Search

PURPOSE Brexucabtagene autoleucel (brexu-cel) is an autologous CD19-directed chimeric antigen receptor (CAR) T-cell therapy approved for relapsed/refractory mantle cell lymphoma (MCL). This therapy was approved on the basis of the single-arm phase II ZUMA-2 trial, which showed best overall and complete response rates of 91% and 68%, respectively. We report clinical outcomes with brexu-cel in the standard-of-care setting for the approved indication. PATIENTS AND METHODS Patients who underwent leukapheresis between August 1, 2020 and December 31, 2021, at 16 US institutions, with an intent to manufacture commercial brexu-cel for relapsed/refractory MCL, were included. Patient data were collected for analyses of responses, outcomes, and toxicities as per standard guidelines. RESULTS Of 189 patients who underwent leukapheresis, 168 (89%) received brexu-cel infusion. Of leukapheresed patients, 79% would not have met ZUMA-2 eligibility criteria. Best overall and complete response rates were 90% and 82%, respectively. At a median follow-up of 14.3 months after infusion, the estimates for 6- and 12-month progression-free survival (PFS) were 69% (95% CI, 61 to 75) and 59% (95% CI, 51 to 66), respectively. The nonrelapse mortality was 9.1% at 1 year, primarily because of infections. Grade 3 or higher cytokine release syndrome and neurotoxicity occurred in 8% and 32%, respectively. In univariable analysis, high-risk simplified MCL international prognostic index, high Ki-67, TP53 aberration, complex karyotype, and blastoid/pleomorphic variant were associated with shorter PFS after brexu-cel infusion. Patients with recent bendamustine exposure (within 24 months before leukapheresis) had shorter PFS and overall survival after leukapheresis in intention-to-treat univariable analysis. CONCLUSION In the standard-of-care setting, the efficacy and toxicity of brexu-cel were consistent with those reported in the ZUMA-2 trial. Tumor-intrinsic features of MCL, and possibly recent bendamustine exposure, may be associated with inferior efficacy outcomes.

We examined the meaning of metabolically active lesions on 1 month restaging nuclear imaging of patients with relapsed/refractory (R/R) large B-cell lymphoma (LBCL) receiving axicabtagene ciloleucel (axi-cel) by assessing the relationship between total metabolic tumor volume (MTV) on positron emission tomography (PET) scans and circulating tumor DNA (ctDNA) in the plasma. In this prospective multicenter sample collection study, MTV was retrospectively calculated via commercial software at baseline, 1 and 3 months post chimeric antigen receptor (CAR) T-cell therapy; ctDNA was available pre and post axi-cel. Spearman correlation coefficient (rs) was used to study the relationship between the variables and a mathematical model was constructed to describe tumor dynamics 1 month post CAR T-cell therapy. The median time between baseline scan and axi-cel infusion was 33 (range, 1-137) days for all 57 patients. For 41 of the patients with imaging within 33 days of axi-cel or imaging before that time but no bridging therapy, the correlation at baseline became stronger (rs 0.61, P< 0.0001) compared to all patients (rs 0.38, P= 0.004). Excluding patients in complete remission with no measurable residual disease, ctDNA and MTV at 1 month did not correlate (rs 0.28, P= 0.11), but did correlate at 3 months (rs 0.79, P= 0.0007). Modeling of tumor dynamics, which incorporated ctDNA and inflammation as part of MTV, recapitulated outcomes of patients with positive radiologic 1-month scans. Our results suggested that non-progressing hypermetabolic lesions on 1 month PET represent ongoing treatment response and their composition may be elucidated by concurrent ctDNA.

Supplemental Tables T1-T3, and Supplemental Figures S1-S5

The treatment landscape of relapsed/refractory (R/R) classic Hodgkin lymphoma (cHL) has evolved significantly over the past decade following the approval of brentuximab vedotin (BV) and the programmed death-1 (PD-1) inhibitors. We evaluated how outcomes and practice patterns have changed for R/R cHL patients who underwent autologous hematopoietic cell transplantation (AHCT) at our institution from 2011-2020 (N=183) compared to 2001-2010 (N=159) and evaluated prognostic factors for progression-free survival (PFS) and overall survival (OS) in both eras. OS was superior in the modern era (4-year estimates 89.1% vs 79.0%, HR 0.53, 95% CI 0.33-0.85, p=0.011) with a trend towards lower non-relapse mortality beyond 2 years post-transplant. Among patients who progressed after AHCT, 4-year post-progression survival increased from 43.3% to 71.4% in the modern era, reflecting increasing use of BV and the PD-1 inhibitors. In multivariable analysis for patients transplanted in the modern era, age ³45 years, primary refractory disease, and lack of complete remission pre-AHCT were associated with inferior PFS, while receipt of a PD-1 inhibitor-based regimen pre-AHCT was associated with superior PFS (HR 0.21, 95% CI 0.05-0.80, p=0.030). Extranodal disease at relapse was associated with inferior OS (HR 3.12, 95% CI 1.25-7.77, p=0.014). Our study demonstrates improved survival for R/R cHL after AHCT in the modern era attributed to more effective salvage regimens allowing for better disease control pre-AHCT and improved outcomes for patients who progressed after AHCT. Excellent outcomes were observed with PD-1 inhibitor-based salvage regimens pre-AHCT and support a randomized trial evaluating immunotherapy in the second line setting.

