Onset Of GVHD Research Articles

A Novel Clinical Risk Factor Based Scoring System to Predict the Severity of Acute Graft Versus Host Disease and Steroid Response in Resource Constrained Settings

INTRODUCTION Acute Graft-versus-host disease (aGVHD) is a common complication of allogeneic hematopoietic cell transplantation (HCT), affecting about 50% of transplants. Grading of aGVHD can serve a variety of purposes, including retrospective assessment of peak severity, real-time assessment of severity at prespecified time points, and determination of the need for treatment. But several problems hamper the application of grading systems to predict outcomes among patients with aGVHD: (1) Assignment of a peak GVHD score is done retrospectively; clinicians cannot use the current grading system for peak score in real-time1. (2) The systems do not account for the time to the response after treatment1. (3) Assignment of grade IV GVHD is often used to indicate that GVHD caused a death, irrespective of the severity. In this situation, the grading reflects the outcome and cannot be used to predict the outcome1. Recently serum biomarkers have emerged as an additional potential measurement of acute GVHD severity. The Mount Sinai Acute GVHD International Consortium (MAGIC) Group, has validated MAGIC algorithm probability (MAP) that combines two GI biomarkers (ST2 and REG3α) into a single value. The MAP predicts response to treatment, GVHD severity. But in resource-limited settings, like transplant centers in India lack testing features. In this study we have developed a risk scoring based on clinical and easily available biochemical parameters to predict the severity of aGVHD. AIMS AND OBJECTIVES To predict the aGVHD severity at the onset based on risk factor score. To assess the steroid response in different risk groups MATERIAL AND METHODS The study included patients who underwent allogeneic HCT at Narayana hrudayalaya hospital, between January 2015 and April 2020 and developed acute GVHD within 100 days of transplant. After taking institutional ethics committee approval, data were collected from medical records. Baseline patient characteristics are mentioned in table 1. The following parameters were analyzed as risk factors for the development of severe GVHD (MAGIC grade 3 and 4): 1. Age >18 yrs, 2. MDR organisms in baseline stool culture, 3. HCT comorbidity index >1, 4. Peripheral blood as a source of stem cells, 5. Female to male transplants, 6. Myeloablative regimens, 7. Suboptimal GVHD prophylaxis, 8. CD34 dose > 6 x 106/kg, 9. Grade 3/4 mucositis 10. Early-onset GVHD (within 28 days), 11. Albumin level at the onset of GVHD, 12. Albumin drop from baseline3, and 13. Bloodstream infection. Risk factors with a p-value of <0.05, were given score 2 and score 1 was given to other parameters. The total score ranged from 0 to 17. The study population was divided into 3 groups (Group 1 with score 0-4, Group 2 with score 5-7, Group 3 with score > 8). The following outcomes were assessed in each group; severity of GVHD (Grade I/II vs Grade III/IV) and response to steroids. RESULTS Out of 148 patients, 35.5% of Group 1, 56.5% of Group 2 patients and 85.5% of Group 3 patients developed Grade 3 or 4 GVHD respectively (P-value <0.001). Positive predictive value of score > 8 to predict Grade 3/4 GVHD is 85.4%, negative predictive value is 50.6%, sensitivity is 50.5%, and specificity is 85.5%. From Group 1 and 2, only 30% of patients were steroid non responders, while 55.3% of Group 3 patients are steroid non responders (P-value - 0.04). CONCLUSION Traditional GVHD scoring systems reflect the outcome and cannot be used to predict the outcome. Various biomarker-based scoring systems are helpful in this situation, but in resource-limited settings, it might not be easily feasible. Clinical scoring systems like risk factor-based scoring systems are very helpful, which can predict the severe GVHD at early time points leading to management decisions such as upfront initiation of aggressive treatments and earlier introduction of second-line agents. References Leisenring WM, et al. An acute graft-versus-host disease activity index to predict survival after hematopoietic cell transplantation with myeloablative conditioning regimens. Blood. 2006;108(2):749-55. 2.Rashidi A, et al. Peritransplant Serum Albumin Decline Predicts Subsequent Severe Acute Graft-versus-Host Disease after Mucotoxic Myeloablative Conditioning. Biol Blood Marrow Transplant. 2016;22(6):1137-41. Disclosures No relevant conflicts of interest to declare.

Early Reconstitution of CD6+ T Cells after Hematopoietic Cell Transplantation Identifies a Suitable Target for Acute Graft Versus Host Disease Treatment Using Anti-CD6 Monoclonal Antibody Itolizumab

Early Reconstitution of CD6+ T Cells after Hematopoietic Cell Transplantation Identifies a Suitable Target for Acute Graft Versus Host Disease Treatment Using Anti-CD6 Monoclonal Antibody Itolizumab

Real World Experience of Etanercept in Steroid Refractory Acute Graft Versus Host Disease from a Tertiary Cancer Centre in India

Introduction Acute graft versus host disease (aGVHD) is an important complication of allogeneic hematopoietic stem cell transplant (AHSCT). Steroid refractory (SR) aGVHD is seen in about 40- 50% of patients with no recommended standard of care. We report our experience with etanercept (ETA) as the initial agent in SR aGVHD. Materials and Methods We retrospectively analysed 43 AHSCT patients from 2010 to 2019 who received ETA as initial agent for SR aGVHD amongst 311 patients who underwent AHSCT in the same period. Patients received myeloablative (MAC) or reduced intensity conditioning (RIC). The transplants were from matched sibling donor (MSD), matched unrelated donor (MUD) and haploidentical donor (Haplo). GVHD prophylaxis was calcineurin inhibitor (CNI) plus methotrexate (MTX)/ mycophenolate mofetil (MMF) in MSD/MUD. Rabbit ATG (2.5-5 mg/kg) was used for MUD. Haplo transplant patients received post-transplant cyclophosphamide (PTCy) with CNI and MMF. aGVHD was graded as per the modified Glucksberg criteria. Additionally, all aGVHD episodes were also classified as per the Minnesota Scoring into High Risk (HR) and Standard Risk (sr) GVHD. First line treatment of aGVHD Grade II-IV was systemic steroids with methylprednisolone 1-2 mg/kg/day. SR aGVHD was defined as per standard criteria. ETA was started as 25 mg (0.4 mg/kg in children) subcutaneously every 72 hours initially and was tapered gradually depending on response. Response of SR aGVHD to ETA was defined as follows:Complete response (CR) was the complete resolution of aGVHD manifestations in all organs. Partial response (PR) was improvement in GVHD stage in at least 1 of the initially involved organs without CR and worsening in any other organs. Very good partial response (VGPR) was improvement in GVHD in all initial organs, with maximum stage I involvement in 1 or more organs (except upper gut). No response (NR)/Progression/Failure of therapy was defined as same grade or progression of GVHD in any organ or the addition of further GVHD therapy beyond ETA. Death prior to 1st response assessment was taken as NR. Response assessment was done at 3 time points - Day 14, Day 28 and Day 56 after initiation of ETA. Reduction of initial systemic steroid dose by ≥30% from baseline on day 28 after start of ETA was analysed as a subgroup to assess outcomes of patients. Results The characteristics of all 43 patients are shown in Table 1. ETA was required in a total of 45 episodes in 43 patients. The overall grade of GVHD and organs involved at the start of first line treatment are as in Table 2. The median day of onset of acute GVHD was 58 days (range 8-738 days). The median day of start of ETA was 86 days (range 16-74 days). The median number of days steroid was given prior to start of ETA was 7 days. The median number of ETA doses given were 11 (range 2-25). Response assessment on the predetermined time points are as in Table 2. The response rates (CR+VGPR+PR) at Day 14, Day 28 and Day 56 were 62%, 69% and 64% respectively. The organ specific response rates (CR+VGPR+PR) were 70% for lower gut, 67% for skin and 50% for liver. A third line agent for aGVHD was added in 10 patients, which included ruxolitinib, cyclosporine, mycophenolate mofetil, basiliximab or low dose weekly cyclophosphamide. The median OS of the whole cohort of SR aGVHD was 5.2 months. The median OS of the responders (CR+PR+VGPR) at Day 28 from ETA start was 6.8 months versus 2.8 months (p=0.0003) in non-responders (Fig 1). The median OS of sr group of Minnesota Grading was 24 months versus 10 months in HR group (p=0.085) (Fig 2). Patients, in whom reduction in the systemic steroid dose ≥ 30% at Day 28 from start of ETA was possible, had a significantly better median OS (9.5 months versus 3.5 months; p=0.000021) (Fig 3). In multivariate analysis, steroid dose reduction on day 28 of start of ETA was the most significant factor (p=0.008; hazard ratio 3.33; CI 1.3-8.1) CMV reactivation was seen in 64% of the patients. Fungal infections in 51% and bacterial infections in 78% patients after start of ETA. The causes of death were infection in 25 (58%) patients, refractory GVHD in 8 (18%), combination of GVHD and infection in 2 (5%) and relapse in 4 (9%) patients. Conclusion ETA used as initial agent has shown acceptable response rates for SR aGVHD in our cohort, especially for lower gut and skin. However, infections were the major cause for mortality for this cohort and early tapering of systemic steroids may be the key to improving outcomes. Disclosures No relevant conflicts of interest to declare.

