Jagasia, Madan

Abstract 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<0.01). Every 2-fold increase in AREG was associated with a 33% decrease in the likelihood of day 28 CR/PR (odds ratio [OR] 0.67, p<0.01, table). Clinical factors alone, as determined by the refined Minnesota Risk Score, were associated with day 28 CR/PR (p=0.02, table). Adding AREG to the Minnesota Risk Score could further risk-stratify patients. Minnesota SR patients with elevated AREG ≥ 33 pg/mL showed a 59% lower odds of day 28 CR/PR than SR patients with low AREG (OR 0.41, p=0.02). Patients with Minnesota HR aGVHD with AREG ≥ 33 pg/mL had the worst outcomes, with an 82% lower odds of day 28 CR/PR in comparison to HR with low AREG (OR 0.18, p<0.01). High AREG was associated with worse OS and NRM in both Minnesota SR (hazard ratios 2-year OS 2.3, p<0.01 and 6 month NRM 2.95, p=0.01, respectively) and HR patients (hazard ratios 2-year OS 3.35, p<0.01 and 6-month NRM 9.38, p<0.01 respectively, figure). Finally, in independent samples from the Chronic GVHD Consortium/MAGIC we confirmed that high AREG was associated with worse day 28 CR/PR (55.2% vs. 79.4%, p=0.02) and significantly worse 6-month survival after the onset of aGVHD (57.1% vs. 82.5%, p=0.01). 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.

BACKGROUND. Cutaneous erythema and histopathology features in patients post allogeneic hematopoietic cell transplantation (HCT) are nonspecific, making it difficult to distinguish acute graft-versus-host disease (aGVHD) from engraftment syndrome, drug reactions, viral infections, and other etiologies. Biopsies from different locations on the same patient frequently differ in aGVHD grade, and serial biopsies of a single lesion are not possible. Proper biopsy site selection, timing, and serial noninvasive microscopic monitoring may improve our understanding and management of aGVHD. In this study, we tested the feasibility of noninvasive reflectance confocal microscopy (RCM) to detect key histopathology features of cutaneous aGVHD. STUDY POPULATION. We enrolled 11 patients with high clinical suspicion of cutaneous aGVHD, as determined by a transplant physician. In total, 16 lesions of cutaneous aGVHD-affected site were imaged by RCM (6x6 mm2en face images at four different depths) and subsequently biopsied (4x4 mm punch biopsy). 9 out of 11 patients had only skin involvement, and 2 patients had skin and gut aGVHD. 5 patients had >50% body surface area (BSA) affected, 1 had 18-50% BSA, and 5 had <18% BSA. 8 out of 11 patients required systemic corticosteroids and 3 patients received topical corticosteroids. All patients were imaged before starting treatment. METHODS. We used a clinical confocal microscope (Vivascope 1500, Caliber I.D.), which enables real-time evaluation of epidermal and superficial dermal tissue at sub-cellular resolution. Four reflectance confocal microscopists blinded to histopathology independently evaluated the presence or absence of 18 RCM features1. Concurrently, four dermatopathologists blinded to clinical and confocal information determined the presence or absence of 19 histopathology features, as well as the Lerner aGVHD grade. A histologic feature was determined as "present" in a lesion when marked by most experts (expert vote) or resolved by a fifth expert in case of a disagreement among four experts. The reflectance confocal microscopist vote was then correlated to the dermatopathologist expert vote for 17 overlapping features. We also evaluated the interrater variability among microscopists and among dermatopathologists. RESULTS. The main aGVHD features by Lerner grade had > 88% correlation between RCM and histopathology: (1) basal vacuolar change, (2) presence of dyskeratotic keratinocytes, and (3) dermal inflammation. By contrast, dyskeratotic cells and inflammation at the adnexal structures had <50% correlation. This may be attributed to the fact that more adnexal structures are typically visible in the 6x6 mm2 RCM horizontal tissue view compared to vertical section histopathology. We found a similar interrater variability among RCM experts (70%) and dermatopathologists (68%). CONCLUSIONS. In this pilot study, we show that noninvasive label-free RCM of skin enables detection of the main Lerner grade features of cutaneous aGVHD. We found a similar interrater variability among reflectance confocal microscopists and among dermatopathologists who each independently evaluated the presence or absence of a list of aGVHD features. Future studies can build on this work to evaluate the feasibility of RCM to determine cutaneous aGVHD grade, as well as distinguish between rash due to aGVHD and drug reactions. ACKNOWLEDGEMENT. This work was supported in part by Career Development Award Number IK2 CX001785 from the United Sates Department of Veterans Affairs Clinical Science R&D (CSRD) Service. Saknite I, Gill M, Alessi-Fox C, Byrne M, Jagasia M, Gonzalez S, Ardigo M, Tkaczyk ER. Features of cutaneous acute graft-versus-host disease by reflectance confocal microscopy. Br J Dermatol (2019). Disclosures Christi: Caliber I.D.: Employment, Equity Ownership. Byrne:Karyopharm: Research Funding. Jagasia:Kadmon: Consultancy; Incyte: Consultancy; Janssen: Research Funding. Tkaczyk:Incyte: Consultancy.

