ALBERT

Description: Abstract 116 Interleukin 7 (IL-7) and its receptor, a heterodimer constituted by IL-7Rα (hereafter referred to as IL7R) and γc subunits, are essential for normal T-cell development and homeostasis. While it is known for long that IL7R genetic inactivation leads to severe combined immunodeficiency, only recently we and others have found IL7R gain-of-function mutations in T-cell acute lymphoblastic leukemia (T-ALL). In agreement with an oncogenic role for deregulated IL-7/IL7R signaling, IL-7 transgenic mice develop lymphomas and we have shown that xenotransplant models of human T-ALL rely on microenvironmental IL-7 for tumor acceleration. These studies have highlighted the importance of keeping IL7R-mediated signaling within physiological levels. Indeed, throughout normal T-cell development IL7R expression at the cell surface is tightly controlled and there is correlative evidence, from studies in AKR/J mice, that high IL7R levels in developing thymocytes can promote the development of T-cell leukemia. However, no studies have yet directly evaluated the impact of IL7R on T-cell oncogenesis. In the present study, we used a mouse model in which expression of an IL7R transgene is controlled by a tetracycline responsive promotor (TreIL7R rtTAhuCD2Il7r−/− mice) to analyze the role of IL7R-mediated signaling in T-cell leukemogenesis. Continuous expression of TreIL7R upon doxycycline feeding induced thymus hypertrophy and hyperplasia due to increased size and hyperproliferation of T-cells, which subsequently infiltrated lymph nodes, spleen and bone marrow, ultimately leading to leukemia/lymphoma-associated death. Adoptive transfer of thymic TreIL7R cells to immunodeficient mice confirmed their malignant origin. Surprisingly, tumors developed in recipient animals even in the absence of doxycycline administration, indicating that they eventually become independent of continuous IL7R expression. Remarkably, the tumors mimicked several features of human T-ALL. First, their immunophenotype varied considerably between animals (from CD4 CD8 double-negative to double positive to CD4 or CD8 single positive cells), reflecting the heterogeneity of human disease. Second, similar to the majority of primary T-ALL cases, most tumors displayed hyperactivation of PI3K/Akt pathway, which sometimes associated with absence or decreased PTEN protein expression. Third, the cell cycle inhibitor p27Kip1 was frequently downregulated, a molecular characteristic associated with some T-ALLs. We next sought to determine the contribution of TCR diversity to IL7R-mediated tumorigenesis by crossing mice to the F5 TCR transgenic background. Tumor development in these mice occurred with similar timing and incidence, suggesting that the tumors arise irrespectively of whether a clonal or polyclonal TCR repertoire exists. Finally, the role of Rag recombinase dependent genomic instability in tumor development was assessed by further crossing mice F5 TreIL-7R rtTAhuCD2Il7r−/− mice to Rag1−/− background. Tumor development occurred independently of Rag1 expression, indicating a dominant role for IL7R elicited signals in tumorigenesis. Altogether, our results reveal that continuous IL7R-mediated signaling promotes T-cell tumorigenesis in vivo, providing further indication that IL7R can act as a critical T-cell oncogene. Disclosures: No relevant conflicts of interest to declare.

Description: We have recently shown that the leukotriene B4 (LTB4)–BLT1 pathway is important in early effector T-cell recruitment in mouse models of inflammation. Here we characterize the phenotype and function of human peripheral blood BLT1+ T cells in health and illustrate their involvement in asthma and acute infection. In healthy individuals, BLT1+ T cells are a rare peripheral blood T-cell population enriched for the activation markers CD38 and HLA-DR. Compared with BLT1– T cells, a larger proportion of peripheral blood BLT1+ T cells express the effector cytokines IFNγ and IL-4 and inflammatory chemokine receptors, CCR1, CCR2, CCR6, and CXCR1. Consequently, in healthy individuals peripheral blood BLT1+ T cells are a rare antigen-primed T-cell subset with unique phenotypic, migratory, and functional properties. BLT1 expression on T cells is tightly regulated by inflammation and only transiently expressed after naive T-cell activation by dendritic cells. Although rare in the peripheral blood of healthy individuals, BLT1+ T cells are markedly increased in frequency in the peripheral blood in response to acute Epstein-Barr virus (EBV) infection and moderately increased in the airways of asymptomatic allergic asthmatics. Our studies provide novel insights into the LTB4-BLT1 lipid chemoattractant pathway in human T-cell responses, and how it may link innate and adaptive immunity.

