ALBERT

Description: Genetic alterations of the mixed-lineage leukemia (MLL) gene are commonly implicated in the development of acute leukemia. In acute myeloid leukemia (AML), a partial tandem duplication (PTD) of MLL occurs in about 5%-11% of patients, mainly in adults and rarely inchildren( Meyer C et al Leukemia 2013) .It is not known whether the initial expression level of MLL-PTD affects the prognosis of AMLs. Therefore, we analyzed the clinical data of 36 AML patients with MLL-PTD treated in our institute from January 2014 to January 2016, and tried to investigate whether the initial level of MLL-PTD has an impact on the prognosis of AML patients. MLL-PTD is present in adult non-leukemichematopoiesisand observed in bone marrow and peripheral blood of adult healthy individuals. In order to identify the threshold of MLL-PTD for AML patients, a control group of bone marrow samples from 29 normal adult donors were tested. The median value of MLL-PTD was 0.046(0.014¡«0.080)%. Patients with AML were considered MLL-PTD positive if their expression level was higher than 0.08%. 430 patients were newly diagnosed AML and treated in institute between January 2014 and January 2016. Among these AML patients, AML patients with MLL-PTD were selected as the subject patients. The following inclusion criteria were applied: (1) more than 16 years old; (2)Bone marrow MLL-PTD/ABL£¾0.08%; (3)receivedmore than 2 cycles of chemotherapy. Our study was approved by the Ethics Committee of Peking UniversityPeople¡¯s Hospital.All patients offered signed informed consent to participate in the study.The cutoff date for follow-up was Match 31, 2016. Of the 430 de novo AML patients, 36(8.4%) patients were MLL-PTD positive. The median age was 48(22¡«72) years. 28 cases were diagnosed M2, and the others included 4 cases of M4, 2 cases of M5 and 2 cases of M6. 5 cases were FLT3-ITD mutation+, 2 cases were NPM1 mutation+,7cases were double-CEBPA mutation+. The complete remission (CR) rate after first induction chemotherapy was 58.3%, the total CR rate after second chemotherapy was 66.7%, 1 case achieved CR after third chemotherapy, and 1 case achieved CR withsorafenibtreatment. 2 cases receivedallo-HSCT. In the 26 cases who achieved CR1, 7 cases relapsed at a median of 5 months. The median follow-up was 4.8 (2¡«17.2) months, and 10 cases were died. According to Hans B. Ommenetal¡äs study (Ommen HB et al Br J Haemato2014), ROC analysis was performed to determine the potential optimal MLL-PTD cutoff level to predict the incidence of CR after second induction chemotherapy. We established MLL-PTD ¡Ý1% as the cut of value. 19 cases were MLL-PTD £¼1% as the low level group and 17 cases were MLL-PTD ¡Ý1% as the high level group. Distribution of FAB type (M2 incidence 100% vs. 53%, P=0.003) and biallelic CEBPA mutation incidence (37% vs. 0%, P=0.008) were significantly different between the two groups. (Table 1) The CR rate after first induction chemotherapy (78.9% VS 35.3%, P=0.008) and CR rate after second induction chemotherapy (84.2% VS 47.1%,P=0.001) were also significantly different between the two groups.(Table 2) Logistic regression analysis showed that high level of MLL-PTD was the only independent risk factor of CR rate after second induction chemotherapy (OR=0.16, P=0.024). There were 5 patients died in each group. The 15-month estimated OS between low-level and high-level groups was not significantly different (51.5% vs .67.2%, P=0.607). Our data indicates that the initial level of MLL-PTD in patients with AML affects clinical prognosis, and patients with high initial level MLL-PTD have a lower CR rate. Low level of MLL-PTD may not play a part in the development of acute leukemia. And it needs further observation to identify the effect of initial MLL-PTD level on survival. Kong Jun and Jiang Hao contributed equally to this work Disclosures No relevant conflicts of interest to declare.

