Although Acute Myeloid Leukemia Research Articles

Low-dose hypomethylating agents cooperate with ferroptosis inducers to enhance ferroptosis by regulating the DNA methylation-mediated MAGEA6-AMPK-SLC7A11-GPX4 signaling pathway in acute myeloid leukemia

BackgroundFerroptosis is a new form of nonapoptotic and iron-dependent type of cell death. Glutathione peroxidase-4 (GPX4) plays an essential role in anti-ferroptosis by reducing lipid peroxidation. Although acute myeloid leukemia (AML) cells, especially relapsed and refractory (R/R)-AML, present high GPX4 levels and enzyme activities, pharmacological inhibition of GPX4 alone has limited application in AML. Thus, whether inhibition of GPX4 combined with other therapeutic reagents has effective application in AML is largely unknown.MethodsLipid reactive oxygen species (ROS), malondialdehyde (MDA), and glutathione (GSH) assays were used to assess ferroptosis in AML cells treated with the hypomethylating agent (HMA) decitabine (DAC), ferroptosis-inducer (FIN) RAS-selective lethal 3 (RSL3), or their combination. Combination index (CI) analysis was used to assess the synergistic activity of DAC + RSL3 against AML cells. Finally, we evaluated the synergistic activity of DAC + RSL3 in murine AML and a human R/R-AML-xenografted NSG model in vivo.ResultsWe first assessed GPX4 expression and found that GPX4 levels were higher in AML cells, especially those with MLL rearrangements, than in NCs. Knockdown of GPX4 by shRNA and indirect inhibition of GPX4 enzyme activity by RSL3 robustly induced ferroptosis in AML cells. To reduce the dose of RSL3 and avoid side effects, low doses of DAC (0.5 µM) and RSL3 (0.05 µM) synergistically facilitate ferroptosis by inhibiting the AMP-activated protein kinase (AMPK)-SLC7A11-GPX4 axis. Knockdown of AMPK by shRNA enhanced ferroptosis, and overexpression of SLC7A11 and GPX4 rescued DAC + RSL3-induced anti-leukemogenesis. Mechanistically, DAC increased the expression of MAGEA6 by reducing MAGEA6 promoter hypermethylation. Overexpression of MAGEA6 induced the degradation of AMPK, suggesting that DAC inhibits the AMPK-SLC7A11-GPX4 axis by increasing MAGEA6 expression. In addition, DAC + RSL3 synergistically reduced leukemic burden and extended overall survival compared with either DAC or RSL3 treatment in the MLL-AF9-transformed murine model. Finally, DAC + RSL3 synergistically reduced viability in untreated and R/R-AML cells and extended overall survival in two R/R-AML-xenografted NSG mouse models.ConclusionsOur study first identify vulnerability to ferroptosis by regulating MAGEA6-AMPK-SLC7A11-GPX4 signaling pathway. Combined treatment with HMAs and FINs provides a potential therapeutic choice for AML patients, especially for R/R-AML.

Sodium Caseinate in Combination With Daunorubicin or Cytarabine Improves Survival of Mice With Long-established Leukemia.

Although acute myeloid leukemia (AML) has traditionally been considered an oncological emergency and initiation of therapy is believed to be crucial to minimizing disease-related morbidity and mortality, it has also been suggested that a certain delay in treatment has no negative consequences in terms of response, early mortality, or survival. We aimed to determine the effect of administration of sodium caseinate (SC), a salt of casein, the main milk protein, with cytarabine or with daunorubicin on survival in mice with well-established leukemia. To assay the time of establishment of leukemia in the bone marrow, Balb/c mice were inoculated with 2.5×10 5 WEHI-3 cells/mouse and after 3, 6 and 9 days were euthanized. Bone marrow mononuclear cells (BM-MNCs) of the femur were obtained and cultured for 120 h with or without rmIL-3 and cell proliferation was evaluated by the crystal violet technique. Then, the effect of administrating SC-cytarabine or SC-daunorubicin on survival rates of mice with well-established leukemia was assayed. Another group of Balb/c mice was inoculated with WEHI-3 cell and after 10 days mice were treated with SC-cytarabine or SC-daunorubicin for 40 days. Survival rates were recorded daily and in surviving mice, the prevalence of bone marrow proliferation after treatment was assayed by the crystal violet technique. The assay on the time of establishment of leukemia shows that in 9 days leukemia cells accumulate in the bone marrow in sufficient quantities to sustain an in vitro culture in the absence of growth factors, and we, thus, used this as a criterion of well-established leukemia. When mice with a burden of leukemic cells of more than 9 days were treated with SC-cytarabine or SC-daunorubicin, this resulted in 55% survival for both treatments, and the proliferation assays showed that the bone marrow retained its normal proliferation capacity. SC-cytarabine or SC-daunorubicin treatment prolonged the survival rate of Balb/c mice with a burden of well-established leukemia, and there was no negative impact on bone marrow functionality; however, SC-cytarabine or SC-daunorubicin combination options need to be sought to increase survival beyond 40 days.

