Qayed, Muna

Abstract Background: CD19 CAR T cell therapies have demonstrated high initial complete remission (CR) rates of 70-80% in relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL) with durable remissions observed in a subset of patients. Despite the remarkable initial CR rate, relapses occur in 20-45% of patients and with increasing use of CD19 CAR T cell therapies in ALL, a key remaining clinical question is the role of post-CAR HCT. Methods: Patients up to 26 years treated with the commercial (i.e., tisagenlecleucel) or investigational CD19 CAR T cell products from 2014 to 2019 in the United States were included. The objectives were to examine the impact of post-CAR HCT on mortality, disease-free survival (DFS), leukemia relapse, GVHD and transplant related mortality among HCT recipients. The intent of HCT was analyzed as consolidation when there was no evidence of post-CAR T cell relapse prior to HCT. Cox regression multivariable analysis used HCT as time-dependent variable along with age, performance status and time from diagnosis to CAR-T cell infusion to evaluate its impact on outcomes. Results: We identified 347 patients who received CD19 CAR T cells from 86 centers. Median age was 13 (range, 0.4-26 years) with 39% of patients from Hispanic or Latino ethnic origin. Patients were high risk and heavily pretreated; 56% had poor cytogenetics, 51% had ≥3 line of prior therapies and 34% had prior HCT. Most of the patients (74%) received tisagenlecleucel. Disease burden at the time of CAR T cell infusion included morphologic disease (≥5% bone marrow blasts) (65%), measurable residual disease (MRD) positive (16%) and negative CR (18%). CRS of all grades was observed in 57.6% and grade 3-4 CRS in 13%; neurotoxicity of all grades was observed in 25% and grade 3-4 in 8%. With a median follow-up of 12.7 months (range, 2.2-60.3), DFS at 3, 6 and 12 months following CAR T cell infusion was 80.9% % (95% CI: 76.6%-84.9%), 71.2% (95% CI: 66.1%-76.0%) and 57.6% (95% CI: 51.8%-63.3%), respectively. OS at 3, 6 and 12 months was 93.6% (95% CI: 90.8%-96.0%), 89.8% (95% CI: 86.4%-92.8%) and 79.4% (95% CI: 74.7%-83.6%), respectively. Incidences of relapse without censoring at subsequent HCT at month 3, 6 and 12 were 18.5% (95% CI: 14.5%-22.8%), 28.2% (95% CI: 23.5%-33.3%), and 40.6% (95% CI: 35.0%-46.3%), respectively. Of the 347 patients who received CD19 CAR therapy, 62 patients (18%) had subsequent HCT in CR as a consolidation post-CAR with a median time from CAR T to HCT of 4.7 months (range, 1.1-18.6). 36% had a prior HCT (i.e., pre-CAR), 87% received myeloablative regimen and among the 57 patients with MRD evaluation 56 were negative (98%). Frequencies of post-CAR HCT did not change during the 5-year study period. Overall, transplant related mortality (TRM) at month 6 was 8.9% (95% CI: 3.6-16.1), and grade 2-4 acute GvHD was observed in 38.0% (95% CI: 28.4-48.2) of patients at month 6. Post-CAR HCT used as a consolidation was associated with lower relapse (hazard ratio (HR) 0.37; p=0.037) but no significant impact on mortality (HR 0.75, p=0.51) or DFS (HR=0.46, p=0.057). When accounting for any HCT performed after CAR T cell, HCT was associated with higher mortality (Figure). Conclusions: This is the largest clinical series to date examining the impact of post-CAR HCT on clinical outcome of R/R B-ALL patients. We found that 18% of the patients had post-CAR HCT as a consolidation therapy with a favorable safety profile of 8.9% TRM rate despite a prior transplant rate of 36%. Consolidation HCT after CAR T cell resulted in significant reduction in leukemia relapse with a trend towards better DFS. Consolidation HCT seem safe and incurred a benefit by reducing disease relapse. Further studies in understanding the role of consolidation HCT as planned, or post-CAR T cell response-based strategy or treatment for early B-cell recovery are needed to further define the best candidates for HCT after tisagenlecleucel . Figure 1 Figure 1. Disclosures Park: BMS: Consultancy; Affyimmune: Consultancy; Kite Pharma: Consultancy; Curocel: Consultancy; Novartis: Consultancy; Minerva: Consultancy; Innate Pharma: Consultancy; PrecisionBio: Consultancy; Intellia: