Outcome of donor-derived TAA-T cell therapy in patients with high-risk or relapsed acute leukemia post allogeneic BMT.

Patients with hematologic malignancies relapsing after allogeneic blood or marrow transplantation (BMT) have limited response to conventional salvage therapies, with an expected 1-year overall survival (OS) of <20%. We evaluated the safety and clinical outcomes following administration of a novel T-cell therapeutic targeting 3 tumor-associated antigens (TAA-T) in patients with acute leukemia who relapsed or were at high risk of relapse after allogeneic BMT. Lymphocytes obtained from the BMT donor were manufactured to target TAAs WT1, PRAME, and survivin, which are over-expressed and immunogenic in most hematologic malignancies. Patients received TAA-T infusions at doses of 0.5 to 4 × 107/m2. Twenty-three BMT recipients with relapsed/refractory (n = 11) and/or high-risk (n = 12) acute myeloid leukemia (n = 20) and acute lymphoblastic leukemia (n = 3) were infused posttransplant. No patient developed cytokine-release syndrome or neurotoxicity, and only 1 patient developed grade 3 graft-versus-host disease. Of the patients who relapsed post-BMT and received bridging therapy, the majority (n = 9/11) achieved complete hematologic remission before receiving TAA-T. Relapsed patients exhibited a 1-year OS of 36% and 1-year leukemia-free survival of 27.3% post-TAA-T. The poorest prognosis patients (relapsed <6 months after transplant) exhibited a 1-year OS of 42.8% postrelapse (n = 7). Median survival was not reached for high-risk patients who received preemptive TAA-T posttransplant (n = 12). Although as a phase 1 study, concomitant antileukemic therapy was allowed, TAA-T were safe and well tolerated, and sustained remissions in high-risk and relapsed patients were observed. Moreover, adoptively transferred TAA-T detected by T-cell receptor V-ß sequencing persisted up to at least 1 year postinfusion. This trial was registered at clinicaltrials.gov as #NCT02203903.

Identification of novel HLA-restricted preferentially expressed antigen in melanoma peptides to facilitate off-the-shelf tumor-associated antigen-specific T-cell therapies.

BACKGROUND AIMS: Preferentially expressed antigen in melanoma (PRAME) is a cancer/testis antigen that is overexpressed in many human malignancies and poorly expressed or absent in healthy tissues, making it a good target for anti-cancer immunotherapy. Development of an effective off-the-shelf adoptive T-cell therapy for patients with relapsed or refractory solid tumors and hematological malignancies expressing PRAME antigen requires the identification of major histocompatibility complex (MHC) class I and II PRAME antigens recognized by the tumor-associated antigen (TAA) T-cell product. The authors therefore set out to extend the repertoire of HLA-restricted PRAME peptide epitopes beyond the few already characterized. METHODS: Peptide libraries of 125 overlapping 15-mer peptides spanning the entire PRAME protein sequence were used to identify HLA class I- and II-restricted epitopes. The authors also determined the HLA restriction of the identified epitopes. RESULTS: PRAME-specific T-cell products were successfully generated from peripheral blood mononuclear cells of 12 healthy donors. Ex vivo-expanded T cells were polyclonal, consisting of both CD4+ and CD8+ T cells, which elicited anti-tumor activity in vitro. Nine MHC class I-restricted PRAME epitopes were identified (seven novel and two previously described). The authors also characterized 16 individual 15-mer peptide sequences confirmed as CD4-restricted epitopes. CONCLUSIONS: TAA T cells derived from healthy donors recognize a broad range of CD4+ and CD8+ HLA-restricted PRAME epitopes, which could be used to select suitable donors for generating off-the-shelf TAA-specific T cells.

Immune Response Following Quadrivalent Human Papillomavirus Vaccination in Women After Hematopoietic Allogeneic Stem Cell Transplant: A Nonrandomized Clinical Trial.

