Impact of rabbit anti-thymocyte globulin (ATG) exposure on outcomes after ex vivo T-cell-depleted hematopoietic cell transplantation in pediatric and young adult patients.

BACKGROUND AIMS: Traditional weight-based dosing of rabbit anti-thymocyte globulin (rATG) used in allogeneic hematopoietic cell transplantation (HCT) to prevent graft-versus-host disease (GVHD) and graft rejection leads to variable exposures. High exposures induce delayed CD4+immune reconstitution (CD4+IR) and greater mortality. We sought to determine the impact of rATG exposure in children and young adults receiving various types of EX-VIVO T-cell-depleted (EX-VIVO-TCD) HCT. METHODS: Patients receiving their first EX-VIVO-TCD HCT (CliniMACS CD34+, Isolex or soybean lectin agglutination), with removal of residual T cells by E-rosette depletion (E-) between 2008 and 2018 at Memorial Sloan Kettering Cancer Center were retrospectively analyzed. rATG exposure post-HCT was estimated (AU*d/L) using a validated population pharmacokinetic model. Previously defined rATG-exposures, <30, 30-55, ≥55 AU*d/L, were related with outcomes of interest. Cox proportional hazard and cause-specific models were used for analyses. RESULTS: In total, 180 patients (median age 11 years; range 0.1-44 years) were included, malignant 124 (69%) and nonmalignant 56 (31%). Median post-HCT rATG exposure was 32 (0-104) AU*d/L. Exposure <30 AU*d/L was associated with a 3-fold greater probability of CD4+IR (P < 0.001); 2- to 4-fold lower risk of death (P = 0.002); and 3- to 4-fold lower risk of non-relapse mortality (NRM) (P = 0.02). Cumulative incidence of NRM was 8-fold lower in patients who attained CD4+IR compared with those who did not (P < 0.0001). There was no relation between rATG exposure and aGVHD (P = 0.33) or relapse (P = 0.23). Effect of rATG exposure on outcomes was similar in three EX-VIVO-TCD methods. CONCLUSIONS: Individualizing rATG dosing to target a low rATG exposure post-HCT while maintaining total cumulative exposure may better predict CD4+IR, reduce NRM and increase overall survival, independent of the EX-VIVO-TCD method.

Interventions and outcomes of adult patients with B-ALL progressing after CD19 chimeric antigen receptor T-cell therapy.

CD19-targeted chimeric antigen receptor (CAR) T-cell therapy has become a breakthrough treatment of patients with relapsed/refractory B-cell acute lymphoblastic leukemia (B-ALL). However, despite the high initial response rate, the majority of adult patients with B-ALL progress after CD19 CAR T-cell therapy. Data on the natural history, management, and outcome of adult B-ALL progressing after CD19 CAR T cells have not been described in detail. Herein, we report comprehensive data of 38 adult patients with B-ALL who progressed after CD19 CAR T therapy at our institution. The median time to progression after CAR T-cell therapy was 5.5 months. Median survival after post-CAR T progression was 7.5 months. A high disease burden at the time of CAR T-cell infusion was significantly associated with risk of post-CAR T progression. Thirty patients (79%) received salvage treatment of post-CAR T disease progression, and 13 patients (43%) achieved complete remission (CR), but remission duration was short. Notably, 7 (58.3%) of 12 patients achieved CR after blinatumomab and/or inotuzumab administered following post-CAR T failure. Multivariate analysis revealed that a longer remission duration from CAR T cells was associated with superior survival after progression following CAR T-cell therapy. In summary, overall prognosis of adult B-ALL patients progressing after CD19 CAR T cells was poor, although a subset of patients achieved sustained remissions to salvage treatments, including blinatumomab, inotuzumab, and reinfusion of CAR T cells. Novel therapeutic strategies are needed to reduce risk of progression after CAR T-cell therapy and improve outcomes of these patients.

Long-Term Follow-up of CD19 CAR Therapy in Acute Lymphoblastic Leukemia.