Consultancy; Amgen: Consultancy; Autolus: Consultancy; Servier: Consultancy; Artiva: Consultancy; Kura Oncology: Consultancy. Nikiforow: Kite/Gilead: Other: ad HOC Advisory Boards; Novartis: Other: ad Hoc Advisory Boards; Iovance: Other: ad Hoc Advisory Boards; Glaxo Smith Kline (GSK): Other: ad Hoc Advisory Boards. Hu: Kite/Gilead: Research Funding; Novartis: Research Funding; Celgene: Research Funding. Ahmed: Merck: Research Funding; Tessa Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Seagen: Research Funding; Xencor: Research Funding. Badar: Pfizer Hematology-Oncology: Membership on an entity's Board of Directors or advisory committees. Cairo: Servier: Speakers Bureau; Sanofi: Speakers Bureau; Amgen: Speakers Bureau; Jazz Pharmaceutical: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sobi: Speakers Bureau; Omeros: Membership on an entity's Board of Directors or advisory committees; Nektar: Membership on an entity's Board of Directors or advisory committees. Dholaria: Janssen: Research Funding; Takeda: Research Funding; Jazz: Speakers Bureau; MEI: Research Funding; Pfizer: Research Funding; Angiocrine: Research Funding; Poseida: Research Funding; Celgene: Speakers Bureau. Grover: Kite: Other: Advisory Board; Genentech: Research Funding; ADC: Other: Advisory Board; Tessa: Consultancy; Novartis: Consultancy. Locke: Gerson Lehrman Group: Consultancy; Emerging Therapy Solutions: Consultancy; Moffitt Cancer Center: Patents & Royalties: field of cellular immunotherapy; Calibr: Consultancy, Other: Scientific Advisory Role; BMS/Celgene: Consultancy, Other: Scientific Advisory Role; Wugen: Consultancy, Other; Legend Biotech: Consultancy, Other; Janssen: Consultancy, Other: Scientific Advisory Role; Iovance Biotherapeutics: Consultancy, Other: Scientific Advisory Role; Novartis: Consultancy, Other, Research Funding; GammaDelta Therapeutics: Consultancy, Other: Scientific Advisory Role; Takeda: Consultancy, Other; EcoR1: Consultancy; Cowen: Consultancy; Umoja: Consultancy, Other; Kite, a Gilead Company: Consultancy, Other: Scientific Advisory Role, Research Funding; Cellular Biomedicine Group: Consultancy, Other: Scientific Advisory Role; Bluebird Bio: Consultancy, Other: Scientific Advisory Role; Amgen: Consultancy, Other: Scientific Advisory Role; Allogene Therapeutics: Consultancy, Other: Scientific Advisory Role, Research Funding. Murthy: CRISPR Therapeutics: Research Funding. Mussetti: GILEAD: Other: Clinical trials participation, Research Funding; TAKEDA: Honoraria; NOVARTIS: Honoraria, Other: Clinical trials participation. Pulsipher: Jasper Therapeutics: Honoraria; Adaptive: Research Funding; Equillium: Membership on an entity's Board of Directors or advisory committees. Qayed: Novartis: Membership on an entity's Board of Directors or advisory committees; Medexus: Membership on an entity's Board of Directors or advisory committees; Mesoblast: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; BMS: Other. Reshef: Gilead, BMS, Precision, Immatics, Atara, Takeda, Shire, Pharmacyclics, Incyte: Research Funding; Gilead and Novartis: Honoraria; Bayer, BMS, Regeneron, TScan, Synthekine, Atara, Jasper: Consultancy. Rizzieri: Kite: Honoraria, Speakers Bureau; Incyte: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Stemline: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Seattle Genetics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Acrotech: Membership on an entity's Board of Directors or advisory committees; Teva: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; AROG: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Karyopharm: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees; Celltrion: Membership on an entity's Board of Directors or advisory committees; Mustang: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Jazz: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Honoraria; Pharmacyclics: Honoraria. Sharma: Vertex Pharmaceuticals/CRISPR Therapeutics: Other: Salary support paid to institution; Medexus Inc: Consultancy; CRISPR Therapeutics: Other, Research Funding; Vindico Medical Education: Honoraria; Spotlight Therapeutics: Consultancy; Novartis: Other: Salary support paid to