ABSTRACT:Integration of torque teno mini virus (TTMV) generating the TTMV::RARA (retinoic acid receptor α) fusion represents a newly recognized subtype of acute promyelocytic leukemia (APL) that merits detailed investigation. We present, to our knowledge, the first comprehensive characterization of its epidemiologic profile, clinical presentation, virologic characteristics, and underlying molecular mechanisms. Our findings indicate that TTMV::RARA is more prevalent in pediatric patients and represents the second most common retinoic acid receptor fusion after PML::RARA. Affected patients exhibit a high incidence of extramedullary involvement, particularly myeloid sarcoma. Cytogenetic abnormalities involving i(17)(q10) or 7q22 were identified in 52.0% of cases, largely in a mutually exclusive manner. Co-occurring mutations in epigenetic regulators were present in 76.9% of patients. Although most patients achieved initial remission, relapse was common and associated with rapid acquisition of all-trans retinoic acid (ATRA)-resistant mutation and secondary chemoresistance. Venetoclax-containing regimens demonstrated encouraging clinical efficacy. Phylogenetic analysis indicated that patient-derived TTMV strains clustered into a distinct clade. TTMV integration consistently occurred within RARA intron 2, involving a consensus fragment of 510 to 610 base pairs encompassing the viral promoter and open reading frame 2 (ORF2) N terminus, likely mediated by microhomology-driven recombination. Tandem RUNX1-binding motifs within the integrated viral promoter may underlie the myelotropism of these TTMV strains and facilitate transcriptional activation of TTMV::RARA. The chimeric protein retains at least the first 56 N-terminal residues of ORF2 and remains transcriptionally responsive to pharmacological concentrations of ATRA. These findings establish TTMV::RARA-APL as a distinct leukemia entity, laying the foundation for future studies on virus-mediated leukemogenesis and therapeutic strategies.

Atypical acute promyelocytic leukemia (aAPL) presents a complex landscape of retinoic acid receptor (RAR) fusion genes beyond the well-known PML::RARA fusion. Among these, 31 individually rare RARA and RARG fusion genes have been documented, often reported in the canonical X::RAR bipartite fusion form. Intriguingly, some artificially mimicked bipartite X::RAR fusions respond well to all-trans retinoic acid (ATRA) in vitro, contrasting with the ATRA resistance observed in patients. To unravel the underlying mechanisms, we conducted a comprehensive molecular investigation into the fusion transcripts in 27 RARA fusion gene-positive aAPL (RARA-aAPL) and 21 RARG-aAPL cases. Our analysis revealed an unexpected novel form of X::RAR::X or X::RAR::Y-type tripartite fusions in certain RARA- and all RARG-aAPL cases, with shared features and notable differences between these two disease subgroups. In RARA-aAPL cases, the occurrence of RARA 3' splices was associated with their 5' fusion partner genes, mapping across the coding region of helix 11_12 (H11_12) within the ligand-binding domain (LBD), resulting in LBD-H12 or H11_12 truncation. In RARG-aAPL cases, RARG 3' splices were consistently localized to the terminus of exon 9, leading to LBD-H11_12 truncation. Significant differences were also observed between RARA and RARG 5' splice patterns. Our analysis also revealed extensive involvement of transposable elements in constructing RARA and RARG 3' fusions, suggesting transposition mechanisms for fusion gene ontogeny. Both protein structural analysis and experimental results highlighted the pivotal role of LBD-H11_12/H12 truncation in driving ATRA unresponsiveness and leukemogenesis in tripartite fusion-positive aAPL, through a protein allosteric dysfunction mechanism.

