Clinical Outcome In Acute Myeloid Leukemia Patients Research Articles

Calreticulin exposure by malignant blasts correlates with robust anticancer immunity and improved clinical outcome in AML patients

AbstractCancer cell death can be perceived as immunogenic by the host only when malignant cells emit immunostimulatory signals (so-called “damage-associated molecular patterns,” DAMPs), as they die in the context of failing adaptive responses to stress. Accumulating preclinical and clinical evidence indicates that the capacity of immunogenic cell death to (re-)activate an anticancer immune response is key to the success of various chemo- and radiotherapeutic regimens. Malignant blasts from patients with acute myeloid leukemia (AML) exposed multiple DAMPs, including calreticulin (CRT), heat-shock protein 70 (HSP70), and HSP90 on their plasma membrane irrespective of treatment. In these patients, high levels of surface-exposed CRT correlated with an increased proportion of natural killer cells and effector memory CD4+ and CD8+ T cells in the periphery. Moreover, CRT exposure on the plasma membrane of malignant blasts positively correlated with the frequency of circulating T cells specific for leukemia-associated antigens, indicating that ecto-CRT favors the initiation of anticancer immunity in patients with AML. Finally, although the levels of ecto-HSP70, ecto-HSP90, and ecto-CRT were all associated with improved relapse-free survival, only CRT exposure significantly correlated with superior overall survival. Thus, CRT exposure represents a novel powerful prognostic biomarker for patients with AML, reflecting the activation of a clinically relevant AML-specific immune response.

Analysis of Prognostic Factors in Chinese Pediatric Acute Myeloid Leukemia

Objective: To describe the epidemiological profile, cytogenetic and molecular aberrations and the survival rate of patients with acute myeloid leukemia (AML) in a province reference pediatric hospital and explore their clinical features and prognosis.Patients and Methods: This is aretrospective Clinical-epidemiological study. The cohort of this study included cases of newly-diagnosed pediatric patients with non-M3- AML between 2010 and 2015, with the age younger than 14 years. The clinical characteristics such as gender, age, subtype of FAB, blood routine, bone marrow blast at the first visit, cytogenetics and molecular markers were analyzed in correlation with their prognosis between different characteristics groups. Survival analyses were calculated by Kaplan-Meier survival curves and the log rank test. Multivariate analyses on categorized data were performed using Cox proportional hazards model.Results: Of the 165 patients studied, 42.4% were females and 57.6%, males, with a age younger than 1 year in 4.8%, from 1 to 10 years in 73.4%, and older than 10 years in 21.8% ( median age 6.8 years ). According to FAB subtype, the majority subtypes were M2, M4 and M5, for 42%, 21.3% , and 26% respectively. 40.6% of patients presented a WBC count below 10 X 109/L at diagnosis while 12.7% of patients higher than 100 X 109/L. 77.0% of patients had less than 90g/L hemoglobin, and 47.9% of patients had less than 50 X 109/L platelets. In 70.0% of patients, the percentage of blasts in bone marrow was higher than 50% at diagnosis. The most common cytogenetic abnormalities in these children, including t(8;21)(q22;q22), inv(16)(p13.1q22) and 11q23/MLL-rearranged abnormalities were detected, with the occurrence of 34.1% , 12.2% and 14.0% respectively. The recurrent gene mutations rates are as the follows: 2.0% FLT3-ITD negative and NPM1 mutated, 4.9% FLT3-ITD positive and NPM1 wild type, 2.0% FLT3-ITDpositive and NPM1mutated, 3.9% FLT3-TKDpositive, 18.6% c-KIT mutation, and 2.9% PTPN11 mutation. Among the 165 cases of non-M3-AML patients, 114 cases achieved complete remission (CR) (69.1%) after one course of chemotherapy, 36 cases were relapsed, and 51 patients accepted hematopoietic stem cell transplantation (HSCT). The 3-year relapse-free survival (RFS) and overall survival (OS) rates were (62.5±3.5)% and (70.6%4±4.6)%, respectively. We failed to correlate the OS with the clinical parameters, such as gender, age, WBC, hemoglobin, blasts in BM, cytogenetic abnormalities except MLL-rearrangements, and gene mutations except FLT3-ITD and PTPN11 mutations . The patients with high platelet count, MLL -rearrangements, FLT3-ITD or PTPN11 mutation exhibited a significantly low OS rate (P£¼£¼(Table 1). Meanwhile, the RFS rate was correlated with the platelet count, FLT3-ITDand HSCT. respectively (Table 1). Multivariate analysis demonstrated that higher platelet count at diagnosis, MLL-rearrangements, FLT3-ITD and PTPN11 mutation were risk prognostic factors in childhood AML, while HSCT was a favorable factor.Conclusion:Higher platelet count at diagnosis, MLL-rearranged abnormalities, FLT3-ITD and PTPN11 mutation were poor prognostic markers in pediatric AML. HSCT could effectively improve the clinical outcome of AML patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Body Mass Index Is a Prognostic Factor in Adults with Newly Diagnosed Acute Myeloid Leukemia: A Retrospective Multi-Institutional Study in Japan

