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Designing effective antileukemic immunotherapy will require understanding mechanisms underlying tumor control or resistance. Here, we report a mechanism of escape from immunologic targeting in an acute myeloid leukemia (AML) patient, who relapsed 1 year after immunotherapy with engineered T cells expressing a human leukocyte antigen A*02 (HLA-A2)–restricted T cell receptor (TCR) specific for a Wilms’ tumor antigen 1 epitope, WT1126–134(TTCR-C4). Resistance occurred despite persistence of functional therapeutic T cells and continuous expression of WT1 and HLA-A2 by the patient’s AML cells. Analysis of the recurrent AML revealed expression of the standard proteasome, but limited expression of the immunoproteasome, specifically the beta subunit 1i (β1i), which is required for presentation of WT1126–134. An analysis of a second patient treated with TTCR-C4demonstrated specific loss of AML cells coexpressing β1i and WT1. To determine whether the WT1 protein continued to be processed and presented in the absence of immunoproteasome processing, we identified and tested a TCR targeting an alternative, HLA-A2–restricted WT137–45epitope that was generated by immunoproteasome-deficient cells, including WT1-expressing solid tumor lines. T cells expressing this TCR (TTCR37–45) killed the first patients’ relapsed AML resistant to WT1126–134targeting, as well as other primary AML, in vitro. TTCR37–45controlled solid tumor lines lacking immunoproteasome subunits both in vitro and in an NSG mouse model. As proteasome composition can vary in AML, defining and preferentially targeting these proteasome-independent epitopes may maximize therapeutic efficacy and potentially circumvent AML immune evasion by proteasome-related immunoediting.

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Supplementary Table 2 from HIF Induces Human Embryonic Stem Cell Markers in Cancer Cells

Supplementary Table 5 from HIF Induces Human Embryonic Stem Cell Markers in Cancer Cells

Supplementary Methods, Figures 1-11, Table Legends 1-7 from HIF Induces Human Embryonic Stem Cell Markers in Cancer Cells

Supplementary Table 3 from HIF Induces Human Embryonic Stem Cell Markers in Cancer Cells

Supplementary Table 1 from HIF Induces Human Embryonic Stem Cell Markers in Cancer Cells

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Axicabtagene ciloleucel (YESCARTA; Kite Pharma, a Gilead Company, Los Angeles CA) and tisagenlecleucel (KYMRIAH; Novartis Pharmaceuticals Corp., Basel, Switzerland) are two CD19-directed chimeric antigen receptor (CAR) T cell products currently approved by the US Food and Drug Administration; the European Medicines Agency; Health Canada; Ministry of Health, Labor and Welfare (Japan); and Therapeutic Goods Administration (Australia) for treatment of specific subtypes of relapsed/refractory aggressive B cell non-Hodgkin lymphoma (NHL). Although this approval has been transformative in the use of cellular immunotherapy in lymphoma, there are concerns regarding appropriate use of this novel therapy and of short- and long-term toxicities. To address these issues, representatives of the American Society of Transplantation and Cellular Therapy convened to recognize and address key issues surrounding the clinical application of CD19 CAR T cell therapy in B cell lymphomas, in collaboration with worldwide experts. The aim of this article is to provide consensus opinion from experts in the fields of hematopoietic cell transplantation, cellular immunotherapy, and lymphoma regarding key clinical questions pertinent to the use of CD19 CAR T cell products for the treatment of NHL. As the clinical practice using CAR T cells grows worldwide, we anticipate that this guidance will be relevant for hematology/oncology physicians who care for patients with lymphomas.

Relapse after allogeneic hematopoietic cell transplantation (HCT) is the leading cause of death in patients with acute myeloid leukemia (AML) entering HCT with poor-risk features1–3. When HCT does produce prolonged relapse-free survival, it commonly reflects graft-versus-leukemia effects mediated by donor T cells reactive with antigens on leukemic cells4. As graft T cells have not been selected for leukemia specificity and frequently recognize proteins expressed by many normal host tissues, graft-versus-leukemia effects are often accompanied by morbidity and mortality from graft-versus-host disease5. Thus, AML relapse risk might be more effectively reduced with T cells expressing receptors (TCRs) that target selected AML antigens6. We therefore isolated a high-affinity Wilms’ Tumor Antigen 1-specific TCR (TCRC4) from HLA-A2+ normal donor repertoires, inserted TCRC4 into Epstein–Bar virus-specific donor CD8+ T cells (TTCR-C4) to minimize graft-versus-host disease risk and enhance transferred T cell survival7,8, and infused these cells prophylactically post-HCT into 12 patients (NCT01640301). Relapse-free survival was 100% at a median of 44 months following infusion, while a concurrent comparative group of 88 patients with similar risk AML had 54% relapse-free survival (P = 0.002). TTCR-C4 maintained TCRC4 expression, persisted long-term and were polyfunctional. This strategy appears promising for preventing AML recurrence in individuals at increased risk of post-HCT relapse. Donor-derived, EBV-specific CD8+ T cells engineered to express a high-affinity WT1-specific TCR established persistent T cell responses that safely prevented post-HCT relapse in patients with high-risk AML.

On August 30, 2017 the US Food and Drug Administration approved tisagenlecleucel (Kymriah; Novartis, Basel, Switzerland), a synthetic bioimmune product of anti-CD19 chimeric antigen receptor T cells (CAR-T), for the treatment of children and young adults with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL). With this new era of personalized cancer immunotherapy, multiple challenges are present, ranging from implementation of a CAR-T program to safe delivery of the drug, long-term toxicity monitoring, and disease assessments. To address these issues experts representing the American Society for Blood and Marrow Transplant, the European Society for Blood and Marrow Transplantation, the International Society of Cell and Gene Therapy, and the Foundation for the Accreditation of Cellular Therapy formed a global CAR-T task force to identify and address key questions pertinent for hematologists and transplant physicians regarding the clinical use of anti CD19 CAR-T therapy in patients with B-ALL. This article presents an initial roadmap for navigating common clinical practice scenarios that will become more prevalent now that the first commercially available CAR-T product for B-ALL has been approved.

BACKGROUND: Hematopoietic cell transplant (HCT) can cause significant distress in patients and their informal caregivers. Despite advances in reduced intensity conditioning and supportive care, few recent studies have reported rates of clinically-significant post-traumatic stress disorder symptomatology (hereafter referred to as PTSD). Goals of the current study were to: 1) examine rates of PTSD and distress in patients and caregivers and 2) identify sociodemographic and clinical risk factors for PTSD. METHODS: As part of an annual survivorship survey, 2,157 HCT recipients and their caregivers were mailed self-report measures of PTSD and distress. Patients also completed self-report measures of sociodemographic information (e.g., age, sex, employment status). Clinical variables (e.g., time since transplant, transplant type) were captured in the transplant database. RESULTS: A total of 691 recipients (56% age 60 or above at the time of survey, 47% female, median 10.1 years post-HCT) and 333 caregivers provided PTSD data and were included in the current analyses. More caregivers reported PTSD (6.6%) than patients (3.3%) (p=0.02). Patients or caregivers who had PTSD reported significantly higher distress related to uncertainty, family strain, medical demands, finances, identity, and health burden (p values < 0.0001) compared to those without PTSD. Patient but not caregiver PTSD was associated with more recent transplant (p=0.01, p=0.16). CONCLUSIONS: Rates of PTSD are relatively low in long-term survivors of HCT and their caregivers. Nevertheless, results are consistent with other studies of cancer caregiving suggesting that caregivers often experience greater distress than patients. Timely referral to psychosocial services should be offered to both HCT recipients and caregivers reporting symptoms of PTSD.

BACKGROUND Little is known about pathogen-specific humoral immunity after chimeric antigen receptor–modified T (CAR-T) cell therapy for B cell malignancies. METHODS We conducted a prospective cross-sectional study of CD19-targeted or B cell maturation antigen–targeted (BCMA-targeted) CAR-T cell therapy recipients at least 6 months posttreatment and in remission. We measured pathogen-specific IgG against 12 vaccine-preventable infections and the number of viral and bacterial epitopes to which IgG was detected (“epitope hits”) using a serological profiling assay. The primary outcome was the proportion of participants with IgG levels above a threshold correlated with seroprotection for vaccine-preventable infections. RESULTS We enrolled 65 children and adults a median of 20 months after CD19- (n = 54) or BCMA- (n = 11) CAR-T cell therapy. Among 30 adults without IgG replacement therapy (IGRT) in the prior 16 weeks, 27 (90%) had hypogammaglobulinemia. These individuals had seroprotection to a median of 67% (IQR, 59%–73%) of tested infections. Proportions of participants with seroprotection per pathogen were comparable to population-based studies, but most individuals lacked seroprotection to specific pathogens. Compared with CD19-CAR-T cell recipients, BCMA-CAR-T cell recipients were half as likely to have seroprotection (prevalence ratio, 0.47; 95% CI, 0.18–1.25) and had fewer pathogen-specific epitope hits (mean difference, –90 epitope hits; 95% CI, –157 to –22). CONCLUSION Seroprotection for vaccine-preventable infections in adult CD19-CAR-T cell recipients was comparable to the general population. BCMA-CAR-T cell recipients had fewer pathogen-specific antibodies. Deficits in both groups support the need for vaccine and immunoglobulin replacement therapy studies. FUNDING Swiss National Science Foundation (Early Postdoc Mobility grant P2BSP3_188162), NIH/National Cancer Institute (NIH/NCI) (U01CA247548 and P01CA018029), NIH/NCI Cancer Center Support Grants (P30CA0087-48 and P30CA015704-44), American Society for Transplantation and Cellular Therapy, and Juno Therapeutics/BMS., In this prospective study, we investigated antibodies against vaccine-preventable infections and other pathogen-specific antibodies in individuals with remission after CAR-T cell therapy for B lineage malignancies.

CD19-targeted chimeric antigen receptor (CAR) T-cell therapy has demonstrated remarkable efficacy in patients with relapsed/refractory B-cell malignancies; however, it is associated with toxicities including cytokine release syndrome (CRS), neurotoxicity, and impaired hematopoietic recovery. The latter is associated with high-grade cytopenias requiring extended growth factor or transfusional support, potentially leading to additional complications such as infection or hemorrhage. To date, the factors independently associated with hematologic toxicity have not been well characterized. To address this deficit, we retrospectively analyzed 173 patients who received defined-composition CD19 CAR T-cell therapy in a phase 1/2 clinical trial (https://clinicaltrials.gov; NCT01865617), with primary end points of absolute neutrophil count and platelet count at day-28 after CAR T-cell infusion. We observed cumulative incidences of neutrophil and platelet recovery of 81% and 75%, respectively, at 28 days after infusion. Hematologic toxicity was noted in a significant subset of patients, with persistent neutropenia in 9% and thrombocytopenia in 14% at last follow-up. Using debiased least absolute shrinkage selector and operator regression analysis for high-dimensional modeling and considering patient-, disease-, and treatment-related variables, we identified increased CRS severity as an independent predictor for decreased platelet count and lower prelymphodepletion platelet count as an independent predictor of both decreased neutrophil and platelet counts after CD19 CAR T-cell infusion. Furthermore, multivariable models including CRS-related cytokines identified associations between higher peak serum concentrations of interleukin-6 and lower day-28 cell counts; in contrast, higher serum concentrations of transforming growth factor-β1 were associated with higher counts. Our findings suggest that patient selection and improved CRS management may improve hematopoietic recovery after CD19 CAR T-cell therapy.