BACHGROUND: Secondary central nervous system lymphoma (SCNSL) occurred in about 5% of patients with diffuse large B-cell lymphoma (DLBCL). The prognosis of SCNSL is very poor. There is no consensus on the treatment of SCNSL and new therapeutic strategies are urgently needed. Zanubrutinib is a new second-generation BTK inhibitor and has showed good efficacy and safety in a variety of B-NHLs. This study attempts to evaluate the efficacy and safety of zanubrutinib combined with rituximab and high-dose methotrexate in the treatment of SCNSL in patients with DLBCL. METHODS: Patients with DLBCL and secondary CNS involvement are eligible if age≥ 18, ≤75, ECOG≤3, and adequate organ function. Patients must have relapsed after ≥1, ≤4 lines of prior systemic lymphoma directed therapy. Zanubrutinib in combination with rituximab and methotrexate will be given as induction therapy for 6 14-day cycles. Zanubrutinib will be given as 160mg bid orally between days 1 and 14 of each cycle; rituximab will be given at 375mg/m2 intravenously on day 1 of each cycle; methotrexate at 3.5g/m2 for patients ≤65 or 1.5g/m2 for patients >65 (standard hydration/leucovorin support) will be given intravenously on day 2 of each cycle. For patients ≤65, autologous hematopoietic stem cell transplantation (ASCT) with conditioning regimen of thiotepa/carmustine will be given as consolidation treatment after induction therapy. For all patients, zanubrutinib will be given continuously after induction therapy or ASCT as maintenance therapy until disease progression, intolerable toxicity, or death. Primary outcome measure is 1-yaer PFS and secondary end points are response, overall survival, and toxicity. RESULTS: Nine patients have been enrolled. Median age was 62 (range 33-72); 5 were men. Median ECOG was 2 (1: 1, 2: 4, 3: 4). Eight had newly diagnosed SCNSL and one had recurrent SCNSL. The median time from first diagnosis to CNS relapse was 2.5 year (range 0.5-15 years). Five patients were isolated CNS disease and 4 patients had synchronous CNS and systemic disease. Seven patients had parenchymal disease, one had additional cerebrospinal fluid (CSF) involvement, and one had spinal disease. Two patients developed CNS progression after 1 and 4 cycles of induction therapy respectively and both had stable systemic disease. The other 7 patients all had received 6 cycles of induction therapy and zanubrutinib maintenance therapy. Two of them received ASCT and then zanubrutinib maintenance. At the end of induction therapy, of 5 patients with isolated CNS disease, 4 achieved CR and 1 achieved PR. Of 2 patients with synchronous CNS and systemic disease, both achieved CNS CR and 1 had systemic CR and 1 had systemic PR. The combined ORR was documented in 7 patients (78%), with CR in 5 (56%) and PR in 2 (22%) patients. After a median follow-up of 9 months, 4 patients progressed (1 of them died) and 5 were still in maintenance therapy. The median PFS was 9 months (range 0.5-15 months). The most common adverse events were leukopenia, thrombocytopenia, and hypokalemia. Grade 3/4 events were only observed in 2 patients (1 grade 4 thrombocytopenia and 1 grade 3 urinary infection). Only these two patients temporarily discontinued therapy due to adverse events. CONCLUSION: Zanubrutinib in combination with rituximab and methotrexate displayed clinical activity in SCNSL in both CNS and systemic disease. The safety and tolerability of this regimen in SCNSL patients were favorable. More clinical data will be updated from this ongoing study (NCT05398224).

