ALBERT

Description: Relapse of malignancy and lethal graft versus host disease (GVHD) are the principal causes of failure of allogeneic hematopoietic cell transplant (HCT). Recently we have shown that at seven days after HCT an algorithm using two serum biomarkers (ST2 and REG3α) can predict severe GVHD (Hartwell et al. JCI Insight 2017). We determined whether serial testing (in the first month following HCT) of patients with low probability biomarkers would improve the predictive accuracy of the algorithm and identify patients with different risks of relapse and lethal GVHD. Patients who received an HCT at 18 centers in the Mount Sinai Acute GVHD International Consortium (MAGIC) for hematologic malignancy and who supplied three blood samples were divided into a training set and validation set with equal numbers of lethal GVHD events, which was defined as death from GVHD or infection during treatment for GVHD. Patients in the training set (n=702) underwent HCT from January 1, 2006 until June 30, 2015, whereas patients in the validation set (n=906) underwent HCT from July 1, 2015 to May 1, 2017. Serum samples were analyzed using the previously published algorithm of two biomarkers up to three times (day 7, day 14, day 28 or GVHD onset, if onset occurred within the first 28 days). The algorithm generates a predicted probability of lethal GVHD between 0 and 1 for each patient. Patients were categorized as either low probability (LP) or high probability (HP) for lethal GVHD. HP thresholds of 0.20 and 0.16 were used to classify patients with and without GVHD symptoms, respectively (once categorized as HP, patients remained in that category and were not retested). All results were similar between training and validation sets, and we present here the validation set results. Serial testing identified 28% of patients as HP with a three-fold greater cumulative incidence of lethal GVHD at one year (13% vs 4%, p

Description: Key Points NOTCH2 activation confers a marked increase in BCR responsiveness by cGVHD patient B cells that associates with increased BLNK. ATRA increases the IRF4-to-IRF8 ratio and blocks aberrant NOTCH2-BCR activation without affecting cGVHD patient B-cell viability/function.

Description: While Notch signaling is being well studied with regard to T cell pathology and graft-versus host disease (GVHD) (Tran IT et al., 2013. J. Clin. Invest.), the role of Notch receptors in the development and activation of B cell subsets in chronic GVHD (cGVHD) genesis remains unknown. We previously identified a subset of Ôpre-germinal centerÕ B cells within the peripheral blood of cGVHD patients that is largely absent in patients without cGVHD. In addition to cell surface characteristics, this extrafollicular B cell subset has potential functional characteristics of marginal zone (MZ)-like B cells, including increased responsiveness to surrogate antigen stimulation. Along with increased proliferative responses to BCR stimulation, B cells from patients with active cGVHD had significantly increased signaling via proximal B cell receptor (BCR) molecules, including Syk and BLNK. In murine models with lymphopenic environments, Notch 2 binds the ligand Delta-like 1 (DLL1/Dll1) and drives maturation of MZ-like B cells. Also, healthy human B cells have increased Notch receptor responsiveness after BCR stimulation. Together previous studies allowed us to hypothesize that a Notch 2 signaling axis underpins B cell hyper-responsiveness in human cGVHD. We found that limiting dose BCR stimulation with surrogate antigen in the presence of Notch ligand over-expressing cells (OP9-DL1) resulted in maintenance of cell surface Notch 2 expression at significantly higher levels on B cells from patients with active cGVHD compared to patients without cGVHD, as assessed by flow cytometry analysis (P 〈 0.01). We also found that in the presence of Notch ligand, B cells from patients with active cGVHD responded to minimal BCR stimulation with surrogate antigen. Using nearly 100x less surrogate antigen than was required to induce proliferation without Notch ligand, cGVHD B cells proliferated to a significantly greater degree than B cells from patients with no cGVHD, as evaluated by Ki-67 staining using flow cytometry (P 〈 0.001 in a two-sided t-test, Figure 1A). Likewise, concomitant BCR- Notch activation of active cGVHD patient B cells was associated with significantly increased B-cell