Measurable residual disease (MRD; previously termed minimal residual disease) is an independent, postdiagnosis, prognostic indicator in acute myeloid leukemia (AML) that is important for risk ...stratification and treatment planning, in conjunction with other well-established clinical, cytogenetic, and molecular data assessed at diagnosis. MRD can be evaluated using a variety of multiparameter flow cytometry and molecular protocols, but, to date, these approaches have not been qualitatively or quantitatively standardized, making their use in clinical practice challenging. The objective of this work was to identify key clinical and scientific issues in the measurement and application of MRD in AML, to achieve consensus on these issues, and to provide guidelines for the current and future use of MRD in clinical practice. The work was accomplished over 2 years, during 4 meetings by a specially designated MRD Working Party of the European LeukemiaNet. The group included 24 faculty with expertise in AML hematopathology, molecular diagnostics, clinical trials, and clinical medicine, from 19 institutions in Europe and the United States.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Summary Background Preclinical data and results from non-randomised trials suggest that the multikinase inhibitor sorafenib might be an effective drug for the treatment of acute myeloid leukaemia. We ...investigated the efficacy and tolerability of sorafenib versus placebo in addition to standard chemotherapy in patients with acute myeloid leukaemia aged 60 years or younger. Methods This randomised, double-blind, placebo-controlled, phase 2 trial was done at 25 sites in Germany. We enrolled patients aged 18–60 years with newly diagnosed, previously untreated acute myeloid leukaemia who had a WHO clinical performance score 0–2, adequate renal and liver function, no cardiac comorbidities, and no recent trauma or operation. Patients were randomly assigned (1:1) to receive two cycles of induction therapy with daunorubicin (60 mg/m2 on days 3–5) plus cytarabine (100 mg/m2 on days 1–7), followed by three cycles of high-dose cytarabine consolidation therapy (3 g/m2 twice daily on days 1, 3, and 5) plus either sorafenib (400 mg twice daily) or placebo on days 10–19 of induction cycles 1 and 2, from day 8 of each consolidation, and as maintenance for 12 months. Allogeneic stem-cell transplantation was scheduled for all intermediate-risk patients with a sibling donor and for all high-risk patients with a matched donor in first remission. Computer-generated randomisation was done in blocks. The primary endpoint was event-free survival, with an event defined as either primary treatment failure or relapse or death, assessed in all randomised patients who received at least one dose of study treatment. We report the final analysis. This trial is registered with ClinicalTrials.gov , number NCT00893373 , and the EU Clinical Trials Register (2008-004968-40). Findings Between March 27, 2009, and Nov 28, 2011, 276 patients were enrolled and randomised, of whom nine did not receive study medication. 267 patients were included in the primary analysis (placebo, n=133; sorafenib, n=134). With a median follow-up of 36 months (IQR 35·5–38·1), median event-free survival was 9 months (95% CI 4–15) in the placebo group versus 21 months (9–32) in the sorafenib group, corresponding to a 3-year event-free survival of 22% (95% CI 13–32) in the placebo group versus 40% (29–51) in the sorafenib group (hazard ratio HR 0·64, 95% CI; 0·45–0·91; p=0·013). The most common grade 3–4 adverse events in both groups were fever (71 53% in the placebo group vs 73 54% in the sorafenib group), infections (55 41% vs 46 34%), pneumonia (21 16% vs 20 14%), and pain (13 10% vs 15 11%). Grade 3 or worse adverse events that were significantly more common in the sorafenib group than the placebo group were fever (relative risk RR 1·54, 95% CI 1·04–2·28), diarrhoea (RR 7·89, 2·94–25·2), bleeding (RR 3·75, 1·5–10·0), cardiac events (RR 3·46, 1·15–11·8), hand-foot-skin reaction (only in sorafenib group), and rash (RR 4·06, 1·25–15·7). Interpretation In patients with acute myeloid leukaemia aged 60 years or younger, the addition of sorafenib to standard chemotherapy has antileukaemic efficacy but also increased toxicity. Our findings suggest that kinase inhibitors could be a useful addition to curative treatment for acute myeloid leukaemia. Overall survival after long-term follow-up and strategies to reduce toxicity are needed to determine the future role of sorafenib in treatment of this disease. Funding Bayer HealthCare.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
