Recurrent Leukemia Research Articles

Genetics of donor cell leukemia in acute myelogenous leukemia and myelodysplastic syndrome.

Allogeneic hematopoietic stem cell transplantation (HSCT) is an important therapeutic modality for patients with acute myelogenous leukemia (AML) with poor risk features. Nonetheless, roughly 30% of such patients have leukemia recurrence and up to 2% of these are donor-derived leukemias, in which malignancy develops in the donor's transplanted cells, despite extremely low rates of leukemia in the donors themselves. Notably, over 20% of these malignancies carry chromosome 7 abnormalities nearly all of which are monosomies. Recent advances in whole exome and genome sequencing have allowed for detection of candidate genes that likely contribute to the development of AML in donor cells (donor leukemia, DCL). These genes include CEBPA, GATA2, JAK2, RUNX1, DDX41, EZH2, IDH1/2, DNMT3A, ASXL1, XPD, XRCC3, and CHEK1. The potential roles of variants in these genes are evaluated based on familial clustering of MDS/AML and corresponding animal studies demonstrating their leukemogenic nature. This review describes the spectrum of genetic aberrations detected in DCL cases in the literature with regard to the character of the individual cases, existing family cohorts that carry individual genes, and functional studies that support etiologic roles in AML development. DCL presents a unique opportunity to examine genetic variants in the donors and recipients with regards to progression to malignancy.

Fe3O4@Pt nanozymes combining with CXCR4 antagonists to synergistically treat acute myeloid leukemia

Abstract Recurrence and drug resistance of acute myeloid leukemia (AML) remained the leading causes of death in blood cancers. The interaction of chemokine receptor 4/chemokine ligand 12 (CXCR4/CXCL12) leads to the trace residue of AML cells in patients after traditional chemotherapy, which hidden security danger for subsequent recurrence. The toxic and side effects of conventional chemotherapy on normal tissues also severely limited the clinical therapeutic efficacy of AML. To overcome these problems, a multifunctional nanoplatform of Fe3O4@Pt composite nanozyme conjugating CXCR4 antagonist was designed, aiming to synergistically treat AML for the first time, in which, the CXCR4 antagonist was used to specifically target AML cells as well as to significantly interfere CXCR4/CXCL12 axis. In the mildly acidic lysosome microenvironment, highly toxic reactive oxygen species (ROS) was generated through the sequential catalytic reactions of Fe3O4@Pt to trigger AML cells apoptosis, leaving the normal cells unharmed. This nanoplatform exhibited a superior synergistic therapeutic efficacy against AML in vitro and in vivo, preventing AML cells from homing to bone marrow and migrating to spleen, lung and liver, which in turn prolonged the survival period of AML mice. Therefore, this designed nanoplatform holds potentials for clinic applications in AML treatment.

Research Status of New Targets MRD Detection in Acute Myeloid Leukemia Recurrence--Review

Although most acute myeloid leukemia (AML) patients can achieve complete remission (CR) induced by standardized chemotherapy, but the relapse rate after remission remains high. The key reason is its high heterogeneity in cytogenetics and molecular biology. There are evidences show that minimal residual disease (MRD) is closely associated with disease recurrence, so that, finding specific genetic and molecular biological changes as new targets for MRD detection has become a research hotspot in recent years. In this review the intrinsic relationship between relapse of AML and MRD detection of specific molecular events, the application of these new targets in MRD detection and their targeted therapies according to the latest guidelines, so as to achieve the optimal treatment in CR phase.

Comparison of prognostic significance between multiparameter flow cytometry and real-time quantitative polymerase chain reaction in the detection of minimal residual disease of Philadelphia chromosome-positive acute B lymphocytic leukemia before allogeneic hematopoietic stem cell transplantation