Importance: Human papillomavirus (HPV) infection is found in about 40% of women who survive allogeneic hematopoietic stem cell transplant and can induce subsequent neoplasms. Objective: To determine the safety and immunogenicity of the quadrivalent HPV vaccine (HPV-6, -11, -16, and -18) in clinically stable women post-allogeneic transplant compared with female healthy volunteers. Interventions: Participants received the quadrivalent HPV vaccine in intramuscular injections on days 1 and 2 and then 6 months later. Design, Setting, and Participants: This prospective, open-label phase-1 study was conducted in a government clinical research hospital and included clinically stable women posttransplant who were or were not receiving immunosuppressive therapy compared with healthy female volunteers age 18 to 50 years who were followed up or a year after first receiving quadrivalent HPV vaccination. The study was conducted from June 2, 2010, until July 19, 2016. After all of the results of the study assays were completed and available in early 2018, the analysis took place from February 2018 to May 2019. Main Outcomes and Measures: Anti-HPV-6, -11, -16, and -18-specific antibody responses using L1 virus-like particle enzyme-linked immunosorbent assay were measured in serum before (day 1) and at months 7 and 12 postvaccination. Anti-HPV-16 and -18 neutralization titers were determined using a pseudovirion-based neutralization assay. Results: Of 64 vaccinated women, 23 (35.9%) were receiving immunosuppressive therapy (median age, 34 years [range, 18-48 years]; median 1.2 years posttransplant), 21 (32.8%) were not receiving immunosuppression (median age, 32 years [range, 18-49 years]; median 2.5 years posttransplant), and 20 (31.3%) were healthy volunteers (median age, 32 years [range, 23-45 years]). After vaccine series completion, 18 of 23 patients receiving immunosuppression (78.3%), 20 of 21 not receiving immunosuppression (95.2%), and all 20 volunteers developed antibody responses to all quadrivalent HPV vaccine types (P = .04, comparing the 3 groups). Geometric mean antibody levels for each HPV type were higher at months 7 and 12 than at baseline in each group (all geometric mean ratios >1; P < .001) but not significantly different across groups. Antibody and neutralization titers for anti-HPV-16 and anti-HPV-18 correlated at month 7 (Spearman ρ = 0.92; P < .001 for both). Adverse events were mild and not different across groups. Conclusions and Relevance: Treatment with the HPV vaccination was followed by strong, functionally active antibody responses against vaccine-related HPV types and no serious adverse events. These findings suggest that HPV vaccination may be safely administered to women posttransplant to potentially reduce HPV infection and related neoplasia. Trial Registration: ClinicalTrials.gov Identifier: NCT01092195.

Framingham Risk Score Is an Ineffective Screening Strategy for Coronary Heart Disease in Long-Term Allogeneic Hematopoietic Cell Transplant Survivors.

Long-term allogeneic hematopoietic cell transplant (allo-HCT) survivors suffer an elevated risk of coronary heart disease (CHD). We conducted a prospective, nonrandomized, cross-sectional study to screen asymptomatic survivors at a single allo-HCT center using cardiac computed tomography (CT) involving coronary CT angiography (CCTA) and the coronary artery calcium (CAC) score. Seventy-nine subjects with a median age of 39 years at allo-HCT and a median follow-up interval of 8 years were evaluated for CHD by Framingham Risk Score (FRS) and cardiac CT. CHD was detected in 33 of 79 (42%) subjects; 91% of lesions were nonobstructive, 19.5% of were noncalcified and 30% had associated valvular calcification. Overall, CAC was significantly superior to FRS in detecting early CHD in allo-HCT survivors [∆C = 0.25; P < 0.0001]. While both FRS and CAC were highly, >95% specific, FRS had a sensitivity, positive and negative predictive values of only 28% (95% CI, 14%-47%), 90% (95% CI, 55%-100%) and 60% (95% CI, 47%-73%), respectively. In contrast, the sensitivity, positive and negative predictive values of CAC were 78% (95% CI, 60%-91%), 96% (95% CI, 80%-100%) and 83% (95% CI, 69%-93%), respectively. Significantly, cardiac CT detected CHD in 23 of the 68 (34%) survivors deemed to have a low Framingham risk. Radiation exposure during cardiac CT was negligible, and there were no adverse events. In conclusion, CAC score with or without CCTA is a safe, feasible and sensitive screening technique for CHD. The FRS greatly underestimates CHD in allo-HCT survivors.