Description: Dickkopf-3 (DKK-3) is a Wnt signaling antagonist, and DKK3 inactivation is associated with poor prognosis in various solid tumors and hematologic malignancies. Epigenetic hypermethylation has been implicated in downregulation of DKK3, but other regulatory mechanisms remain to be investigated. In this study, we examined DKK3 expression and the role of microRNA in the regulation of DKK3 in adult B-ALL. Our results showed low levels of DKK3 expression in Nalm-6 and BALL-1 cell lines, and leukemia cells from adult B-ALL patients. DKK3 expression was remarkably lower in the initial diagnosis and relapse samples than in the matched samples during remission. In addition to hypermethylation, low levels of DKK3 were associated with high expression of miR-708. This finding was similar to that in childhood ALL. The miR-708 was predicted to bind to the 3'-UTR of DKK3. By using miR-708 mimics, we found that miR-708 targets DKK3 to promote B-ALL cell proliferation through cell cycle promotion and apoptosis inhibition. The treatment with 5-aza-2'-deoxycytidine significantly increased DKK3 and decreased p-GSK3β, cyclin D1 and nuclear and cytoplasmic β-catenin protein expression. A synergistic effect was seen when the miR-708 inhibitor and 5-aza were used simultaneously. Although miR-708 was reported to either promote or suppress tumorigenecity in some solid tumors, our findings indicate that suppression of miR-708 increases DKK3 expression to inhibit the Wnt/β- catenin signaling pathway in B-ALL. Disclosures No relevant conflicts of interest to declare.

Description: Background: Acute graft-versus-host disease(aGVHD) remains a major complication following allogeneic hematopoietic stem cell transplantation(allo-HSCT). The pathogenesis of aGVHD is commonly considered to be caused by exaggerated and undesirable immune responses. Metabolism not only provide energy and substrates for T cell growth and survival, but also instruct effector functions, differentiation, and gene expression of T cells. In this regard, the metabolic profile of T cells was reported to play a critical role in the occurrence and development of many immunological disorders such as systemic lupus erythematosus and rheumatoid arthritis. Murine studies found that alloreactive T cells use aerobic glycolysis as the predominant metabolic process to meet activation and proliferation demand after allo-HSCT. However, the metabolic profile of T cells and the approach for regulating T cell metabolism in aGVHD patients remains to be elucidated. Aims: To determine the metabolic state in T cells of patients with aGVHD. Moreover, to investigate the effect of the novel approach targeting the abnormal metabolism in T cells of aGVHD patients, which may provide a potential therapeutic target for aGVHD patients after allo-HSCT. Methods: In this prospective case-control study, a total of 25 patients with aGVHD and 25 matched patients without aGVHD(non-aGVHD) after allo-HSCT were enrolled. T cell subsets were analyzed in aGVHD and non-aGVHD patients by flow cytometry. Th1, Th2, Th17, and Treg cells were identified as CD4+IFN-γ+, CD4+IL-4+, CD4+IL17A+, and CD4+CD25+Foxp3+, respectively. Tc1 and Tc2 cells were identified as CD8+IFN-γ+ and CD8+IL-4+, respectively. In order to determine the metabolic state in T cells of patients with aGVHD and non-aGVHD, the metabolic profile was determined using a Seahorse XF96 Analyzer. The glucose consumption and lactate production rates were detected by glucose assay kit and lactate assay kit. The mitochondrial mass, the mitochondrial membrane potential, the protein expressions for the lipid metabolism enzyme CTP1a and the glycolytic activator PFKFB3 were measured by flow cytometry. To further understand the metabolic state of T cells in aGVHD and non-aGVHD patients and investigate its mechanism, RNA sequencing (RNA-Seq) was performed to analyze the gene expression profiles of T cells. Subsequently, to explore the potential way of targeting the abnormal metabolism in T cells, the glycolysis inhibitor 3-PO was administrated to T cells from aGVHD patients. Results: When compared with T cells in non-aGVHD patients, T cells in aGVHD patients were polarized towards pro-inflammatory T cells, characterized by an elevated proportion of Tc1, Th1 and Th17. Furthermore, T cells isolated from aGVHD patients exhibited higher extracellular acidification rate, as well as the increased glucose consumption rate and lactate production rate compared to those in non-aGVHD patients. Moreover, elevated expression of PFKFB3 was observed in T cells, especially in naïve T cells of aGVHD