Immune effector cell-associated neurotoxicity syndrome (ICANS) is a prevalent condition seen after treatment with chimeric antigen receptor T-cell (CAR T) therapy and other cancer cell therapies. The underlying pathophysiology and neuropathology of the clinical syndrome are incompletely understood due to the limited availability of brain tissue evaluation from patient cases, and a lack of high-fidelity preclinical animal models for translational research. Here, we present the cellular and tissue neuropathologic analysis of a patient who experienced grade 4 ICANS after treatment with anti-CD19 CAR T therapy for mantle cell lymphoma. Our pathologic evaluation reveals a pattern of multifocal demyelinating leukoencephalopathy associated with a clinical course of severe ICANS. A focused analysis of glial subtypes further suggests region-specific oligodendrocyte lineage cell loss as a potential cellular and pathophysiologic correlate in severe ICANS. We propose a framework for the continuum of neuropathologic changes thus far reported across ICANS cases. Future elucidation of the mechanistic processes underlying ICANS will be critical in minimizing neurotoxicity following CAR T-cell and related immunotherapy treatments across oncologic and autoimmune diseases.

Background Fine-needle aspiration (FNA) is used to diagnose malignancies, recurrences, and metastases. The procedure is quick and well tolerated and can be facilitated by ultrasound guidance. Methods This article describes the authors' experience in using serial FNA to harvest cellular material during 4 clinical trials of immunotherapy by in situ vaccination in patients with low-grade lymphoma. Results Two hundred ninety-six FNA samples were collected from 44 patients over a span of approximately 6 weeks for each patient. Samples were sufficient in quantity and quality to be analyzed by flow cytometry and/or single-cell messenger RNA sequencing. FNA samples yielded an average of 12 × 106 cells with a mean cellular viability of 86%. Material collected from the tumor lymph nodes differed significantly in the proportions and phenotypes of cellular populations in comparison with matched peripheral blood samples. A comparison of flow cytometry results obtained by FNA directly from the patient and by FNA performed ex vivo and a dissociation of the same lymph node after surgical excision confirmed that FNA sampling of the patient accurately represented the tumor and the microenvironment. An analysis of the FNA samples from immunotherapy-treated target lymph nodes versus nodes from nontreated tumor sites provided insight into the impact of specific immunotherapy regimens. Conclusions This is the largest study describing the use of serial FNA sampling to harvest cellular material during immunotherapy clinical trials. The success of this technique opens the door for FNA sampling to expand significantly future investigations of the dynamic effects of investigational agents, be they immunotherapies or targeted therapies.

PURPOSE Although the majority of patients with relapsed or refractory large B-cell lymphoma respond to axicabtagene ciloleucel (axi-cel), only a minority of patients have durable remissions. This prospective multicenter study explored the prognostic value of circulating tumor DNA (ctDNA) before and after standard-of-care axi-cel for predicting patient outcomes. METHODS Lymphoma-specific variable, diversity, and joining gene segments (VDJ) clonotype ctDNA sequences were frequently monitored via next-generation sequencing from the time of starting lymphodepleting chemotherapy until progression or 1 year after axi-cel infusion. We assessed the prognostic value of ctDNA to predict outcomes and axi-cel–related toxicity. RESULTS A tumor clonotype was successfully detected in 69 of 72 (96%) enrolled patients. Higher pretreatment ctDNA concentrations were associated with progression after axi-cel infusion and developing cytokine release syndrome and/or immune effector cell–associated neurotoxicity syndrome. Twenty-three of 33 (70%) durably responding patients versus 4 of 31 (13%) progressing patients demonstrated nondetectable ctDNA 1 week after axi-cel infusion ( P < .0001). At day 28, patients with detectable ctDNA compared with those with undetectable ctDNA had a median progression-free survival and OS of 3 months versus not reached ( P < .0001) and 19 months versus not reached ( P = .0080), respectively. In patients with a radiographic partial response or stable disease on day 28, 1 of 10 patients with concurrently undetectable ctDNA relapsed; by contrast, 15 of 17 patients with concurrently detectable ctDNA relapsed ( P = .0001). ctDNA was detected at or before radiographic relapse in 29 of 30 (94%) patients. All durably responding patients had undetectable ctDNA at or before 3 months after axi-cel infusion. CONCLUSION Noninvasive ctDNA assessments can risk stratify and predict outcomes of patients undergoing axi-cel for the treatment of large B-cell lymphoma. These results provide a rationale for designing ctDNA-based risk-adaptive chimeric antigen receptor T-cell clinical trials.

Monitoring of measurable residual disease (MRD) is essential to the management of acute lymphoblastic leukemia (ALL) and is typically performed through repeated bone marrow (BM) assessments. Using a next-generation sequencing (NGS) MRD platform, we performed a prospective observational study evaluating the correlation between peripheral blood (PB) and BM MRD in adults with ALL receiving cellular therapies (hematopoietic cell transplantation [HCT] and chimeric antigen receptor T-cell [CAR-T] therapies). Among the study cohort (N = 69 patients; 126 paired PB/BM samples), we found strong correlation between PB and BM MRD (r = 0.87; P < .001), with a sensitivity and specificity of MRD detection in the PB of 87% and 90%, respectively, relative to MRD in the BM. MRD became detectable in the PB in 100% of patients who subsequently relapsed following HCT, with median time from MRD+ to clinical relapse of 90 days, and in 85% of patients who relapsed following CAR T, with median time from MRD+ to clinical relapse of 60 days. In adult patients with ALL undergoing cellular therapies, we demonstrate strong concordance between NGS-based MRD detected in the PB and BM. Monitoring of ALL MRD in the PB appears to be an adequate alternative to frequent invasive BM evaluations in this clinical setting.