Acute myeloid leukaemia and the immune system: implications for immunotherapy.

The recognition of the curative potential of the graft-versus-leukaemia effect for patients with acute myeloid leukaemia (AML) undergoing stem cell transplantation, and the emergence of immunotherapy as a powerful weapon to treat cancer, has spurred the exploration of the immune landscape of AML to apply immunotherapeutic approaches to curing the disease. While current concepts of cancer immunology and immunotherapy have relevance, there are also unique aspects of immune dysregulation in AML to be considered when designing rational immunotherapy for this leukaemia. This is timely because rapid advances in cancer immunobiology, together with technological developments have opened up the field of immunotherapy for malignant disease. Here the current knowledge of AML immunobiology is summarized together with a description of new immunotherapies to counter immunosuppression and immune evasion by AML. Recent advances in treatment with recombinant antibodies, adoptive cell therapy and vaccines and their future promise in AML treatment are reviewed.

T-cell receptor sequencing demonstrates persistence of virus-specific T cells after antiviral immunotherapy.

Viral infections are a serious cause of morbidity and mortality following haematopoietic stem cell transplantation (HSCT). Adoptive cellular therapy with virus-specific T cells (VSTs) has been successful in preventing or treating targeted viruses in prior studies, but the composition of ex vivo expanded VST and the critical cell populations that mediate antiviral activity in vivo are not well defined. We utilized deep sequencing of the T-cell receptor beta chain (TCRB) in order to classify and track VST populations in 12 patients who received VSTs following HSCT to prevent or treat viral infections. TCRB sequencing was performed on sorted VST products and patient peripheral blood mononuclear cells samples. TCRB diversity was gauged using the Shannon entropy index, and repertoire similarity determined using the Morisita-Horn index. Similarity indices reflected an early change in TCRB diversity in eight patients, and TCRB clonotypes corresponding to targeted viral epitopes expanded in eight patients. TCRB repertoire diversity increased in nine patients, and correlated with cytomegalovirus (CMV) viral load following VST infusion (P = 0·0071). These findings demonstrate that allogeneic VSTs can be tracked via TCRB sequencing, and suggests that T-cell receptor repertoire diversity may be critical for the control of CMV reactivation after HSCT.

Over-expression of PD-1 Does Not Predict Leukemic Relapse after Allogeneic Stem Cell Transplantation.

Blockade of the T-cell exhaustion marker PD-1 to re-energize the immune response is emerging as a promising cancer treatment. Relapse of hematologic malignancy after allogeneic stem cell transplantation limits the success of this approach, and PD-1 blockade may hold therapeutic promise. However, PD-1 expression and its relationship with post-transplant relapse is poorly described. Because the donor immunity is activated by alloresponses, PD-1 expression may differ from nontransplanted individuals, and PD-1 blockade could risk graft-versus-host disease. Here we analyzed T-cell exhaustion marker kinetics and their relationship with leukemia relapse in 85 patients undergoing myeloablative T-cell-depleted HLA-matched stem cell transplantation. At a median follow-up of 3.5 years, 35 (44%) patients relapsed. PD-1 expression in CD4 and CD8 T cells was comparably elevated in relapsed and nonrelapsed cohorts. Helios+ regulatory T cells and CD8 effector memory cells at day 30 emerged as independent predictors of relapse. Although leukemia antigen-specific T cells did not overexpress PD-1, single-cell analysis revealed LAG3 and TIM3 overexpression at relapse. These findings indicate that PD-1 is an unreliable marker for leukemia-specific T-cell exhaustion in relapsing patients but implies other exhaustion markers and suppressor cells as relapse biomarkers.

Cytomegalovirus Infection Incidence and Risk Factors Across Diverse Hematopoietic Cell Transplantation Platforms Using a Standardized Monitoring and Treatment Approach: A Comprehensive Evaluation from a Single Institution.