Bone health disturbances commonly occur after hematopoietic cell transplantation (HCT) with loss of bone mineral density (BMD) and avascular necrosis (AVN) foremost among them. BMD loss is related to pretransplantation chemotherapy and radiation exposure and immunosuppressive therapy for graft-versus-host-disease (GVHD) and results from deficiencies in growth or gonadal hormones, disturbances in calcium and vitamin D homeostasis, as well as osteoblast and osteoclast dysfunction. Although the pathophysiology of AVN remains unclear, high-dose glucocorticoid exposure is the most frequent association. Various societal treatment guidelines for osteoporosis exist, but the focus is mainly on menopausal-associated osteoporosis. HCT survivors comprise a distinct population with unique comorbidities, making general approaches to bone health management inappropriate in some cases. To address a core set of 16 frequently asked questions (FAQs) relevant to bone health in HCT, the American Society of Transplant and Cellular Therapy Committee on Practice Guidelines convened a panel of experts in HCT, adult and pediatric endocrinology, orthopedics, and oral medicine. Owing to a lack of relevant prospective controlled clinical trials that specifically address bone health in HCT, the answers to the FAQs rely on evidence derived from retrospective HCT studies, results extrapolated from prospective studies in non-HCT settings, relevant societal guidelines, and expert panel opinion. Given the heterogenous comorbidities and needs of individual HCT recipients, answers to FAQs in this article should be considered general recommendations, with good medical practice and judgment ultimately dictating care of individual patients. Readers are referred to the Supplementary Material for answers to additional FAQs that did not make the core set.

CD19-targeted chimeric antigen receptor-engineered (CD19 CAR) T-cell therapy has shown significant efficacy for relapsed or refractory (R/R) B-cell malignancies. Yet, CD19 CAR T cells fail to induce durable responses in most patients. Second infusions of CD19 CAR T cells (CART2) have been considered as a possible approach to improve outcomes. We analyzed data from 44 patients with R/R B-cell malignancies (acute lymphoblastic leukemia [ALL], n = 14; chronic lymphocytic leukemia [CLL], n = 9; non-Hodgkin lymphoma [NHL], n = 21) who received CART2 on a phase 1/2 trial (NCT01865617) at our institution. Despite a CART2 dose increase in 82% of patients, we observed a low incidence of severe toxicity after CART2 (grade ≥3 cytokine release syndrome, 9%; grade ≥3 neurotoxicity, 11%). After CART2, complete response (CR) was achieved in 22% of CLL, 19% of NHL, and 21% of ALL patients. The median durations of response after CART2 in CLL, NHL, and ALL patients were 33, 6, and 4 months, respectively. Addition of fludarabine to cyclophosphamide-based lymphodepletion before the first CAR T-cell infusion (CART1) and an increase in the CART2 dose compared with CART1 were independently associated with higher overall response rates and longer progression-free survival after CART2. We observed durable CAR T-cell persistence after CART2 in patients who received cyclophosphamide and fludarabine (Cy-Flu) lymphodepletion before CART1 and a higher CART2 compared with CART1 cell dose. The identification of 2 modifiable pretreatment factors independently associated with better outcomes after CART2 suggests strategies to improve in vivo CAR T-cell kinetics and responses after repeat CAR T-cell infusions, and has implications for the design of trials of novel CAR T-cell products after failure of prior CAR T-cell immunotherapies.

Caregivers are critical to recipient recovery after hematopoietic cell transplant (HCT); however, little is known about their long-term health and quality of life (QoL). In this study we surveyed 4446 caregiver–recipient pairs in the post-HCT period to describe their QoL and its determinants. In total, 849 caregiver–recipient pairs at a median of 6 years after autologous or allogeneic HCT responded. Among 849 responding caregivers at a median of 6 years post-HCT, 67% of caregivers were women and 68% indicated they were still providing care to the recipient. Mean and median QoL measures of caregivers were at or above general population norms; however, approximately 20% of caregivers reported poor QoL relative to general population norms. Multivariate analysis revealed that caregiver characteristics, including age, gender, and educational attainment, were important determinants of caregiver QoL. Additional determinants of caregiver QoL included recipient QoL, relapse after autologous HCT, and ongoing use of immunosuppression after allogeneic HCT. Additionally, the prevalence of depression and sleep disorders appear to be higher in caregivers than in the general population. We have identified a population of caregivers who may benefit from interventions aimed at improving QoL and health outcomes. HCT clinical practice should also consider caregiver well-being.

Moderate to severe chronic graft-versus-host disease (GVHD) is treated with potent immunosuppressive therapy (IST) to modulate the allo-immune response, control symptoms, and prevent further organ damage. We sought to understand the types of treatments used in clinical practice and the likelihood of successful treatment associated with each. A chart review was performed for 250 adult patients at Fred Hutchinson Cancer Research Center enrolled in a prospective observational study. After a median follow-up of 5.6 years for survivors, approximately one-third were still on IST (of whom half were on fourth or greater line of therapy), one-third were alive and off IST, and one-third had relapsed or died. Approximately half of survivors stopped all IST at least once, although half of these restarted IST after a median of 3.4 months (interquartile range, 2.3 to 8.0) off therapy. Successful discontinuation of IST for at least 9 months was associated with myeloablative conditioning (P = .04), more years since transplant (P = .009), and lack of oral (P

In a multicenter, prospective, phase II study we evaluated the safety and efficacy of pentostatin followed by donor lymphocyte infusion (DLI) in patients with low donor Tcell chimerism after allogeneic hematopoietic cell transplantation (HCT). Thirty-six patients with low donor blood CD3 chimerism were enrolled in this study. Thirty-five patients received a total of 41 DLIs after a dose of pentostatin, and 1 patient received pentostatin only. Median donor CD3 chimerism prompting the initiation of pentostatin and DLI was 28% (range, 5% to 47%). Responses (defined by increases in donor CD3 chimerism ≥10% maintained to day 56 post-DLI) were seen in 16 patients (44.4%) with a median rise in CD3 donor chimerism to 64% (range, 48% to 100%). There was a trend for better responses among 21 patients who received first treatment within 100 days after transplant (57% response rate) compared with15 patients who received first treatment more than 100 days after HCT (27% response rate, P = .07). Fourteen patients (39%) developed grades II to IV acute graft-versus-host disease (GVHD) at a median of 10 days (range, 0 to 83) after DLI. Ten patients (28%) developed extensive chronic GVHD. Seventeen patients (47%) developed new grade 4 cytopenias after DLI. There was no difference in relapse between nonresponders and responders. Twenty-eight patients (78%) died, most (n = 21) because of relapse. Five of 16 responders (31%) are alive, all disease-free, at a median of 60 months (range, 21 to 132) after DLI. Six of 20 nonresponders (30%) are alive at a median of 47 months (range, 16 to 100) after DLI, 3 in complete remission. Pentostatin and DLI had acceptable toxicity and appeared to increase low donor CD3 chimerism after HCT but had no impact on mortality.