目的探讨多参数流式细胞术（MFC）与实时定量聚合酶链反应技术（RQ-PCR）两种方法检测费城染色体阳性（Ph+）急性B淋巴细胞白血病（B-ALL）患者异基因造血干细胞移植（allo-HSCT）前微小残留病（MRD）的预后意义。方法回顾性分析2014年7月至2018年2月在北京大学血液病研究所接受allo-HSCT的280例Ph+ B-ALL患者，同时用MFC和RQ-PCR法（检测BCR-ABL融合基因表达）检测移植前MRD。结果RQ-PCR与MFC检测MRD具有相关性（rs＝0.435，P<0.001）。MFC、RQ-PCR法检测移植前MRD的阳性率分别为25.7％（72/280）、60.7％（170/280）。移植前MFC-MRD阳性组患者移植后白血病3年累积复发率（CIR）明显高于MFC-MRD阴性组（23.6％对8.6％，P<0.001）。RQ-PCR检测BCR/ABL融合基因阳性组（RQ-PCR MRD阳性组）的3年CIR、非复发死亡（NRM）、无白血病生存（LFS）、总生存（OS）与BCR/ABL融合基因阴性组（RQ-PCR MRD阴性组）相比差异均无统计学意义（P>0.05）。移植前RQ-PCR MRD≥1％组比<1％组具有更高的3年CIR（23.1％对11.4％，P＝0.032）、更低的LFS率（53.8％对74.4％，P＝0.015）与OS率（57.7％对79.1％，P＝0.009）。多因素分析显示，移植前MFC-MRD阳性是影响移植后CIR的危险因素（HR＝2.488，95％CI1.216～5.088，P＝0.013），移植前RQ-PCR MRD≥1％是影响LFS（HR＝2.272，95％CI 1.225～4.215，P<0.001）、OS（HR＝2.472，95％ CI 1.289～4.739，P＝0.006）的危险因素。MFC检测MRD预测复发的敏感性、特异性、阳性预测值（PPV）、阴性预测值（NPV）分别为48.50％、77.56％、23.62％、87.16％。以RQ-PCR MRD≥1％预测复发的敏感性、特异性、PPV、NPV分别为23.00％、88.59％、17.15％、91.84％。移植前MFC-MRD阳性或RQ-PCR MRD≥1％二者任一成立为指标预测移植后复发的敏感性、特异性、PPV、NPV分别为54.29％、73.88％、45.70％、91.87％。结论MFC和RQ-PCR法检测移植前MRD水平均可预测Ph+ B-ALL患者移植预后。移植前MFC-MRD阳性是移植后复发的危险因素。联合使用两种方法（移植前MFC-MRD阳性状态或RQ-PCR MRD≥1％成立）可提高预测移植后复发的敏感性、阳性预测值与阴性预测值，有助于更好筛选出高危患者。

A case report of fundus hemorrhage in recurrent acute myeloid leukemia

A case report of fundus hemorrhage in recurrent acute myeloid leukemia

LOPH and D-MHC in the Treatment of FeLV Associated Acute Leukemia in a Cat

Background: Bone marrow primary malignancies are denominated leukemias, classified as myeloid or lymphoid, according to the cell lineage, and acute or chronic, according to the cell´s state of maturation. In cats, acute lymphoid leukemia is the most common form, especially in regions endemic for feline leukemia virus and / or feline immunodeficiency virus. A new treatment protocol for lymphomas, called LOPH, has been described for animals with FeLV persistent viremia. This study aimed to report a case of a cat presenting with FeLV associated acute leukemia and treated with the LOPH protocol, and, in the rescue phase, a modification of the D-MAC protocol, denominated D-MHC.Case: A 4-year-old mixed breed intact queen was attended due to lethargy and inappetence. The patient did not present any relevant abnormalities in the clinical exam and complementary exams were performed including complete blood count, biochemical profile, SNAP Feline Triple Test, chest radiographs and abdominal ultrasound. Imaging tests and biochemical values were unremarkable, but the patient presented a reagent result for FeLV and severe leukocytosis due to lymphocytosis. The morphological evaluation of the blood smear revealed the presence of blasts, in a concentration greater than 20% of the nucleated cells, which allowed the characterization of a leukemic state, probably lymphoid. First-line treatment was based on the LOPH protocol, including Lomustine, Vincristine, Prednisoloneand Doxorubicin, in four-week cycles. Nevertheless, during the third cycle, 66 days after the institution of this protocol, the patient presented a febrile condition along with marked leukocytosis due to lymphocytosis, confirming leukemia recurrence. A rescue attempt was performed with a modification of the D-MAC protocol, originally consisting of the combination of dexamethasone, melphalan, actinomycin-D and cytarabine, but with replacement of actinomycin-D by doxorubicin, and therefore nominated D-MHC. After the three cycles there was a return of the febrile condition associated with severe pancytopenia and euthanasia was elected due to poor clinical condition, resulting in a survival time of 124 days. The hematological toxicity of the induction protocol included anemia and neutropenia, with mainly grade I events, but with the occurrence of a grade IV event. The adverse effects of the rescue protocol were similar, but with a greater number of grade IV events.Discussion: FeLV is considered the most lethal retrovirus of the domestic cat, with a major impact on health and life expectancy. Persistent FeLV antigenemia increases the risk of hematopoietic neoplasms in 62.1 times due to a direct insertional mutagenesis. In endemic regions, approximately 70% of cats with acute leukemia have persistent FeLV antigenemia, as the patient in this report. The diagnosis was made through association of retroviral status and the identification of more than 20% of blasts, possibly lymphoblasts, in the blood stream, sparing the need for a myelogram. Considering the poor prognosis for acute lymphoid leukemias and the patient's retroviral status, treatment was initiated with the LOPH protocol, including lomustine, as a potent agent to induce remission, and doxorubicin, which can result in longer remission intervals. After 66 days free of the disease, the patient presented recurrence of the leukemic condition, starting the rescue protocol D-MHC. Remission was again obtained with duration similar to the first protocol, however, on the occasion of a new leukemia recurrence, euthanasia was elected. The treatment adopted for the patient in this report resulted in a longer survival time than reported in other studies. Despite the aggressiveness of the protocols, especially the D-MHC, it was possible to perform it using the monocyte and granulocyte stimulation factor to reverse neutropenia.

