patients with severe GVHD. Our data indicate reactive release of kynurenines in GVHD-associated inflammation.

Previous randomized graft-versus-host disease (GVHD)-prophylaxis trials have failed to demonstrate reduced incidence and severity of chronic GVHD (cGVHD). Here we reanalyzed and updated a randomized phase 3 trial comparing standard GVHD prophylaxis with or without pretransplantation ATG-Fresenius (ATG-F) in 201 adult patients receiving myeloablative conditioning before transplantation from unrelated donors. The cumulative incidence of extensive cGVHD after 3 years was 12.2% in the ATG-F group versus 45.0% in the control group (P < .0001). The 3-year cumulative incidence of relapse and of nonrelapse mortality was 32.6% and 19.4% in the ATG-F group and 28.2% and 33.5% in the control group (hazard ratio [HR] = 1.21, P = .47, and HR = 0.68, P = .18), respectively. This nonsignificant reduction in nonrelapse mortality without increased relapse risk led to an overall survival rate after 3 years of 55.2% in the ATG-F group and 43.3% in the control group (HR = 0.84, P = .39, nonsignificant). The HR for receiving immunosuppressive therapy (IST) was 0.31 after ATG-F (P < .0001), and the 3-year probability of survival free of IST was 52.9% and 16.9% in the ATG-F versus control, respectively. The addition of ATG-F to standard cyclosporine, methotrexate GVHD prophylaxis lowers the incidence and severity of cGVHD, and the risk of receiving IST without raising the relapse rate. ATG-F prophylaxis reduces cGVHD morbidity.

The role of allogeneic stem cell transplantation in chronic myeloid leukemia is being reevaluated. Whereas drug treatment has been shown to be superior in first-line treatment, data on allogeneic hematopoietic stem cell transplantation (allo SCT) as second-line therapy after imatinib failure are scarce. Using an interim safety analysis of the randomized German CML Study IV designed to optimize imatinib therapy by combination, dose escalation, and transplantation, we here report on 84 patients who underwent consecutive transplantation according to predefined criteria (low European Group for Blood and Marrow Transplantation [EBMT] score, imatinib failure, and advanced disease). Three-year survival after transplantation of 56 patients in chronic phase was 91% (median follow-up: 30 months). Transplantation-related mortality was 8%. In a matched pair comparison of patients who received a transplant and those who did not, survival was not different. Three-year survival after transplantation of 28 patients in advanced phase was 59%. Eighty-eight percent of patients who received a transplant achieved complete molecular remissions. We conclude that allo SCT could become the preferred second-line option after imatinib failure for suitable patients with a donor. The study is registered at the National Institutes of Health, http://clinicaltrials.gov: NCT00055874.

Toxicity-reduced conditioning is being used for allogeneic stem cell transplantation in older and/or comorbid patients. We report on the treatment of 133 patients (median age: 55.6 years [23-73 years]) with acute myeloid leukemia (AML)/myelodysplastic syndrome (MDS; n = 81), myeloproliferative syndromes (MPS; n = 20), and lymphoid malignancies (n = 32) using conditioning with FBM: fludarabine (5 × 30 mg/m2), 1,3-bis(2-chloroethyl)-1-nitrosourea (or carmustine, BCNU; 2 × 200 mg/m2), and melphalan (140 mg/m2). Patients 55 years or older received fludarabine with reduced BCNU (2 ×150 mg/m2) and melphalan (110 mg/m2). After engraftment, chimerism analyses revealed complete donor hematopoiesis in 95.7% of patients. With a median follow-up of 58.5 months, 3- and 5-year overall survival (OS) was 53.0% and 46.1%, event-free survival (EFS) was 46.4% and 41.9%. No significant differences in OS and EFS were evident considering disease status (early vs advanced), patient age (

Three patients with hematologic relapse after bone marrow transplantation for chronic myelogenous leukemia were treated with interferon alpha and transfusion of viable donor buffy coat. All had complete hematologic and cytogenetic remission, which persisted 32 to 91 weeks after treatment. In two patients graft-versus-host disease developed and was treated by immunosuppression. These results are an example of adoptive immunotherapy without cytoreductive chemotherapy or radiotherapy in human chimeras.