Introduction: The gut microbiome is a potentially modifiable factor in treatment related outcomes in allogeneic hematopoietic cell transplant (HCT). Prior studies have linked pre- or mid-treatment gut microbiome diversity with risk for treatment related morbidity and mortality. However, these studies have been limited by the inclusion of one or only a few institutions and the lack of longitudinal sampling with high quality metadata. These limitations complicate the interpretation of microbiome alterations over the course of HCT. Methods: To overcome these, we devised and implemented a large-scale biospecimen collection protocol in conjunction with BMT CTN 1703, a randomized, multicenter, Phase III trial of tacrolimus/methotrexate vs. post-transplant cyclophosphamide/tacrolimus/mycophenolate mofetil in reduced intensity conditioning (RIC) allogeneic HCT (NCT03959241). Patients enrolled on 1703 were optionally co-enrolled in the companion immune and microbiome profiling study, 1801 (MI-IMMUNE). This involved blood, urine and stool sampling before conditioning (PCON), then weekly starting on day 0 through day 77 (through day 84 for blood), and on days 98, 180, 270, 365 and 730. For all enrolled participants where the donor consented, residual donor cells were saved from the empty hematopoietic stem cell product bag for later analysis. Additionally, the protocol included one-time blood, urine and stool sample collection from consented matched related donors (MRDs) prior to stem cell collection. Starting with protocol version 4.0 on February 1, 2021, participation in 1801 stool collection was required for the first six out of eighteen sample collection timepoints. Participation in later stool and urine timepoint collections remained optional. Here we review the feasibility of creating a multi-institutional biobank. Additionally, we assess the success of our strategies by calculating sample collection compliance and standard deviation in compliance across centers for each timepoint. Results: On June 18, 2021, BMT CTN 1703/1801 closed to accrual with 431 patients enrolled on 1703; 323 patients from 36 centers were co-enrolled on 1801. 304 (94%) provided study samples, making this the largest prospective microbiome and immune profiling study in HCT patients to date. As of July 6, 2021, 3,683 blood, 2,668 urine, and 2,098 stool samples had been collected. Across the first 6 timepoints for all participating centers, blood, urine and stool sample collection averaged 93%, 82%, and 74% compliance, respectively. Of the 99 (30%) patients enrolled on 1801 with a MRD, 34 (34%) donors consented to sample collection. Sample collection compliance was lower for MRDs than for patients on the study with 76%, 74%, and 62% of expected blood, urine and stool samples collected, respectively, from this group. For stool collection exclusively, a median of 5 samples were collected per patient across the first 6 timepoints (median of 6 across all timepoints) with 93 (31%) of patients completing a full sample set through Day 28. 139 (46%) patients provided at least one sample after day 28; these represented 37% of the total samples collected to date. The PCON sample, which provides an important measure of pre-treatment gut microbiome diversity, had the third highest compliance with 74% of patients providing a sample. Surprisingly, Day 28 had the lowest compliance (66%) and highest standard deviation (37%) possibly because this timepoint often falls around the time of hospital discharge. Between PCON and day 28, the standard deviation between sites in the average collection compliance (24%) and number of samples collected per patient (1.1) was small indicating the successful adoption of stool collection across institutions. Table 1 summarizes sample collection statistics. Conclusion: Overall this study has resulted in a large, novel biobank of blood, urine and stool samples from patients undergoing RIC allogeneic HCT at 36 centers across the US. This will serve as a valuable resource for investigating the role of the gut microbiome in long term health outcomes following HCT. Although the results of 1801 are forthcoming given ongoing sample collection, the size and composition of the biobank to date clearly demonstrate the feasibility of implementing multi-institutional stool collection. This study represents a critical step towards the large-scale adoption of microbiome sampling as a diagnostic tool. Figure 1 Figure 1. Disclosures Chhabra: GSK: Honoraria. Clark: Kadmon: Consultancy. Horowitz: Mesoblast: Research Funding; Shire: Research Funding; Vertex: Research Funding; Stemcyte: Research Funding; Vor Biopharma: Research Funding; Janssen: Research Funding; Miltenyi Biotech: Research Funding; Kiadis: Research Funding; Sobi: Research Funding; Kite/Gilead: Research Funding; Pfizer, Inc: Research Funding; Jazz Pharmaceuticals: Research Funding; Magenta: Consultancy, Research Funding; Medac: Research Funding; Novartis: Research Funding; GlaxoSmithKline: Research Funding; Daiicho Sankyo: Research Funding; Xenikos: Research Funding; Omeros: Research Funding; Orca Biosystems: Research Funding; Pharmacyclics: Research Funding; Regeneron: Research Funding; Tscan: Research Funding; Takeda: Research Funding; CSL Behring: Research Funding; Genentech: Research Funding; Gamida Cell: Research Funding; Chimerix: Research Funding; Bristol-Myers Squibb: Research Funding; bluebird bio: Research Funding; Astellas: Research Funding; Amgen: Research Funding; Allovir: Consultancy; Actinium: Research Funding; Sanofi: Research Funding; Seattle Genetics: Research Funding. Jenq: Microbiome DX: Consultancy; Merck: Consultancy; Prolacta: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kaleido: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seres: Consultancy, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; LisCure: Consultancy, Membership on an entity's Board of Directors or advisory committees; MaaT Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Karius: Consultancy. Levine: Equillium Bio: Membership on an entity's Board of Directors or advisory committees; Incyte: Consultancy, Research Funding; Kamada: Research Funding; Biogen: Research Funding; Omeros: Membership on an entity's Board of Directors or advisory committees; Symbio: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Talaris Therapeutics: Membership on an entity's Board of Directors or advisory committees; Viracor: Patents & Royalties: GVHD biomarker patent with royalties from Viracor; Mesoblast: Consultancy, Research Funding; X4 Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees. Murthy: CRISPR Therapeutics: Research Funding. Riches: ATARA Biotherapeutics: Other: Payment; BioIntelect: Membership on an entity's Board of Directors or advisory committees; Jazz Pharmaceuticals: Other: Payment. Sung: Merck: Research Funding; Novartis: Research Funding; Enterome: Research Funding; Seres: Research Funding; AVROBIO: Consultancy; Abbott Nutrition: Honoraria; Clasado: Other: Research Product; DSM: Other: Research Product. Al Malki: Neximmune: Consultancy; Jazz Pharmaceuticals, Inc.: Consultancy; Rigel Pharma: Consultancy; Hansa Biopharma: Consultancy; CareDx: Consultancy. Rezvani: Kaleido: Other: One-time scientific advisory board; Nohla Therapeutics: Other: One-time scientific advisory board; Pharmacyclics-Abbvie: Research Funding; US Department of Justice: Consultancy. Bolaños-Meade: Incyte Corp: Consultancy. Holtan: Incyte: Consultancy, Research Funding; Generon: Consultancy. Saber: Govt. COI: Other. Hamadani: Sanofi, Genzyme, AstraZeneca, BeiGene: Speakers Bureau; Janssen, Incyte, ADC Therapeutics, Omeros, Morphosys, Kite: Consultancy; Takeda, Spectrum Pharmaceuticals and Astellas Pharma: Research Funding. Kean: Bluebird Bio: Research Funding; Bristol Myers Squibb: Patents & Royalties: From clinical trial data, Research Funding; Vertex: Consultancy; Novartis: Consultancy; Gilead: Research Funding; Regeneron: Research Funding; EMD Serono: Consultancy. Perales: Cidara: Honoraria; Servier: Honoraria; Incyte: Honoraria, Other; Equilium: Honoraria; Takeda: Honoraria; Novartis: Honoraria, Other; Nektar Therapeutics: Honoraria, Other; NexImmune: Honoraria; MorphoSys: Honoraria; Omeros: Honoraria; Karyopharm: Honoraria; Sellas Life Sciences: Honoraria; Merck: Honoraria; Miltenyi Biotec: Honoraria, Other; Medigene: Honoraria; Bristol-Myers Squibb: Honoraria; Kite/Gilead: Honoraria, Other; Celgene: Honoraria.

Background Chimeric antigen receptor-modified T (CAR-T) cell immunotherapy for B cell hematologic malignancies results in prolonged B cell depletion. Little is known about the effects of CAR-T cell therapy on pre-existing pathogen-specific humoral immunity. Methods We conducted a prospective, cross-sectional study of children and adults treated with CD19- or BCMA-CAR-T cell therapy. Eligible patients were ≥ 6 months post-CAR-T cell infusion and in remission without subsequent chemoimmunotherapy. We measured total immunoglobulin G (IgG), pathogen-specific IgG levels for 12 vaccine-preventable infections, and B cell subsets from blood. Seroprotective antibody titers were based on standard thresholds. We described the proportion of patients with seroprotective titers and tested for associations between clinical factors and seroprotection using generalized estimating equations. Results We enrolled 65 patients who received CD19- (n=54) or BCMA- (n=11) CAR-T cell therapy. Seven patients were < 18 years old. Samples were collected a median of 20 months (range, 7–68) after CAR T cell infusion. Seroprotection to vaccine-preventable pathogens was generally comparable to the U.S. population (Fig 1) even though blood CD19+ B cell counts were low (< 20 cells/mm3) in 60% of patients. Among 30 patients without IgG replacement in the prior 16 weeks (4 half-lives of IgG), 27 (90%) had hypogammaglobulinemia. Despite this, these individuals had seroprotection to a median of 67% (IQR, 59%-73%) of tested pathogens (Fig 2A). The proportion of patients with seroprotection was lowest for mumps, hepatitis A and B, H. influenzae type B (Hib), S. pneumoniae, and B. pertussis. Patients receiving BCMA-CAR-T cells had seroprotection to fewer pathogens than those receiving CD19-CAR-T cells (Fig 2B), but the difference did not reach statistical significance (Fig 3). There were no significant differences by other variables. Figure 1. Proportion of CAR-T cell recipients with seroprotection to vaccine-preventable infections compared to the U.S. population, stratified by receipt of IgG replacement in the previous 16 weeks. Figure 2 A-B. Percentage of pathogens with seroprotective antibody titers among patients without IgG replacement in the previous 16 weeks. Figure 3. Association of clinical factors with seroprotection to vaccine-preventable infections among patients without IgG replacement in the previous 16 weeks (n=30) Conclusion Seroprotection for vaccine-preventable infections after CD19-CAR-T cell therapy was comparable to the general population. BCMA-CAR-T cell recipients may benefit most from replacement IgG. Vaccinations after CAR-T cell therapy should be considered and prioritized for S. pneumoniae, Hib, hepatitis viruses, and B. pertussis. Disclosures Justin J. Taylor, PhD, Vir Biotechnology (Grant/Research Support) Damian J. Green, MD, Cellectar Biosciences (Grant/Research Support)GSK (Advisor or Review Panel member)Juno Therapeutics (Grant/Research Support, Advisor or Review Panel member, Other Financial or Material Support, Royalities)Seattle Genetics (Grant/Research Support, Advisor or Review Panel member) Michael Boeckh, MD PhD, AlloVir (Consultant)EvrysBio (Advisor or Review Panel member, Other Financial or Material Support, share options)Gilead (Consultant, Grant/Research Support)GSK (Consultant)Helocyte (Advisor or Review Panel member, Shareholder)Lophius (Grant/Research Support)Merck (Consultant, Grant/Research Support)SymBio (Consultant)VirBio (Consultant, Grant/Research Support) David G. Maloney, MD, PhD, A2 Biotherapeutics (Consultant, Other Financial or Material Support, Stock Options)Bioline Rx (Consultant)Celgene (Consultant, Grant/Research Support)Gilead (Consultant)Juno Therapeutics (Consultant, Research Grant or Support, Other Financial or Material Support, four pending patents, not issued, licensed, no royalities, no licensees)Kite Pharma (Consultant, Grant/Research Support)Novartis (Consultant)Pharmacyclics (Consultant) Cameron J. Turtle, MBBS, PhD, Allogene (Other Financial or Material Support, Ad hoc advisory board (last 12 months))ArsenalBio (Advisor or Review Panel member, Other Financial or Material Support, Stock/options)AstraZeneca (Grant/Research Support, Other Financial or Material Support, Ad hoc advisory board (last 12 months))Caribou Biosciences (Advisor or Review Panel member, Other Financial or Material Support, Stock/options)Century Therapeutics (Advisor or Review Panel member)Eureka Therapeutics (Advisor or Review Panel member, Other Financial or Material Support, Stock/options)Juno Therapeutics (Grant/Research Support, Other Financial or Material Support, Patent: Licensed to Juno Therapeutics)Myeloid Therapeutics (Advisor or Review Panel member, Other Financial or Material Support, Stock/options)Nektar Therapeutics (Grant/Research Support, Other Financial or Material Support, Ad hoc advisory board (last 12 months))PACT Pharma (Other Financial or Material Support, Ad hoc advisory board (last 12 months))Precision Biosciences (Advisor or Review Panel member, Other Financial or Material Support, Stock/options)TCR2 Therapeutics (Grant/Research Support)T-CURX (Advisor or Review Panel member) Joshua A. Hill, MD, Allogene (Consultant)Allovir (Consultant)Gilead (Consultant)Karius (Grant/Research Support, Scientific Research Study Investigator)Takeda (Grant/Research Support, Scientific Research Study Investigator)

