Postinduction molecular MRD identifies patients with NPM1 AML who benefit from allogeneic transplant in first remission.

ABSTRACT: Selection of patients with NPM1-mutated acute myeloid leukemia (AML) for allogeneic transplant in first complete remission (CR1-allo) remains controversial because of a lack of robust data. Consequently, some centers consider baseline FLT3-internal tandem duplication (ITD) an indication for transplant, and others rely on measurable residual disease (MRD) status. Using prospective data from the United Kingdom National Cancer Research Institute AML17 and AML19 studies, we examined the impact of CR1-allo according to peripheral blood NPM1 MRD status measured by quantitative reverse transcription polymerase chain reaction after 2 courses of induction chemotherapy. Of 737 patients achieving remission, MRD was positive in 19%. CR1-allo was performed in 46% of MRD+ and 17% of MRD- patients. We observed significant heterogeneity of overall survival (OS) benefit from CR1-allo according to MRD status, with substantial OS advantage for MRD+ patients (3-year OS with CR1-allo vs without: 61% vs 24%; hazard ratio [HR], 0.39; 95% confidence interval [CI], 0.24-0.64; P < .001) but no benefit for MRD- patients (3-year OS with CR1-allo vs without: 79% vs 82%; HR, 0.82; 95% CI, 0.50-1.33; P = .4). Restricting analysis to patients with coexisting FLT3-ITD, again CR1-allo only improved OS for MRD+ patients (3-year OS, 45% vs 18%; compared with 83% vs 76% if MRD-); no interaction with FLT3 allelic ratio was observed. Postinduction molecular MRD reliably identifies those patients who benefit from allogeneic transplant in first remission. The AML17 and AML19 trials were registered at www.isrctn.com as #ISRCTN55675535 and #ISRCTN78449203, respectively.

Molecular, clinical and therapeutic determinants of outcome in NPM1 mutated AML.

Although NPM1-mutated acute myeloid leukemia (AML) carries a generally favorable prognosis, many patients still relapse and die. Previous studies identified several molecular and clinical features associated with poor outcome, however only FLT3-ITD mutation and adverse karyotype are currently used for risk stratification due to inconsistent results and uncertainty around how other factors should influence treatment, particularly given the strong prognostic impact of post-induction measurable residual disease (MRD). Here we analyzed a large group of patients with NPM1mut AML enrolled in prospective trials (NCRI AML17 and AML19, n=1357) to delineate the impact of baseline molecular and clinical features, post induction MRD status and treatment intensity on outcome. FLT3-ITD (HR 1.28, 95%CI 1.01-1.63), DNMT3A (HR 1.65, 95%CI 1.32-2.05), WT1 (HR 1.74, 95%CI 1272-2.38) and non-ABD NPM1 mutations (HR 1.64, 95%CI 1.22-2.21) were independently associated with poorer overall survival (OS). These factors were also strongly associated with MRD positivity. For patients achieving MRD negativity, these mutations (except FLT3-ITD) were associated with an increased cumulative incidence of relapse (CIR) and poorer OS. However, apart from the few patients with adverse cytogenetics, we could not identify any group of MRD negative patients with a CIR >40% or with benefit from allograft in first remission. Intensified chemotherapy with the FLAG-Ida regimen was associated with improved outcomes in all subgroups, with greater benefits observed in the highest risk molecular subgroups.

Droplet digital PCR for sensitive relapse detection in acute myeloid leukaemia patients transplanted by reduced intensity conditioning.

INTRODUCTION: Follow-up after allogeneic transplantation in acute myeloid leukaemia (AML) is guided by measurable residual disease (MRD) testing. Quantitative polymerase chain reaction (qPCR) is the preferred MRD platform but unfortunately, 40%-60% of AML patients have no high-quality qPCR target. This study aimed to improve MRD testing by utilising droplet digital PCR (ddPCR). ddPCR offers patient-specific monitoring but concerns of tracking clonal haematopoiesis rather than malignant cells prompt further validation. METHODS: Retrospectively, we performed MRD testing on blood and bone marrow samples from AML patients transplanted by reduced-intensity conditioning. RESULTS: The applicability of ddPCR was 39/42 (92.9%). Forty-five ddPCR assays were validated with a 0.0089% median sensitivity. qPCR targeting NPM1 mutation detected relapse 46 days before ddPCR (p = .03). ddPCR detected relapse 34.5 days before qPCR targeting WT1 overexpression (p = .03). In non-relapsing patients, zero false positive ddPCR MRD relapses were observed even when monitoring targets associated with clonal haematopoiesis such as DNMT3A, TET2, and ASXL1 mutations. CONCLUSION: These results confirm that qPCR targeting NPM1 mutations or fusion transcripts are superior in MRD testing. In the absence of such targets, ddPCR is a promising alternative demonstrating (a) high applicability, (b) high sensitivity, and (c) zero false positive MRD relapses in non-relapsing patients.