Abstract Introduction Acute graft-versus-host disease (aGVHD) is a significant cause of mortality post allogeneic stem cell transplantation (SCT). First line therapy is corticosteroid based for patients with aGVHD ≥grade (Gd) II. Steroid refractory (SR) aGVHD is associated with a dismal prognosis. Although experimental therapeutics is an unmet need, there is no recent prospective real world data to establish a benchmark for outcomes of best available therapy. We undertook a prospective observational multi-centre international clinical trial of outcomes of second line therapy of steroid refractory aGVHD. The aim was to measure recently defined endpoints including Day 28 (D28) overall response rate (ORR), and 6 month freedom from treatment failure (6mFFTF; defined as a patient (pt) being alive, without relapse of underlying disease or addition of new systemic therapy for aGVHD, prior to the development of chronic(c) GVHD. Methods Data was collected prospectively in a longitudinal observational study in patients starting second line therapy for SR aGVHD. Twelve UK and 2 US sites participated. Adult pts with Gd II-IV aGVHD refractory to corticosteroids were enrolled within 5 days of starting second line therapy. Modified Glucksberg criteria were used to grade aGVHD. Expert panel consensus recommendations (Martin et al 2009) were used for complete response (CR), very good partial response (VGPR), partial response (PR), stable disease (SD) and progressive disease (PD). Kaplan-Meier survival curves and log rank tests for comparison were done on Prism7. Results Between 2014-2018, 64pts were enrolled; 4pts were excluded due to incomplete data. Baseline characteristics of the cohort are outlined in Table 1. GVHD prophylaxis varied according to centre. Forty-seven percent had T depletion; 32% ATG and 25% Alemtuzumab. GVHD was donor lymphocyte infusion (DLI) induced in 7%. At start of second line therapy,5% had Gd I, 27% Gd II and 68% Gd III-IV aGVHD. All patients had received corticosteroids at a minimum dose of 1mg/kg. Second line therapies varied, 60% received ECP and 40% non-ECP therapy including anti-TNF-alpha antibodies (20%), mycophenolate mofetil (MMF) (8%), mesenchymal stem cells (4%) and 'other' (8%). The cumulative incidence of 6mFFTF was 32% (n=19), with 68% (n=41) failing to meet the 6mFFTF endpoint. The causes of failure were, addition of third line therapy in 40% (n=24), relapse 8%(n=5), cGVHD 7% (n=4) and death 13% (n=8). The 6m incidence of death was 45% (n=27); death due to GVHD 30%, infection 8% and relapse 7%. D28 responses were: 56% ORR (8% CR, 28% VGPR, 20% PR), 13% SD, 18% PD and 13% died before D28. Using Kaplan-Meier analysis, median OS was 7.3m with a median follow up of 1 year. The overall cumulative incidence of death was 63% (n=38); GVHD was the leading cause of death 40%, infection 8% and relapse 8%. D28 ORR was associated with a significantly improved median OS not reached versus (vs) 5.2m (p=0.03) in pts who had SD or PD at D28. In pts that achieved 6mFFTF, median survival beyond 6m was not yet reached vs median survival of 3.9m in those that did not achieve 6mFFTF (p=0.0004). The trend was for a longer median OS of 8.2m in the ECP arm vs 5.3m in non ECP (p=0.79). GVHD Gd at start of second line therapy was associated with a longer median survival, not reached in Gd II cohort vs 5.7m in Gd III-IV cohort (p=0.25). Discussion Our multi-centre prospective study illustrates 'real-life' data of clinical outcomes in pts starting second line therapy for SR aGVHD. Median survival from start of second line therapy was 7.3m with a high mortality rate of 63%. D28 ORR was 56% but only 32% met the composite 6mFFTF endpoint, although both outcomes predicted survival. The leading cause for failure of 6mFFTF was starting third line therapy which may be subject to clinician bias, however the 6m mortality was 45%, thus pts went on to die after failing for another cause. This data can be used as a contemporary data while planning studies of experimental intervention vs. best available therapy. Disclosures Radia: Mallinckrodt: Research Funding. Alfred:Mallinckrodt: Speakers Bureau. Dignan:Mallinckrodt: Research Funding, Speakers Bureau. Jagasia:Incyte Corporation: Membership on an entity's Board of Directors or advisory committees.

