Rapid and highly specific screening for NPM1 mutations in acute myeloid leukemia

NPM1 mutations, the most frequent molecular alterations in acute myeloid leukemia (AML), have become important for risk stratification and treatment decisions for patients with normal karyotype AML. Rapid screening for NPM1 mutations should be available shortly after diagnosis. Several methods for detecting NPM1 mutations have been described, most of which are technically challenging and require additional laboratory equipment. We developed and validated an assay that allows specific, rapid, and simple screening for NPM1 mutations. FAST PCR spanning exons 8 to 12 of the NPM1 gene was performed on 284 diagnostic AML samples. PCR products were visualized on a 2 % agarose E-gel and verified by direct sequencing. The FAST PCR screening method showed a specificity and sensitivity of 100 %, i.e., all mutated cases were detected, and none of negative cases carried mutations. The limit of detection was at 5-10 % of mutant alleles. We conclude that the FAST PCR assay is a highly specific, rapid (less than 2 h), and sensitive screening method for the detection of NPM1 mutations. Moreover, this method is inexpensive and can easily be integrated in the routine molecular diagnostic work-up of established risk factors in AML using standard laboratory equipment.

BIRC6 (APOLLON) is down-regulated in acute myeloid leukemia and its knockdown attenuates neutrophil differentiation

Background Inhibitors of apoptosis (IAPs) were intensively investigated in the context of cancer where they promote tumor growth and chemoresistence. Overexpression of the IAP BIRC6 is associated with unfavorable clinical features and negatively impacts relapse-free survival in childhood acute myeloid leukemia (AML). Currently, BIRC6 levels in adult primary AML have not been compared to the expression in normal myeloid cells. Thus, we compared for the first time BIRC6 levels in adult primary AML patient samples to normal myeloid cells and studied its regulation and function during neutrophil differentiation. Findings We found significantly lower BIRC6 levels in particular AML subtypes as compared to granulocytes from healthy donors. The lowest BIRC6 expression was found in CD34+ progenitor cells. Moreover, BIRC6 expression significantly increased during neutrophil differentiation of AML cell lines and knocking down BIRC6 in NB4 acute promyelocytic leukemia (APL) cells significantly impaired neutrophil differentiation, but not cell viability. Conclusion Together, we found an association of low BIRC6 levels with an immature myeloid phenotype and describe a function for BIRC6 in neutrophil differentiation of APL cells.

Azacytidine for acute myeloid leukemia in elderly or frail patients: A phase II trial (SAKK 30/07)

Abstract This phase II trial treated elderly or frail AML patients with single agent subcutaneous azacytidine at 100 mg/m(2), on 5 of 28 days for up to 6 cycles. Treatment was stopped for lack of response, or continued to progression in responders. Primary endpoint was response within 6 months. A response rate >34% was considered a positive trial outcome. From 9/2008-4/2010, 45 patients from 10 centres (median age 74 (55-86) years) were accrued. Patients received 4 (1-21) cycles. Best response was CR/CRi in 8 (18%; 95% CI: 8%-32%.), 0 (0%) PR, 7 (16%) hematologic improvement, 17 (38%) stable disease. Three nonresponding patients stopped treatment after 6 cycles, 31 patients had stopped early and 11 patients continued treatment for 8-21 cycles. Adverse events (grade >III) were infections (13), febrile neutropenia (14), thrombocytopenia (7), dyspnea (6), bleeding (5) and anemia (4 patients). Median overall survival was 6 months. Peripheral blood blast counts, grouped at 30% had a borderline significant association with response (p = 0.07). This modified azacytidine schedule is feasible for elderly or frail AML patients in an outpatient setting with moderate, mainly hematologic, toxicity and response in a proportion of patients, although the primary objective was not reached.