BACKGROUND: CD19-specific chimeric antigen receptor (CAR) T cells induce high rates of initial response among patients with relapsed B-cell acute lymphoblastic leukemia (ALL) and long-term remissions in a subgroup of patients. METHODS: We conducted a phase 1 trial involving adults with relapsed B-cell ALL who received an infusion of autologous T cells expressing the 19-28z CAR at the Memorial Sloan Kettering Cancer Center (MSKCC). Safety and long-term outcomes were assessed, as were their associations with demographic, clinical, and disease characteristics. RESULTS: A total of 53 adults received 19-28z CAR T cells that were manufactured at MSKCC. After infusion, severe cytokine release syndrome occurred in 14 of 53 patients (26%; 95% confidence interval [CI], 15 to 40); 1 patient died. Complete remission was observed in 83% of the patients. At a median follow-up of 29 months (range, 1 to 65), the median event-free survival was 6.1 months (95% CI, 5.0 to 11.5), and the median overall survival was 12.9 months (95% CI, 8.7 to 23.4). Patients with a low disease burden (<5% bone marrow blasts) before treatment had markedly enhanced remission duration and survival, with a median event-free survival of 10.6 months (95% CI, 5.9 to not reached) and a median overall survival of 20.1 months (95% CI, 8.7 to not reached). Patients with a higher burden of disease (≥5% bone marrow blasts or extramedullary disease) had a greater incidence of the cytokine release syndrome and neurotoxic events and shorter long-term survival than did patients with a low disease burden. CONCLUSIONS: In the entire cohort, the median overall survival was 12.9 months. Among patients with a low disease burden, the median overall survival was 20.1 months and was accompanied by a markedly lower incidence of the cytokine release syndrome and neurotoxic events after 19-28z CAR T-cell infusion than was observed among patients with a higher disease burden. (Funded by the Commonwealth Foundation for Cancer Research and others; ClinicalTrials.gov number, NCT01044069 .).

CD19 CAR T cells following autologous transplantation in poor-risk relapsed and refractory B-cell non-Hodgkin lymphoma.

High-dose chemotherapy and autologous stem cell transplantation (HDT-ASCT) is the standard of care for relapsed or primary refractory (rel/ref) chemorefractory diffuse large B-cell lymphoma. Only 50% of patients are cured with this approach. We investigated safety and efficacy of CD19-specific chimeric antigen receptor (CAR) T cells administered following HDT-ASCT. Eligibility for this study includes poor-risk rel/ref aggressive B-cell non-Hodgkin lymphoma chemosensitive to salvage therapy with: (1) positron emission tomography-positive disease or (2) bone marrow involvement. Patients underwent standard HDT-ASCT followed by 19-28z CAR T cells on days +2 and +3. Of 15 subjects treated on study, dose-limiting toxicity was observed at both dose levels (5 × 106 and 1 × 107 19-28z CAR T per kilogram). Ten of 15 subjects experienced CAR T-cell-induced neurotoxicity and/or cytokine release syndrome (CRS), which were associated with greater CAR T-cell persistence (P = .05) but not peak CAR T-cell expansion. Serum interferon-γ elevation (P < .001) and possibly interleukin-10 (P = .07) were associated with toxicity. The 2-year progression-free survival (PFS) is 30% (95% confidence interval, 20% to 70%).  Subjects given decreased naive-like (CD45RA+CCR7+) CD4+ and CD8+ CAR T cells experienced superior PFS (P = .02 and .04, respectively). There was no association between CAR T-cell peak expansion, persistence, or cytokine changes and PFS. 19-28z CAR T cells following HDT-ASCT were associated with a high incidence of reversible neurotoxicity and CRS. Following HDT-ASCT, effector CD4+ and CD8+ immunophenotypes may improve disease control. This trial was registered at www.clinicaltrials.gov as #NCT01840566.

Toxicity and response after CD19-specific CAR T-cell therapy in pediatric/young adult relapsed/refractory B-ALL.

Chimeric antigen receptor (CAR) T cells have demonstrated clinical benefit in patients with relapsed/refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). We undertook a multicenter clinical trial to determine toxicity, feasibility, and response for this therapy. A total of 25 pediatric/young adult patients (age, 1-22.5 years) with R/R B-ALL were treated with 19-28z CAR T cells. Conditioning chemotherapy included high-dose (3 g/m2) cyclophosphamide (HD-Cy) for 17 patients and low-dose (≤1.5 g/m2) cyclophosphamide (LD-Cy) for 8 patients. Fifteen patients had pretreatment minimal residual disease (MRD; <5% blasts in bone marrow), and 10 patients had pretreatment morphologic evidence of disease (≥5% blasts in bone marrow). All toxicities were reversible, including severe cytokine release syndrome in 16% (4 of 25) and severe neurotoxicity in 28% (7 of 25) of patients. Treated patients were assessed for response, and, among the evaluable patients (n = 24), response and peak CAR T-cell expansion were superior in the HD-Cy/MRD cohorts, as compared with the LD-Cy/morphologic cohorts without an increase in toxicity. Our data support the safety of CD19-specific CAR T-cell therapy for R/R B-ALL. Our data also suggest that dose intensity of conditioning chemotherapy and minimal pretreatment disease burden have a positive impact on response without a negative effect on toxicity. This trial was registered at www.clinicaltrials.gov as #NCT01860937.