BackgroundAnti-B-cell maturation antigen (BCMA) chimeric antigen receptor T-cell (CAR T) therapy showed remarkable efficacy in patients with relapsed or refractory multiple myeloma (RRMM). This phase 1 dose-escalation and expansion study developed C-CAR088, a novel second-generation humanized anti-BCMA CAR T-cell therapy, and assessed the safety and efficacy of three dosages of C-CAR088 in patients with RRMM.MethodsPatients received lymphodepletion with three doses of cyclophosphamide (300 mg/m2) and three doses of fludarabine (30 mg/m2) on days –5, –4, and –3, followed by an infusion of C-CAR088 on day 0. Doses of 1.0×106, 3.0×106, and 6.0×106 CAR T cells/kg (±20%) were tested in the dose-escalation cohorts and expansion cohorts. The primary endpoint was treatment safety, including the rate of treatment-emergent adverse events after cell infusion. Secondary endpoints were the overall response rate and progression-free survival. The exploratory endpoints were the quantification of C-CAR088 CAR T cells, selection of cytokines and chemokines in blood, and measurement of tumor BCMA expression.ResultsAs of July 2, 2021, 31 patients had been infused with C-CAR088. Any grade cytokine release syndrome (CRS) occurred in 29 patients (93.5%), and grade 3 CRS occurred in 3 patients (9.7%). One patient from the high-dose group (4.5–6.0×106 CAR T cells/kg) developed grade 1 neurotoxicity. No dose-limiting toxicities were observed in any dose group, and all adverse events were reversible after proper management. The overall response, stringent complete response, complete response (CR), and very good partial response rates were 96.4%, 46.4%, 10.7%, and 32.1%, respectively. The CR rate in the medium-dose (3.0×106 CAR T cells/kg) and high-dose (4.5–6.0×106 CAR T cells/kg) groups was 54.5% and 71.4%, respectively. In the CR group, 15 (93.7%) patients achieved minimal residual disease (MRD) negativity (test sensitivity >1/10−5). All seven patients with double-hit or triple-hit multiple myeloma achieved MRD-negative CR.ConclusionsThe present study demonstrated that C-CAR088 had a good safety profile and high antitumor activity in patients with RRMM, constituting a promising treatment option for RRMM.Trial registration numberNCT03815383,NCT03751293,NCT04295018, andNCT04322292.

Objective:To investigate the molecular genetic and clinical characteristics of MEF2D-BCL9 fusion gene-positive acute B-cell lymphoblastic leukemia (B-ALL), and to provide the reference for the diagnosis and treatment of the disease.Methods:The medical record and experimental examination data of a 18-year-old female MEF2D-BCL9 fusion gene-positive B-ALL patient were retrospectively analyzed. The clinical manifestations and biological characteristics of MEF2D-BCL9 fusion gene-positive B-ALL were summarized.Results:This 18-year-old female patient was treated in a local hospital in December 2018 and was diagnosed as B-ALL. She achieved complete remission after chemotherapy and recurred at 6 months after the initial onset, and then she was admitted to Hebei Yanda Ludaopei Hospital in the 9 months after the initial onset.MEF2D-BCL9 fusion gene was detected through RNA-sequencing (RNA-seq) and verified by using polymerase chain reaction and Sanger sequencing. Bone marrow cell morphology was similar to mature B cells with vacuoles but without characteristic chromosome karyotype abnormalities. The patient achieved remission after VLD regimen chemotherapy, chimeric antigen receptor T-cell (CAR-T) therapy and bridged to allogeneic hematopoietic stem cell transplantation (allo-HSCT). She has maintained complete remission for 2 years at the last follow-up in February 2022.Conclusions:MEF2D-BCL9 fusion gene-positive B-ALL is characterized with high risk, early relapse and poor prognosis. These patients may benefit from CAR-T and allo-HSCT. It further emphasizes the importance of taking MEF2D-BCL9 fusion gene into the detection or identification by using RNA-seq, particularly for those newly diagnosed B-ALL patients in children and adolescents with specific bone marrow morphology.