Cytoreductive treatment and association with platelet function and maturity in patients with essential thrombocythaemia.

Patients with essential thrombocythaemia (ET) have an increased risk of thromboembolic events, which may differ according to different cytoreductive drugs. We investigated the effect of cytoreductive treatment on platelet function and turnover in ET patients. Blood samples were obtained at 1 and 24 h after aspirin intake. Platelet function was evaluated by platelet aggregation and flow cytometry. Platelet turnover was assessed by immature platelet count, immature platelet fraction (IPF) and mean platelet volume (MPV). A total of 47 ET patients were included and grouped into 21 patients not receiving cytoreductive treatment, 15 patients receiving hydroxycarbamide and 11 patients receiving pegylated interferon alpha (peg-IFN). Patients receiving peg-IFN had significantly higher IPF and MPV than the other ET groups. Patients not receiving cytoreductive treatment had significantly higher platelet aggregation 24 h after aspirin intake than the other ET groups (p-values from 0.03 to 0.0002). Patients receiving hydroxycarbamide had significantly higher expression of platelet granule makers, P-selectin and CD63, than patients receiving peg-IFN (p-values ≤0.003). Cytoreduction provides more consistent platelet inhibition compared with no cytoreductive treatment. Moreover, peg-IFN provides superior inhibition of platelet activation markers than hydroxycarbamide, which in part may explain differences in risk of thromboembolic events in ET patients.

Molecular MRD status and outcome after transplantation in NPM1-mutated AML.

Relapse remains the most common cause of treatment failure for patients with acute myeloid leukemia (AML) who undergo allogeneic stem cell transplantation (alloSCT), and carries a grave prognosis. Multiple studies have identified the presence of measurable residual disease (MRD) assessed by flow cytometry before alloSCT as a strong predictor of relapse, but it is not clear how these findings apply to patients who test positive in molecular MRD assays, which have far greater sensitivity. We analyzed pretransplant blood and bone marrow samples by reverse-transcription polymerase chain reaction in 107 patients with NPM1-mutant AML enrolled in the UK National Cancer Research Institute AML17 study. After a median follow-up of 4.9 years, patients with negative, low (<200 copies per 105ABL in the peripheral blood and <1000 copies in the bone marrow aspirate), and high levels of MRD had an estimated 2-year overall survival (2y-OS) of 83%, 63%, and 13%, respectively (P < .0001). Focusing on patients with low-level MRD before alloSCT, those with FLT3 internal tandem duplications(ITDs) had significantly poorer outcome (hazard ratio [HR], 6.14; P = .01). Combining these variables was highly prognostic, dividing patients into 2 groups with 2y-OS of 17% and 82% (HR, 13.2; P < .0001). T-depletion was associated with significantly reduced survival both in the entire cohort (2y-OS, 56% vs 96%; HR, 3.24; P = .0005) and in MRD-positive patients (2y-OS, 34% vs 100%; HR, 3.78; P = .003), but there was no significant effect of either conditioning regimen or donor source on outcome. Registered at ISRCTN (http://www.isrctn.com/ISRCTN55675535).

Venetoclax combined with low dose cytarabine compared to standard of care intensive chemotherapy for the treatment of favourable risk adult acute myeloid leukaemia (VICTOR): Study protocol for an international, open-label, multicentre, molecularly-guided randomised, phase II trial.