Human cytomegalovirus (CMV) infection and disease remains a significant cause of morbidity and mortality for hematopoietic cell transplantation (HCT) recipients. Disruption of or weak reconstitution of virus-specific cellular immune function, such as with certain HCT approaches, poses significant risk for CMV-related complications. The incidence of and risk factors for CMV infection and the nature of CMV disease were evaluated retrospectively among 356 consecutive HCT recipients transplanted at the National Institutes of Health using all graft sources, including bone marrow, peripheral blood stem cell (PBSC), and umbilical cord blood (UCB), and a range of in vivo and ex vivo approaches for graft-versus-host disease (GVHD) prophylaxis. The cumulative incidence of CMV infection was higher for CMV-seropositive recipients at 33%, regardless of donor CMV serostatus. Patients transplanted with CMV-seropositive donors had a significantly shorter duration of antiviral therapy. Among graft sources UCB was associated with the highest cumulative incidence of CMV infection at 65% and significantly longer treatment duration at a median of 36days, whereas PBSC HCT was associated with the lowest incidence at 26% and the shortest CMV treatment duration at a median of 21days. There were significant differences in the cumulative incidence of CMV infection by T cell manipulation strategy when systemic steroids were included as a risk-modifying event. Over one-third of CMV infections occurred in the setting of systemic steroid administration. CMV disease occurred in 5% of HCT recipients, with 70% of cases in the setting of treatment for GVHD. Although factors related to serostatus, graft source, and GVHD prophylaxis were associated with varied CMV infection incidence, unplanned post-HCT corticosteroid therapy contributed greatly to the incidence of both CMV infection and disease across HCT approaches, highlighting this post-HCT intervention as a key time to potentially tailor the approach to monitoring, preemptive therapy, and even prophylaxis.

Transfusion support for matched sibling allogeneic hematopoietic stem cell transplantation (1993-2010): factors that predict intensity and time to transfusion independence.

BACKGROUND: Patients undergoing allogeneic hematopoietic stem cell transplant require variable, often extensive transfusion support. Identification of factors that predict urgent, intensive, or special needs should improve management of these patients. STUDY DESIGN AND METHODS: This is a retrospective study of red blood cell (RBC) and platelet transfusion support provided for sequential matched sibling donor allogeneic transplants conducted at the Clinical Center, National Institutes of Health, from 1993 through 2010. Factors potentially important for predicting quantity of RBC and platelet transfusions, and time to transfusion independence through Day 200 following hematopoietic stem cell transplantation were evaluated. RESULTS: Subjects (n = 800) received 10,591 RBC and 10,199 platelet transfusions. Multivariable analysis demonstrated that the need for RBC pretransplant, CD34+ dose, transplant year, diagnostic category, and ABO match were significantly independently associated with quantity of RBC transfusions during Days 0 through 30. Only pretransplant need for RBCs, CD34+ dose, and transplant year had significance during Days 0 through 100. Similar analyses for quantity of platelet transfusions demonstrated that for both Days 0 through 30 and 0 through 100 significant factors were need for platelet support before transplant, CD34+ dose, transplant year, and transplant regimen. Of note, long term, during Days 101 through 200, only CD34+ dose remained significant for quantity of RBC and platelet transfusions. Analysis of time to transfusion independence demonstrated that patients with ABO major mismatches required longer to achieve freedom from RBC transfusion support compared to identical matches or those with minor mismatches. CONCLUSION: Patient-specific factors including CD34+ dose and ABO match of the graft should be given particular consideration by transfusion services when planning support of patients receiving allogeneic hematopoietic stem cell transplant.

Divergent roles for antigenic drive in the aetiology of primary versus dasatinib-associated CD8+ TCR-Vß+ expansions.