Description: Bone marrow is the preferred stem cell source for allogeneic transplantation in the treatment of severe aplastic anemia (AA) due to a higher risk of GVHD associated with peripheral blood stem cell allografting. Higher doses or longer durations of administration of lympholytic agents such as antithymocyte globulin (ATG) or alemtuzumab may prevent GVHD and reduce graft failure rate. We treated a 19 y/o AA woman with transfusion and antibiotic dependent severe AA associated with a small PNH clone using 12/12 HLA-match allogeneic peripheral blood stem cells (allo-PBSCT) from her brother. Conditioning regimen consisted of Cyclophosphamide (CY) at 50 mg/kg/day intravenously on day -5 through day -2 before transplant. Twelve hours after each doses of CY, she was given horse ATG (Atgam; Upjohn, Kalamazoo, MI) at 30 mg/kg IV per dose infused over a period of 10 hours. ATG was given for 4 days, instead of 3 days as in the original study (R. Storb et al, Blood 1994;84:941-9). She received premedication with oral acetaminophen, intravenous diphenhydramine, and methylprednisolone at 100 mg, 30 minutes prior to each dose of ATG. During the first infusion of ATG, the patient received hydrocortisone 50 mg IV twice for infusion-related reactions, but tolerated all subsequent infusions.She received prophylaxis with acyclovir, levofloxacin, and fluconazole. Unmanipulated, G-CSF-mobilized allo-PBSCs at dose of 4.125 x 106 CD34+cells/kg were transplanted fresh in 48 hours after the last dose of CY. She received GVHD prophylaxis with tacrolimus and low dose methotrexate (5 mg/kg/day) on Days +1, +3, +6, and +11. Her transplant course was notable for breakthrough vaginal spotting despite leuprolide administration, and neutropenic fever with negative infectious workup. On day +14, the patient was given a single dose of G-CSF to expedite WBC recovery. Based on CIBMTR criteria, neutrophil and platelet engraftments occurred on day +15 and day +20, respectively. She was started on steroids with a quick taper for suspected engraftment syndrome with ongoing fevers with negative cultures. She was discharged on day +16 after transplant with a total length of hospital stay of 22 days. Her outpatient course was completely uneventful with no GVHD, infection or any other transplant-related complications. The patient was never readmitted. Her blood counts remained within normal range and blood chimerism analysis showed 100% donor in myeloid and lymphoid on day+30 and day+100 evaluations. She is currently day+120 on continuous tacrolimus. This case illustrates a successful early outcome after peripheral blood allogeneic stem cell transplantation in a young patient with aplastic anemia using modified ATG protocol. The concept of T cell in vivo purging after allo-PBSCT with extended administration of ATG in aplastic anemia warrants a prospective trial. Disclosures Seddon: Sanofi: Speakers Bureau.

Description: The role for IL-7R expression in the differentiation of effector T cells into resting memory remains controversial. Here, using a conditional IL-7R transgenic model, we were able to test directly whether CD8 effector T cells require IL-7R expression for their differentiation into resting memory cells. In the absence of IL-7R expression, effector cells transferred into “full” hosts underwent a protracted and unremitting contraction compared with IL-7R–expressing control cells and were unable to develop into long-term resting memory cells. Surprisingly, when the same effector cells were transferred into empty T-cell–deficient hosts, they could generate long-lived fully functional resting memory cells independently of IL-7R expression. Formation of these latter cells was found to be dependent on IL-15, because the same IL-7R–deficient effector cells were rapidly lost from IL-15–deficient hosts, having a half-life of less than 40 hours. Therefore, our data suggest that, under physiological conditions, both IL-7 and IL-15 synergize to promote the formation of memory cells directly by limiting the contraction of effectors that occurs following an immune response and that reexpression of IL-7R is a key checkpoint in the regulation of this process.