A Favorable Outcome With CPX-351 Liposome (Vyxeos) in the Management of A Sporadic Monosomy 7 Myelodysplastic Syndrome Related Acute Myeloid Leukemia

Introduction: Although Acute Myeloid Leukemia (AML) has been associated with several environmental factors, in some patients the evolution to AML is preceded by myelodysplastic syndrome (MDS). We report a case of 63-year-old male who was diagnosed with sporadic monosomy 7 MDS/AML who achieved good response to treatment with CPX-351 (Vyxeos) induction chemotherapy. Case information: A 63-year-old male initially presented to the ED for further evaluation of pancytopenia that was discovered incidentally during a routine office visit. Further evaluation revealed his blast count was elevated with presence of Auer rods, myelocytes and basophilia. He was admitted for further work-up of pancytopenia with suspicion being for MDS/AML and bone marrow biopsy for flow cytometric evaluation of the bone marrow was strongly suggestive of acute myeloid leukemia, with morphology suggesting a predisposing MDS. Subsequent molecular cytogenetic [FISH] report confirmed -7/7q abnormality in 80% of nuclei with loss of 1 copy of chromosome 7. The following week, patient was initiated on CPX–351 [Vyxeos; daunorubicin and cytarabine liposome for injection]. Bone marrow biopsy following induction chemotherapy showed 11 to 12% of CD34 positive myeloid blasts with abnormal myeloid maturation, representing notable improvement with greater than 50% reduction in his blast count. Conclusion: Sporadic monosomy 7 MDS/AML that arises without evidence of germline genetic predisposition and in the absence of other predisposing medical conditions or treatments is an uncommon subset of AML. Vyxeos induction chemotherapy may be an excellent option in the management of sporadic monosomy 7 MDS/AML.

This Is a Title in Chidamide, a Novel HDAC Inhibitor, Is Effective for Treating Leukemia Relapse Post Allogeneic Stem Cell Transplantation and Results in Th1 Polarization in AML Patients with AML1-ETO