patients, but oxygen consumption rate, CPT1A, mitochondrial mass or membrane potential showed no significant differences in T cells between aGVHD and non-aGVHD patients. These results implied higher glycolytic activity of T cells in aGVHD patients when compared with those in non-aGVHD patients. Consistent with the increased glycolytic activity observed in T cells from aGVHD patients, the mRNA levels of genes involved in the glycolytic pathway were substantially elevated in T cells of aGVHD patients compared to those in non-aGVHD patients. Importantly, in vitro treatment with glycolysis inhibitor 3-PO improved the activity of T cells derived from aGVHD patients through down-regulating glycolytic activity of T cells. Summary/Conclusion: The current study demonstrated that T cells in aGVHD patients preferentially depend on glycolysis to meet activation and proliferation demands. Furthermore, the activity of T cells from aGVHD patients could be ameliorated by glycolysis inhibitor 3-PO in vitro. Although further validation is required, T cell glycolysis promises to be a novel therapeutic target for aGVHD patients after allo-HSCT. Disclosures No relevant conflicts of interest to declare.

Description: Introduction: T-cell based therapies have emerged as one of the major breakthroughs in anticancer treatment: Immune checkpoint inhibitors, chimeric antigen receptor gene-modified T-cells (CAR-T-cells), and T-cell engaging bispecific antibodies (BsAb) are leading the advances. In the era of personalized medicine, T-cells offer alternative strategies to overcome resistance to chemotherapy or small molecules. Yet, hurdles for such therapy can be crippling, such as inability of T cells to infiltrate "cold tumors", cytokine release syndrome following T cell-based therapies, neurologic toxicity, and on-target off-tumor effects. To address these hurdles, polyclonal T-cells armed with GD2xCD3 or HER2xCD3 BsAb for cytotherapy hold promise. Ganglioside GD2 and HER2 are tumor associated surface antigens expressed in a broad spectrum of aggressive malignancies, while being restricted in normal tissues. Phase I trials of T-cells armed with a chemical conjugate of hu3F8 x mouse OKT3 (NCT02173093) or trastuzumab x mouse OKT3 (NCT00027807) demonstrated the safety of 160 x 106/kg/injection x 8 doses (or 1.28 x 109/cycle) with suggestion of clinical benefit. Here, we report the safety and efficacy of adoptive T-cell therapy armed with the recombinant forms of these BsAb for the treatment of GD2(+) and/or HER2(+) tumors in preclinical models. Methods: Recombinant anti-GD2 BsAb and anti-HER2 BsAb were made using the IgG(L)-scFv platform (Can Immunol Res, 3:266, 2015, Oncoimmunology, PMID:28405494). T-cells from normal volunteer donors were isolated, activated and expanded by CD3/CD28 beads in the presence of 100 IU/mL of interleukin 2 (IL-2). Between day 7 and day 14, activated T cells (ATCs) were harvested and armed for 20 minutes at room temperature with -GD2-BsAb or HER2-BsAb. After washing, armed ATCs were tested for cell surface density of BsAb and antibody dependent T cell mediated cytotoxicity (ADTC) in vitro. In vivo anti-tumor potencies of armed T cells were tested against GD2(+) or HER2(+) cell lines or patient derived xenografts (PDXs) in BALB-Rag2-/-IL-2R-γϲ-KO (DKO) mice. Results: GD2-BsAb of the IgG(L)-scFv form showed superior potency over other bispecific platforms in vitro and in vivo. GD2-BsAb or HER2-BsAb armed ATCs showed potent antigen-specific cytotoxicity against GD2 or HER2 positive tumors such as neuroblastoma, melanoma and osteosarcoma in vitro over a range of antibody dose (5 to 500 ng/106 cells). Optimal arming per T cell required 25,000 to 45,000 idiotype(+) molecules. There was no evidence of activation induced cell death when confronted by antigen or tumor target. In vivo GD2-BsAb or HER2-BsAb armed ATCs could ablate neuroblastoma, malignant melanoma, and osteosarcoma tumors over a range of cell doses (10x106, 20x106 and 40x106 per injection, one to three times a week for 2 to 4 weeks) with a range of BsAb doses (5 ng to 500 ng/million of T-cells) without significant toxicities in DKO mice. By immunohistochemistry, the frequency of tumor infiltrating CD3(+) T-cells strongly correlated with tumor response. Conclusions: Using the IgG(L)-scFv format, GD2-BsAb or HER2-BsAb armed ATCs could provide a potent and economical cytotherapy platform against GD2(+) or HER2(+) tumors without the complexity of gene modification (as in chimeric antigen receptor modified T cells). At such low arming doses, where BsAb is T cell bound, where ADTC is not induced, and T cell expansion is not required for anti-tumor effect, clinical toxicity is expected to be low. Disclosures Cheung: Ymabs: Patents & Royalties.