Background: Chimeric antigen receptor T-cell (CAR-T) therapy is an effective strategy for patients (pts) with B-cell non-Hodgkin lymphomas (B-NHLs). However, long-term remissions are only observed in ~40% of pts with large cell lymphomas (LCL) treated with FDA-approved CD19-targeted products and there is currently no approved CAR-T for other histologies other than mantle cell lymphoma (MCL). CD20 is a proven therapeutic target for B-NHL, supported by previously approved naked and radiolabeled anti-CD20 antibodies and promising results from bispecific antibodies. CD20-targeted CAR-T is another potential adoptive immunotherapy option that could be utilized in combination or in sequence with CD19 CAR-T. We present interim results of our ongoing phase I/II clinical trial investigating safety and efficacy CD20 CAR-T for high-risk B-NHLs (NCT03277729). Methods: MB-106 is a fully human third-generation CD20-targeted CAR-T with both 4-1BB and CD28 costimulatory domains. We use a continuous reassessment method dose escalation design to find the maximally tolerated dose. Lymphodepletion (LD) consists of cyclophosphamide (Cy) ± fludarabine (Flu). CAR-T cells are administered at one of 4 dose levels (DL): DL1: 3.3 x 105, DL2: 1 x106, DL3: 3.3x106, DL4: 1x107 CAR T cells/kg. CAR-T is infused in the outpatient setting except for the first pts of each dose cohort (overnight observation). Pts with CD20+ B-NHL are eligible, including but not limited to LCL, follicular lymphoma (FL)/indolent histologies and MCL. Prior treatment with CD19 CAR-T is permitted. Treatment response is assessed on day 28 using 2014 Lugano criteria. Cytokine release syndrome (CRS) and immune effector cell-associated neurotoxicity syndrome (ICANS) were graded per 2014 Lee criteria and the ASTCT consensus grading, respectively. Results: Between 2018-2020, 12 pts underwent leukapheresis (LA) and 11 (92%) were treated on the protocol. The first 7 pts (3 FL, 3 MCL, 1 hairy cell variant) were treated using the original cell manufacturing method with separate culturing of CD4+ and CD8+ cells ("original process"). First 4 of those 7 pts (2 treated at DL1 and 2 at DL2) only received Cy for LD and the other 3 (all at DL1) were treated after LD with Cy-Flu. There was no evidence of ICANS in those 7 pts and only 1 pt developed CRS (grade 3 - unexplained elevated alkaline phosphatase (ALP) in the setting of fever). Responses at 4 weeks for pts treated with the "original process" included stable disease (SD) in 4 and progressive disease (PD) in 3 pts. Given the challenges in meeting the protocol requirements for CD4+ and CD8+ specific doses, poor CAR-T expansion, and lack of clinical responses, enrollment was placed on a hold and cell manufacturing process underwent a major revision which included changing to a combined culture of CD4+ and CD8+ cells, among other modifications ("modified process"). Since December 2019 enrollment was reinitiated at DL1 under the "modified process." Four pts were treated after LD with Cy-Flu at DL1 (2 FL) and dose level 2 (1 FL and 1 MCL) (details in Table 1). Target cell doses were achieved in all 4 patients and treatment was tolerated with no occurrence of CRS (any grade) or ICANS (any grade). Responses were observed at both dose levels and included complete remissions (CRs) in 2 FL pts (1 treated at DL1 and 1 at DL2), partial remission (PR) in a MCL pt treated at DL2 and PD in a FL pt who was treated at DL1. Robust CAR-T expansion was seen in 3 of the 4 patients, with peak blood levels of CAR+ T cells of 161, 5.5, and 401 CAR+ cells/µl, corresponding to 80%, 23%, and 72% of all CD8+ cells. Combining all 11 pts treated under the "original" and "modified" processes, no dose-limiting toxicity (DLT) was observed. Grade ≥ 3 toxicities included: anemia (n=4, 36%), lymphopenia (n=3,27%), febrile neutropenia (n=2, 18%), hypertension (n=1, 9%) , hypotension (n=1, 9%), thromboembolic event (n=1, 9%), neutropenia (n=1, 9%), elevated ALP (n=1, 9%), pneumonia (n=1, 9%), bacteremia (n=1, 9%), hyperglycemia (n=1, 9%), pleural effusion (n=1, 9%) and generalized pain (n=1, 9%). Conclusion: Early results of our ongoing study of CD20 CAR-T for high-risk NHLs suggests an extremely favorable safety profile (no CRS or ICANS of any grades) with the modified manufacturing process. There is also evidence of clinical activity even at low dose levels with observed CRs at DLs 1 and 2. Enrollment at higher DLs is currently ongoing and the data will be updated at the time of presentation. Disclosures Shadman: Abbvie, Genentech, Astra Zeneca, Sound Biologics , Pharmacyclics, Verastem, ADC therapeutics, Beigene, Cellectar, BMS, Morphosys and Atara Biotherapeutics: Consultancy; Mustang Bio, Celgene, Pharmacyclics, Gilead, Genentech, Abbvie, TG therapeutics, Beigene, Astra Zeneca, Sunesis, Beigene: Research Funding. Yeung:OBI;Pfizer: Research Funding; Adaptive Biotechnology;Eli Lilly;Merck: Consultancy. Ramachandran:Mustang Bio: Current Employment. Graf:TG Therapeutics: Research Funding; BeiGene: Research Funding; MorphoSys: Consultancy; Acerta Pharma: Research Funding. Gopal:IgM bio, BMS, merck: Research Funding; Seattle Genetics; Janssen; IMab Bio; TG Therapeutics; Astra Zeneca; Merck; Gilead; ADC Therapeutics; Nurix; TG therapeutics, Cellectar; Actinium: Consultancy; imab bio, takeda,astrazeneca,gilead: Research Funding; Seattle Genetics; Janssen; Takeda; IgM Bio; IMab Bio; BMS; Astra Zeneca; Merck; Gilead: Research Funding. Gauthier:JMP, Eusapharma, Multerra Bio: Honoraria. Cassaday:Pfizer: Honoraria, Research Funding; Seattle Genetics: Current Employment, Current equity holder in publicly-traded company; Vanda Pharmaceuticals: Research Funding; Kite/Gilead: Consultancy, Research Funding; Merck: Research Funding; Amgen: Consultancy, Research Funding. Kiem:Magenta Therapeutics, CSL,Homology Medicines, Vor Biopharma , Enochian, Umoja, Rocket Pharma: Consultancy. Turtle:Novartis: Consultancy; Myeloid Therapeutics: Current equity holder in private company, Honoraria, Membership on an entity's Board of Directors or advisory committees; Allogene: Consultancy; PACT Pharma: Consultancy; Arsenal Bio: Current equity holder in private company, Honoraria, Membership on an entity's Board of Directors or advisory committees; Caribou Biosciences: Current equity holder in private company, Honoraria, Membership on an entity's Board of Directors or advisory committees; Eureka Therapeutics: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees; Precision Biosciences: Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees; Physician Education Resource: Consultancy; AstraZeneca: Consultancy, Research Funding; Nektar Therapeutics: Consultancy, Research Funding; Juno/BMS: Patents & Royalties, Research Funding; Century Therapeutics: Honoraria, Membership on an entity's Board of Directors or advisory committees; T-CURX: Membership on an entity's Board of Directors or advisory committees; Humanigen: Consultancy; Kite/Gilead: Consultancy. Maloney:Pharmacyclics: Consultancy, Honoraria; Juno Therapeutics: Consultancy, Honoraria, Patents & Royalties: Patents are pending, but not issued, licensed, no royalties, no licensees., Research Funding; Celgene: Consultancy, Honoraria, Research Funding; Kite, a Gilead Company: Consultancy, Honoraria, Research Funding; Gilead Sciences: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; MorphoSys: Consultancy, Honoraria; Bioline Rx: Consultancy, Honoraria; A2 Biotherapeutics: Consultancy, Current equity holder in publicly-traded company, Honoraria; Genentech: Consultancy, Honoraria. Till:Mustang: Patents & Royalties, Research Funding.

Background: Donor T cells specific for minor histocompatibility (H) antigens can deliver potent, selective anti-leukemic effects after allogeneic HCT when the antigen is negligibly or not expressed by non-hematopoietic tissues, not present in the donor, and expressed by the recipient. We reported a new minor H antigen-directed T-cell therapy that can be deployed after HCT to manage persistent or recurrent measurable residual hematologic malignancies or overt relapse (Blood 2018;131(1):108). We developed a transgene with 4 components: 1) a high-affinity T-cell receptor (TCR) specific for the hematopoietic-restricted minor H antigen, HA-1 that is presented on HLA-A*02:01; 2) a CD8 co-receptor to enhance function of the class I-restricted TCR in CD4+ T cells so they promote cytotoxic CD8+ T cell function and survival; 3) an inducible caspase-9 safety switch, which can be triggered by the drug rimiducid in case of in vivo toxicity; and 4) a CD34-CD20 epitope to facilitate selection of the engineered product during manufacturing and track HA-1 TCR T cells in the recipient. The 21-day manufacturing process entails CD45RA+ naïve T cell depletion (minimizes the risk of GvHD), and subsequent CD4+ and CD8+ separation (provides a consistent 1:1 CD4:CD8 ratio). The separate cultures are transduced with the lentivirus construct iCasp9-HA1-TCR2-RQR-CD8, expanded, and selected using the CD34 marker to ensure removal of untransduced T cells. Study Design and Methods: The single-center phase I trial (NCT03326921) evaluates the feasibility and safety of infusion of HA-1 TCR T-cell immunotherapy. Primary end points are 1) Feasibility of manufacturing and administering HA-1 TCR CD8+ and CD4+ memory T cells and 2) Dose-limiting toxicity of HA-1 TCR T cells. Major inclusion criteria are: HLA-A*02:01-positive, HA-1-positive patients who underwent HCT for acute leukemia, myelodysplastic syndrome, BPDCN, CML, CMML or JMML from a HLA-A*-02:01+/HA-1-negative donor or HLA-A*02:01-negative haploidentical or mismatched donor (excluding umbilical cord). HA-1 genotype screening is performed on patient and donor blood, hair follicle or cheek swab samples shipped to Fred Hutchinson Cancer Research Center. To be eligible for treatment, patients must develop measurable residual disease or overt relapse after HCT but may receive other standard or investigational therapies prior to treatment with HA-1 TCR T-cell immunotherapy if clinically indicated. Some systemic immunosuppression may be continued, but prior grade IV acute GVHD and prior severe chronic GVHD are key exclusions. Two groups, Recruitment and Patient Characteristics: To date, 3 subjects have been treated on the phase I clinical trial and received a total of 5 infusions (Table 1). HA-1 TCR T cell persistence in blood and bone marrow has been documented from >3 months to >13 months. Clear in vivo anti-leukemic activity was observed at the first dose level, including in a subject with aggressive, highly refractory T-ALL and early post-HCT relapse. Outlook: Minor H antigen-specific T-cell immunotherapy may offer effective management of post-HCT relapse while avoiding GvHD and other off-target effects. Due to population genetics of HA-1 and HLA-A*02:01, HA-1 TCR T-cell immunotherapy is applicable to 10-15% of HCT recipients with various hematological malignancies. The ongoing phase I trial is actively recruiting patients. Development of T-cell immunotherapy targeting other minor H antigen/HLA combinations is also underway to increase the broad applicability of minor H antigen-targeted T-cell immunotherapy. Disclosures Krakow: HighPass Bio: Research Funding. Cunningham:HighPass Bio: Research Funding. Vartanian:HighPass Bio: Research Funding. Bleakley:HighPass Bio: Current equity holder in private company, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding.