institution. Schultz: BMS DSMB: Other: payment; Sobi: Membership on an entity's Board of Directors or advisory committees. Turtle: Century Therapeutics Ad hoc advisory boards (last 12 months): Nektar Therapeutics, Allogene, PACT Pharma, Astra Zeneca, Amgen: Membership on an entity's Board of Directors or advisory committees; Precision Biosciences: Membership on an entity's Board of Directors or advisory committees; Eureka Therapeutics: Membership on an entity's Board of Directors or advisory committees; Caribou Biosciences: Membership on an entity's Board of Directors or advisory committees; T-CURX: Membership on an entity's Board of Directors or advisory committees; Myeloid Therapeutics: Membership on an entity's Board of Directors or advisory committees; ArsenalBio: Membership on an entity's Board of Directors or advisory committees; Minerva: Other; Nektar Therapeutics: Other; Juno Therapeutics/BMS: Other: Payments, Patents & Royalties: Patent licensed to Juno Therapeutics.; Precision Biosciences, Eureka Therapeutics, Caribou Biosciences, Myeloid Therapeutics, ArsenalBio: Current holder of stock options in a privately-held company. Pasquini: Kite Pharma: Research Funding; Novartis: Research Funding; Bristol Myers Squibb: Consultancy, Research Funding; GlaxoSmithKline: Research Funding. Perales: MorphoSys: Honoraria; Cidara: Honoraria; Bristol-Myers Squibb: Honoraria; Miltenyi Biotec: Honoraria, Other; Sellas Life Sciences: Honoraria; Takeda: Honoraria; Karyopharm: Honoraria; Celgene: Honoraria; Omeros: Honoraria; Nektar Therapeutics: Honoraria, Other; Equilium: Honoraria; Novartis: Honoraria, Other; NexImmune: Honoraria; Incyte: Honoraria, Other; Kite/Gilead: Honoraria, Other; Merck: Honoraria; Servier: Honoraria; Medigene: Honoraria.

Abstract Background: A major cause of mortality in patients receiving hematopoietic stem cell transplantation (HCT) is acute graft-versus-host disease (GVHD), a multiorgan disorder that includes the skin, liver and gastrointestinal tract. We have previously identified elafin, a protease inhibitor overexpressed in inflamed epidermis, as a diagnostic biomarker of GVHD in the skin, the most commonly involved GVHD organ. However, our initial study was limited to a subset of patients with isolated skin GVHD. The main driver of nonrelapse mortality (NRM) in HCT patients is GI GVHD. Two biomarkers, Regenerating islet-derived 3a (REG3α) and Suppressor of tumorigenesis 2 (ST2), have since been validated as biomarkers of GI GVHD that predict long-term outcomes in patients treated for GVHD. We undertook this study to determine the utility of elafin as a prognostic biomarker of acute GVHD in the general population of previously unstudied acute GVHD patients, and to compare it to ST2 and REG3α. Study Design: 526 patients who received systemic corticosteroid treatment for skin GVHD were analyzed from the Mount Sinai Acute GVHD International Consortium (MAGIC), which includes patients from 25 HCT centers. We used ELISA to measure serum concentrations of elafin, ST2 and REG3α. Patients were divided randomly into equal training and validation sets; and we developed a competing risk regression model for 6-month NRM using elafin concentration in the training set. We developed additional models for 6-month NRM using concentrations of ST2 and REG3α, or the combination of all three biomarkers as predictors. We then constructed ROC curves to evaluate the predictive accuracy of each model and to analyze the ability of each model to stratify patients into high- and low-risk groups. We analyzed the cumulative incidence of 6-month NRM and overall survival in each model and compared the accuracy of each model in the validation set. Results: The area under the receiver operating curve (AUROC) for elafin alone was 0.55 whereas it was 0.75 and statistically superior (P = 0.02) for the combination of ST2 and REG3α. The combination of 3 biomarkers produced an AUROC of 0.76 that was not significantly better than the two biomarker model (P = 0.10). Elafin concentrations, either alone or in combination with ST2 and REG3α, did not produce higher hazard ratios of NRM (data not shown). Patients in the low-risk elafin group paradoxically demonstrated