MicroRNA-1246 by Targeting AXIN2 and GSK-3β Overcomes Drug Resistance and Induces Apoptosis in Chemo-resistant Leukemia Cells.

Background and objective: Chemotherapy plays an important role in the treatment of leukemia. Multidrug resistance (MDR) induced by chemotherapy always leads to treatment failure and disease recurrence. MicroRNAs (miRNAs) have been verified as crucial components in carcinogenesis, including chemo-resistance of tumor cells, which has not been fully understood. In this study, we aimed to identify the potential candidate miRNA, miR-1246, and reveal its regulatory role in chemo-resistance of leukemia cells.Methods: Candidate miRNAs were selected by microarray analysis, screened by bioinformatics tools and verified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Chemo-resistant phenotypes, including cell viability, apoptosis, adriamycin (ADM) efflux and in vivo oncogenicity of leukemia cells following transfected with miR-1246 mimics or inhibitor were checked with or without ADM treatment to make clear the relationship between miR-1246 and chemo-resistance. RT-qPCR, western blot and dual luciferase reporter assay were performed to measure the expression of related genes and address the potential regulatory mechanism of miR-1246 in chemo-resistance.Results: The expression of miR-1246 was significantly higher in chemo-resistant leukemia K562/ADM cells, HL-60/RS cells and recurrent primary leukemia cells. Loss of miR-1246 inhibited proliferation, induced apoptosis, altered cell cycle distribution, inhibited ADM efflux in chemo-resistant leukemia cells, while overexpression of miR-1246 showed the opposite role in chemo-sensitive leukemia cells. Both bioinformatics prediction and luciferase assay indicated that AXIN2 and glycogen synthase kinase 3 beta (GSK-3β) were the direct targets of miR-1246 in leukemia cells. Inhibition of miR-1246 could up-regulate AXIN2 and GSK-3β and inactivate Wnt/β-catenin pathway, accompanied with inhibiting the expression of β-catenin and further influencing the expression of P-glycoprotein (P-gp) in the chemo-resistant leukemia cells.Conclusions: Chemo-resistant ability of MDR leukemia cells is attenuated by loss of miR-1246 via negatively regulating AXIN2 and GSK-3β to inactivate Wnt/β-catenin pathway and suppress P-gp expression, these mean that targeting miR-1246-AXIN2/GSK-3β-Wnt/β-catenin axis may be beneficial to overcome the chemo-resistance in relapse and refractory leukemia patients.

Gene Modified CAR-T Cellular Therapy for Hematologic Malignancies

With advances in the understanding of characteristics of molecules, specific antigens on the surface of hematological malignant cells were identified and multiple therapies targeting these antigens as neoplasm treatments were developed. Among them, chimeric antigen receptor (CAR) T-cell therapy, which got United States Food and Drug Administration (FDA) approval for relapsed/refractory (r/r) diffuse large B-cell lymphoma (DLBCL) as well as for recurrent acute lymphoblastic leukemia (ALL) within the past five years, and for r/r mantle cell lymphoma (MCL) this year, represents one of the most rapidly evolving immunotherapies. Nevertheless, its applicability to other hematological malignancies, as well as its efficacy and persistence are fraught with clinical challenges. Currently, more than one thousand clinical trials in CAR T-cell therapy are ongoing and its development is changing rapidly. This review introduces the current status of CAR T-cell therapy in terms of the basic molecular aspects of CAR T-cell therapy, its application in hematological malignancies, adverse reactions during clinical use, remaining challenges, and future utilization.