Cellular and molecular profiling of T-cell subsets at the onset of human acute GVHD

AbstractThe cellular and molecular processes involved in acute graft-versus-host disease (aGVHD) development early after allogeneic hematopoietic cell transplantation (HCT) in humans remain largely unknown. We have performed multiparameter immunophenotyping and molecular profiling of CD4+and CD8+T cells in 2 independent cohorts of patients undergoing HCT, as well as in their HLA-identical sibling donors. Cellular profiling using spectral flow cytometry showed an incomplete reconstitution of the T-cell compartment in recipients without aGVHD early after transplantation, as well as a shift toward an effector memory phenotype, paralleled by depletion of the naive T-cell pool. Molecular profiling of T-cell populations in donors vs recipients without aGVHD revealed increased pathway activity of >40 gene modules in recipients. These pathways were associated in particular with T-cell activation, adhesion, migration, and effector functions. Cellular profiles from recipients developing aGVHD displayed an enrichment of cells with a T memory stem cell–like phenotype compared with recipients without aGVHD. Comparison of gene profiles from these recipients revealed that transforming growth factor-β (TGF-β) signaling was most significantly downregulated, whereas the pathway activity of NF-κB–associated transcription factors and signaling pathways were increased, at aGVHD onset. This study suggests that the integration of cellular and molecular profiles provides new insights into the development of aGVHD in humans.

Abstract 4512: Mouse model for in vivo evaluation of efficacy and potential cytokine release syndrome of chimeric antigen receptor (CAR) T cell therapy

Abstract Chimeric antigen receptor (CAR) T-cell therapy holds great promise as cancer immunotherapy. While impressive responses have been achieved in patients with hematological malignancies there are challenges for CAR T therapy such as toxicity complications due to cytokine release syndrome (CRS) and limitations due to CAR T cell exhaustion. As increasing number of novel CAR T products are in the discovery and preclinical phase of development, preclinical models to assess efficacy as well as potential toxicity of CAR T in vivo are needed. Here, we describe a new NSG™ mice model to examine CAR T cells for efficacy and potential CRS. Immunodeficient NSG™ mice are commonly used to evaluate CAR T functions. However, human T cells can cause acute xeno-GVHD in NSG™ limiting the use of the model system. A system to study human T cell function in the absence of xeno-GVHD is required. A new NSG™ derivative NSG-MHC Class I/II knockout (KO) strain has MHC class I and MHC class II deficiency in addition to B, T and NK cell deficiency. NSG-MHC Class I/II KO mice are known to have a delayed onset of GVHD and can provide a useful background to study human immunity in the absence of GVHD [M. A. Brehm et al., FASEB J. 33, 3137–3151 (2019)]. Human B-cell lymphoma Raji tumor cells express high levels of CD19 and CD22 which are targets for CAR T therapy. Using NSG-MHC Class I/II KO mice and luciferase-tagged Raji-Luc tumor model we found that compared to controls CD22 CAR T and CD19 CAR T showed significant tumor growth inhibition by Xenogen imaging analysis. No body weight loss was observed with the CAR T treatments. As high tumor burdens are usually associated with the potential of causing CRS we examined CD19 CAR T cells on low and high tumor burdens and the data will be presented in context of efficacy. We have established a new mouse model using NSG-MHCI/II KO mice to enable evaluation of CAR T efficacy and propensity to induce cytokines simultaneously in vivo. Furthermore PBMC-humanized NSG-MHCI/II KO mice can also be established and used to examine CAR T cells prepared from the same PBMC donor to mimic autologous CAR T cell therapy. Citation Format: Jing Jiao, Chunting Ye, Danying Cai, Mingshan Cheng, James Keck. Mouse model for in vivo evaluation of efficacy and potential cytokine release syndrome of chimeric antigen receptor (CAR) T cell therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4512.

Sustained Immunogenic and Clinical Effects of Low-Dose Interleukin-2 Therapy with an Intermittent Maintenance Method for Refractory Chronic Graft-Versus-Host Disease: Results of Phase1/2a LDIL2-01 Study

Introduction and Objectives Regulatory T cells (Treg) play a central role in the maintenance of immune tolerance. We previously reported that the altered Treg homeostasis in the prolonged lymphopenia after HSCT is one of the major pathogenesis of impaired Treg recovery leading to the onset of chronic GVHD and low-dose IL-2 can restore normal Treg homeostasis by a PD-1-dependent mechanism, resulting in the improvement of clinical GVHD symptoms. In these years, the tolerogenic effects of IL-2 have been tested for various autoimmune diseases by clinical trials. However, the schedules of IL-2 dosing in each trial are different and the optimal strategy to expand Treg is still unclear. Our murine study has shown that daily administration of IL-2 seems to be optimal for the initial expansion of Treg but less frequent dosing within the threshold could be preferable for the following maintenance of expanded Treg. Methods Based on the findings, we conducted a phase1/2a clinical trial of low-dose IL-2 for steroid-refractory chronic GVHD with a distinctive dosing schedule consisting of an initial induction phase (IL-2 dosing everyday for the first 4 weeks) and a subsequent maintenance phase (IL-2 dosing 3 times a week for the following 8 weeks). Patients who obtained clinical benefit by the 3 months could proceed to the extended therapy up to total 12 months. The appropriate single dose of IL-2 was examined by a three-step dose escalation design. Results A total of 12 patients were enrolled. None had progression of chronic GVHD or recurrent hematologic malignancy. Dose-limiting toxicity did not occur even at the maximum dose. All patients were evaluable for response, and 7 had clinically significant responses involving multiple sites. Response was observed most often in the joints (67%). One patient with oral GVHD and another patient with gastrointestinal GVHD achieved complete response. One patient with severe lung GVHD markedly improved pulmonary functional test (%FEV1; from 24.8% to 64.8 %) and activity of daily life (from bedridden to return to work). Treg were preferentially increased in all patients, with a peak median value almost 10 times the baseline value (P Conclusion These results indicate that the sustained efficacy of IL-2 against chronic GVHD does not necessarily require that Treg is maintained at a high level by continuing daily IL-2 administration over the long term. Our data provide important information for reconsidering IL-2 dosing methods from the viewpoint of long-term safety and possible combination with other treatments.

Long Term Outcomes of Patients with Aggressive T-Cell Non-Hodgkin Lymphoma Undergoing Allogeneic Stem Cell Transplantation: Retrospective Results from Single Center.

