Influence of Germline Genetics on Tacrolimus Pharmacokinetics and Pharmacodynamics in Allogeneic Hematopoietic Stem Cell Transplant Patients.

Tacrolimus exhibits high inter-patient pharmacokinetics (PK) variability, as well as a narrow therapeutic index, and therefore requires therapeutic drug monitoring. Germline mutations in cytochrome P450 isoforms 4 and 5 genes (CYP3A4/5) and the ATP-binding cassette B1 gene (ABCB1) may contribute to interindividual tacrolimus PK variability, which may impact clinical outcomes among allogeneic hematopoietic stem cell transplantation (HSCT) patients. In this study, 252 adult patients who received tacrolimus for acute graft versus host disease (aGVHD) prophylaxis after allogeneic HSCT were genotyped to evaluate if germline genetic variants associated with tacrolimus PK and pharmacodynamic (PD) variability. Significant associations were detected between germline variants in CYP3A4/5 and ABCB1 and PK endpoints (e.g., median steady-state tacrolimus concentrations and time to goal tacrolimus concentration). However, significant associations were not observed between CYP3A4/5 or ABCB1 germline variants and PD endpoints (e.g., aGVHD and treatment-emergent nephrotoxicity). Decreased age and CYP3A5*1/*1 genotype were independently associated with subtherapeutic tacrolimus trough concentrations while CYP3A5*1*3 or CYP3A5*3/*3 genotypes, myeloablative allogeneic HSCT conditioning regimen (MAC) and increased weight were independently associated with supratherapeutic tacrolimus trough concentrations. Future lines of prospective research inquiry are warranted to use both germline genetic and clinical data to develop precision dosing tools that will optimize both tacrolimus dosing and clinical outcomes among adult HSCT patients.

Evaluation of a Test Dose Strategy for Pharmacokinetically-Guided Busulfan Dosing for Hematopoietic Stem Cell Transplantation.

Targeted busulfan dosing helps limit chemotherapy-related toxicity and optimize disease outcomes in hematopoietic stem cell transplantation (HCT). The objective of this study was to evaluate busulfan exposure from a pharmacokinetic (PK)-guided dosing strategy using a test dose. This retrospective evaluation included adult patients who underwent HCT at our institution with busulfan-based myeloablative (>9 mg/kg) conditioning between January 2014 and October 2015. A weight-based test dose of 0.8 mg/kg was used with PK assessments to predict area under the curve (AUCpred) achieved with weight-based dosing, with a target AUC of 4800 µM*minute (AUCtarget). PK from the test dose was then used to calculate a PK-guided first myeloablative busulfan dose. PK assessments were also done after the first dose to assess if the goal area under the curve (AUC) had been achieved (AUCfirst). A PK-guided first dose resulted in achievement of target AUC with target ranges of ±10% in 50% of patients, ±15% in 75%, and ±20% in 94%. This was an improved rate of target achievement compared with the 33%, 44%, and 63% of patients who achieved the desired AUC for these respective target ranges when using weight-based dosing (P = .12, .004, and <.001, respectively). The PK-guided strategy also decreased the variability of AUC from 3.6-fold in AUCpred from the weight-based test doses (2700.8 to 9631 µM*minute; SD, 1211.6 µM*minute) to 1.8-fold in AUCfirst from the PK-guided first doses (3672.1 to 6609.8 µM*minute; SD, 574.7 µM*minute). This reflects a 2-fold improvement in AUC variability with a PK-guided dosing strategy. This is also improved from the 3-fold variability in AUC reported in other studies. Weight and body surface area were significantly associated with the likelihood of AUCfirst being within the ±10% target range (P = .04 for both associations). There was no significant association between AUCfirst and death, relapse, or a composite of the two. These results demonstrate a significant improvement in target AUC attainment and less interpatient variability with PK-guided dosing using a test dose strategy compared with weight-based dosing.

Effectiveness of an algorithm-based approach to the utilization of plerixafor in patients undergoing chemotherapy-based stem cell mobilization.