OBJECTIVE:To study the effect and safety of G-CSF combined with Plerixafor on the mobilization of peripheral blood hematopoietic stem cells from healthy related donors of allogeneic hematopoietic stem cell transplantation (allo-HSCT). METHODS:It was analyzed retrospectively that the data of peripheral blood hematopoietic stem cells from 33 (observation group) related donors mobilized by G-CSF plus Plerixafor in Hebei Yanda Lu Daopei Hospital from April 2019 to April 2021. Bone marrow and peripheral blood hematopoietic stem cells (PBSCs) of these donors were respectively collected on the fourth and fifth day of G-CSF-induced mobilization. Following the administration of Plerixafor on the night of the fifth day, PBSCs were collected on the sixth day once again. 46 donors using "G-CSF only" mobilization method in the same period were randomly selected as the control and respectively analyzed the differences of CD34+ cell counts on the fifth and the sixth day in two groups. And the donors' adverse reaction to Plerixafor in the form of questionnaire was also observed. Then it was compared that the patients who underwent allo-HSCT in "G-CSF+Plerixafor" group and "G-CSF only" group in terms of acute GVHD at grade I-IV or III-IV, CMV reactivation and EBV reactivation. RESULTS:CD34+ cells count (M±Q) among PBSCs collected on the fifth and the sixth day in the observation group were (1.71±1.02)×106/kg and (4.23±2.33)×106/kg, respectively. CD34+ cell counts on the sixth day was significantly higher than that of the fifth day (P<0.001); While the counterparts in the control group were (2.47±1.60)×106/kg and (1.87±1.37)×106/kg, respectively. By statistical analysis, CD34+ cell counts on the sixth day was significantly less than that of the fifth day (P<0.001). The adverse reaction to Plerixafor for the donors in the study were all grade 1 or 2 (mild or moderate) according to CTCAE 5.0 and disappeared in a short time. The patients who underwent allo-HSCT in the "G-CSF+Plerixafor" group and "G-CSF only" group were not statistically significant in terms of acute GVHD at grade I-IV or III-IV, CMV reactivation and EBV reactivation (P>0.1). CONCLUSION:The cell mobilization program of G-CSF combined with Plerixafor is safe and effective for being applied to allo-HSCT. The addition of Plerixafor can significantly increase the number of CD34 postive cells in the PBSC collection. Key words ; ;

Minimal residual disease (MRD) detection is an important prognostic parameter in patients with refractory or relapsed B-cell acute lymphoblastic leukemia (R/R B-ALL). CD79a has been reported to exhibit a high degree of linage-specificity for B-cell differentiation, with a specificity of 88% and a sensitivity of 100%. In this study, we investigated the efficiency and prognostic role of cytoplasmic CD79a (cCD79a) antibody-gated multicolor flow cytometry (MFC) in MRD detection in patients with B-ALL who received CD19-targeted chimeric antigen receptor (CAR) T-cell therapy bridging to allogeneic hematopoietic stem cell transplantation (allo-HSCT). The retrospective analysis was carried on to 59 patients who accepted allo-HSCT after CD19-CAR-T infusion from June 2016 to May 2017. The MFC MRD statuses before and after allo-HSCT were both strongly correlated with the transplantation prognosis, the MFC panel with cCD79a gating can effectively monitor MRD after CD19 CAR T-cell therapy and predict the prognosis after allo-HSCT. Trial registration: ClinicalTrials#: ChiCTR-IIh-16008711.gov: NCT03173417. Registered 30 May 2017 - retrospectively registered, https://www.clinicaltrials.gov/.