<h3>Introduction</h3> Peripheral T-cell lymphomas (PTCL) comprise 15-20% of adult non-Hodgkin lymphoma and have a poor prognosis; 5-year survival is less than 30% for the most aggressive subtypes. Allogeneic HCT (allo-HCT) is offered to eligible patients as a potentially curative modality in the salvage setting or in high risk patients to consolidate an initial response to frontline therapy. <h3>Objective</h3> To report clinical outcomes derived from large sample size and long-term follow up data. <h3>Methods</h3> We retrospectively reviewed medical records of 87 consecutive patients with PTCL who underwent allo-HCT at City of Hope from January 2000 to June 2018. Baseline patient demographic, treatment, and disease characteristics were summarized by descriptive statistics. Overall survival (OS) and progression-free survival (PFS) were evaluated by Kaplan-Meier curves and the log-rank test. Cumulative incidences of time to relapse and time to non-relapse mortality (NRM) were calculated with relapse and NRM as competing risks. Cumulative incidences of acute and chronic GVHD were calculated as time to onset of GVHD with relapse and death as competing events for GVHD. <h3>Results</h3> Median age at allo-HCT was 49 (range 2-70) years. Histologies were PTCL-NOS (n=21); transformed CTCL (n=19); NK TCL (n=17); AITL (n=15), ALCL (n=7); γδTCL (n=6) and other rare subtypes (n=2). No patients had a prior auto transplant. 42 patients (48%) had myeloablative conditioning; FTBI-based (n=39), or BEAM regimen (n=3). 45 patients (52%) had reduced intensity conditioning with a fludarabine/melphalan based regimen in 39. Sibling HCT was done in 47 (54%) patients. MUD HCT was done in 36 (41%); donors were fully HLA matched for 15 (17%) patients and mismatched in 21 (24%); 4 (5%) got haploidentical HCT. The most common GVHD prophylaxis was tacrolimus/sirolimus (n=54). Stem cell source was PBSC in 77 (88%), bone marrow in 5 (6%), and cord blood in 5 (6%) patients. At allo-HCT 25 (29%) patients were in complete remission, 25 (29%) in partial remission, 22 (25%) with induction failure and 14 (16%) with relapsed disease. The median follow-up among survivors was 6.9 years (range 1.1-15.5). The 5- and 10-year PFS was 47% (95% CI: 36%-58%) and 38% (95% CI: 26%-50%), respectively. The 5- and 10-year OS was 53% (95% CI: 41%-63%) and 42% (95% CI: 29%-54%), respectively (Fig.1). At day 100 after allo-HCT, the rate of acute GVHD grade II-IV was 41% (95% CI: 30%-51). Chronic GVHD rates at 3 years were 62% (95% CI: 51%-72%), with extensive GVHD of 55% (95% CI: 44%-65%). On univariate analysis, age (> 60 or not), sex, TBI-based conditioning, donor type, stem cell source or remission status prior to allo-HCT did not predict for OS. <h3>Conclusions</h3> This large single-institution series with a long-term follow-up on allo-HCT outcomes in patients with high-risk, aggressive T-cell NHL shows encouraging survival outcomes for these patients with limited treatment options.

Enteral Nutrition in Pediatric Patients Undergoing Hematopoietic SCT Promotes the Recovery of Gut Microbiome Homeostasis.

Hematopoietic stem cell transplantation (HSCT) is the first-line immunotherapy to treat several hematologic disorders, although it can be associated with many complications reducing the survival rate, such as acute graft-versus-host disease (aGvHD) and infections. Given the fundamental role of the gut microbiome (GM) for host health, it is not surprising that a suboptimal path of GM recovery following HSCT may compromise immune homeostasis and/or increase the risk of opportunistic infections, with an ultimate impact in terms of aGvHD onset. Traditionally, the first nutritional approach in post-HSCT patients is parenteral nutrition (PN), which is associated with several clinical adverse effects, supporting enteral nutrition (EN) as a preferential alternative. The aim of the study was to evaluate the impact of EN vs. PN on the trajectory of compositional and functional GM recovery in pediatric patients undergoing HSCT. The GM structure and short-chain fatty acid (SCFA) production profiles were analyzed longitudinally in twenty pediatric patients receiving HSCT—of which, ten were fed post-transplant with EN and ten with total PN. According to our findings, we observed the prompt recovery of a structural and functional eubiotic GM layout post-HSCT only in EN subjects, thus possibly reducing the risk of systemic infections and GvHD onset.

Dll1 and Dll4 Notch Ligands Prime T Cell Alloimmunity and Are Expressed in Non-Overlapping Populations of Fibroblastic Stromal Cells in Spleen and Lymph Nodes at the Onset of Gvhd

Notch signaling drives graft-versus-host-disease (GVHD) pathogenesis in preclinical models of allogeneic hematopoietic cell transplantation (allo-HCT). Short-term systemic blockade of the Notch ligands Delta-like1 (Dll1) and Delta-like4 (Dll4) within two days of allo-HCT conferred long-term protection from acute GVHD lethality in mice, with a dominant impact of Dll4. Surprisingly, critical Notch ligands in GVHD were provided by radioresistant non-hematopoietic fibroblastic stromal cells lineage traced by a Ccl19-Cre transgene in secondary lymphoid organs (SLO) (Chung, JCI 2017). Our discovery revises prevailing GVHD models and identifies a pathogenic role for a stromal cell niche expressing Notch ligands in SLOs. However, little is known about the distribution and regulation of Dll1/Dll4-expressing cells among SLO stromal cells. To address this question, we deployed genetic and biochemical tools to map expression of Notch ligands in SLO stromal cells, characterize the transcriptional landscape of these cells, and identify putative regulators of Notch ligand expression. To map Dll1 and Dll4 expression, we combined a Ccl19-Cre transgene with a ROSA26-YFP Cre-activated allele and Dll1-mCherry or Dll4-mCherry BAC reporters. Using this model, we fluorescently traced all cells derived from Ccl19-Cre+ cells which contain the essential Dll1/Dll4 source in GVHD, while detecting Dll1 vs. Dll4 expression with high sensitivity. Within the lymph node (LN) Ccl19-Cre+ROSA26-YFP+ compartment, Dll4-mCherry was present predominantly among CD157hi CD45-Ter119-CD31-gp38+ fibroblastic stromal cells, including fibroblastic reticular cells (FRCs), marginal reticular cells (MRCs) and follicular dendritic cells (FDCs). In the spleen, Dll4-mCherry was detected among both CD157+ cells (predominant in the T-zone) and CD157- cells. In contrast, Dll1-mCherry was abundant among spleen CD157+ and CD157- cells, but not detected in the corresponding LN compartments. Thus, Dll1 and Dll4 are expressed with a non-overlapping distribution pattern in different SLOs and stromal cell subsets. Next, we studied expression of the dominant Dll4 ligand after MHC-mismatched allo-HCT (BALB/c into C57BL/6). Although Dll4-mCherry fluorescence did not increase, we detected a rise in surface Dll4 protein, peaking 12 hrs after allo-HCT in LN Ccl19-Cre+ CD157hi FRCs, MRCs and FDCs. Increased Dll4 abundance coincided with the critical timing of pathogenic Notch signals within days after allo-HCT and required irradiation conditioning as well as infusion of allogeneic T cells, suggesting a crosstalk between alloreactive T cells and Dll4+ stromal cells. The mechanisms underlying increased surface Dll4 abundance are under investigation, but could involve post-transcriptional effects as the Dll4-mCherry transcriptional reporter did not change. We next characterized transcriptomic features of Dll4+ fibroblastic stromal cells. We performed RNA-Seq on rare SLO stromal cells sort-purified from LNs at baseline and 12 hrs after allo-HCT. We compared CD157hi FRCs that do or do not express Dll4-mCherry, while including CD157lo FRCs that do not express Dll4-mCherry and lymphatic endothelial cells that express Dll4 at high levels as controls. These populations were sort-purified for RNA-Seq analysis, using the data to produce a comprehensive picture of gene expression and regulatory machinery in the SLO niche in alloimmunity, as well as identify new putative regulators of Notch ligand expression. Interestingly, we found only 13 differentially expressed genes between Ccl19-Cre-expressing CD157hi fibroblastic stromal cells that do and do not express the Dll4-mCherry reporter 12 hrs after transplant (log-fold change ≥ 1, corrected p ≤ 0.01). The list of up-regulated transcripts included Cxcl13, encoding a chemokine produced by FDCs, and Rankl, encoding a TNF-family member important in LN development and homeostasis. In summary, our data uncover key new features of the specialized fibroblastic stromal cells expressing Delta-like Notch ligands in SLOs. Ongoing work is testing how qualitative and quantitative differences in Dll1 and Dll4's effects during GVHD may relate to their distribution, regulated expression, or biochemical properties as they interact with Notch receptors in T cells. Our ultimate goal is to design strategies that target Notch ligand expression in SLOs as new prophylactic approaches in GVHD. Disclosures Blazar: Magenta Therapeutics and BlueRock Therapeuetics: Membership on an entity's Board of Directors or advisory committees; Fate Therapeutics, Inc.: Research Funding; Tmunity: Other: Co-Founder; KidsFirst Fund: Research Funding; Childrens' Cancer Research Fund: Research Funding; Leukemia and Lymphoma Society: Research Funding; Abbvie Inc: Research Funding; Alpine Immune Sciences, Inc.: Research Funding; RXi Pharmaceuticals: Research Funding; Regeneron Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Five Prime Therapeutics Inc: Co-Founder, Membership on an entity's Board of Directors or advisory committees; Kamon Pharmaceuticals, Inc: Membership on an entity's Board of Directors or advisory committees; BlueRock Therapeutics: Membership on an entity's Board of Directors or advisory committees. Maillard:Genentech: Consultancy; Regeneron: Consultancy.