Chimeric antigen receptor (CAR)-T cell therapy is a novel treatment with promising results for patients with hematological malignancies. Currently three FDA approved products are available for treatment of relapsed/refractory lymphoid malignancies, and several products are in different stages of clinical investigations for treatment of multiple myeloma and other hematological malignancies. CAR-T cell therapy has known early toxicities of cytokine release syndrome (CRS) and neurotoxicity, but little is known about long-term neuropsychiatric adverse effects. We previously reported neuropsychiatric patient-reported outcomes (PROs) of long-term survivors after CAR-T cell therapy (Ruark at al. BBMT 2020; 26: 34-43), but without baseline PROs before treatment for comparison. In this study we have utilized PROs, including PROMIS® measures, to assess symptoms and quality of life (QOL) of patients with relapse/refractory non-Hodgkin lymphoma (NHL), acute lymphoblastic leukemia (ALL), chronic lymphocytic leukemia (CLL), and multiple myeloma (MM) at time of arrival to our cellular immunotherapy clinic for CAR-T cell therapy. Between February 2019 and March 2020, patients received a PRO questionnaire at time of their arrival to our immunotherapy clinic. The questionnaire included the PROMIS Scale v1.2-Global Health, PROMIS-29 Profile v2.1, PROMIS Cognitive Function Short-Form v2.0, as well as 30 additional questions, including questions pertaining to cognitive function. As of March 30, 2020, 58 questionnaires were returned (59% response rate for online questionnaire versus 80% for paper questionnaire) and included in the analysis. Patients' characteristics are summarized in Table 1. PROMISmeasures are standardized to a T-score metric, with a score of 50 representing the general US population mean. Clinically meaningful differences were defined as a 5-point difference in scores (1/2 standard deviation). Cognitive function was assessed for all patients by asking if patients had experienced difficulties with concentration, finding words, memory, or solving problems; answering "yes" to each of the questions received "1" point to determine the total cognitive difficulty score (0-4). The PROMIS Cognitive Function Short-Form was added later to the PRO questionnaire and data are available for 28 patients. The cohort's self-reported cognitive difficulties and PROMIS mean T scores are shown in Table 2. Mean T score of PROMIS Global Physical Health was clinically meaningfully different from the mean in the general US population (43.7), while the mean T scores of all the other domains, including Global Mental Health, Social Function, Anxiety, Depression, Fatigue, Pain Interference, Sleep Disturbance and Cognitive Function were not clinically meaningfully different from the mean in the general US population. 23 of the 56 participants for whom complete data were available (41%) reported at least one cognitive difficulty and/or clinically meaningful depression and/or anxiety (Figure 1). On univariate risk factor analysis, older age was found to be associated with worse Global Physical Health (p=0.04) but with a trend for less depression (p=0.07). Diagnosis of ALL was found to be a risk factor for depression (p Our study demonstrates overall worse physical function in our cohort of 58 patients with hematological malignancies at time of CAR-T cell therapy, compared to the general population, but no clinically meaningful difference was found in neuropsychiatric status. 41% of the participants reported depression, anxiety or cognitive difficulties at time of CAR-T cell therapy, compared to 47.5% of participants 1-5 years after CAR-T cell therapy in our prior published work (Ruark at al. BBMT 2020; 26: 34-43). Ten participants (17.2%) reported 1-4 cognitive difficulties at time of CAR-T cell therapy compared to 37.5% of participants 1-5 years after CAR-T cell therapy. However, conclusions cannot be drawn from these two independent cohorts, and longitudinal data are needed to compare patients' neuropsychiatric status before and after CAR-T cell therapy in order to determine the treatment effect. Such study is currently ongoing in our institution. Disclosures Maloney: Celgene: Consultancy, Honoraria, Research Funding; Gilead Sciences: Consultancy, Honoraria; Juno Therapeutics: Consultancy, Honoraria, Patents & Royalties: Patents are pending, but not issued, licensed, no royalties, no licensees., Research Funding; Amgen: Consultancy, Honoraria; A2 Biotherapeutics: Consultancy, Current equity holder in publicly-traded company, Honoraria; Kite, a Gilead Company: Consultancy, Honoraria, Research Funding; Genentech: Consultancy, Honoraria; MorphoSys: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Pharmacyclics: Consultancy, Honoraria; Bioline Rx: Consultancy, Honoraria.

On August 30, 2017, the U.S. Food and Drug Administration (US-FDA) approved tisagenlecleucel (KYMRIAH, Novartis, Basel, Switzerland), a synthetic bioimmune product of anti-CD19 chimeric antigen receptor-T cells (CAR-T), for the treatment of children and young adults with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). With this new era of personalized cancer immunotherapy, multiple challenges are present ranging from implementation of a CAR-T program to safe delivery of the drug, long-term toxicity monitoring and disease assessments. To address these issues, experts representing the American Society for Blood and Marrow Transplant (ASBMT), the European Group for Blood and Marrow Transplantation (EBMT), the International Society of Cell and Gene Therapy (ISCT), and the Foundation for the Accreditation of Cellular Therapy (FACT), formed a global CAR-T task force to identify and address key questions pertinent for hematologists and transplant physicians regarding the clinical use of anti CD19 CAR-T therapy in patients with B-ALL. This article presents an initial roadmap for navigating common clinical practice scenarios that will become more prevalent now that the first commercially available CAR-T product for B-ALL has been approved.

CD19-targeted chimeric antigen receptor-modified T cell (CAR-T cell) therapy has shown excellent antitumor activity in patients with relapsed/refractory B cell malignancies, with very encouraging response rates and outcomes. However, the late effects following this therapy remain unknown. Here we report late adverse events-defined as starting or persisting beyond 90 days after CAR-T cell infusion-in patients who survived at least 1 year after therapy. The median duration of follow-up was 28.1 months (range, 12.5 to 62.6 months). At last follow-up, 73% of patients were still alive and 24% were in ongoing complete remission (CR). The most common late adverse event was hypogammaglobulinemia (IgG400 mg/dL or i.v immunoglobulinm (IVIG) replacement, observed in 67% of the patients with available data. Infection density was .55 infection/100 days at risk (2.08 per patient-year). The majority (80%) of the infections were treated in the outpatient setting, and 5% necessitated admission to the intensive care unit (ICU). Subsequent malignancies occurred in 15% of patients, including 5% with myelodysplastic syndrome (MDS). Among patients with ongoing CR and with no MDS, 16% experienced prolonged cytopenia requiring transfusions or growth factor support. Graft-versus-host disease occurred in 3 of 15 patients (20%) who had undergone previous allogeneic hematopoietic cell transplantation. Most of the late events observed in this cohort were not severe, and many could be related to previous or subsequent therapies, suggesting a safe long-term profile of CD19-targeted CAR-T cell immunotherapy.

Although differences in the incidence of chronic graft-versus-host disease (GVHD) across the races have been suggested, these have not been systematically investigated. This study compared the incidence, sites, severity, and outcomes of late acute GVHD and chronic GVHD according to National Institutes of Health (NIH) consensus criteria between Japanese (n = 413) and white (n = 708) patients after first allogeneic hematopoietic cell transplantation. Analysis was stratified according to bone marrow transplantation (BMT) or peripheral blood stem cell transplantation (PBSCT). Japanese patients, compared with white patients, had a similar incidence of late acute GVHD (BMT, 19% vs 16%; PBSCT, 19% vs 16%) but experienced more frequent liver late acute GVHD as defined by transaminase elevation (BMT, 79% vs 8%; PBSCT, 92% vs 33%) and less frequent gastrointestinal late acute GVHD (BMT, 11% vs 58%; PBSCT, 20% vs 68%). Japanese patients were more likely to discontinue systemic immunosuppression after late acute GVHD than white patients (hazard ratio, 3.68; 95% confidence interval, 1.96-6.94; P < .001). Japanese patients, compared with white patients, had a lower incidence of chronic GVHD (BMT, 15% vs 30% [P = .002]; PBSCT, 37% vs 45% [P < .001]) and experienced more frequent chronic GVHD of the mouth, eyes, and liver and less frequent gastrointestinal chronic GVHD. The duration of immunosuppressive treatment of NIH chronic GVHD was similar between the races. These differences could not be entirely attributed to practice variation between the centers. This study shows that the incidence, affected sites, severity, and clinical outcomes of late acute GVHD and NIH chronic GVHD differ between Japanese and white patients.

On August 30, 2017, the U.S. Food and Drug Administration (US-FDA) approved tisagenlecleucel (KYMRIAH, Novartis, Basel, Switzerland), a synthetic bioimmune product of anti-CD19 chimeric antigen receptor-T cells (CAR-T), for the treatment of children and young adults with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). With this new era of personalized cancer immunotherapy, multiple challenges are present ranging from implementation of a CAR-T program to safe delivery of the drug, long-term toxicity monitoring and disease assessments. To address these issues, experts representing the American Society for Blood and Marrow Transplant (ASBMT), the European Group for Blood and Marrow Transplantation (EBMT), the International Society of Cell and Gene Therapy (ISCT), and the Foundation for the Accreditation of Cellular Therapy (FACT), formed a global CAR-T task force to identify and address key questions pertinent for hematologists and transplant physicians regarding the clinical use of anti CD19 CAR-T therapy in patients with B-ALL. This article presents an initial roadmap for navigating common clinical practice scenarios that will become more prevalent now that the first commercially available CAR-T product for B-ALL has been approved.

Hematopoietic stem cell transplantation (HSCT) has the potential to provide long-term survival and potential cure in patients with hematological malignancies and nonmalignant hematologic and autoimmune disorders. Conditioning regimens are designed to make room for the stem cell allograft by ablation of hosts’ immune system with the objective of sustained foreign immune cell engraftment. However, a number of short-term and long-term complications are associated with regimens used for preparatory conditioning in patients prior to allogenic stem cell infusion. Myeloablative conditioning regimens may cause significant toxicities, which include pancytopenia and injury to the orointestinal tract, brain, heart, liver, kidneys, and lungs. In order to mitigate such adverse events, reduced-intensity regimens have been developed. Emesis, anorexia, mucositis, and diarrhea are common orointestinal tract adverse events; sinusoidal obstruction syndrome is the main hepatotoxicity, and kidney injury may be either acute, or chronic. Treatment-induced defects in innate and adaptive immune function make patients susceptible to a variety of infections. Neurologic complications such as encephalopathy, seizures, cerebrovascular events, cognitive changes, and peripheral neuropathy are also not uncommon. Whereas early cardiac complications include congestive heart failure, arrhythmias, pericardial effusions, and endocarditis; whereas late cardiac complication also includes valvular heart disease. Lungs are a frequent site for such complications and include, acute pneumonitis, noncardiogenic pulmonary edema, diffuse alveolar hemorrhage, idiopathic pneumonia, as well as late obstructive and restrictive lung disease. Increased risk for bleeding may occur due to severe thrombocytopenia; additionally, hemolytic complications secondary to thrombotic microangiopathy may also be seen. Conditioning regimens may adversely affect the endocrine function such as, growth and skeletal development, metabolic functions, gonadal and sexual function, and fertility. Finally, high-dose chemotherapy and radiation therapy used in preparatory regimen may contribute towards an increased risk for cancers, mostly noted late after transplantation. In this chapter, a detailed review of complications associated with conditioning regimens given in preparation for stem cell allograft transplantation is presented.