size compared to patients without disease (P 〈 0.01). BLNK expression in active cGVHD B cells was also maintained at higher levels under these conditions, suggesting a mechanistic link between the BCR and Notch pathways in cGVHD. Strikingly, targeting Notch 2 with an antagonistic monoclonal antibody (mAb) (Wu Y et al., 2010. Nature; kindly provided by Genentech, Inc.) completely abrogated the BCR-Notch axis hyper-responsiveness of active cGVHD B cells without affecting B-cell survival (P 〈 0.001, Figure 1B). In this in vitro system, using nanoString Technologies¨ gene profiling, we found that two, well-defined effector genes downstream of Notch signaling were significantly decreased in active cGVHD B cells after exposure to the anti-Notch 2 mAb (P = 0.0006 and P 〈 0.02, respectively, compared to isotype control mAb). Also consistent with a Notch 2-driven activation pathway, the expression of multiple genes involved in homeostasis/cell cycle regulation were altered in active cGVHD B cells exposed to anti-Notch 2 mAb (P 〈 0.01). Finally, ongoing in vivo analyses of the Notch 2 mAb in a pre-clinical mouse model of cGVHD indicates that Notch 2 blockade does not negatively impact early B cell recovery following bone marrow transplantation. These results may reveal that therapeutic targeting of Notch 2 alone would be sufficient to quell B cell hyper-responsiveness in active cGVHD, while preserving protective humoral immunity. In summary, our data suggest a working model in which Notch-mediated aberrant B cell maturation contributes to cGVHD pathophysiology. In this model, Notch 2 stimulation along with a combination of complex B-cell selection and tolerance mechanisms afford production of pathological B cells. Given that Notch 2 is a cell surface receptor expressed by activated B cell subsets of pathological relevance, and Notch 2 blockade has been shown to be well-tolerated in pre-clinical models, our findings support an important clinical opportunity: Targeting Notch 2 on B cells in active cGVHD represents a viable future therapeutic strategy worthy of continued investigation. This work was supported by National Institutes of Health grant 5K08-HL107756, and a Translational Research Program grant from the Leukemia & Lymphoma Society. Figure 1. Figure 1. Disclosures Rizzieri: Teva: Other: ad board, Speakers Bureau; Celgene: Other: ad board, Speakers Bureau.

Description: Introduction: Umbilical cord blood (UCB) extends the curative potential of stem cell transplantation to adult patients without a suitable donor. The most commonly used myeloablative preparative regimen results in an unacceptably high 6-month treatment related mortality rate of approximately 32% (Barker J et al. Br J Haematol. 2015). In an attempt to reduce treatment related mortality, we piloted a modified myeloablative regimen with total body irradiation (TBI), fludarabine, and thiotepa. We report clinical outcomes from a cohort of patients who received single or double UCBT after conditioning with this regimen. Methods: Thirty-one consecutive adult patients ≥ 18 years old with hematologic malignancies who underwent single or double umbilical cord blood transplantation at Duke University from 2010 to 2015 were included in this study. The conditioning regimen consisted of thiotepa 5 mg/kg/day i.v. x 2 days (days -11 to -10), TBI 150 cGy twice a day for total nine fractions (1350 cGy days -9 to -5), and fludarabine 40 mg/m2/day i.v. x 4 days (days -5 to -2). Cord blood units were matched to the recipient at 4 or more HLA loci (intermediate-resolution for A and B, high-resolution for DRB1). Graft versus host disease (GVHD) prophylaxis was with tacrolimus (target level 10-15 ng/ml) and mycophenolate mofetil 1000 mg TID. Antimicrobial prophylaxis and supportive care measures including GCSF administration until ANC 〉 1000 were conducted per institutional protocol. Probabilities of neutrophil and platelet recovery, acute and chronic GVHD, and treatment-related mortality were estimated by the cumulative incidence method. Relapse-free and overall survival rates were estimated by the Kaplan-Meier method. Results: Thirty-one patients (median age 46 years; range, 19-65) with hematologic malignancies were evaluated. Twenty-four patients (77%) had acute leukemia or myelodysplastic syndrome, while 7 patients (23%) had non-Hodgkin's lymphoma or multiple myeloma. By the "Disease Risk Index" (Armand P et al. Blood. 