Single Cell Analysis of Circulating Endothelial Cells in Allogeneic Hematopoietic Stem Cell Transplant; To Whom Do They Belong: Host or Donor?

Background. We recently reported that Circulating Endothelial Cell (CEC) count changes represent a promising marker to monitor endothelial damage in patients undergoing allogeneic hematopoietic stem cell transplant (allo-HSCT), potentially becoming a valuable tool in the diagnostic definition of GVHD. Besides confirming an increase of CEC counts at GVHD onset, we repeatedly documented at time of engraftment statistically significant higher numbers of CEC in patients who will not manifest GVHD in comparison to patients in which GVHD will be diagnosed (Transplantation 2014,98:706-12; Bone Marrow Transplantation 2017,52:1637-42; Scientific Reports 2019,9:1-12). Recent knowledges in organ transplant pointed out that endothelial cells from the grafted organ, besides being a continuous source of alloantigens, can downregulate alloreactivity exerting tolerogenic responses. By inference to the allo-HSCT field, it could be envisaged that presence of donor CEC could induce protective effects on alloreactivity. Methods. We planned a study to test the hypothesis that at time of engraftment, CEC present in peripheral blood (PB), besides coming from cells shedding from patient vasculature, could partly belong to donor, originating from the cellular graft. Therefore, in an exploratory set, we performed FISH analysis on flowcytometry-sorted CEC (CD45neg/CD34bright/CD146pos, Lyotube #623920, BD Biosciences) (n=3) and on whole PB derived culture-expanded CEC (n=3) (EGM-2 BulletKit, Lonza), obtained at engraftment in sex-mismatched allo-HSCT. In the confirmatory set (n=15), single CEC were recovered from PB, at engraftment (T1) and at 90 days (T2) after allo-HSCT, through the DEPArrayTM technology (Menarini Silicon Biosystems), after preliminary bulk separation step carried out with the CellSearch® System. Single recovered CEC was whole genome amplified (Ampli1™ WGA Kit) and short tandem repeat (STR) profile determined (Ampli 1TM STR kit) on each single CEC. To confirm host/donor origin, single CEC STR profile was compared to that determined on patient and donor cells before allo-HSCT. Moreover, donor CEC presence was evaluated by CISH analysis on formaline fixed and paraffin-embedded biopsy sections obtained at least three months after sex mismatched allo-HSCT. Results. By positive findings of the exploratory set, we proved, at the single cell level in the confirmatory set, the presence of donor CEC at engraftment (T1) in 4 out of 15 patients (Table 1). Of them, 2 did not manifested GVHD, despite a GVHD risk score of 2, and the other 2 presented GVHD grade I. On the contrary, among the 10 patients in whom no donor CEC were detected, 6 experienced GVHD grade II-III, while 4 did not manifested GVHD, despite a 1-3 GVHD risk score. Conclusions. Our data represent the proof of principle that donor CEC may flow in host PB early on from hematopoietic recovery and seldom persist thereafter at steady-state conditions, being potentially embedded in host vascular wall. These puzzling findings suggest that neovascularization takes place in parallel with hematopoietic engraftment and could provide further clues on shedding light on tissue tolerance in the context of GVHD, opening up paradoxical scenarios on the protective role potentially played by donor CEC. Disclosures Fontana: Menarini Silicon Biosystem: Employment. Rotta:BD Biosciences Italia: Employment. Manaresi:Menarini SIlicon Biosystem: Employment, Membership on an entity's Board of Directors or advisory committees.

Improved Survival of Patients Diagnosed with Severe (Grade 3-4) Acute GVHD or Severe NIH Grade Chronic GVHD in the Current Era Compared to Historic Controls

Severe (grade 3-4) acute GVHD (aGVHD) and NIH grade severe chronic GVHD (cGVHD) have historically been associated with poor survival. However, earlier diagnosis, improved treatment and supportive care, may result in an improvement in outcomes for patients recently diagnosed with severe GVHD compared to prior cohorts. Our institution has prospectively documented onset, grading and therapy of patients with GVHD using a single dedicated practitioner since 2005. We assessed 851 consecutive patients who received allografts at our center between 1/2005 and 12/ 2016 and identified 130 patients (15.3%) who developed severe aGVHD through Dec 2017 without prior relapse of malignancy. Characteristics were: median age 52.5, 51% male, race-81% white -17% black, diagnosis- AML 31% - MDS/MPD 20%- NHL/HL/CLL-23%, -ALL 11%, donor - MRD 21% - MUD 49% - haplo 30%, PBSC 78%, BM 17%, CBU 5%, preparative regimen MAC 44% - RIC/NST 56%, median time from BMT to onset of severe aGVHD was 49 days (7-376). Maximum grade = gd 3 (85%), gd 4 (15%), Median follow-up for survivors from date of onset was 59 months (23-155m). Survival estimates for the entire cohort from date of onset of grade 3-4 aGVHD at 1, 2 and 3 years were 62%, 49% and 47% respectively. Patients who developed severe aGVHD in 2016-2017 had significantly improved overall survival compared to patients who developed severe acute GVHD in prior years (2005-2015) -1 and 2 yr survival 86% &amp; 79% vs 55% &amp; 41% respectively, p=0.002 log-rank test, Fig 1). No significant differences were found between earlier cohorts. On multivariable analysis assessing the following variables: age, gender, race, diagnosis, donor type, cell source, regimen intensity, DRI, HCT-CI, CMV status, grade of aGVHD (3 vs 4), days from transplant to gd 3-4 aGVHD onset, year of development of severe acute GVHD (2016-2017 vs earlier) remained significant for survival (HR 0.36, p=0.018). Other significant variables were grade 4 vs 3 GVHD (HR 3.78, p&lt;0.001) and DRI (high/very high vs low/intermediate, HR 1.84, p=0.011). For cGVHD we assessed 522 consecutive patients who underwent allografts between 4/2011 (start date of prospective documentation of NIH grade cGVHD )and 12/2016, and identified 146 (28%) patients who developed moderate to severe NIH grade chronic GVHD without prior relapse by Dec 2017 (85 severe, 61 moderate). Patient characteristics were: median age 53, 55% male, race-74% white -22% black, AML 40% - MDS/MPD 31%- NHL/HL/CLL-15%, -ALL 12% , donor - MRD 34% - MUD 40% - haplo 25%, graft- PBSC 82%, BM 18%, preparative regimen MAC 54% - RIC/NST 44%, median time from BMT to onset of moderate/severe cGVHD was 289 days (27-1364). Median follow-up for survivors from date of onset of moderate to severe GVHD was 48 months (19-94m). Estimated rates of survival from date of onset of moderate to severe cGVHD for the entire cohort at 1, 2 and 3 years were 82%, 73% and 71% respectively. For patients who developed severe cGVHD the corresponding survival estimates were 77%, 67% and 63%. No significant difference in post-onset survival was encountered when comparing patients developing moderate cGVHD in 2011-2013, 2014-2015 and 2016-2017. However, for severe cGVHD, 1 and 2 year estimated survival rates for the three cohorts were : 53% &amp; 41%, 79% &amp; 73% and 87% and 74% respectively (p=0.004 for 2011-2013 vs 2014-2015 but p=NS for 2014-2015 vs 2016-2017, Fig 2). On a multivariable analysis considering age, gender, race, diagnosis, donor type, graft source, regimen intensity, DRI, HCT-CI, and days from transplant to cGVHD onset, none of these variables was significantly associated with post-cGVHD survival. In the Cox model including year of onset of severe cGVHD, onset in recent years was linked to lower risk of mortality compared to onset in earlier years (onset 2014-2015 vs 2011-2013 - HR=0.34, p-0.012; onset 2016-2017 vs 2011-2013 - HR=0.28, p=0.006). These data suggest that survival of patients developing either severe aGVHD or severe NIH grade cGVHD has significantly improved in recent years compared to historical controls. More than two-thirds of such patients now survive two years from onset of severe GVHD. This must be taken into account when evaluating novel therapies for severe GVHD. Disclosures Solh: ADC Therapeutics: Research Funding; Celgene: Speakers Bureau; Amgen: Speakers Bureau.