Autologous stem cell transplantation remains a mainstay of therapy for diseases such as multiple myeloma and relapsed lymphoma. The use of plerixafor has been shown to augment the ability to collect adequate stem cells, but the optimal use of this agent when used with chemotherapy is not yet clear. We utilized an algorithm-based approach with the addition of plerixafor to 54 patients undergoing chemomobilization with reduced-dose etoposide who had a less than optimal preapheresis CD34(+) cell count. We used a CD34(+) precount of 20 cells/µL as a threshold to initiate stem cell apheresis. Ninety-four percent of patients were successfully collected and proceeded to transplantation. Fourteen of 51 (28%) patients who successfully collected required plerixafor to augment stem cell yield. Of the patients who successfully collected, 94% (89% of the entire population) were able to collect in 2 or fewer days. Compared with previous data from our institution, the rate of patients collecting > 4 × 10(6) CD34(+) cells/kg in a single collection was increased from 39% to 69%. The safety profile of this approach was acceptable. The use of this algorithm-based method to determine when and whether to add plerixafor to chemomobilization was shown to be a successful and cost-effective approach to stem cell collection.

Anti-CD30 CAR T cells as consolidation after autologous haematopoietic stem-cell transplantation in patients with high-risk CD30+ lymphoma: a phase 1 study.

BACKGROUND: Chimeric antigen receptor (CAR) T cells targeting CD30 are safe and have promising activity when preceded by lymphodepleting chemotherapy. We aimed to determine the safety of anti-CD30 CAR T cells as consolidation after autologous haematopoietic stem-cell transplantation (HSCT) in patients with CD30+ lymphoma at high risk of relapse. METHODS: This phase 1 dose-escalation study was performed at two sites in the USA. Patients aged 3 years and older, with classical Hodgkin lymphoma or non-Hodgkin lymphoma with CD30+ disease documented by immunohistochemistry, and a Karnofsky performance score of more than 60% planned for autologous HSCT were eligible if they were considered high risk for relapse as defined by primary refractory disease or relapse within 12 months of initial therapy or extranodal involvement at the start of pre-transplantation salvage therapy. Patients received a single infusion of CAR T cells (2 × 107 CAR T cells per m2, 1 × 108 CAR T cells per m2, or 2 × 108 CAR T cells per m2) as consolidation after trilineage haematopoietic engraftment (defined as absolute neutrophil count ≥500 cells per µL for 3 days, platelet count ≥25 × 109 platelets per L without transfusion for 5 days, and haemoglobin ≥8 g/dL without transfusion for 5 days) following carmustine, etoposide, cytarabine, and melphalan (BEAM) and HSCT. The primary endpoint was the determination of the maximum tolerated dose, which was based on the rate of dose-limiting toxicity in patients who received CAR T-cell infusion. This study is registered with ClinicalTrials.gov (NCT02663297) and enrolment is complete. FINDINGS: Between June 7, 2016, and Nov 30, 2020, 21 patients were enrolled and 18 patients (11 with Hodgkin lymphoma, six with T-cell lymphoma, one with grey zone lymphoma) were infused with anti-CD30 CAR T cells at a median of 22 days (range 16-44) after autologous HSCT. There were no dose-limiting toxicities observed, so the highest dose tested, 2 × 108 CAR T cells per m2, was determined to be the maximum tolerated dose. One patient had grade 1 cytokine release syndrome. The most common grade 3-4 adverse events were lymphopenia (two [11%] of 18) and leukopenia (two [11%] of 18). There were no treatment-related deaths. Two patients developed secondary malignancies approximately 2 years and 2·5 years following treatment (one stage 4 non-small cell lung cancer and one testicular cancer), but these were judged unrelated to treatment. At a median follow-up of 48·2 months (IQR 27·5-60·7) post-infusion, the median progression-free survival for all treated patients (n=18) was 32·3 months (95% CI 4·6 months to not estimable) and the median progression-free survival for treated patients with Hodgkin lymphoma (n=11) has not been reached. The median overall survival for all treated patients has not been reached. INTERPRETATION: Anti-CD30 CAR T-cell infusion as consolidation after BEAM and autologous HSCT is safe, with low rates of toxicity and encouraging preliminary activity in patients with Hodgkin lymphoma at high risk of relapse, highlighting the need for larger studies to confirm these findings. FUNDING: National Heart Lung and Blood Institute, University Cancer Research Fund at the Lineberger Comprehensive Cancer Center.

Three-Year Outcomes in Recipients of Mismatched Unrelated Bone Marrow Donor Transplants Using Post-Transplantation Cyclophosphamide: Follow-Up from a National Marrow Donor Program-Sponsored Prospective Clinical Trial.

