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Despite worldwide promising clinical outcome of CD19 CAR-T therapy, relapse after this therapy is associated with poor prognosis and has become an urgent problem to be solved. We conducted a CD22 CAR T-cell therapy in 34 relapsed or refractory (r/r) B-ALL pediatric and adult patients who failed from previous CD19 CAR T-cell therapy. Complete remission (CR) or CR with incomplete count recovery (CRi) was achieved in 24 of 30 patients (80%) that could be evaluated on day 30 after infusion, which accounted for 70.5% of all 34 enrolled patients. Most patients only experienced mild cytokine-release syndrome and neurotoxicity. Seven CR patients received no further treatment, and 3 of them remained in remission at 6, 6.6, and 14 months after infusion. Eleven CR patients were promptly bridged to transplantation, and 8 of them remained in remission at 4.6 to 13.3 months after transplantation, resulted in 1-year leukemia-free survival rate of 71.6% (95% CI, 44.2–99.0). CD22 antigen loss or mutation was not observed to be associated with relapsed patients. Our study demonstrated that our CD22 CAR T-cells was highly effective in inducing remission in r/r B-ALL patients, and also provided a precious window for subsequent transplantation to achieve durable remission.

The prognosis of relapsed acute lymphoblastic leukemia (ALL) after allogeneic transplantation is dismal when treated with conventional approaches. While single-target CD19 or CD22 chimeric antigen receptor (CAR) T-cell therapy has achieved high complete remission (CR) rates in refractory/relapsed B-ALL, it could not maintain a durable remission in most patients. To prolong relapse-free survival, we sequentially combined CD19 and CD22 CAR-T cells to treat post-transplant relapsed B-ALL patients with both CD19/CD22 antigen expression on lymphoblasts. Patient-derived donor cells were collected to produce CAR-T cells that were transfected by lentiviral vectors encoding second generation CARs composed of CD3ζ and 4-1BB. The second T-cell infusion was scheduled at least 1 month, and usually within 6 months after the first CAR-T treatment. Twenty-seven adult and pediatric patients, including 11 (41%) with extramedullary diseases (EMD), received the first CD19 CAR-T and 23 (85%) achieved CR. Subsequently, 21 out of 27 patients received the second CD22 CAR-T and were followed-up for a median of 19.7 (range, 5.6-27.3) months; 14 cases remained in CR, seven relapsed and two of them died from disease progression; Kaplan-Meier survival analysis showed overall survival and event-free survival rates of 88.5% and 67.5%, respectively, at both 12 months and 18 months. CAR-T associated graft-versus-host disease (GVHD) occurred in 23% of patients, with 8% new-onset acute GVHD and 15% persistent or worsened pre-existing cGVHD before CAR-T. This combination strategy of sequential CD19 and CD22 CAR-T therapy significantly improved the long-term survival in B-ALL patients who relapsed after transplantation.

Refractory or relapsed B lymphoblastic leukemia (B-ALL) patients have a dismal outcome with current therapy. We treated 42 primary refractory/hematological relapsed (R/R) and 9 refractory minimal residual disease by flow cytometry (FCM-MRD+) B-ALL patients with optimized second generation CD19-directed CAR-T cells. The CAR-T-cell infusion dosages were initially ranged from 0.05 to 14 × 105/kg and were eventually settled at 1 × 105/kg for the most recent 20 cases. 36/40 (90%) evaluated R/R patients achieved complete remission (CR) or CR with incomplete count recovery (CRi), and 9/9 (100%) FCM-MRD+ patients achieved MRD-. All of the most recent 20 patients achieved CR/CRi. Most cases only experienced mild to moderate CRS. 8/51 cases had seizures that were relieved by early intervention. Twenty three of twenty seven CR/CRi patients bridged to allogeneic hematopoietic stem cell transplantation (allo-HCT) remained in MRD- with a median follow-up time of 206 (45-427) days, whereas 9 of 18 CR/CRi patients without allo-HCT relapsed. Our results indicate that a low CAR-T-cell dosage of 1 × 105/kg, is effective and safe for treating refractory or relapsed B-ALL, and subsequent allo-HCT could further reduce the relapse rate.