Background: Although acute myeloid leukemia (AML) with t(8; 21) carries a favorable prognosis, relapse still occursin up to 40% of patients who require allogeneic stem cell transplantation (alloSCT). However, 10-20% of this patients suffer post-transplant relapse. AML1-ETO fusion protein can cause leukemogenesis via epigenetic modulation of AML1 target genes. In addition, emerging evidence support a role of this fusion protein in the downregulation of immune modulators on leukemia and subsequent immune evasion. Both process are histone deacetylase (HDAC)-dependent. Therefore we hypothesize that inhibition of HDAC may represent an effective treatment for AML with AML1-ETO. Chidamide is a novel HDAC inhibitor that has been CFDA approved for PTCL. In this study, we aim to test the safety and efficacy of Chidamide in post-transplant relapse AML with AML1-ETO.Method: This is a single arm, open label phase I study administrating Chidamide in AML patients with AML1-ETO who has molecular relapse post alloSCT. Chidamide is given 0.5mg/kg PO twice a week for children or 30mg PO twice a week for adults. All patients are treated for 3 months before analysis of molecular study. Treatment is continued for patients with persistently positive AML1-ETO, while discontinued in patients who achieved complete molecular remission (CMR). For patients not able to achieve CMR, DLI is added in addition to Chidamide. The primary objective is to determine the safety of Chidamide in AML post-transplant. The secondary objective is to evaluate overall response. Blood and bone marrow samples were collected prior to the treatment, monthly during the treatment, and 3 months after discontinuation of the treatment. Correlative studies for immune markers were performed by flow cytometry.Results: Seven patients have been enrolled in this study. No severe adverse event was observed. The most common adverse event was CTCAE grade 2 thrombocytopenia (7/7). Other adverse events that were possibly or probably related to Chidamide included grade 1 constitutional symptoms (7/7), reversible grade 2 transaminitis (3/7), grade 1 renal dysfunction (1/7), grade 1 digestive tract symptoms (4/7), grade 2 neutropenia (2/7), and grade 1 skin rash (4/7). No GVHD was observed in patients received Chidamide alone, whereas 2 patients received Chidamide and DLI developed grade 3 GVHD. Strikingly all 7 patients responded to Chidamide with decreased AML1-ETO level (ORR 100%). Among them, 6 patients achieved complete response (CR 85.7%), 1 patient had partial response (PR 14.3%) and eventually deteriorated.We also performed correlative studies and made several important findings. First, we assessed the distribution of each immune component from these 7 patients prior to Chidamide treatment, at which point all patients had molecular relapse. Samples from 4 patients who remain CMR post-transplant were used as controls. We observed a significantly decreased absolute counts of CD3+CD4-CD8+T cells in the patients with relapse, compared with that of patients remaining remission (272.0±63.7 vs. 2087.5±536.5, P=0.042), whereas NK cells, B cells, and CD3+CD4+CD8- T cells showed no statistical difference. Importantly there was a significant increase in percentages of Th2 and Th17 cells, while no difference of Th1 and FOXP3+Treg cells was observed. We next examined the level of Th1, Th2, and Th17 from the 7 relapse patients prior to treatment, compared with that from the same patient 1, 2, or 3 months post Chidamide treatment. We found that both percentages and absolute counts of Th1 cells increased gradually post treatment (26.0±12.8 vs. 190.8±43.4, P=0.027). In contrast, Th2 and Th17 cells decreased. Interestingly, at the time of 3 months after Chidamide discontinuation, the increased absolute counts of Th1 cells and decreased percentages of Th2 and Th17 remained. Furthermore, the Th1/Th2 ratio was low in patients with relapse disease. It increased gradually during Chidamide treatment, correlated with the down trend of AML1-ETO titer.Conclusion: Chidamide is safe and tolerable in AML patients with AML1-ETO who has post-transplant relapse. Although limited by sample size, our study showed promising clinical efficacy of Chidamide in AML. Importantly our study demonstrated a polarization for Th1 and the significant elevated ratio of Th1/Th2 in patients received Chidamide, which may contribute to the treatment efficacy of Chidamide for AML patients with AML1-ETO. DisclosuresWang:Meryx: Equity Ownership; University of North Carolina: Patents & Royalties.

Inhibition of mitochondrial complex III induces differentiation in acute myeloid leukemia

Although acute myeloid leukemia (AML) is a highly heterogeneous disease with diverse genetic subsets, one hallmark of AML blasts is myeloid differentiation blockade. Extensive evidence has indicated that differentiation induction therapy represents a promising treatment strategy. Here, we identified that the pharmacological inhibition of the mitochondrial electron transport chain (ETC) complex III by antimycin A inhibits proliferation and promotes cellular differentiation of AML cells. Mechanistically, we showed that the inhibition of dihydroorotate dehydrogenase (DHODH), a rate-limiting enzyme in de novo pyrimidine biosynthesis, is involved in antimycin A-induced differentiation. The activity of antimycin A could be reversed by supplement of excessive amounts of exogenous uridine as well as orotic acid, the product of DHODH. Furthermore, we also found that complex III inhibition exerts a synergistic effect in differentiation induction combined with DHODH inhibitor brequinar as well as with the pyrimidine salvage pathway inhibitor dipyridamole. Collectively, our study uncovered the link between mitochondrial complex III and AML differentiation and may provide further insight into the potential application of mitochondrial complex III inhibitor as a mono or combination treatment in differentiation therapy of AML.