Long Term Outcomes of Patients with Aggressive T-Cell Non-Hodgkin Lymphoma Undergoing Allogeneic Stem Cell Transplantation: Retrospective Results from a Single Center

Background: Mature T cell and NK cell neoplasms collectively known as peripheral T-cell lymphomas (PTCL) comprise 15-20% of Non-Hodgkin lymphomas in adults and have a poor prognosis with a 5-year survival of less than 30% for the most aggressive subtypes. Allogeneic HCT (allo-HCT) is offered to eligible patients as a potentially curative modality in the salvage setting or in high risk patients to consolidate an initial response to frontline therapy. There are few studies that report clinical outcomes derived from large sample size and long-term follow up data. Methods: We retrospectively reviewed medical records of 87 consecutive patients with PTCL including transformed mycosis fungoides and NK/T-cell lymphoma without prior autologous transplant who underwent allo-HCT at City of Hope from January 2000 to June 2018 after IRB approval was obtained. Descriptive statistics were used to summarize baseline patient demographic, treatment, and disease characteristics. Kaplan-Meier curves and the log-rank test were used to evaluate the overall survival (OS) and progression-free survival (PFS). Cumulative incidences of time to relapse and time to non-relapse mortality (NRM) were calculated with relapse and NRM as competing risks. Cumulative incidences of acute and chronic GVHD were calculated as time to onset of GVHD with relapse and death as competing events for GVHD. Results: 87 patients were included for the analysis. Median age at the time of allo-HCT was 49 years (range 2-70 years). Histologies were PTCL-NOS (n=21); transformed CTCL (n=19); NK T-cell lymphoma (n=17); AITL (n=15), ALCL (n=7); gamma delta T-cell lymphoma (n=6) and other rare subtypes (n=2). None of the patients had a prior auto transplant. 42 patients (48%) received myeloablative conditioning, with the majority of patients receiving FTBI based conditioning (n=39) and three patients received BEAM regimen for conditioning. 45 patients (52%) received reduced intensity conditioning; fludarabine/melphalan based-conditioning was the most common regimen used (n=39). Sibling HCT was performed in 47 patients (54%), while MUD HCT was performed in 36 patients (41%) with fully matched HLA unrelated donor in 15 (17%) and HLA mismatched in 21 (24%) patients; 4 (5%) received haploidentical HCT. GVHD prophylaxis consisted of tacrolimus/sirolimus (n=54), tacrolimus/sirolimus/MTX (n=11), tacrolimus or cyclosporine/MTX (n=7), tacrolimus or cyclosporine/MMF (n=7), post-transplant cyclophosphamide/tacrolimus/MMF (n=5) and other (n=3). Source of stem cells was PBSC in 77 (88%), bone marrow in 5 (6%), and cord blood in 5 (6%) patients. At the time of allo-HCT, there were a total of 25 (29%) patients in complete remission (CR1 n=15, CR2+ n=10), 25 (29%) patients in partial remission 22 (25%) with induction failure and 14 (16%) with relapsed disease. The median follow-up among survivors was 6.9 years (range 1.1-15.5). The 5- and 10-year PFS was 47% (95% CI: 36%-58%) and 38% (95% CI: 26%-50%), respectively. The 5- and 10-year OS was 53% (95% CI: 41%-63%) and 42% (95% CI: 29%-54%), respectively (Fig.1). Relapses at 5 and 10 years were both 24% (95% CI: 16%-34%), while NRM at 5 and 10 years was 28% (95% CI: 19%-39%) and 37% (95% CI: 25%-50%), respectively. At day 100 after allo-HCT, the rates of acute GVHD grade II-IV were 41% (95% CI: 30%-51%) and grade III-IV of 16% (95%CI: 9%-25%). Chronic GVHD rates at 3 years were 62% (95% CI: 51%-72%), with extensive GVHD of 55% (95% CI: 44%-65%). On univariate analysis, age (&gt; 60 or not), sex, TBI-based conditioning, donor type (MSD vs MUD), stem cell source or remission status prior to allo-HCT did not predict for overall survival in our study. Conclusions: Our results constitute the largest reported single-institution series with a long-term follow-up on allo-HCT outcomes in patients with aggressive T-cell NHL. The 5-year PFS and OS of 47 and 53%, respectively, are encouraging for the high-risk T-cell NHL patients with limited treatment options. Disclosures Salhotra: Celgene: Other: Research Support; Kadmon Corporation: Other: Non paid consultant. Popplewell:City of Hope: Employment. Herrera:Adaptive Biotechnologies: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Gilead Sciences: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; AstraZeneca: Research Funding; Merck: Consultancy, Research Funding; Genentech, Inc.: Consultancy, Research Funding; Pharmacyclics: Research Funding; Immune Design: Research Funding; Kite Pharma: Consultancy, Research Funding. Mei:Seattle Genetics, Inc.: Research Funding. Zain:spectrum: Honoraria; Seattle Genetics: Honoraria, Speakers Bureau.