Addition of bevacizumab to chemotherapy in acute myeloid leukemia at older age: a randomized phase 2 trial of the Dutch-Belgian Cooperative Trial Group for Hemato-Oncology (HOVON) and the Swiss Group for Clinical Cancer Research (SAKK)

An urgent need for new treatment modalities is emerging in elderly patients with acute myeloid leukemia (AML). We hypothesized that targeting VEGF might furnish an effective treatment modality in this population. Elderly patients with AML were randomly assigned in this phase 2 study (n = 171) to receive standard chemotherapy (3 + 7) with or without bevacizumab at a dose of 10 mg/kg intravenously at days 1 and 15. In the second cycle, patients received cytarabine 1000 mg/m(2) twice daily on days 1-6 with or without bevacizumab. The complete remission rates in the 2 arms were not different (65%). Event-free survival at 12 months was 33% for the standard arm versus 30% for the bevacizumab arm; at 24 months, it was 22% and 16%, respectively (P = .42). The frequencies of severe adverse events (SAEs) were higher in the bevacizumab arm (n = 63) compared with the control arm (n = 28; P = .043), but the percentages of death or life-threatening SAEs were lower in the bevacizumab arm (60% vs 75% of SAEs). The results of the present study show that the addition of bevacizumab to standard chemotherapy does not improve the therapeutic outcome of older AML patients. This trial is registered as number NTR904 in The Nederlands Trial Register (www.trialregister.nl).

Deregulated expression of EVI1 defines a poor prognostic subset of MLL-rearranged acute myeloid leukemias: a study of the German-Austrian Acute Myeloid Leukemia Study Group and the Dutch-Belgian-Swiss HOVON/SAKK Cooperative Group

To evaluate the prognostic value of ecotropic viral integration 1 gene (EVI1) overexpression in acute myeloid leukemia (AML) with MLL gene rearrangements.

Comparative analysis of the value of allogeneic hematopoietic stem-cell transplantation in acute myeloid leukemia with monosomal karyotype versus other cytogenetic risk categories

To determine to what extent allogeneic hematopoietic stem-cell transplantation (alloHSCT) quantitatively reduces relapse in acute myeloid leukemia with monosomal karyotype (MK-AML) compared with alternative postremission therapy and how it compares with other cytogenetic subcategories.

ATRA resolves the differentiation block in t(15;17) acute myeloid leukemia by restoring PU.1 expression

Tightly regulated expression of the transcription factor PU.1 is crucial for normal hematopoiesis. PU.1 knockdown mice develop acute myeloid leukemia (AML), and PU.1 mutations have been observed in some populations of patients with AML. Here we found that conditional expression of promyelocytic leukemia-retinoic acid receptor alpha (PML-RARA), the protein encoded by the t(15;17) translocation found in acute promyelocytic leukemia (APL), suppressed PU.1 expression, while treatment of APL cell lines and primary cells with all-trans retinoic acid (ATRA) restored PU.1 expression and induced neutrophil differentiation. ATRA-induced activation was mediated by a region in the PU.1 promoter to which CEBPB and OCT-1 binding were induced. Finally, conditional expression of PU.1 in human APL cells was sufficient to trigger neutrophil differentiation, whereas reduction of PU.1 by small interfering RNA (siRNA) blocked ATRA-induced neutrophil differentiation. This is the first report to show that PU.1 is suppressed in acute promyelocytic leukemia, and that ATRA restores PU.1 expression in cells harboring t(15;17).

C/EBPalpha and the pathophysiology of acute myeloid leukemia

PURPOSE OF REVIEW: The transcription factor C/EBPalpha controls differentiation and proliferation in normal granulopoiesis in a stage-specific manner. Loss of C/EBPalpha function in myeloid cells in vitro and in vivo leads to a block to myeloid differentiation similar to that which is observed in malignant cells from patients with acute myeloid leukemia. The finding of C/EBPalpha alterations in subgroups of acute myeloid leukemia patients suggests a direct link between critically decreased C/EBPalpha function and the development of the disorder. RECENT FINDINGS: Conditional mouse models provide direct evidence that loss of C/EBPalpha function leads to the accumulation of myeloid blasts in the bone marrow. Targeted disruption of the wild type C/EBPalpha protein, while conserving the dominant-negative 30 kDa isoform of C/EBPalpha, induces an AML-like disease in mice. In hematopoietic stem cells C/EBPalpha serves to limit cell self-renewal. Finally, C/EBPalpha function is disrupted at different levels in specific subgroups of acute myeloid leukemia patients. SUMMARY: There is evidence that impaired C/EBPalpha function contributes directly to the development of acute myeloid leukemia. Normal myeloid development and acute myeloid leukemia are now thought to reflect opposite sides of the same hematopoietic coin. Restoring C/EBPalpha function represents a promising target for novel therapeutic strategies in acute myeloid leukemia.