The use of post-transplantation cyclophosphamide (PTCy) as graft-versus-host disease (GVHD) prophylaxis has resulted in reductions in GVHD and improved outcomes in allogeneic hematopoietic cell transplantation (HCT) using HLA-mismatched related donors. We report the 3-year outcomes of the first multicenter prospective clinical trial using PTCy in the setting of mismatched unrelated donor (MMUD) bone marrow HCT. The study enrolled 80 patients, treated with either myeloablative conditioning (MAC; n = 40) or reduced-intensity conditioning (RIC; n = 40), with the primary endpoint of 1-year overall survival (OS). The median follow-up for this study was 34 months (range, 12 to 46 months) in the RIC group and 36 months (range, 18 to 49 months) in the MAC group. Three-year OS and nonrelapse mortality were 70% and 15%, respectively, in the RIC group and 62% and 10% in the MAC group. No GVHD was reported after 1 year. The incidence of relapse was 29% in the RIC group and 51% in the MAC group. OS did not differ based on HLA match grade (63% in the 7/8 strata and 71% in the 4 to 6/8 strata). These encouraging outcomes, which were sustained for 3 years post-HCT, support the continued exploration of MMUD HCT using a PTCy platform. Important future areas to address include relapse reduction and furthering our understanding of optimal donor selection based on HLA and non-HLA factors.

Phase II trial of parathyroid hormone after double umbilical cord blood transplantation.

Transplantation of 1 or 2 umbilical cord blood products is a useful alternative stem cell source. However, the limited number of stem cells in each infusion results in slow engraftment. In mouse models, administration of parathyroid hormone (PTH) is an effective way to enhance the ability of limited numbers of hematopoietic stem cells to support hematopoiesis. In this study, patients received either a myeloablative or a reduced-intensity double umbilical cord blood transplantation, followed by PTH at 100 µg/day for 28 days. Thirteen patients (median age, 42 years) were enrolled. All patients engrafted; the median time to neutrophil and platelet engraftment of >20 × 10(9) cells/L was 30 days and 61 days, respectively. The incidence of grade II-IV acute GVHD was 38.5% at day 100. Four deaths occurred before day 100, prompting early study closure. No patient who received a myeloablative regimen relapsed. Overall survival at 6 months after transplantation was 62%, and disease-free survival at 2 years was 39%. At the dose and schedule studied, there was no evidence that PTH influenced blood count recovery.

Phase Ib trial of lenalidomide as post-remission therapy for older adults with acute myeloid leukemia: Safety and longitudinal assessment of geriatric functional domains.

BACKGROUND AND OBJECTIVES: Novel, non-cytotoxic agents are driving a paradigm shift for treatment of older adults with acute myeloid leukemia (AML). Older patients who initially receive intensive cytotoxic induction may choose to not proceed with cytotoxic consolidation therapy. Lenalidomide is an orally-administered immunomodulatory small molecule with activity in AML and a favorable safety profile in older adults with active leukemia. We conducted a phase Ib study of lenalidomide as post-remission therapy in older adults and assessed its impact on geriatric functional domains. MATERIALS AND METHODS: Participants were patients with AML over age 60 years who had undergone induction therapy and were poor candidates for cytotoxic consolidation. Lenalidomide was administered for 28 days in three dose cohorts. A Bayesian dose-escalation method determined cohort assignment and maximum tolerated dose (MTD). Geriatric assessment (GA) was performed before and after the cycle of lenalidomide. RESULTS: Nineteen patients with median age 68 were treated with at least one 28-day course of lenalidomide. Dose-limiting toxicities were observed in three participants at 25 mg, zero participants at 35 mg, and one participant at 50 mg. MTD was 35 mg. Median relapse-free survival was 4.3 months. GA was completed before and after treatment in fifteen patients, demonstrating improved cognitive function and no changes in physical, psychological, or social function after lenalidomide. CONCLUSION: Lenalidomide can be safely administered to older adults with AML with preservation of functional domains important to older patients. Serial GA can be performed in a novel drug study as a tool to characterize treatment tolerability.

Phase II Trial of Pembrolizumab after High-Dose Cytarabine in Relapsed/Refractory Acute Myeloid Leukemia.