Outcomes of Patients with T-Lymphoblastic Lymphoma Undergoing Allogeneic Stem Cell Transplantation: Retrospective Results from a Single Center

Background: T-lymphoblastic lymphoma (T-LBL) is a highly aggressive non-Hodgkin lymphoma seen in young adults that usually presents with a mediastinal mass and less than 20% bone marrow involvement with neoplastic cells. T-LBL and T-cell ALL are morphologically and immmunophenoytpically similar and are thus frequently grouped together. However there are differences in clinical presentation, cytogenetic features and molecular pathogenesis indicating biologic differences. Patients with relapsed refractory T-LBL have poor prognosis and there is a paucity of data on allogeneic HCT (allo-HCT) outcomes in this setting. Methods: We retrospectively reviewed medical records of 25 consecutive patients with T lymphoblastic lymphoma without prior auto-HCT who underwent allo-HCT at City of Hope from January 2000 to June 2018 after IRB approval was obtained. Descriptive statistics were used to summarize baseline patient demographic, treatment, and disease characteristics. Kaplan-Meier curves and the log-rank test were used to evaluate the overall survival (OS) and progression-free survival (PFS). Cumulative incidences of time to relapse and time to non-relapse mortality (NRM) were calculated with relapse and NRM as competing risks. Cumulative incidences of acute and chronic GVHD were calculated as time to onset of GVHD with relapse and death as competing events for GVHD. Results: 25 patients were included for the analysis. Median age at the time of allo-HCT was 25 years (range 7-70 years). 21 patients (80%) received myeloablative conditioning: FTBI containing regimens (n=20) with FTBI/VP-16 being most commonly used(n=16) and one patient received Bu/Cy based conditioning; 4 patients received reduced intensity conditioning with Flu/Mel. Sibling HCT was performed in 12 patients (48%), while MUD HCT was performed in 13 patients (52%) with fully matched HLA unrelated donor in 9 (36%) and HLA mismatched in 4 (16%) patients. GVHD prophylaxis consisted of tacrolimus/sirolimus (n=10), tacrolimus or cyclosporine/MTX (n=7), tacrolimus/sirolimus/MTX (n=4) and others (n=4) Source of stem cells was PBSC in 19 (76%), bone marrow in 5 (20%), and cord blood in 1 (4%) patients. At the time of allo-HCT, there were a total of 11 (44%) patients in complete remission (CR1 n=4, CR2+ n=7), 9 (36%) patients in partial remission and unknown remission status (n=5). The median follow-up among survivors was 8.9 years (range 2.3-12.1). The 5- and 10-year PFS was 31% (95% CI: 14%-50%). The 5- and 10-year OS was 31% (95% CI: 14%-49%) (Fig.1). Cumulative incidence of relapses at 5 and 10 years were both 36% (95% CI: 18%-55%) while NRM at 5 and 10 years was 33% (95% CI: 15%-52%). At day 100 after allo-HCT, the rates of acute GVHD grade II-IV were 63% (95% CI: 39%-79%) and grade III-IV of 25% (95%CI: 10%-44%). Chronic GVHD rates at 3 years were 60% (95% CI: 37%-77%). In univariate analysis, preHCT remission status did not improve PFS on long term follow up. Conclusions: Our results in patients with T-LBL shows 5-year PFS and OS of 31%, respectively, indicating that a subset of patients can be cured with allo-HCT in the high-risk setting. Disclosures Salhotra: Celgene: Other: Research Support; Kadmon Corporation: Other: Non paid consultant. Popplewell:City of Hope: Employment. Herrera:Kite Pharma: Consultancy, Research Funding; Merck: Consultancy, Research Funding; Genentech, Inc.: Consultancy, Research Funding; Pharmacyclics: Research Funding; AstraZeneca: Research Funding; Immune Design: Research Funding; Bristol-Myers Squibb: Consultancy, Research Funding; Adaptive Biotechnologies: Consultancy; Gilead Sciences: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding. Mei:Seattle Genetics, Inc.: Research Funding. Aldoss:AUTO1: Consultancy; Helocyte: Consultancy, Honoraria, Other: travel/accommodation/expenses; Jazz Pharmaceuticals: Honoraria, Other: travel/accommodation/expenses, Speakers Bureau; Agios: Consultancy, Honoraria. Zain:Seattle Genetics: Honoraria, Speakers Bureau; spectrum: Honoraria.

Early Broad-Spectrum Antibiotics and Risk of Acute Graft-Versus-Host Disease in Children: An Analysis from the Center for International Blood and Marrow Transplantation Research (CIBMTR) and the Pediatric Health Information System (PHIS)

Background Variation in the gastrointestinal microbiota after hematopoietic cell transplantation (HCT) has been associated with acute graft-versus-host disease (aGVHD). The significant immunosuppression associated with HCT leads to frequent broad-spectrum antibiotic exposures that in turn drive host microbiota composition. We sought to explore the association of different antipseudomonal antibiotic classes, commonly administered as empiric therapy for neutropenic fever, with subsequent aGVHD-risk in a large, multicenter cohort of children undergoing HCT for acute leukemia. We hypothesized that antibiotic classes that retain broad anti-anaerobic activity would increase aGVHD due to the greater potential impact of these agents on the microbiota. Methods Children 1-21 years old who underwent first HCT for acute leukemia from 2004 to 2017 were identified in 2 databases: CIBMTR, an observational database with demographic, transplant and outcomes information, and PHIS, an administrative database with resource utilization data from freestanding children's hospitals. Patients were then merged based on date of birth, date of transplant, sex and disease. The outcome was grade 2-4 aGVHD by day +100. The primary exposures were 3 distinct classes of antibiotics commonly used for empiric therapy: (1) antipseudomonal cephalosporins, (2) antipseudomonal penicillins and (3) carbapenems. They were captured as days of therapy from conditioning start to day +7, and classified as no, low (1-3 days), or high (&amp;gt;3 days) exposure. Logistic regression was applied to evaluate the association between aGVHD and each exposure, controlling for exposure to the other two classes. For the subset of patients with data on date of GVHD onset, a time to event analysis was performed considering death without GVHD as a competing event. For both analyses, we constructed multivariable models that were informed by a directed acyclic graph and included covariates representing demographics, transplant characteristics and GVHD risk factors associated with both exposure and outcome. The adjusted models also included exposure to other antibiotics within the same window, captured as fluoroquinolones, intravenous vancomycin and other adjunctive antibiotics, with and without anti-anaerobic activity. Generalized estimating equations with robust variance estimates were employed to account for clustering at the hospital level. Results The assembled cohort included 2,501 patients at 35 hospitals. One-third of patients (36.6%) developed aGVHD, 4.4% died without GVHD and no patients were lost to follow-up. The proportion of patients exposed to at least 1 day of an antipseudomonal cephalosporin was 52.2%, but was lower for antipseudomonal penicillins (24.1%) and carbapenems (18.2%). Table 1 shows patient characteristics by exposure to the antibiotic classes of interest. The unadjusted and adjusted associations between the three antibiotic classes and aGVHD are shown in table 2. In the unadjusted model, high exposure to cephalosporins and carbapenems was statistically associated with increased aGVHD risk. For antipseudomonal penicillins, the direction of association was the same but not statistically significant. After adjustment, most point estimates reverted towards the null. The time to event analysis included 1,146 patients that were similar to the overall cohort with a similar incidence of aGVHD (35.5%) diagnosed a median of 28 (range: 8-100) days after HCT. This group had a higher proportion of mismatched donors (40.9 vs 30.2%), cord blood transplants (40.0 vs 23.5%) and receipt of growth factors (50.5% vs 38.9%). Table 2 also shows the unadjusted and adjusted associations for this analysis. As above, the unadjusted point estimates of association between cephalosporins and carbapenems and aGVHD were suggestive of an association but reverted towards the null with adjustment. Conclusions After controlling for other antibiotic exposures and GHVD-risk factors, there was not a statistically significant association between antibiotic exposure and aGVHD for any of three antibiotic classes commonly used for empiric therapy. However, the adjusted point estimates suggest exposure to carbapenems may confer some additional aGHVD risk. Further exploration of different exposure windows is planned to understand if the identified associations change with capture of earlier or later antibiotic exposures. Disclosures Jagasia: Kadmon: Consultancy; Incyte: Consultancy; Janssen: Research Funding. MacMillan:Alpine Immune Sciences: Consultancy; Angiocrine: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy; Equillium: Membership on an entity's Board of Directors or advisory committees. Fisher:Pfizer: Research Funding; Astellas: Other: Data Safety Monitoring Board Chair for an antifungal study; Merck: Research Funding.