Abstract Background: C-CAR088, an anti-BCMA CAR T-cell therapy, is a novel 2nd generation 4-1BB chimeric antigen receptor T (CAR-T) cell therapy targeting BCMA. Previously presented results from an ongoing study of C-CAR088 in R/R MM (NCT03751293, NCT03815383, NCT04322292, NCT04295018) included a 95.7% overall response rate (ORR) for the dose of 1.0~6.0x10 6 CAR-T cells/kg with a favorable safety profile (Lu, 2020 ASH Oral Presentation #182). Here we present the updated results of the study, with more patients and longer follow up time. Methods: Dose escalation and expansion studies were conducted at four medical centers in China to evaluate the safety and efficacy of C-CAR088 in patients with R/R MM who were previously treated with at least 2 lines of therapy, including proteasome inhibitors (PIs) and IMiDs. C-CAR088 was administered to patients as a single infusion after lymphodepletion with fludarabine (30 mg/m 2) and cyclophosphamide (300 mg/m 2) daily for 3 days. The primary endpoint was the incidence of adverse events (AEs), including dose-limiting toxicities (DLTs), and the secondary endpoints included overall response rate(ORR), duration of response (DOR), and progression-free survival (PFS) by IMWG Uniform Response Criteria. Results: As of July 2nd, 2021, 31 patients had been infused with C-CAR088. The median vein-to-vein time was 18 days. The manufacturing success rate was 100%. 4, 13 and 14 patients were infused with 1.0, 3.0 and 4.5~6.0 x10 6 CAR+ T cells/kg respectively. The median follow-up time for all patients was 8.0 months (0.1-24.2). The median age of patients was 61 years (45-74). The median number of prior lines of therapy was 4 (2-13). There were 25 (80.6%) patients with at least one high risk cytogenetic abnormality and 17 (54.8%) patients with at least two high risk cytogenetic abnormalities. 7 patients (22.6%) received bridging therapy before C-CAR088 therapy. Cytokine release syndrome (CRS) developed in 29/31 (93.5%) patients, grade 1 in 18/31 (58.1%), grade 2 in 8/31 (25.8%) and grade 3 in 3/31 (9.7%) respectively. The median time to the first onset of CRS was 6 days (1-11) and the median duration of CRS was 5 days (2-14). 9/31 (29%) patients used tocilizumab and 6/31 (19.4%) patients used corticosteroids to manage CRS. Only one patient developed a grade 1 neurotoxicity. No DLTs were observed and all adverse events were reversible. One patient died of septic shock on day 2 after receiving C-CAR088. Clinical efficacy was assessed in 28 patients with ≥ 1 month of follow up. Among the 28 patients, 3, 11 and 14 patients were infused with the dose of 1.0 x 10 6 CAR+ T cells/kg 3 x10 6 CAR+ T cells/kg, and 4.5~6x10 6 CAR+ T cells/kg respectively. The ORR was 27/28 (96.4%): 4 (14.3%) achieved CR, 12 (42.9%) achieved sCR and 9 (32.1%) achieved very good partial response (VGPR). At the dose level of 1.0 x10 6 CAR+ T cells/kg, 3(100%) patients achieved VGPR. The median DOR was 3.7 months (1.8-5.8), and the median PFS was 4.6 months (2.7-6.2). The CR rate was 54.5% (6/11) and 71.4% (10/14) in the 3.0 and 4.5~6.0 x10 6 CAR+ T cells/kg cohorts respectively. The median time to CR was 2.0 (0.5-9.5) months. Minimal residual disease (MRD) was testedbyEuroFlow-based flow cytometric analysis in 16 patients who had CR, 15/16 (93.7%) patients were MRD negative with the sensitivity of 10 -5. With a median follow-up of 9.5 months (1.9-24.2) in ≥ 3.0x10 6 CAR+ T cells/kg cohorts, the median DOR and PFS had not been reached. The Kaplan-Meier estimation of PFS at 6 and 12 months was 81.1% (95% CI:65.9% ~99.8%) and 69.5 % (95% CI:51.6 % ~93.6%) respectively. 8 patients in the ≥ 3.0x10 6 CAR+ T cells/kg cohorts discontinued the study. 7 discontinued due to disease progression (PD), and 1 discontinued for other anticancer therapy. 4 progressed within 6 months, 2 progressed within 6-12 months, and 1 progressed within 12-24 months. C-CAR088 proliferated and expanded well in patients' blood. The median C max was 734,868 copies/μg gDNA. The median AUC 0~28day was 7,468,779 day·copies/μg gDNA. The median T max was 14 days. The median T last was 84 days. 71% (95% CI: 42%~92%) of patients with C max equal to or greater than the median C max achieved CR/sCR. Conclusion: C-CAR088 has a manageable safety profile, which includes low neurotoxicity rates (with no gr ≥3 events). Deep and durable responses were observed in ≥ 3.0x10 6 CAR-T cells/kg cohorts. Doses of 3.0 and 6.0×10 6 CAR T cells/kg were selected for further study. Figure 1 Figure 1. Disclosures Zhu: CBMG: Current Employment. Huang: CBMG: Current Employment. Li: CBMG: Current Employment. Lan: CBMG: Current Employment. Chen: CBMG: Current Employment. Humphries: CBMG Ltd: Current Employment. Yao: CBMG: Current Employment, Current holder of stock options in a privately-held company.