Background: The prevalence of obesity has more than doubled between 1980 and 2014, worldwide. In Japan, 25% of adults aged ≥20 years (29 % of males and 20% of females) were overweight in 2013. Body mass index (BMI) assesses the proportion of weight versus height, and is commonly used to stratify underweight, normal, overweight, and obesity in adults. However, the prognostic impact of BMI in acute myeloid leukemia (AML) is debatable. In this retrospective study, we aimed to assess whether BMI was associated with clinical outcomes in AML patients in Japan.Patients and Methods: We identified 374 patients with newly diagnosed AML who had been administered either daunorubicin or idarubicin in combination with cytarabine as induction chemotherapy at any of the seven Japanese hospitals that collaborate to form the Yokohama City University Hematology Group from January 2000 to March 2015. Patients with acute promyelocytic leukemia were excluded from this study. BMI is defined as a person's weight in kilograms divided by the square of his height in meters (kg/m2). All patients were categorized in one of two groups according to their BMI: underweight (BMI <18.5) and normal weight (BMI, 18.5-24.9) designed as NW, and overweight (BMI, 25.0-29.9) and obese (BMI ≥30.0) designed as OW. We analyzed complete remission (CR) rate, primary induction failure (PIF) which was defined as not achieving CR in two cycles of chemotherapy, and overall survival (OS) in each group.Results: This study included 227 males and 147 females, with median age at diagnosis of 49 years (range, 15-77 years). In BMI classification, 273 were NW (47, underweight; 226, normal weight), 101 were OW (83, overweight; 18, obese). Median BMI was 22.4 (range, 15.9-39). There were not significant differences in age, sex, Performance status (PS), cytogenetic risk, and comorbidities such as diabetes, hypertension, and ischemic heart disease requiring treatment on diagnosis of AML in the two BMI groups. In this cohort, 283 patients (75.7%) achieved CR and 81 (21.7%) experienced PIF, and 10 subjects (2.8%) had an early death (ED) as death occurring within 30 days of chemotherapy initiation. Only one patient received reduced intensity of chemotherapy ( ≥20%) because of obesity. Relapse during the first CR occurred in 173 subjects (46.3%), and death occurred in 139 patients (37.2%). There was no significant difference in CR rate between the NW and OW groups (73.3% and 82%, respectively, P = 0.079). All 10 patients who experienced ED were in NW (3.7%, P = 0.0068). Causes of ED were as follows: infection in 5 subjects; 3 from cerebral bleeding; and 2 deaths from alveolar bleeding. With a median follow-up of 42 months (1-176 months), OS was 52.4% and 64% at 3 years for the NW and OW groups, respectively (P = 0.022; Figure 1). There was no significant difference in PIF and adverse event between theNW and OW groups. Multivariate analysis showed that a better OS was associated with OW (HR 0.65, 95% CI 0.43-0.97, P = 0.033) and the other prognostic factors of age, sex, PS, and cytogenetic risk.Conclusions: The results of this study show that AML patients with BMI ≥25 had better survival. There was no difference in the toxicity of chemotherapy between the different BMI groups. Out study suggests that Increased BMI should not be a criterion for reducing the dose of chemotherapy administered to patients of newly diagnosed AML. [Display omitted] DisclosuresFujita:Chugai Pharmaceutical CO.,LTD.: Honoraria.

Low expression of circulating microRNA-328 is associated with poor prognosis in patients with acute myeloid leukemia.

BackgroundDysregulation of circulating miR-328 has been identified in several tumors and is associated with prognosis of patients. However, the expression pattern of miR-328 and the impact on prognosis has not yet been studied in acute myeloid leukemia (AML). The purpose of this study is to investigate the expression status of miR-328 and its clinical significance in AML patients.MethodsRNA was extracted from plasma of 176 patients with newly diagnosed AML and 70 healthy volunteers. The miR-328 expression was examined by Realtime quantitative PCR. The association of circulating miR-328 expression with clinicopathological factors and prognosis of AML patients was statistically analyzed.ResultsThe expression of miR-328 was significantly downregulated in AML patients (median value 22.99, range: 3.63-242.0) compared with those of healthy controls (median value 89.17, range: 12.05-397.7; P < 0.001), and miR-328 expression was markedly increased in patients after treatment than before (23.40 ± 1.76 vs. 46.61 ± 3.83, P < 0.001). Moreover, low levels of miR-328 were associated with a higher white blood cell count and BM blast count (P = 0.026 and P = 0.003, respectively), and lower hemoglobin and platelet count (P = 0.004 and P = 0.022, respectively). Patients with low miR-328 expression had a relatively poor overall survival (P = 0.022) and shorter relapse-free survival (P = 0.008) than those with high miR-328 expression. In addition, low miR-328 expression was an independent prognostic factors for both OS (P = 0.017) and RFS (P = 0.023).ConclusionsCirculating miR-328 downregulation is a common event and is associated with poor clinical outcome in AML patients.