Despite worldwide promising clinical outcome of CD19 CAR-T therapy, relapse after this therapy is associated with poor prognosis and has become an urgent problem to be solved. We conducted a CD22 CAR T-cell therapy in 34 relapsed or refractory (r/r) B-ALL pediatric and adult patients who failed from previous CD19 CAR T-cell therapy. Complete remission (CR) or CR with incomplete count recovery (CRi) was achieved in 24 of 30 patients (80%) that could be evaluated on day 30 after infusion, which accounted for 70.5% of all 34 enrolled patients. Most patients only experienced mild cytokine-release syndrome and neurotoxicity. Seven CR patients received no further treatment, and 3 of them remained in remission at 6, 6.6, and 14 months after infusion. Eleven CR patients were promptly bridged to transplantation, and 8 of them remained in remission at 4.6 to 13.3 months after transplantation, resulted in 1-year leukemia-free survival rate of 71.6% (95% CI, 44.2-99.0). CD22 antigen loss or mutation was not observed to be associated with relapsed patients. Our study demonstrated that our CD22 CAR T-cells was highly effective in inducing remission in r/r B-ALL patients, and also provided a precious window for subsequent transplantation to achieve durable remission.

Cytokine release syndrome (CRS) is the most prominent and potentially life-threatening toxicity caused by chimeric antigen receptor (CAR) T cell therapy, therefore, effectively controlling severe CRS is critical to ensure patient safety. Tocilizumab, an interleukin-6 receptor antagonist, has been widely used to treat CRS, whereas it is not clear if corticosteroids could be as another optimal choice for managing CRS. We applied corticosteroids instead of tocilizumab as the first-line agent to control CRS in patients with relapsed/refractory B-cell acute lymphoblastic leukemia during CAR-T therapy. The impacts of steroids on treatment efficiency and kinetics of CAR-T cells were assessed by comparing two groups of patients who did (42 cases) or did not (26 cases) receive steroids. Patients followed up less than one month (went to other hospitals for transplantation or died within one month) were excluded. Treatment effects were evaluated on day 30 after T-cell infusion and then monthly in follow-up patients. Minimal residual disease (MRD) was detected by multiparameter flow cytometry (FCM) and quantitative PCR for fusion genes. The dynamic monitoring of CAR-T cells was performed through flow cytometric quantitation of FITC+CD3+ T cells. B-cell aplasia (BCA) was assayed by FCM. Dexamethasone or methylprednisolone or both (alternately) were administrated. Dexamethasone was used in most cases especially for patients with neurologic symptoms; methylprednisolone was preferred for patients with pulmonary or liver dysfunction, and patients accepting high dose steroids. Steroids started with low dose and could be increased if symptoms were not resolved, for severe CRS, steroids would be escalated up to dexamethasone 20mg/m2/d or more higher up to methylprednisolone 10mg/kg/d. Once CRS was improved, steroids were rapidly reduced and stopped. A total of 68 patients (28 adults and 40 children younger than 18 years) were included, 22 (32.4%) presented with extramedullary diseases (EMD), bone marrow blasts in patients without EMD varied between 5%-96.5%, 31 (45.6%) patients had an allogeneic transplantation, 54 (79.4%) cases received CD19-specific and 14 (20.6%) received CD22-specific CAR-T therapy. Forty-two (61.8%) cases, including all (10) of grade III CRS, 68.2% (30/44) of grade II CRS and 2 patients with no CRS but with GVHD (1 case) or neurotoxicity (1 case), were administered steroids, among them, 23/42 (54.8%) received high dose steroids (>10mg/m2/d dexamethasone or equivalent), the duration of steroid use was 1-16 days (78.6% We found that there was no difference either in complete remission (CR) rate (95.2% vs 92.3%, p=.344) or in MRD negative CR rate (80.0% vs 79.2%, p=.249) between steroid and non-steroid group, verified that corticosteroids even high dose steroids did not influence the treatment response. Furthermore, we investigated the dynamics of CAR-T cells. Firstly, the expansion of CAR-T cells in peripheral blood (PB) was evaluated, the average CAR-T cell counts in steroid group were significantly higher than those in non-steroid group on D11 (p=.0302), D15 (p=.0053), D20 (p=.0045) and D30 (p=.0028), except for D7 when CAR-T cells began to expand (p=.9815), this demonstrated that steroids did not suppress the proliferation of CAR-T cells in PB. Secondly, the percentages of patients with detectable CAR-T cells in bone marrow (BM) and cerebrospinal fluid (CSF) were compared between steroid and non-steroid group, there were no differences both in BM (85.2% vs 78.6%, p=.923) and in CSF (68.6% vs 57.9%, p=.433), which implied steroids did not influence the trafficking of T-cells to BM and CSF. Thirdly, we monitored B-cell aplasia (BCA) in part of patients followed-up more than 2 months without further treatments, the percentages of patients with BCA in steroid group had no significant differences compared to non-steroid group at 2-month (p=.086) and 3-month (p=.146). Later, although limited cases left, in the steroid group, 100% of patients (4-month, 7/7; 5-month, 7/7; 6-month, 5/5) still maintained BCA and CR, indicating that corticosteroids did not impact the duration of functional CAR-T cells. In conclusion, corticosteroids do not compromise the treatment efficacy and kinetics of CAR-T cells, could be as a feasible and effective approach to manage CAR-T associated CRS. Disclosures No relevant conflicts of interest to declare.