ASTCT Consensus Grading for Cytokine Release Syndrome and Neurologic Toxicity Associated with Immune Effector Cells.

Chimeric antigen receptor (CAR) T cell therapy is rapidly emerging as one of the most promising therapies for hematologic malignancies. Two CAR T products were recently approved in the United States and Europe for the treatment ofpatients up to age 25years with relapsed or refractory B cell acute lymphoblastic leukemia and/or adults with large B cell lymphoma. Many more CAR T products, as well as other immunotherapies, including various immune cell- and bi-specific antibody-based approaches that function by activation of immune effector cells, are in clinical development for both hematologic and solid tumor malignancies. These therapies are associated with unique toxicities of cytokine release syndrome (CRS) and neurologic toxicity. The assessment and grading of these toxicities vary considerably across clinical trials and across institutions, making it difficult to compare the safety of different products and hindering the ability to develop optimal strategies for management of these toxicities. Moreover, some aspects of these grading systems can be challenging to implement across centers. Therefore, in an effort to harmonize the definitions and grading systems for CRS and neurotoxicity, experts from all aspects of the field met on June 20 and 21, 2018, at a meeting supported by the American Society for Transplantation and Cellular Therapy (ASTCT; formerly American Society for Blood and Marrow Transplantation, ASBMT) in Arlington, VA. Here we report the consensus recommendations of that group and propose new definitions and grading for CRS and neurotoxicity that are objective, easy to apply, and ultimately more accurately categorize the severity of these toxicities. The goal is to provide a uniform consensus grading system for CRS and neurotoxicity associated with immune effector cell therapies, for use across clinical trials and in the postapproval clinical setting.

Building a CAR Garage: Preparing for the Delivery of Commercial CAR T Cell Products at Memorial Sloan Kettering Cancer Center.

Two commercial chimeric antigen receptor (CAR) T cell therapies for CD19-expressing B cell malignancies, Kymriah and Yescarta, have recently been approved by the Food and Drug Administration. The administration of CAR T cells is a complex endeavor involving cell manufacture, tracking and shipping of apheresis products, and management of novel and severe toxicities. At Memorial Sloan Kettering Cancer Center, we have identified 8 essential tasks that define the CAR T cell workflow. In this review, we discuss practical aspects of CAR T cell program development, including clinical, administrative, and regulatory challenges for successful implementation.

Successful reduced-intensity conditioning hematopoietic stem cell transplantation for paroxysmal nocturnal hemoglobinuria with aplastic anemia in two children.

Paroxysmal nocturnal hemoglobinuria (PNH) is an extremely rare cause of bone marrow failure in children. We report two children who presented with pancytopenia, and were diagnosed with PNH with severe aplastic anemia. Both children underwent upfront, successful hematopoietic stem cell transplantation with reduced-intensity conditioning. One patient had a syngeneic donor, and one patient had a 10/10 matched unrelated donor. Neither patient developed graft versus host disease, infections, or recurrent PNH. Reduced-intensity conditioning hematopoietic stem cell transplantation is a reasonable therapy for PNH with marrow failure in children.

Enhancing antitumor efficacy of chimeric antigen receptor T cells through constitutive CD40L expression.

Adoptive cell therapy with genetically modified T cells expressing a chimeric antigen receptor (CAR) is a promising therapy for patients with B-cell acute lymphoblastic leukemia. However, CAR-modified T cells (CAR T cells) have mostly failed in patients with solid tumors or low-grade B-cell malignancies including chronic lymphocytic leukemia with bulky lymph node involvement. Herein, we enhance the antitumor efficacy of CAR T cells through the constitutive expression of CD40 ligand (CD40L, CD154). T cells genetically modified to constitutively express CD40L (CD40L-modified T cells) demonstrated increased proliferation and secretion of proinflammatory TH1 cytokines. Further, CD40L-modified T cells augmented the immunogenicity of CD40(+) tumor cells by the upregulated surface expression of costimulatory molecules (CD80 and CD86), adhesion molecules (CD54, CD58, and CD70), human leukocyte antigen (HLA) molecules (Class I and HLA-DR), and the Fas-death receptor (CD95). Additionally, CD40L-modified T cells induced maturation and secretion of the proinflammatory cytokine interleukin-12 by monocyte-derived dendritic cells. Finally, tumor-targeted CD19-specific CAR/CD40L T cells exhibited increased cytotoxicity against CD40(+) tumors and extended the survival of tumor-bearing mice in a xenotransplant model of CD19(+) systemic lymphoma. This preclinical data supports the clinical application of CAR T cells additionally modified to constitutively express CD40L with anticipated enhanced antitumor efficacy.