CD47 protein expression in acute myeloid leukemia: A tissue microarray-based analysis

Binding of CD47 to signal regulatory protein alpha (SIRPα), an inhibitory receptor, negatively regulates phagocytosis. In acute myeloid leukemia (AML), CD47 is overexpressed on peripheral blasts and leukemia stem cells and inversely correlates with survival.Aim of the study was to investigate the correlation between CD47 protein expression by immunohistochemistry (IHC) in a bone marrow (BM) tissue microarray (TMA) and clinical outcome in AML patients. CD47 staining on BM leukemia blasts was scored semi-quantitatively and correlated with clinical parameters and known prognostic factors in AML. Low (scores 0–2) and high (score 3) CD47 protein expression were observed in 75% and 25% of AML patients. CD47 expression significantly correlated with percentage BM blast infiltration and peripheral blood blasts. Moreover, high CD47 expression was associated with nucleophosmin (NPM1) gene mutations. In contrast, CD47 expression did not significantly correlate with overall or progression free survival or response to therapy.In summary, a BM TMA permits rapid and reproducible semi-quantitative analysis of CD47 protein expression by IHC. While CD47 expression on circulating AML blasts has been shown to be a negative prognostic marker for a very defined population of AML patients with NK AML, CD47 expression on AML BM blasts is not.

Clinical and prognostic implications of Roundabout 4 (robo4) in adult patients with acute myeloid leukemia.

BackgroundRobo4 is involved in hematopoietic stem/progenitor cell homeostasis and essential for tumor angiogenesis. Expression of Robo4 was recently found in solid tumors and leukemia stem cells. However, the clinical implications of Robo4 expression in patients with acute myeloid leukemia (AML) remain unclear.MethodsWe investigated the clinical and prognostic relevance of mRNA expression of Robo4 in bone marrow (BM) mononuclear cells from 218 adult patients with de novo AML. We also performed immunohistochemical staining to assess the Robo4 protein expression in the BM biopsy specimens from 30 selected AML patients in the cohort.ResultsHigher Robo4 expression was closely associated with lower white blood cell counts, expression of HLA-DR, CD13, CD34 and CD56 on leukemia cells, t(8;21) and ASXL1 mutation, but negatively correlated with t(15;17) and CEBPA mutation. Compared to patients with lower Robo4 expression, those with higher expression had significantly shorter disease-free survival (DFS) and overall survival (OS). This result was confirmed in an independent validation cohort. Furthermore, multivariate analyses showed that higher Robo4 expression was an independent poor prognostic factor for DFS and OS in total cohort and patients with intermediate-risk cytogenetics, irrespective of age, WBC count, karyotype, and mutation status of NPM1/FLT3-ITD, and CEBPA.ConclusionsBM Robo4 expression can serve as a new biomarker to predict clinical outcomes in AML patients and Robo4 may serve as a potential therapeutic target in patients with higher Robo4 expression.

In-Depth Quantitative Multiplex Proteomics Reveal Subtype-Specific Differences Among Functionally Validated AML Stem Cell Populations

Acute Myeloid Leukemia (AML) is a heterogeneous group of myeloid malignancies. The classification of AML subtypes is based on recurrent genetic abnormalities and typical histopathological features, which impact on the patient’s prognosis. AML is also the model disease for the cancer stem cell model with leukemia stem cells (LSCs) residing at the top of a hierarchical organization. LSCs have self-renewal activity and generate leukemic progeny, which make up the majority of leukemic cells. Because LSCs can be quiescent and reside in specific niches in the bone marrow, rendering them resistant to conventional chemotherapy approaches, they are considered the source of relapse. Hence, further strategies to eradicate LSCs are pivotal to improve patient outcomes of this dismal disease.LSCs present within cell populations can be detected by their capacity to re-initiate the leukemia after xenotransplantation into immuno-compromised mice. However, using current methods, it is neither possible to prospectively isolate pure functional LSCs nor to distinguish them reliably from normal hematopoietic stem cells (HSCs). To define functional LSCs we FACS-sorted primary patient samples of different AML subtypes according to surface-expression of CD34 and CD38 and transplanted each of the resulting four cell populations into conditioned NSG recipients. Several AML samples showed human leukemic engraftment in at least one of the subsets, dissecting LSC-containing and LSC-free subpopulations within the same patient. AML engraftment was mainly observed within the CD34+CD38- fraction, but several cases showed LSC activity also in the CD34+CD38+ fraction or even in the CD34- subsets. As age-matched healthy controls, we collected bone marrow hematopoietic stem and progenitor cells (HSPCs, Lineage -CD34+CD38-) from individuals older than 60 years, undergoing hip replacement surgery.In-depth quantitative multiplex proteomic analysis was performed by employing tandem mass tag (TMT) labeling and high-resolution mass spectrometry. Using this approach, more than 7,000 proteins were quantified from 10 LSC-containing and 8 LSC-free fractions from six individual AML samples of different subtypes. Importantly, our data include many low abundance proteins or others that tend to be difficult to detect by mass spectrometry, such as transcription factors and membrane proteins. We identified up to 1500 differentially expressed proteins between LSC-containing and LSC-free fractions. Most interestingly, differentially expressed proteins also clustered according to AML subtype, indicating subtype-specific proteome differences at the level of AML LSCs. Along the same lines, Gene Ontology and Gene Set Enrichment Analyses showed distinct (in some instances even opposing) differences between AML subtypes. For example, interferon- and integrin-signaling were enriched in LSC-fractions of FLT3-ITD, NPM1-mutated AMLs while reduced in LSC-fractions of FLT3-wt, NPM1-wt AML samples. In summary, our data indicate that also at the level of functionally validated LSC populations, subtype-specific differences in protein expression are remarkably evident. This heterogeneity should be taken into account with respect to the development of targeting strategies for LSCs aiming to improve the clinical outcome of AML patients. DisclosuresNo relevant conflicts of interest to declare.

