Prognostic Factor In Acute Myeloid Leukemia Research Articles

Mutation Profiling and Prediction of Outcome in Acute Myeloid Leukemia

2014 Biomarkers in acute myeloid leukemia Acute myeloid leukemia (AML) is a heterogeneous disease characterized by accumulation of somatically acquired genetic alterations in hematopoietic progenitor cells. Cytogenetic analysis has been used for more than three decades and has allowed classifications of AML patients into distinct subsets that differ in biologic and prognostic characteristics [1,2]. Patients with cytogenetic profiles associated with ‘favorable risk’ (i.e., PML-RARA, RUNX1-RUNX1T1 or MYH11-CBFB) have relatively good outcomes without allogeneic hematopoietic cell transplantation (HCT) whereas patients with ‘unfavorable risk’ cytogenetics (e.g., at least three cytogenetic abnormalities) are thought to require HCT during the first remission to improve their outcome [3–5]. Although cytogenetics has long been recognized as the single most powerful prognostic factor in AML, nearly 50% and 25% of adult and pediatric patients, respectively, have no cytogenetic abnormalities [1,6]. Some of these patients with normal cytogenetic AML (CN-AML) do well with standard chemotherapy without HCT, but others do not. Given the current prognostic uncertainties in CN-AML, recent studies have focused on establishing new biomarkers to improve disease risk stratification and, accordingly, allocation to standard or investigational treatments. Rapid advances in genomics technologies have led to identification of an increasing number of molecular alterations in AML. For instance, targeted sequencing has identified recurrent mutations in FLT3, NPM1, c-KIT, CEBPA and TET2 [7,8]. Massively parallel sequencing enabled the discovery of additional recurrent mutations in DNMT3A and IDH1 [9,10]. More recent genome-wide studies have identified an increasing number of other recurrent somatic mutations in AML, including mutations in EZH2, PTPN-11, ASXL1 and PHF6, among others. Several studies retrospectively investigated the prevalence and prognostic significance of some of these molecular markers in relatively large cohorts of AML patients treated with standard regimens, in particular containing anthracycline and cytarabine

DDX43 promoter is frequently hypomethylated and may predict a favorable outcome in acute myeloid leukemia

DEAD box polypeptide 43 (DDX43), a cancer/testis antigen (CTA), has been found to be overexpressed in various solid tumors and some hematologic malignancies. In the present work hypomethylation of the DDX43 gene was detected in 15% (32/214) of primary acute myeloid leukemia (AML) using real-time quantitative methylation-specific PCR (RQ-MSP). The level of DDX43 expression was correlated with DDX43 hypomethylation (R=0.277, P=0.014). Moreover, bisulfite sequencing confirmed the significant correlation between the methylation density and the level of DDX43 hypomethylation. Additionally, restoration of DDX43 expression in the K562 cell line by 5-aza-2'-deoxycytidine treatment confirmed a direct contribution of methylation in regulating the DDX43 gene. DDX43 hypomethylation was observed more frequently in favorable group (21.4%) and intermediate group (15.8%) than in poor group (0%) (P=0.009). AML patients with DDX43 hypomethylation had a better overall survival (median not obtained) than those with DDX43 methylation (median 8 months, 95% confidence interval 5.6-10.4 months) (P=0.014). In summary, the DDX43 gene is activated by promoter hypomethylation and DDX43 hypomethylation may be a favorable prognostic factor in AML.