a higher incidence of 6-month NRM, although this difference was not statistically significant (17% vs. 11%, P=0.19), and both overall survival at 6 months (68% vs. 68%, P>0.99) and four-week response (78% vs. 78%, P=0.98) were similar in the low- and high-risk elafin groups (Figure 1). As demonstrated in previous data sets, the combination of ST2 and REG3α divided patients into two groups with a nearly five-fold difference in NRM (6.7% vs. 31%, P <0.001). Conclusion: We demonstrated that serum elafin concentrations measured at the initiation of systemic treatment for acute GVHD in a multicenter population of patients treated systemically for acute GVHD do not predict 6-month NRM, overall survival, or treatment response. As seen in previous studies, serum concentrations of the GI GVHD biomarkers ST2 and REG3α were significant predictors of NRM and the addition of elafin levels did not improve their accuracy. These results underscore the importance of GI disease in driving NRM in patients who develop acute GVHD. Figure 1. Cumulative incidence of nonrelapse mortality and overall survival in high and low risk groups Six-month cumulative incidences of nonrelapse mortality (NRM) in high (solid line) and low (dotted line) risk groups defined by optimized biomarker thresholds (upper panels) and six-month overall survival estimated using the Kaplan-Meier method (lower panels). (A) Cumulative incidence of NRM (14%) and overall survival (75%) in the total validation set (N=263). (B) Cumulative incidence of NRM in the low (N=150) and high (N=113) elafin group (17% vs. 11%, P=0.19). Overall survival in the low and high elafin group (68% vs. 68%, P > 0.99). (C) Cumulative incidence of NRM in the low (N=175) and high (N=88) ST2 + REG3a group (6.7 vs. 31%, P < 0.001). Overall survival in the low and high ST2 + REG3a group (77% vs. 51%, P < 0.001). (D) Cumulative incidence of NRM in the low (N=180) and high (N=83) elafin + ST2 + REG3a group (7.0 vs. 30%, P < 0.001). Overall survival in the low and high elafin + ST2 + REG3a group (79% vs. 64%, P < 0.001). Figure 1 Figure 1. Disclosures Ozbek: Viracor: Patents & Royalties: GVHD biomarker patent with royalties from Viracor. DeFilipp: Omeros, Corp.: Consultancy; Incyte Corp.: Research Funding; Regimmune Corp.: Research Funding; Syndax Pharmaceuticals, Inc: Consultancy. Grupp: Novartis, Kite, Vertex, and Servier: Research Funding; Jazz Pharmaceuticals: Consultancy, Other: Steering committee, Research Funding; Novartis, Roche, GSK, Humanigen, CBMG, Eureka, and Janssen/JnJ: Consultancy; Novartis, Adaptimmune, TCR2, Cellectis, Juno, Vertex, Allogene and Cabaletta: Other: Study steering committees or scientific advisory boards. Hexner: Blueprint medicines: Membership on an entity's Board of Directors or advisory committees, Research Funding; Tmunity Therapeutics: Research Funding; PharmaEssentia: Membership on an entity's Board of Directors or advisory committees. Kitko: Co-investigator on two NIH grants as part of the cGVHD consortium: Research Funding; Vanderbilt University Medical Center: Current Employment; PER: Other: PER - CME educational talks about GVHD; Horizon: Membership on an entity's Board of Directors or advisory committees. Qayed: Novartis: Honoraria; Mesoblast: Honoraria; Medexus: Honoraria. Reshef: ilead, BMS, Precision, Immatics, Atara, Takeda, Shire, Pharmacyclics, Incyte: Research Funding; Bayer: Consultancy; Gilead and Novartis: Honoraria; BMS, Regeneron, TScan, Synthekine, Atara, Jasper, Bayer: Consultancy. Levine: Incyte: Consultancy, Research Funding; Viracor: Patents & Royalties: GVHD biomarker patent with royalties from Viracor; Mesoblast: Consultancy, Research Funding; Equillium Bio: Membership on an entity's Board of Directors or advisory committees; X4 Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Talaris Therapeutics: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Omeros: Membership on an entity's Board of Directors or advisory committees; Symbio: Membership on an entity's Board of Directors or advisory committees; Biogen: Research Funding; Kamada: Research Funding. Ferrara: Eurofins Viracor: Consultancy, Other: Royalties. Chen: Incyte: Consultancy; Gamida: Consultancy.