2014), 20 patients (65%) had low or intermediate risk disease, while 11 patients (35%) had high or very high risk disease. 30 patients underwent double UCB and 1 patient received single UCB transplantation. The median cryopreserved total nucleated cell dose was 5.4 x 107/kg (range: 3.2-8.4 x 107/kg). The cumulative incidence of neutrophil engraftment was 90% (95% CI, 82%-99%; Figure 1) at a median time of 21 days (95% CI, 19-26). Three patients did not have neutrophil engraftment; two patients had early death at days 7 and 14 prior to engraftment, while one patient had graft failure requiring second transplant. The cumulative incidence of platelet engraftment was 86% (95% CI, 75%-97%) at a median time of 47 days (95% CI, 37-73). Cumulative incidences of grades II-IV and grades III-IV acute GVHD were 48% (95% CI, 34%-69%) and 10% (95% CI, 3%-28%), respectively. The overall incidence of chronic GVHD was 40% (95% CI, 27%-59%), with 17% (95% CI, 8%-37%) of patients experiencing moderate to severe chronic GVHD. Treatment-related mortality at 6 months was 13% (Figure 2), while at 1 year and 3 years was 27% and 33%, respectively. With a median follow-up of 35.5 months (95% CI, 12.7-52.2), disease-free and overall survival at 3 years was 51% (95% CI, 29%-69%) and 57% (95% CI, 36%-73%), respectively. Conclusion: UCB transplantation with the modified myeloablative conditioning regimen of TBI, fludarabine, and thiotepa results in reliable neutrophil engraftment with reduced early treatment related mortality as compared to standard myeloablative conditioning consisting of TBI, fludarabine, and cyclophosphamide. It provides a promising disease-free and overall survival in an older (median age 46), heterogeneous patient population. This regimen represents a reasonable alternative to standard conditioning with TBI, fludarabine, and cyclophosphamide and warrants further study. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

Description: Our group previously showed that B cells signal aberrantly through the B cell receptor (BCR) in allogeneic hematopoietic stem cell transplant (HCT) patients with active chronic graft-versus-host disease (cGVHD). Preclinical mouse studies have demonstrated the importance of the proximal BCR molecule, spleen tyrosine kinase (SYK), in cGVHD development. Hypothesizing that the oral small molecule SYK inhibitor, fostamatinib, would safely target aberrant BCR-activated B cells in HCT patients, we are conducting a single-center, investigator-initiated phase 1 trial (NCT02611063). Our primary objective is to evaluate the safety and tolerability of fostamatinib in patients early after HCT and in those with refractory active cGVHD. Secondary objectives include assessment of cGVHD manifestations, B cell activation, and immune recovery. Methods: All patients receive HCT treatment per program standards at Duke University. Prophylaxis (P-cGVHD) subjects enroll 80-150 days after HCT and have no evidence of cGVHD. P-cGVHD subjects receive drug for up to 1 year post-transplant (215-285 fostamatinib days). Steroid-refractory cGVHD (SR-cGVHD) subjects enroll with active cGVHD that persists despite systemic high-dose steroids. SR-cGVHD subjects receive drug for up to 365 days total. For all enrollees, modified continual reassessment criteria are used to determine starting dose (100mg daily, 150mg daily, or 100mg twice BID) and any needed dose modifications. We monitor for drug-limiting toxicities (DLTs), adverse events (AEs), and cGVHD manifestations using NIH cGVHD consensus criteria at up to 12 follow-up visits. Results: 15 of a planned 18 total patients have enrolled. In the P-cGVHD group (n=5), of the 4 patients who completed treatment (mean 239 fostamatinib days), 1 patient developed cGVHD while enrolled and 2 patients subsequently developed cGVHD, 4 and 6 weeks after study completion. The fifth P-cGVHD subject discontinued therapy on study day 155 (provider decision to initiate donor lymphocyte infusion for low CD3+ chimerism). In the SR-cGVHD group (n=10), 2 patients completed treatment (mean 365 fostamatinib days); 3 patients withdrew (mean 132 fostamatinib days), for non-cardiac chest pain, progression of cGVHD, and moved away; and 5 patients are actively enrolled (mean 207 fostamatinib days). Both SR-cGVHD patients who completed the study clinically improved while on fostamatinib and requested continuation of drug. A total of 2, 9, and 4 