Objective:To analyze the incidence and mutation characteristics of FLT3 gene mutation and clinical efficacy of tyrosine kinase inhibitor (TKI) in patients with mixed phenotype acute leukemia (MPAL).Methods:A total of 48 patients with MPAL who were admitted to Hebei Yanda Lu Daopei Hospital from June 2015 to February 2018 were retrospectively analyzed. The common mutated 58 genes in hematologic malignancies were detected by using amplicon-targeted next generation sequencing, of which internal tandem duplication (ITD) and point mutation occurred in the hotspot region of exon 14, 15 and 20 in FLT3 gene. Multiplex polymerase chain reaction (PCR) analysis was used to detect 35 gene fusions in hematological neoplams.Results:There were 7 cases of FLT3 mutation in 48 MPAL patients, which were all ITD mutations. The median length of the inserts of FLT3-ITD was 48 bp, and one MPAL patient carried 2 multiple length inserts simultaneously, and the median variant allele frequency (VAF) was 40.5% (7.9%-84.7%). There were no statistically significant differences in clinical and genetic characteristics between FLT3 mutation-positive and FLT3 mutation-negative MPAL patients (both P > 0.05). Among 7 FLT3 mutation-positive MPAL patients, 4 cases were often accompanied with RUNX1 mutation. A total of 4 MPAL patients with FLT3-ITD-positive received sorafenib or sunitinib combined chemotherapy, and 3 of them achieved complete remission. Conclusions:ITD mutation is the main part in the FLT3 mutation of MPAL patients. FLT3-ITD-positive MPAL patients are often accompanied with RUNX1 mutation, which may benefit from targeted therapy with FLT3 kinase inhibitor.

Background: C-CAR088, an anti-BCMA CAR T-cell therapy is a novel 2nd generation 4-1BB chimeric antigen receptor T (CAR-T) cell therapy targeting BCMA which is specifically and highly expressed on multiple myeloma (MM) cells. C-CAR088 is manufactured in a serum-free, automated and digital, closed system. Initial, early clinical trial results in patients with R/R MM supported preclinical findings and showed promising efficacy and manageable safety profile (Yao, Blood (2019) 134 (Supplement_1): 50.) Methods: The dose escalation and expansion studies have been conducted at four medical centers in China to evaluate the safety and efficacy of C-CAR088 in patients with R/R MM who were previously treated with at least 2 lines of therapy including proteasome inhibitors (PIs) and IMiDs. C-CAR088 is administered to patients as a single intravenous dose after a standard 3-day cyclophosphamide/fludarabine conditioning regimen. Results: As of July 15, 2020, 24 patients were infused and 21 patients had evaluable data for safety and clinical response at dose levels of 1.0 x 106 CAR-T cells/kg (n=3), 3 x106 CAR-T cells/kg (n=11) and 4.5~6x106 CAR-T cells/kg (n=7). The median vein to vein time was 16 days. The manufacturing success rate was 100%. The median age of patients dosed was 60 years (range: 45-74 years).The median number of prior lines of therapy was 4 (range: 2-12 prior therapies). There were 17 (81%) patients with at least one and 12 (57.1%) patients with at least two high risk cytogenetic tumor changes. Five patients (23.8%) had bridging therapy. C-CAR088 treatment was well tolerated. 20 of 21 (95%) patients had Grade 1-2 CRS and one patient experienced Grade 3 CRS. Median time to CRS was 6.5 days (range: 1-11 days) and median duration of CRS was 5 days (range: 2-10 days). Four patients (19%) received tocilizumab for CRS treatment. Only one patient experienced a Grade 1 neurotoxicity event. No dose-limiting toxicities were observed and all adverse events were reversible. The best overall response (BOR) included 6 complete responses (CRs), 10 very good partial responses (VGPRs) and 4 partial responses (PRs). Median follow-up was 182 days (range: 30-375 days). The median duration of response has not been reached. In the 3 x106 CAR-T cells/kg dose group, 5/11(45%) patients achieved a CR. The C-CAR088 PK profile in peripheral blood showed a trend of a dose dependent profile. AUC0~28day and Cmax increased and Tmax decreased with dose (P<0.05). Conclusion: The clinical trial results in patients with R/R MM treated with C-CAR088 show a favorable safety profile and