Use of Venetoclax in Patients with Relapsed or Refractory Acute Myeloid Leukemia: The Pethema Registry Experience

Introduction The prognosis of patients with recurrent or refractory acute myeloid leukemia (AML-RR) is very poor, especially if they are not candidates for allogeneic transplantation (allo-SCT) after a second complete response (CR). Venetoclax, a potent and selective inhibitor of the antiapoptotic protein BCL-2, was approved by the FDA in combination with hypomethylating agents (HMAs) or low-dose cytarabine (LDAC) in patients with newly diagnosed AML of age ≥ 75 years, or who have comorbidities that preclude the use of intensive chemotherapy. However, the evidence in AML-RR patients is still scarce. For this reason, the objective of our study is to retrospectively analyze the efficacy of the off-label use of venetoclax in patients with AML-RR. Methods We conducted a retrospective, multicenter, observational study of a cohort of patients with AML-RR who were treated with venetoclax in the hospitals of the PETHEMA group. We evaluated efficacy, CR/CRi rate and overall survival (OS). We performed a descriptive analysis. Overall survival (OS) was calculated using the Kaplan-Meier method. Results A total of 41 patients were included, 25 men and 16 women, with a median age of 68 years (25 - 82 years) and an ECOG ≥ 2 at the beginning of the venetoclax treatment in 52% of the cases. Seventy-five percent of patients had AML with myelodysplasia-related changes. 25 patients (61%) were at high risk according to the European Leukemia Net 2017. Sixty-six percent of patients received ≥2 previous lines (range, 1-4), 29 patients (71%) received intensive first line chemotherapy, 10 (25%) received a previous transplant and 18 (44%) received previous treatment with HMA. Venetoclax median treatment duration was 40 days, and it was administered in 54% with azacitidine, 34% with decitabine and 12% with LDAC. In all, 11% of patients achieved CR/CRi. Only 10% of patients received subsequent salvage treatment. With a median follow-up time of 166 days (range, 21 - 311), 65% of the patients died. The median OS from diagnosis was 15 months (1 - 67 months) and the median from venetoclax initiation was 78 days (2 - 311 days). Those patients who achieved CR/CRi had higher OS (median not reached vs. 78 days, p= 0.048). Regarding toxicity, it was the expected in these patients. Twenty-eight percent of the patients required discontinuation of treatment due to toxicity. Sixty percent of the patients were admitted at some time during treatment with venetoclax, mainly because of infections (53%), 12% because of bleeding and other causes in 15%. Conclusions Our real-life series depicts a marginal probability of CR/CRi and poor OS after venetoclax-based salvage. Patients treated with this regimen had very poor-risk features, and were heavily pre-treated, which could explain in part the observed poor outcomes. Although follow-up is still short, the small proportion of responders did not reach the median overall survival. Further studies will help to identify those patients potentially benefiting from venetoclax-based salvage regimens. Disclosures Sanchez: Amgem: Other: travel grants; Janssen: Other: travel grants; Roche: Other: travel grants; Abbvie: Other: travel grants; Celgene: Other: travel grants. Tormo:Roche: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Servier: Honoraria; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; MSD: Honoraria; Daiichi Sankyo: Honoraria. OffLabel Disclosure: The objective of our study is to retrospectively analyze the efficacy of the off-label use of venetoclax in patients with recurrent or refractory acute myeloid leukemia

Analysis of Adct-602 Pre-Clinical Activity in B-Cell Lymphoma Models and Identification of Potential Biomarkers for Its Activity

Analysis of Adct-602 Pre-Clinical Activity in B-Cell Lymphoma Models and Identification of Potential Biomarkers for Its Activity

Allogeneic Hematopoietic Cell Transplantation Is Critical to Maintain Remissions after CD19-CAR T-Cell Therapy for Pediatric ALL: A Single Center Experience