Immune suppression, exhaustion, and senescence are frequently seen throughout disease progression in acute myeloid leukemia (AML). We conducted a phase II study of high-dose cytarabine followed by pembrolizumab 200 mg i.v. on day 14 to examine whether PD-1 inhibition improves clinical responses in relapsed/refractory (R/R) AML. Overall responders could receive pembrolizumab maintenance up to 2 years. Among 37 patients enrolled, the overall response rate, composite complete remission (CRc) rate (primary endpoint), and median overall survival (OS) were 46%, 38%, and 11.1 months, respectively. Patients with refractory/early relapse and those receiving treatment as first salvage had encouraging outcomes (median OS, 13.2 and 11.3 months, respectively). Grade ≥3 immune-related adverse events were rare (14%) and self-limiting. Patients who achieved CRc had a higher frequency of progenitor exhausted CD8+ T cells expressing TCF-1 in the bone marrow prior to treatment. A multifaceted correlative approach of genomic, transcriptomic, and immunophenotypic profiling offers insights on molecular correlates of response and resistance to pembrolizumab. SIGNIFICANCE: Immune-checkpoint blockade with pembrolizumab was tolerable and feasible after high-dose cytarabine in R/R AML, with encouraging clinical activity, particularly in refractory AML and those receiving treatment as first salvage regimen. Further study of pembrolizumab and other immune-checkpoint blockade strategies after cytotoxic chemotherapy is warranted in AML.See related commentary by Wei et al., p. 551. This article is highlighted in the In This Issue feature, p. 549.

National Marrow Donor Program-Sponsored Multicenter, Phase II Trial of HLA-Mismatched Unrelated Donor Bone Marrow Transplantation Using Post-Transplant Cyclophosphamide.

PURPOSE: Hematopoietic cell transplantation (HCT) is curative for hematologic disorders, but outcomes are historically inferior when using HLA-mismatched donors. Despite unrelated donor registries listing > 38 million volunteers, 25%-80% of US patients lack an HLA-matched unrelated donor, with significant disparity across ethnic groups. We hypothesized that HCT with a mismatched unrelated donor (MMUD) using post-transplant cyclophosphamide (PTCy), a novel strategy successful in overcoming genetic disparity using mismatched related donors, would be feasible and increase access to HCT. PATIENTS AND METHODS: We performed a prospective phase II study of MMUD bone marrow HCT with PTCy for patients with hematologic malignancies. The primary end point was 1-year overall survival (OS), hypothesized to be 65% or better. 80 patients enrolled at 11 US transplant centers (December 2016-March 2019). Following myeloablative or reduced-intensity conditioning-based HCT, patients received PTCy on days +3, +4, with sirolimus and mycophenolate mofetil starting on day +5. We compared outcomes to Center for International Blood and Marrow Transplant Research contemporary controls receiving PTCy. RESULTS: Notably, 48% of patients enrolled were ethnic minorities. 39% of pairs were matched for 4-6 out of 8 HLA alleles. The primary end point was met, with 1-year OS of 76% (90% CI, 67.3 to 83.3) in the entire cohort, and 72% and 79% in the myeloablative and reduced-intensity conditioning strata, respectively. Secondary end points related to engraftment and graft-versus-host-disease were reached. Multivariate analysis comparing the study group with other mismatched HCT controls found no significant differences in OS. CONCLUSION: Our prospective study demonstrates the feasibility and effectiveness of HCT with an MMUD in the setting of PTCy. Remarkably, nearly half of the study participants belonged to an ethnic minority population, suggesting this approach may significantly expand access to HCT.

A Comparison of Clofarabine-based (GCLAC) and Cladribine-based (CLAG) Salvage Chemotherapy for Relapsed/Refractory AML.