Description: Background: The current standard of care for the treatment of patients with newly diagnosed AML is an anthracycline plus cytarabine. Both anthracyclines and cytarabine have been associated with the development of typhlitis, a serious adverse event characterized by inflammation of the bowel wall in patients with profound neutropenia, diagnosed via abdominal CT imaging and clinical symptoms. Given the paucity of available data, the aim of our study was to determine the incidence of typhlitis among AML patients receiving induction with either idarubicin 12 mg/m2 (IDA), daunorubicin 60 mg/m2 (DNA60), or daunorubicin 90 mg/m2 (DNA90). Methods: Adult patients with AML or MDS receiving either daunorubicin or idarubicin along with cytarabine as part of their induction regimen between 1/1/2009 and 6/30/2013 were included. A definition of typhlitis required CT confirmation of inflammation of the cecum, according to CTCAE version 4.03 along with clinical symptoms. Two radiologists (OA, JC) blinded to the treatment and outcomes independently reviewed CT scans. Two additional definitions including inflammation of the ileocecal region only and enterocolitis were also evaluated. All statistical analyses were performed on SAS software version 9.3. P values were calculated using Fisher Exact and Wilcoxon tests. Inter-rater reliability was assessed with Cohen’s Kappa. Results: Baseline characteristics were similar among the 3 treatment groups with the exception of age. The median age was lower in the DNA90 arm (79 years, 74 years, and 49 years in the IDA group, DNA60 group, and DNA90 group, respectively). A pre-existing GI disorder was reported in 24.1% IDA, 25.7% DNA60, and 22.4% DNA90 patients. Of the 202 total patients, the two radiologists determined that 40 (20%) and 38 (19%) developed typhlitis, based on the predefined standard. Tables 1 and 2 illustrate the relationship between treatment arm and associated incidence of typhlitis. The incidence in each treatment group did not statistically differ (p=0.23 and p=0.29). When the definition was broadened to include ileocecal region and enterocolitis, the incidence increased (Tables 1 and 2). The inter-reliability ratings of the 2 radiologists’ evaluations for each definition indicated substantial agreement (0.803 cecum, 0.834 ileocecal region only, and 0.752 enterocolitis). Neither the anthracycline chosen, nor the dose had a statistically significant impact on the incidence of typhlitis. Of all patient and treatment specific risk factors assessed for association with development of typhlitis (pre-existing GI disorder, rheumatologic disorder or cancer, prior RT, stem cell transplant or anthracycline exposure, cytarabine regimen or AML risk group), none were found to be statistically significant. In patients that developed typhlitis, approximately 56% and 51% had a concurrent documented infection around the typhlitis episode (Tables 1-2). Conclusion: To our knowledge, this is the first study to compare the incidence of typhlitis in adult patients receiving idarubicin or daunorubicin for the treatment of AML. While the cumulative incidence of typhlitis was higher than in published literature, the incidence was similar irrespective of the anthracycline chosen or dose. Of the potential factors that may have contributed to the development of typhlitis, none were significantly associated with typhlitis. All patients were managed conservatively with broad-spectrum antibiotics. A more definitive definition of typhlitis may help clinicians identify affected patients sooner and choose appropriate targeted therapy. Abstract 3725. Table 1. Association of treatment to Radiologist 1-rated typhlitis Enterocolitis Ileocecal region only Cecum No Yes P No Yes P No Yes P Treatment 0.52 0.68 0.23 DNA60 58 (78%) 16 (22%) 58 (78%) 16 (22%) 61 (82%) 13 (18%) DNA90 34 (69%) 15 (31%) 35 (71%) 14 (29%) 35 (71%) 14 (29%) IDA 58 (73%) 21 (27%) 60 (76%) 19 (24%) 66 (84%) 13 (16%) Concomitant infection Yes 11 (26%) 32 (74%) 13 (30%) 30 (70%) 19 (44%) 24 (56%) Abstract 3725. Table 2. Association of treatment to Radiologist 2-rated typhlitis Enterocolitis Ileocecal region only Cecum No Yes P No Yes P No Yes P Treatment 0.86 0.98 0.29 DNA60 57 (77%) 17 (23%) 57 (77%) 17 (23%) 62 (84%) 12(16%) DNA90 36 (73%) 13 (27%) 37 (76%) 12 (24%) 36 (73%) 13 (27%) IDA 58 (73%) 21 (27%) 61 (77%) 18 (23%) 66 (84%) 13 (16%) Concomitant infection Yes 11 (26%) 32 (74%) 13 (30%) 30 (70%) 21 (49%) 22 (51%) Disclosures No relevant conflicts of interest to declare.

Description: Given advanced age, comorbidities, and immune dysfunction, chronic lymphocytic leukemia (CLL) patients may be at particularly high risk of infection and poor outcomes related to coronavirus disease 2019 (COVID-19). Robust analysis of outcomes for CLL patients, particularly examining effects of baseline characteristics and CLL-directed therapy, is critical to optimally manage CLL patients through this evolving pandemic. CLL patients diagnosed with symptomatic COVID-19 across 43 international centers (n = 198) were included. Hospital admission occurred in 90%. Median age at COVID-19 diagnosis was 70.5 years. Median Cumulative Illness Rating Scale score was 8 (range, 4-32). Thirty-nine percent were treatment naive (“watch and wait”), while 61% had received ≥1 CLL-directed therapy (median, 2; range, 1-8). Ninety patients (45%) were receiving active CLL therapy at COVID-19 diagnosis, most commonly Bruton tyrosine kinase inhibitors (BTKi’s; n = 68/90 [76%]). At a median follow-up of 16 days, the overall case fatality rate was 33%, though 25% remain admitted. Watch-and-wait and treated cohorts had similar rates of admission (89% vs 90%), intensive care unit admission (35% vs 36%), intubation (33% vs 25%), and mortality (37% vs 32%). CLL-directed treatment with BTKi’s at COVID-19 diagnosis did not impact survival (case fatality rate, 34% vs 35%), though the BTKi was held during the COVID-19 course for most patients. These data suggest that the subgroup of CLL patients admitted with COVID-19, regardless of disease phase or treatment status, are at high risk of death. Future epidemiologic studies are needed to assess severe acute respiratory syndrome coronavirus 2 infection risk, these data should be validated independently, and randomized studies of BTKi’s in COVID-19 are needed to provide definitive evidence of benefit.