159 Plasma Elafin as a biomarker for Skin Graft Versus Host Disease

Skin is the most common organ involved in GVHD following hematopoietic stem cell transplant (HSCT). It is often difficult to clinically distinguish a GVHD rash from skin rash due to drugs, infection or engraftment. Plasma elafin has been found to be a diagnostic and prognostic biomarker of skin GVHD. We performed a prospective study to evaluate the role of plasma elafin as a biomarker. Consecutive allogeneic HSCT recipients over a 2 year period were recruited. Heparinised blood samples were collected at baseline and at first episode of skin rash after HSCT. Skin biopsies were obtained on day of rash. Patients were categorised into 3 groups: Patients with GVHD rash (group 1), non GVHD rash (Group 2) and Controls (group 3). Control samples were collected on Day 28 from 25 patients with no skin rash. Two hundred and sixty one patients with a median age of 16 years (1-61) and a male predominance (175:86) underwent HSCT. There were 56 patients in group 1, 49 in group 2 and 156 in group 3. Median baseline elafin value for the entire cohort was 16915 pg/ml (1205-158586). Elafin values at onset of rash between GVHD and non-GVHD were no different [34549 vs. 32077pg/ml;p=0.11]. Median rise =in elafin values [(value at rash–baseline value)/baseline value] were similar between group 1 (1.03) and group 2 (0.818; p+0.497) except at the highest quartile of elafin levels >61545 [p = 0.030]. Baseline elafin values >33329 were associated with lower overall survival [52.3% vs. 71.4%] [p=0.005] while elafin values >30248 at the time of onset of skin rash was associated with poorer outcomes [62.4 vs. 79.6%] [p=0.011]. Plasma elafin increased at the time of GVHD rash but could distinguish between GVHD and non-GVHD rash only at higher levels. There is a significant correlation between elafin levels at the time of onset of skin GVHD and overall survival. We conclude that plasma elafin can be a good prognostic marker.

Abstract 4984: Development of an improved humanized patient-derived xenograft, Hu-PDX, mouse model for evaluation of antitumor immune response in lung cancer

Abstract Current preclinical models of non-small cell lung cancer (NSCLC) do not recapitulate the human tumor microenvironment. Mice reconstituted with a human immune system and bearing human patient derived xenografts may be advantageous in evaluating human anti-tumor immune response. We developed an improved NOD scid gamma (NSG) mouse model derived from non-expanded CD34+ stem cells, without CD3+ T cell contamination, to evaluate antitumor responses to immunotherapy in NSCLC. Using fresh CD34+ from umbilical cord blood reduced humanization time significantly. Human CD45+ cell reconstitution with increased functional human lymphoid (B, T, monocytes and NK cells) and myeloid (macrophages and MDSCs) lineage repopulation, without the onset of GvHD, was achieved as early as 4 weeks post-stem cell engraftment. Published studies using expanded CD34+ derived humanization reveal compromised purity of CD34+ stem cells with an increasing number of mononuclear cells. Reconstitution of CD8+ and CD4+T cells is not achieved until 12 to 15 weeks post-engraftment at much lower levels than fresh CD34+ humanization. Single cell suspension analysis shows levels of human reconstituted T, B, NK, DC and MDSC cells at 4 weeks, which increased significantly at 6 and 9 weeks in peripheral blood, spleen and bone marrow. Human repopulated T cells were functionally active in secretion of IFN-γ by mitogenic stimuli such as PMA and IL-2 and by allogenic human cancer cells. Antigen specific CTL responses were observed when reconstituted human T cells from PDX bearing humanized mice were challenged with PDX tumor. No non-antigen specific responses were observed when T cells were co-cultured with HLA-matched human bronchial epithelial cells (HBEC). To evaluate the applicability of the humanized mouse in lung cancer translational research, we combined it with Hu-PDX or Hu-xenograft tumors and analyzed tumor growth and treatment response to the anti-PD1 checkpoint inhibitor pembrolizumab. We found that efficient engraftment of PDXs and xenograft tumors were not dependent on donor HLA-status. Similar to the clinical outcome, treatment with pembrolizumab, inhibited tumor growth significantly in both Hu-PDX, and Hu-xenograft mice regardless of donor HLA-types, increasing cytotoxic T cells and decreasing MDSC levels. Pembrolizumab had no effect on the non-humanized NSG controls. In concordance with our previous study with a syngeneic mouse tumor, the antitumor effect of check point blockade was significantly enhanced when combined with nanoparticle systemically deliveredTUSC2, a tumor suppressor and immunomodulatory gene, in a KRAS mutant lung metastasis humanized mouse model. In conclusion, fresh CD34+ are more effective than their expanded counterparts in humanizing mice, do so in much reduced time, and recapitulate the immune response to cancer. Citation Format: Ismail M. Meraz, Mourad Majidi, Feng Meng, RuPing Shao, Min Jin Ha, Shinya Neri, Bingliang Fang, Steven H. Lin, Peggy T. Tinkey, Elizabeth J. Shpall, Jeffrey Morris, Jack A. Roth. Development of an improved humanized patient-derived xenograft, Hu-PDX, mouse model for evaluation of antitumor immune response in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4984.

α4β7 Integrin Is Upregulated on CD8+ Effector Memory T-Cells in Children with Gut Gvhd Prior to Clinical Symptoms and Represents a Therapeutic Target in Pediatric Allogeneic HSCT Patients

Background α4β7 integrin is expressed on T-cells which migrate to gut-associated lymphoid tissues. Expression of α4β7 integrin is elevated on memory T-cells in adult hematopoietic stem cell transplant (HSCT) recipients at diagnosis of GI GVHD. No pediatric data exist regarding α4β7 integrin expression before or at onset of GVHD. We hypothesized that α4β7 integrin expression on effector memory T-cells (TEMs) would be elevated in patients before acute GI GVHD symptoms compared to patients without GVHD and may be a useful therapeutic target for the prevention of acute GI GVHD. Methods Cryopreserved T-cells were characterized in 48 children who developed acute GI GVHD (n=22), isolated skin GVHD (n=12) and no GVHD (n=14). In patients with GVHD, T-cells were characterized prior to HSCT, one week before GVHD and at GVHD diagnosis. In patients without GVHD, samples were analyzed at baseline and day+30 after HSCT. Samples were incubated with fluorochrome conjugated monoclonal antibodies directed against CD3, CD8, CCR7, α4β7 integrin and CD45RA. TEMs were defined as CD3+, CCR7-, CD45RA- lymphocytes. Co-expression of trafficking markers implicated in GI GVHD (CCR5, CCR9), and liver GVHD (CXCR6 and CCR5) were also assessed. Samples were analyzed on an LSR Fortessa flow cytometer (BD Biosciences). Data were analyzed using FCS Express (De Novo Software). Surface expression of all markers were compared using the Mann Whitney U-test. Patients with skin GVHD and no GVHD were grouped for analysis. Patients with at least 200 absolute CD8+ T-cell events were deemed evaluable. Results Twenty patients with GI GVHD, 8 with skin GVHD and 10 patients without GVHD had at least 200 absolute CD8+ events and were evaluable. Demographics of patients are shown in Figure 1. Expression of α4β7 integrin on CD8+ TEMs was upregulated in patients with GI GVHD compared to the No GI GVHD (skin GVHD + no GVHD) group one week prior to clinical symptoms (p=0.02; Figure 2A). Co-expression of CCR5 and CCR9 with α4β7 integrin was also higher on CD8+ TEM cells in the GI GVHD group compared to No GI GVHD group (p Conclusion α4β7 integrin is elevated on CD8+ TEMs prior to acute GI GVHD symptoms and co-expression of intestinal trafficking-specific markers such as CCR5 and CCR9 are also elevated prior to acute GVHD in pediatric patients. Our data suggest a rationale for the blockade of α4β7 integrin or the dual blockade of α4β7 integrin and CCR5 with clinically available therapeutics (maraviroc and vedolizumab) for acute GI GVHD prevention in pediatric allogeneic HCT patients.

The Easix Score Is Associate with Overall Survival and Non-Relapse Mortality after Ex-Vivo CD34+-Selected Allogeneic HCT.