In acute myeloid leukemia (AML), therapy resistance frequently occurs, leading to high mortality among patients. However, the mechanisms that render leukemic cells drug resistant remain largely ...undefined. Here, we identified loss of the histone methyltransferase EZH2 and subsequent reduction of histone H3K27 trimethylation as a novel pathway of acquired resistance to tyrosine kinase inhibitors (TKIs) and cytotoxic drugs in AML. Low EZH2 protein levels correlated with poor prognosis in AML patients. Suppression of EZH2 protein expression induced chemoresistance of AML cell lines and primary cells in vitro and in vivo. Low EZH2 levels resulted in derepression of HOX genes, and knockdown of HOXB7 and HOXA9 in the resistant cells was sufficient to improve sensitivity to TKIs and cytotoxic drugs. The endogenous loss of EZH2 expression in resistant cells and primary blasts from a subset of relapsed AML patients resulted from enhanced CDK1-dependent phosphorylation of EZH2 at Thr487. This interaction was stabilized by heat shock protein 90 (HSP90) and followed by proteasomal degradation of EZH2 in drug-resistant cells. Accordingly, inhibitors of HSP90, CDK1 and the proteasome prevented EZH2 degradation, decreased HOX gene expression and restored drug sensitivity. Finally, patients with reduced EZH2 levels at progression to standard therapy responded to the combination of bortezomib and cytarabine, concomitant with the re-establishment of EZH2 expression and blast clearance. These data suggest restoration of EZH2 protein as a viable approach to overcome treatment resistance in this AML patient population.
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IJS, NUK, SBMB, UL, UM, UPUK
The pathogenesis and progression of many tumors, including hematologic malignancies is highly dependent on enhanced lipogenesis. De novo fatty-acid synthesis permits accelerated proliferation of ...tumor cells by providing membrane components but these may also alter physicochemical properties of lipid bilayers, which can impact signaling or even increase drug resistance in cancer cells. Cancer type-specific lipid profiles would permit us to monitor and interpret actual effects of lipid changes, potential fingerprints of individual tumors to be explored as diagnostic markers. We have used the shotgun MS approach to identify lipid patterns in different types of acute myeloid leukemia (AML) patients that either show no karyotype change or belong to t(8;21) or inv16 types. Differences in lipidomes of t(8;21) and inv(16) patients, as compared to AML patients without karyotype change, presented mostly as substantial modulation of ceramide/sphingolipid synthesis. Furthermore, between the t(8;21) and all other patients we observed significant changes in physicochemical membrane properties. These were related to a marked alteration in lipid saturation levels. The discovered differences in lipid profiles of various AML types improve our understanding of the pathobiochemical pathways involved and may serve in the development of diagnostic tools.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Mutations of the NPM1 gene (NPM1mut) are among the most common genetic alterations in acute myeloid leukemia and are suitable for minimal residual disease detection. We retrospectively investigated ...the prognostic impact of NPM1mut-based minimal residual disease detection from bone marrow for development of relapse by using a newly developed real-time polymerase chain reaction based on locked nucleic acid–containing primers in 174 patients, 155 of whom were treated within prospective protocols. The prognostic value of 5 cutoff values after completion of treatment or after allogeneic transplantation was studied by using cause-specific hazard models. Subsequent validation using cross-validated partial likelihood analysis revealed that an increase of more than 1% NPM1mut/ABL1 was most prognostic for relapse after chemotherapy, whereas an increase of more than 10% NPM1mut/ABL1 was most prognostic for relapse after allogeneic transplantation. Univariate and multivariate analysis of disease-free survival and overall survival revealed a significantly worse outcome in patients with >1% NPM1mut/ABL1 and >10% NPM1mut/ABL1, respectively, which remained significant after adjustment for FLT3–internal tandem duplication status. Our results in a large data set define and optimize cutoff values for early diagnosis of molecular relapse. These results may be especially important for defining triggers for early therapeutic intervention.