BACKGROUND: For patients with acute myeloid leukaemia (AML), the only potentially curative treatment is intensive chemotherapy (IC). This is highly toxic, particularly for patients > 60 years, potentially leading to prolonged hospitalisations requiring intensive supportive care, and sometimes treatment-related death. This also results in extensive healthcare costs and negatively impacts quality of life (QoL). Venetoclax with low-dose cytarabine (VEN + LDAC) is a novel, low-intensity treatment for AML patients who cannot receive IC. VEN + LDAC is given as an outpatient and toxicity appears significantly lower than with IC. Analysis of clinical trials performed to date are promising for patients with the genotype NPM1mutFLT3 ITDneg, where remission and survival rates appear comparable to those achieved with IC. METHODS: VICTOR is an international, two-arm, open-label, multi-centre, non-inferiority, randomised-controlled phase II trial to assess VEN + LDAC compared to standard of care (IC) as first-line treatment in older patients (initially aged ≥ 60 years) with newly diagnosed AML. The trial will recruit patients with a NPM1mutFLT3 ITDneg genotype; those with a favourable risk in relation to the experimental treatment. University of Birmingham is the UK co-ordinating centre, with national hubs in Aarhus University Hospital, Denmark, and Auckland District Health Board, New Zealand. The primary outcome is molecular event-free survival time where an event is defined as failure to achieve morphological complete response (CR) or CR with incomplete blood count recovery after two cycles of therapy; molecular persistence, progression or relapse requiring treatment change; morphological relapse, or; death. Secondary outcomes include cumulative resource use at 12- and 24-months, and QoL as assessed by EORTCQLQ-C30 and EQ-5D-3L at 3-, 6-, 12-, 18- and 24-months. The trial employs an innovative Bayesian design with target sample size of 156 patients aged > 60 years. DISCUSSION: The principle underpinning the VICTOR trial is that the chance of cure for patients in the experimental arm should not be compromised, therefore, an adaptive design with regular checks on accumulating data has been employed, which will allow for a staged expansion of the trial population to include younger patients if, and when, there is sufficient evidence of non-inferiority in older patients. TRIAL REGISTRATION: EudraCT: 2020-000,273-24; 21-Aug-2020. ISRCTN: 15,567,173; 08-Dec-2020.

Altered Fibrinolysis in Hematological Malignances.

Bleeding and thrombosis are well-known complications to hematological malignancies, and changes in fibrinolysis impact both these issues. In the present systematic review, we provide an overview and discussion of the current literature in regards to clinical manifestations, diagnosis, and treatment of altered fibrinolysis in patients suffering from hematological malignancies, beyond acute promyelocytic leukemia. We performed a systematic literature search employing the databases Pubmed, Embase, and Web of Science to identify original studies investigating fibrinolysis in hematological malignancies. Studies investigating fibrinolysis in acute promyelocytic leukemia or disseminated intravascular coagulation were excluded. We identified 32 studies fulfilling the inclusion criteria. A majority of the studies were published more than two decades ago, and none of the studies examined all available markers of fibrinolysis or used dynamic clot lysis assays. In acute leukemia L-asparaginase treatment induced a hypofibrinolytic state, and prior to chemotherapy there seemed to be little to no change in fibrinolysis. In studies examining fibrinolysis during chemotherapy results were ambiguous. Two studies examining multiple myeloma indicated hypofibrinolysis prior to chemotherapy, and in another plasma cell disease, amyloid light chain-amyloidosis, clear signs of hyperfibrinolysis were demonstrated. In myeloproliferative neoplasms, the studies reported signs of hypofibrinolysis, in line with the increased risk of thrombosis in this disease. Only one study regarding lymphoma was identified, which indicated no alterations in fibrinolysis. In conclusion, this systematic review demonstrated that only sparse, and mainly old, evidence exists on fibrinolysis in hematological malignancy. However, the published studies showed a tendency toward hypofibrinolysis in myeloproliferative disorders, an increased risk of hyperfibrinolysis, and bleeding in patients with AL-amyloidosis, whereas studies regarding acute leukemias were inconclusive except with regard to L-asparaginase treatment, which induced a hypofibrinolytic state.

Platelet Function and Turnover in Essential Thrombocythemia: A Systematic Review.