Systemic glucocorticoids are the principal treatment for acute graft-versus-host disease (GVHD), which remains the major cause of non-relapse mortality (NRM) after allogeneic hematopoietic cell transplantation (HCT). However, there are no validated biomarkers that measure a patient's response to glucocorticoid therapy, and thus response is evaluated by the change in clinical symptom severity. A major weakness in the predictive power of clinical responses is that changes to all organs are weighted equally even though the major driver of NRM is irreversible damage to the crypts of the GI tract. Recent studies from the Mount Sinai Acute GVHD International Consortium (MAGIC) have validated an algorithm probability (MAP) that combines serum concentrations of two biomarkers of GVHD (REG3α and ST2) to generate an estimated probability of 6 month NRM for individual patients. The MAP has been considered a "liquid biopsy" that estimates the damage caused by GVHD to crypts throughout the lower GI tract (Hartwell et al., JCI Insight, 2017; Major-Monfried et al., Blood, 2018). We hypothesized that the change in MAP between start of treatment and 28 days later could serve as a response biomarker for GVHD and might compare favorably to the change in clinical symptoms that measures response to GVHD treatment, which is widely used as a surrogate for long term survival and is the primary endpoint in most GVHD treatment trials (Martin et al., BBMT, 2009; MacMillan et al., Blood, 2010). We prospectively collected serum samples and clinical staging from 368 sequential HCT patients who received systemic treatment for acute GVHD in one of 20 MAGIC centers between January 2016 and February 2018. We measured the serum concentrations of REG3α and ST2 before and after systemic therapy for acute GVHD and computed MAPs, the changes in MAPs, and clinical responses for each patient. MAPs of patients who experienced 6 month NRM showed significantly greater increases than MAPs of patients who survived (p=0.0004). In patients whose MAPs at the start of treatment were low (Ann Arbor 1, MAP < 0.141) or intermediate (Ann Arbor 2, 0.141 ≤ MAP ≤ 0.290), 6 month NRM clustered among those who had the greatest increases in MAP after 28 days (Fig 1A,B). In patients with high MAPs at the start of treatment (Ann Arbor 3, MAP > 0.290), those who survived tended to have the largest decreases in MAP (Fig 1C). These changes in MAP suggested crossing a single threshold could predict risk of mortality. We found that patients whose MAPs rose above a threshold MAP of 0.290 (5% of Ann Arbor 1, 27% of Ann Arbor 2) had significantly worse survival compared to those who remained below it, whereas the large number patients with initially high MAPs that remained above the threshold (66% of Ann Arbor 3) had a large increases in mortality (Fig 2). When measured at day 28, the MAP was significantly more accurate in predicting NRM than the gold standard of the clinical response, with areas under the receiver operating characteristic curve (AUC) of 0.86 and 0.70, respectively (p<0.0001). An algorithm that combined clinical response with biomarkers generated the same AUC as the MAP alone (0.83 v 0.86, p = NS). We next tested whether the same MAP threshold of 0.290 could predict risk within clinical response subsets. A significant minority (10%) of clinical responders had high MAPs and experienced three-fold greater NRM than those with low MAPs (40% v 12%, p<0.0001) whereas the majority (57%) of non-responders had low MAPs and experienced almost three-fold lower NRM than those with high MAPs (24% v 65%, p<0.0001) (Fig 