Abstract Abstract 897 Recent changes in allogeneic hematopoietic cell transplantation (HCT) including increased use of reduced-intensity conditioning, peripheral blood cells as the graft source (PB) and unrelated donors (URD) warrant re-evaluation of risk factors for acute graft-versus-host disease (aGVHD) and its impact on overall survival (OS). Risk factors for these outcomes were analyzed using more recent data from CIBMTR observational database. Methods: The cohort included adult patients ≥ 20 y transplanted from an HLA-identical sibling (SD) (n=3191) or URD (N=2370) for acute myelogenous leukemia (AML), acute lymphoid leukemia (ALL), chronic myeloid leukemia (CML), or myelodsyplastic syndrome (MDS) using a T-cell replete graft from 1999–2005. Six groups were created to evaluate the impact of conditioning [(myeloablative (MA) and reduced-intensity or non-myeloablative (RIC)], total-body irradiation (TBI) and graft source [(bone marrow (BM) or PB] as follows: MA+TBI+PB (group 1), MA+TBI+BM (group 2) MA+ no TBI+PB (group 3), MA+ no-TBI+BM (group 4), RIC+PB (group 5) and RIC+BM (group 6). Separate analyses were performed for SD and URD. IBMTR grade was used to classify aGVHD. A p-value of ≤ 0.01 was considered significant. Results: Among the SD cohort, the probability of aGVHD grade B-D and grade C-D at 100 days was 39% (95% CI, 37–41%) and 16% (95% CI, 14–17%). In multivariate analyses, non-TBI based MA regimens with a BM graft (group 4), RIC conditioning with PB (group 5) and tacrolimus plus methotrexate aGVHD prophylaxis were associated with lower odds ratio of grade B-D aGVHD (Table 1). The probability of OS was 51% (95% CI: 49–53%) at 3 y and 46% (95% CI: 44–49%) at 5 y. In multivariate analyses, grade B-D aGVHD was associated with a higher risk of death. Other independent risk factors for OS are shown in Table 2. Among the URD cohort, the probability of aGVHD grade B-D and C-D at 100 days was 59% (95%, CI 57–61%) and 32% (30-34%). In multivariate analysis, BM with MA (TBI and no TBI) or RIC conditioning (groups 2, 4 and 6) were significantly associated with lower odds ratio of grade B-D aGVHD (Table 1). Other independent risk factors included a diagnosis of CML. HCT from a 7/8 HLA-mismatched URD showed a trend for higher incidence of aGVHD (p=0.02). The probability of OS was 38% (95% CI: 35–40%) at 3 y and 33% (95% CI: 31–35%) at 5 y. In multivariate analysis, grade B-D aGVHD was associated with a higher mortality. Table 2 shows other independent risk factors for OS. Conclusion: Intensity of the conditioning regimen, TBI and the graft source has a combined effect on the risk of aGVHD. In both SD and URD cohorts, BM with MA, non-TBI regimens were associated with lower risk of aGVHD. In URD cohort, BM after a RIC also was associated with a reduced risk of aGVHD. Modulation of these risk factors is needed to reduce acute GVHD incidence and death after allogeneic transplantation. Disclosures: No relevant conflicts of interest to declare.