patients have been initiated on 100mg daily, 150mg daily, and 100mg BID, respectively. At the 100mg daily dose, no DLTs were noted. At the 150mg daily dose, 1 patient developed liver function test (LFT) elevation. At the 100mg BID dose, 2 patients developed LFT elevation and 1 patient developed non-cardiac chest pain. One patient required dose adjustment: 100mg BID to 150mg daily, for LFT elevation. Two serious AEs possibly related to fostamatinib occurred: 1 patient developed non-cardiac chest pain and 1 patient developed a deep venous thrombosis. No probably- or definitely-related serious AEs occurred. To assess whether fostamatinib effectively targets aberrantly activated B cells, we examined subjects' whole blood using flow cytometry. When comparing CD19+ B cells on study day 1 versus study day 60 in the SR-cGVHD group (n=7), we found the relative proportion of CD19+CD38+IgDlow plasmablast-like cells was decreased (p=0.03, Fig 1A-B), suggesting fostamatinib 'hit target.' Importantly, in the P-cGVHD group, total lymphocyte and B cell counts did not decrease during day 1 to day 225 (Fig 1C-D), suggesting fostamatinib did not affect immune recovery when given early after HCT. Further investigations with functional assays are underway. Conclusions: This study demonstrates for the first time that fostamatinib is safe and tolerated in HCT recipients both early after transplant and in those with active cGVHD. Importantly, fostamatinib does not appear to hinder lymphocyte or B cell recovery when initiated between days 80-150 after HCT. Additionally, fostamatinib may effectively target aberrantly activated B cells in patients with active SR-cGVHD. Fostamatinib, now FDA-approved for treatment of immune thrombocytopenia, merits a phase 2, randomized controlled trial to assess efficacy as a prophylactic agent against cGVHD. This work was supported by a National Institutes of Health grant, NIH (NHLBI) R01 HL 129061. Fostamatinib was supplied by Rigel. Disclosures Horwitz: Abbvie Inc: Membership on an entity's Board of Directors or advisory committees. Gasparetto:BMS: Consultancy, Honoraria, Other: Travel, accommodations, or other expenses paid or reimbursed ; Celgene: Consultancy, Honoraria, Other: Travel, accommodations, or other expenses paid or reimbursed ; Janssen: Consultancy, Honoraria, Other: Travel, accommodations, or other expenses paid or reimbursed . Sung:Novartis: Research Funding; Merck: Research Funding; Seres: Research Funding. Rizzieri:Celgene, Gilead, Seattle Genetics, Stemline: Other: Speaker; AbbVie, Agios, AROG, Bayer, Celgene, Gilead, Jazz, Novartis, Pfizer, Sanofi, Seattle Genetics, Stemline, Teva: Other: Advisory Board; AROG, Bayer, Celgene, Celltron, Mustang, Pfizer, Seattle Genetics, Stemline: Consultancy; Stemline: Research Funding.

Description: Allogeneic hematopoietic stem cell transplantation (HCT)-related immune pathology severely limits patient (Pt) survival, largely due to infections. Chronic graft versus host disease (cGVHD) Pts are particularly immune incompetent with impaired memory B cell responses. Paradoxically, cGVHD Pts are hypogammaglobulinemic and yet are variably immune tolerant with potentially pathogenic alloantibodies frequently produced. B cells with marginal zone (MZ)-like properties are expanded in cGVHD Pts with high BAFF/B-cell ratios (rev. Sarantopoulos, Blood 2015), similar to findings in BAFF-overexpressing mice. We previously found that the increased BCR responsiveness of cGVHD B cells depended on NOTCH2 signaling, since a specific monoclonal antibody (Genentech) blocked hyperactivation (ASH 2015 oral abstract: Blood. 2015. 126:145). Our current objective is to define the molecular defects underlying this aberrant NOTCH2-BCR axis. Increased NOTCH2 expression/activity is found in mice genetically deficient in the transcription factor IRF4 (J. Exp. Med. 2013. 210:2887-2902). Likewise, IRF4 levels are subnormal in unstimulated B cells from active cGVHD Pts relative to healthy donors (Blood. 2014. 