Refractory Acute Myeloid Leukemia Patients Research Articles

Prognostic Value of IGF2BP3 Gene Expression Levels in Patients with Acute Myeloid Leukemia

To investigate the relationship between IGF2BP3 gene expression and prognosis in patients with acute myeloid leukemia (AML). High throughput transcriptome sequencing was performed on bone marrow primary leukemia cells from 27 patients with AML in our center, the relationship between IGF2BP3 expression levels and clinical characteristics were analyzed and verify the samples from patients with newly treated AML and refractory AML. The expression level of IGF2BP3 gene were analyzed in 20 healthy subjects and 26 patients with AML. The expression of IGF2BP3 in two anthracycline-resistant cell lines (HL60/ADR, K562/ADR) was detected by RT-qPCR and Western blot, and the expression difference of IGF2BP3 was compared with that in sensitive cells (HL60, K562). The relationship between the expression level of IGF2BP3 in patients with AML and prognostic were analyzed through data analysis of 746 patients with AML, and the prognostic value of IGF2BP3 in AML was analyzed by multivariate Cox regression analysis. In the bone marrow primary leukemia cells of 27 AML patients in our center, the expression level of IGF2BP3 in patients with refractory AML was significantly higher than that in chemotherapy sensitive patients (P =0.0343). The expression of IGF2BP3 in leukemia patients with extramedullary infiltration (EMI) was significantly higher than that in AML patients without extramedullary infiltration (P =0.0049). Compared with healthy subjects (n=20), IGF2BP3 expression in AML patients (n=26) was higher (P =0.0009). The expression of IGF2BP3 mRNA in the anthracycline resistant cell lines (HL60/ADR, K562/ADR) was significantly higher than that in the sensitive cell lines (K562/ADR vs K562,P =0.0430; HL60/ADR vs HL60, P =0.7369). Western blot results showed that the expression of IGF2BP3 protein in mycin resistant cells was significantly higher than that in sensitive cells (P < 0.001). qPCR results showed that the expression level of IGF2BP3 mRNA in refractory AML patients was significantly higher than that in patients with chemotherapy sensitive (P =0.002). High expression of IGF2BP3 was associated with poor prognosis in AML (P < 0.05) in 3 large sample cohorts of AML patients. Univariate and multivariate prognostic analyses demonstrated that high expression of IGF2BP3 was significantly associated with shorter event-free survival (EFS, HR=1.887, P =0.024) and overall survival (OS, HR=1.619, P =0.016). The high expression of IGF2BP3 gene may be an important factor in the poor prognosis of AML, suggesting that IGF2BP3 gene may be a new molecular marker for the clinical prognosis evaluation and treatment strategy of AML.

Pursuing dynamics of minimal residual leukemic subclones in relapsed and refractory acute myeloid leukemia during conventional therapy.

Acute myeloid leukemia (AML) is characterized by clonal heterogeneity, leading to frequent relapses and drug resistance despite intensive clinical therapy. Although AML's clonal architecture has been addressed in many studies, practical monitoring of dynamic changes in those subclones during relapse and treatment is still understudied. Fifteen longitudinal bone marrow (BM) samples were collected from three relapsed and refractory (R/R) AML patients. Using droplet digital polymerase chain reaction (ddPCR), the frequencies of patient's leukemic variants were assessed in seven cell populations that were isolated from each BM sample based on cellular phenotypes. By quantifying mutant clones at the diagnosis, remission, and relapse stages, the distribution of AML subclones was sequentially monitored. Minimal residual (MR) leukemic subclones exhibit heterogeneous distribution among BM cell populations, including mature leukocyte populations. During AML progression, these subclones undergo active phenotypic transitions and repopulate into distinct cell population regardless of normal hematopoiesis hierarchic order. Of these, MR subclones in progenitor populations of patient BM predominantly carry MR leukemic properties, leading to more robust expansion and stubborn persistence than those in mature populations. Moreover, a minor subset of MR leukemic subclones could be sustained at an extremely low frequency without clonal expansion during relapse. In this study, we observed treatment persistent MR leukemic subclones and their phenotypic changes during the treatment process of R/R AML patients. This underscores the importance of preemptive inhibition of clonal promiscuity in R/R AML, proposing a practical method for monitoring AML MR subclones.

Abstract 7123: Biomarker-targeted alkylator and DNA damage repair inhibitor combination for refractory and relapsed acute myeloid leukemia (AML)