Description: Interleukin-7 (IL-7) plays a central role in the homeostasis of the T-cell compartment by regulating T-cell survival and proliferation. Whether IL-7 can influence T-cell receptor (TCR) signaling in T cells remains controversial. Here, using IL-7–deficient hosts and TCR-transgenic T cells that conditionally express IL-7R, we examined antigen-specific T-cell responses in vitro and in vivo to viral infection and lymphopenia to determine whether IL-7 signaling influences TCR-triggered cell division events. In vitro, we could find no evidence that IL-7 signaling could costimulate T-cell activation over a broad range of conditions, suggesting that IL-7 does not directly tune TCR signaling. In vivo, however, we found an acute requirement for IL-7 signaling for efficiently triggering T-cell responses to influenza A virus challenge. Furthermore, we found that IL-7 was required for the enhanced homeostatic TCR signaling that drives lymphopenia-induced proliferation by a mechanism involving efficient contacts of T cells with dendritic cells. Consistent with this, saturating antigen-presenting capacity in vivo overcame the triggering defect in response to cognate peptide. Thus, we demonstrate a novel role for IL-7 in regulating T cell–dendritic cell interactions that is essential for both T-cell homeostasis and activation in vivo.

Description: Introduction: Patients (pts) with CLL may be at particular risk of severe COVID-19 given advanced age and immune dysregulation. Two large series with limited follow-up have reported outcomes for pts with CLL and COVID-19 (Scarfò, et al. Leukemia 2020; Mato, et al. Blood 2020). To provide maximal clarity on outcomes for pts with CLL and COVID-19, we partnered in a worldwide effort to describe the clinical experience and validate predictors of survival, including potential treatment effects. Methods: This international collaboration represents a partnership between investigators at 141 centers. Data are presented in two cohorts. Cohort 1 (Co1) includes pts captured through efforts by European Research Initiative on CLL (ERIC), Italian CAMPUS CLL Program, and Grupo Español de Leucemia Linfática Crónica. The validation cohort, Cohort 2 (Co2), includes pts from US (66%), UK (23%), EU (7%), and other countries (4%). There is no overlap in cases between cohorts. CLL pts were included if COVID-19 was diagnosed by PCR detection of SARS-CoV-2 and they required inpatient hospitalization. Data were collected retrospectively 2/2020 - 5/2020 using standardized case report forms. Baseline characteristics, preexisting comorbidities (including cumulative illness rating scale (CIRS) score ≥6 vs.

Description: Tight regulation of IL-7Rα expression is essential for normal T-cell development. IL-7Rα gain-of-function mutations are known drivers of T-cell acute lymphoblastic leukemia (T-ALL). Although a subset of patients with T-ALL display high IL7R messenger RNA levels and cases with IL7R gains have been reported, the impact of IL-7Rα overexpression, rather than mutational activation, during leukemogenesis remains unclear. In this study, overexpressed IL-7Rα in tetracycline-inducible Il7r transgenic and Rosa26 IL7R knockin mice drove potential thymocyte self-renewal, and thymus hyperplasia related to increased proliferation of T-cell precursors, which subsequently infiltrated lymph nodes, spleen, and bone marrow, ultimately leading to fatal leukemia. The tumors mimicked key features of human T-ALL, including heterogeneity in immunophenotype and genetic subtype between cases, frequent hyperactivation of the PI3K/Akt pathway paralleled by downregulation of p27Kip1 and upregulation of Bcl-2, and gene expression signatures evidencing activation of JAK/STAT, PI3K/Akt/mTOR and Notch signaling. Notably, we also found that established tumors may no longer require high levels of IL-7R expression upon secondary transplantation and progressed in the absence of IL-7, but remain sensitive to inhibitors of IL-7R–mediated signaling ruxolitinib (Jak1), AZD1208 (Pim), dactolisib (PI3K/mTOR), palbociclib (Cdk4/6), and venetoclax (Bcl-2). The relevance of these findings for human disease are highlighted by the fact that samples from patients with T-ALL with high wild-type IL7R expression display a transcriptional signature resembling that of IL-7–stimulated pro-T cells and, critically, of IL7R-mutant cases of T-ALL. Overall, our study demonstrates that high expression of IL-7Rα can promote T-cell tumorigenesis, even in the absence of IL-7Rα mutational activation.