CD34-related coexpression of MDR1 and BCRP indicates a clinically resistant phenotype in patients with acute myeloid leukemia (AML) of older age

Clinical resistance to chemotherapy in acute myeloid leukemia (AML) is associated with the expression of the multidrug resistance (MDR) proteins P-glycoprotein, encoded by the MDR1/ABCB1 gene, multidrug resistant-related protein (MRP/ABCC1), the lung resistance-related protein (LRP), or major vault protein (MVP), and the breast cancer resistance protein (BCRP/ABCG2). The clinical value of MDR1, MRP1, LRP/MVP, and BCRP messenger RNA (mRNA) expression was prospectively studied in 154 newly diagnosed AML patients >or=60 years who were treated in a multicenter, randomized phase 3 trial. Expression of MDR1 and BCRP showed a negative whereas MRP1 and LRP showed a positive correlation with high white blood cell count (respectively, p < 0.05, p < 0.001, p < 0.001 and p < 0.001). Higher BCRP mRNA was associated with secondary AML (p < 0.05). MDR1 and BCRP mRNA were highly significantly associated (p < 0.001), as were MRP1 and LRP mRNA (p < 0.001) expression. Univariate regression analyses revealed that CD34 expression, increasing MDR1 mRNA as well as MDR1/BCRP coexpression, were associated with a lower complete response (CR) rate and with worse event-free survival and overall survival. When adjusted for other prognostic actors, only CD34-related MDR1/BCRP coexpression remained significantly associated with a lower CR rate (p = 0.03), thereby identifying a clinically resistant subgroup of elderly AML patients.

Drug treatment is superior to allografting as first-line therapy in chronic myeloid leukemia

Early allogeneic hematopoietic stem cell transplantation (HSCT) has been proposed as primary treatment modality for patients with chronic myeloid leukemia (CML). This concept has been challenged by transplantation mortality and improved drug therapy. In a randomized study, primary HSCT and best available drug treatment (IFN based) were compared in newly diagnosed chronic phase CML patients. Assignment to treatment strategy was by genetic randomization according to availability of a matched related donor. Evaluation followed the intention-to-treat principle. Six hundred and twenty one patients with chronic phase CML were stratified for eligibility for HSCT. Three hundred and fifty four patients (62% male; median age, 40 years; range, 11-59 years) were eligible and randomized. One hundred and thirty five patients (38%) had a matched related donor, of whom 123 (91%) received a transplant within a median of 10 months (range, 2-106 months) from diagnosis. Two hundred and nineteen patients (62%) had no related donor and received best available drug treatment. With an observation time up to 11.2 years (median, 8.9 years), survival was superior for patients with drug treatment (P = .049), superiority being most pronounced in low-risk patients (P = .032). The general recommendation of HSCT as first-line treatment option in chronic phase CML can no longer be maintained. It should be replaced by a trial with modern drug treatment first.

[Retinal abnormalities in adult acute myeloid leukemia: semeiological features and prognostic correlations. Analysis of 178 cases]

INTRODUCTION: Adult patients with acute myeloid leukemia (AML) frequently present retinal abnormalities. We tried to find a relationship between fundus lesions and treatment responsiveness, prognosis, and several hematologic parameters. PATIENTS AND METHODS: We examined 178 adult patients with newly diagnosed AML. All patients were assigned to two groups regarding retinal parameters (1 or 2) and age (A or B). Group 1 included cases with retinal dysfunction classified as retinal abnormalities with impaired visual acuity; group 2 included cases with no or only minor retinal changes. Subgroup A included patients younger than 60 years (n=97), subgroup B patients older than 60 years (n=81). RESULTS: In this study, higher age and a lower Hb value were associated with retinal findings (group 1). Among the younger patients (subgroup A), 78% of those with complete remission had no retinal findings (group 2) compared to 18% of the nonresponders. In the elderly population (subgroup B), this ratio was 58% versus 19%. In the younger patients (subgroup A), the mean overall survival was 50 months if they had no retinal abnormalities (group 2) and 7 months in the case of retinal changes (group 1). In the older population (subgroup B), the ratio was 15 months versus 3 months, respectively. CONCLUSION: Retinal abnormalities in AML are generally associated with higher age, although they correlate with a shorter survival in both age groups. This association is stronger in younger patients.