Essential thrombocythemia (ET) is a myeloproliferative neoplasm characterized by increased platelet counts. ET has an incidence of 0.6 to 2.5 per 100,000 per year in Europe and North America. The disease is characterized by an increased thromboembolic risk, possibly caused by increased platelet counts. Furthermore, increased platelet function and/or increased platelet turnover may play a role. We aimed to explore: (1) whether platelet function and platelet turnover are increased in ET patients compared with healthy controls, and (2) whether these parameters are associated with increased thromboembolic risk and, therefore, may support decision-making on treatment in ET patients. We performed a systematic literature search on March 20, 2020 in Embase and PubMed following the Preferred Reporting Items for Systematic and Meta-Analysis (PRISMA) guidelines. In total, 1,923 articles were identified, 38 of which were included according to prespecified inclusion and exclusion criteria. Among the 38 studies, platelet activation (CD36 and CD62P) was investigated in 18 studies and was found to be increased in 12 of these. Platelet aggregation was investigated in 21 studies and was reported to be reduced in 20 of them. Platelet turnover (immature platelet count and mean platelet volume) was investigated in five studies with inconclusive results. No parameters were reported to predict the risk of thromboembolic events. In conclusion, platelet activation was increased in ET patients, but platelet aggregation was reduced. Future studies exploring markers of thromboembolic risk in ET patients are warranted.

Clinical Use of Measurable Residual Disease in Acute Myeloid Leukemia.

OPINION STATEMENT: Treatment of acute myeloid leukemia (AML) remains a high-risk venture for the patient suffering from the disease. There is a real risk of succumbing to the treatment rather than the disease, and even so, cure is much less than certain. Since the establishment of complete remission as a prerequisite for cure in the 1960s, a number of years passed before advanced techniques for detecting minute amounts of disease matured sufficiently for clinical implementation. The two main techniques for detection of measurable residual disease (MRD) remain qPCR and multicolor flow cytometry. When performed in expert laboratories, both these modalities offer treating physicians excellent opportunity to follow the amount of residual disease upon treatment and offer unparalleled prognostication. In some AML and age group subsets, evidence now exist to support the choice of both proceeding to allogeneic transplant and not doing so. In other AML subgroups, MRD has sufficient discriminative power to identify patients likely to benefit from allogeneic transplant and patients likely not to. After treatment or transplantation, follow-up by molecular techniques can, with high certainty, predict relapse months before bone marrow function deterioration. On the other hand, options upon so-called molecular relapse are less well tested but recent evidence supports the use of azacitidine both in transplanted patients and patients consolidated with chemotherapy. In conclusion, MRD testing during treatment is a superb prognosticator and a major tool when choosing whether a patient should be transplanted or not. The exact use of MRD testing after treatment is less well defined but evidence is mounting for the instigation of treatment upon rising MRD levels (pre-emptive treatment) before morphologically detectable relapse.

Treatment of the elderly patient with haematological cancer.

During the last two decades, novel targeted therapies, in particular, ¼small molecules« for oral administration and monoclonal antibodies, have revolutionized the treatment and prognosis of haematological cancers. Generally, these treatments are well tolerated and therefore suitable for elderly patients. This review presents a short update on the current standard-of-care treatment of elderly patients with haematological cancer.

Towards individualized follow-up in adult acute myeloid leukemia in remission.

An increasing body of data has demonstrated that the traditional concept of morphologic complete remission in acute myeloid leukemia, in which less than 5% myeloblasts is regarded as a sufficient response criterion, is not biologically sound. Fortunately, the quantitative reverse-transcribed polymerase chain reaction (RT-PCR) method seems to be a promising alternative because of its high degree of preclinical standardization and extreme sensitivity on the background of an accurate day-to-day estimate of sample quality. Widespread implementation of this has, however, to some extent been hampered by the lack of knowledge of how and when to measure minimal residual disease levels and, even more importantly, how to react preemptively on a molecular relapse defined by a PCR reversal. Thus, only few prospective studies have been published to date to clinically validate this assay. Here, we discuss outstanding issues in the clinical implementation of RT-PCR for fusion transcripts, mutated and overexpressed genes in acute myeloid leukemia patients in complete remission, and propose a set of guidelines, which can be used when designing prospective trials aimed at validating the use of RT-PCR as well as for following these patients based on mathematical models for disease recurrence recently developed in our laboratory.

Presence of FLT3-ITD and high BAALC expression are independent prognostic markers in childhood acute myeloid leukemia.