A modular and controllable T cell therapy platform for acute myeloid leukemia

Targeted T cell therapy is highly effective in disease settings where tumor antigens are uniformly expressed on malignant cells and where off-tumor on-target-associated toxicity is manageable. Although acute myeloid leukemia (AML) has in principle been shown to be a T cell-sensitive disease by the graft-versus-leukemia activity of allogeneic stem cell transplantation, T cell therapy has so far failed in this setting. This is largely due to the lack of target structures both sufficiently selective and uniformly expressed on AML, causing unacceptable myeloid cell toxicity. To address this, we developed a modular and controllable MHC-unrestricted adoptive T cell therapy platform tailored to AML. This platform combines synthetic agonistic receptor (SAR) -transduced T cells with AML-targeting tandem single chain variable fragment (scFv) constructs. Construct exchange allows SAR T cells to be redirected toward alternative targets, a process enabled by the short half-life and controllability of these antibody fragments. Combining SAR-transduced T cells with the scFv constructs resulted in selective killing of CD33+ and CD123+ AML cell lines, as well as of patient-derived AML blasts. Durable responses and persistence of SAR-transduced T cells could also be demonstrated in AML xenograft models. Together these results warrant further translation of this novel platform for AML treatment.

Genetic biomarkers identify a subgroup of high-risk patients within low-risk NPM1-mutated acute myeloid leukemia

Although acute myeloid leukemia (AML) with NPM1 mut /FLT3-ITDneg is a low-risk entity, its relapse rate remains high. Out of 333 AML patients, 27 were NPM1 mut, and were analyzed in greater detail in order to find associations between clinical and molecular features and cumulative incidence of relapse. Next-generation sequencing (NGS) was performed on diagnosis and remission samples using two capture-based panels. The presence of the FLT3 D835 variant at diagnosis and a qPCR value of NPM1 mut ≥0.1% after induction chemotherapy were associated with an increased probability of relapse, especially if both conditions are present together. By contrast, patients in which the main clone found at diagnosis harbored NPM1 variant had a lower risk of relapse. Nineteen of the 85 variants found at diagnosis were detected by NGS in remission. AML Subgroup with NPM1 mut/FLT3-ITDneg is a heterogeneous entity, which can be further risk-stratified based on molecular biomarkers.

Identification of chlorprothixene as a potential drug that induces apoptosis and autophagic cell death in acute myeloid leukemia cells.

Although acute myeloid leukemia (AML) is a highly heterogeneous malignance, the common molecular mechanisms shared by different AML subtypes play critical roles in AML development. It is possible to identify new drugs that are effective for various AML subtypes based on the common molecular mechanisms. Therefore, we developed a hypothesis-driven bioinformatic drug screening framework by integrating multiple omics data. In this study, we identified that chlorprothixene, a dopamine receptor antagonist, could effectively inhibit growth of AML cells from different subtypes. RNA-seq analysis suggested that chlorprothixene perturbed a series of crucial biological processes such as cell cycle, apoptosis, and autophagy in AML cells. Further investigations indicated that chlorprothixene could induce both apoptosis and autophagy in AML cells, and apoptosis and autophagy could act as partners to induce cell death cooperatively. Remarkably, chlorprothixene was found to inhibit tumor growth and induce insitu leukemic cell apoptosis in the murine xenograft model. Furthermore, chlorprothixene treatment could reduce the level of oncofusion proteins PML-RARα and AML1-ETO, thus elevate the expression of apoptosis-related genes, and lead to AML cell death. Our results provided new insights for drug repositioning of AML therapy and confirmed that chlorprothixene might be a potential candidate for treatment of different subtypes of AML by reducing expression of oncofusion proteins. DATABASE: RNA-seq data are available in GEO database under the accession number GSE124316.

Targeting Hedgehog (Hh) Pathway for the Acute Myeloid Leukemia Treatment.

The Hedgehog (Hh) pathway, containing the Patched (PTCH) and Smoothened (SMO) multitransmembrane proteins, is the main regulator of vertebrate embryonic development. A non-canonical Hh pathway was recently observed in numerous types of solid cancers and hematological malignancies. Although acute myeloid leukemia (AML) is a common and lethal myeloid malignancy, the chemotherapy for AML has not changed in the last three decades. The Hh pathway and other intracellular signaling pathways are important for the tumor cells’ cycle or therapeutic resistance of AML cells. In this article, we will review the current trends in Hh pathway inhibitors for treating AML.