CD19-CAR T-cell therapy has shown remarkable efficacy in pediatric patients with relapsed and/or refractory B-cell acute lymphoblastic leukemia (r/r ALL). Despite high short-term remission rates, many responses are not durable and the best management of patients who achieve a complete response (CR) post-CAR T-cell therapy remains controversial. In particular, it is unclear if these patients should be observed or proceed to consolidative allogeneic hematopoietic cell transplantation (HCT). To address this question, we reviewed the clinical course of all patients (n=22) who received either an investigational CAR T-cell product (Phase I study: SJCAR19 [NCT03573700]; n=12) or tisagenlecleucel (n=10) at our institution. The investigational CD19-CAR T cells were generated by a standard cGMP-compliant procedure using a lentiviral vector encoding a 2nd generation CD19-CAR with a FMC63-based CD19 binding domain, CD8a stalk and transmembrane domain, and 41BB.ζ signaling domain. Patients received therapy between 8/2018 and 3/2020. All products met manufacturing release specifications. Within the entire cohort, median age at time of infusion was 12.3 years old (range: 1.8-23.5) and median pre-infusion marrow burden using flow-cytometry minimal residual disease (MRD) testing was 6.8% (range: 0.003-100%; 1 patient detectable by next-generation sequencing [NGS] only). All patients received lymphodepleting chemotherapy (fludarabine, 25mg/m2 daily x3, and cyclophosphamide, 900mg/m2 daily x1), followed by a single infusion of CAR T-cells. Phase I product dosing included 1x106 CAR+ T-cells/kg (n=6) or 3x106 CAR+ T-cells/kg (n=6). Therapy was well tolerated, with a low incidence of cytokine release syndrome (any grade: n=10; Grade 3-4: n=4) and neurotoxicity (any grade: n=8; Grade 3-4: n=3). At 4-weeks post-infusion, 15/22 (68.2%) patients achieved a CR in the marrow, of which 13 were MRDneg (MRDneg defined as no detectable leukemia by flow-cytometry, RT-PCR and/or NGS, when available). Among the 2 MRDpos patients, 1 (detectable by NGS only) relapsed 50 days after CAR T-cell infusion and 1 died secondary to invasive fungal infection 35 days after infusion. Within the MRDneg cohort, 6/13 patients proceeded to allogeneic HCT while in MRDneg/CR (time to HCT, range: 1.8-2.9 months post-CAR T-cell infusion). All 6 HCT recipients remain in remission with a median length of follow-up post-HCT of 238.5 days (range 19-441). In contrast, only 1 (14.3%) patient out of 7 MRDneg/CR patients who did not receive allogeneic HCT, remains in remission with a follow up of greater 1 year post-CAR T-cell infusion (HCT vs. no HCT: p&amp;lt;0.01). The remaining 6 patients developed recurrent detectable leukemia within 2 to 9 months post-CAR T-cell infusion (1 patient detectable by NGS only). Notably, recurring leukemia remained CD19+ in 4 of 5 evaluable patients. All 4 patients with CD19+ relapse received a 2nd CAR T-cell infusion (one in combination with pembrolizumab) and 2 achieved MRDneg/CR. There were no significant differences in outcome between SJCAR19 study participants and patients who received tisagenlecleucel. With a median follow up of one year, the 12 month event free survival (EFS) of all 22 patients is 25% (median EFS: 3.5 months) and the 12 month overall survival (OS) 70% (median OS not yet reached). In conclusion, infusion of investigational and FDA-approved autologous CD19-CAR T cells induced high CR rates in pediatric patients with r/r ALL. However, our current experience shows that sustained remission without consolidative allogeneic HCT is not seen in most patients. Our single center experience highlights not only the need to explore maintenance therapies other than HCT for MRDneg/CR patients, but also the need to improve the in vivo persistence of currently available CD19-CAR T-cell products. Disclosures Sharma: Spotlight Therapeutics: Consultancy; Magenta Therapeutics: Other: Research Collaboration; CRISPR Therapeutics, Vertex Pharmaceuticals, Novartis: Other: Clinical Trial PI. Velasquez:St. Jude: Patents &amp; Royalties; Rally! Foundation: Membership on an entity's Board of Directors or advisory committees. Gottschalk:Patents and patent applications in the fields of T-cell &amp; Gene therapy for cancer: Patents &amp; Royalties; TESSA Therapeutics: Other: research collaboration; Inmatics and Tidal: Membership on an entity's Board of Directors or advisory committees; Merck and ViraCyte: Consultancy.

Optimal Interval for Detection of Molecular Relapse after Stop of Tyrosine Kinase Inhibitor (TKI) in Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia (Ph+ALL) Caliculated By Kinetics of BCR-ABL Transcripts