• NPM1 RT-PCR levels >1% are associated with poor overall and disease-free survival in AML patients treated with chemotherapy.• NPM1 MRD levels >10% are associated with poor overall and disease-free survival in AML patients after allogeneic transplantation.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The prognosis of elderly patients with acute myeloid leukemia (AML) is still dismal even with intensive chemotherapy. In this trial, we compared the antileukemic activity of standard induction and ...consolidation therapy with or without the addition of the kinase inhibitor sorafenib in elderly patients with AML.
All patients received standard cytarabine and daunorubicin induction (7+3 regimen) and up to two cycles of intermediate-dose cytarabine consolidation. Two hundred one patients were equally randomly assigned to receive either sorafenib or placebo between the chemotherapy cycles and subsequently for up to 1 year after the beginning of therapy. The primary objective was to test for an improvement in event-free survival (EFS). Overall survival (OS), complete remission (CR) rate, tolerability, and several predefined subgroup analyses were among the secondary objectives.
Age, sex, CR and early death (ED) probability, and prognostic factors were balanced between both study arms. Treatment in the sorafenib arm did not result in significant improvement in EFS or OS. This was also true for subgroup analyses, including the subgroup positive for FLT3 internal tandem duplications. Results of induction therapy were worse in the sorafenib arm, with higher treatment-related mortality and lower CR rates. More adverse effects occurred during induction therapy in the sorafenib arm, and patients in this arm received less consolidation chemotherapy as a result of higher induction toxicity.
In conclusion, combination of standard induction and consolidation therapy with sorafenib in the schedule investigated in our trial is not beneficial for elderly patients with AML.
Gene expression profiling has been used extensively to characterize cancer, identify novel subtypes, and improve patient stratification. However, it has largely failed to identify transcriptional ...programs that differ between cancer and corresponding normal cells and has not been efficient in identifying expression changes fundamental to disease etiology. Here we present a method that facilitates the comparison of any cancer sample to its nearest normal cellular counterpart, using acute myeloid leukemia (AML) as a model. We first generated a gene expression-based landscape of the normal hematopoietic hierarchy, using expression profiles from normal stem/progenitor cells, and next mapped the AML patient samples to this landscape. This allowed us to identify the closest normal counterpart of individual AML samples and determine gene expression changes between cancer and normal. We find the cancer vs normal method (CvN method) to be superior to conventional methods in stratifying AML patients with aberrant karyotype and in identifying common aberrant transcriptional programs with potential importance for AML etiology. Moreover, the CvN method uncovered a novel poor-outcome subtype of normal-karyotype AML, which allowed for the generation of a highly prognostic survival signature. Collectively, our CvN method holds great potential as a tool for the analysis of gene expression profiles of cancer patients.
•This study describes a method for the comparison of gene expression data of any type of cancer cells with their corresponding normal cells.•Our analyses reveal novel disease entities, identify common deregulated transcriptional networks, and predict survival.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
The current European LeukemiaNet (ELN) recommendations for acute myeloid leukemia (AML) propose a new risk reporting system, integrating molecular and cytogenetic factors and subdividing the large ...heterogenous group of intermediate-risk patients into intermediate-I (IR-I) and intermediate-II (IR-II). We assessed the prognostic value of the new risk classification in a large cohort of patients.
Complete data for classification were available for 1,557 of 1,862 patients treated in the AML96 trial. Patients were assigned to the proposed genetic groups from the ELN recommendations, and survival analyses were performed using the Kaplan-Meier method and log-rank test for significance testing.
The median age of all patients was 67 years. With a median follow-up of 8.3 years, significant differences between all risk categories were observed in patients age ≤ 60 years regarding the time to relapse, relapse-free survival, and overall survival (OS). Patients in the IR-II group had a better prognosis than patients in the IR-I group. The median OS times in young patients with favorable risk (FR), IR-I, IR-II, and adverse risk (AR) were 5.3, 1.1, 1.6, and 0.5 years, respectively. Separate analyses in the age group older than 60 years revealed significant differences between FR, AR, and IR as a whole, but not between IR-I and IR-II.