Acute graft-versus-host disease, interstitial pneumonitis, endothelial leakage syndrome, and veno-occlusive disease are major complications of bone marrow transplantation. Though several new regimens for prophylaxis and treatment of these syndromes have been introduced, the overall incidence has been only slightly reduced over the last few years. We retrospectively analyzed tumor necrosis factor alpha (TNF alpha) serum levels between day -8 and day 100 after bone marrow transplantation in 56 patients transplanted in our unit for a variety of hematological diseases. In 34 patients with uneventful courses, mean TNF alpha levels rose to a maximum of 76 +/- 29 pg/mL. In contrast, 22 patients with major transplant related complications showed mean increases of TNF alpha of 492 +/- 235 pg/mL (P less than .0001). Increases of TNF alpha occurred before interstitial pneumonitis and severe acute graft-versus-host disease with a latency of 25 to 54 days. Early complications such as endothelial leakage syndrome and veno- occlusive disease were closely associated with increases of TNF alpha serum levels. Our study suggests two pathways of TNF alpha release: activation of host macrophages and stimulation of donor cells in the course of acute graft-versus-host disease. Cytokine monitoring should be helpful for prediction and earlier treatment of major transplant related complications.

Acute graft-versus-host disease (aGvHD) contributes significantly to morbidity and mortality after allogeneic hematopoietic stem cell transplantation (allo-HSCT). Diagnosis of GvHD is mainly based on clinical features and tissue biopsies. A noninvasive, unbiased laboratory test for GvHD diagnosis does not exist. Here we describe the application of capillary electrophoresis coupled online with mass spectrometry (CE-MS) to 13 samples from 10 patients with aGvHD of grade II or more and 50 control samples from 23 patients without GvHD. About 170 GvHD-specific polypeptides were detected and a tentatively aGvHD-specific model consisting of 31 polypeptides was chosen, allowing correct classification of 13 of 13 (sensitivity 100.0% [95% confidence interval {CI} 75.1 to 100.0]) aGvHD samples and 49 of 50 (specificity 98.0% [95% CI 89.3 to 99.7]) control samples of the training set. The subsequent blinded evaluation of 599 samples enabled diagnosis of aGvHD greater than grade II, even prior to clinical diagnosis, with a sensitivity of 83.1% (95% CI 73.1 to 87.9) and a specificity of 75.6% (95% CI 71.6 to 79.4). Thus, high-resolution proteome analysis represents an unbiased laboratory-based screening method, enabling diagnosis, and possibly enabling preemptive therapy.

The JAK2-V617F mutation occurs in about 50% of patients with myelofibrosis and might be a reliable marker to monitor residual disease after allogeneic stem cell transplantation. We describe a new, highly sensitive (≥ 0.01%) real-time polymerase chain reaction (PCR) to monitor and quantify V617F-JAK2–positive cells after dose-reduced allogeneic stem cell transplantation. After 22 allogeneic stem cell transplantation procedures in 21 JAK2-positive patients with myelofibrosis, 78% became PCR negative. In 15 of 17 patients (88%), JAK2 remained negative after a median follow-up of 20 months. JAK2 negativity was achieved after a median of 89 days after allograft (range, 19-750 days). A significant inverse correlation was seen for JAK2 positivity and donor-cell chimerism (r: −0.91, P < .001). Four of 5 patients who never achieved JAK2 negativity fulfilled during the entire follow-up all criteria for complete remission recently proposed by the International Working Group, suggesting a major role for JAK2 measurement to determine depths of remission. In one case, residual JAK2-positive cells were successfully eliminated by donor lymphocyte infusion. In conclusion, allogeneic stem cell transplantation after dose-reduced conditioning induces high rates of molecular remission in JAK2-positive patients with myelofibrosis, and quantification of V617F-JAK2 mutation by real-time PCR allows the detection of minimal residual disease to guide adoptive immunotherapy.