Background The EASIX (Endothelial Activation and Stress Index) score has been shown to be associated with NRM and overall survival (OS) after reduced intensity alloHCT. We aimed to validate EASIX in patients who underwent a calcineurin-inhibitor (CNI)-free alloHCT using ex-vivo CD34-selection. Design We included 364 adult patients who underwent first CD34-selected alloHCT between July 2006-December 2016 using the CliniMACS CD34 Reagent System (Miltenyi Biotech) as GVHD prophylaxis. The EASIX score (LDH*Creatinine/platelet counts) was calculated at several timepoints (pre-HCT [day -30 to day -10], day 30, day 100 and onset of acute GVHD). For EASIX scores post-HCT, a landmark analysis was conducted at the given timepoint. A log transformation using base 2 (log2) was applied to all EASIX variables to reduce skew. The Kaplan-Meier and cumulative incidence methods were used to estimate OS and NRM/acute GVHD, respectively. Cox models (cause-specific for NRM and acute GVHD) were used to evaluate differences in outcomes. Relapse and death or relapse, were considered competing risks for NRM and acute GVHD, respectively. Results Median age was 55 years (range 19-73). Most patients had myeloid diseases (47% AML, 28% MDS and 4% CML) and all patients received myeloablative conditioning (MAC). With a median follow-up among survivors of 4.7 years (range, 0.7-11.8), 1 and 3-year OS was 78.6% (95% confidence interval [CI], 74.0-82.4) and 61.8% (95% CI, 56.5-66.7), respectively. The cumulative incidence of NRM at 1 and 3 years was 15.1% (95% CI, 11.7-19.0) and 24.2% (95% CI, 19.9-28.8), respectively. The 1-year cumulative incidences of grades 1-4, 2-4, and 3-4 acute GVHD were 40.1% (95% CI, 35.1-45.1), 29.4% (95% CI, 24.8-34.1) and 11.0% (95% CI, 8.0-14.5), respectively. Pre-HCT EASIX was not significantly associated with OS, NRM, or acute GVHD. A higher day 30 EASIX score was significantly associated with increased risk of death (HR=1.35 [95% CI, 1.19-1.52], p Conclusions EASIX score at day 30 and at day 100 post-ex-vivo CD34-selected alloHCT is associated with higher NRM and inferior OS. Despite a CNI-free platform, endothelial injury is an important contributor to poorer outcomes after MAC. EASIX provides a simple, complimentary tool to predict allo-HCT outcomes in these patients.

Comparison of Gvhd Biomarker Algorithms for Predicting Lethal Gvhd and Non-Relapse Mortality

Acute GVHD biomarkers predict long-term outcomes after GVHD diagnosis. Our group has validated the MAGIC algorithm which uses the concentrations of 2 GVHD biomarkers, ST2+REG3a, to predict lethal GVHD (defined as death from GVHD without relapse) [Hartwell 2017]. Other published biomarker combinations that predict GVHD outcomes include ST2+ REG3a+TNFR1 [Levine 2015], ST2+TIM3 [Abu Zaid 2017], ST2+TNFR1 [McDonald 2015], TIM3+TNFR1+IL6 [McDonald 2015], as well as AREG alone [Holtan 2018]. It is not clear which biomarker combination best predicts lethal GVHD because these algorithms were developed using different patient cohorts with different endpoints. To answer this question, we compared the predictive accuracy of different biomarker combinations as well as these six specific combinations in the same patient cohort. We studied 522 patients with serum samples at GVHD diagnosis who were transplanted at 19 Mount Sinai Acute GVHD International Consortium (MAGIC) centers between January 1, 2004 and April 30, 2017. Patients were divided into training (n = 253) and validation (n = 269) sets; validation patients were transplanted after November 1, 2015 and had not previously been used to generate an algorithm. Biomarkers were measured by ELISA and log-transformed values were used to predict 1-year lethal GVHD by competing risk regression. Four of the 6 biomarkers (ST2, REG3a, TNFR1, and TIM3) independently predicted lethal GVHD in the training set. We then developed algorithms that predicted lethal GVHD using all possible combinations of these 4 biomarkers as well as the published combination of TIM3+TNFR1+IL6 and AREG alone. The best algorithms of 1-4 biomarkers for predicting lethal GVHD were REG3a alone, ST2+REG3a, ST2+REG3a+TNFR1, and ST2+REG3a+TNFR1+TIM3. While ST2+REG3a was the most accurate combination based on the lowest Akaike Information Criterion (AIC), the other published biomarker combinations produced similar AICs. Therefore, we used the independent validation set to compare all six published algorithms. We generated area under the receiver operating characteristic curves (AUC) for each algorithm (FIG 1). We next determined the threshold that maximized sensitivity and specificity for each algorithm and calculated the cumulative incidence of lethal GVHD in the resulting high and low risk strata (FIG 2). Highly similar results were obtained when the cumulative incidence of non-relapse mortality was used as the endpoint. In a validation set of previously unanalyzed patients, several biomarker combinations reproducibly stratified patients with GVHD for risk of death. The best 4 algorithms, all of which included ST2, produced comparable outcomes. Adding TNFR1 to ST2+ REG3a did not improve accuracy. The ST2+REG3a algorithm best identifies patients at onset of GVHD for high risk of lethal GVHD and NRM, and thus remains a standard for biomarker-based risk prediction.

Carfilzomib for Treatment of Refractory Chronic Gvhd: A Chronic GVHD Consortium Pilot Trial

Advances are needed in the therapy of treatment-refractory chronic graft-vs.-host disease (GVHD). Prior experimental and clinical data suggest proteasome inhibition may have immunomodulatory activity relevant to GVHD. Carfilzomib is a specific, irreversible, next-generation proteasome inhibitor with extensive experience in the therapy of multiple myeloma. To explore the safety and activity of carfilzomib in advanced chronic GVHD, we conducted a pilot multi-center phase II trial through the Chronic GVHD Consortium. Carfilzomib was administered at 20 mg/m2 on day 1, and then 36mg/m2 on days 8 and 15 of a 28-day treatment cycle (cycle 1), and then 36 mg/m2 on days 1, 8, and 15 of a 28-day treatment cycle (cycles 2-6). The primary endpoint was 6 month treatment failure, a composite endpoint including death, relapse, and requirement for an additional line of systemic immune suppressive (IS) therapy. Considering a historical 6 month treatment failure rate of 44%, this 20 subject pilot trial allowed estimation of the 6 month treatment failure rate with a standard error of 10 percentage points. Secondary objectives included safety, physician- and NIH-based response rates, failure-free survival, and patient-reported outcomes. Key eligibility criteria included age ≥ 18, NIH Consensus defined chronic GVHD, and failure of at least one prior line of systemic IS therapy. A total of 20 subjects were enrolled at 4 institutions. Median age was 53 years. Median time from transplant to enrollment was 2.8 years (IQR 1.7-5), and from chronic GVHD onset to enrollment 1.5 years (IQR 0.5-3.7). Chronic GVHD was NIH moderate (30%) or severe (70%), predominantly classic (90% vs. overlap 10%), and involved multiple diverse organ sites. Prior lines of systemic therapy for chronic GVHD were ≤ 2 (n=6, 30%) or ≥ 3 (n=14, 70%). Doses were held primarily for infection (50% of total held doses); only 4 patients (20%) completed all planned 6 months of carfilzomib. SAEs occurred in 40% of subjects, and 7 deaths occurred among study participants occurring between 0.3-9 months after last carfilzomib dose (none attributed to carfilzomib). The 6 month treatment failure rate was not significantly improved vs. the historical benchmark (40% vs. 44%, p=0.36), figure 1. NIH response rates at 3, 6, and 12 months are presented in figure 2. Overall survival at 6 and 12 months was 79% and 58%. Failure-free survival at 12 months was 32%. These pilot phase II data suggest that carfilzomib therapy in this very advanced chronic GVHD population did not improve expected 6 month treatment failure rates achieved under conventional practices and is not recommended for further study for this indication.