BACKGROUND: Salvage regimens for patients with relapsed/refractory acute myeloid leukemia (rrAML) lack comparative data for superiority. Thus, we conducted a retrospective analysis of clofarabine-based (GCLAC; granulocyte colony-stimulating factor [filgrastim], clofarabine, high-dose cytarabine) versus cladribine-based (CLAG; cladribine, cytarabine, granulocyte colony-stimulating factor [filgrastim]) regimens in rrAML. PATIENTS AND METHODS: We identified 41 consecutive patients with rrAML who had received either GCLAC or CLAG from 2011 to 2014. The primary outcome measure was the complete remission (CR) rate defined according to the International Working Group criteria. The secondary outcomes included the proportion of patients who underwent allogenic stem cell transplantation and the rate of relapse-free survival and overall survival. RESULTS: We found no significant differences in the baseline characteristics of the patients treated with GCLAC (n = 22) or CLAG (n = 19). The outcomes with these 2 regimens were not significantly different. Patients treated with GCLAC had a CR/CR with incomplete blood count recovery rate of 64% compared with 47% for the patients treated with CLAG (P = .36). Of the GCLAC patients, 45% underwent allogeneic stem cell transplantation compared with 26% of the CLAG patients (P = .32). The median relapse-free survival after GCLAC and CLAG was 1.59 years and 1.03 years, respectively (P = .75). The median overall survival after GCLAG and CLAG was 1.03 years and 0.70 years, respectively (P = .08). The drug costs were significantly different for GCLAC versus CLAG. Using an average wholesale price, the cost per patient per cycle was $60,821.60 for GCLAC and $4910.60 for CLAG. CONCLUSION: A single-institutional retrospective analysis found no significant differences in the outcomes between GCLAC and CLAG for rrAML patients, although formal comparisons should be performed in a randomized clinical trial. The cost of GCLAC was greater than that of CLAG, which should be considered when evaluating the choice for the salvage chemotherapy options.

Increasing Rates of Fluoroquinolone Resistance in Escherichia coli Isolated From the Blood and Urine of Patients with Hematologic Malignancies and Stem Cell Transplant Recipients.

Fluoroquinolone (FQ) antibiotics have been shown to reduce mortality and the number of febrile episodes when used as prophylaxis during neutropenia. Prior studies suggest that prophylaxis may result in increasing rates of FQ resistance. Fluoroquinolone non-susceptibility trends in Escherichia coli isolated from blood and urine cultures were evaluated over a 16-year period during which prophylaxis was initiated in patients with hematologic malignancies and stem cell transplants. Non-susceptibility rates increased after the introduction of prophylaxis, with yearly non-susceptibility rates rising from 30%-33% to 40%-88% in blood isolates. The high rates of non-susceptibility now observed raise concerns about the continued efficacy of FQ prophylaxis. This concern exists particularly in those patients undergoing stem cell transplants where the total FQ non-susceptibility rates over the study period were 82.3%. Further evaluation of the effect of FQ prophylaxis on antibiotic resistance and its efficacy in the setting of increased rates of resistance is warranted.

Profile of sapacitabine: potential for the treatment of newly diagnosed acute myeloid leukemia in elderly patients.

Acute myeloid leukemia (AML) is a hematopoietic stem cell disorder that affects approximately 14,000 persons each year in the US. AML occurs at all ages but the incidence increases with age with the median age at diagnosis being 67 years. Advances in the treatment of AML over the past decades have led to improved survival, albeit mostly in younger patients. The prognosis of older patients with this disease over the same time span has not changed much and remains dismal. This review focuses on the epidemiology and characteristics of AML in elderly patients, the rationale for treating elderly AML patients, and the currently available and potential future treatment options such as sapacitabine. Elderly AML patients treated with intensive chemotherapy have a higher mortality rate, and a lower rate of complete remission and overall survival when compared to the younger population. This is due to both the different biology of the disease and the number of patient-specific factors. However, elderly AML patients treated with aggressive chemotherapy can achieve durable remissions, which offer prolonged survival and improved quality of life. Recent data also indicates that elderly AML patients deemed unfit for intensive chemotherapy benefit from leukemia-specific attenuated dose chemotherapy compared to supportive care alone. This has led to renewed interest to look for anti-leukemic therapies designed specifically for older patients. Sapacitabine, a novel oral nucleoside analog, promises good efficacy, favorable toxicity profile, and ease of administration; all of which makes it very appealing. Results from pre-clinical and clinical studies have been very encouraging and sapacitabine is currently being evaluated in a Phase III study, of which the results are eagerly awaited.

Interim analysis of safety and efficacy of ruxolitinib in patients with myelofibrosis and low platelet counts.