Network-based sub-network signatures unveil the potential for acute myeloid leukemia therapy.

Although gene expression profiling studies of acute myeloid leukemia (AML) patients have provided key insights into potential diagnostic and prognostic markers and therapeutic targets, it is not clear that the patterns of molecular heterogeneity affect the tumor biology and respond to the treatment. We hypothesized that network-based gene expression signatures of AML represent the mechanistically important genes and may improve the predicted performance of prognosis and clinical outcome. We provided the random walk with restart (RWR) analysis to discover the sub-network of genomic alterations. The RWR approach integrates the signature genes derived from the random forest (RF) analysis as "seeds" to identify genes critical to the AML recurrence phenotype. To test whether the 81-gene biomarkers could predict AML recurrence, we developed Survival Support Vector Machine (SSVM) models using a gene expression dataset and test on an independent dataset. The random forest classifier was built based on 81-gene biomarkers to separate the AML patients into "recurrence" and "non-recurrence" groups. The 81-gene biomarkers showed significant enrichment related to cancer pathophysiology and provided good coverage of sub-network biomarkers and AML-related signaling pathways. The SSVM-based score was significantly associated with overall survival (hazard ratio [HR], 2.16; 95% confidence interval [CI], 1.18-3.97; p = 0.01). Similar results were obtained with reversed training and testing datasets (hazard ratio [HR], 1.6; 95% confidence interval [CI], 1.08-2.37; p = 0.02). The 81-gene biomarker based RF classifier improved classification performance. Overall, 81-gene biomarkers might be useful prognostic and predictive molecular markers to predict the clinical outcome of AML patients.

Obatoclax potentiates the cytotoxic effect of cytarabine on acute myeloid leukemia cells by enhancing DNA damage

Resistance to cytarabine and anthracycline-based chemotherapy is a major cause of treatment failure for acute myeloid leukemia (AML) patients. Overexpression of Bcl-2, Bcl-xL, and/or Mcl-1 has been associated with chemoresistance in AML cell lines and with poor clinical outcome of AML patients. Thus, inhibitors of anti-apoptotic Bcl-2 family proteins could be novel therapeutic agents. In this study, we investigated how clinically achievable concentrations of obatoclax, a pan-Bcl-2 inhibitor, potentiate the antileukemic activity of cytarabine in AML cells. MTT assays in AML cell lines and diagnostic blasts, as well as flow cytometry analyses in AML cell lines revealed synergistic antileukemic activity between cytarabine and obatoclax. Bax activation was detected in the combined, but not the individual, drug treatments. This was accompanied by significantly increased loss of mitochondrial membrane potential. Most importantly, in AML cells treated with the combination, enhanced early induction of DNA double-strand breaks (DSBs) preceded a decrease of Mcl-1 levels, nuclear translocation of Bcl-2, Bcl-xL, and Mcl-1, and apoptosis. These results indicate that obatoclax enhances cytarabine-induced apoptosis by enhancing DNA DSBs. This novel mechanism provides compelling evidence for the clinical use of BH3 mimetics in combination with DNA-damaging agents in AML and possibly a broader range of malignancies.

Expression of musashi-2 gene in leukemia stem cells from acute myeloid leukemia patients

This study was aimed to detect the expression of Musashi-2 (Msi2) in acute myeloid leukemia (AML) and investigate the relationship between Msi2 and other clinical parameters, especially CD34. A total RNA was extracted from bone marrow of newly diagnosed AML patietns. The Msi2 mRNA expression in newly diagnosed AML patients was detected with real-time fluorescence quantitative RT-PCR. The expression level of CD34 in above-menthioned patients was detected by flow cytometry (FCM). The relationship between the expression of Msi2 mRNA and clinical outcome in AML patients was analysed. The results showed that (1)the expression of Msi2 mRNA in newly diagnosed AML patients was much higher than that in healthy volunteers (P < 0.05) , especially in M1, M4 and M5 patients; (2)the expression level of Msi2 did not correlate with age, sex, white blood cell count of peripheral blood, AML1/ETO and PML/RARa fusion gene (P > 0.05); (3) Msi2 expression level in patients with CD34(+) cells was significantly higher than that in patients with CD34(-) cells (P < 0.05). It is concluded that the Msi2 mRNA expresses in leukamia stem cells, the high expression of Msi2 mRNA has been found in newly diagnosed AML patients, especially in M1, M4 and M5 patients, the high expression also has been observed in patients with CD34(+).