Allogeneic hematopoietic cell transplantation (HCT) is a potentially curative treatment for patients with hematological malignancies. However, is associated with substantial rates of morbidity and mortality. We and others have shown that malglycemia is associated with adverse transplant outcome. Therefore, improving glycemic control may improve transplant outcome. In this prospective study we evaluated the feasibility of using Glucommander (a Computer-Guided Glucose Management System; CGGM) in order to achieve improved glucose control in hospitalized HCT patients. Nineteen adult patients contributed 21 separate instances on CGGM. Patients were on CGGM for a median of 43 h. Median initial blood glucose (BG) on CGGM was 244 mg/dL, and patients on 20 study instances reached the study BG target of 100-140 mg/dL after a median of 6 h. After BG reached the target range, the median average BG level per patient was 124 mg/dL. Six patients had a total of 10 events of BG

Background Allogeneic Hematopoietic Cell Transplantation (allo HCT) is currently the only curative therapy for high-risk hematologic malignancies due to graft versus tumor effect (GVT), but with the cost of Graft Versus Host Disease (GVHD). Despite extensive research, very few predictors of GVHD have been identified to date. To study the mechanisms of GVHD and GVT and to identify potential GVHD markers we applied a novel approach, called Transcriptome Fingerprint Assay (TFA), relying on high frequency sampling and blood transcriptome profiling. The TFA is a multiplex microfluidics q-PCR based assay, linked to a computational model for functional analyses, uniquely tailored to answer complex questions on immune perturbations through frequent profiling of gene expression signatures (Chaussabel et a 2014, Speake et a 2017). This approach has been successfully applied to stratify patients' prognosis in autoimmune and infectious diseases. Herein we report the discovery of an association between the expression abundance of a set of genes pre-transplant and the development of acute or chronic GVHD. Methods Seventy-eight patients candidate to allo-HCT were enrolled on the study. Patients donated micro-quantities of blood (50-600 microliters) prior to transplant conditioning, weekly until day 100 post-transplant and every 2 weeks thereafter until 2 years after transplant. Detailed clinical, laboratory and therapy annotations are captured during the follow-up. Gene expression of 264 immune-related genes for each sample are measured through Fluidigm BioMark high-throughput qPCR system. Data interpretation is performed through TFA modular analyses and correlated with the clinical annotations. Here we report the univariate analysis of gene expression data and its association with GVHD in 31 patients for whom data are available until at least day 169 post-transplant (169-672, median 513). SIgnificant genes were tested in a longitudinal analysis with 4 combined linear-mixed models, measuring the parameters: 1) groups (GVHD NO-GVHD) 2) time 3) interaction of groups over time. Results Patients' characteristics are summarized in Fig 1; 26 patients developed GVHD (12 acute GVHD only, 3 late acute GVHD, 1 overlap syndrome, 5 acute and chronic GVHD and 5 chronic GVHD only). By TFA analysis of pre-transplant samples, we identified 3 genes significantly downregulated among patients who developed GVHD (GPSM3, LRG1 and EPHX4), and 2 genes borderline (1.5 fold upregulated in the GVHD group, but with a non-significant p-value (Volcano-plot Fig.1). Of note, 12 of them were >4 fold upregulated, and their transcriptome pertains to B cells, T lymphocytes, monocytes, IFN, TNF, other cytokines and neutrophil activation. Moreover, among these genes IFN-genes were consistently represented by multiple IFN-modules, emerging as promising candidates. Longitudinal analyses confirmed that EPHX4 and GPSM3 distinguished the cohorts for all the measured parameters in the post-transplant time. EPHX4 increased over time in both groups but significantly more in the NO-GVHD group (with a fixed effect estimate of 2.05); GPSM3 instead decreased over time, again significantly more in the NO-GVHD group (with a fixed effect estimate of -0.95). LRG3 showed significance only for the parameter "time", resulting after transplant more abundant in the GVHD group, thus confirming previous literature data. IFN modules were also tested longitudinally, showing very distinct patterns in the cohorts, and several significant genes (IFI27, NT5C3, CCR1, LBA1, Fig.1) Conclusion Our data demonstrate that TFA may be used to assess expression of immune related genes in transplant patients and to identify specific pathways that may be predictive for GVHD development. Additional work is required to validate our data in a larger cohort and for better understanding of the role of those genes in triggering or protecting against GVHD. Upon completion of the study and the longitudinal analyses, we aim to define in depth transcriptome signatures of GVHD and relapse. Disclosures No relevant conflicts of interest to declare.

Cancer care advances have led to increased numbers of cancer survivors and to improved understanding of late effects of cancers and their therapies and survivorship issues. Long-term follow-up of cancer patients is crucial in preventing and managing many of the late effects of cancers and their therapies. However, the literature has highlighted the high rates of loss to follow-up (loss to FU) after cancer treatment, particularly in patients with hematologic malignancies. In this review, we performed a systematic search of published literature on issues pertaining to loss to FU in survivors of hematologic malignancies, highlighting the predictors of increased or decreased rates of loss to FU. We found that the literature on survivors of adulthood cancers is very limited, in contrast to articles discussing young adult survivors of childhood cancers. Predictors and barriers of loss to FU were found to be variable in different studies; however, they shared some common themes, including disease-related, logistic, financial and educational factors. Furthermore, we discuss the potential interventions to mitigate the loss to FU, along with discussing research priorities in this area.

Introduction Despite promising results in patients with relapsed/refractory B cell malignancies, CD19 CAR-T cell therapy can be associated with significant toxicities. Specifically, impaired hematopoietic recovery can be observed in a subset of patients. Objectives We aimed to describe hematopoietic recovery following CD19 CAR-T cell therapy, and to identify factors independently associated with this complication using multivariable modeling. Methods We retrospectively analyzed 125 adult patients with R/R acute lymphoblastic leukemia (ALL), non-Hodgkin lymphoma (NHL), and chronic lymphocytic leukemia (CLL), treated with CD19 CAR-T cells on a phase 1/2 clinical trial (characteristics summarized in Table 1). Criteria for neutropenia, thrombocytopenia, and recovery were defined as per the CIBMTR reporting guidelines. For competing risk analysis, an event was defined as ANC or platelet (Plt) recovery, with death, new cytotoxic therapy, relapse with marrow involvement in the absence of ANC or platelet recovery considered as competing events. Patient, disease, and CAR-T cell therapy-related variables were included in multivariable analyses. Results ANC and Plt recovery after CD19 CAR-T cell therapy were observed in 91% (ALL, 86%; CLL, 92%; NHL, 95%) and 86% (ALL, 86%; CLL, 86%; NHL, 84%) of patients, respectively. Median time to ANC recovery was 9 days and the probability of ANC recovery at day 28, 60, and 90 was 80% (95%CI, 73-87), 86% (95%CI, 80-92) and 89% (95%CI, 83-94), respectively. The probability of platelet recovery on the day of CAR-T cell infusion was 55% (95%CI, 46-64); rising to 74% (95%CI, 67-82), 83% (95%CI, 76-90), and 84% (95%CI, 77-90) on days 28, 60, and 90, respectively. A competing event was always observed in patients without ANC or Plt recovery. In multivariable analysis (Table 2), higher pre-lymphodepletion (LD) Plt count and higher peak CD8+ CAR-T cells in blood were associated with faster ANC recovery. A diagnosis of ALL was associated with slower ANC recovery. (CLL vs ALL, HR=1.60, p=0.02; NHL vs ALL, HR=2.07, p=0.007). Higher CRS grade was also associated with slower Plt recovery (HR=0.67 per grade increase, p Conclusion We observed impaired ANC and Plt recovery in a significant fraction of patients undergoing CD19 CAR-T cell therapy. Moreover, we identified factors independently associated with impaired hematopoietic recovery: disease type, prelymphodepletion Plt count, in vivo CAR-T cell expansion, and CRS grade. In addition to providing a benchmark for future CAR-T cell research, our findings suggest CRS prevention might improve hematopoietic recovery after CD19 CAR-T cell therapy.

Topic Significance & Study Purpose/Background/Rationale In 2016, a Comprehensive Cancer Center opened an ambulatory cellular immunotherapy clinic, partnered with an inpatient facility for hospitalization. The center provides FDA Approved CAR T Cell therapies as well investigational immune effector cell (IEC) products under Phase I/II clinical trials. To optimize patient care, a multidisciplinary workgroup developed IEC specific standard of care guidelines. These standards facilitate alignment of care between sites and promotes collaboration between nurses and providers to enhance patient outcomes. Methods, Intervention, & Analysis A multidisciplinary workgroup of content experts, including medical providers, clinical pharmacists, and nursing leaders collaborated to define best practices for patients undergoing cellular immunotherapy using current literature and clinical experience. As nurses play an integral role in providing direct patient care and symptom recognition, their collaboration in the development of the standard processes was essential. The guidelines helped define nursing policies, such as a focused nursing assessment for Cytokine Release Syndrome (CRS) and Neurotoxicity. This has been coupled with CRS & Neurotoxicity orders to ensure prompt and comprehensive treatment. Utilizing comprehensive standards which encompass organizational training, policy development, and patient care orders, ensures best care for patients and better health outcomes. Findings & Interpretation Implementation of novel IEC treatments is a multidisciplinary effort, with nurses playing a pivotal role in direct patient assessment and recognition of life-threatening complications. Standardization of practice is imperative to the delivery of consistent and comprehensive care to patients receiving these treatments. Discussion & Implications Nurses are on the forefront of caring for patients receiving innovative IEC treatments. Implementation of standard guidelines of care for this unique patient population aligns practices and streamlines patient care. The rapid development of novel IEC therapies requires defined evidence-based care practices be implemented with concurrent guidelines mandatorily assessed and updated based on emerging scientific data. Clinical standardization for IEC science implementation assures consistent best patient outcomes.

Contains fulltext : 190745.pdf (Publisher’s version ) (Closed access) Individuals with acute myeloid leukemia (AML) harboring an internal tandem duplication (ITD) in the gene encoding Fms-related tyrosine kinase 3 (FLT3) who relapse after allogeneic hematopoietic cell transplantation (allo-HCT) have a 1-year survival rate below 20%. We observed that sorafenib, a multitargeted tyrosine kinase inhibitor, increased IL-15 production by FLT3-ITD(+) leukemia cells. This synergized with the allogeneic CD8(+) T cell response, leading to long-term survival in six mouse models of FLT3-ITD(+) AML. Sorafenib-related IL-15 production caused an increase in CD8(+)CD107a(+)IFN-gamma(+) T cells with features of longevity (high levels of Bcl-2 and reduced PD-1 levels), which eradicated leukemia in secondary recipients. Mechanistically, sorafenib reduced expression of the transcription factor ATF4, thereby blocking negative regulation of interferon regulatory factor 7 (IRF7) activation, which enhanced IL-15 transcription. Both IRF7 knockdown and ATF4 overexpression in leukemia cells antagonized sorafenib-induced IL-15 production in vitro. Human FLT3-ITD(+) AML cells obtained from sorafenib responders following sorafenib therapy showed increased levels of IL-15, phosphorylated IRF7, and a transcriptionally active IRF7 chromatin state. The mitochondrial spare respiratory capacity and glycolytic capacity of CD8(+) T cells increased upon sorafenib treatment in sorafenib responders but not in nonresponders. Our findings indicate that the synergism of T cells and sorafenib is mediated via reduced ATF4 expression, causing activation of the IRF7-IL-15 axis in leukemia cells and thereby leading to metabolic reprogramming of leukemia-reactive T cells in humans. Therefore, sorafenib treatment has the potential to contribute to an immune-mediated cure of FLT3-ITD-mutant AML relapse, an otherwise fatal complication after allo-HCT.