Chimeric antigen receptors and bispecific antibodies to retarget T cells in pediatric oncology.

Cancer immunotherapy using antigen-specific T cells has broad therapeutic potential. Chimeric antigen receptors and bispecific antibodies can redirect T cells to kill tumors without human leukocyte antigens (HLA) restriction. Key determinants of clinical potential include the choice of target antigen, antibody specificity, antibody affinity, tumor accessibility, T cell persistence, and tumor immune evasion. For pediatric cancers, additional constraints include their propensity for bulky metastatic disease and the concern for late toxicities from treatment. Nonetheless, the recent preclinical and clinical developments of these T cell based therapies are highly encouraging.

A robust quality infrastructure is key to safe and effective delivery of immune effector cells: how FACT-finding can help.

ABSTRACT: Immune effector cells (IECs) include a broad range of immune cells capable of modulating several disease states, including malignant and nonmalignant conditions. The growth in the use of IECs as both investigational and commercially available products requires medical institutions to develop workflows/processes to safely implement and deliver transformative therapy. Adding to the complexity of this therapy are the variety of targets, diseases, sources, and unique toxicities that a patient experiences following IEC therapy. For over 25 years, the Foundation for the Accreditation of Cellular Therapy (FACT) has established a standard for the use of cellular therapy, initially with hematopoietic cell transplantation (HCT), and more recently, with the development of standards to encompass IEC products such as chimeric antigen receptor (CAR)-T cells. To date, IEC therapy has challenged the bandwidth and infrastructure of the institutions offering this therapy. To address these challenges, FACT has established a programmatic framework to improve the delivery of IEC therapy. In this study, we outline the current state of IEC program development, accreditation, and solutions to the challenges that programs face as they expand their application to novel IEC therapy.

T-cell depleted allogeneic hematopoietic stem cell transplant for the treatment of Fanconi anemia and MDS/AML.

The only curative approach for myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) arising in patients with Fanconi anemia (FA) is allogeneic hematopoietic stem cell transplantation (HCT); however, HCT approaches are inconsistent and limited data on outcomes exist. We retrospectively evaluated outcomes of thirty patients with FA and MDS/AML who underwent first allogeneic HCT with a T-cell depleted (TCD) graft at our institution. Patients were transplanted on successive protocols with stepwise changes in cytoreduction and GVHD prophylaxis. All but two patients (93%) experienced durable hematopoietic engraftment. With median follow-up of 8.7 years, 5-year OS was 66.8% and DFS 53.8%. No significant differences in survival were found in patients with high-risk prognostic features (age ≥20 years, AML diagnosis, alternative donor graft) or when stratified by conditioning regimen. The 5-year cumulative incidences of relapse and NRM were 24.3% and 21.9%, respectively. NRM was higher in patients ≥20 years at HCT but did not otherwise differ. We herein demonstrate promising outcomes following allogeneic HCT for patients with FA and MDS/AML using TCD grafts, particularly in a cohort of high-risk patients with 50% ≥20 years and a majority receiving mismatched grafts. Future prospective studies are needed to compare this approach with other HCT platforms.

INSPIRED Symposium Part 1: Clinical Variables Associated with Improved Outcomes for Children and Young Adults treated with Chimeric Antigen Receptor T cells for B cell Acute Lymphoblastic Leukemia.