Autologous T cells genetically engineered to express a chimeric antigen receptor (CAR) targeting CD19 and/or CD22 have achieved high complete response rates in patients with hematologic malignancies, but >50% of patients progress following therapy. Here, we sought to understand key T cell intrinsic factors impacting efficacy: CAR T cell expansion, persistence, and homing to the tumor. Using an endogenous T cell receptor (TCR) sequence as a ‘barcode’, we followed individual T cell clonotypes at the single-cell level from pre-manufacture apheresis and infusion products to tumor-involved lymph node and blood at peak and late expansion in 22 adult patients with relapsed or refractory large B cell lymphoma (LBCL) or acute lymphoblastic leukemia (ALL) treated with axicabtagene ciloleucel, an FDA-approved CD19-CAR T cell immunotherapy, or bispecific CD19/CD22 CAR T cells on an investigator-initiated trial (NCT03233854). The resulting CAR T cell atlas comprises matched transcriptome (scRNA-seq) and surface protein expression (CITE-seq) for 846,344 cells from 97 samples, with 215,045 unique TCR clonotypes identified, including 8,747 clonotypes that could be traced across 2+ timepoints in CAR mRNA+ cells. This atlas enabled us to ask: “What were the phenotypes of ‘successful’ CAR T cell clonotypes with optimal homing, expansion, and persistence properties at the time of infusion or pre-manufacture apheresis?” We found that successful T cell clonotypes at apheresis had juvenile features, including IL7R expression. Conversely, successful clonotypes in the infusion product had elevated interferon pathway activity and effector signatures, including GZMB expression. Further, we built a cell-cell interactome using all live cells from on-treatment biopsies and identified a set of 149 specific ligand-receptor pairs significantly enriched in patients who progressed. Finally, we defined dynamics of TCR clonotypes with predicted specificities for viral and self-antigens. These analyses pinpoint the identities of source T cells and infusion CAR T cells with properties impacting efficacy, and also identify ligand-receptor pairs that could be modulated to enhance CAR T cell response in the tumor at the genetic or pharmacological level. This work was supported in part by the Parker Institute for Cancer Immunotherapy, California Institute for Regenerative Medicine, Kite Pharma, and Stanford Cancer Institute. Citation Format: Zinaida Good, Mark P. Hamilton, Jay Y. Spiegel, Sreevidya Kurra, Moksha H. Desai, Snehit Prabhu, Shin-Heng Chiou, Christine Y. Yeh, Yiyun Chen, Eric Yang, Michael G. Ozawa, Fang Wu, Matthew J. Frank, Lori Muffly, Gursharan K. Claire, Juliana Craig, Maria I. Iglesias, Sushma Bharadwaj, Katherine A. Kong, Dhananjay Wagh, John Coller, Mark M. Davis, Sylvia K. Plevritis, Bita Sahaf, David B. Miklos, Crystal L. Mackall. Lineage tracing of CAR T cells in patients with B cell malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1128.

Background: Multiple myeloma (MM) is an incurable disease with a heterogenous clinical course and genomic landscape. Autologous anti-BCMA chimeric antigen receptor (CAR) T-cells are a promising new therapy, but determinants of response and resistance are not well known. Cell-free DNA (cfDNA) is a useful tool to study MM as it allows for repeated, non-invasive tumor assessment. We apply a novel method for simultaneously tracking tumor mutations and CAR T-cells from cfDNA using Cancer Personalized Profiling by Deep Sequencing (CAPP-Seq). Methods: We designed a 480kb CAPP-Seq hybrid capture panel to identify mutations, track tumor burden and minimal residual disease, and detect cfDNA derived from the CAR transgene (CAR-cfDNA) in patients receiving idecabtagene vicleucel (ide-cel). Flow cytometry (FC) for enumeration of CAR T-cells was performed from peripheral blood mononuclear cells (PBMCs) when available. Results: We profiled 153 biologic samples, including plasma, PBMCs, and bone marrow mononuclear cells, from 15 patients receiving ide-cel and 18 healthy controls. We observed a median of 84 SNVs (range 30-277) prior to therapy. Patients with prolonged responses (>90 days) had significantly lower circulating tumor DNA (ctDNA) at day 28 post-infusion than patients with early progression ( Conclusions: Cell-free DNA is a promising biomarker for mutational genotyping, disease monitoring, and tracking CAR T-cells in MM. The persistence of CAR-cfDNA has particular prognostic importance and novel strategies to increase CAR persistence should be explored. Citation Format: Mia Carleton, Hitomi Hosoya, Kailee L. Tanaka, Brian Sworder, Vanna Hovanky, Bita Sahaf, Matthew J. Frank, George E. Duran, Tian Y. Zhang, Sally Arai, David Iberri, Michaela Liedtke, David B. Miklos, Michael S. Khodadoust, Surbhi Sidana, David M. Kurtz. Tumor and immune determinants of response to anti-BCMA CAR T-cell therapy in multiple myeloma using cell-free DNA. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5707.

The prognosis of patients with large B-cell lymphoma (LBCL) that progresses after treatment with chimeric antigen receptor (CAR) T-cell therapy targeting CD19 (CAR19) is poor. We report on the first 3 consecutive patients with autologous CAR19-refractory LBCL who were treated with a single infusion of autologous 1 × 106 CAR+ T cells per kilogram targeting CD22 (CAR22) as part of a phase 1 dose-escalation study. CAR22 therapy was relatively well tolerated, without any observed nonhematologic adverse events higher than grade 2. After infusion, all 3 patients achieved complete remission, with all responses continuing at the time of last follow-up (mean, 7.8 months; range, 6-9.3). Circulating CAR22 cells demonstrated robust expansion (peak range, 85.4-350 cells per microliter), and persisted beyond 3 months in all patients with continued radiographic responses and corresponding decreases in circulating tumor DNA beyond 6 months after infusion. Further accrual at a higher dose level in this phase 1 dose-escalation study is ongoing and will explore the role of this therapy in patients in whom prior CAR T-cell therapies have failed. This trial is registered on clinicaltrials.gov as #NCT04088890.