123: 2108-15). Thus, we hypothesized that a B cell-intrinsic IRF4 deficiency contributes to the NOTCH2-BCR axis in cGVHD. We studied multi-center (Duke Univ., NCI-NIH, Dana Farber Cancer Inst.) de-identified clinical samples from 38 Pts with or without NIH diagnostic criteria of cGVHD who were 〉12 months post-HCT and not receiving high dose steroids. We employed our in vitro culture system utilizing NOTCH ligand-expressing feeder cells and measured proliferative responses (Ki-67% by flow cytometry) to surrogate Ag. First, adding to our previous findings, B cells from active cGVHD Pts (n=8) proliferated robustly to minimal BCR ligation when NOTCH was activated compared to B cells from Pts without cGVHD (n=7) (38.1% vs 15.3%, P =0.006). To test our new hypothesis, IRF4 levels were determined by quantitative PCR analysis in B cells isolated from HCT Pts with active cGVHD and with no cGVHD stimulated ex vivo for 24 hours (h) with a limiting amount of surrogate Ag (anti-IgM). BCR-engaged B cells from active cGVHD Pts (Figure 1A, open bars) maintained significantly lower IRF4 levels compared to B cells from Pts with no cGVHD (Figure 1A, filled bars; n=4 in each group, P =0.01 in a two-sided t-test). Importantly, this IRF4 deficiency may be correctable therapeutically in cGVHD Pts, since the ex vivo treatment of human B cells with all-trans retinoic acid (ATRA) increases IRF4 expression (J. Immunol. 2015. 195:2601-11), and ATRA has demonstrated efficacy in a mouse model of cGVHD (Blood. 2012. 119:285-95). When ATRA (100 nM) was added to cultures of surrogate Ag-stimulated B cells (Figure 1A, right), IRF4 levels increased significantly in both active cGVHD B cells (n = 4, P

Description: Increased B cell-activating factor (BAFF) and aberrant B cell survival and activation are associated with chronic graft versus host disease (cGVHD) in patients. Whether excessive BAFF production has a pathologic role in the development of cGVHD after allogeneic hematopoietic stem cell transplantation (allo-HCT) remains unknown. Herein, we address this hypothesis by employing BAFF knockout (KO) and BAFF overexpressing (Tg) donor mice in the major MHC mismatched C57BL/6 (B6)-to-BALB/c cGVHD model (Wu et al J Immunol 2013;191:488), except we increased the T cell depleted bone marrow (TCDBM) dose to 1x107 per recipient to improve engraftment. BALB/c recipients receiving TCDBM plus 1x106 splenocytes (Sp) are the disease group, and those receiving TCDBM only are controls. After verifying cGVHD phenotypic manifestations (including longevity, weight loss, eye and lung findings), we examined whether cGVHD mice had the increased BAFF and B cell findings as occur in human cGVHD (Sarantopoulos et al, Blood 2009; 113: 3865). We observed that cGVHD mice had higher BAFF/B cell ratios compared to controls (Fig. 1A, n=10 each). We fully characterized the peripheral B cell compartment in these mice, and found that the relative composition of B cell subsets was significantly altered in diseased mice. CD93 cell surface expression has been used in mice to define B cells that emigrate from BM and circulate through secondary lymphoid organs. In cGVHD mice, we found a significant increase in the relative proportion of CD93+ B cells and a significant decrease in the proportion of CD93- B cells (Fig1B). Also, a significant increase in the frequency of cells positive for the germinal center and activation marker GL7 was found only within CD93- B cell subset of cGVHD mice (Fig 1C). When stimulated via BCR ex vivo, this GL7+ mature B cell subset had significantly increased Syk and BLNK activation, measured by phosphoflow (n=5, p=0.008). We next aimed to determine whether high BAFF alone or high BAFF and alloantigen together lead to altered B cell homeostasis and to the promotion of GL7+ BCR-activated B cells. C57BL/6 (B6) BAFF Tg TCDBM cells were used as donor to afford excessive and persistent BAFF levels after HCT in either syngeneic or BALB/c recipients. After B6 BAFF Tg TCDBM to BALB/c HCT, body weight decreased significantly. By contrast, B6 BAFF Tg TCDBM to B6 syngeneic HCT recipients remained healthy (Fig.1D, left panel,*p=0.004). Notably, the frequency of the CD93-GL7+ B cell subset was increased in BALB/c mice that received B6 BAFF Tg TCDBM only, compared to recipients of syngeneic BAFF Tg or WT TCDBM only (Fig.1D, right panel, p=0.008). Thus, both BAFF and alloantigen are required for B cell activation after HCT. Together, our data also suggest that GL7+ B cells identify aberrantly BCR-activated B cells in murine cGVHD, arising in the setting of BAFF-driven altered B cell homeostasis. While polymorphisms in either donor or recipient BAFF genes are associated with human GVHD (Clark et al Blood 2011; 118: 1140), the source of excess BAFF after allo-HCT remains unknown. We addressed whether pathologic BAFF was derived