Introduction: Recently, chimeric antigen receptor (CAR) T-cell (CART) targeting CD19 has resulted in high complete remission (CR) rate in patients with refractory or relapsed B cell acute lymphoblastic leukemia (r/r B-ALL). Our previous clinical study has achieved more than 90% CR rate with CD19-CART in r/r B-ALL, but approximately 10% of patients have failed even CD19 high expression on their leukemia cells (Pan J, et al. Leukemia 2017; 31: 2587-2593). In current study, we investigate whether leukemia burden in peripheral blood (PB) has any influence in CART preparation ex vivo and its clinical efficacy. Patients and Methods: From June 2017 to June 2019, 143 r/r B-ALL patients received CD19-CART therapy. The median age was 8 years old (10 months to 65 years old). Thirty-six patients had leukemia cells in PB (positive group) and the median leukemia cells proportion after lymphocyte separation was 32.7% (3.23% to 83.2%); 107 patients had no leukemia cells detectable in PB (negative group). After apheresis, peripheral blood mononuclear cells (PBMCs) were isolated using lymphocyte separation liquid and leukemia cells proportion were determined by flow cytometry (FCM). PBMCs were activated with CD3 and CD28 antibodies for 24 hours, then transduced with the lentivirus encoding anti-CD19-CD3zeta-4-1BB CAR and cultured for 4-6 days in serum-free media containing IL2, IL7, IL15, IL21. Mycoplasma, endotoxin, bacteria and fungi were strictly controlled during the culture. Cell viability, expanding fold, percentage of CD3+ T cells, transduction efficiency and B cells were all examined before CART infusion. The patients received cyclophosphamide (250 mg/m2) and fludarabine (30 mg/m2) all for 3 days, and followed by CART infusion with a median number of 4.7 (0.6 to 43) X 105cells/kg. CD19-CART cells' expansion capacity in vivo was analyzed by FCM. Clinical efficacy was evaluated by bone marrow morphology and FCM on day 30. Results: Both normal B cells and leukemia B cells were all undetectable in infused CD19-CART. The median percentage of CD3+ T cells and transduction efficiency in positive and negative groups were 94.1% (80.3% to 98.0%) vs. 96.6% (80.1% to 99.5%) (P=0.056), and 35.6% (4.82% to 56.1%) vs. 52.4% (9.44% to 82%) (P Table Disclosures No relevant conflicts of interest to declare.