Profiling of circulating tumor DNA (ctDNA) in the bloodstream shows promise for noninvasive cancer detection. Chromatin fragmentation features have previously been explored to infer gene expression profiles from cell-free DNA (cfDNA), but current fragmentomic methods require high concentrations of tumor-derived DNA and provide limited resolution. Here we describe promoter fragmentation entropy as an epigenomic cfDNA feature that predicts RNA expression levels at individual genes. We developed 'epigenetic expression inference from cell-free DNA-sequencing' (EPIC-seq), a method that uses targeted sequencing of promoters of genes of interest. Profiling 329 blood samples from 201 patients with cancer and 87 healthy adults, we demonstrate classification of subtypes of lung carcinoma and diffuse large B cell lymphoma. Applying EPIC-seq to serial blood samples from patients treated with PD-(L)1 immune-checkpoint inhibitors, we show that gene expression profiles inferred by EPIC-seq are correlated with clinical response. Our results indicate that EPIC-seq could enable noninvasive, high-throughput tissue-of-origin characterization with diagnostic, prognostic and therapeutic potential.

Chimeric antigen receptor (CAR) T-cell therapy targeting CD19 has significantly improved outcomes in the treatment of refractory or relapsed large B-cell lymphoma (LBCL). We evaluated the long-term course of hematologic recovery, immune reconstitution, and infectious complications in 41 patients with LBCL treated with axicabtagene ciloleucel (axi-cel) at a single center. Grade 3+ cytopenias occurred in 97.6% of patients within the first 28 days postinfusion, with most resolved by 6 months. Overall, 63.4% of patients received a red blood cell transfusion, 34.1% of patients received a platelet transfusion, 36.6% of patients received IV immunoglobulin, and 51.2% of patients received growth factor (granulocyte colony-stimulating factor) injections beyond the first 28 days postinfusion. Only 40% of patients had recovered detectable CD19+ B cells by 1 year, and 50% of patients had a CD4+ T-cell count

Allogeneic hematopoietic cell transplantation (HCT) remains an important treatment for adults with acute lymphoblastic leukemia (ALL). We hypothesized that advances in ALL and transplantation have resulted in improved HCT outcomes in recent years. In this study, we evaluated the characteristics and outcomes of adult ALL patients undergoing allogeneic HCT over the last decade. Patients with ALL aged 18 years and older who underwent allogeneic HCT at Stanford University between 2008 and 2019 were included in this study. Patients were divided into 2 eras based on year of HCT: 2008 to 2013 (earlier era) and 2014 to 2019 (later era). A total of 285 patients were included: 119 patients underwent HCT in the earlier era and 166 in the later era. Patients who underwent transplantation in the later era were more likely to be Hispanic (38% versus 21%) and to have an HCT-comorbidity index ≥3 (31% versus 18%). Donor source for HCT also differed with an increase in the use of HLA-mismatched donor sources (38% versus 24%), notably umbilical cord blood in the later era (16% versus 0%). Patients in the later era were less likely to undergo transplantation with active disease (4% versus 16%); pre-HCT rates of measurable residual disease were similar across the eras (38% versus 40%). In unadjusted analyses, overall survival (OS) improved across eras, with 2-year estimates for the later and earlier eras of 73% (95% confidence interval [CI], 66%-80%) versus 55% (95% CI, 46%-64%), respectively. Multivariable analysis confirmed the association between later era and OS (hazard ratio = 0.52, 95% CI, 0.34-0.78). Finally, among patients relapsing after HCT (25% in later era and 33% in earlier era), the use of novel immunotherapies increased in the later era (44% versus 3%), as did the median OS after post-HCT relapse (16 months versus 8 months, P.001). OS after HCT for adult ALL has improved in recent years. This is due, in part, to a significant improvement in the ability to effectively salvage adults with ALL relapsing after HCT.

We evaluated 79 patients with multiple myeloma (MM) ≥70 years referred to our blood and marrow transplant clinic, within 1 year of diagnosis from 2010 to 2019, for consideration of autologous stem cell transplant (ASCT). Thirty-eight (48%) of 79 patients underwent ASCT. ASCT was not pursued in 41 (52%) patients due to: patient or physician preference in 80% (n = 33) or ineligibility in 20% (n = 8). Baseline characteristics of patients in the two groups were similar. Median PFS from treatment start amongst patients undergoing ASCT (n = 38) vs. not (n = 41) was 41 months vs. 33 months, p = 0.03. There was no difference in OS, with estimated 5-year OS of 73% vs. 83%, respectively (p = 0.86). Day +100 transplant-related mortality (TRM) was 0%. ASCT was an independent favorable prognostic factor for PFS in multivariate analysis, after accounting for HCT-CI score, performance status, hematologic response, and maintenance. Finally, patients ≥70 years undergoing ASCT had similar PFS compared to a contemporaneous institutional cohort of patients