Mutation status of FLT3, NPM1, CEBPA, and WT1 genes and gene expression levels of ERG, MN1, BAALC, FLT3, and WT1 have been identified as possible prognostic markers in acute myeloid leukemia (AML). We have performed a thorough prognostic evaluation of these genetic markers in patients with pediatric AML enrolled in the Nordic Society of Pediatric Hematology and Oncology (NOPHO) 1993 or NOPHO 2004 protocols. Mutation status and expression levels were analyzed in 185 and 149 patients, respectively. Presence of FLT3-internal tandem duplication (ITD) was associated with significantly inferior event-free survival (EFS), whereas presence of an NPM1 mutation in the absence of FLT3-ITD correlated with significantly improved EFS. Furthermore, high levels of ERG and BAALC transcripts were associated with inferior EFS. No significant correlation with survival was seen for mutations in CEBPA and WT1 or with gene expression levels of MN1, FLT3, and WT1. In multivariate analysis, the presence of FLT3-ITD and high BAALC expression were identified as independent prognostic markers of inferior EFS. We conclude that analysis of the mutational status of FLT3 and NPM1 at diagnosis is important for prognostic stratification of patients with pediatric AML and that determination of the BAALC gene expression level can add valuable information.

A novel RT-qPCR assay for quantification of the MLL-MLLT3 fusion transcript in acute myeloid leukaemia.

OBJECTIVES: Patients with acute myeloid leukaemia (AML) of the monocytic lineage often lack molecular markers for minimal residual disease (MRD) monitoring. The MLL-MLLT3 fusion transcript found in patients with AML harbouring t(9;11) is amenable to RT-qPCR quantification but because of the heterogeneity of translocation break points, the MLL-MLLT3 fusion gene is a challenging target. We hypothesised that MRD monitoring using MLL-MLLT3 as a RT-qPCR marker is feasible in the majority of patients with t(9;11)-positive AML. METHODS: Using a locked nucleic acid probe, we developed a sensitive RT-qPCR assay for quantification of the most common break point region of the MLL-MLLT3 fusion gene. Five paediatric patients with t(9;11)-positive AML were monitored using the MLL-MLLT3 assay. RESULTS: A total of 43 bone marrow (BM) and 52 Peripheral blood (PB) samples were collected from diagnosis until follow-up. Two patients relapsed, and both were MRD positive in BM after first induction course. A total of three relapses occurred, and they were detected by RT-qPCR 3 wks before haematological relapse was diagnosed. CONCLUSION: This MLL-MLLT3 RT-qPCR assay could be useful in MRD monitoring of a group of patients with AML who often lack reliable MRD markers.

Impaired fibrinolysis and increased clot strength are potential risk factors for thrombosis in lymphoma.

Thrombosis and bleeding are significant contributors to morbidity and mortality in patients with hematological cancer, and the impact of altered fibrinolysis on bleeding and thrombosis risk is poorly understood. In this prospective cohort study, we investigated the dynamics of fibrinolysis in patients with hematological cancer. Fibrinolysis was investigated before treatment and 3 months after treatment initiation. A dynamic clot formation and lysis assay was performed beyond the measurement of plasminogen activator inhibitor 1, tissue- and urokinase-type plasminogen activators (tPA and uPA), plasmin-antiplasmin complexes (PAP), α-2-antiplasmin activity, and plasminogen activity. Clot initiation, clot propagation, and clot strength were assessed using rotational thromboelastometry. A total of 79 patients were enrolled. Patients with lymphoma displayed impaired fibrinolysis with prolonged 50% clot lysis time compared with healthy controls (P = .048). They also displayed decreased clot strength at follow-up compared with at diagnosis (P = .001). A patient with amyloid light-chain amyloidosis having overt bleeding at diagnosis displayed hyperfibrinolysis, indicated by a reduced 50% clot lysis time, α-2-antiplasmin activity, and plasminogen activity, and elevated tPA and uPA. A patient with acute promyelocytic leukemia also displayed marked hyperfibrinolysis with very high PAP, indicating extreme plasmin generation, and clot formation was not measurable, probably because of the extremely fast fibrinolysis. Fibrinolysis returned to normal after treatment in both patients. In conclusion, patients with lymphoma showed signs of impaired fibrinolysis and increased clot strength, whereas hyperfibrinolysis was seen in patients with acute promyelocytic leukemia and light-chain amyloidosis. Thus, investigating fibrinolysis in patients with hematological cancer could have diagnostic value.

Prophylaxis with low dose tranexamic acid in acute myeloid leukemia patients undergoing intensive chemotherapy.