Abstract Isocitrate dehydrogenase 1 and 2 (IDH1/2) convert isocitrate to α-ketoglutarate in the tricarboxylic acid (TCA) cycle. A missense mutation in IDH1/2 leads to accelerated conversion of α-ketoglutarate into 2-hydroxyglutarate (2-HG), an oncometabolite that competitively inhibits α-ketoglutarate-dependent dioxygenases including Ten-Eleven Translocation (TET) enzymes. TET enzymes are DNA demethylases that convert 5-methylcytosine to 5-carboxycytosine and regulate CpG island promoter methylation signatures. In IDH1/2 mutant glioma, inhibition of TET2 enzyme by 2-HG can lead to promoter hypermethylation of O6-methylguanine methyltransferase (MGMT) and reduce its expression. Approximately 10-15% of de novo or treatment-related AML harbor mutually exclusive mutations in IDH1/2 or TET2, and emerging resistance towards clinical IDH1/2 inhibitors, therefore warrants novel therapeutic options. We performed a pan-cancer TCGA analysis and observed MGMT promoter hypermethylation and reduced expression in IDH1/2 AML. Our independent study on 10 AML patients (5 IDH1/2 and 5 TET2 mutant) corroborated the TCGA data and showed a significantly reduced expression of MGMT in comparison to 7 wild-type AML samples. As MGMT resolves O6-alkylguanine lesions during DNA damage, we performed an exhaustive alkylator screen, where temozolomide (TMZ) and our novel compound KL-50 emerged as the top hits that imparted a maximum therapeutic index in multiple MGMT (MGMT+/–) isogenic AML cell line models. Both TMZ and KL-50 induced MGMT-dependent DNA strand breaks and replication stress and activated ATM and ATR DNA damage response pathways in a time-dependent manner. Inhibition of ATR activation with Ceralasertib, an ATR inhibitor (ATRi) currently in clinical trials for chronic myelomonocytic leukemia and myelodysplastic syndromes, imparted a profound MGMT-dependent synergy with TMZ and KL-50. Researchers have established in glioma that mismatch repair (MMR) loss confers resistance to TMZ, and reported studies also show evidence of MMR loss in refractory or relapsed AML patients. Thus, we developed numerous MMR-deficient isogenic AML cell lines in an MGMT+/– background and observed MMR loss-mediated resistance to TMZ and abrogation of its synergy with ATRi. Unlike TMZ, KL-50 retained a profound synergy with ATRi in an MGMT-dependent manner irrespective of MMR status. We are currently investigating our novel KL-50 and ATRi combination in MGMT/MMR isogenic cell line-based in vivo AML models and in AML patient-derived xenografts to establish it as a promising novel biomarker-targeted therapy for AML patients. Citation Format: Prateek Bhardwaj, Ranjini Sundaram, Amro Baassiri, Martin Matthews, Jennifer VanOudenhove, Susan Gueble, Stephanie Halene, Ranjit Bindra. Biomarker-targeted alkylator and DNA damage repair inhibitor combination for refractory and relapsed acute myeloid leukemia (AML) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7123.

Integrating Full Bayesian Inference and Student's t-Distribution Method for Enhanced Outlier Handling in Caffeine Population Pharmacokinetics: Assessing Drug-Drug Interactions with Enasidenib in Relapsed or Refractory AML and MDS Patients.

As the first-in-class, selective, and potent inhibitor of the isocitrate dehydrogenase-2 (IDH2) mutant protein, enasidenib was approved by the US Food and Drug Administration (FDA) in 2017 for the treatment of adult patients with relapsed or refractory acute myeloid leukemia (AML) with an IDH2 mutation. Known for its interactions with various cytochrome P450 (CYP)enzymes and transporters in vitro, a clinical pharmacokinetics (PK) trial was initiated to assess the impact of multiple doses of enasidenib on the single-dose PK of sensitive probe substrates of several cytochrome P450 enzymes and transporters. In this study, a population pharmacokinetic analysis approach was employed to address challenges posed by high, nonzero baseline caffeine concentrations. Moreover, we integrated full Bayesian inference into this approach innovatively for a more detailed understanding of parameter uncertainty and greater modeling flexibility, alongside Student's t-distribution for robust error modeling in handling the abnormal outlier caffeine concentration data observed in this trial. Our analyses demonstrated that multiple doses of enasidenib altered caffeine clearance to a clinically meaningful extent, as evidenced by an approximate 8-fold decrease. This finding led to a specific recommendation in the package insert to avoid the concurrent use of certain CYP1A2 substrates with enasidenib, unless directed otherwise in the prescribing information. Furthermore, this research underlines the technical benefits of integrating full Bayesian inference and incorporating Student's t-distribution for residual error modeling in the PK field.

ABC transporters are predictors of treatment failure in acute myeloid leukaemia

IntroductionTo date, no chemoresistance predictors are included in acute myeloid leukaemia (AML) prognostic scoring systems to distinguish responding and refractory AML patients prior to chemotherapy. ABC transporters have been described as altering AML chemosensitivity; however, a relevant study investigating their role at various molecular levels was lacking. MethodsGene expression, genetic variants, methylation and activity of ABCA2, ABCA5, ABCB1, ABCB6, ABCC1, ABCC3 and ABCG2 were analysed in AML blasts and healthy myeloblasts. Differences between responding and refractory AML in a cohort of 113 patients treated with 3 + 7 induction therapy were explored. ResultsABCC3 variant rs2301837 (p = 0.049), ABCG2 variant rs11736552 (p = 0.044), higher ABCA2 (p = 0.021), ABCC1 (p = 0.017), and ABCG2 expression (p = 0.023) and a higher number of concurrently overexpressed transporters (p = 0.002) were predictive of treatment failure by multivariate analysis. Expression of ABCA5 (p = 0.003), ABCB6 (p = 0.001) and ABCC3 (p < 0.0001) increased significantly after chemotherapy. Higher ABCG2 promoter methylation correlated with lower ABCG2 expression (p = 0.0001). ABCC1 was identified as the most active transporter in AML blasts by functional analysis. ConclusionsABC transporters, especially ABCC1 seem to contribute substantially to AML chemoresistance. A detailed understanding of chemoresistance mechanisms and the clinical implications of chemosensitivity predictors may lead to alternative therapeutic approaches for AML patients with unveiled chemoresistance signatures.