CD8+ T-cell expansions are the primary manifestation of T-cell large granular lymphocytic leukemia (T-LGLL), which is frequently accompanied by neutropenia and rheumatoid arthritis, and also occur as a secondary phenomenon in leukemia patients treated with dasatinib, notably in association with various drug-induced side-effects. However, the mechanisms that underlie the genesis and maintenance of expanded CD8+ T-cell receptor (TCR)-Vß+ populations in these patient groups have yet to be fully defined. In this study, we performed a comprehensive phenotypic and clonotypic assessment of expanded (TCR-Vß+) and residual (TCR-Vß-) CD8+ T-cell populations in T-LGLL and dasatinib-treated chronic myelogenous leukemia (CML) patients. The dominant CD8+ TCR-Vß+ expansions in T-LGLL patients were largely monoclonal and highly differentiated, whereas the dominant CD8+ TCR-Vß+ expansions in dasatinib-treated CML patients were oligoclonal or polyclonal, and displayed a broad range of memory phenotypes. These contrasting features suggest divergent roles for antigenic drive in the immunopathogenesis of primary versus dasatinib-associated CD8+ TCR-Vß+ expansions.

Reprint of: Virus-Specific T Cells: Broadening Applicability.

Virus infection remains an appreciable cause of morbidity and mortality after hematopoietic stem cell transplantation (HSCT). Although pharmacotherapy and/or antibody therapy may help prevent or treat viral disease, these drugs are expensive, toxic, and often ineffective due to primary or secondary resistance. Further, effective treatments are limited for many infections (eg, adenovirus, BK virus), which are increasingly detected after alternative donor transplants. These deficiencies in conventional therapeutics have increased interest in an immunotherapeutic approach to viral disorders, leading to adoptive transfer of virus-specific cytotoxic T lymphocytes (VSTs), which can rapidly reconstitute antiviral immunity post-transplantation without causing graft-versus-host disease. This review will explore how the VST field has improved outcomes for many patients with life-threatening viral infections after HSCT, and how to broaden applicability beyond the "patient-specific" products, as well as extending to other viral diseases even outside the context of HSCT.

Distinct Biomarker Profiles in Ex Vivo T Cell Depletion Graft Manipulation Strategies: CD34+ Selection versus CD3+/19+ Depletion in Matched Sibling Allogeneic Peripheral Blood Stem Cell Transplantation.

Various approaches have been developed for ex vivo T cell depletion in allogeneic stem cell transplantation to prevent graft-versus-host disease (GVHD). Direct comparisons of T cell depletion strategies have not been well studied, however. We evaluated cellular and plasma biomarkers in 2 different graft manipulation strategies, CD3+CD19+ cell depletion (CD3/19D) versus CD34+ selection (CD34S), and their associations with clinical outcomes. Identical conditions, including the myeloablative preparative regimen, HLA-identical sibling donor, GVHD prophylaxis, and graft source, were used in the 2 cohorts. Major clinical outcomes were similar in the 2 groups in terms of overall survival, nonrelapse mortality, and cumulative incidence of relapse; however, the cumulative incidence of acute GVHD trended to be higher in the CD3/19D cohort compared with the CD34S cohort. A distinct biomarker profile was noted in the CD3/19D cohort: higher levels of ST2, impaired Helios- FoxP3+Treg reconstitution, and rapid reconstitution of naïve, Th2, and Th17 CD4 cells in the early post-transplantation period. In vitro graft replication studies confirmed that CD3/19D disproportionately depleted Tregs and other CD4 subset repertoires in the graft. This study confirms the utility of biomarker monitoring, which can be directly correlated with biological consequences and possible future therapeutic indications.

Virus-Specific T Cells: Broadening Applicability.

Virus infection remains an appreciable cause of morbidity and mortality after hematopoietic stem cell transplantation (HSCT). Although pharmacotherapy and/or antibody therapy may help prevent or treat viral disease, these drugs are expensive, toxic, and often ineffective due to primary or secondary resistance. Further, effective treatments are limited for many infections (eg, adenovirus, BK virus), which are increasingly detected after alternative donor transplants. These deficiencies in conventional therapeutics have increased interest in an immunotherapeutic approach to viral disorders, leading to adoptive transfer of virus-specific cytotoxic T lymphocytes (VSTs), which can rapidly reconstitute antiviral immunity post-transplantation without causing graft-versus-host disease. This review will explore how the VST field has improved outcomes for many patients with life-threatening viral infections after HSCT, and how to broaden applicability beyond the "patient-specific" products, as well as extending to other viral diseases even outside the context of HSCT.