Central nervous system (CNS) involvement is considered rare in adults with acute myeloid leukemia (AML). Therefore the cerebrospinal fluid (CSF) is typically examined only in patients with CNS symptoms. In our institution all AML patients considered candidates for allogeneic hematopoietic cell transplantation (HCT) undergo routine CSF examination as part of pre-transplant evaluation, allowing a relatively unbiased look at the incidence of AML in CSF pre-HCT. The primary objectives of this study were to assess the rate of CNS involvement in AML patients undergoing HCT, evaluate potential risk factors for CNS disease, and examine the effect of CNS involvement on transplant outcome. We retrospectively evaluated 327 adults with AML who underwent ablative HCT between January 2007 and December 2012. Median age was 49 (range 19-73). Twenty eight (8%) patients had favorable karyotype, 215 (66%) had intermediate-risk karyotype, and 84 (26%) had unfavorable karyotype at diagnosis. 206 patients had received a high intensity induction regimen (daily ARA-C dose ≥ 500mg/m2). At time of CSF evaluation pre-HCT, 166 patients were in CR without minimal residual disease (MRD), 65 had CR with MRD, 41 had CRp or CRi, and 54 were in relapse. Median follow-up time was 630 days. 22 patients (7%) had CSF AML involvement, as assessed morphologically or using multiparameter flow cytometry, at their pre-HCT evaluation. 5 of these patients had had prior (treated) CNS AML, 3 had had prior extramedullary disease (EMD) not involving the CNS, and 2 had had both prior CNS and other extramedullary disease (Fig. 1). The incidence of CSF AML at pre-HCT evaluation was 7/20 in patients with past CNS involvement vs. 15/ 307 in patients without history of CNS disease (p Classification and regression tree (CART) analysis identified 3 risk groups: (1) high = prior CNS disease (20 pts, 35% with CSF involvement at pre-HCT evaluation), (2) intermediate = no prior CNS involvement but in systemic relapse at pre-HCT CSF evaluation (51 pts, 16% with CSF involvement at pre-HCT evaluation), (3) low = no prior CNS disease and either CR (+/- MRD) or CRp/CRi (254 pts, 3% with CSF involvement at pre-HCT evaluation). All 22 patients with CSF involvement at pre-HCT evaluation received CNS-directed treatment (intrathecal chemotherapy +/- intracranial irradiation) and cleared their CNS disease prior to transplant. 18 patients also received CNS-directed therapy after HCT (between 1-6 intrathecal treatments). Nine and 8 patients with prior history of CNS disease, but with no evidence of CSF involvement at time of pre-HCT evaluation, received CNS-directed therapy before and after transplant respectively. Two of 35 patients (6%) with CNS disease at any time pre-HCT had documented CNS disease after transplant. While patients with CSF involvement at pre-HCT evaluation had shorter post-HCT survival (p = 0.002) (Fig. 2), multivariate analysis indicated that this reflected the association of CNS disease with poorer systemic response to therapy and evidence of systemic disease at time of transplant; specifically multivariate hazard rates were 1.48 for CNS involvement vs no CNS involvement (p = 0.17), contrasted with 3.62 for CR with MRD vs CR without MRD (p We conclude that CNS AML involvement pre-HCT is relatively uncommon (7%), is primarily associated with a history of prior CNS disease, and, when controlled, is not an independent factor in determining survival after HCT. Figure 1. CNS and extramedullary disease among 327 AML patients undergoing ablative HCT Figure 1. CNS and extramedullary disease among 327 AML patients undergoing ablative HCT Figure 2. Overall survival since transplant Figure 2. Overall survival since transplant Disclosures No relevant conflicts of interest to declare.

Is there a need for morphologic exam to detect relapse in AML if multi-parameter flow cytometry is employed?

Central nervous system (CNS) relapse after allogeneic hematopoietic stem cell transplantation (HSCT) confers a poor prognosis in adult patients with acute lymphoblastic leukemia (ALL). Preventing CNS relapse after HSCT remains a therapeutic challenge, and criteria for post-HSCT CNS prophylaxis have not been addressed. In a 3-center retrospective analysis, we reviewed the data for 457 adult patients with ALL who received a first allogeneic HSCT in first or second complete remission (CR). All patients received CNS prophylaxis as part of their upfront therapy for ALL, but post-transplantation CNS prophylaxis practice varied by institution and was administered to 48% of the patients. Eighteen patients (4%) developed CNS relapse after HSCT (isolated CNS relapse, n = 8; combined bone marrow and CNS relapse, n = 10). Patients with a previous history of CNS involvement with leukemia had a significantly higher rate for CNS relapse (P = .002), and pretransplantation CNS involvement was the only risk factor for post-transplantation CNS relapse found in this study. We failed to find a significant effect of post-transplantation CNS prophylaxis to prevent relapse after transplantation. Furthermore, no benefit for post-transplantation CNS prophylaxis could be detected when a subgroup analysis of patients with (P = .10) and without previous CNS involvement (P = .52) was performed. Finally, we could not find any significant effect for intensity of the transplantation conditioning regimen on CNS relapse after HSCT. In conclusion, CNS relapse is an uncommon event after HSCT for patients with ALL in CR1 or CR2, but with higher risk among patients with CNS involvement before transplantation. Furthermore, neither the use of post-HSCT CNS prophylaxis nor the intensity of the HSCT conditioning regimen made a significant difference in the rate of post-HSCT CNS relapse.

Improved survival after allogeneic hematopoietic cell transplantation (allo-HCT) enables us to learn more about potential late complications after HCT, one of which is metabolic syndrome. There are no studies investigating the prevalence or development of metabolic syndrome within the first year post-HCT in adult myeloablative transplant recipients.In this retrospective study, we evaluated the prevalence of and risk factors associated with metabolic syndrome early post-HCT in human subjects. Due to lack of complete information regarding all the factors that define metabolic syndrome, we evaluated metabolic characteristics using available objective data referred to as modified metabolic syndrome (MMS). The cohort included 785 patients.We demonstrated that the incidence of MMS was 34% pre-HCT, 48% at day 80 post-HCT, and 40% at 1 year post-HCT. MMS at day 80 post-HCT was predictive of having MMS at 1 year post-HCT.These results support the need for nutrition and lifestyle intervention to prevent and treat metabolic abnormalities among patients who survive the acute transplant period.

Introduction Allogeneic Hematopoietic Cell Transplantation (allo HCT) is currently the only curative therapy for high-risk hematologic malignancies due to the immune response of the donor cells against the malignant cells (graft versus tumor effect; GVT), but with the cost of Graft Versus Host Disease (GVHD). Despite extensive research, very few predictors of GVHD and GVT have been identified to date. Additionally, clinical GVHD diagnosis can be challenging due to chemotherapy-related or infection-related organ toxicity manifestations, which further complicate prediction and treatment stratification algorithms. In order to study the mechanisms of GVHD and GVT and to identify potential GVHD markers we apply a novel approach, called Transcriptome Fingerprint Assay (TFA), relying on high frequency sampling and blood transcript profiling. The TFA is a multiplex microfluidics q-PCR based assay linked with a computational model for modular functional transcriptome analyses, uniquely tailored to answer complex questions on immune perturbations through frequent profiling of gene expression signatures from < 1 ml of blood (Chaussabel and Baldwin. Nat Rev Immunol 2014, Speake et al. Clin Exp Immunol 2017). This approach has been successfully applied to stratify patients' prognosis in autoimmune and infectious diseases (Banchereau R et al. Cell 2016, Dunning et al. Nat Immunol 2018). In our study we use the TFA to capture longitudinal immune signatures as dynamic "snapshots" of the patient's immune system after HCT. Hypotheses Fluctuations over-time in gene expression of allo HCT patients' immune system reflect the pathologic/disease control programs (GVHD/GVT) and may be used to identify diagnostic and predictive biomarkers. GVHD/GVT control immune programs depend on the "inner" interface between the donor immune-system and the recipient, and are influenced by external variables, as infections or drugs. These variables can affect the immune system-related gene expression and can be measured. Objectives To systematically measure gene expression signatures in immune perturbations post-allo HCT, in order to: Identify GVHD-related immune signatures consistent with clinical diagnosis of GVHD.Predict and stratify therapy-resistant GVHD and severe chronic GVHD, according to immune signatures.Identify links (causative and consequential) between GVHD, GVT, relapse, and other post-transplant immune perturbations (e.g. infections). Methods Enroll 250 allo HCT patients to populate a "GVHD cohort" and a "non-GVHD cohort" of 50 patients each, and 50 donors (healthy controls cohort) . Patients donate micro-quantities of blood (50 to 600 microliters), weekly until day 100 post-transplant and every 2 weeks thereafter until 2 years after transplant. Detailed clinical, laboratory and therapy annotations are captured during the follow-up. Gene expression of 264 immune-related genes for each sample are measured through Fluidigm BioMark high throughput qPCR system, and normalized to the geometric mean Ct of 8 housekeeping genes. Data interpretation is performed through TFA modular analyses and correlated with the clinical annotations. Results Results of three series of patients' samples are shown to exemplify the potential of TFA as a method to study the mechanisms of GVHD and GVT. All three patients underwent myeloablative peripheral blood stem cell transplant from an HLA identical sibling donor. Two patients developed steroid responsive-acute GVHD (patient #1: GVHD stage I was diagnosed on day 38 post HCT, patient #4: GVHD stage III was diagnosed on day 21 post HCT). One patient (Patient #6) did not develop clinical GVHD, but routine skin biopsy on day 80 revealed apoptotic cells consistent with subclinical skin GVHD. Principal Component Analysis (PCA) of the three patients' series is shown in Figure 1, the dynamic transcriptomes according to TFA modules of patients #1 and #6 are shown in Figure 2, and representative TFA modular fingerprints are shown in Figure 3. Conclusion We anticipate that using the TFA approach will help to fill knowledge gaps instrumental to solve clinical dilemmas related to allo HCT complications, and to improve the clinical outcomes of allo HCT patients. Disclosures No relevant conflicts of interest to declare.