A Novel Anticancer Quinolone, ( R)-Wac-224 Monotherapy or Combination with Cytarabine/Venetoclax Confers Promising Activities Against Acute Myeloid Leukemia

Introduction Approximately 60%-80% of patients with acute myeloid leukemia (AML) who achieve complete remission eventually relapse after conventional chemotherapy and have poor prognoses despite the recent advances of novel anticancer agents. Targeting topoisomerase 2 (Top2) is important to treat patients with AML, as anthracycline is widely used in general practice. However, anthracycline-induced cardiotoxicity or anthracycline resistance due to P-glycoproteinoverexpression often becomes major obstacles to successful treatment. Therefore, continuing development of more effective novel treatments for AML is necessary. Quinolones, antibacterials that attack DNA gyrase and topoisomerase IV (functional analog of mammalian Top2), possess a broad spectrum chemotherapeutic activities including anti-leukemic effects (e.g. vosaroxin). In this study, we developed and characterized a novel quinolone that demonstrates promising activities against human AML as a single agent and in combination with other agents. Methods and Results We developed 5-methyl-7-{3-[(oxan-2-ylmethyl) carbamoyl] azetidin-1-yl}-4-oxo-1-(1,3-thiazol-2-yl)-1,4-dihydro-1,8-naphthyridine-3-carboxylic acid [racemic WAC-224 (WAC) or (R)-WAC-224 (R-WAC), which is a novel anticancer quinolone, targeting Top 2. WAC inhibited Top2α comparable with vosaroxin (a known Top2 inhibitor) in a topoisomerase relaxation assay. IC 50 of vosaroxin and WAC were 1.10 μM and 2.26 μM, respectively. R-WAC significantly inhibited the human AML cell line proliferation (MV4-11, HL60, and KG1a), which was comparable to daunorubicin and cytarabine, in a manner not affected by P-glycoprotein overexpression. WAC also elicited cell apoptosis against MV4-11 cells comparable with vosaroxin. Whereas, WAC did neither increase serum troponin-T nor decrease the crypt numbers in the small intestine, indicating WAC was less toxic than doxorubicin. R-WAC monotherapy demonstrated prolonged survival in the AML mice model and inhibited tumor growth in the MV4-11 xenograft mice model. Moreover, the combination of R-WAC and cytarabine demonstrated more active anti-leukemia effects than daunorubicin and cytarabine in the AML mice model. R-WAC and cytarabine or daunorubicin and cytarabine did not exhibited decreased body weight in mice. Next, we investigated R-WAC combination with venetoclax, selective BCL-2 inhibitor, for anti-leukemia effects. R-WAC and venetoclax exhibited increased cell apoptosis against MV4-11 and KG1a cells in vitro, and more active anti-leukemia effects in xenotranplant MV4-11 mouse model than each monotherapy. These results suggest that R-WAC and combination with cytarabine or venetoclax has strong anti-leukemia effects without increased toxicities. Finally, R-WAC significantly suppressed the colony-forming capacity of bone marrow CD34 + stem/progenitor cells in refractory AML patients, indicating R-WAC depleted AML stem cells. Conclusion R-WAC has promising anti-leukemia effects with less toxicities compared with conventional anthracycline possibly due to the different inhibitory mechanisms of Top2 isoform (anthracycline inhibits Top2β). These results indicate that R-WAC is a promising therapeutic agent for AML and support its clinical development, especially as a combination of R-WAC with cytarabine or venetoclax.

Significance of the Apoptotic Regulators BAX, BCL2, BCL- XL and MCL1 in Newly Diagnosed AML