Most relapsed/refractory large B cell lymphoma (r/rLBCL) patients receiving anti-CD19 chimeric antigen receptor (CAR19) T cells relapse. To characterize determinants of resistance, we profiled over 700 longitudinal specimens from two independent cohorts (n = 65 and n = 73) of r/rLBCL patients treated with axicabtagene ciloleucel. A method for simultaneous profiling of circulating tumor DNA (ctDNA), cell-free CAR19 (cfCAR19) retroviral fragments, and cell-free T cell receptor rearrangements (cfTCR) enabled integration of tumor and both engineered and non-engineered T cell effector-mediated factors for assessing treatment failure and predicting outcomes. Alterations in multiple classes of genes are associated with resistance, including B cell identity (PAX5 and IRF8), immune checkpoints (CD274), and those affecting the microenvironment (TMEM30A). Somatic tumor alterations affect CAR19 therapy at multiple levels, including CAR19 T cell expansion, persistence, and tumor microenvironment. Further, CAR19 T cells play a reciprocal role in shaping tumor genotype and phenotype. We envision these findings will facilitate improved chimeric antigen receptor (CAR) T cells and personalized therapeutic approaches.

ImportanceDetermining whether neurofilament light chain (NfL) elevations in patients who develop immune effector cell–associated neurotoxicity syndrome (ICANS) occur before or after infusion of cellular product is important to identify high-risk patients and inform whether neuroaxonal injury is latent or a consequence of treatment.ObjectiveTo quantify serial NfL levels in patients undergoing cellular therapy.Design, Setting, and ParticipantsThis retrospective 2-center study examined plasma NfL levels in 30 patients with detailed medical and treatment history, including all major pretreatment and posttreatment risk factors. Exclusion criteria included dementia and severe, symptomatic central nervous system (CNS) involvement.Main Outcomes and MeasuresPatients’ NfL levels were measured at 7 time points: baseline (prelymphodepletion), during lymphodepletion, postinfusion day (D) 1, D3, D7, D14, and D30. Prediction accuracy for the development of ICANS was next modeled using receiver operating characteristic (ROC) classification. Finally, univariate and multivariate modeling examined the association between NfL levels, ICANS, and potential risk factors including demographic (age, sex), oncologic (tumor burden, history of CNS involvement), neurologic (history of nononcologic CNS disease or neuropathy), and neurotoxic exposure histories (vincristine, cytarabine, methotrexate, or CNS radiotherapy).ResultsA total of 30 patients (median [range] age, 64 [22-80] years; 12 women [40%] and 18 men [60%]) were included. Individuals who developed ICANS had elevations in NfL prior to lymphodepletion and chimeric antigen receptor T-cell infusion compared with those who did not develop ICANS (no ICANS: 29.4 pg/mL, vs any ICANS: 87.6 pg/mL; P Conclusions and RelevanceIn a subset of patients in this cross-sectional study, the risk of developing ICANS was associated with preexisting neuroaxonal injury that was quantifiable with plasma NfL level. This latent neuroaxonal injury was present prior to drug administration but was not associated with historic neurotoxic therapies or nononcologic neurologic disease. Preinfusion NfL may further permit early screening and identification of patients most at risk for ICANS. Additional studies are needed to determine NfL’s utility as a predictive biomarker for early (preemptive or prophylactic) intervention and to delineate the origin of this underlying neural injury.

Background As a result of the COVID-19 widespread pandemic, cryopreservation of allogeneic donor apheresis products was implemented to mitigate the challenges of donor availability and product transport. Although logistically beneficial, the impact of cryopreservation on clinical outcomes and graft composition remains unclear. Objectives To compare the outcomes and graft composition with cryopreserved versus fresh allografts in the setting of allogeneic hematopoietic cell transplantation (allo-HCT). Study design We retrospectively analyzed the clinical outcomes of 30 consecutive patients who received cryopreserved allografts between March and August 2020 as compared to 60 consecutive patients who received fresh allografts prior to the COVID-19 pandemic. Primary endpoints were hematopoietic engraftment, graft failure (GF) and secondary outcomes were overall survival (OS), relapse free survival (RFS) and non-relapse mortality (NRM). In addition, extended immunophenotype analysis was performed on cryopreserved versus prospectively collected fresh apheresis samples. Results Compared to fresh allografts, both neutrophil and platelet recovery were delayed in recipients of cryopreserved reduced intensity conditioning (RIC) allo-HCT with median times to engraftment of 24 days vs 18 days (P = .01) and 27 days vs 18 days (P = .069), respectively. We observed primary GF in 4 of 30 patients in the cryopreserved cohort (13.3%) vs only one of 60 patients (1.7 %) in the fresh cohort (P = .03). Cryopreserved RIC allo-HCT was associated with significantly lower median total, myeloid and T-cell donor chimerism at 1 month. OS and RFS were inferior for cryograft recipients with hazard ratio [HR (95%Cl)]: 2.16 (1.00, 4.67) and 1.90 (0.95, 3.79), respectively. Using an extended immunophenotype analysis we compared 14 samples from the cryopreserved cohort to 6 prospectively collected fresh apheresis donor samples. These analyses showed both decrease in total cell viability and significantly reduced absolute numbers of NK cells (CD3−CD56+) in the cryopreserved apheresis samples. Conclusion In this single institution study we note delayed engraftment and a trend toward clinical inferiority of cryopreserved vs fresh allografts. Further evaluation of the use of cryopreserved allografts and their impact on clinical and laboratory outcomes is warranted.