Highlights of This Issue

Clinical differentiation between follicular thyroid carcinoma and adenoma presents a diagnostic challenge that often leads to unnecessary thyroidectomies. Levi and colleagues explored the utility of molecular photoacoustic imaging as a noninvasive method for detection of follicular thyroid cancer. Dual wavelength imaging of the matrix-metalloproteinase–activated photoacoustic probe revealed efficient activation and high accumulation of the probe in follicular tumors in mice. The combination of the intrinsic qualities of photoacoustic imaging and specificity that a molecular imaging agent imparts promises a safe, noninvasive, and inexpensive technique that can allow early diagnosis of follicular thyroid carcinomas.Pronounced interindividual pharmacokinetic variability is common with tyrosine kinase inhibitors (TKI) and may have implications for their clinical efficacy and toxicity. TKIs are extensively metabolized in the liver, but mechanisms of hepatocyte uptake remain unclear. After initial evaluation of TKI transport by OATP1B uptake carriers in vitro, Zimmerman and colleagues investigated the influence of these transporters on sorafenib elimination in transfected cell models as well as in transporter knockout and humanized mice. This study sheds light on factors affecting the elimination of sorafenib and its glucuronide conjugate and provides a framework for future investigations aimed at elucidating mechanisms of TKI pharmacologic variability.Radiation therapy is an accepted treatment for prostate cancer; however, no commonly used factors predict outcome specifically following this treatment. Schoenfeld and colleagues identified an association between genetic variation in the inflammation-related RNASEL gene and outcome following prostate radiotherapy in a large cohort of men followed for cancer-specific outcomes. This association was not found in patients derived from the same cohort treated with radical prostatectomy. These data suggest that inherited variation in RNASEL may predict outcome following radiation therapy in a treatment-specific manner that could eventually be used to help guide treatment.Gemtuzumab-ozogamicin (GO), an anti-CD33 monoclonal antibody conjugated to calicheamicin, is a promising agent for treatment of acute myeloid leukemia (AML). Although GO has shown efficacy in a subset of patients, clinical response to it is heterogeneous, emphasizing the need for a better understanding of factors to identify patients who will or will not benefit from this treatment. Mortland and colleagues evaluated genetic variation in CD33 and identified coding polymorphisms predictive of clinical outcome in AML patients receiving a combination of the ADE regimen (cytarabine/daunorubicin/etoposide) and GO. Integration of this information as an independent prognostic marker into current cytogenetic/molecular-based risk classification models would present an opportunity to increase our accuracy in forecasting therapeutic outcome in AML.

JS5-4 - Targeting Chemotherapy-Resistant AML Stem Cells

ABSTRACT Hematopoietic stem cells supplying all mature hematopoietic and immune cells throughout life provide protection from various diseases. However, in some hematopoietic malignancies, hematopoietic stem/progenitor cells are hit by genetic and/or epigenetic oncogenic events and transform into leukemic stem cells. As is proposed in solid cancer, leukemia stem cells (LSCs) may be important in initiation, resistance to therapy, and post-remission relapse in patients with acute myeloid leukemia (AML). We aimed to clarify the role of LSCs in AML relapse and to develop therapeutic strategies targeting human LSCs. To do so, we established a mouse model recapitulating human AML by transplantation of purified human leukemic cells into NOD/SCID/IL2rgKO newborns. Using this xenogeneic transplantation system, we demonstrated that CD34 + CD38- AML cells satisfy the criteria for LSCs: in vivo leukemia initiation, propagation of non-stem leukemic cells, and self-renewal capacity. Chemotherapy treatment of AML-engrafted recipients demonstrated that LSCs within the endosteal region are resistant to chemotherapy. Cell cycle analysis by flow cytometry and immuno-fluorescence studies further revealed that AML LSCs at the niche are cell cycle quiescent and non-stem AML cells are actively cycling. These findings suggest that cell cycle quiescence of LSCs within the endosteal niche may, at least partially, account for their chemo-resistance. To improve the clinical outcome in AML patients with unfavorable prognosis factors, elimination of chemotherapy-resistant AML stem cells through targeting LSC-specific molecules could be a novel therapeutic strategy.

Identification and Characterization of Human Leukemia Stem Cell Functional Regulators