Chimeric antigen receptor (CAR) T cell therapy (CAR-T) targeting the CD19 antigen on B cell acute lymphoblastic leukemia (B-ALL) has transitioned from a highly investigational therapy with limited access to a commercial therapy with established toxicities, response and survival rates, and access in numerous countries. With more than a decade of clinical study and 5 years of commercial access, data showing associations with success and failure have emerged. To address functional limitations of CAR-T and overcome constrained sample sizes when studying single-trial or single-center data, collaborative groups, including the Pediatric Real World CAR Consortium, the CAR-Multicenter Analysis, the Center for International Blood and Marrow Transplant Research, and the International BFM Study Group, among others, have been retrospectively interrogating the amassed clinical experience. The high patient numbers and varied clinical experiences compiled by these groups have defined clinical variables impacting CAR-T outcomes. Here we review published CAR-T trials and consortium/collaborative outcomes to establish variables associated with optimal response to CAR-T in children and young adults with B-ALL. We focus on findings with clinical relevance that have emerged, including data implicating pretreatment disease burden, presence of extramedullary disease, nonresponse to prior CD19 antigen targeting (blinatumomab therapy), CAR T cell dose, and fludarabine pharmacokinetics as factors impacting post-CAR-T survival. Additionally, we address the role of collaborative efforts going forward in guiding clinical practice evolution and further optimizing post-CAR-T outcomes.

Excellent leukemia control after second hematopoietic cell transplants with unrelated cord blood grafts for post-transplant relapse in pediatric patients.

Background: Patients with leukemia relapse after allogeneic hematopoietic cell transplant (HCT) have poor survival due to toxicity and disease progression. A second HCT often offers the only curative treatment. Methods: We retrospectively reviewed our bi-institutional experience (MSKCC-USA; Utrecht-NL) with unrelated cord blood transplantation (CBT) for treatment of post-transplant relapse. Overall survival (OS) and event-free survival (EFS) were evaluated using the Kaplan-Meier method, treatment-related mortality (TRM) and relapse were evaluated using the competing risk method by Fine-Gray. Results: Twenty-six patients age < 21 years received a second (n=24) or third (n=2) HCT with CB grafts during the period 2009-2021. Median age at first HCT (HCT1) was 11.5 (range: 0.9-17.7) years and all patients received myeloablative cytoreduction. Median time from HCT1 to relapse was 12.8 (range 5.5-189) months. At CBT, median patient age was 13.5 (range 1.4-19.1) years. Diagnoses were AML: 13; ALL: 4, MDS: 5, JMML: 2; CML: 1; mixed phenotype acute leukemia: 1. Sixteen patients (62%) were in advanced stage, either CR>2 or with active disease. Median time from HCT1 to CBT was 22.2 (range 7-63.2) months. All patients engrafted after CBT. Thirteen patients developed acute GvHD; 7 had grade III or IV. With a median survivor follow-up of 46.6 (range 17.4-155) months, 3-year OS was 69.2% (95% CI 53.6-89.5%) and 3-year EFS was 64.9% (95% CI 48.8-86.4%). Eight patients died, 3 of AML relapse and 5 due to toxicity (respiratory failure [n=4], GvHD [n=1]) at a median time of 7.7 (range 5.9-14.4) months after CBT. Cumulative incidence of TRM at 3 years was 19.2% (95% CI 4.1-34.4%). Notably, all TRM events occurred in patients transplanted up to 2015; no toxicity-related deaths were seen in the 16 patients who received CBT after 2015. Cumulative incidence of relapse was 15.9% (95% CI 1.6-30.2%) at 3 years, remarkably low for these very high-risk patients. Conclusions: Survival was very encouraging following CB transplants in pediatric patients with recurrent leukemia after first HCT, and TRM has been low over the last decade. CBT needs to be strongly considered as a relatively safe salvage therapy option for post-transplant relapse.

Validation of a High-Sensitivity Assay for Detection of Chimeric Antigen Receptor T-Cell Vectors Using Low-Partition Digital PCR Technology.

Although in vivo engraftment, expansion, and persistence of chimeric antigen receptor (CAR) T cells are pivotal components of treatment efficacy, quantitative monitoring has not been implemented in routine clinical practice. We describe the development and analytical validation of a digital PCR assay for ultrasensitive detection of CAR constructs after treatment, circumventing known technical limitations of low-partitioning platforms. Primers and probes, designed for detection of axicabtagene, brexucabtagene, and Memorial Sloan Kettering CAR constructs, were employed to validate testing on the Bio-Rad digital PCR low-partitioning platform; results were compared with Raindrop, a high-partitioning system, as reference method. Bio-Rad protocols were modified to enable testing of DNA inputs as high as 500 ng. Using dual-input reactions (20 and 500 ng) and a combined analysis approach, the assay demonstrated consistent target detection around 1 × 10-5 (0.001%) with excellent specificity and reproducibility and 100% accuracy compared with the reference method. Dedicated analysis of 53 clinical samples received during validation/implementation phases showed the assay effectively enabled monitoring across multiple time points of early expansion (day 6 to 28) and long-term persistence (up to 479 days). CAR vectors were detected at levels ranging from 0.005% to 74% (vector versus reference gene copies). The highest levels observed in our cohort correlated strongly with the temporal diagnosis of grade 2 and 3 cytokine release syndrome diagnosis (P < 0.005). Only three patients with undetectable constructs had disease progression at the time of sampling.