To assess the role of NOD2/CARD15 variants on the long-term outcome of allogeneic stem cell transplantation in a genetically homogeneous group, we extended our previous study (cohort I, n = 78) and typed DNA for NOD2/CARD15 single nucleotide polymorphisms (SNPs) from an additional 225 recipients and their HLA-identical sibling donors (cohort II) treated at four other European centers. Results of genotyping were compared with clinical outcome. The strong association of NOD2/CARD15 variants with transplantation-related mortality (TRM) was confirmed in univariate and multivariate analysis; TRM increased from 20% in cohort I/22% in cohort II in recipient/donor pairs without any NOD2/CARD15 variants to 47% in cohort I/32% in cohort II in the presence of one variant in either donor or recipient and further to 57% in cohort I/74% in cohort II in the presence of 2 or more variants (P < .002 in both cohorts). NOD2/CARD15 SNPs were not associated with relapse rate but had a strong impact on overall survival. In an analysis of center effects, the type of gastrointestinal decontamination was the only factor interfering with the prognostic significance of NOD2/CARD15 SNPs. Our data further support an interaction between gastrointestinal defense mechanisms, activation of the innate immune system, and specific transplant-related complications.

Background In multiple myeloma (MM), the introduction of novel compounds into first-line intensive treatment pathways has clearly improved patients’ prognosis. Very recently however, specific molecular cytogenetic abnormalities, lactate dehydrogenase elevation and International Staging System 3 disease were identified to be associated with dismal prognosis despite upfront autologous (auto) stem cell transplant (SCT). Consolidative allogeneic (allo) following initial auto SCT was shown to extend progression-free survival (PFS) as well as overall survival (OS) in some prospective studies on newly diagnosed MM patients (pts). Relatively little is known on the impact of cytogenetic features other than chromosome 13q deletion (del13q) on the outcomes of pts undergoing upfront auto followed by allo (auto/allo) SCT. Patients and methods When the DSMM V treatment program was designed del13q detected by fluorescence in situ hybridisation (FISH) was accepted as one of the distinct risk factors in MM. We therefore used FISH del13q to define the study’s “high-risk” group and aimed to compare tandem high-dose melphalan 200 mg/m² (Mel) with one cycle of Mel followed by reduced-intensity conditioning (RIC) allo SCT. Allocation to either transplant regimen was by availability of an HLA-matched (at least 9/10 matches) related (MRD) or unrelated donor (MUD). Initially, all pts underwent non-novel compound cytoreduction and chemomobilization of peripheral blood stem cells (PBSC). RIC allo SCT was prepared by fludarabine and melphalan (plus ATG in MUD cases). PFS was the primary endpoint. The study was powered to detect an improvement of 2-year PFS from 20% (tandem Mel) to 40.3% (HR, 1.769). Results 199 out of 225 del13q pts with a median age of 53 (range, 30 – 60) yrs who had been enrolled between 10/2001 and 03/2007, were included in the intent-to treat population. Allo SCT was performed in 126/199 pts (63%), 74 of whom (59%) received MUD allografts. At a median follow-up of 49.2 months (mo), 2-year PFS (calculated from day 1 of second SCT) was 59% with auto/allo SCT versus 47% with tandem Mel. Median PFS with auto/allo SCT was 34.5 mo versus 21.8 mo, respectively (p=.005). Two-year non-relapse mortality (NRM) associated with auto/allo SCT was 11.9%. As of yet, there is no difference in OS between the groups, with the median not yet reached for either transplant modality. PFS/OS in auto/allo SCT were independent of donor source (MRD vs MUD). As definitions of cytogenetic risk have evolved over time, we analyzed further FISH abnormalities in pts’ baseline samples: in addition to uniform del13q, 13.6% of pts displayed del17p. Median PFS for del13q/del17p pts after HD Mel was 6 mo versus not reached with auto/allo SCT, respectively (p=.0002). Median OS in del13q/del17p after HD Mel was 23.4 mo versus not reached, respectively (p=.011). In translocation (4;14)/del13q pts (20.7%), median PFS with tandem Mel was 19.3 mo versus 19.1 with auto/allo SCT, respectively (p=.251). Conclusions This prospective trial shows auto/allo SCT to significantly extend PFS when compared to tandem HD Mel in a large cohort of del13q MM pts. It is the first study to demonstrate allo SCT in MM can be safely performed from matched unrelated donors at a reasonable rate of NRM. Utilizing a comprehensive set of FISH cytogenetics, our data for the first time demonstrate allo SCT to specifically benefit patients with high-risk features (del13q/del17p). Incremental gain of PFS when compared to tandem Mel was more than 20 months. Extended OS data on the whole study will be presented. Disclosures No relevant conflicts of interest to declare.