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 Background: Chronic GVHD contributes to morbidity and mortality after allogeneic transplantation. Several phase 2 and 3 open label studies suggested that ATLG (formerly referred to as ATG-F) reduces cGVHD without impacting relapse or survival. This report describes the first prospective randomized double blind phase 3 trial to assess the effect of ATLG (Neovii) when added to tacrolimus / methotrexate prophylaxis on cGVHD free survival. Patients and Methods: This study was conducted at 27 sites in the United States and Australia. 260 patients were randomized (132 placebo, 128 ATLG). 6 patients who were randomized never received ATLG/placebo (4 placebo and 2 ATLG). Data presented include the 128 placebo and 126 ATLG patients who underwent treatment and transplant. Patients were ages 18-65 years with a diagnosis of acute myeloid (AML, 64%) or lymphoblastic (ALL, 21%) leukemia in first or subsequent complete remission or myelodysplastic syndrome (MDS, 15%) with less than 10% marrow blasts. Median age was 48 years (18-65) with 55% males. All patients received 8/8 HLA (A, B, C, DR) allele matched unrelated products (80% mobilized peripheral blood, 20% bone marrow). Myeloablative conditioning was with cyclophosphamide and total body irradiation (Cy/TBI, 27%), busulfan and cyclophosphamide (Bu/Cy, 33%), or busulfan and fludarabine (Bu/Flu, 40%). All patients received tacrolimus starting on Day -1 and standard mtx on days 1, 3, 6, and 11 for GVHD prophylaxis. Patients received ATLG or placebo on Days -3, -2, -1 at an ATLG dose of 20 mg/kg per day (60 mg/kg total). The primary endpoint of the study was moderate/severe chronic GVHD free survival. cGVHD was scored by the investigator and then confirmed or overturned by an independent adjudication committee. Secondary endpoints included engraftment, acute GVHD , moderate/severe cGVHD, non-relapse mortality, GVHD and relapse free survival (GRFS), progression free (PFS) and overall survival (OS). The impact of ATLG on immune reconstitution was assessed in 91 patients. Results: Treatment arms were balanced with respect to age, diagnosis, remission status, cytogenetics, graft source (PB or BM), CMV serostatus and conditioning regimen. Median follow up of survivors was 745 days (61, 1425). Regarding the primary endpoint, there was no difference in 2 year moderate/severe cGVHD free survival between ATLG and placebo (48 vs 44%, p = 0.57), nor was there a difference in GRFS (Table 1). Neutrophil engraftment at Day 30 was less in the ATLG arm (85% vs 95%, p=0.00001). Grade 2 or higher infusion reactions were higher in the ATLG arm (16% vs 2.3%, p=0.0001). Reactivation of CMV was similar (27% vs 31%, p=0.58). The Day 180 cumulative incidence of grades 2-4 acute GVHD was lower in the ATLG arm (23 vs 40%, p = 0.003) as was the 2-year cumulative incidence of moderate/severe cGVHD (12 vs 33%, p = 0.000006). However, both 2-year OS and PFS were lower in ATLG treated patients (58 v 76% and 47 vs 67%, respectively) due in part to a higher incidence of relapse (32 vs 19%, p=0.068). Multivariable models confirmed that ATLG was associated with inferior OS, HR 1.85 (1.1-2.9, p=.0075) and PFS, HR 1.63 (1.1-2.41, p=0.015). We then conducted an unplanned post-hoc analysis and discovered a striking influence of conditioning regimen on outcomes. There were no differences in OS or PFS between ATLG and placebo arms in patients receiving Bu/Cy or Bu/Flu but a dramatic difference in patients receiving Cy/TBI conditioning (OS, 88% vs 48% p =0.006 and PFS, 75 vs 29%, p=0.007 in placebo and ATLG arms, respectively). 91 patients (44 ATLG, 47 placebo) participated in the immune reconstitution study. ATLG treatment was associated with lower CD3, CD4, and CD8 counts at Days 30, 100, and 360, respectively. In turn lower CD3, CD4, and CD8 counts assessed as time dependent variables were all associated with inferior OS and PFS as well as higher NRM (data not shown). Conclusion: In this first ever prospective, randomized, double blind trial of ATLG added to tacrolimus /methotrexate in recipients of matched unrelated donor grafts after myeloablative conditioning, there was no significant difference in 2 year moderate/severe cGVHD free survival or GRFS. ATLG did significantly reduce grades 2-4 acute and moderate/severe chronic GVHD. However, overall survival was higher in the placebo arm, driven mainly by patients who received Cy/TBI conditioning. Further analyses are needed to understand the proper role for ATLG in HCT. Disclosures Soiffer: Juno: Membership on an entity's Board of Directors or advisory committees; Kiadis: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Porter:Genentech: Employment; Novartis: Patents & Royalties, Research Funding. Jagasia:Therakos: Consultancy. Szer:Alnylam: Honoraria, Membership on an entity's Board of Directors or advisory committees; Ra Pharma: Honoraria, Membership on an entity's Board of Directors or advisory committees; Alexion Pharmaceuticals, Inc.: Honoraria, Membership on an entity's Board of Directors or advisory committees. Ritz:Kiadis: Membership on an entity's Board of Directors or advisory committees. Glavin:Neovii Biotech: Employment. Chen:Incyte Corporation: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding.