In younger patients with AML, the ELN classification seems to be the best available framework for prognostic estimations to date. Caution is advised concerning its use for prospective treatment allocation before it has been prospectively validated. In elderly patients, alternative prognostic factors are desirable for further risk stratification of IR.
Summary Background About 50% of patients (age ≥60 years) who have acute myeloid leukaemia and are otherwise medically healthy (ie, able to undergo intensive chemotherapy) achieve a complete remission ...(CR) after intensive chemotherapy, but with a substantially increased risk of early death (ED) compared with younger patients. We verified the association of standard clinical and laboratory variables with CR and ED and developed a web-based application for risk assessment of intensive chemotherapy in these patients. Methods Multivariate regression analysis was used to develop risk scores with or without knowledge of the cytogenetic and molecular risk profiles for a cohort of 1406 patients (aged ≥60 years) with acute myeloid leukaemia, but otherwise medically healthy, who were treated with two courses of intensive induction chemotherapy (tioguanine, standard-dose cytarabine, and daunorubicin followed by high-dose cytarabine and mitoxantrone; or with high-dose cytarabine and mitoxantrone in the first and second induction courses) in the German Acute Myeloid Leukaemia Cooperative Group 1999 study. Risk prediction was validated in an independent cohort of 801 patients (aged >60 years) with acute myeloid leukaemia who were given two courses of cytarabine and daunorubicin in the Acute Myeloid Leukaemia 1996 study. Findings Body temperature, age, de-novo leukaemia versus leukaemia secondary to cytotoxic treatment or an antecedent haematological disease, haemoglobin, platelet count, fibrinogen, and serum concentration of lactate dehydrogenase were significantly associated with CR or ED. The probability of CR with knowledge of cytogenetic and molecular risk (score 1) was from 12% to 91%, and without knowledge (score 2) from 21% to 80%. The predicted risk of ED was from 6% to 69% for score 1 and from 7% to 63% for score 2. The predictive power of the risk scores was confirmed in the independent patient cohort (CR score 1, from 10% to 91%; CR score 2, from 16% to 80%; ED score 1, from 6% to 69%; and ED score 2, from 7% to 61%). Interpretation The scores for acute myeloid leukaemia can be used to predict the probability of CR and the risk of ED in older patients with acute myeloid leukaemia, but otherwise medically healthy, for whom intensive induction chemotherapy is planned. This information can help physicians with difficult decisions for treatment of these patients. Funding Deutsche Krebshilfe and Deutsche Forschungsgemeinschaft.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK
Cell-free DNA (cfDNA) is proposed to be a valuable source of biomarkers in liquid biopsies for various diseases as it is supposed to partially originate from tumor cells. However, data about the ...diagnostic implications of cfDNA in urine for the detection of bladder cancer (BCa) is sparse.
We evaluated the usability of urinary cfDNA for diagnostic purposes compared to urine sediment DNA (sDNA) in 53 BCa patients and 36 control subjects by analyzing two abundant point-mutations (C228T/C250T) in the TERT promoter using Next-Generation Sequencing.
Mutations were detected in 77% of the urinary sDNA compared to 63% of the cfDNA samples. Moreover, the TERT mutation allele frequencies (MAF) were highly correlated in cfDNA and sDNA. In comparison, the accuracy of the TERT assay was higher in sDNA (84%) compared to cfDNA or voided urine cytology (both 77%). Interestingly, MAFs from leukocyte-rich urines were higher in cfDNA than in sDNA, indicating a diagnostic advantage of cfDNA in such urines.
Urine-based mutation detection has the ability to augment and surpass voided urine cytology as the current gold-standard for the non-invasive detection and surveillance of BCa. The analysis of cell-free DNA provides no general diagnostic advantage compared to urine sediment DNA.
•Detection of TERTp mutations in urinary cfDNA/sDNA is feasible with high sensitivities.•Variant allele frequencies were highly correlated in cfDNA and sDNA.•The accuracy of the TERT assay was higher using sDNA, compared to cfDNA and VUC.•NGS-based mutation detection has the ability to surpass VUC for detection of bladder cancer.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
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