Assesment of Frailty Scores Validating Fitness of Elderly AML Patients for Intensive Chemotherapy: A Single Centre Study

Background: Although acute myeloid leukemia (AML) occurs most commonly in adults ≥60 years, the treatment of AML in older patients remains a significant challenge due to both, more aggressive disease biology as well as patient-related risk factors that limit tolerance of intensive chemotherapy. Several studies demonstrated improved survival for older patients receiving intensive induction chemotherapy. Therefore intensive chemotherapy, as long as patient fitness allows, is aimed. Defining the subset of patients that is eligible or “fit” for intensive chemotherapy involves a great deal of subjectivity. Criteria yet have to be standardized across or within institutions.Aim: The aim of the present study was to investigate the power of three validation scores in assessing patient fitness at diagnosis in parallel to physician evaluation. Further patient outcome according the respective assessment was compared.Methods: A total of 130 clinically and molecularly well characterized consecutive elderly (≥60 years) patients with newly diagnosed AML were treated from 2012 to 2018 according to age, performance status and co-morbidities in a single hematology center. Initial haematologist evaluation followed by discussion of the patient case in an interdisciplinary board led to decision of therapy intensity of each patient. In parallel, independently from the medical board decision, three scores were performed: i) a local geriatric G8 screening tool, consisting of seven items from the Mini Nutritional Assessment (MNA) questionnaire and age, ii) the Sorror Index used for hematopoietic stem cell transplantation (HSCT) evaluation as well as iii) the AML score proposed by the German Acute Myeloid Leukemia Cooperative Group, predicting the probability of complete remission (CR) and early death (ED). Therapy response was defined according to ELN criteria. Overall survival from diagnosis was compared between groups using the Cox model.Results: Median age was 71,2 (range: 60-86) years. A total of 75 (57,7%) patients were evaluated “fit” by the medical board and treated by intensive chemotherapy (“7+3” regimen), whereas 41 patients (31,5%) underwent semi-intensive therapy (based on hypomethylating agents or low-dose Cytarabine s.c.) and 14 patients (10,7%) received best supportive care. Fifty patients (38,5%) achieved a complete remission after induction chemotherapy, could follow consolidation chemotherapy and ten of them underwent allogeneic HSCT. Sixty-four (49,2%) were non responders and 16 patients (12,3%) died during the first cycle.Overall, the median survival time was 11,2 months (95% CI 7,9-17,8). Primary physician care evaluation was able to define a “fit” from an “unfit” patient in a statistically significantly way. Median survival time from the “fit” patients was 17,6 months (95%CI 9-28,9) compared to the “unfit” evaluated patients with 3,6 months (95%CI 1,8-8,8), p<0.001 with a HR (unfit vs. fit) of 3,04 (95% CI of 1,80-5,15). Even the distinction by the local G8 screening tool resulted significant distinguishing “fit” with median OS of 18,7 months from “unfit” patients with a median OS of 7,9 months, HR (unfit vs. fit) 2,1 (95% CI 1,23-3,58), p=0,007. The Sorror Index (HR 2,14 with a 95% CI of 1,27-3,59) as well as the AML score ED (HR 1,94 with 95%CI of 1,16-3,24) resulted also significant in their power of separating “fit” from “unfit” patients, p=0,004 and p=0,011, respectively. Although a certain degree of correlation is expected between scores, this was limited to levels below 0.35 according to the pairwise spearman correlation coefficient.Conclusion: In conclusion, the frailty scores G8, Sorror Index and the AML Score, that have been applied to elderly patients at AML diagnosis seem to discriminate patients quite well in terms of overall survival in this single centre patient cohort and may represent objective instruments for the haematologist in the validation of the fitness of the elderly AML patients for intensive chemotherpy. In order to validate the discrimination ability arising from the performed analysis, a multi-centre study is planned. DisclosuresVitolo:Gilead: Speakers Bureau; Roche: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Janssen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Speakers Bureau; Sandoz: Speakers Bureau.