from donor or recipient hematopoietic cells using TCDBM from B6 BAFF KO mice vs WT as donor cells for HCT to BALB/c recipients. Consistent with the known requirement of recipient BAFF for recovery of the B cell compartment after syngeneic transplant (Gorelik et al JEM 2003; 198:937), recovery of a donor peripheral B cell pool was not significantly different between groups. Remarkably, plasma BAFF levels were also not different between BALB/c recipients of B6 BAFF KO TCDBM +Sp vs recipients of WT TCDBM +Sp, suggesting that radioresistant recipient cells produced BAFF in cGVHD. Chronic GVHD-associated weight loss was also not different between groups (Fig.1E, representative of 3 separate experiments). We found that BAFF production in the engrafted KO recipient BM was uniformly low. Thus, we have now determined that non-BM, radioresistant recipient cells are the principal source of soluble pathologic BAFF in murine cGVHD. Further characterization of BAFF-producing cells is underway in order to identify therapeutic targets. In summary, we have now demonstrated that recipient-derived BAFF has a mechanistic role in aberrant activation of B cells in cGVHD. Our findings will lead to the development of anti-BAFF and BCR targeting agents for patients. This work was supported the National Institutes of Health (NHLBI R01 HL 129061-01). Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Description: Background Graft versus host disease (GVHD) is the principal cause of non-relapse mortality after allogeneic hematopoietic stem cell transplantation (allo HCT) and is associated with intestinal microbial dysbiosis. Administration of microbial metabolite butyrate or microbial cocktails of butyrogenic bacteria reduce the severity of acute GVHD in mice. Furthermore, this intestinal microbiome-metabolome axis can be manipulated via dietary intervention in healthy humans with supplementation by defined quantities of resistant potato starch (RPS), an indigestible carbohydrate that is metabolized by intestinal anaerobic commensal bacteria to produce butyrate1. Hereon we aimed to study the feasibility and tolerability of RPS in allo HCT recipients to test the hypothesis that the patients' intestinal microbiome and butyrate levels could be rationally modified by administration of defined quantities of RPS. Methods Between May 8, 2017 and September 30, 2018, we performed a single-center prospective, single arm, pilot study. We recruited adults who were undergoing human leukocyte antigen-matched, related-donor myeloablative allo HCT. Participants received RPS (Bob's Red Mill®) 20 g package orally, once/day for the first 3 days followed by twice daily, from day -7 through day 100 after allo HCT. Feasibility was defined as adherence to ≥ 70 % of scheduled doses in ≥ 60 % of patients. The primary objective was to test adherence to scheduled doses of RPS. Secondary endpoints included assessing tolerability of RPS and its ability to alter representation of RPS-degrading and butyrate-producing bacteria as well as butyrate levels in the intestines of allo HCT recipients. Stool samples were collected in the OMNIgene-Gut® (DNA Genotek) collection kit at baseline before intake of RPS, at time of nadir (day 7-10), engraftment (day 12-18), at day 100, and additional samples were also collected. Fecal microbiome was determined by 16S rRNA gene sequencing and butyrate by liquid chromatography. Results Ten subjects were enrolled. The median age was 57 years (range 52-62 years). All subjects received GVHD prophylaxis with tacrolimus and methotrexate as well as antibiotic prophylaxis with levaquin, and were treated for neutropenic fever with IV cefepime (90%) or IV vancomycin along with IV aztreonam (10%). One patient developed biopsy proven stage I acute GI GVHD with overall grade II acute GVHD (10%). Feasibility exceeded the preset goal of ≥ 70% adherence to scheduled dosages in ≥ 60 % of patients as 8 of the 10 patients (80%) received ≥ 70 % of scheduled doses (Figure 1A). No adverse effects/toxicities attributed to RPS were observed and longitudinal specimens were collected successfully. There was greater abundance of intestinal RPS-degraders such as Ruminococcus bromii, R. lactaris, R. gnavus, and Bifidobacterium spp and butyrate-producers such as Roseburia spp, Faecalibacterium prausnitzii, Eubacterium rectale, and Anaerostipes spp in the 10 patients receiving RPS compared to 15 historical controls after allo HCT (Figure 1B). Butyrate levels were significantly higher in the participants when they were on RPS as compared to pre RPS intake [median (interquartile range): 10.76 (7.62, 19.05) vs. 3.06 (2.32, 6.21) mmoL/kg, p