Allogeneic HSCT has been established as the only curative treatment option for patients with chronic myeloid leukemia (CML). However, after the advent of tyrosine kinase inhibitors (TKI) the proportion of transplanted patients has decreased dramatically. After imatinib failure, most patients receive second or third line therapy with alternative TKIs. In an important minority of patients, SCT is performed too late as more patients are transplanted after disease progression to accelerated phase or blast crisis than in first chronic phase (CP, Saussele et al. BMT 2012). A possible reason is the uncertainty on long-term outcome after SC T in the imatinib-era as reports are scarce and accurate comparative data on the impact of salvage TKI therapy vs allogeneic transplantation are missing. We therefore investigated the outcome of transplanted patients within the CML study IV. Preliminary data were published (Saussele et al. BLOOD 2010). Here, we sought to re-evaluate the outcome of these patients with a longer follow-up. In July 2002, the German CML-Study Group activated a prospective randomized trial comparing different imatinib based strategies in CP CML. Elective early HSCT was considered for patients with EBMT score 0–1 for those with high disease risk, and after imatinib failure. By the end of March 2012, 1551 patients were randomized. In 2008, HSCT was documented in 84 patients. One patient was not evaluable any more due to withdrawal of consent. 52 patients were male (65%), 23 high risk patients (28%) according to the Euro CML score. Median age at diagnosis was 37 years (range, 16-62), median time to HSCT was 12.6 months (range, 3.5-54). EBMT score was 0-1 in 8 (10%), 2 in 10 (12%), 3-4 in 44 (55%), and >=5 in 18 patients (23%), three patients were missing. Median follow-up after HSCT was 86.9 months (range, 0.3-122). Based on the indication for HSCT three groups are defined: 1) early HSCT, n= 19 (23%; low EBMT score (n=9), high risk patients (n=7), patient request (n=3); 2) HSCT after imatinib failure or intolerance in first CP (n=36 patients, 43%), and 3) HSCT in second CP or higher, accelerated phase or blast crisis (n=28 patients, 34%). 26 patients died, 13 deaths were transplant related, 9 CML related 4 either unrelated or unknown. Overall survival rate at 6 years after HSCT was 89% (95%-confidence interval (CI): 72-99%) for group 1, 80% (95%-CI: 66-91%) for group 2, and 49% (31-68%) for group 3. A matched pair analysis could be performed for 53 transplanted patients of group 1 and 2. To each of the transplanted patients two imatinib-treated patients could be matched with regard to age, sex, risk profile, disease phase, and interval to transplantation. Median follow up of this population was 87 months. Overall survival after 8 years was 83% (95%-CI: 71-92%) for transplanted and 89% (95%-CI: 82-94%) for imatinib treated patients without any statistical difference. Data from this update with a longer follow-up support the role of HSCT as an attractive and important salvage therapy for CML patients with imatinib failure or intolerance. In a matched pair comparison of transplanted and non-transplanted patients, we did not find significant differences. Disclosures Saussele: Novartis: Honoraria, Research Funding, Travel Other; Bristol-Myers Squibb: Honoraria, Research Funding, Travel, Travel Other; Pfizer: Honoraria, Travel, Travel Other. Müller:Novartis: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; ARIAD: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Hanfstein:Novartis: Research Funding; Bristol-Myers Squibb: Honoraria. Hochhaus:Novartis: Consultancy, Honoraria, Research Funding; Bristol-Myers Squibb: Consultancy, Honoraria; ARIAD: Honoraria, Research Funding; Pfizer: Consultancy, Research Funding. Pfirrmann:Novartis: Consultancy; Bristol-Myers Squibb: Honoraria. Hehlmann:Bristol-Myers Squibb: Research Funding; Novartis: Research Funding.