Defining and Treating Older Adults with Acute Myeloid Leukemia Who Are Ineligible for Intensive Therapies.

Although acute myeloid leukemia (AML) is primarily a disease of older adults (age ≥60 years), the optimal treatment for older adults remains largely undefined. Intensive chemotherapy is rarely beneficial for frail older adults or those with poor-risk disease, but criteria that define fitness and/or appropriateness for intensive chemotherapy remain to be standardized. Evaluation of disease-related and patient-specific factors in the context of clinical decision making has therefore been largely subjective. A uniform approach to identify those patients most likely to benefit from intensive therapies is needed. Here, we review currently available objective measures to define older adults with AML who are ineligible for intensive chemotherapy, and discuss promising investigational approaches.

Concentrative nucleoside transporter 3 as a prognostic indicator for favorable outcome of t(8;21)-positive acute myeloid leukemia patients after cytarabine-based chemotherapy.

Although acute myeloid leukemia (AML) exhibits diverse responses to chemotherapy, patients harboring the t(8;21) translocation are part of a favorable risk group. However, the reason why this subgroup is more responsive to cytarabine-based therapy has not been elucidated. In the present study, we analyzed expression levels of cytarabine metabolism-related genes in patients diagnosed with AML with or without t(8;21) and investigated their correlation with clinical outcomes after cytarabine-based therapy. Among the 8 genes studied, expression of the concentrative nucleoside transporter 3 (CNT3) gene was significantly higher in t(8;21)-positive patients compared to the others in the test population and the validation cohort (P<0.001 in Mann-Whitney U test; P<0.002 in Pearson's correlation analysis). Additionally, in both multivariate and univariate analyses, t(8;21)-positive patients categorized in a higher CNT3 expression tertile had longer disease-free survival [hazard ratio (HR), 0.117; 95% confidence interval (CI), 0.025-0.557; P=0.008] and overall survival (HR, 0.062; 95% CI, 0.007-0.521; P=0.010) compared to t(8;21)-positive patients in a lower CNT3 expression tertile. Notably, these trends did not occur in t(8;21)-negative patients. Our results demonstrate that CNT3 expression is associated with overall favorable outcomes and is predictive of clinical outcomes in AML patients with t(8;21). This suggests that CNT3 expression can be used to optimize treatment strategies for AML patients.

Acute myeloid leukemia in the pregnant patient.

Although acute myeloid leukemia (AML) mostly occurs in older patients, it could be seen in women of childbearing age. It is therefore not surprising that in some patients, the management of AML will be complicated by a coexistent pregnancy. However, the association of leukemia and pregnancy is uncommon. Its incidence is estimated to be 1 in 75,000-100,000 pregnancies. During pregnancy, most leukemias are acute: two-thirds are myeloid and one-third are lymphoblastic. There is no standard approach for this clinical dilemma, in part because of variables such as the type of AML, the seriousness of the symptoms, and the patient's personal beliefs. In many cases, the diagnostic workup has to be altered because of the pregnancy, and often available treatments have varying risks to the fetus. While chemotherapy is reported to have some risks during the first trimester, it is admitted that it can be administered safely during the second and the third trimesters.

Acute myeloid leukemia in the pregnant patient.

Although acute myeloid leukemia (AML) mostly occurs in older patients, it could be seen in women of childbearing age. It is therefore not surprising that in some patients the management of AML will be complicated by a coexistent pregnancy. However, the association of leukemia and pregnancy is uncommon. Its incidence is estimated to be 1 in 75,000 to 100,000 pregnancies. During pregnancy, most leukemias are acute: two thirds are myeloid and one third are lymphoblastic. There is no standard approach for this clinical dilemma, in part because of variables such as the type of AML, the seriousness of the symptoms, and the patient's personal beliefs. In many cases, the diagnostic work up has to be altered because of the pregnancy, and often available treatments have varying risks to the fetus. While chemotherapy is reported to have some risks during the first trimester, it is admitted that it can be administered safely during the second and the third trimesters. This article is protected by copyright. All rights reserved.