3). Thus the MAP provides important prognostic information over and above the change in clinical symptoms, further stratifying both responders and non-responders at four weeks of treatment. The MAP threshold classified patients both with and without significant lower GI symptoms because the MAP is a more specific measure of irreversible cryptic damage in patients with copious diarrhea and more sensitive in patients with less than 0.5 liters of daily diarrhea (Fig 4). We conclude that the MAP is, to our knowledge, the first validated laboratory test to serve as response biomarker for the treatment for acute GVHD and a more accurate predictor of survival than clinical response after four weeks of treatment. The MAP may serve as a novel endpoint and an important complement to changes in clinical symptom severity in future trials of GVHD treatment. Disclosures Srinagesh: National Institutes of Health: Research Funding. Ozbek:Viracor: Patents & Royalties: Biomarker Patent. Ayuk:Novartis: Honoraria, Other: Advisory Board, Research Funding. Aziz:Doris Duke Charitable Foundation: Research Funding. Defilipp:Incyte: Research Funding. Grupp:Novartis: Consultancy, Research Funding; Roche: Consultancy; GSK: Consultancy; CBMG: Consultancy; Novartis: Research Funding; Kite: Research Funding; Servier: Research Funding; Jazz: Other: study steering committees or scientific advisory boards; Adaptimmune: Other: study steering committees or scientific advisory boards; Cure Genetics: Consultancy; Humanigen: Consultancy. Hexner:novartis: Research Funding. Kitko:Mallinckrodt: Honoraria; Novartis: Consultancy, Honoraria. Mielke:EBMT/EHA: Other: Travel support; ISCT: Other: Travel support; Miltenyi: Consultancy, Honoraria, Other: Travel and speakers fee (via institution), Speakers Bureau; Jazz Pharma: Honoraria, Other: Travel support, Speakers Bureau; IACH: Other: Travel support; Kiadis Pharma: Consultancy, Honoraria, Other: Travel support (via institution), Speakers Bureau; DGHO: Other: Travel support; Bellicum: Consultancy, Honoraria, Other: Travel (via institution); GILEAD: Consultancy, Honoraria, Other: travel (via institution), Speakers Bureau; Celgene: Honoraria, Other: Travel support (via institution), Speakers Bureau. Merli:Sobi: Consultancy; Amgen: Honoraria; Novartis: Honoraria; Bellicum: Consultancy. Pulsipher:Amgen: Other: Lecture; Miltenyi: Research Funding; Bellicum: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Jazz: Other: Education for employees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Medac: Honoraria. Qayed:Bristol-Myers Squibb: Honoraria. Reshef:Pfizer: Consultancy; Magenta: Consultancy; Kite: Consultancy, Research Funding; Atara: Consultancy, Research Funding; BMS: Consultancy; Pharmacyclics: Consultancy, Research Funding; Incyte: Consultancy, Research Funding; Celgene: Research Funding; Shire: Research Funding. Levine:Incyte: Consultancy, Research Funding; Biogen: Other: non-financial support; Viracor: Patents & Royalties: biomarker patent; Ironwood: Honoraria; bluebird bio: Consultancy; National Cancer Institute: Research Funding; Novartis: Honoraria; Kamada: Research Funding. Ferrara:National Institutes of Health: Research Funding; ViraCor: Consultancy; Incyte: Consultancy; Kamada: Consultancy; Mallinckrodt: Consultancy; Enlivex: Consultancy; Xenikos: Consultancy; CSL Behring: Consultancy.

Key Points The MAGIC algorithm probability, computed from 2 serum biomarkers, predicts mortality in all GVHD grades after 4 weeks of treatment. Dynamic changes in the MAGIC algorithm probability occur within all biomarker risk groups and can guide therapy.