Study of CD123 (interleukin-3 receptor alpha Chain) and nuclear factor kappa B in adult acute myeloid leukemia patients

Background: The genomic alterations in acute myeloid leukemia (AML) affect the function of signaling molecules, transcription factors, and growth factor receptors besides they influence the response to treatment. Interleukin-3 receptor alpha chain (CD123) is overexpressed in about 45% of AML, this phenomenon was associated with high blast cell counts at diagnosis, with a worse prognosis. The transcription nuclear factor kappa B (NF-κB) can intervene in oncogenesis through its capacity to regulate the expression of a large number of genes that regulate apoptosis, cell proliferation, and differentiation. This study aimed to assess the relationship between CD123 expression and NF-κB level, in adult patients with AML at presentation and after induction therapy together with a well-known diagnostic and prognostic factors in AML. The study was conducted on forty adult newly diagnosed AML (group A) as well as twenty adult subjects who were hemato-oncologically free, as a control group (group B). Bone marrow (BM) examination and immunophenotyping by flow cytometry (FCM) on BM aspirate, for CD123 and quantitation of NF-κB in BM samples using ELISA (at three levels: Plasma, cytoplasmic and nuclear fraction of blasts, accordingly cytoplasmic/nuclear ratio) were performed for all studied subjects. In addition, levels of CD123 on blast cells and NF-κB were evaluated at D28 for remitted and resistant cases. All patients were positive for CD123 by FCM on BM blasts at time of diagnosis. The median cell counts expressing CD123 was 52.9 (34.4-75.6). Positive expression of CD123 was ≥20%, while in the control subjects CD123 expression was 25.1 (16.9-59.3), with significant decrease in CD123 expression after therapy, Besides, CD123 was also significantly higher in resistant patients compared to remitted patients (P = 0.009). As regard NF-κB, it was significantly higher in AML patients compared to control subjects at the three levels (plasma - cytoplasmic fraction - nuclear fraction) with a significant decrease after therapy. It was found that those NF-κB levels detected at follow-up (D28) on BM expression were significantly higher in resistant patients compared to remitted patients. While only The follow-up results, showed a highly significant positive correlation between CD123 expression on blast cells level and NF-κB plasma level alone (r = 0.587, P = 0.007) while there was not any correlation between CD123 and other two NF-κB levels neither at diagnosis nor at the follow-up. Conclusion: These data suggest that the correlation between CD123 and NF-κB was identified in variable results, and further elucidation of this role is likely to have important implications.

ABR, a Novel Inducer Of Transcription Factor C/EBPα, Is Necessary For Myeloid Differentiation and a Prognostic Factor In Acute Myeloid Leukemia

Abstract ABR (Active BCR-related) is the only protein in humans and mice closely homologous to BCR. BCR acts as a tumor suppressor in different cancers, such as chronic myeloid leukemia and meningiomas. A putative anti-oncogenic role of ABR has been shown in tumors of the central nervous system, such as medulloblastoma and astrocytomas, in which deletion of ABR was found. However, the role of ABR in hematopoiesis or leukemia remains unclear. We hypothesized that ABR might be important for myelopoiesis via increasing the expression of C/EBPα, a transcription factor known to be pivotal for myeloid differentiation and functionally impaired in acute myeloid leukemia (AML). In fact, we found that ABR expression is dramatically down-regulated (Figure 1, p=0.01) in bone marrow from AML patients (pts; n=63) compared to bone marrow (BM) mononuclear cells from healthy donors (n=3). In agreement with this finding, Abr is significantly increased during M-CSF and G-CSF-stimulated differentiation of primary wild type mouse BM cells (p<0.05). Additionally, we observe that ABR is necessary for monopoiesis induced by PMA (phorbol 12-myristate 13-acetate), since ABR knockdown in leukemic U937 cells results in a significant reduction of about 50% in the number of CD11b+ cells 48h after PMA treatment (p<0.05). Enforced ABR expression induces C/EBPα and its targets M-CSFR, G-CSFR and microRNA (miR)-223 in U937 cells (p<0.01). Moreover, we prove that ABR knockdown prevents induction of CEBPA, M-CSFR and G-CSFR during PMA-mediated differentiation (p<0.05). ABR overexpression blocks cell-cycle progression and down-regulates the known C/EBPα inhibitor E2F1 (p<0.01) in U937 cells, indicating the functional role of ABR as tumor suppressor. Those data suggest that ABR might induce CEBPA expression via inhibition of cell cycle activator E2F1. Finally, we are the first to identify ABR as a good prognostic factor in AML: patients with high ABR expression (median cut) survive significantly longer after allogeneic hematopoietic stem cell transplantation (Figure 2, p=0.04, log-rank test). Furthermore, high ABR expression associates with a low percentage of blasts in the peripheral blood (p=0.006) and high levels of antileukemic miR-181a (p<0.001). In conclusion, these data indicate that ABR, a novel inducer of C/EBPα, is necessary for myelopoiesis and a prognostic factor in AML. Raising ABR levels might be a goal for future therapeutics in AML. Disclosures: No relevant conflicts of interest to declare.