Description: Background Therapy-related myelodysplastic syndrome (MDS) and acute leukemias represent a major cause of non-relapse morbidity and mortality in childhood cancer survivors, and have been associated with exposure to cytotoxic therapies (e.g. radiation, alkylators, topoisomerase inhibitors). Neuroblastoma (NB) patients receive multimodality therapy with intensive chemotherapy, radiation, and immunotherapy, and have had high rates of treatment-related leukemias (Kushner, Cheung et al. 1998). Whilst specific therapeutic modalities have been associated with distinct cytogenetic and molecular abnormalities, our understanding of the relationships between timing of mutation acquisition, dynamics of clonal selection in relation to specific therapeutic modalities, and how these in unison result in overt leukemia, remains limited. Motivated to study these relationships and inform future screening guidelines, we characterized serial bone marrow (BM) samples obtained during surveillance for NB recurrence and therapy-related leukemias. Methods We studied a total of 219 serial samples from 55 NB patients treated at MSKCC over a 21-year period. These included 19 patients with MDS or leukemic transformation (median time following NB diagnosis 4.4 years), 15 with transient cytogenetic abnormalities (median time to abnormality 3.1 years), and 21 matched controls (median disease-free follow-up 8.1 years). On average, we analyzed 4 samples per transformation patient, representative of pre-treatment timepoints at NB diagnosis, during NB treatment, and throughout follow-up, with a lead time of 18 years - 1 month prior to transformation, and at time of leukemic transformation. Comprehensive genomic profiling with targeted gene sequencing (MSK-IMPACT Heme), RNA-seq, and Archer FusionPlex was performed to capture acquired gene mutations, chromosome-level copy number alterations (CNA), and fusion genes at the time of diagnosis. Backtracking studies were performed in longitudinal samples with complete molecular and clonal characterization. Results We detected at least one disease-defining alteration in all cases with MDS or leukemic transformation at time of diagnosis, with a total of 61 putative oncogenic events across all patients (median 3 alterations per patient, range 1-12). As expected, the most frequent events were MLL fusions (n=6 patients), and mutations in TP53 (n=5 patients). The remaining cases harbored chromosomal aneuploidies or acquired gene mutations in NPM1, IDH1, PTPN11, NRAS, BCOR, CUX1, STAG2, WT1, amongst other genes, at a median variant allele frequency 24% (range 4-68%). Backtracking studies identified at least one of these mutations in 81% of patients at a sampling time point prior to diagnosis. In contrast, only two patients (2/15) from the cohort with transient cytogenetic abnormalities had acquired somatic mutations detected at a median VAF of 3%, with resolution of the molecular alterations in subsequent samples. One of the control cases also had an identified mutation, though this patient died of NB with limited hematologic follow-up. The median time of detection of a putative driver alteration was 6 months prior to leukemic transformation, with the earliest identified at 2.8 years prior to disease transformation. At least 3 cases of MLL fusions were detected 2.2, 14.5, and 20.9 months prior to diagnosis. Mutations in TP53 co-occurred with CNAs in all patients in our cohort, and has been shown to be predictive of chemoresistant disease. Mutations in TP53 were also identified in at least 2 pre-leukemic samples per patient in 4 of 5 cases, at a median VAF 5% (range 5-20%). In all of these cases, TP53 mutations preceded clinically detectable CNA. Genetic evolution led clonal dominance, which, intriguingly, often preceded disease presentation in the context of normal hematopoeisis. We also found evidence of clonal drifts, possibly as a consequence of treatment effects. Conclusions Our preliminary data demonstrate that NB patients at risk of developing secondary leukemia can be identified by molecular profiling of BM aspirates obtained during routine disease surveillance for NB. These findings present an opportunity for the development of early detection studies for patients with pediatric malignancies undergoing intensive therapy and importantly inform studies into mechanisms of leukemic transformation and specific gene-treatment effects. Disclosures Cheung: Ymabs: Patents & Royalties.