Minimal residual disease monitoring in t(8;21) acute myeloid leukemia based on RUNX1‐RUNX1T1 fusion quantification on genomic DNA

Although acute myeloid leukemia (AML) with t(8;21) belongs to the favorable risk AML subset, relapse incidence may reach 30% in those patients. RUNX1-RUNX1T1 fusion transcript is a well-established marker for minimal residual disease (MRD) monitoring. In this study, we investigated the feasibility and performances of RUNX1-RUNX1T1 DNA as MRD marker in AML with t(8;21). In 17/22 patients with t(8;21)-positive AML treated in the French CBF-2006 trial, breakpoints in RUNX1 and RUNX1T1 were identified using long-range PCR followed by next-generation sequencing. RUNX1-RUNX1T1 DNA quantification was performed by real-time quantitative PCR using patient-specific primers and probe. MRD levels were evaluated in 71 follow-up samples from 16 patients, with a median of four samples [range 2-7] per patient. RUNX1 breakpoints were located in intron 5 in all cases. RUNX1T1 breakpoints were located in intron 1b in 15 cases and in intron 1a in two cases. RUNX1-RUNX1T1 MRD levels measured on DNA and RNA were strongly correlated (r = 0.8, P < 0.0001). Discordant MRD results were observed in 10/71 (14%) of the samples: in three samples from two patients who relapsed, RUNX1-RUNX1T1 was detectable only on DNA, while RUNX1-RUNX1T1 was detectable only on RNA in seven samples. MRD monitoring on genomic DNA is feasible, but with sensitivity variations depending on the patient breakpoint sequence and the qPCR assay efficiency. Although interpretation of the results is easier because it is closely related to the number of leukemic cells, this method greatly increases time, cost and complexity, which limits its interest in routine practice.

Class-II Associated Invariant Chain Peptide (CLIP) Expression on AML Blasts Adversely Affects Alloreactive CD4+ T Cell Recognition.

Although acute myeloid leukemia (AML) can be cured with intensive treatment including myeloablative chemotherapy and haematopoietic stem cell transplantation, relapses occur in the majority of cases. A common feature of tumor cells is their ability to escape immune surveillance through adapted intrinsic mechanisms. Thus, it is a great challenge to develop optimal strategies that direct a specific cellular immune response against residual AML blasts in vivo. As CD4+ T cells are needed to initiate a strong anti-leukemic CD8+ T cell response, the mechanism through which HLA class-II restricted (leukemia-specific) antigens are presented on AML blasts could be an essential factor in immune surveillance. Previously, we showed that the self peptide Class-II Associated Invariant Chain Peptide (CLIP) important in HLA class-II antigen presentation appeared to be disadvantageous, as its expression on AML blasts predicted a shortened disease-free survival (Chamuleau et al. Canc. Res. 2004; 64(16):5546–50). We hypothesized that CLIP interferes with the presentation of specific tumor antigens on HLA class-II molecules, thereby preventing recognition of AML blasts by CD4+ T cells. To investigate whether CLIP expression indeed has a functional effect on leukemia-specific T cell activation in patients, an AML cell line model with CLIP+ and CLIP− leukemic blasts was set up. The Kasumi-1 and THP-1 AML cell lines were selected as both stained positive for extracellular HLA-DR (89%; MFI=31.3 and 91%; MFI=37.5 respectively) and CLIP expression (88%; MFI=37.2 and 91%; MFI=34.0 respectively) by flow cytometric analysis. These DR+CLIP+ cell lines were specifically silenced for Invariant Chain (Ii) expression using RNA interference to down-modulate CLIP presentation on the cell surface. Indeed, Ii siRNA-treated cells not only showed a significant decrease of intracellular Ii expression (MFI decrease of 87.7% for Kasumi-1 and 82.7% for THP-1), but also a marked downregulation of relative CLIP amount per HLA-DR molecule (fold decline in CLIP/DR ratio of 1.4 for Kasumi-1 and 2.0 for THP-1). Wild type (DR+CLIP+) and modulated (DR+CLIP−) cells of Kasumi-1 or THP-1 origin acted as stimulators for alloreactive CD4+ T cells in mixed leukocyte reactions using different stimulator to responder (S/R) ratios. Modulated DR+CLIP− Kasumi-1 and THP-1 cells induced a strong increase in alloreactive CD4+ T cell proliferation as compared to DR+CLIP+ wild type controls, both in an HLA-DR-specific and a S/R-dependent manner. At the highest S/R ratio, mean proliferation increases of 2.58-fold for Kasumi-1 (n=3) and 1.71-fold for THP-1 (n=2) were observed. These data support our hypothesis that the expression of CLIP on AML blasts plays an important role in immune surveillance, which might have impact on cellular immunotherapy with dendritic cell-based vaccines in AML.