We evaluated the outcomes of 168 patients undergoing delayed or second autologous stem cell transplant (ASCT) for relapsed multiple myeloma (MM) from 2010 to 2019. Overall, 21% (n = 35) patients had received a prior transplant and 69% (n = 116) underwent transplant at first relapse. Overall, 27% patients had high-risk cytogenetics and 15% had ISS stage III disease. Stem cell collection was performed after relapse in 72% and 35% of patients received maintenance therapy. Median PFS from salvage treatment and transplant were 28 and 19 months, respectively. Median OS from salvage treatment and transplant was 69 and 55 months. Multivariate analysis revealed that ASCT in first relapse was associated with superior PFS (HR 0.63, p = 0.03) and OS (HR 0.59, p = 0.04) compared to later lines of therapy. In addition, PFS of ≥36 months with prior therapy was associated with improved PFS (HR 0.62, p = 0.04) and OS (HR 0.41, p = 0.01). Ninety-five patients underwent delayed transplant at first relapse, median PFS and OS from start of therapy was 30 and 69 months, and median OS from diagnosis was 106 months. These data may serve as a guide when counseling patients undergoing ASCT for relapsed MM and provide a benchmark in designing clinical trials of transplantation/comparative treatments for relapsed MM.

Autologous T cells genetically engineered to express a chimeric antigen receptor targeting CD19 (CD19-CAR) have achieved high complete response rates in patients with hematologic malignancies, but >50% of patients progress following therapy. Here, we sought to understand key T-cell intrinsic factors impacting efficacy, namely CAR T-cell expansion, persistence, and homing to the tumor. Using an approach called reverse fate mapping, we followed individual T-cell clones at the single-cell level from pre-manufacture apheresis to the infusion product, tumor-involved lymph node, and blood at peak and late expansion in 12 adult patients with relapsed or refractory large B-cell lymphoma treated with axicabtagene ciloleucel, an FDA-approved CD19-CAR T-cell immunotherapy. The resulting CAR T-cell atlas comprises matched transcriptome (scRNA-seq) and surface protein expression (CITE-seq) for 322,028 cells from 44 samples, with 119,397 unique T-cell receptor (TCR) clonotypes identified. This atlas enabled us to ask questions like: “What were the phenotypes of the most successful CAR T-cell clones at the time of infusion or pre-manufacture apheresis?” We found that T-cell clonotypes with juvenile features at apheresis, including IL7R expression, were the most successful at expansion to higher frequencies in the infusion product, while clones with effector gene expression programs, such as those encoding perforin and granzymes, contracted between apheresis and product. Conversely, it was GZMK-expressing T cells in pre-manufacture apheresis that were dominant in the tumor early following CAR T-cell infusion. Further, T-cell clonotypes with active effector programs at infusion dominated at peak expansion. Finally, we defined active expression modules and pathways in the infusion product for CAR T-cell clones that homed to the tumor or became dominant at late expansion. These analyses pinpoint the molecular mechanisms that could be modulated to rationally steer CAR T-cell differentiation trajectories at the genetic or pharmacological level. This work was supported in part by the Parker Institute for Cancer Immunotherapy, California Institute for Regenerative Medicine, Kite Pharma, and Stanford Cancer Institute. Citation Format: Zinaida Good, Mark P. Hamilton, Jay Y. Spiegel, Sreevidya Kurra, Moksha Desai, Snehit Prabhu, Eric Yang, Michael G. Ozawa, Paul J. Hanson, Fang Wu, Matthew J. Frank, John H. Baird, Lori Muffly, Gursharan K. Claire, Juliana Craig, Katherine A. Kong, Dhananjay Wagh, John Coller, Sylvia K. Plevritis, Bita Sahaf, David B. Miklos, Crystal L. Mackall. Reverse fate mapping of CD19-targeted CAR T cells in patients with large B-cell lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3603.

Key Points Clinically significant bleeding occurred in 9% of patients after CAR T therapy and was associated with features of systemic coagulopathy.Low baseline platelets and possibly high-grade ICANS are risk factors for bleeding and require close monitoring for bleeding up to 1 month., Visual Abstract, Bleeding and thrombotic events are an emerging toxicity associated with chimeric antigen receptor (CAR) therapies. To determine their incidence, we retrospectively analyzed consecutive adult patients (N = 127) with large B-cell lymphoma (LBCL) or B-cell acute lymphoblastic leukemia (B-ALL) treated from 2017 through 2020 with axicabtagene ciloleucel (axi-cel; n = 89) or a bispecific CD19/CD22 CAR (n = 38). Twelve (9.4%) and 8 (6.3%) patients developed bleeding and thrombosis within the first 3 months, respectively. In the axi-cel subgroup, these occurred in 11.2% and 6.7%, respectively. Bleeding occurred between days 8 and 30 (median, 17.5) and thrombosis between days 2 and 91 (median, 29). Bleeding sites included genitourinary, soft tissue, intracranial, gastrointestinal, and pulmonary and were associated with features of consumptive coagulopathy. On univariate analysis, patients with bleeding were older, had lower baseline platelets (86 × 103/μL vs 178 × 103/μL; P < .01), lower platelet and fibrinogen nadirs , and elevated lactate dehydrogenase. Immune effector cell (IEC)–associated neurotoxicity syndrome (ICANS) grade ≥3 was associated with increased bleeding (50% vs 15%; P = .01), thrombosis (50% vs 16%; P = .04), prothrombin time prolongation, hypofibrinogenemia, and elevated D-dimer. Low pretreatment platelet counts were associated with bleeding in a multivariate logistic regression model. Patients with thrombocytopenia or severe ICANS are at increased risk of bleeding and should be closely monitored, particularly within the first month after CAR therapy. Future studies in larger cohorts should assess risk factors for systemic coagulopathies in CAR T therapy, including their association with neurotoxicity.