The severity of symptoms at the onset of graft versus host disease (GVHD) does not accurately define risk, and thus most patients (pts) are treated alike with high dose systemic steroids. We hypothesized that concentrations of one or more plasma biomarkers at the time of GVHD diagnosis could define distinct non-relapse mortality (NRM) risk grades that could guide treatment in a multicenter setting. We first analyzed plasma that was prospectively collected at acute GVHD onset from 492 HCT pts from 2 centers, which we randomly divided into training (n=328) and validation (n=164) sets; 300 HCT pts who enrolled on multicenter BMT CTN primary GVHD therapy clinical trials provided a second validation set. We measured the concentrations of 3 prognostic biomarkers (TNFR1, REG3α, and ST2) and used competing risks regression to create an algorithm from the training set to compute a predicted probability (p) of 6 mo NRM from GVHD diagnosis where log[-log(1-p)] = -9.169 + 0.598(log2TNFR1) - 0.028(log2REG3α) + 0.189(log2ST2). We then rank ordered p from lowest to highest and identified thresholds that met predetermined criteria for 3 GVHD grades so that NRM would increase 15% on average with each grade. A range of thresholds in the training set met these criteria, and we chose one near each median to demarcate each grade. In the resulting grades, risk of NRM significantly increased with each grade after the onset of GVHD in both the training and validation sets (FIG 1A,B). Most (80%) NRM was due to steroid-refractory GI GVHD, even though surprisingly only half of these pts presented with GI symptoms. We next applied the biomarker algorithm and thresholds to the second multicenter validation set (n=300) and observed similarly significant differences in NRM (FIG 1C). Relapse, which was treated as a competing risk for NRM, did not differ among the three GVHD grades (Figure 1D-F). The differences in NRM thus translated into significantly different overall survival for each GVHD grade (Figure 1G-I). These differences in survival are explained by primary therapy response at day 28, which was highly statistically different for each of Ann Arbor grade (grade 1, 81%; grade 2, 68%; grade 3, 46%; p We performed additional analyses on the multicenter validation set of pts that developed GVHD after treatment with a wide spectrum of supportive care, conditioning and GVHD prophylaxis practices. As expected, the Glucksberg grade at GVHD onset did not correlate with NRM (data not shown). Despite small sample sizes, the same biomarker algorithm and thresholds defined three distinct risk strata for NRM within each Glucksberg grade (FIG 2A-C). Pts with the higher Ann Arbor grades were usually less likely to respond to treatment. Unexpectedly, approximately the same proportion of pts were assigned to each Ann Arbor grade (~25% grade 1, ~55% grade 2, ~20% grade 3) regardless of the Glucksberg grade (FIG 2D-F). Several clinical risk factors, such as donor type, age, conditioning, and HLA-match, can predict treatment response and survival in patients with GVHD. Using Ann Arbor grade 2 as a reference, we found that Ann Arbor grade 1 predicted a lower risk of NRM (range 0.16-0.32) and grade 3 a higher risk of NRM (range 1.4-2.9), whether or not any of these clinical risk factors were present. To directly compare Ann Arbor grades to Glucksberg grades, we fit a multivariate model with simultaneous adjustment for both grades. FIG 3 shows that Ann Arbor grade 3 pts had significantly higher risk for NRM (p=0.005) and Ann Arbor grade 1 pts had significantly less risk for NRM (p=0.002) than pts with Ann Arbor grade 2. By contrast, the confidence intervals for the HRs of the Glucksberg grades encompassed 1.0, demonstrating a lack of statistical significance between grades. In conclusion, we have developed and validated an algorithm of plasma biomarkers that define three grades of GVHD with distinct risks of NRM and treatment failure despite differences in clinical severity at presentation. The biomarkers at GVHD onset appear to reflect GI tract disease activity that does not correlate with GI symptom severity at the time. This algorithm may be useful in clinical trial design. For example, it can exclude pts who are likely to respond to standard therapy despite severe clinical presentations, thus limiting the exposure of low risk pts to investigational agents while also identifying the high risk pts most likely to benefit from investigational approaches. Figure 1 Figure 1. Figure 2 Figure 2. Figure 3 Figure 3. Disclosures Levine: University of Michigan: GVHD biomarker patent Patents & Royalties. Braun:University of Michigan: GVHD biomarker patent Patents & Royalties. Ferrara:University of Michigan: GVHD biomarker patent Patents & Royalties.