Description: Key Points EBV-induced DLBLs are characterized by genomic and transcriptomic alterations in the Rho pathway. Targeting the Rho pathway using a ROCK inhibitor, fasudil, inhibited tumor growth in EBV-positive DLBL patient-derived xenograft models.

Description: Introduction: The autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy, axicabtagene ciloleucel (Axi-cel) improved long-term survival of patients with relapsed/refractory diffuse large B-cell lymphoma (r/r DLBCL). Long-term analysis of the pivotal ZUMA-1 trial indicates a 2-year PFS of ~40% (Locke, Lancet Oncology 2018). Early identification of patients with increased relapse risk may allow for early intervention and improved outcomes. In a pilot study of 6 ZUMA-1 patients, minimal residual disease (MRD) evaluation via a next-generation sequencing MRD assay (Adaptive Biotechnologies, Seattle, WA) to assess for circulating tumor (ct)DNA, mirrored clinical outcome as assessed by PET-CT (Hossain et. al. Leukemia & Lymphoma 2019). Based on these promising results, a multi-institutional prospective study utilizing cell-free MRD assessments to predict outcomes in r/r DLBCL patients after Axi-cel therapy was initiated. Methods: To identify tumor clonotype(s), tumor DNA extracted from archival paraffin-embedded tissue underwent PCR amplification of IgH-VDJ, IgH-DJ and IgKappa/Lambda regions using universal consensus primers. CtDNA levels were measured pre-LD, 0, 7, 14, 21, 28, 56, 90, 180, 270, and 365 days following Axi-cel infusion. PET-CT scans were obtained at baseline, Day 28, Month 3, 6, and 12 with response assessed per Lugano criteria. Deauville 1-3 was considered PET-negative. The protocol prespecified that patients with less than Day 28 follow-up be excluded from analysis. Any detectable ctDNA was considered MRD positive. Results: Here we report on the pre-planned analysis of the first 50 study patients with at least a Day 28 MRD assessment and 3 months of follow up. An additional 4 patients with at least 3 months of follow-up but who did not have a Day 28 MRD assessment were also included. Baseline characteristics and clinical outcomes of patients were similar to ZUMA-1 and a real-world analysis of 295 patient who received Axi-cel (Nastoupil et al ASH 2018). The median age was 61 years old (range 19-76) (53.7% male, 46.3% female) and 59% of patients received 3 or more prior lines of therapy (range 1-6). After a median follow-up of 7.5 months, the best overall response rate was 87% (47 of 54) and complete response rate was 57% (31 of 54). The median OS was not reached and median PFS was 4.6 months (panel A). At Day 28, 56% (28 of 50) of patients were MRD negative (MRD-neg) and 44% (22 of 50) were MRD positive (MRD-pos). As compared to MRD-pos, MRD-neg correlated with improved median PFS (not reached vs. 2.96 months, p

Description: Background JAK2 expression and activity increased in classical Hodgkin lymphoma (HL) and primary mediastinal large B-cell lymphoma (PMBCL) because both of them were reported to have chromosome 9p24.1/JAK2 amplification. Thus, JAK2 has been suggested as a potential therapeutic target in both disease having similar clinical and genetic features, and a previous in vitro and in vivo study showed cHL and PMBCL with JAK2 amplification were sensitive to JAK2 inhibition. However, the efficacy of JAK2 inhibitor has never been evaluated in clinical trials with cHL and PMBCL patients. We performed a prospective pilot proof of concept study with JAK2 inhibitor, ruxolitinib in relapsed or refractory cHL and PMBCL to evaluate the effect of