Growth factor and chemotherapy-based stem cell mobilization strategies are commonly used to treat patients with multiple myeloma. We retrospectively compared 398 patients mobilized between 2017 and 2020 using either cyclophosphamide (4 g/m(2)) plus granulocyte colony-stimulating factor (G-CSF) or G-CSF alone, with on demand plerixafor (PXF) in both groups. Although total CD34(+) yield was higher after chemomobilization compared with G-CSF +/− PXF (median, 13.6 × 10(6)/kg versus 4.4 × 10(6)/kg; P < .01), achievement of ≥2 × 10(6) CD34(+) cells (95% versus 93.7%; P =.61) and rates of mobilization failure (5% versus 6.3%; P =.61) were similar. Fewer patients required PXF with chemomobilization (12.3% versus 49.5%; P < .01), and apheresis sessions were fewer (median, 1 [range, 1 to 4] versus 2 [range, 1 to 5]). The rate of complications, including neutropenic fever, emergency department visits, and hospitalizations, was higher after chemomobilization (30% versus 7.4%; P < .01). Previous use of ≤6 cycles of lenalidomide did not impair cell yield in either group. The median cost of mobilization was 17.4% lower in the G-CSF +/− PXF group (P = .01). Between group differences in time to engraftment were not clinically significant. Given similar rates of successful mobilization, similar engraftment time, and less toxicity and lower costs compared with chemomobilization, G-CSF with on-demand PXF may be preferable in myeloma patients with adequate disease control and limited lenalidomide exposure.

Background: MGTA-145, a CXCR2 agonist, has shown promising activity for hematopoietic stem cell (HSC) mobilization with plerixafor in pre-clinical models and healthy volunteers. It has the potential to become the first GCSF free regimen for HSC mobilization/apheresis in preparation for transplant, with fewer side effects, better patient experience and optimal resource utilization. Methods: We conducted a single center phase 2 trial of MGTA-145 + plerixafor for HSC mobilization in patients with multiple myeloma (MM), NCT04552743. Primary endpoint was collection of ≥2 x 10 6 CD34+ cells/kg in up to 2 days of mobilization/apheresis. Secondary endpoints were collection of 4 and 6 x 10 6 CD34+ cells/kg, safety and engraftment. Patients with MM, 18-70 years of age, within 1 year of starting treatment & CrCl > 30 ml/min were eligible. Patients received plerixafor 0.24 mg/kg (0.16 mg/kg if renal dysfunction) SQ, followed 2 hours later by MGTA-145 (0.03 mg/kg) IV over 3-10 minutes and apheresis within 30 minutes. Mobilization and collection were repeated for a second consecutive day if day 1 yield was < 6 x 10 6 CD34+ cells/kg. The study was open-label single arm trial of 15 patients. If 13 or more patients met primary endpoint, an expansion cohort of 10 patients was planned. The trial has 85% power at a 5% one-sided type I error rate. Our analysis is based on aggregated results from total cohort of 25 patients. Results: Median age was 62 years, 52% were female, 24% had ISS stage 3, 57% had high-risk FISH (primarily gain1q). Induction therapy was VRD in 68% (17), daratumumab VRD in 24% (6), CyBorD in 8% (2) patients. Median duration of induction was 4 months (3-6) and median lenalidomide exposure was 5 cycles (1-8), with > VGPR in 88% of patients. (Table 1) Plerixafor 0.24 mg/kg was given in 24 (96%) patients. Median pre-apheresis CD34 count was day 1, 24/uL (3-99) & day 2, 15/uL (5-46). Median total HSC cell yield (CD34+ cells/kg x 10 6) was 5.0 (1.1-16.2), day 1 yield was 3.4 (0.3-16.2) and day 2 yield was 1.9 (0.5-4.6) (Fig1). 88% (n=22) of patients met the primary endpoint of collecting sufficient HSCs in < 2 days of mobilization + apheresis to proceed to transplant, 68% (17) in 1 day (2 x 10 6 CD34+ cells/kg). 3 patients who did not meet primary endpoint successfully collected HSCs with standard GCSF + plerixafor dosing & 2-3 apheresis sessions. Secondary endpoints of 4 and 6 x 10 6 CD34+ cells/kg in < 2 days were met in 68% (17) and 40% (10) patients. MGTA-145+plerixafor was well tolerated. At least 1 treatment emergent AE (TEAE) with MGTA-145 was seen in 60% of patients (grade 1, n= 14, grade 2, n=1). Pain (all grade 1) was most common, seen in 44% (11), with 38% (9) patients experiencing acute onset transient bone pain with MGTA-145 (duration: 7 minutes, range: 3-28). Using the validated Brief Pain Inventory, 56% (14) patients self-reported pain with mobilization vs 40% (10) at baseline. 7/15 patients without baseline pain reported pain with mobilization. An increase for worst pain was seen on mobilization day 1, that returned to the baseline, but no difference for aggregate score of pain severity/interference (linear mixed effects model). At last follow-up, 18 patients have completed transplant with MGTA-145 mobilized graft, with melphalan 200 mg/m 2 in 15 (83%) patients. Median of 3.5 (2.2-8.1) x 10 6 CD34+ cells/kg were infused. All patients have engrafted. Median time to neutrophil engraftment of 12 days (range: 11-15) & platelet engraftment (platelets > 20,000, no transfusion in 7 days) of 17.5 days (15-33) are comparable to historical data (DiPersio et al. 2009). RBC transfusion was needed in 3 (17%) patients. 14 patients have day 100 follow-up, all with durable engraftment. MGTA-145 + plerixafor mobilized grafts had a favorable graft composition. We observed high enrichment for CD90+CD45RA- among CD34+ cells, a CD34 subset of long term engrafting HSCs (median: 36% of CD34+ cells, range 10-66%, N=25). 74% (17 of 23) grafts were minimal residual disease negative with next generation flow cytometry. Conclusions: This is the first study to evaluate the novel G-CSF-free regimen of MGTA-145 + plerixafor for HSC cell mobilization & collection for hematologic malignancies. The study cohort was representative of transplant eligible patients with MM, with 88% patients meeting the primary endpoint. The regimen was well tolerated. Patients achieved timely & durable engraftment. Our data support further development of this regimen for rapid HSC mobilization. Figure 1 Figure 1. Disclosures Sidana: Janssen: Consultancy, Research Funding; Allogene: Research Funding; Magenta Therapeutics: Consultancy, Research Funding; BMS: Consultancy. Kumar: Bluebird Bio: Consultancy; Adaptive: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Carsgen: Research Funding; Astra-Zeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Merck: Research Funding; Oncopeptides: Consultancy; KITE: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Tenebio: Research Funding; Takeda: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Beigene: Consultancy; Antengene: Consultancy, Honoraria; Amgen: Consultancy, Research Funding; Roche-Genentech: Consultancy, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; BMS: Consultancy, Research Funding; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Research Funding. Muffly: Pfizer, Amgen, Jazz, Medexus, Pfizer: Consultancy; Astellas, Jasper, Adaptive, Baxalta: Research Funding; Adaptive: Honoraria, Other: fees for non-CME/CE services: , Research Funding. Arai: Magenta Therapeutics: Research Funding. Meyer: Indee, Jura: Consultancy; Orca Biosystems: Research Funding; GigaImmune: Current holder of stock options in a privately-held company; Triursus Therapeutics: Current holder of stock options in a privately-held company. Rezvani: US Department of Justice: Consultancy; Kaleido: Other: One-time scientific advisory board; Nohla Therapeutics: Other: One-time scientific advisory board; Pharmacyclics-Abbvie: Research Funding. Weng: Kite Pharma: Research Funding. Frank: Kite-Gilead: Membership on an entity's Board of Directors or advisory committees; Allogene Therapeutics: Research Funding; Adaptive Biotechnologies: Research Funding. Shiraz: Kite Pharma-Gilead: Research Funding. Girgenti: Magenta Therapeutics: Current Employment. Goncalves: Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Schmelmer: Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company, Patents & Royalties. Davis: Magenta Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Shizuru: Jasper Therapeutics, Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Other: Chair of scientific advisory board; Forty seven Inc: Other: Inventor on a patent licenses by Forty Seven. Forty seven was acquired by Gilead in 2020. Miklos: Pharmacyclics: Patents & Royalties; Kite, a Gilead Company, Amgen, Atara, Wugen, Celgene, Novartis, Juno-Celgene-Bristol Myers Squibb, Allogene, Precision Bioscience, Adicet, Pharmacyclics, Janssen, Takeda, Adaptive Biotechnologies and Miltenyi Biotechnologies: Consultancy; Pharmacyclics, Amgen, Kite, a Gilead Company, Novartis, Roche, Genentech, Becton Dickinson, Isoplexis, Miltenyi, Juno-Celgene-Bristol Myers Squibb, Allogene, Precision Biosciences, Adicet, Adaptive Biotechnologies: Research Funding; Adaptive Biotechnologies, Novartis, Juno/Celgene-BMS, Kite, a Gilead Company, Pharmacyclics-AbbVie, Janssen, Pharmacyclics, AlloGene, Precision Bioscience, Miltenyi Biotech, Adicet, Takeda: Membership on an entity's Board of Directors or advisory committees.