Abstract 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<0.001, Figure 1). Relapse rates were the same in both probability groups, and thus LP patients experienced significantly better relapse free survival (RFS) (69% vs 53%, p<0.001). As expected, significantly fewer LP patients experienced severe GVHD at onset (as measured by Minnesota risk), maximum grade III/IV GVHD, or steroid resistant GVHD by day 180 after HCT (Figure 2, p<0.001 for all three parameters). To measure the accuracy of prediction, at each timepoint we calculated the area under the curve (AUC) of receiver operating characteristic curves; the AUC increased significantly with each subsequent evaluation, from 0.59 at one timepoint (dotted line) to 0.74 at the third timepoint (solid line) (p<0.001), with a final sensitivity of 65% and specificity of 74% (Figure 3A). Early development of GVHD (by day 28) is a risk factor for lethal GVHD. Therefore, we next plotted RFS (dashed line), relapse (solid line), and lethal GVHD (dotted line) rates in patients who developed GVHD by day 28. 25% of patients with GVHD were categorized as HP and had a cumulative incidence of lethal GVHD more than four times higher (28%) than that of relapse (6%); however the risks were reversed for the 75% of patients who were LP, where relapse (15%) occurred twice as often as lethal GVHD (7%) (Figure 3B). In patients who did not develop GVHD in the first month, this reversal of risks was even more dramatic. Approximately half (53%) of the entire validation cohort did not develop GVHD by day 28 and was LP at all three evaluations. These patients had an exceptionally low risk of lethal GVHD and thus they relapsed (25%) much more often than they died from GVHD (3%). When malignancies were classified according to risk for relapse by the disease risk index (DRI) (Figure 3C), the probability of relapse was three fold higher than lethal GVHD in malignancies with a low DRI (12%), six fold higher for intermediate DRI (20%), and eleven fold higher for high/very high DRI (33%). We conclude that a serial monitoring strategy using GVHD biomarkers for one month after HCT is able to identify two groups of patients with very different risks of lethal GVHD and relapse. For these patients, the intensity of immunosuppression after day 28 could be tailored according to the probabilities of developing lethal GVHD and relapse in the context of clinical trials. Disclosures Aziz: Doris Duke Charitable Foundation: Research Funding. Ayuk:Therakos (Mallinckrodt): Honoraria; Novartis: Honoraria; Celgene: Consultancy; Gilead: Consultancy. Chen:REGiMMUNE: Consultancy; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees; Magenta Therapeutics: Consultancy; Takeda Pharmaceuticals: Consultancy. Merli:Neovii Biotech: Honoraria; AMGEN: Honoraria. Roesler:Sanofi: Other: Travel, Accommodations, Expenses; Amgen: Equity Ownership; Jazz Pharmaceuticals: Other: Travel, Accommodations, Expenses; Immunomedics: Equity Ownership; Biogen: Equity Ownership; Merck: Consultancy; Pfizer: Consultancy. Kitko:Novartis: Consultancy, Honoraria; Mallinckrodt: Honoraria, Other: Travel, Accommodations, Expenses. Qayed:Novartis: Consultancy. Wölfl:Bristol-myers Squibb: Equity Ownership; Novartis: Equity Ownership; Taheda: Equity Ownership; Juno: Equity Ownership; Neovii: Other: Travel, Accommodations, Expenses. Mielke:Celgene: Speakers Bureau; DGHO: Speakers Bureau; EHA: Speakers Bureau; Kiadis Pharma: Speakers Bureau; Miltenyi: Speakers Bureau. Wudhikarn:Takeda Oncology: Other: Travel, Accommodations, Expenses. Nakamura:Celgene: Honoraria; Molmed: Honoraria; Merck: Consultancy; Pharmacyclics: Consultancy; Atara: Consultancy; Jazz Pharmaceuticals: Consultancy. Pulsipher:CSL Behring: Consultancy; Novartis: Consultancy, Honoraria, Speakers Bureau; Adaptive Biotech: Consultancy, Research Funding; Amgen: Honoraria. Reshef:Pfizer: Consultancy; Atara Biotherapeutics: Consultancy; Kite Pharma: Consultancy; Takeda Pharmaceuticals: Consultancy; Bristol-Myers Squibb: Consultancy; Incyte: Consultancy. Levine:Therakos: Consultancy; Novartis: Consultancy; Bluebird: Consultancy; Incyte: Consultancy; Kamada: Research Funding; Viracor: Patents & Royalties. Ferrara:Incyte: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses; Xenikos: Consultancy, Other: Travel, Accommodations, Expenses; Kamada: Consultancy, Research Funding; Viracor: Consultancy, Patents & Royalties.