Description: Background Graft-versus-host disease (GVHD) is a serious, potentially fatal complication of allogeneic hematopoietic cell transplantation (HCT). Ruxolitinib (RUX), a Janus kinase (JAK) 1/JAK2 inhibitor, is in phase 3 development for patients (pts) with steroid-refractory chronic GVHD (SR cGVHD), and is approved for the treatment of steroid-refractory acute GVHD (SR aGVHD) in the United States. Outside of clinical trials, access to RUX is provided to pts with SR cGVHD through an expanded access program (EAP) sponsored by Incyte Corporation in the United States. The primary objective of this analysis was to report safety data from pts with SR cGVHD who received RUX in the Incyte-sponsored US EAP. Study Design and Methods Patients eligible to enroll in the open-label, multicenter US EAP from September 2017-May 2020 were ≥12 years of age, developed SR aGVHD or SR cGVHD after allogeneic HCT, and had an ECOG PS of 0-3. Pts with SR aGVHD and those with incomplete or missing data were excluded from the analysis. Based on clinical experience, the recommended starting dose of oral RUX for pts with cGVHD was 10 mg twice daily (BID). Doses could not be escalated above 10 mg BID, but dose reductions were permitted during treatment based on safety and laboratory assessments. The dose of RUX could be re-escalated if toxicity management thresholds were met or if pts experienced GVHD flares and had adequate hematologic parameters. Any GVHD treatments received before RUX initiation were recorded, and concurrent therapy with other cGVHD treatments was permitted. Serious adverse events (SAEs) were assessed according to National Cancer Institute Common Terminology Criteria for Adverse Events v4.03 from the time of consent until 30 days after end of treatment. Pt characteristics, prior treatments, and RUX dosing were summarized using descriptive statistics. Overall survival (OS) was assessed using Kaplan-Meier methodology. Results The analysis included 481 pts with SR cGVHD and complete data (data cutoff, 08 May 2020). Median (range) age was 60.0 (15-81) years; 51.8% of pts were male. At the time of RUX initiation, the organs involved included the skin (70.7%), eyes (59.3%), mouth (55.7%), joints (38.0%), lungs (34.9%), gastrointestinal tract (28.1%), liver (18.9%), and genitals (11.0%). Most pts (65.3%) received ≥2 prior treatments for GVHD, including systemic corticosteroids (54.9%), calcineurin inhibitors (25.6%), sirolimus (20.8%), mycophenolate mofetil (14.6%), ibrutinib (7.1%), and RUX (3.1%); 26.6% of pts received topical corticosteroids and 12.3% received other topical treatments. Most pts initially received RUX 5 mg BID (n=228 [47.4%]) or 10 mg BID (n=229 [47.6%]); 276 pts (57.4%) received RUX 10 mg BID as their last dose at the time of discontinuation or data cutoff. At data cutoff, 332 pts (69.0%) were still receiving RUX. Primary reasons for treatment discontinuations were death (7.1%), GVHD progression (5.4%), malignancy relapse (3.5%), and adverse events (2.5%). The median (range) duration of RUX treatment was 7.2 (0.03-33.0) months. SAEs, regardless of causality, were reported in 162 pts (33.7%). Median (range) time to first SAE from RUX initiation was 77.0 (1-693) days. The most common SAEs were sepsis (n=18 [3.7%]), pyrexia (n=9 [1.9%]), dyspnea (n=8 [1.7%]), respiratory failure (n=8 [1.7%]), acute kidney injury (n=7 [1.5%]), failure to thrive (n=7 [1.5%]), influenza (n=7 [1.5%]), diarrhea (n=6 [1.2%]), fall (n=6 [1.2%]), pulmonary embolism (n=6 [1.2%]), and upper respiratory tract infection (n=6 [1.2%]). One pt (0.2%) had a cytomegalovirus viremia SAE. There were few SAEs reported for cytopenias (febrile neutropenia, n=2 [0.4%]) or fungal infections (fungal pneumonia, n=1 [0.2%]); SAEs for neoplasms were reported for 8 pts (1.7%). There were no SAEs of thrombotic microangiopathy. Forty-six pts (9.6%) had fatal SAEs, most commonly attributed, at least in part, to infections (n=13 [28.3% of SAE-related fatalities]). Thirty-six pts (7.5%) had SAEs deemed related to RUX. The OS rates (95% CI) were 88% (84-91) at 1 year and 82% (74-88) at 2 years (Figure). Conclusions Patients with SR cGVHD in the RUX EAP program were heavily