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.

Abstract Abstract 1940 Background: Chronic graft-versus-host disease (GVHD) is a significant source of morbidity, mortality, impaired patient-reported quality of life (QOL), greater symptom burden, and prolonged duration of immune suppressive therapy following allogeneic hematopoietic cell transplantation (HCT). While available data support the adverse prognosis of overlap subtype of chronic GVHD, the relative importance of site of gastrointestinal (GI) and type of hepatic involvement is not known. Methods: We analyzed prospectively acquired observational cohort data to examine whether the site of GI involvement (esophageal, upper GI, lower GI) and type of hepatic laboratory test abnormality (bilirubin, alkaline phosphatase (AP), alanine aminotransferase (ALT) elevation over the upper limit of normal based on study site-specific laboratory reference ranges) among chronic GVHD-affected patients are associated with overall survival (OS) and non-relapse mortality (NRM), symptoms (Lee Symptom Scale), and quality of life (QOL) per SF-36 and FACT-BMT instruments. Results: This analysis included 567 individual subjects with baseline and 1548 follow up visits. The majority were incident cases (59%), adults (98%), and predominantly White/Non-Hispanic. Median time from HCT to cohort enrollment was 11.9 months (range 3–294 months). Overall NIH chronic GVHD severity was mild in 10%, moderate in 52%, and severe in 38% at enrollment. Prior acute GVHD was noted in 66%, KPS < 80% in 17%, and platelet count at chronic GVHD onset of < 100 K/uL in 23%. At enrollment, GI involvement was seen in 40% (upper GI 20%, esophagus 16%, lower GI 13%), and liver involvement was seen in 52% (AP 38%, ALT 38%, bilirubin 10%). Time from transplant to cGVHD onset was not different for patients with or without GI involvement or hepatic involvement. In multivariate analysis utilizing data from enrollment visits only and adjusting for patient and transplant variables, lower GI involvement (HR 1.7, p=0.03) and elevated bilirubin (HR 2.36, p=0.001) were associated with OS; both were also associated with NRM. In multivariable analysis using all visits (time-dependent covariates), GI score greater than zero (HR 1.7, p=0.01) and elevated bilirubin (HR 3.7, p<0.001) were associated with OS; results were similar for NRM. Separate models were constructed to examine the relationship of severity of GI and hepatic involvement (0–3 severity score per NIH consensus) and mortality: Increasing lower GI severity at enrollment was significantly associated with non-relapse mortality, with progressively increasing HR for greater severity levels (lower GI score 2/3 vs. 0: HR 5.5, 95% CI 2.3–13.1, p = 0.0001). Increasing bilirubin was also associated with NRM (bilirubin score 2/3 vs. 0: HR 3.3, 95% CI 1.3–8.4, p=0.01). A similar association was observed for bilirubin elevation and NRM in the time-dependent model (bilirubin 2/3 vs. 0: HR 8.9, 95% CI 3.3–24, p < 0.0001). When analyzing all visits, any esophageal involvement and GI score greater than zero were associated with both symptoms and QOL while elevated bilirubin was associated with QOL. We found no consistent evidence that upper GI involvement, AP, ALT, or NIH liver score add prognostic value for survival, overall symptom burden, or quality of life. Conclusion: These data support important differences in patient-reported outcomes according to GI and hepatic involvement among chronic GVHD affected patients, and identify those with elevated bilirubin or higher GI score at any time, or lower GI involvement at cohort enrollment, as patients at greater risk for mortality under current treatment approaches. Disclosures: No relevant conflicts of interest to declare.

Key PointsHigh-circulating AREG (≥33 pg/mL) reclassifies additional patients into HR categories and further refines the Minnesota aGVHD risk score. Patients with aGVHD and AREG ≥33 pg/mL have lower rates of steroid response, higher NRM, and poorer OS.