Abstract 2955Emerging evidence shows that many cancers are organized as cellular hierarchies sustained by a subpopulation of cancer stem cells (CSC). It is thought that CSC properties influence therapy response, disease relapse, and overall survival. However, little is currently known about the molecular pathways that control stem cell behaviour. Our goal is to identify and characterize the molecular regulatory networks that govern the self-renewal and developmental program of human leukemia stem cells (LSC).To expand our understanding of LSC function, we selected genes from our previously established LSC transcriptional program as novel candidate regulators of stem cell function. The LSC transcriptional program was identified by fractionation of 16 primary human acute myeloid leukemia (AML) samples into four populations that were subjected to sensitive in vivo LSC assays. mRNA expression analysis was performed on each fraction and a global LSC-specific signature was determined from functionally defined LSC fractions. Using similar methodology, a hematopoietic stem cell (HSC) enriched gene signature was established from human cord blood. Bioinformatic analysis identified a core transcriptional program that LSCs and HSCs share, revealing the molecular machinery that underlies stemness properties. The LSC and HSC signatures have prognostic significance independent of other factors when validated on a large cohort of cytogenetically normal AML patients. We have begun experiments to determine the role of 17 candidate stem cell regulators using functional in vivo and in vitro assays.We first examined the effect of candidate gene overexpression in normal HSC using xenograft assays. Lineage negative cord blood cells were transduced with lentiviral overexpression vectors, injected into immune-deficient mice and engraftment of human cells was measured after 12 weeks. In the preliminary round of in vivo analysis, over-expression of GPR56 increased the engraftment capability of HSCs in immune-deficient recipients compared to control (p=0.0019). GPR56 is involved in cell adhesion and differentiation and evidence is accumulating for a role in various cancers such as melanoma, esophageal cancer, and glioma. By qPCR analysis we observed that GPR56 mRNA is more highly expressed in both LSC-enriched and primitive normal hematopoietic cell populations, including highly purified HSC, compared to more mature cell populations such as AML blasts and normal lineage committed progenitor cells.In conclusion, our LSC and HSC data established that determinants of stemness influence clinical outcome of AML patients. We have identified multiple novel candidate stem cell-related genes and provided evidence for a role for GPR56 in hematopoietic stem cell regulation. Disclosures:No relevant conflicts of interest to declare.

Mutation of FLT3 gene in acute myeloid leukemia with normal cytogenetics and its association with clinical and immunophenotypic features

Acute myeloid leukemia (AML) with normal karyotype represents a clinically and molecularly heterogeneous disease. Molecular markers with prognostic significance have been examined to improve risk profile characterization of this group. Activating mutations on FLT3 receptor are one of the most common genetic alterations reported. However, the prevalence and prognostic significance of FLT3 genetic alterations in AML patients with cytogenetically normal karyotype is still controversial. In this study, FLT3/ITD and FLT3/D835 mutations were analyzed in 133 patients with de novo AML with normal cytogenetics by genomic PCR assay. Of 133 patients with AML with normal cytogenetics, FLT3 internal tandem duplication (ITD) and FLT3/D835 mutations were detected in 27 (20%) and 4 (3%) samples, respectively. Although statistically insignificant, the frequency of FLT3/ITD was higher in >15 year age group when compared to <15 year group (23 vs. 13%, P = 0.2). The white blood count was found to be significantly higher in patients with FLT3/ITD mutation when compared to those without the mutation (40 × 10⁹/L vs. 20 × 10⁹/L, P = < 0.002) or those with FLT3/D835 mutations (30 × 10⁹/l). Aberrant expression of CD7 was observed more frequently in patients with FLT3/ITD mutation (P < 0.002). There was no significant difference in the response rate to chemotherapy in patients with or without FLT3/ITD mutation (67 and 64%, respectively). FLT3/ITD mutation was found to be associated with the age, leukocytosis and aberrant expression of CD7, although no influence of FLT3/ITD mutation was seen on the clinical outcome of AML patients with normal cytogenetics.

Gene-specific and global methylation patterns predict outcome in patients with acute myeloid leukemia

This study was designed to analyze the effect of global and gene-specific DNA methylation patterns on the outcome of patients with acute myeloid leukemia (AML). Methylation of CDKN2B (p15), E-cadherin (CDH) and hypermethylated in cancer 1 (HIC1) promoters and global DNA methylation by luminometric methylation assay (LUMA) was analyzed in 107 AML patients and cytogenetic and molecular mutational analysis was performed. In addition, genome-wide promoter-associated methylation was assessed using the Illumina HumanMethylation27 array in a proportion of the patients. Promoter methylation was discovered in 66, 66 and 51% of the patients for p15, CDH and HIC1, respectively. In multivariate analysis, low global DNA methylation was associated with higher complete remission rate (hazard ratio (HR) 5.9, P=0.005) and p15 methylation was associated with better overall (HR 0.4, P=0.001) and disease-free survival (HR 0.4, P=0.016). CDH and HIC1 methylation were not associated with clinical outcome. Mutational status and karyotype were not significantly associated with gene-specific methylation or global methylation. Increased genome-wide promoter-associated methylation was associated with better overall and disease-free survival as well as with LUMA hypomethylation. We conclude that global and gene-specific methylation patterns are independently associated with the clinical outcome in AML patients.