Elderly Acute Myeloid Leukemia: A Retrospective Analysis of 89 Cases in China.

Although acute myeloid leukemia (AML) is generally responsive to chemotherapy, most patients with this disease are older than 60 years and have relatively resistant neoplasms. AML in the older patient is distinctive, both biologically and clinically, from that occurring in patients younger than 60. The intrinsic resistance of AML in the older patient is exemplified by the higher incidence of chromosomal abnormalities. Furthermore, blasts obtained in elderly patients with AML are more likely to express proteins that mediate multidrug resistance, such as the p-glycoprotein encoded by the MDR1 gene. A combination of an anthracycline and a cytarabine is the most commonly used induction regimen in acute myeloid leukemia (AML), but most of Chinese older adults with AML didn't agree with the standard doses of induction therapy. The study is to analysis the attenuated therapy for Chinese older adults. 89cases of newly diagnosed aged over 60 were observed from February 2000 to July 2005, including 50 male and 39 female, media age is 68yr. Patients were accepted attenuated therapy, either mitoxantrone per day on days 1, 2(MA);35 mg/m2 pirarubicin(TA) 7–8mg/m2 idarubicin per day on days 1, 2 (IA). Each patient was given 100mg/m2 cytarabine intravenously per day for 7 days. Total CR rate was 34% after 2 regimens, 2 years survival rate was 12.5%. The CR and 2-year survival rates of acute leukemia in Chinese elderly AML were low. Physicians should be encouraged to offer standard doses of induction therapy.

Expression of the Neural Cell Adhesion Molecule CD56 Is Associated With Short Remission Duration and Survival in Acute Myeloid Leukemia With t(8; 21)(q22; q22)

Although acute myeloid leukemia (AML) with t(8;21) (q22;q22) is associated with a high complete remission (CR) rate and prolonged disease-free survival, treatment outcome is not universally favorable. Identifying factors that predict for treatment outcome might allow therapy to be optimized based on risk. AML with t(8;21) has a distinctive immunophenotype, characterized by expression of the myeloid and stem cell antigens CD13, CD15, CD34, and HLADr, and frequent expression of the B-cell antigen CD19 and the neural cell adhesion molecule CD56, a natural killer cell/stem cell antigen. Because CD56 expression has been associated with both extramedullary leukemia and multidrug resistance, we sought to correlate CD56 expression with treatment outcome in AML with t(8;21). Pretreatment leukemia cells from 29 adult de novo AML patients with t(8;21) treated on Cancer and Leukemia Group B (CALGB) protocols were immunophenotyped by multiparameter flow cytometry as part of a prospective immunophenotyping study of adult AML (CALGB 8361). CD56 was expressed in 16 cases (55%). There was no correlation between CD56 expression and age, sex, white blood cell count, granulocyte count, the presence of additional cytogenetic abnormalities, or the presence of extramedullary disease at diagnosis. The CR rate to standard-dose cytarabine and daunorubicin was similar for cases with and without CD56 expression (88% v 92%; P = 1.0). Post-CR therapy included at least one course of high-dose cytarabine in 24 of 26 patients who achieved CR; numbers of courses administered were similar in cases with and without CD56 expression. Although post-CR therapy did not differ, CR duration was significantly shorter in cases with CD56 expression compared with those without (median, 8.7 months v not reached; P = .01), as was survival (median, 16.5 months v not reached; P = .008). We conclude that CD56 expression in AML with t(8;21) is associated with significantly shorter CR duration and survival. Our results suggest that CD56 expression may be useful in stratifying therapy for this subtype of AML.