Abstract 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<0.0001) (Fig 1A). Analysis of the validation cohort using the same TS definitions showed similar differences in response rates (22% vs 61%, p<0.0001) (Fig 1B). Further, nearly four times as many pts with high scores in both cohorts died within 6 months from non-relapse causes compared to pts with low scores (test: 57% vs 17%, p<0.0001; validation: 57% vs 14%, p<0.0001) (Fig 1C/D). As expected, the majority of non-relapse deaths in pts treated for GVHD were directly attributable to GVHD (test: 95%; validation: 89%). Relapse rates for high and low score pts were similar (data not shown), and thus pts with a high TS experienced significantly worse overall survival in both cohorts (test: 37% vs 72%, p<0.0001; validation: 38% vs 79%, p<0.0001) (Fig 1E/F). 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<0.0001) (Fig 2B). Two thirds of early non-responders comprised this more favorable group. These highly significant results reproduced in the independent validation cohort in similar proportions (CR+PR: 45% vs 6%, p=0.0003; NR: 61% vs 22%, p=0.0001) (Fig 2C/D). Finally, a subset analysis revealed that pts classified as NR after one week of steroids due to isolated, yet persistent, grade I skin GVHD (24/378, 6%) overwhelmingly had low treatment scores (22/24, 92%) and experienced rates of NRM (9%) comparable to responders with low scores, thus forming a distinct, albeit small, subset of pts with non-responsive GVHD that fares particularly well (Fig 3). 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.

Abstract 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<0.001) (Fig 1A). The relapse rates did not differ between risk groups so that overall survival (OS) was 60% for HR and 84% for LR (p<0.001) (Fig 1B). When applied to the test set (Fig 1C/D), the algorithm identified 54/309 (17%) of the patients as HR with an NRM of 33% vs 7% for LR patients (p<0.001) and 6-month OS of 57% and 81% for HR and LR patients, respectively (p<0.001). In the independent validation set (Fig 1 E/F), the algorithm identified 72/358 (20%) of the patients as HR with an NRM of 26% vs 10% for LR patients (p<0.001) and OS of 68% and 85% for HR and LR patients, respectively (p<0.001). High risk patients were three times more likely to die from GVHD than LR patients in each cohort (p<0.001) (Fig 2). The GI tract is the GVHD target organ that is most resistant to treatment and represents a major cause of NRM, and we observed twice as much severe GI GVHD (stage 3 or 4) in HR patients as in LR patients (p<0.001, data not shown). The algorithm successfully separated HR and LR strata for 6 month NRM in several groups with differing risks for GVHD and NRM, including donor type, degree. of HLA-match, age group, and conditioning regimen intensity (Fig 3). 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.

Abstract 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 <50kg) based upon a chart review of 300 patients with post-BMT diarrhea from any cause. Non-GVHD rectal bleeding that results in flecks or streaks of blood in the stool are ignored for staging. We use the highest daily stool output in the 3 days prior to the diagnosis of lower GI GVHD to determine onset stage and, when available, a 3-day average stool volume for subsequent staging to smooth daily variability. 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 <3 episodes/day Child: < 10 ml/kg/day or <4 episodes/day 1 Rash < 25% BSA 2-3 mg/dl Persistent nausea, vomiting or anorexia Adult: 500-999 ml/day or 3-4 episodes/day Child: 10 -19.9 ml/kg/day or 4-6 episodes/day 2 Rash 25 - 50% BSA 3.1-6 mg/dl - Adult: 1000-1500 ml/day or 5-7 episodes/day Child: 20 - 30 ml/kg/day or 7-10 episodes/day 3 Rash > 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.

Key Points Compared with parous female sibling donors, male URDs confer more aGVHD in all patients and more cGVHD in females. There was no difference in survival, relapse, or transplant mortality between recipients of parous female sibling or male URD grafts.

Key Points Biomarker scores generated after 1 week of steroid treatment of GVHD are prognostic. Biomarkers reflect prognosis better than early clinical response to GVHD treatment.