CD19-targeted chimeric antigen receptor (CAR)-T cell therapy is a novel treatment with promising results for patients with relapsed/refractory lymphoid malignancies. CAR-T cell therapy has known early toxicities of cytokine release syndrome (CRS) and neurotoxicity, but little is known about long-term neuropsychiatric adverse effects. We have utilized patient-reported outcomes (PROs), including PROMIS®measures, to assess outcomes of patients with relapse/refractory chronic lymphocytic leukemia (CLL), non-Hodgkin lymphoma (NHL), and acute lymphoblastic leukemia (ALL) who were treated with CD19-targeted CAR-T cells on a clinical trial in our institution (NCT01865617) and survived at least one year after treatment. Between October 2018 to February 2019, 52 patients (at their 1-5 year anniversary after CAR-T cell therapy) were sent a questionnaire. The questionnaire included the PROMIS Scale v1.2-Global Health and the PROMIS-29 Profile v2.1, as well as 30 additional questions, including questions pertaining to cognitive function. As of February 28, 2019, 40 questionnaires were returned (76.9% response rate) and were included in the analysis. Patients' characteristics are summarized in Table 1. Cognitive function was assessed by asking if patients had experienced difficulties with concentration, finding words, memory, or solving problems since their CAR-T cell therapy; answer "yes" to each of the questions received "1" point to determine the total cognitive difficulty score (0-4). PROMIS measures are standardized to a T-score metric, with a score of 50 representing the general US population mean. Clinically meaningful differences were defined as a 5-point difference in scores (1/2 standard deviation). The cohort's self-reported cognitive difficulties and PROMIS mean T scores are shown in Table 2. Mean T scores of PROMIS domains of Global Mental health, Global Physical Health, Social Function, anxiety, depression, fatigue, pain and sleep disturbance were not clinically meaningfully different from the mean in the general US population. However, 19 participants (47.5%) reported at least one cognitive difficulty and/or clinically meaningful depression and/or anxiety (Figure 1), and 7 participants (17.5%) scored ≤ 40 in Global Mental Health, indicating at least one standard deviation worse than the general population mean. On risk factor analysis, younger age was found to be associated with worse Global Mental Health (p=0.02), anxiety (p=0.01) and depression (p=0.01). Anxiety prior to CAR-T cell therapy was associated with increased likelihood of anxiety after CAR-T cell therapy (p=0.001). Multivariate analysis confirmed association between age and PROMIS Global Mental Health score (p=0.03). 15 participants (37.5%) reported cognitive difficulties post CAR-T cell therapy. On multivariate analysis, depression prior to CAR-T cell therapy was statistically significantly associated with higher likelihood of self-reported cognitive difficulties after CAR-T therapy (p=0.02) and there was a trend for association between acute neurotoxicity after CAR-T cell infusion and self-reported long-term cognitive difficulties (p=0.08). Having more cognitive difficulties was associated with worse Global Mental Health (p=0.0001) and worse Global Physical Health (p= 0.01). Similarly, worse scores for pain interference, sleep disturbance, fatigue, depression, anxiety, physical function, and social function were associated with more long-term self-reported cognitive difficulties (p=0.007,p=0.0003, p=0.00006, p=0.01, p=0.0007, p=0.003, p=0.0004 respectively). Our study demonstrates overall good neuropsychiatric outcomes in 40 long-term survivors after CAR-T cell therapy. However, despite good overall mean scores, nearly 50% of patients in the cohort reported at least one negative neuropsychiatric outcome (anxiety, depression or cognitive difficulty), and almost 20% scored at least one standard deviation lower than the general US population mean in Global Mental Health, indicating that there is a significant number of patients who would likely benefit from mental health services following CAR-T cell therapy. Younger age, anxiety and depression pre-CAR-T cell therapy, and acute neurotoxicity after CAR-T cell infusion may be risk factors for long-term neuropsychiatric problems in this patient population. Larger studies are needed to confirm these findings. Disclosures Shaw: Therakos: Other: Speaker Engagement. Lee:AstraZeneca: Research Funding; Incyte: Research Funding; Syndax: Research Funding; Amgen: Research Funding; Novartis: Research Funding; Takeda: Research Funding; Kadmon: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding. Turtle:T-CURX: Membership on an entity's Board of Directors or advisory committees; Kite/Gilead: Other: Ad hoc advisory board member; Caribou Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Allogene: Other: Ad hoc advisory board member; Humanigen: Other: Ad hoc advisory board member; Juno Therapeutics: Patents & Royalties: Co-inventor with staff from Juno Therapeutics; pending, Research Funding; Nektar Therapeutics: Other: Ad hoc advisory board member, Research Funding; Precision Biosciences: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Eureka Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Novartis: Other: Ad hoc advisory board member. Maloney:Juno Therapeutics: Honoraria, Patents & Royalties: patients pending , Research Funding; A2 Biotherapeutics: Honoraria, Other: Stock options ; Celgene,Kite Pharma: Honoraria, Research Funding; BioLine RX, Gilead,Genentech,Novartis: Honoraria.

Effective cellular therapy for human malignancies requires first identifying and validating an appropriate antigenic target, and then establishing in each patient a tumor-reactive T cell response of high avidity and high magnitude that is safe and can infiltrate and retain function in the tumor microenvironment. We have been exploring in preclinical models and clinical trials methods to reproducibly provide such responses by transfer of genetically engineered T cells that acquire target specificity by virtue of an introduced high affinity TCR. To identify candidate antigens in leukema, we examined purified human leukemic stem cells for over-expression of genes based on comparisons to purified human hematopoietic stem cells as well as normal somatic tissues. Our analysis revealed that WT1, a gene known to be associated with promoting leukemic transformation, is expressed in comparative abundance in human leukemic stem cells. Preclinical studies were then performed in a mouse model, and revealed that CD8 T cells specific for this oncogene with even higher avidity than can be detected in normal repertoires could be safely administered, with no evidence of toxicity to the normal tissues known to express low but detectable levels of WT1. For our initial clinical trial, poor prognosis leukemia patients who relapsed after hematopoietic cell transplant (HCT) were treated with transfer of WT1-specific CD8 T cells clones isolated and expanded in vitro from the HCT donor. This study demonstrated that such T cells were safe, mediated in vivo anti-leukemic activity, and were associated with maintenance of long-term remissions in some patients, but generating sufficient numbers of WT1-specific CD8 T cells with high avidity for the target in each patient represented a substantive problem. Therefore, to create a more predictably effective standardized reagent for treatment of patients with a tumor that expresses the target antigen and shares the associated MHC restricting allele, we pursued methods to genetically engineer patient T cells to acquire high avidity for the tumor target. This requires identifying a high affinity TCR and producing a vector that can achieve high-level expression of the genes encoding the Vα and Vβ genes of a TCR demonstrated to have high affinity for the target epitope. Therefore, we screened a large number of normal repertoires for the presence of high avidity WT1-specific CD8 T cells, and selected the T cell clone expressing the highest affinity TCR. We then incorporated changes in the TCR genes such as codon optimization to enhance expression, and introduced a point mutation in each chain to create a disulfide bond that minimizes the potential problem of mispairing of the introduced TCR chains with the endogenous TCR chains. We have now have now initiated a trial in which this high affinity, WT1-specific, HLA-A2-restricted TCR is being introduced into patient CD8 T cells with a lentiviral vector and the transduced cells are being infused into the patient. The early results from this trial appear promising in terms of both evidence of antileukemic activity and the capacity for the transferred cells to persist in patients, and we plan to begin very shortly another trial in patients with non-small cell lung cancer (NSCLC) utilizing this same TCR, as WT1 is also commonly overexpressed in NSCLC as well as many other malignancies. For many candidate target antigens that are also normal self-antigens, isolating high affinity TCRs may not be readily achieved from normal repertoires. Therefore, we have developed strategies to enhance the affinity of isolated TCRs with retention of specificity, including saturation mutagenesis of CDR3 regions and an in vitro thymic selection system that allows for capture of a more diverse set of high affinity specific TCR genes during TCR gene rearrangement. These approaches induce modifications to the TCR region that predominantly makes contacts with the peptide epitope rather than MHC, which is necessary to minimize the risk of off-target toxicity from promiscuous peptide/MHC recognition. However, it remains essential that such modified TCRs do not induce unanticipated tissue damage, and we are using bioinformatics as well as modeling in the mouse to uncover any potential for off-target toxicity. Unfortunately, providing a high avidity T cell response does not necessarily result in tumor eradication, as there are other substantive obstacles that can preclude even a T cell expressing a high affinity TCR from being effective. These impediments include the development of T cell dysfunction, particularly within the microenvironment of solid tumors, and we are using genetically engineered mouse models to elucidate the cellular and molecular pathways that need to be modulated to achieve meaningful therapeutic benefit in a variety of solid tumor settings, including pancreatic and ovarian cancer. Our preclinical therapy studies, particularly in a pancreatic ductal adenocarcinoma (PDA) model, already appear very promising, as we have demonstrated that T cells expressing a high affinity TCR targeting a tumor antigen expressed by PDA cells can infiltrate the tumor, mediate tumor lysis, modify the tumor stroma, and provide therapeutic benefit. We have already identified high affinity human TCRs specific for this tumor antigen, and plan to use the insights derived from these studies to initiate within the next 1-2 years clinical trials in human pancreatic and ovarian cancers. The genetically-engineered mouse models of spontaneously developing tumors we are using, which recapitulate many aspects of the analogous human cancer, are also making it possible to assess strategies to improve the efficacy of T cell therapy. These models have helped elucidate the importance of not only cell extrinsic mechanisms of regulation and dysfunction that render T cells unresponsive, particularly via inhibitory cells commonly present in the tumor microenvironment that interfere with an effector response such as the accumulation of regulatory CD4 T cells (Treg), myeloid derived suppressor cells (MDSC), and tumor-associated macrophages (TAM), but also the cell intrinsic mechanisms that derive in large part from persistent stimulation by the tumor antigen and ultimately can render T cells progressively dysfunctional, leading to epigenetic modifications that eventually result in non-responsive cells that cannot be readily rescued. These cumulative mechanisms highlight the difficulties eliciting and/or sustaining responses to tumor antigens. Strategies to disrupt inhibitory pathways by systemic administration of mAbs or cytokines are currently being pursued clinically, but such reagents globally disrupt inhibitory pathways which can have significant toxicity to the host. Therefore, we are evaluating strategies to sustain function and anti-tumor activity by genetically modifying T cells to enhance function and to be resistant to obstacles that prevent tumor eradication. As different tumor types exhibit unique characteristics and are capable of engaging distinct inhibitory pathways, improved understanding of the immunobiology of the tumor type to be treated will likely prove essential for designing effective therapies. However, the relatively straightforward means to use synthetic biology to genetically engineer T cells to acquire novel capacities to overcome inhibitory signals and function in the tumor microenvironment suggests that cancer therapy with engineered T cells will likely find an increasing role in the treatment of human cancers. Citation Format: Philip D. Greenberg, Tom M. Schmitt, Andrea Schietinger, Ingunn M. Stromnes, Sunil R. Hingorani, Shannon K. Oda, Rachel Perret, Kristin G. Anderson, Merav Bar, Aude G. Chapuis. Employing TCRs in engineered T cells to develop therapeutic reagents for effectively targeting malignancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr SY31-03. doi:10.1158/1538-7445.AM2015-SY31-03

Comparisons without hematopoietic cell transplantation (HCT) to myeloablative (MAC) or reduced-intensity HCT (RIC) for adults with acute lymphoblastic leukemia (ALL) in first minimal-residual-disease negative remission (MRD(Neg) CR1) are limited. Further, the importance of MRD(Neg) following salvage therapy (MRD(Neg) CR2+) is unknown. We evaluated 89 patients in MRD(Neg) CR1 after adult-inspired treatment: 33 received MAC (12 Philadelphia chromosome [Ph]+), 17 received RIC (13 Ph+), and 39 Deferred HCT (3 Ph+). Three-year overall survival (OS) estimates for MAC, RIC, and Deferred HCT were 71%, 69%, and 68%, while 3-year event-free survival (EFS) estimates were 65%, 54%, and 28%, respectively. Further, HCT in MRD(Neg) CR1 performed similarly to MRD(Neg) CR2+: 3-year OS estimates were 70% and 69%, and 3-year EFS estimates were 62% and 62%, respectively. In conclusion, adults with ALL in MRD(Neg) CR1 following adult-inspired therapy had similar OS with or without HCT, and HCT in MRD(Neg) CR2 + can yield long-term survival.