Tisagenlecleucel utilisation and outcomes across refractory, first relapse and multiply relapsed B-cell acute lymphoblastic leukemia: a retrospective analysis of real-world patterns.

Background: Tisagenlecleucel was approved by the Food and Drug Administration (FDA) in 2017 for refractory B-cell acute lymphoblastic leukemia (B-ALL) and B-ALL in ≥2nd relapse. Outcomes of patients receiving commercial tisagenlecleucel upon 1st relapse have yet to be established. We aimed to report real-world tisagenlecleucel utilisation patterns and outcomes across indications, specifically including patients treated in 1st relapse, an indication omitted from formal FDA approval. Methods: We conducted a retrospective analysis of real-world tisagenlecleucel utilisation patterns across 185 children and young adults treated between August 30, 2017 and March 6, 2020 from centres participating in the Pediatric Real-World CAR Consortium (PRWCC), within the United States. We described definitions of refractory B-ALL used in the real-world setting and categorised patients by reported Chimeric Antigen Receptor (CAR) T-cell indication, including refractory, 1st relapse and ≥2nd relapse B-ALL. We analysed baseline patient characteristics and post-tisagenlecleucel outcomes across defined cohorts. Findings: Thirty-six percent (n = 67) of our cohort received tisagenlecleucel following 1st relapse. Of 66 evaluable patients, 56 (85%, 95% CI 74-92%) achieved morphologic complete response. Overall-survival (OS) and event-free survival (EFS) at 1-year were 69%, (95% CI 58-82%) and 49%, (95% CI 37-64%), respectively, with survival outcomes statistically comparable to remaining patients (OS; p = 0.14, EFS; p = 0.39). Notably, toxicity was increased in this cohort, warranting further study. Interestingly, of 30 patients treated for upfront refractory disease, 23 (77%, 95% CI 58-90%) had flow cytometry and/or next-generation sequencing (NGS) minimum residual disease (MRD)-only disease at the end of induction, not meeting the historic morphologic definition of refractory. Interpretation: Our findings suggested that tisagenlecleucel response and survival rates overlap across patients treated with upfront refractory B-ALL, B-ALL ≥2nd relapse and B-ALL in 1st relapse. We additionally highlighted that definitions of refractory B-ALL are evolving beyond morphologic measures of residual disease. Funding: St. Baldrick's/Stand Up 2 Cancer, Parker Institute for Cancer Immunotherapy, Virginia and D.K. Ludwig Fund for Cancer Research.

Association between busulfan exposure and survival in patients undergoing a CD34+ selected stem cell transplantation.

Busulfan is an alkylating drug routinely used in conditioning regimens for allogeneic hematopoietic cell transplantation (allo-HCT). A myeloablative conditioning regimen, including busulfan, is commonly used in patients undergoing T-cell depletion (TCD) and allo-HCT, but data on optimal busulfan pharmacokinetic (PK) exposure in this setting are limited. Between 2012 and 2019, busulfan PK was performed to target an area under the curve exposure between 55 and 66 mg × h/L over 3 days using a noncompartmental analysis model. We retrospectively re-estimated busulfan exposure following the published population PK (popPK) model (2021) and correlated it with outcomes. To define optimal exposure, univariable models were performed with P splines, wherein hazard ratio (HR) plots were drawn, and thresholds were found graphically as the points at which the confidence interval crossed 1. Cox proportional hazard and competing risk models were used for analyses. 176 patients were included, with a median age of 59 years (range, 2-71). Using the popPK model, the median cumulative busulfan exposure was 63.4 mg × h/L (range, 46.3-90.7). The optimal threshold was at the upper limit of the lowest quartile (59.5 mg × h/L). 5-year overall survival (OS) with busulfan exposure ≥59.5 vs <59.5 mg × h/L was 67% (95% CI, 59-76) vs 40% (95% CI, 53-68), respectively (P = .02), and this association remained in a multivariate analyses (HR, 0.5; 95% CI, 0.29; 0.88; P = .02). In patients undergoing TCD allo-HCT, busulfan exposure is significantly associated with OS. The use of a published popPK model to optimize exposure may significantly improve the OS.