Introduction: In acute myeloid leukemia (AML), cytogenetics and molecular mutations remain important prognostic tools for tailoring treatment after induction therapy. Abnormalities in multiple components of apoptotic pathways have been identified as potential drivers of AML. Identifying other biomarkers may help us further refine prognosis and identify other therapeutic strategies. B-cell lymphoma 2 (Bcl2), Bcl-xL and myeloid cell leukemia-1 (Mcl1) are commonly expressed prosurvival proteins in hematologic malignancies, whereas Bax acts as a promoter of apoptosis. We retrospectively evaluated the significance of Bax, Bcl2, Bcl-xL and Mcl1 expression in newly diagnosed AML patients using the Beat AML program 1. Methods: We analyzed RNA-expression data from The Beat AML study (672 tumor specimens collected from 562 patients) 1. Data was extracted from the public database. Different expression levels of the above genes were compared using t-tests. Results were considered significant with p-value &amp;lt; .05. Kaplan-Meier curves were used to estimate survival. Results: For the correlative survival analyses, Bax, Bcl2, Bcl-xL and Mcl1 mRNA expression and clinical information were both obtained from the BeatAML cohort 1. Patients were categorized into high expressers (≥ median) and low expressers (&amp;lt;median). In total, 461 patients had available gene expression data. The mean age of diagnosis was 57 years, and 56.6% of patients were male. The most common specific diagnosis at inclusion was AML with mutated NPM1 (22%), followed by AML with myelodysplasia related changes (21%) and AML, NOS (11%). Complete response to induction was seen in more than half (56%) of the patients for which response was recorded (n=369). Others developed refractory AML (32%). No data was available for 12%. Standard chemotherapy (induction therapy with idarubicin and cytarabine (7+3) and consolidation therapy with high dose cytarabine) was used in 98.4% of these patients. Over half of the analyzed samples were peripheral blood (52%), and the remaining samples were either bone marrow aspirate (46%) or from leukapheresis (2%). Most of the specimens were collected at initial acute leukemia diagnosis (62.2%), others were collected with residual disease or post chemotherapy (25.1%). The mean survival for this cohort was 13.7 months. Above median expression of Mcl1 was associated with longer survival (33 vs. 20 months; p = 0.0262). Above median expression of Bax was also associated with longer survival (32 vs. 18.2 months; p = 0.0114). In the Bax analysis, gene expression was further subdivided into 4 quartiles which showed correlation of expression levels with length of survival (Figure 1). The prognostic significance of the ratio Bax/ (Mcl1+Bcl2+Bcl-xL) was also analyzed given the mechanism of action of these various regulators. A higher ratio was associated with longer survival (40.7 vs. 18.7 months; p = 0.0095) (Figure 2) Conclusions Bcl-2, Bcl-xL and Mcl-1 are commonly expressed antiapoptotic proteins, whereas Bax is a promoter of apoptosis. The results with Bax are consistent with previous literature. Our results with Mcl1 expression differed from what we may have expected. This may be due to the sample size or methodology. However, it may also be due to the fact that a ratio of the actors is needed to help predict prognosis (as we analyzed in Figure 2). The overall survival between Bcl-2 and Bcl-xL was compared independently, however, they did not appear to have a significant difference in survival between high and low expression groups. We are currently working to see if this ratio can be validated in a prospective cohort of relapsed/ refractory AML patients treated on clinical trial and this data will be presented at the meeting. The Bcl-2 inhibitor venetoclax has demonstrated promising results in treating AML. However, venetoclax appears to have limited activity as a single agent. Our study suggests that potentially a combination of agents targeting factors such as Bcl-2, Bcl-xL and Mcl-1 may be associated with improved survival in patients with AML. Further validation of these biomarkers is warranted, and Mcl-1 inhibitors are currently in clinical trials.

Sequential Conditioning Regimen with Thiotepa, Clofarabine and Busulfan (TEC/CloB2A2) and Post-Transplant Cyclophosphamide in Adults with Refractory AML: A Retrospective Monocentric Study

Background : Since almost 20 years, sequential allogeneic stem cell transplantation is proposed to fit and relatively young patients with refractory hematologic malignancies (mainly acute myeloid leukemia (AML)) and who will otherwise die from the disease. This procedure is characterized by the use of a renforced conditioning regimen combining a debulking chemotherapy followed after 3 days of rest by the conditioning regimen itself. However, the results overall remain dismal as only a small proportion of patients are finally cured. Recently, a combination of thiotepa, etoposide, cyclophosphamide (TEC) followed by fludarabine/busulfan/ATG (FB2A2) using any type of donor for refractory hematologic malignancies have shown some promising results (Dulery, 2018). As clofarabine may have better antileukemic effect than fludarabine, we have investigated in our patients with refractory AML a new sequential approach combining TEC+clofarabine/busulfan/ATG (TEC/CloB2A2). Methods : We have investigated in our Hematology Department a new sequential conditioning regimen replacing fludarabine by clofarabine as part of a TEC/FB2A2 regimen with the hope to obtain less relapse and better survival in a cohort of patients with refractory AML. The TEC/CloB2A2 regimen consisted of Thiotepa 5 mg/kg at day(d)-13, Cyclophosphamide 400 mg/m²/d from d-12 to d-9 and Etoposide phosphate 100 mg/m²/d from d-12 to d-9, followed after 3 days of rest, by Clofarabine 30 mg/m²/d from d-5 to d-1, Busulfan 3,2 mg/kg/d d-5 and d-4 and Thymoglobuline 2,5 mg/kg/d d-3 and d-2. GVHD prophylaxis consisted of high-dose of post-transplant Cyclophosphamide (PTCY) 50 mg/kg/d at d+3 and d+5 in combination with mycophenolate mofetyl and cyclosporine starting at d+6. Any type of donor have been considered. Results : Between February 2020 and August 2022, 12 patients have been treated. Patient characterisitcs are given in the Table. Median age was 58 years (y) old. The majority of patients had primary refractory AML (n=9). This was the first transplant for all patients. One patient needed desensibilization before conditioning due to high level of anti-HLA antibodies directed against the donor. All but 2 donors were haploidentical. One patient with a matched donor did not receive PTCY after transplant. Two patients died during the aplasia phase, one at d+6 of sepsis and multiple organ failure (MOF) and one at d+30 of disseminated fungal infection. The median time of neutrophils (&amp;gt; 1 Giga/L) and platelets (&amp;gt; 50 Giga/L) recoveries were 22 days (range : 12-137) and 34 days (range : 16-241), respectively. There were no primary or secondary graft failures. Responses were evaluated at a median of 57 days (range : 26-70) after transplant. Five complete remission (CR), 3 CR with incomplete platelet recovery (CRp) and two failures were documented for an overall CR/CRp rate of 67%. Only one cutaneous grade 2 acute GVHD occured after transplant but no chronic GVHD. Six out of 8 CR/CRp patients received maintenance treatment after transplant to prevent relapse using 5'azacytidine alone in 1, 5 azacytidine+ donor lymphocyte infusion (DLI) in 2, DLI alone in 1. The last two patients received maintenance as part of a trial. The three patients receiving DLI developped acute GVHD thereafter including 2 grade 2 and one grade 3. Six of the patients achieving CR/CRp relapsed (75%) at a median of 160 days (range : 48-344) post-transplant. At last follow-up (July 2023), only one patient is alive in CR (8%) at 795 days from transplant (SET/CLoB2A2 +PTCY and sibling donor, grade 3 acute GVHD after DLI). Causes of deaths were relapse in 6, and infections in five (disseminated fungal infection n=1, COVID-19 n=2, bacterial infection + MOF n=1 and + distress respiratory syndrome n=1). Non-relapse mortality (NRM) was 25% and 41% at 1y and 2y, respectively. 1-y and 2y overall survival and leukemia-free survival were both at 25% (9.3-66.6) and 8.3% (1.2-54.4), respectively. Conclusion : The sequential TEC/CloB2A2 conditioning regimen is not associated with better outcomes compared to the TEC/FB2A2 sequential conditioning regimen. Other sequential approaches and/or GVHD/maintenance strategies have to be considered in the future for these very high-risk refractory AML patients in order to obtain better graft-versus-leukemia effect and lesser toxicity.