With traditional therapies, the prognosis of relapsed acute lymphoblastic leukemia (ALL) after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is extremely poor. Chimeric antigen receptor (CAR) T cell therapy targeting at CD19 has demonstrated a significant efficacy on refractory/relapsed (r/r) B-ALL, but single-target CART could not maintain a long-term remission. Recently, CD22-CART has also shown an exciting result in r/r B-ALL. Here we sequentially applied CD19- and CD22-specific CART cells to treat relapsed B-ALL post-HSCT and observed the therapeutic effect. From June 30,2017 through May 31,2018, twenty-four B-ALL patients (pts) relapsing after allo-HSCT with both antigens CD19 and CD22 expression on blasts were enrolled, the median age was 24 (2.3-55) years. Seventeen pts had hematologic relapse, 6 with both bone marrow and extramedullary (EM) involvements and 1 with EM disease (EMD) only. Fourteen pts had failed to previous therapies including chemotherapy, donor lymphocyte infusion, interferon and even murinized CD19-CART in other hospitals. Recipient-derived donor T cells were collected for producing CAR-T cells, which were transfected by a lentiviral vector encoding the CAR composed of CD3ζ and 4-1BB. Eighteen pts were initially infused with murinized CD19-CART, then humanized CD22-CART; while 6 pts (5 failed to prior murinized CD19-CART and 1 had bright CD22-expression) were initially infused with humanized CD22-CART, then humanized CD19-CART. The time interval between two infusions was 1.5-6 months based on patients' clinical conditions. The average dose of infused CAR T cells was 1.4×105/kg (0.4-9.2×105/kg) for CD19 and 1.9×105/kg (0.55-6.6×105/kg) for CD22. All patients received fludarabine with or without cyclophosphamide prior to each infusion, some pts accepted additional chemo drugs to reduce the disease burden. Treatment effects were evaluated on day 30 and then monthly after each CART, minimal residual disease (MRD) was detected by flow cytometry (FCM) and quantitative PCR for fusion genes, EMD was examined by PET-CT, CT or MRI. Sixteen patients finished sequential CD19- and CD22-CART therapies. Three cases could not undergo the second round of CART infusion (1 died, 1 gave up and 1 developed extensive chronic graft-versus-host disease (GVHD)). The rest of 5 pts are waiting for the second CART. After first T-cell infusion, 20/24 (83.3%) pts achieved complete remission (CR) or CR with incomplete count recovery (CRi), MRD-negative was 100% in CR or CRi pts, 3 (12.5%) cases with multiple EMD obtained partial remission (PR), and 1 (4.2%) died of severe cytokine release syndrome (CRS) and severe acute hepatic GVHD. Sixteen patients (15 CR and 1 PR) underwent the second CART therapy. Before second infusion, 3/15 pts in CR became MRD+ and others remained MRD-. On day 30 post-infusion, 1 of 3 MRD+ pts turned to MRD-, 1 maintained MRD+ ( BCR/ABL+) and 1 had no response then hematologic relapse later. The PR patient still had not obtained CR and then disease progressed. As of 31 May 2018, at a median follow-up of 6.5 (4-10) months, among 16 pts who received sequential CD-19 and CD-22 CART therapies, 1 had disease progression, 2 presented with hematological relapse and 2 with BCR/ABL+ only, the overall survival (OS) rate was 100% (16/16), disease-free survival (DFS) was 81.3% (13/16) and MRD-free survival was 68.8% (11/16). CRS occurred in 91.7% (22/24) pts in the first round of T-cell infusion, most of them were mild-moderate (grade I-II), merely 2 pts experienced severe CRS (grade III-IV). The second CART only caused grade I or no CRS since the leukemia burden was very low. GVHD induced by CART therapy was a major adverse event in these post-HSCT patients. After the first CART, 7/24 (29.2%) pts experienced GVHD, of them, 4 presented with mild skin GVHD, 2 with severe hepatic GVHD (1 recovered and 1 died), and 1 developed extensive chronic GVHD. No severe GVHD occurred in the second infusion. Our preliminary clinical study showed that for B-ALL patients who relapsed after allo-HSCT, single CD19- or CD22- CART infusion resulted in a high CR rate of 83.3%, sequentially combined CD19- and CD22-CART therapies significantly improved treatment outcome with the rate of OS, DFS and MRD-free survival being 100%, 81.3% and 68.8%, respectively, at a median follow-up of 6.5 months. The effect of CART on multiple EMD was not good and CART induced GVHD needs to be cautious. Disclosures No relevant conflicts of interest to declare.