Acquired RhD mosaicism identifies fibrotic transformation of thrombopoietin receptor-mutated essential thrombocythemia.

BACKGROUND: Acquired copy-neutral loss of heterozygosity has been described in myeloid malignant progression with an otherwise normal karyotype. CASE REPORT: A 65-year-old woman with MPL-mutated essential thrombocythemia and progression to myelofibrosis was noted upon routine pretransplant testing to have mixed field reactivity with anti-D and an historic discrepancy in RhD type. The patient had never received transfusions or transplantation. RESULTS: Gel immunoagglutination revealed group A red blood cells and a mixed-field reaction for the D phenotype, with a predominant D-negative population and a small subset of circulating red blood cells carrying the D antigen. Subsequent genomic microarray single nucleotide polymorphism profiling revealed copy-neutral loss of heterozygosity of chromosome 1 p36.33-p34.2, a known molecular mechanism underlying fibrotic progression of MPL-mutated essential thrombocythemia. The chromosomal region affected by this copy-neutral loss of heterozygosity encompassed the RHD, RHCE, and MPL genes. We propose a model of chronological molecular events that is supported by RHD zygosity assays in peripheral lymphoid and myeloid-derived cells. CONCLUSION: Copy-neutral loss of heterozygosity events that lead to clonal selection and myeloid malignant progression may also affect the expression of adjacent unrelated genes, including those encoding for blood group antigens. Detection of mixed-field reactions and investigation of discrepant blood typing results are important for proper transfusion support of these patients and can provide useful surrogate markers of myeloproliferative disease progression.

Cellular immune profiling after sequential clofarabine and lenalidomide for high risk myelodysplastic syndromes and acute myeloid leukemia.

Patients with high risk myelodysplastic syndromes (MDS) and acute myelogenous leukemia (AML) are commonly older with multiple co-morbidities, rendering them unsuitable for intensive induction chemotherapy or transplantation. We report preliminary cellular immune profiling of four cases receiving sequential clofarabine and lenalidomide for high risk MDS and AML in a phase I study. Our results highlight the potential of immune profiling for monitoring immune-modifying agents in high risk MDS and AML.

Ex vivo T-cell-depleted allogeneic stem cell transplantation for hematologic malignancies: The search for an optimum transplant T-cell dose and T-cell add-back strategy.

BACKGROUND: T-cell depletion (TCD) of allogeneic stem cell transplants (SCT) can reduce graft-versus-host disease but may negatively affect transplant outcome by delaying immune recovery. To optimize TCD in HLA-matched siblings with hematologic malignancies, we explored varying the transplant CD3+ T-cell dose between 2 and 50 × 104/kg (corresponding to 3-4 log depletion) and studied the impact of 0-6 × 107/kg CD3+ donor lymphocyte infusion (DLI) "add-back" on immune recovery post-SCT. METHODS: Two hundred seventeen consecutive patients (age range, 10-75 years) with hematologic malignancy (excluding chronic leukemias) underwent ex vivo TCD SCT from HLA-identical sibling donors from 1994-2015. Ninety-four patients had standard-risk disease (first remission acute leukemia [AL] and early stage myelodysplastic syndromes [MDS]) and 123 had high-risk disease (AL beyond first complete remission, advanced MDS or refractory B-cell malignancy). RESULTS: Median follow-up was 8.5 years. At 20 years post-SCT, overall survival (OS) was 40%, nonrelapse mortality (NRM) was 27% and relapse incidence was 39%. Factors affecting outcome in multivariate analysis were transplantation era, with OS increasing from 38% in the period 1994-2000 to 58% in 2011-2015, disease risk (hazard ratio [HR], 1.68 for high risk) and increasing age (HR, 1.19 per decade). Neither the T-cell dose or the add back of T cells in the first 100 days had any effect on OS, NRM and relapse. CONCLUSIONS: Outcomes for TCD SCT have greatly improved. However, our data do not support the need to precisely manipulate transplant CD3+ T-cell dose provided at least 3-log depletion is achieved or the use of T-cell add-back. Future improvements for TCD SCT await better strategies to prevent relapse, especially in high-risk recipients.