Comparison of HLA antibody identification methods for the selection of platelet products for HLA-mediated platelet refractory patients.

In an ineffective transfusion context, solid-phase immunoassays using the Luminex platform for the detection and characterization of HLA antibodies are currently used to select HLA-compatible platelet products. A new HLA antibody identification method, the HISTO SPOT® HLA AB test (BAG Health care GmbH, Lich, Germany), based on the detection of antibodies directed against a recombinant single antigen (SA) by colored spots detected by HISTO MATCH HLA AB module software, runs fully automated on the MR.SPOT®. The aim of this study was to compare the ability of the HISTO SPOT HLA AB and C1qScreen™ (C1q SAB) assays with that of the Labscreen single antigen class I (OL SAB) assay to detect anti-HLA class I antibodies in 56 serum samples from 54 platelet refractory acute myeloid leukemia patients who received HLA mismatch platelet concentrates at a single oncohematology center. In total, 1414 class I specificities, 433 HLA-A and 981 HLA-B, were detected by the OL SAB test. The mean fluorescence intensity (MFI) was >5000 for 874 antigens and <5000 for 655 antigens. The HISTO SPOT® HLA AB and C1q SAB tests identified 85% and 79% of OL SA-detected antigens with an MFI >5000, respectively, but did not identify 34% and 44% of OL SAB-detected antigens, highlighting the lower sensitivity of these techniques. Interestingly, the donor-specific antibodies (DSAs) identified by the HISTO SPOT® HLA AB and C1q SAB assays reacted against HLA mismatch platelet concentrates with the same specificity (86%) and positive predictive (77%) value as in the OL SAB test when the MFI threshold was >2000 for DSA detection. Although the HISTO SPOT® HLA AB test is less sensitive than the OL SAB test, this test could be used for the selection of HLA-compatible platelet products.

Real-life data of azacitidine-venetoclax combination in acute myeloid leukemia patients: a single center experience

Aims: To evaluate real-life data on the efficacy and safety of Venetoclax (Ven) therapy used in combination with hypomethylating agent (HMA) in patients with acute myeloid leukemia (AML).&#x0D; Methods: The records of newly diagnosed, relapsed or refractory (RR) AML patients over 18 years of age who were planned to be treated with Azacitidine (AZA) combined with Ven because they were not suitable for intensive chemotherapy and patients who received AZA combined with Ven maintenance therapy after achieving remission were retrospectively analyzed. The standard protocol for patients is subcutaneous or intravenous AZA 75 mg/m2 on days 1-7/ every 28 days + oral Ven treatment 100-400 mg/day for 28 days. The treatment response rates, survival times, and side effect profiles of 18 newly diagnosed patients, 12 RR patients, and 4 patients receiving AZA+Ven as maintenance treatment between January 2021 and March 2022 were evaluated.&#x0D; Results: It was found that 8 of the 34 patients (23.5%) who were examined in the present study died before the first response could be evaluated. When the response rates were evaluated, complete response (CR) or complete remission with incomplete blood count recovery (CRi) (CR+CRi) was found to be 61% in the group receiving AZA+Ven in the first line, and CR+CRi was 50% in the group receiving AZA+Ven because of RR AML. In the group receiving AZA+Ven in the first line, the average Overall Survival (OS) was 8.00 months (95% CI: 1.58-14.41), and 7.00 months in the RR group (95% CI: 1.78-12, 21). All patients in the group receiving AZA+Ven for maintenance purposes were alive and the median follow-up period was 12.50±6.02 months in this group (Mean±SD). The most common side effect was neutropenia, and the most common cause of death was disease progression.&#x0D; Conclusion: In AML patients ineligible for intensive treatment due to advanced age or comorbidities, real-life data of AZA+Ven therapy with effective CR+CRi rates and a manageable spectrum of side effects promise hope.