Abstract Introduction: Patients (pts) with relapsed refractory B-ALL are mostly incurable by chemotherapy. The disease free survival (DFS) is low even treatment with allogeneic hematopoietic stem cell transplantation (allo-HSCT). We explored treatment of 47 cases of relapsed refractory B-ALL with low dose CD19 CAR-T cells and assessed the clinical safety and efficacy. Patients and Methods: (6-8)X107 pts' peripheral blood mononuclear cells(PBMCs) were activated with CD3 and CD28 antibodies for 24h, then transduced with the lentivirus encoding anti-CD19-CD3zeta-4-1BB CAR (Image1/Picture1) and cultured for 5-6 days in serum-free media containing IL2,IL7,IL15,IL21. All pts except one who had persistent cytopenia received cyclophosphamide 250 mg/m2/d X 3d, and fludarabine 30 mg/m2/d X 3d, then CAR-T cell infusion. Between Jul.31 2015 and Jul. 15 2016, a total of 47 cases were treated with CAR-T cells (Chart1). 37/47 cases had frank hematologically relapsed refractory B-ALL, who could not achieve complete remission (CR) after more than 1 cycles of chemotherapy. The median prior chemotherapy duration was 18 months. The median pre-treatment bone marrow blast percentage was 67% (6.5-98.5%). The most recently treated 15 cases had their PB blasts Chart 1: Characteristics of Patients Pre-CAR-T Image 1/Picture 1: Results: The pts received a median of 10 (0.5-140) X104cells/kg CAR-T cells, and the most recently treated 15 cases all received 10 X 104cells/kg. The median observation period was 201 days (20-368 days). On day 16-20 after CAR-T infusion, 31/35 (88.6%) relapsed hematological refractory cases achieved CR or incomplete CR(Cri), and 29/35 cases (82.9%) achieved negative MRD by FCM(CMR: complete molecular remission). No extramedullary leukemia was detected in any cases with residual disease. The most recently treated 15 consecutive cases all achieved CR with only mild ( Conclusion: Our anti-CD19 CAR-T cell therapy can result in a high CR/CRi /CMR rate in pts with refractory B-ALL and could overcome pre-existing risk factors for poor outcomes, including complex chromosome abnormalities, poor gene mutations, inherited predisposing gene mutation, extramedullary leukemia etc. Pts could be safely bridged into allo-HSCT for potential cure. The dose of infused CAR-T cells in our patients was far lower than previously reported in the literature and the culture period was only 6-7 days, which could dramatically reduce the cost of the CAR-T therapy. 10X104cells/kg of CAR-T cells was a safe and effective number for treating B-ALL. The major complication was CRS and the severity of CRS was directly correlated with the number of malignant B cells in the PB. The efficacy of CAR-T therapy was correlated to the infused number of CAR-T cells. The most recently treated 15 consecutive cases all achieved CR without severe CRS suggesting that the optimal number of CAR-T cells and patient selection are important for the efficacy and safety. Table. Table. Figure. Figure. Disclosures No relevant conflicts of interest to declare.

e19015Background: Some lymphoma patients may harbor mutations in PRF1, UNC13D, STX11, STXBP2 or SH2D1A, which cause function defects of cytotoxic lymphocytes. Data of the association between geneti...

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.

Abstract 1661 Minimal residual disease (MRD) is the most important factor to predict the prognosis after chemotherapy in the patients with acute leukemia(AL). Among the techniques for MRD detection, flowcytometry (FCM) is both sensitive and feasible tool, which can be used in almost all patients no matter with or without cytogenetic or molecular marker. It has been widely recognized that the MRD by FCM can predict the prognosis of the patients with ALL after several cycles of chemotherapy, but there are few reports on the value of MRD by FCM in AML. To investigate the clinical prognostic value of monitoring MRD regularly with FCM, the correlation of MRD and leukemia-free survival (LFS) in patients with AL without initial high risk factors was studied. From April 2005 to July 2009, 119 newly diagnosed patients (AML 85, ALL 34) and 39 treated cases (AML 19, ALL 20) were included. Those treated patients had attained the first complete remission (CR1) for more than 1 year after chemotherapy in other hospital and then were transferred to our hospital and detected MRD by FCM regularly since then. MRD in bone marrow (BM) was detected every 1 to 3 months in the first year after CR1, and every 2 to 6 months thereafter until hematological or extramedullary relapse or by July 2010. The special antibody combinations were employed for each patient according to aberrant expression of leukemia cells detected by the primary immune phenotype, or antigen drift in the follow-up. MRD was analyzed by the same doctor and assessed according to some abnormal characteristics, including antigen over-expression, antigen low-expression or even loss, co-expression of different lineages or different stage antigens on a single cell, and abnormal patterns in light scatters. MRD+ was defined as the aberrant cells more than 0.01% in BM at the twelfth month for AML and the fifth month for ALL. Among 85 newly diagnosed AML, 33 cases were MRD+ with the median ages 29(4-73)years old, 52 cases were MRD- with the median ages 35(9-68)years old. A total of 2/33 patients in MRD+ group and 40/52 patients in MRD- group remained in LFS. The probability of LFS at 12 months and 24 months was 56% and 16% in MRD+ group, 100% and 84% in MRD- group, respectively (see Figure 1), the difference between MRD+ and MRD- groups was significant statistically (p Disclosures: No relevant conflicts of interest to declare.