Patients suffering from acute myeloid leukemia (AML) carry a high risk of serious bleeding complications due to severe thrombocytopenia for long periods of time during treatment. Prior to prophylactic platelet transfusion becoming the standard of care, intracranial bleeding was a major contributor to death in AML patients. However, despite prophylactic platelet transfusions, up to 79% of patients with AML experience clinically significant bleeding during treatment. Antifibrinolytics are effective and well tolerated hemostatic agents widely used in many patient groups, and in this study, we investigated the effect of low dose tranexamic acid (TXA) in patients with AML and thrombocytopenia. We compared bleeding and thrombosis between 113 thrombocytopenic AML patients receiving TXA 500 mg three times daily (n = 36) versus no-TXA (n = 77). Clinical information was obtained systematically from electronic medical records, and laboratory data were collected from the laboratory information system. No difference was demonstrated in number of patients with at least one bleeding episode (TXA: 89% vs. no-TXA: 93%, p = 0.60), median number of bleeding days (TXA: 2.5 days vs. no-TXA 2.0 days, p = 0.30), bleeding location or transfusion needs between the two groups. However, platelet count was found to be a significant risk factor for bleeding, with a probability of bleeding of 35% with a platelet count below 5 × 109/L (logistic regression, p < 0.01). We found no difference in thromboembolic events between the two groups (TXA: 8% vs. no-TXA 10%, p = 0.99). In conclusion, treatment with low dose TXA is safe, but we found no evidence to suggest that it reduces bleeding in AML patients with thrombocytopenia.

Sensitivity of minimal residual disease in acute myeloid leukaemia in first remission--methodologies in relation to their clinical situation.

The concept of minimal residual disease in acute myeloid leukaemia has been steadily developed pre-clinically, with quantitative polymerase chain reaction (qPCR) leading the way with highly validated assays for patient-based risk stratification at post-treatment time points, which are being integrated in clinical trials both at evaluation of first complete remission (CR1) and after attaining CR1. Moreover, multicolour flow cytometry (MFC) has been increasingly employed in identifying leukaemia-associated immunophenotypes (LAIPs) with significant progress being made in standardization. In translating these widely varying methodologies to parameters useful for individualized patient decision-making, one of the obstacles has been that the assays entail varying sensitivities dependent on a number of variables. For qPCR, sensitivity depends on target type (i.e. fusion transcript, mutated gene or even overexpressed gene) and - in the case of overexpressed genes - on expression in healthy haematopoiesis. For MFC, sensitivity is likewise largely a function on whether the same phenotype is seen in normal immature cells and, in addition, antigen drift/shift with LAIPs changing at relapse is a well-known problem. In considering which sensitivity to opt for, a further variable is the situation of patient, most importantly the level of cytoreduction intended. Here we will attempt to give an overview of these pertinent questions intended for the practicing haematologist, focusing on where the field is heading at the clinical level.

Strikingly different molecular relapse kinetics in NPM1c, PML-RARA, RUNX1-RUNX1T1, and CBFB-MYH11 acute myeloid leukemias.

Early relapse detection in acute myeloid leukemia is possible using standardized real-time quantitative polymerase chain reaction (RQ-PCR) protocols. However, optimal sampling intervals have not been defined and are likely to vary according to the underlying molecular lesion. In 74 patients experiencing hematologic relapse and harboring aberrations amenable to RQ-PCR (mutated NPM1 [designated NPM1c], PML-RARA, RUNX1-RUNX1T1, and CBFB-MYH11), we observed strikingly different relapse kinetics. The median doubling time of the CBFB-MYH11 leukemic clone was significantly longer (36 days) than that of clones harboring other markers (RUNX1-RUNX1T1, 14 days; PML-RARA, 12 days; and NPM1c, 11 days; P < .001). Furthermore, we used a mathematical model to determine frequency of relapse detection and median time from detection of minimal residual disease to hematologic relapse as a function of sampling interval length. For example, to obtain a relapse detection fraction of 90% and a median time of 60 days, blood sampling every sixth month should be performed for CBFB-MYH11 leukemias. By contrast, in NPM1c(+)/FLT3-ITD(-), NPM1c(+)/FLT3-ITD(+), RUNX1-RUNX1T1, and PML-RARA leukemias, bone marrow sampling is necessary every sixth, fourth, and fourth and second month, respectively. These data carry important implications for the development of optimal RQ-PCR monitoring schedules suitable for evaluation of minimal residual disease-directed therapies in future clinical trials.

Genetic and epigenetic similarities and differences between childhood and adult AML.