pretreated and primarily had organ involvement of the skin, eyes, and mouth. SAEs were reported in one-third of pts, including 7% of pts with RUX-related SAEs. Ten percent of pts had fatal SAEs, primarily due to infectious complications of cGVHD. No new or unexpected SAEs were reported. Figure 1 Disclosures Hari: Incyte Corporation: Consultancy; Takeda: Consultancy; BMS: Consultancy; Amgen: Consultancy; GSK: Consultancy; Janssen: Consultancy. Ali:Incyte Corporation: Consultancy. Chen:Takeda: Consultancy; Incyte Corporation: Consultancy; Equillium: Other: Data and Safety Monitoring Board Member; Actinium: Other: Data and Safety Monitoring Board Member; Magenta: Consultancy; Kiadis: Consultancy; AbbVie: Other: Data and Safety Monitoring Board Member. Fazal:Agios: Consultancy, Speakers Bureau; Jazz Pharma: Consultancy, Speakers Bureau; Jansen: Speakers Bureau; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Glaxosmith Kline: Consultancy, Speakers Bureau; Stemline: Consultancy, Speakers Bureau; Incyte Corporation: Consultancy, Honoraria, Speakers Bureau; Takeda: Consultancy, Speakers Bureau; Karyopham: Speakers Bureau; Gilead/Kite: Consultancy, Speakers Bureau; Celgene: Speakers Bureau; Novartis: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau. Gregory:Bluebird: Research Funding; Celgene: Research Funding; BMS: Research Funding; CURIS: Research Funding; Celularity: Research Funding; Constellation: Research Funding; CRISP Therapeutics: Research Funding; Acetylon: Research Funding; AbbVie: Research Funding; Amgen: Research Funding; Incyte Corporation: Consultancy; Vivolux: Research Funding; Teva: Research Funding; Takeda: Research Funding; Sanofi: Research Funding; Poseida: Research Funding; Novartis: Research Funding; Kesios: Research Funding; Lilly: Research Funding; Janssen: Research Funding; Glenmark: Research Funding; Genentech: Research Funding; EMD Sorono: Research Funding. Anand:AltruBio: Research Funding; CSL Behring: Research Funding; Incyte Corporation: Research Funding; Equillium: Research Funding; Kadmon: Research Funding. Naim:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Paranagama:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Bhatt:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. Blithe:Incyte Corporation: Current Employment, Current equity holder in publicly-traded company. DiPersio:Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Ruxolitinib is a JAK1/JAK2 inhibitor approved by the FDA for the treatment of adults with steroid-refractory acute GVHD. Ruxolitinib is in phase 3 testing for the treatment of steroid-refractory chronic GVHD but is currently not approved for this indication.

Description: Chronic graft versus host disease (cGVHD) patients have increased B cell-activating factor (BAFF) levels, but whether BAFF promotes disease after allogeneic bone marrow transplantation (allo-BMT) remains unknown. In a major MHC-mismatched model with cGVHD-like manifestations we first examined B-lymphopenic mMT allo-BMT recipients and found that increased BAFF levels in cGVHD mice were not merely a reflection of B cell number. Mice that later developed cGVHD, had significantly increased numbers of recipient fibroblastic reticular cells (FRCs) with higher BAFF transcript levels. Increased BAFF production by donor cells also likely contributed to cGVHD since BAFF transcript in CD4+ T cells from diseased mice and patients was increased. Chronic GVHD manifestations in mice associated with high BAFF/B cell ratios and persistence of B Cell Receptor (BCR)-activated B cells in peripheral blood and lesional tissue. By employing BAFF transgenic (Tg) mice donor cells, we addressed whether high BAFF contributed to BCR activation in cGVHD. BAFF increased NOTCH2 expression on B cells, augmenting BCR-responsiveness to surrogate antigen and NOTCH ligand. BAFF-Tg B cells had significantly increased protein levels of the proximal BCR signaling molecule SYK, and high SYK protein was maintained by BAFF after in vitro BCR-activation or when alloantigen was present in vivo. Using T-cell depleted (BM only) BAFF-Tg donors, we found that BAFF promoted cGVHD manifestations, circulating GL7+ B cells and alloantibody production. We demonstrate that pathological production of BAFF promotes an altered B-cell compartment and augments BCR-responsiveness. Our findings compel studies of therapeutic targeting of BAFF and BCR pathways in cGVHD patients.