Fludarabine, cytarabine, and idarubicin with or without venetoclax in patients with relapsed/refractory acute myeloid leukemia.

Treatment options for relapsed and refractory acute myeloid leukemia patients (R/R AML) are limited. This retrospective cohort study compares safety and efficacy of fludarabine, cytarabine, and idarubicin (FLA-IDA) without or with venetoclax (FLAVIDA) in patients with R/R AML. Thirty-seven and 81 patients received one course FLA-IDA with or without a 7-day course of venetoclax, respectively. The overall response rate (ORR) was significantly higher in FLAVIDA compared to FLAIDA- treated patients (78% vs. 47%; P=0.001), while measurable residual disease was negative at a similar proportion in responding patients (50% vs. 57%), respectively. Eighty-one percent and 79% of patients proceeded to allogeneic hematopoietic cell transplantation or donor lymphocyte infusion after FLAVIDA and FLA-IDA, respectively. Event-free and overall survival were similar in FLAVIDA- and FLA-IDA-treated patients. Refractory patients could be salvaged more successfully after FLA-IDA compared to FLAVIDA pretreatment. Neutrophil and platelet recovery times were similar in the venetoclax and the control group. In conclusion, short-term venetoclax in combination with FLA-IDA represents an effective treatment regimen in R/R AML identifying chemosensitive patients rapidly and inducing measurable residual disease-negative remission in a high proportion of R/R AML patients.

Co‐Targeting c‐Myc and Bcl‐2 by Oral Small Molecule Combination of WBC100 and Venetoclax Effectively Controls Acute Myeloid Leukemia in Preclinical Models

AbstractUncontrolled proliferation and apoptosis evasion are two hallmarks of acute myeloid leukemia (AML), but the molecular mechanisms remain poorly understood. In this study, it is demonstrated that the double over‐expresser of oncoprotein c‐Myc and anti‐apoptotic protein Bcl‐2 is a critical “genetic overdrive” of proliferation and apoptosis evasion in AML and is associated with poor genetic alterations. Double‐knockdown of c‐Myc/ Bcl‐2 synergistically kills AML cells in vitro and in vivo. Moreover, a novel oral small molecule combination co‐targeting c‐Myc and Bcl‐2 with WBC100 and Venetoclax (VEN) at low doses are developed. Importantly, the study shows that this combination results in deep and durable remissions of AML, and its efficacy is superior to the frontline combination of Venetoclax and hypomethylation azacitidine (AZA) in AML mouse models and PDX models from relapsed or refractory AML patients. Mechanically, Bcl‐2 knockdown induces mitochondrial outer membrane permeabilization and c‐Myc‐knockdown impairs mitochondria biogenesis. Co‐targeting c‐Myc/Bcl‐2 reciprocally abrogates over‐proliferation and apoptosis resistance via forming a double hit to mitochondrial biogenesis and apoptosis machinery. The findings for the first time demonstrate that co‐targeting c‐Myc/Bcl‐2 by the novel oral small molecule combination of WBC100/Venetoclax is a promising and convenient therapy for AML and support its clinical trial.

CAG (cytarabine, aclarubicin and granulocyte colony-stimulating factor) regimen for core binding factor acute myeloid leukaemia with measurable residual disease.

Acute myeloid leukaemia (AML) with t (8;21) or inv (16), called core binding factor (CBF) AML, has a favourable prognosis. However, some CBF-AML patients have persistent measurable residual disease (MRD) and are more likely to relapse after standard chemotherapy treatment. The CAG regimen, composed of cytarabine, aclarubicin and granulocyte colony-stimulating factor, has been proven to be effective and safe in treating refractory AML patients. We performed a retrospective study to evaluate the efficacy of the CAG regimen to eliminate MRD detected by RUNX1::RUNX1T1 and CBFβ::MYH11 transcript levels by quantitative polymerase chain reaction (Q-PCR) among 23 patients. Molecular response was defined as the ratio of fusion transcript after treatment to that before treatment less than or equal to 0.5. The molecular response rate and median decrease ratio of fusion transcripts at the molecular level of the CAG regimen were 52% and 0.53, respectively. The median fusion transcripts before CAG treatment was 0.25% whereas after CAG was 0.11%. Among the 15 patients who had a poor molecular response to the high/intermediate-dose cytarabine regimen, the median decrease ratios of transcripts at the molecular level of high/intermediate-dose cytarabine and CAG were 1.55 and 0.53 (P = 0.028), respectively, and 6 of 15 patients achieved a molecular response to CAG (40%). The median disease-free survival was 18months, and the overall survival rate at 3years among all patients was 72.7% ± 10.7%. The common grades 3-4 adverse events were nausea (100%), thrombocytopenia (39%) and neutropenia (37.5%). The CAG regimen may have activity in CBF-AML patients and could provide a new option for patients who have a poor molecular response to high/intermediate-dose cytarabine.