BACKGROUND: The biology of acute myeloid leukemia (AML) is complex and includes both genetic and epigenetic aberrations. We addressed the combined consequences of promoter hypermethylation of p15, CDH1, ER, MDR1, and RARB2 and mutation of NPM1, CEBPA, FLT3, and WT1 in a Danish cohort of 70 pediatric and 383 adult AML patients. PROCEDURE: Mutation analysis was done by fragment analysis followed by sequencing or by sequencing alone. Methylation status was determined using methylation-sensitive melting curve analysis (MS-MCA) after initial bisulfite modification. RESULTS: Among pediatric AMLs, we found promoter hypermethylation in p15 (47%), CDH1 (64%), ER (62%), MDR1 (8%), and RARB2 (22%) and mutations in NPM1 (11%), CEBPA (3%), FLT3ITD (4%), FLT3D835 (7%), and WT1 (7%). Promoter hypermethylation was significantly more frequent in core binding factor leukemias (CBF) compared to AMLs with abnormalities involving 11q23 (P = 0.024). Compared to adult AML we found a significant difference in p15 (47% vs. 73%, P < 0.001) and RARB2 (22% vs. 42%, P = 0.003) methylation, as well as in NPM1 (11% vs. 31%, P = 0.001) and FLT3ITD (4% vs. 26%, P < 0.001) mutation. CONCLUSION: Age-related differences exist in the frequency of mutations and it appears that promoter hypermethylation occurs in a non-random pattern in childhood AML accompanying specific genetic aberrations, and might represent an important step in the leukemogenic transformation.

A national Danish proof of concept on feasibility and safety of home -based intensive chemotherapy in patients with acute myeloid leukemia.

Technological advances have made it possible to offer home-based chemotherapy to patients without health care professionals being present. Prior studies on effects of home-based treatment lack inclusion of patients with hematologic malignancies. We present data from a multicenter single-arm feasibility and safety study of home-based intensive chemotherapy in patients with newly diagnosed acute myeloid leukemia and their quality of life and psychological wellbeing. This national study included patients from six sites in Denmark who received intensive chemotherapy on programmed CADD Solis infusion pumps through a central venous catheter and were also managed as outpatients during treatment-induced pancytopenia. Data are presented from 104 patients, receiving 272 treatments with 1.096 (mean 4.57, SD 3.0) home infusion days out of 1.644 treatment days (67 %). Sixty-two of 168 (36.9 %) reinduction and consolidation treatment cycles ensuing pancytopenia phases were solely handled in the outpatient clinic. Patients reported high satisfaction with home-based treatment, which had a positive influence on their ability to be involved in their treatment and be socially and physically active. No unexpected events occurred during the intervention. Overall, patients improved in all quality of life outcomes over time. Home-based intensive chemotherapy treatment was feasible and safe in this population. ClinicalTrials.gov identifier: NCT04904211.

Disruption of the NHR4 domain structure in AML1-ETO abrogates SON binding and promotes leukemogenesis.

AML1-ETO is generated from t(8;21)(q22;q22), which is a common form of chromosomal translocation associated with development of acute myeloid leukemia (AML). Although full-length AML1-ETO alone fails to promote leukemia because of its detrimental effects on cell proliferation, an alternatively spliced isoform, AML1-ETO9a, without its C-terminal NHR3/NHR4 domains, strongly induces leukemia. However, full-length AML1-ETO is a major form of fusion product in many t(8;21) AML patients, suggesting additional molecular mechanisms of t(8;21)-related leukemogenesis. Here, we report that disruption of the zinc-chelating structure in the NHR4 domain of AML1-ETO by replacing only one critical amino acid leads to rapid onset of leukemia, demonstrating that the NHR4 domain with the intact structure generates inhibitory effects on leukemogenesis. Furthermore, we identified SON, a DNA/RNA-binding domain containing protein, as a novel NHR4-interacting protein. Knock-down of SON by siRNA resulted in significant growth arrest, and disruption of the interaction between AML1-ETO and endogenous SON rescued cells from AML1-ETO-induced growth arrest, suggesting that SON is an indispensable factor for cell growth, and AML1-ETO binding to SON may trigger signals inhibiting leukemogenesis. In t(8;21) AML patient-derived primary leukemic cells and cell lines, abnormal cytoplasmic localization of SON was detected, which may keep cells proliferating in the presence of full-length AML1-ETO. These results uncovered the crucial role of the NHR4 domain in determination of cellular fate during AML1-ETO-associated leukemogenesis.