Despite impressive progress, more than 50% of patients treated with CD19-targeting chimeric antigen receptor T cells (CAR19) experience progressive disease. Ten of 16 patients with large B cell lymphoma (LBCL) with progressive disease after CAR19 treatment had absent or low CD19. Lower surface CD19 density pretreatment was associated with progressive disease. To prevent relapse with CD19− or CD19lo disease, we tested a bispecific CAR targeting CD19 and/or CD22 (CD19-22.BB.z-CAR) in a phase I clinical trial (NCT03233854) of adults with relapsed/refractory B cell acute lymphoblastic leukemia (B-ALL) and LBCL. The primary end points were manufacturing feasibility and safety with a secondary efficacy end point. Primary end points were met; 97% of products met protocol-specified dose and no dose-limiting toxicities occurred during dose escalation. In B-ALL (n = 17), 100% of patients responded with 88% minimal residual disease-negative complete remission (CR); in LBCL (n = 21), 62% of patients responded with 29% CR. Relapses were CD19−/lo in 50% (5 out of 10) of patients with B-ALL and 29% (4 out of 14) of patients with LBCL but were not associated with CD22−/lo disease. CD19/22-CAR products demonstrated reduced cytokine production when stimulated with CD22 versus CD19. Our results further implicate antigen loss as a major cause of CAR T cell resistance, highlight the challenge of engineering multi-specific CAR T cells with equivalent potency across targets and identify cytokine production as an important quality indicator for CAR T cell potency., Bispecific CAR T cells targeting CD19 and CD22 exhibit clinical activity and low toxicity in patients with large B cell lymphoma and B cell acute lymphoblastic leukemia, with relapses associated with loss of CD19 but not CD22.