JAK2 inhibition. Methods This study enrolled only relapsed or refractory cHL and PMBCL. Subjects should experience at least two times of relapse or treatment failure before enrollment. We enrolled subjects who were more than 18 years and had at least one lesion of FDG-PET positive measureable disease. Ruxolitinib was administered orally at a dose of 20 mg twice a day of a 28-day cycle. Treatment cycles were repeated up to 16 cycles until disease progression or unacceptable toxicity occurred. The primary objective was to assess the overall disease control rate including the achievement of complete response (CR), partial response (PR) and stable disease (SD). Response evaluation was done according to the 2007 Cheson criteria, and the planned number of subjects was 20. Pathology review was done by the Korean Lymphoma Pathology Review Board after the enrollment was completed. The JAK2 amplification in tumor cells was independently analyzed by the Cancer Institute Hospital of Japanese Foundation for Cancer Research without clinical information. Details of the study were registered at ClinicalTrials.gov (NCT01965119). Results We enrolled 20 patients (median age: 43, range: 19 - 76) including 14 cHL and 6 PMBCL between 2013 and 2015. The median number of prior therapies was four (range: 2 - 10). Eight patients underwent autologous stem cell transplantation, and ten patients received involved field radiotherapy during their treatment period. Four cHL (29%, 4/14) and four PMBCL (67%, 4/6) were primarily refractory to induction treatment. At the time of enrollment, 18 patients were refractory to salvage treatment, and two patients relapsed five years after their last treatment. The first response evaluation after the 2nd cycle showed 40% (7/20) of overall disease control rate (1 CR, 6 PR, and 1 SD). However, one cHL case with PR was excluded from the analysis after the final diagnosis was changed to chronic active EBV infection by the pathology review board. The comparison between cHL and PMBCL showed more efficacy of ruxolitinib in cHL because all patients with PMBCL rapidly progressed after 1st or 2nd cycle whereas disease was controlled in seven patients with cHL (1 CR, 5 PR and 1 SD) out of 13 patients with cHL (54%, 7/13). Although the median number of treatment cycle of all patients was two (range: 1-10), six responders received on the average five cycles of treatment (range: 4-10), and the median duration of response was 5 months (range: 4 - 12+ months). The toxicity was manageable without any grade 3 or 4 hematologic and non-hematologic toxicity. Most non-hematologic toxicities including liver enzyme elevation and abdominal pain were grade 1 or 2. The analysis of JAK2 amplification by FISH was done in 9 patients whose tissue sample was available. Among three patients with high amplification (more than 10 signals), only one SD was observed whereas two patients achieved PR out of six cases with low or absent amplification. Conclusions The efficacy of ruxolitinib in heavily pretreated patients with relapsed or refractory cHL suggested JAK2 inhibition might be more effective for the treatment of cHL than PMBCL. A phase I/II study with larger study population should be warranted based on our results of pilot study. Disclosures Kim: Celltrion, Inc.: Consultancy, Honoraria.

Description: Key Points USP22 deficiency in Ras-driven myeloproliferative neoplasm blocks myeloid differentiation promoting acute myeloid leukemia. USP22 is a PU.1 deubiquitylase that positively regulates PU.1 stability and the expression of myeloid-differentiation genes.