High p53 Protein Expression LEVEL Independent Of Mutational Status Is An Adverse Prognostic Factor For Survival In ACUTE Myeloid Leukemia

Abstract Background The tumor suppressor p53 is frequently mutated in human cancer, including acute myeloid leukemia (AML), particularly in cases with high-risk cytogenetics. It has been shown that p53 stabilization, which frequently occurs when the protein is mutated, can compromise its function. We have shown that p53 stabilization, regardless of the presence of mutations, suggesting alterations of other components in the p53 pathway. Methodology p53 expression was determined using high-throughput reverse phase protein array (RPPA) technology in 719 samples from 511 pts. Eleven CD34+ bone marrow (BM) and 10 normal peripheral blood (PB) lymphocyte samples were used as controls. Samples were printed as 5 serial 1:2 dilutions in duplicate using an Aushon 2470 Arrayer. Mutational status of p53 alleles was assessed by Sanger sequencing of exons 5 through 9. Expression of components of the p53 pathway was determined using standard immunohistochemical techniques. Nutlin-3a was used in in vitro culture experiments. Results Paired PB- and BM-derived AML samples expressed similar p53 levels (p=0.25). A trend towards higher p53 expression at relapsed was observed among 47 paired diagnosis/relapse samples (p=0.07). p53 expression correlated directly with CD34 (p=0.001) and inversely correlated with WBC (p=0.007), PB and BM blast burden (p=0.0001), and survival (p=0.01). High p53 (p53high) expression was more associated with unfavorable cytogenetics, particularly -5 (p=0.00001). p53high resulted in lower complete remission (CR) rates (51% vs 56%; p=??), higher relapsed rates (82% vs 62%; p=??), and shorter median overall survival (OS; 29.8 vs. 51 wks, p=0.009) compared to p53low pts. Most cases with p53high had unfavorable cytogenetics. We next correlated p53 stabilization with the presence of p53 mutations in 68 pts. p53 mutations were detected in 20/54 (37%) p53high pts and in 0/14 (0%) pts with p53low. p53high, either in the presence (29 wks) or in the absence (24 wks) of p53 mutations (p=1.0), was associated with significantly shorter OS compared with p53low pts (56 wks; p=0.05). Multivariate analysis revealed p53 expression to be an independent risk factor for survival in AML (p=0.02). p53high was positively correlated with p53pSER15 (p=0.00001), Rbp807p811 (p=0.0002), BAD (p=0.0001), cleaved PARP (p=0.002), and cleaved PARP (p=0.01), and negatively with p21 (p=0.01), and MDM2 (p=0.001).Given the similar OS in p53high pts carrying mutant or wild-type p53, we scored the immunohistochemical expression of MDM2, MDM4, and p21 in 30 p53high pts (9 p53 mutated, 21 wild-type p53). Overexpression of MDM2 was observed in 44% vs 48% pts with mutant vs wild-type p53, respectively, whereas rates were 67% vs 62% for MDM4, and 0% vs 19% for p21, for each respective genotype. Overall, of the 21 p53high pts carrying wild-type p53, 15 (71%) had overexpression of MDM2 and/or MDM4, whereas 81% had no p21 expression, indicating deficient activation of the p53 pathway similar to those cases carrying mutant p53. We are currently assessing response to nutlin-3a therapy in 24 primary AML samples (4 mutant p53, 20 wild-type p53). Results showing the impact of p53 mutation and/or stabilization, and expression levels of MDM2, MDM4, and p21 on nutlin-3a therapy will be presented. Conclusions p53 stabilization (p53high) is a powerful predictive and prognostic factor in AML, which is independent of the presence of mutant p53 alleles. Poor outcomes in pts with p53high lacking p53 mutations are very frequently associated with overexpression of negative regulators of p53 such as MDM2 and/or MDM4 and p21 downregulation, indicating a functionally altered p53 pathway. These findings may have implications for therapies targeting the MDM2/p53 axis in AML. Disclosures: No relevant conflicts of interest to declare.