BACKGROUND: Ruxolitinib, a Janus kinase 1 and 2 inhibitor, demonstrated improvements in spleen volume, symptoms, and survival over placebo and best available therapy in intermediate-2 or high-risk myelofibrosis patients with baseline platelet counts ≥100 × 109/L in phase III studies. The most common adverse events were dose-dependent anemia and thrombocytopenia, which were anticipated because thrombopoietin and erythropoietin signal through JAK2. These events were manageable, rarely leading to treatment discontinuation. Because approximately one-quarter of MF patients have platelet counts <100 × 109/L consequent to their disease, ruxolitinib was evaluated in this subset of patients using lower initial doses. Interim results of a phase II study of ruxolitinib in myelofibrosis patients with baseline platelet counts of 50-100 × 109/L are reported. METHODS: Ruxolitinib was initiated at a dose of 5 mg twice daily (BID), and doses could be increased by 5 mg once daily every 4 weeks to 10 mg BID if platelet counts remained adequate. Additional dosage increases required evidence of suboptimal efficacy. Assessments included measurement of spleen volume by MRI, MF symptoms by MF Symptom Assessment Form v2.0 Total Symptom Score [TSS]), Patient Global Impression of Change (PGIC); EORTC QLQ-C30, and safety/tolerability. RESULTS: By week 24, 62% of patients achieved stable doses ≥10 mg BID. Median reductions in spleen volume and TSS were 24.2% and 43.8%, respectively. Thrombocytopenia necessitating dose reductions and dose interruptions occurred in 12 and 8 patients, respectively, and occurred mainly in patients with baseline platelet counts ≤75 × 109/L. Seven patients experienced platelet count increases ≥15 × 109/L. Mean hemoglobin levels remained stable over the treatment period. Two patients discontinued for adverse events: 1 for grade 4 retroperitoneal hemorrhage secondary to multiple and suspected pre-existing renal artery aneurysms and 1 for grade 4 thrombocytopenia. CONCLUSIONS: Results suggest that a low starting dose of ruxolitinib with escalation to 10 mg BID may be appropriate in myelofibrosis patients with low platelet counts.

Late-phase investigational approaches for the treatment of relapsed/refractory acute myeloid leukemia.

INTRODUCTION: Acute myeloid leukemia (AML) is a malignant hematologic disorder that affects more than 13,000 adults each year in the USA. Despite continued advances in the understanding of the molecular pathogenesis of the disease and patient management, the cure rate for AML remains relatively low, largely due to a high rate of relapsed or refractory disease. AREAS COVERED: The purpose of this review is to provide an understanding of the unmet needs in relapsed/refractory AML, and to assess promising investigational agents with ongoing or planned Phase III clinical trials. EXPERT OPINION: Although the treatment of relapsed/refractory AML remains a challenge, numerous new chemotherapeutics are currently in development. Enrollment in clinical trials should be strongly considered for patients with relapsed/refractory disease, and emerging data may help identify new agents with significant activity in this setting.

Single cycle of arsenic trioxide-based consolidation chemotherapy spares anthracycline exposure in the primary management of acute promyelocytic leukemia.

PURPOSE Event-free survival following all-trans-retinoic acid (ATRA) -based therapy for acute promyelocytic leukemia (APL) averages 70% at 5 years. While arsenic trioxide (ATO) can induce remissions in 95% of relapsed patients, few studies have addressed the integration of ATO into the primary management of APL. This study examines the efficacy of a single cycle of ATO-based consolidation therapy in a treatment regimen designed to decrease exposure to other cytotoxic agents. PATIENTS AND METHODS After induction with ATRA and daunorubicin (DRN), untreated patients with APL received 3 days of cytarabine and DRN followed by 30 doses of ATO beginning on day 8. Molecular remitters received 2 years of risk-based maintenance therapy. Results Forty-one of 45 patients receiving induction therapy achieved remission; four patients died (one before treatment was initiated). Thirty-seven patients received consolidation and maintenance; of these one patient relapsed (CNS) and one died in remission during maintenance therapy (hepatic sickle cell crisis). With a median follow-up of 2.7 years, estimated disease-free survival was 90%; overall survival for all patients was 88%. Despite a total anthracycline dose of only 360 mg/m(2), cardiac ejection fraction decreased by > or = 20% in 20% of patients. CONCLUSION These data, combined with other recent studies using ATO in the primary management of APL, demonstrate the important role that ATO can play in the primary management of this curable disease. Future studies should continue to focus on reducing the toxicity of treatment without increasing the relapse rate.