Somatic CEBPA mutations are a frequent second event in families with germline CEBPA mutations and familial acute myeloid leukemia

PURPOSE: The transcription factor CCAAT/enhancer binding protein-alpha (CEBPA) is crucial for normal myeloid differentiation. Mutations in the CEBPA gene are found in subsets of patients with acute myeloid leukemia (AML). Recently, three families were reported in whom several family members had germline CEBPA mutations and subsequently developed AML. Whereas familial AML is considered a rare event, the frequency of CEBPA germline mutations in AML is not known. PATIENTS AND METHODS: In this study, we screened 187 consecutive AML patients for CEBPA mutations at diagnosis. We detected 18 patients (9.6%) with CEBPA mutations. We then analyzed remission samples and constitutive DNA from these patients. RESULTS: We found that two (11.1%) of 18 AML patients with CEBPA mutations carried a germline N-terminal frameshift CEBPA mutation. Interestingly, additional members in the families of both of these patients have been affected by AML, and the germline CEBPA mutations were also observed in these patients. Additional somatic mutations in AML patients with germline CEBPA mutations in the two families comprised in-frame C-terminal CEBPA mutations in two patients, two nonsilent CEBPA point mutations in one patient, and monosomy 7 in one patient. CONCLUSION: This study shows, for the first time to our knowledge, that germline CEBPA mutations are frequently observed among AML patients with CEBPA mutations. Including the families with germline CEBPA mutations reported previously, additional somatic CEBPA mutations represent a frequent second event in AML with germline CEBPA mutations. Our data strongly indicate that germline CEBPA mutations predispose to AML and that additional somatic CEBPA mutations contribute to the development of the disease.

Modeling of C/EBPalpha mutant acute myeloid leukemia reveals a common expression signature of committed myeloid leukemia-initiating cells

Mutations in the CEBPA gene are present in 7%-10% of human patients with acute myeloid leukemia (AML). However, no genetic models exist that demonstrate their etiological relevance. To mimic the most common mutations affecting CEBPA-that is, those leading to loss of the 42 kDa C/EBPalpha isoform (p42) while retaining the 30kDa isoform (p30)-we modified the mouse Cebpa locus to express only p30. p30 supported the formation of granulocyte-macrophage progenitors. However, p42 was required for control of myeloid progenitor proliferation, and p42-deficient mice developed AML with complete penetrance. p42-deficient leukemia could be transferred by a Mac1+c-Kit+ population that gave rise only to myeloid cells in recipient mice. Expression profiling of this population against normal Mac1+c-Kit+ progenitors revealed a signature shared with MLL-AF9-transformed AML.

Programmed death 1 signaling on chronic myeloid leukemia-specific T cells results in T-cell exhaustion and disease progression

Chronic myeloid leukemia (CML) is a malignant myeloproliferative disease with a characteristic chronic phase (cp) of several years before progression to blast crisis (bc). The immune system may contribute to disease control in CML. We analyzed leukemia-specific immune responses in cpCML and bcCML in a retroviral-induced murine CML model. In the presence of cpCML and bcCML expressing the glycoprotein of lymphocytic choriomeningitis virus as a model leukemia antigen, leukemia-specific cytotoxic T lymphocytes (CTLs) became exhausted. They maintained only limited cytotoxic activity, and did not produce interferon-gamma or tumor necrosis factor-alpha or expand after restimulation. CML-specific CTLs were characterized by high expression of programmed death 1 (PD-1), whereas CML cells expressed PD-ligand 1 (PD-L1). Blocking the PD-1/PD-L1 interaction by generating bcCML in PD-1-deficient mice or by repetitive administration of alphaPD-L1 antibody prolonged survival. In addition, we found that PD-1 is up-regulated on CD8(+) T cells from CML patients. Taken together, our results suggest that blocking the PD-1/PD-L1 interaction may restore the function of CML-specific CTLs and may represent a novel therapeutic approach for CML.