Background In Ph+ALL patients, prophylactic/maintenance administrations of TKI are used after the protocol treatment. Unlike Chronic myelogenous leukemia (CML), after TKI stopped, there is no consensus about how often to monitor to detect molecular relapse. In the current study, from clinical data of 31 patients we tried to determine the optimal frequency of MRD monitor for better prognosis using a mathematical model. Methods Doubling time (DT) and Growth rate (GR) were retrospectively calculated by the increase of BCR-ABL from 99 kinetic data from 31 patients with Ph+ALL at molecular relapse. Measurement of amount BCR-ABL was performed by RQ-PCR. Mimicking CML, we defined BCR-ABL/ABL ratio of 0.1% as "MR3 (Major molecular response)" and undetectable levels of BCR-ABL transcript as "MR5 (Complete molecular response)". In order to investigate the relationship between tumor burden (BCR/ABL) at the time of MRD detection and prognosis, it was divided into 3 groups, BCR/ABL&amp;lt;0.1% (MR3), 0.1%&amp;lt;BCR/ABL&amp;lt;1% (MR2) and BCR/ABL&amp;gt;1% (MR1). This study was approved by the institutional review committee. Results The doubling time was 1.3 days (GR 0.7%/day) to 95.4 days (70.8%/day) with a median of 12.3 days (5.8%). The rate of increase was compared by three groups of tumor burden. The DT among patients in MR1 was shorter than those in MR3 and MR2 (7.7 days vs 15.2 days in median, t-test p&amp;lt;0.01). There were no differences of DT in sex, age, treatments and BCR/ABL mutation. Most patient had multiple kinetic data and the smallest amount of BCR/ABL was used in each patient. Among 8 patients who showed MR3 (MR3pt) and 11 who showed MR2 (MR2pt), 14 are alive at this analysis, while among 10 patients who showed MR1 (MR1pt), only 1 patient is alive.(Log-rank test, p&amp;lt;0.1) (Figure) Median survival time is 882 days, 330 days and 16 days in MR3pt, Mr2pt and MR1pt, respectively. All patients died of progression of the disease. Finding molecular relapse before MR2 may related to better results. Optimal interval of MRD detection We set several hypotheses to determine the optimal frequency to detect early recurrence of leukemia. We estimated that patients with MR2 and MR5 have 1010 and 107 Ph+ cells in body, respectively. We defined "MR2" and "MR5" as "optimal intervention threshold" and "detection threshold". From our clinical data, we tentatively determined that doubling time of leukemia growth is distributed between 1 day (GR 100%/day) and 100days (0.7%). Also, we assumed that single cell has a relapse potency and the GR is constant during observation in each patient. The number of Ph+ cells in a MR5 patient who would potentially relapse might be distributed between 1 cell and 1 x 107 (MR5). "Success" was defined as if molecular relapse is detected between M5 and MR2 and "Failure" was defined as if detected more than MR2. According to the daily clinical practice, the optimal examination interval is tentatively every 7 days, every 14 days, every 28 days, every 56 days, every 84 days, every 6 months, and every year. First, in order to calculate the growth rate that would be successful at 7-day intervals, the rate of 168%/day for 1 cell to increase 107 (MR5) in 7 days was calculated. This rate is faster than the maximum rate of 100%/day in this study, so an interval of 7 days is good for the initial period. Similarly, at 14-day intervals, 68%/day is calculated, and in this case, it requires 32 days to increase 107 (MR5) from 1 cell. As a result, after 32.7 days, 14-day intervals are acceptable. Similarly, the calculated results for 28 days, 56 days, 84 days, 6 months, and 1 year are shown in the Table. MRD can be found before M3 (success) with 28-day intervals after 65 days, 56-day intervals after 131 days, 84-day intervals after 196 days, 6-month intervals after 419 days and 1-year intervals after 852 days. Conclusion Taken together, soon after stop of TKI, more frequent monitoring of MRD than in "treatment free remission" in CML is needed. The interval can be prolonged with the passage of time. In several patients, hematopoietic stem cell transplantation (HSCT) was possible due to early intervention by changing TKI and chemotherapy. After HSCT, TKI combined with rapid reduction of immune-suppressants and donor lymphocyte infusion successfully related with long term survival. (data will be shown in ASH) Thus, the tight monitoring according to the mathematical model is important. The current strategy may be applied to other leukemia in which MRD monitoring by PCR is established. Disclosures Miyamura: Bristol-Myers Squibb Co., Ltd.:Honoraria;Celgene Co., Ltd.:Honoraria;Daiichi-Sankyo Co., Ltd.:Honoraria;Otsuka Co., Ltd.:Honoraria;Pfizer Co., Ltd.:Honoraria;Novartis Co., Ltd.:Honoraria.Goto:Takeda Pharmaceutical Co., Ltd:Honoraria;Novartis Pharma Co., Ltd.:Honoraria.Morishita:Bristol-Myers Squibb Co., Ltd.:Honoraria.Ozawa:Novartis Co., Ltd.:Honoraria.

Risk-Adapted Intensive Chemotherapy for Primary ACUTE Myeloid Leukemia during the Last 25 YEARS: Increase in Complete Remission RATE, Hematopoietic Cell Transplantation Access and Decrease in Relapse Incidence Have LED to Improved Survival

Risk-Adapted Intensive Chemotherapy for Primary ACUTE Myeloid Leukemia during the Last 25 YEARS: Increase in Complete Remission RATE, Hematopoietic Cell Transplantation Access and Decrease in Relapse Incidence Have LED to Improved Survival

Detection of residual disease in chronic myeloid leukemia utilizing genomic next generation sequencing reveals persistence of differentiated Ph+ B cells but not bone marrow stem/progenitors