promising signs of efficacy. We will continue to evaluate these patients to understand the long-term effect of C-CAR088 in multiple myeloma patients. Clinical trial information: NCT04322292、NCT03815383、NCT03751293、NCT04295018 Research Sponsor: Cellular Biomedicine Group, Inc. Disclosures Zhu: Cellular Biomedicine Group Inc: Current Employment, Current equity holder in publicly-traded company. Zheng:Cellular Biomedicine Group Inc: Current Employment, Current equity holder in publicly-traded company. Yan:Cellular Biomedicine Group Inc: Current Employment, Current equity holder in publicly-traded company. Lv:Cellular Biomedicine Group Inc: Current Employment, Current equity holder in publicly-traded company. Lan:Cellular Biomedicine Group Inc: Current Employment, Current equity holder in publicly-traded company. Yang:Cellular Biomedicine Group Inc: Current Employment, Current equity holder in publicly-traded company. Huo:Cellular Biomedicine Group Inc: Current Employment, Current equity holder in publicly-traded company. Han:Cellular Biomedicine Group Inc: Current Employment, Current equity holder in publicly-traded company. Zhao:Cellular Biomedicine Group Inc: Current Employment, Current equity holder in publicly-traded company. Qin:Cellular Biomedicine Group Inc: Current Employment, Current equity holder in publicly-traded company. Wu:Cellular Biomedicine Group Inc: Current Employment, Current equity holder in publicly-traded company. Yao:Cellular Biomedicine Group Inc: Current Employment, Current equity holder in publicly-traded company. Zhu:Cellular Biomedicine Group Inc: Current Employment, Current equity holder in publicly-traded company. Ren:Cellular Biomedicine Group Inc: Current Employment, Current equity holder in publicly-traded company. Zhang:Cellular Biomedicine Group Inc: Current Employment, Current equity holder in publicly-traded company. Huang:Cellular Biomedicine Group Inc: Current Employment, Current equity holder in publicly-traded company. Humphries:Cellular Biomedicine Group Inc: Current Employment, Current equity holder in publicly-traded company. Yao:Cellular Biomedicine Group Inc: Current Employment, Current equity holder in publicly-traded company.

Objective: To assess the efficacy of immunotherapy based on autologous dendritic cells-cytokine induced killer cells (DC-CIK) and allogeneic natural killer cells (NK) in treating low-risk and intermediate-risk acute myeloid leukemia (AML). The study was conducted over a period of 11 years (April 1, 2006 to April 1, 2017) across the Beijing Ludaopei hospital systems. Methods: DC-CIK cells were derived by culturing patients9 peripheral blood mononuclear cells (PBMCs) in vitro with cytokines for 8-12 days. To generate NK cells, allogenic PBMCs were cultured for 3 to 6 days. After patients completed intensified chemotherapy consisting of 4 cycles of high-dose Ara-C within 6 months, DC-CIK or NK cells were infused and given once every 3 months for 2-4 cycles along with chemotherapy of Fludarabine, Cyclophosphamide and Ara-C given few days prior to each cycle of immunotherapy. Results: From April 1, 2006 to April 1, 2017, a total 152 patients in the low and intermediate risk groups of AML (except APL) underwent combined immunotherapy and chemotherapy in our center. Overall survival (OS) rate was 82% and disease-free survival (DFS) rate was 67% for the cohort. Beginning in June 2012, the low and intermediate risk groups were further stratified into very-good risk, good risk, and intermediate risk groups according to the specific AML related gene mutations. The OS rate of the 2012-2017 group was significantly better than that of the 2006-2011 group (91.7% vs. 71.6% p= 0.003). For patients treated prior to 2012, there were no significant differences between the low and intermediate risk group in OS (72.2% vs 71.4%) and DFS (72.2% vs 61.2%). From 2012 to 2017, the OS rates were 94.4%, 86.3%, and 93.3%, and the DFS rates were 83.3%, 81.8%, and 62.2%, (p=0.15) for the very-good risk, good-risk & intermediate risk groups, respectively, again suggesting no statistical differences among the groups. Side effects were mild with some fever, chills and fatigue. Patients who were treated with 2-4 cycles of immunotherapy were further