Description: Axicabtagene ciloleucel (Axi-cel) is an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy approved for the treatment of relapsed or refractory diffuse large B-cell lymphoma (r/r DLBCL). Long-term analysis of the ZUMA-1 phase 1-2 clinical trial showed that ~40% of Axi-cel patients remained progression-free at 2 years (Locke et al., Lancet Oncology 2019). Those patients who achieved a complete response (CR) at 6 months generally remained progression-free long-term. The biological basis for achieving a durable CR in patients receiving Axi-cel remains poorly understood. Here, we sought to identify CAR T-cell intrinsic features associated with CR at 6 months in DLBCL patients receiving commercial Axi-cel at our institution. Using mass cytometry, we assessed expression of 33 surface or intracellular proteins relevant to T-cell function on blood collected before CAR T cell infusion, on day 7 (peak expansion), and on day 21 (late expansion) post-infusion. To identify cell features that distinguish patients with durable CR (n = 11) from those who developed progressive disease (PD, n = 14) by 6 months following Axi-cel infusion, we performed differential abundance analysis of multiparametric protein expression on CAR T cells. This unsupervised analysis identified populations on day 7 associated with persistent CR or PD at 6 months. Using 10-fold cross-validation, we next fitted a least absolute shrinkage and selection operator (lasso) model that identified two clusters of CD4+ CAR T cells on day 7 as potentially predictive of clinical outcome. The first cluster identified by our model was associated with CR at 6 months and had high expression of CD45RO, CD57, PD1, and T-bet transcription factor. Analysis of protein co-expression in this cluster enabled us to define a simple gating scheme based on high expression of CD57 and T-bet, which captured a population of CD4+ CAR T cells on day 7 with greater expansion in patients experiencing a durable CR (mean±s.e.m. CR: 26.13%±2.59%, PD: 10.99%±2.53%, P = 0.0014). In contrast, the second cluster was associated with PD at 6 months and had high expression of CD25, TIGIT, and Helios transcription factor with no CD57. A CD57-negative Helios-positive gate captured a population of CD4+ CAR T cells was enriched on day 7 in patients who experienced progression (CR: 9.75%±2.70%, PD: 20.93%±3.70%, P = 0.016). Co-expression of CD4, CD25, and Helios on these CAR T cells highlights their similarity to regulatory T cells, which could provide a basis for their detrimental effects. In this exploratory analysis of 25 patients treated with Axi-cel, we identified two populations of CD4+ CAR T cells on day 7 that were highly associated with clinical outcome at 6 months. Ongoing analyses are underway to fully characterize this dataset, to explore the biological activity of the populations identified, and to assess the presence of other populations that may be associated with CAR-T expansion or neurotoxicity. This work demonstrates how multidimensional correlative studies can enhance our understanding of CAR T-cell biology and uncover populations associated with clinical outcome in CAR T cell therapies. This work was supported by the Parker Institute for Cancer Immunotherapy. Figure Disclosures Muffly: Pfizer: Consultancy; Adaptive: Research Funding; KITE: Consultancy. Miklos:Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Membership on an entity's Board of Directors or advisory committees; Kite-Gilead: Membership on an entity's Board of Directors or advisory committees, Research Funding; AlloGene: Membership on an entity's Board of Directors or advisory committees; Precision Bioscience: Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotech: Membership on an entity's Board of Directors or advisory committees; Becton Dickinson: Research Funding; Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Membership on an entity's Board of Directors or advisory committees. Mackall:Vor: Other: Scientific Advisory Board; Roche: Other: Scientific Advisory Board; Adaptimmune LLC: Other: Scientific Advisory Board; Glaxo-Smith-Kline: Other: Scientific Advisory Board; Allogene: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Apricity Health: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Unum Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Obsidian: Research Funding; Lyell: Consultancy, Equity Ownership, Other: Founder, Research Funding; Nektar: Other: Scientific Advisory Board; PACT: Other: Scientific Advisory Board; Bryologyx: Other: Scientific Advisory Board.