Persistence of leukemic stem cells (LSCs) results in the recurrence of chronic myeloid leukemia (CML) after the administration of tyrosine kinase inhibitors (TKIs). Thus, the detection of minimal residual disease (MRD) with LSC potential can improve prognosis. Here, we analyzed 115 CML patients and found that CD25 was preferentially expressed on the phenotypic stem and progenitor cells (SPCs), and TKI therapy decreased the number of CD25-positive cells in the SPC fraction. To detect MRD harboring BCR-ABL1 fusion DNA, we developed a highly-sensitive method using patient-specific primers and next-generation sequencing. By using this method, we identified that in patients who achieved molecular remission, almost all residual CD25-positive SPCs were BCR-ABL1-negative. Moreover, in some patients BCR-ABL1 was detectable in peripheral B cells but not in SPCs. We conclude that CD25 marks LSCs at diagnosis but does not mark MRD following TKI treatment and that analysis of peripheral B cells can allow sensitive detection of MRD.

Случай развития переднего увеита на фоне острого лимфобластного лейкоза

A clinical case of the development of a rare form of ocular manifestation of acute lymphoblastic leukemia, one of the most common oncological diseases in childhood, is described. A seven-year-old child initially had weakness, decreased appetite, and a single episode of subfebrile condition for two days without catarrhal symptoms. Further, an increase in the cervical lymph nodes was noticed. Then a hemorrhagic rash appeared all over the body. In the complete blood count (CBC) test: erythrocytes (RBC) – 3.8 * 1012 / l, hemoglobin (HGB) – 115 g/L, leukocytes (WBC) – 121*109/L, platelets (PLT) – 22*109/L. The child was admitted to the regional children’s clinical hospital. On examination, 90% of blast cells were detected in the myelogram. Against the background of a confirmed remission of the oncological disease, iridocyclitis of the left eye was diagnosed. It could not be stopped by standard antibacterial and antiinflammatory therapy. After a while, the right eye was also involved in the pathological process. Hematologists confirmed the recurrence of acute lymphoblastic leukemia after bone marrow puncture. The presented clinical case demonstrates the fact that the treatment of ocular manifestations of acute lymphoblastic leukemia can be effective only in the treatment of the underlying disease.

Case Report: IgG multiple myeloma and chronic myeloid leukemia in a single patient

A 58-year-old man presented with recurrence of chronic myeloid leukemia (CML) after complete molecular remission in the setting of non-compliance with imatinib. He was restarted on imatinib and was also noted to have IgG kappa monoclonal gammopathy of undetermined significance (MGUS). The patient re-achieved molecular remission after resumption of imatinib, but his MGUS progressed to smoldering myeloma and he was eventually diagnosed with multiple myeloma (MM) and initiated on treatment for MM with thalidomide, bortezomib and dexamethasone. He has responded well to treatment of the myeloma and continues concurrent maintenance imatinib treatment for CML and is being evaluated for bone marrow transplant. The association of two concurrent hematological malignancies, CML and MM, is very rare and has been infrequently reported in literature. The pathophysiology of this has not yet been fully understood. This case report reviews the various theories to explain this and discusses the potential challenges of simultaneous treatment of MM and CML.

Tisagenlecleucel in Children and Young Adults: Reverse Translational Research by Using Real-World Safety Data.

Tisagenlecleucel has revolutionized the pharmacological approach of relapsed or refractory B-cell acute lymphoblastic leukaemialeukaemia in paediatrics. The safety profile of tisagenlecleucel still needs to be better defined. The aim of this study was a post-marketing evaluation of the safety of tisagenlecleucel through the analysis of the Eudravigilance database with focus on the paediatric population. From 2017 to 2020, one third of Individual Case Safety Reports referring to tisagenlecleucel (117/364) have been collected in paediatrics, on average nine year-old boys. Overall, 92% of the638 adverse events were serious and caused or prolonged hospitalisation. A total of 55 adverse events presented a fatal outcome, mainly due to progression of malignant neoplasm (N = 10; 18.2%), recurrence of acute lymphocytic leukaemia (N = 6; 10.9%) or occurrence of acute lymphocytic leukaemia (N = 5; 9.1%). Cytokine release syndrome was commonly reported after tisagenlecleucel infusion (54/638), followed by pyrexia (45/638) and hypotension (27/638). Only 18/638 events referred to neurotoxicity, none of them resulted in death. More than one third of cases (41/117) were suggestive of therapeutic failure. This first post-marketing analysis confirms pre-approval evidence of the safety profile of tisagenlecleucel in paediatrics. Since only a few years of marketing is available, further followed-up studies need to be performed to investigate longer-term safety of tisagenlecleucel.