divided into the DC-CIK alone group and the DC-CIK alternating with NK group. Importantly, the OS and DFS rates of 67 patients in the DC-CIK alternating with NK group were significantly better than those of 53 patients in the DC-CIK alone group (OS 95.5% vs 71.4%, p=0.0003), (DFS 85% vs 63.5%, p =0.0099). . Twenty-nine of 48 relapsed patients from both groups underwent allo-hematopoietic stem cell transplantation (allo-HSCT). The long-term OS after HSCT was 65.5%, closed to the OS of AML patients who transplanted in their first complete remission (CR) in our center. OS of 12 patients transplanted after 2012 was significantly better than that of 17 patients transplanted before 2012 (88.2% vs. 33.3%, p = 0.015). This because that after 2012, all patients have been checked minimal residual disease (MRD) by flow cytometry. Once patient was found to have relapse with MRD,immediately pursued HSCT while some patients transplanted prior to 2012 had morphological relapse. Conclusion: When combined with chemotherapy, DC-CIK/NK cells-based immunotherapy significantly improves the long-term OS and DFS rates of good and intermediate risk groups of AML. Auto DC-CIK alternating with allo NK is superior to auto DC-CIK alone. Relapsed patients could be safely and effectively treated with HSCT. If relapse patients with only positive MRD, then the outcome of HSCT will be comparable to those who received the HSCT during their first CR. Figure 1. OS of 152 patients treated with DC-CIK/NK immunotherapy from 2006 to 2017. Figure 2, OS of 67 patients treated from 2006 to 2012 vs 85 patients from 2012 to 2017 (71.6% vs 91.7%, p= 0.003). Figure 3, OS & DFS of DC-CIK+NK vs DC-CIK treated groups from 2012 to 2017 (p= 0.0003, p= 0.0099) Disclosures No relevant conflicts of interest to declare.

Abstract Background Acute myeloid leukemia (AML) is a heterogeneous disease with respect to presentation and clinical outcome. In recent years, more and more somatic mutations and their clinical significance were identified in AML. Most AML patients carry multiple gene mutations and the repertoire of mutations changing during the disease process, which determine the patient's unique clinical manifestations. Herein we recommend using the novel word ”mutaome¡± for representing the repertoire of somatic gene mutations in a specific tumor tissue, and aimed to establish a panel of mutation profiling protocol and retrospective profiling using archived bone marrow smear for clinical use in AML. Methods and Cases Mutation profiling protocol for CEBPA, DNMT3A, FLT3-ITD/TKD, IDH1, IDH2, KIT, NPM1, PHF6 and TET2 by PCR and Sanger sequencing was established, with the detection sensitivity about 15% to 20%. Bone Marrow (BM) or peripheral blood (PB) was collected form patients with newly diagnosed or relapsed. Archived BM smear on glass slides at the time of newly diagnosed were used for retrospective mutation analysis. Results 1) Totally 61 archived smear, 106 fresh BM or PB and 2 paraffin-embedded pathological specimens from 157 patients were analyzed. Age ranged from 1 to 77 years old, with the median age of 27 years old. 2) 60.4% (102/169) samples carrying at least one mutation, 51.0% (53/102) of them carrying 2 or more mutations, 30.4% (31/102) carrying mutations within 2 or more different genes. The number of mutated sample for each gene is 33 for CEBPA¡¢9 DNMT3A¡¢35 FLT3¡¢6 IDH1¡¢7 IDH2¡¢7 KIT¡¢19 NPM1¡¢8 PHF6¡¢and 21 TET2. 3) Paired archived smear and relapsed samples were analyzed for 11 cases, 9 of them showing difference, detailed results shown in Table 1. 4) Totally 53 patients come to our hospital after certain treatment, with the tumor cells below 15% and without AML gene mutation results. For these patients, archived BM smear samples were used for retrospective mutation profiling and then to guide targeted therapy and stratified evaluation. Conclusions Panel testing for gene mutations is effective method for detection of AML molecular markers. For the patients came with partial remission and without gene mutation result, archived bone marrow smear sample can be used for retrospective mutation analysis and then help guiding targeted therapy and stratified evaluation. Disclosures: No relevant conflicts of interest to declare.