Palbociclib in Acute Leukemias With KMT2A-rearrangement: Results of AMLSG 23-14 Trial

Palbociclib in Acute Leukemias With KMT2A-rearrangement: Results of AMLSG 23-14 Trial

Knockdown of let-7b in leukemia associated macrophages inhibit acute myeloid leukemia progression.

Macrophages, critical components of bone marrow microenvironment, are reported to be remodeled into leukemia-associated macrophages (LAMs) in leukemic microenvironment where they contribute to leukemia development, characterized as M2 macrophages with pro-tumor effects. However, how leukemic microenvironment transforms macrophages into LAMs remains unknown. Here, we analyzed the clinical relevance of LAMs and profiled their RNA-Seq from acute myeloid leukemia (AML) patients with complete remission (CR) after induction treatment and refractory AML patients. Our results showed that the proportion and number of LAMs in refractory AML patients was higher than that in CR patients and LAM was a poor prognostic factor of AML patients. Furthermore, let-7b was a potentially aberrant gene in LAMs contributed to M2-subtype characteristics. Knockdown of let-7b in LAMs could inhibit the development of AML by repolarizing LAMs toward M1-subtype characteristics through the activation of Toll-like receptor and NF-κB pathway. Our study provides insight for future LAM-based immunotherapy strategies for AML.

Targeting regulated cell death pathways in acute myeloid leukemia.

The use of the BCL2 inhibitor venetoclax has transformed the management of patients with acute myeloid leukemia (AML) who are ineligible for intensive chemotherapy. By triggering intrinsic apoptosis, the drug is an excellent illustration of how our greater understanding of molecular cell death pathways can be translated into the clinic. Nevertheless, most venetoclax-treated patients will relapse, suggesting the need to target additional regulated cell death pathways. To highlight advances in this strategy, we review the recognized regulated cell death pathways, including apoptosis, necroptosis, ferroptosis and autophagy. Next, we detail the therapeutic opportunities to trigger regulated cell death in AML. Finally, we describe the main drug discovery challenges for regulated cell death inducers and their translation into clinical trials. A better knowledge of the molecular pathways regulating cell death represents a promising strategy to develop new drugs to cure resistant or refractory AML patients, particularly those resistant to intrinsic apoptosis.

FLAG Regimen and Bortezomib: A Study of Eight Cases with Relapsed Leukaemia

Historically, relapsed and refractory Acute Lymphoblastic Leukaemia (ALL) and Acute Myeloid Leukaemia (AML) have been associated with a dismal prognosis. Relapse-refractory ALL is associated with cure rates of &lt;10% in adults and 30% in the paediatric population. Similarly, the five-year survival rates for relapsed and refractory AML are only 10%. This case series describes eight patients with relapsed/refractory leukaemia (6 with ALL and 2 with AML) who received the FLAG-Bortezomib protocol from January 2021 to August 2022. In this case series, the authors investigated the remission rate and toxicity of the regimen, including Intensive Care Unit (ICU) admissions and the incidence of neutropenic sepsis. In the present study, the rates of culture-positive sepsis and High Dependency Unit (HDU) admission were 50% (4/8) and 37.5% (3/8), respectively, with no post-chemotherapy mortality in this cohort. Morphologic remission was documented in 87.5% (7/8) of cases, and negative minimal residual disease was achieved in 62.5% (5/8), with 100% (2/2) remission in those with AML. One patient with ALL experienced disease progression during treatment. The FLAG with Bortezomib protocol induces remission in relapsed and refractory ALL and AML patients with an acceptable toxicity profile. This protocol requires rigorous blood product support in the form of packed red blood cells, platelet-rich plasma, and single-donor platelet apheresis. It serves as a bridge to stem cell transplant.

Characteristics and treatment results of refractory and relapsed acute myeloid leukaemia in paediatric patients treated in Polish Paediatric Leukaemia/Lymphoma Study Group institutions according to the Protocol Acute Myeloid Leukaemia Berlin-Frankfurt-Munster 2012 and a review of novel treatment possibilities in paediatric acute myeloid leukaemia.