CAR T-cell bridging to allo-HSCT for relapsed/refractory B-cell acute lymphoblastic leukemia: the follow-up outcomes

目的研究嵌合抗原受体T细胞（CAR-T）细胞治疗桥接异基因造血干细胞移植（allo-HSCT）治疗复发/难治急性B淋巴细胞白血病（R/R B-ALL）的疗效及安全性。方法回顾性分析2017年1月至2019年5月于苏州大学附属第一医院行CAR-T细胞治疗桥接allo-HSCT的50例R/R B-ALL患者的临床资料，分析CAR-T细胞治疗前、后及allo-HSCT前不同骨髓微小残留病（MRD）水平患者总生存（OS）率、无事件生存（EFS）率、累积复发率（CIR）、移植相关死亡率（TRM）。结果全部患者共行55例次CAR-T细胞治疗，CAR-T细胞治疗反应率、严重细胞因子释放综合征（CRS）发生率分别为92％、28％。未发生治疗相关死亡。CAR-T细胞输注至allo-HSCT的中位时间为54（26～232）d。CAR-T细胞输注后中位随访637（117～1097）d，1年OS、EFS率分别为（80.0±5.7）％、（60.0±6.9）％。移植后1年CIR、TRM分别为（28.0±0.4）％、（8.0±0.2）％。CAR-T细胞输注后及allo-HSCT前骨髓MRD<0.01％患者1年EFS率更高［（70.0±7.2）％、（20.0±12.6）％，P<0.001；（66.7±7.5）％对（36.4±14.5）％，P＝0.008］，CIR更低［（25.0±0.5）％、（70.0±2.6）％，P<0.001；（23.1±0.5）％、（45.4±2.6）％，P＝0.038］。结论CAR-T细胞治疗桥接allo-HCT对于复发/难治B-ALL是一种安全有效的治疗策略。

The regulatory role of exosomes in leukemia and their clinical significance.

Recurrence is a primary cause of death in patients with leukemia. The interactions of tumor cells with the microenvironment and tumor stem cells hidden in bone marrow promote the recurrence and metastasis of leukemia to lymphoid tissue. Exosomes, membrane-coated nanovesicles secreted by living cells, perform biomaterial transfer and information exchange between cells. Exosomes contain various other biological components derived from parental cells, and they remotely regulate the function of target cells through body fluid flow. Recent studies revealed that exosomes participate in the development of leukemia and play important roles in its diagnosis and treatment by influencing cell proliferation and apoptosis, regulating bone marrow microenvironment, promoting angiogenesis, and inhibiting hematopoiesis. Exosomes are potential biomarkers and therapeutic targets for leukemia, and they can influence drug resistance. Leukemia-derived exosomes present leukemia-related antigens to target cells, promote the proliferation of leukemic cells, help these cells escape immunity, protect them from the cytotoxic effects of chemotherapeutics, and promote angiogenesis and tumor migration. Therefore, exosomes are closely related to the metastasis, treatment, and prognosis of leukemia, and they can be used to detect and monitor the progression of leukemia. This paper reviews the regulatory roles of exosomes in leukemia and their clinical significance.

Detection of Fusion Genes to Determine Minimal Residual Disease in Leukemia Using Next-Generation Sequencing.

Measuring minimal residual disease (MRD), the persistence of leukemic cells after treatment, is important for monitoring leukemia recurrence. The current methods for monitoring MRD are flow cytometry, to assess aberrant immune phenotypes, and digital droplet PCR (ddPCR), to target genetic aberrations such as single-nucleotide variants and gene fusions. We present the performance of an RNA-based next-generation sequencing (NGS) method for MRD gene fusion detection compared with ddPCR. This method may have advantages, including the capacity to analyze different genetic aberrations and patients in 1 experiment. In particular, detection at the RNA level may be highly sensitive if the genetic aberration is highly expressed. We designed a probe-based NGS panel targeting the breakpoints of 11 fusion genes previously identified in clinical patients and 2 fusion genes present in cell lines. Blocking probes were added to prevent nonspecific enrichment. Each patient RNA sample was diluted in background RNA, depleted for rRNA and globin mRNA, converted to cDNA, and prepared for sequencing. Unique sequence reads, identified by unique molecular identifiers, were aligned directly to reference transcripts. The same patient and cell-line samples were also analyzed with ddPCR for direct comparison. Our NGS method reached a maximum sensitivity of 1 aberrant cell in 10000 cells and was mostly within a factor of 10 compared with ddPCR. Our detection limit was below the threshold of 1:1000 recommended by European Leukemia Net. Further optimizations are easy to implement and are expected to boost the sensitivity of our method to diagnostically obtained ddPCR thresholds.