Bone marrow angiogenesis magnetic resonance imaging in patients with acute myeloid leukemia: peak enhancement ratio is an independent predictor for overall survival

AbstractEmerging evidence suggests that progression of hematologic malignancies is associated with angiogenesis. Dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) can provide global and functional imaging of tumor angiogenesis. In this study, we performed bone marrow DCE-MRI prospectively at diagnosis and after induction chemotherapy in 78 de novo acute myeloid leukemia (AML) patients and correlated it with treatment outcome. An algorithm to assess bone marrow angiogenesis by measuring the DCE-MRI time-intensity curve pixel by pixel was developed using 3 distinct parameters: peak enhancement ratio (Peak) to indicate tissue blood perfusion; amplitude (Amp) to reflect vascularity; and volume transfer constant (K trans) to indicate vascular permeability. The Peak and Amp decreased significantly at remission status after induction chemotherapy. Patients with higher Peak or Amp at diagnosis had shorter overall survival and disease-free survival than others. Cox multivariate analysis identified higher Peak value (hazard ratio, 9.181; 95% confidence interval, 1.740-48.437; P = .009) as an independent predictor for overall survival in addition to unfavorable karyotype and old age. Our findings provide evidence that increased bone marrow angiogenesis measured by DCE-MRI can predict adverse clinical outcome in AML patients. DCE-MRI may help to select high-risk phenotype AML patients for tailored antiangiogenic therapy and to monitor treatment response.

MicroRNA-29a Is An Oncogene in Acute Myeloid Leukemia

microRNAs (miRNAs) are short, non-protein encoding RNAs that bind to the 3′UTR's of target mRNAs and negatively regulate gene expression by facilitating mRNA degradation or translational inhibition. Aberrant miRNA expression is well-documented in both solid and hematopoietic malignancies, and a number of recent miRNA profiling studies have identified miRNAs associated with specific human acute myeloid leukemia (AML) cytogenetic groups as well as miRNAs that may prognosticate clinical outcomes in AML patients. Unfortunately, these studies do not directly address the functional role of miRNAs in AML. In fact, there is no direct functional evidence that miRNAs are required for AML development or maintenance. Herein, we report on our recent efforts to elucidate the role of miRNAs in AML stem cells. miRNA expression profiling of AML stem cells and their normal counterparts, hematopoietic stem cells (HSC) and committed progenitors, reveals that miR-29a is highly expressed in human hematopoietic stem cells (HSC) and human AML relative to normal committed progenitors. Ectopic expression of miR-29a in mouse HSC/progenitors is sufficient to induce a myeloproliferative disorder (MPD) that progresses to AML. During the MPD phase of the disease, miR-29a alters the composition of committed myeloid progenitors, significantly expedites cell cycle progression, and promotes proliferation of hematopoietic progenitors at the level of the multipotent progenitor (MPP). These changes are manifested pathologically by marked granulocytic and megakaryocytic hyperplasia with hepatosplenomegaly. Mice with miR-29a-induced MPD uniformly progress to an AML that contains a leukemia stem cell (LSC) population that can serially transplant disease with as few as 20 purified LSC. Gene expression analysis reveals multiple tumor suppressors and cell cycle regulators downregulated in miR-29a expressing cells compared to wild type. We have demonstrated that one of these genes, Hbp1, is a bona fide miR-29a target, but knockdown of Hbp1 in vivo does not recapitulate the miR-29a phenotype. These data indicate that additional genes are required for miR-29a's leukemogenic activity. In summary, our data demonstrate that miR-29a regulates early events in normal hematopoiesis and promotes myeloid differentiation and expansion. Moreover, they establish that misexpression of a single miRNA is sufficient to drive leukemogenesis, suggesting that therapeutic targeting of miRNAs may be an effective means of treating myeloid leukemias.

Expression of Tumor-Associated Antigens (TAAs) in Acute Myeloid Leukemia (AML) Correlated with Specific T Cell Responses and Survival.

Several Tumor-associated antigens (TAAs) are expressed in acute myeloid leukemia (AML) and elicit specific immune responses of CD8 positive T cells. These specific T cell responses against leukemic blasts expressing TAAs might play a critical role in the control of minimal residual disease (MRD) in AML. Therefore, we investigated whether TAAs inducing specific immune responses in AML patients were associated with the clinical outcome. A DNA-microarray analysis of 116 AML samples was performed to correlate expression of TAAs to the clinical outcome. In these AML patients specific T cell responses to TAAs were assessed by ELISPOT analysis, tetramer staining and chromium release assays. Quantitative RT-PCR based validation of our results demonstrated the power of DNA microarray technology. We found a significant correlation of high mRNA expression of the TAA G250/CA9 with a longer overall survival (P=0.022), a trend for better outcome in patients with high expression levels of PRAME (P=0.103), and a hint for RHAMM/HMMR. In contrast, for other TAAs like WT1, TERT, PRTN3, BCL2, and LAMR1 we found no correlation with clinical outcome of AML patients. Moreover, co-expression of RHAMM/HMMR, PRAME and G250/CA9 provided a favorable prognostic effect (P=0.005). We found specific T cell responses at high frequency for these three antigens in AML patients. Positive immune reactions were detected in 8/17 (47%) AML patients for RHAMM/HMMR-R3-derived, in 7/10 (70%) for PRAME-P3-derived, and in 6/10 (60%) for newly characterized G250/CA9-G2-derived peptides. We detected a significant increased immune response of AML patients in complete remission compared to AML patients with refractory disease (P<0.001). Furthermore, we could demonstrate specific lysis of T2 cells and AML blasts presenting these epitope peptides RHAMM/HMMR-R3, PRAME-P3 and G250/CA9-G2. In conclusion, the expression of the TAAs RHAMM/HMMR, PRAME and G250/CA9 can induce strong anti-leukemic immune responses of CD8 positive T cells possibly enabling the control of MRD in AML patients. Thus, the antigens RHAMM/HMMR, PRAME and G250/CA9 represent interesting target structures for polyvalent immunotherapeutic approaches in AML.