This study aimed to present the clinical features and results of treatment of patients diagnosed with refractory or relapsed acute myeloid leukaemia (AML) in Polish Paediatric Leukaemia/Lymphoma Study Group (PPL/LSG) institutions, treated in accordance with the Protocol Acute Myeloid Leukaemia Berlin-Frankfurt-Munster 2012, as their first-line therapy. The outcome data of 10 patients with refractory AML (median age 9.5 years) and 30 with relapsed AML (median age 12 years) were analysed retrospectively. Re-induction was usually based on idarubicin, fludarabine, and cytarabine along with allogeneic haematopoietic stem cell transplant (allo-HSCT) in 5 patients with refractory AML and 7 relapsed AML children. 37.5% (3/8) of refractory AML patients achieved second complete remission second complete remission (CRII). One of ten patients (1/10; 10%) was alive and stayed in complete remission for 34 months after the allo-HSCT. The probability of 3-year event-free survival (pEFS) in this group was 0.125 ±0.11. In the group of relapsed AML patients, the CRII was achieved in 9 patients (34%), and the probability of survival was: pEFS = 0.24 ±0.08; probability overall survival (pOS) = 0.34 ±0.09, with significantly better results achieved in patients who underwent allo-HSCT (pOS = 0.54 ±0.14 vs. 0.08 ±0.08, p < 0.0001). The prognosis of refractory AML and the first AML recurrence in children who were first-line treated in PPL/LSG centres according to Protocol Acute Myeloid Leukaemia Berlin-Frankfurt-Munster 2012 is poor. Failures of re-induction treatment particularly result from difficulties in achieving remission. Allogeneic HSCT improves prognosis in children with refractory and first recurrent AML, under the condition it is performed in complete remission. Novel therapeutic approaches are needed to increase the remission rate and improve the outcomes.

Anti-tumor effect of antibody drug conjugate ASP1235 targeting Fms-like tyrosine kinase 3 with venetoclax plus azacitidine in an acute myeloid leukemia xenograft mouse model.

Antibody drug conjugates (ADC) are one of the attractive modalities for the treatment of acute myeloid leukemia (AML). Previously, we have developed ASP1235, a novel ADC targeting Fms-like tyrosine kinase 3 (FLT3) which is widely expressed on the leukemic blasts of AML patients. In this study, we sought to evaluate the therapeutic effect of ASP1235 in combination with venetoclax plus azacitidine, a novel standard-of-care treatment for elderly AML patients, in ASP1235 poor sensitive AML cells. To identify the suitable preclinical model, we first evaluated the growth inhibitory effect of ASP1235 on several leukemia cell lines expressing FLT3 and found that THP-1 cells were partially sensitive to ASP1235 in vitro. Furthermore, ASP1235 showed marginal anti-tumor activity in a THP-1 xenograft model. Compared to the leukemic blasts in most of the relapsed or refractory (R/R) AML patients tested, THP-1 cells expressed equivalent protein levels of Bcl-2, suggesting that ASP1235 in combination with venetoclax plus azacitidine is a rational treatment in the THP-1 model. In vitro, ASP1235 showed a cytotoxic effect on THP-1 cells in combination with venetoclax, and the combination effect was greater than the additive effect. Furthermore, ASP1235 also showed a combination effect with venetoclax plus azacitidine treatment. Similarly, the combination of ASP1235, venetoclax and azacitidine showed a superior anti-tumor effect in a THP-1 xenograft model without obvious body weight loss. These findings provide supportive evidence that the triple combination of ASP1235, venetoclax and azacitidine would improve the clinical outcome of ASP1235 monotherapy and venetoclax plus azacitidine regimen in AML patients.

Naturally selected CD7 CAR-T therapy without genetic editing demonstrates significant antitumour efficacy against relapsed and refractory acute myeloid leukaemia (R/R-AML)

BackgroundThe survival rate for patients with relapsed and refractory acute myeloid leukaemia (R/R-AML) remains poor, and treatment is challenging. Chimeric antigen receptor T cells (CAR-T cells) have been widely used for haematologic malignancies. Current CAR-T therapies for acute myeloid leukaemia mostly target myeloid-lineage antigens, such as CD123 and CD33, which may be associated with potential haematopoietic toxicity. As a lineage-specific receptor, CD7 is expressed in acute myeloid leukaemia cells and T cells but is not expressed in myeloid cells. Therefore, the use of CD7 CAR-T cells for R/R-AML needs to be further explored.MethodsIn this report, immunohistochemistry and flow cytometry were used to analyse CD7 expression in clinical samples from R/R-AML patients and healthy donors (HDs). We designed naturally selected CD7 CAR-T cells to analyse various functions and in vitro antileukaemic efficacy based on flow cytometry, and xenograft models were used to validate in vivo tumour dynamics.ResultsWe calculated the percentage of cells with CD7 expression in R/R-AML patients with minimal residual disease (MRD) (5/16, 31.25%) from our institution and assessed CD7 expression in myeloid and lymphoid lineage cells of R/R-AML patients, concluding that CD7 is expressed in T cells but not in myeloid cells. Subsequently, we designed and constructed naturally selected CD7 CAR-T cells (CD7 CAR). We did not perform CD7 antigen knockdown on CD7 CAR-T cells because CD7 molecule expression is naturally eliminated at Day 12 post transduction. We then evaluated the ability to target and kill CD7+ acute myeloid leukaemia cells in vitro and in vivo. Naturally selected CD7 CAR-T cells efficiently killed CD7+ acute myeloid leukaemia cells and CD7+ primary blasts of R/R-AML patients in vitro and significantly inhibited leukaemia cell growth in a xenograft mouse model.ConclusionNaturally selected CD7 CAR-T cells represent an effective treatment strategy for relapsed and refractory acute myeloid leukaemia patients in preclinical studies.