Predictable Prognostic Factor of CD56 Expression in Acute Myeloid Leukemia with t(8:21) Including Allogeneic Hematopoietic Stem Cell Transplantation.

CD56 expression in acute myeloid leukemia (AML) has been associated with extramedullary leukemia and multi-drug resistance, but the clinical and prognostic significances are not yet clearly defined. Recently, some investigators reported that AML patients with t(8;21) showed more frequent CD56 expression rate and the expression of CD56 antigen adversely affected disease-free survival (DFS). It could explain a diverse clinical outcome in AML patients with favorable cytogenetics. This study investigated CD56 expression in 37 adult de novo AML patients with t(8:21) between November 1996 and June 2005 at three institutions. Immunophenotyping was performed with flow cytometry (Coulter EPICS XL) and considered positive if at least 20% of blasts expressed. CD56 was expressed in 25 cases (67.6%). There was no statistically significant differences in age, sex, leukocyte count, the percentage of bone marrow blasts or the presence of additional cytogenetic abnormalities between the CD56+ and the CD56- group. The complete remission (CR) rate to standard dose cytarabine or N4-behenoyl-1-D-arabinofuranosylcytosine (BH-AC) and idarubicin was similar in both groups (91.7% v 88.7%; P=0.73), but the relapse rate to high-dose cytarabine or allogeneic hematopoietic stem cell transplantation (HST) was quite different (60% v 25%; P=0.08). Allogeneic HST was performed from siblings in 15 patients (40.5%) who achieved CR, 8 patients (32.0%) in CD56+ and 7 patients (58.3%) in CD56- group (P=0.16). The median durations of DFS were significantly shorter in CD56+ (median, 12.2 months) than in the CD56- group (median, not reached) (P =0.02). Also, the median durations of survival showed the same results in the CD56+ group (median, 14.9 months) compared with the CD56- group (median, not reached) (P=0.01). Within fifteen transplanted patients, the median durations of DFS in eight CD56+ patients was significantly shorter than seven CD56- patients (median, 24.4 months v not reached; P=0.02)(Fig.1 and 2).We concluded that CD56 expression was associated with reduced DFS and survival for AML patients with t(8:21) including transplanted patients. Although further larger studies are needed, we suggested that CD56 expression at diagnosis is a predictable prognostic factor in AML with t(8:21). [Display omitted] [Display omitted]

FLT3 ITD and FLT3 Tyrosine Kinase Domain Mutants Induce Different Phenotypes in a Murine Bone Marrow Transplant Model.

Activating mutations of FLT3 are frequent in patients with acute myeloid leukemia (AML). Two distinct types of FLT3 mutations are most common: Internal tandem duplication (ITD) of the juxtamembrane domain-coding sequence in approximately 30% of patients with AML and point mutations within the second tyrosine kinase domain (TKD) in about 7% of AML patients. Patients carrying the FLT3 ITD mutation seem to have a significantly worse prognosis, whereas the impact of TKD mutations on clinical outcome has not yet been determined. Recently, point mutations within the activation loop of FLT3 were also found in a significant percentage of infant and childhood acute lymhoblastic leukemia (ALL).Previous studies demonstrate that mice receiving transplants of bone marrow retrovirally infected with FLT3 ITD develop a myeloproliferative disease. The effect of FLT3 TKD mutations in vivo has not yet been investigated.To examine the transforming properties of FLT3 TKD mutants in primary hematopoietic cells, we used a bone marrow transplant model (BMT). Therefore we transduced bone marrow with retrovirus expressing either FLT3 D835Y or FLT3 I836M+R and transplanted it to lethally irradiated syngeneic recipient mice. As control we also transplanted mice with FLT3 WT and ITD infected bone marrow, respectively. We found that mice transplanted with FLT3 ITD developed a myeloproliferative disorder in mice, as previously described. In contrast, mice transplanted with FLT3 TKD mutants developed a lymphoid disease with distinct hematologic manifestation.Most recipients of FLT3 TKD transduced bone marrow developed T lymphoma syndrome, characterized by massive enlargement of thymus and lymph nodes. Some mice developed a B lymphoid leukemia with splenomegaly and enlarged lymph nodes. Interestingly, the disease latency of 53 to 183 days (median 102 days) of FLT3 TKD mutants contrasted with FLT3 ITD mice, which succumbed myeloproliferative disease within 53 to 70 days (median 58 days).The lymphoid manifestation and longer latency of FLT3 TKD in a murine BMT model together with the absent influence of FLT3 TKD mutations on clinical outcome of AML patients suggest differences in cell signaling between FLT3 TKD mutants and FLT3 ITD. The TKD mutants seem to require lymphoid cell context for full malignant transformation, whereas FLT3 ITDs transform myeloid cells.