Expression Of Hedgehog Pathway Mediator Gli2 Represents a Clinically Negative Prognostic Marker In Acute Myeloid Leukemia and Its Inhibitor GANT61 Exerts Anti-Leukemic Effects In Vitro

Abstract Background Leukemic stem cells depend on signals provided by bone marrow (BM) niche cells. Due to its function in stem cell biology, we investigated expression and prognostic impact of the Hedgehog pathway in acute myeloid leukemia (AML). Methods and results Pre-treatment samples from 104 patients with newly diagnosed AML (AMLSG 07-04 trial) were analyzed by quantitative PCR. Expression of receptors Smoothened and Patched and downstream transcription factors Gli1, Gli2 and Gli3 was found in 69%, 41%, 73%, 20% and 26% of cases, respectively. However, no expression of Hedgehog ligands was observed. Gli2 expression had a significant influence on event-free, relapse-free and overall survival (p=0.037, p=0.026 and p=0.013, respectively) and was furthermore correlated to FLT3 mutational status (p<0.001). To verify our findings in a second, independent patient cohort, microarray-based gene expression data of patients published by Valk et.al.(N Engl J Med. 2004 Apr 15;350(16):1617-28) was used. Clinical data of 291 AML patients from this cohort were available and the negative prognostic impact of Gli2 expression on overall and event-free survival could be confirmed (p=0.003 for overall and p=0.007 for event-free survival, respectively; no data for relapse-free survival available). Additionally, the correlation of Gli2 expression to FLT3 mutational status was also observed in this patient cohort (p=0.003). Although Hedgehog ligands were not expressed in AML bone marrow aspirates, Desert Hedgehog (DHH) plasma levels were significantly increased in AML patients compared to healthy subjects (p=0.002). Provision of Desert Hedgehog could be ascribed to BM niche cells including endothelial cells and osteoblasts, since DHH expression was found in outgrowth endothelial cells (OECs) and osteoblasts in vitro and on immunohistochemistry of leukemic bone marrow samples in patients. Next, we investigated the effect of Gli inhibition in vitro. AML cell lines KG-1, OCI-AML5 and UKE-1 as well as freshly isolated primary AML cells were treated with the specific Gli inhibitor GANT61. GANT61 dose-dependently induced apoptosis in all three cell lines investigated. Furthermore, GANT61 treatment resulted in significantly increased apoptosis in primary AML cells upon treatment with 10 µM, 30 µM and 90 µM GANT61 (n=10 pts). Additionally, a pronounced growth inhibitory effect on primary AML cells was observed with a significant reduction in cell count to 82 ± 13%, to 54 ± 11% and to 47 ± 13% upon treatment with 10 µM, 30 µM and 90 µM of GANT61, respectively (n=9 pts). Incubation of KG-1, UKE-1 and OCI-AML5 cells for 7 days with 30 µM GANT61 resulted in reductions in cell numbers compared to the control to 14 ± 2%, to 44 ± 6% and to 31 ± 0%, respectively. Moreover, GANT61 mediated a strong and significant inhibitory effect on the colony formation capacity of AML cell lines and primary AML cells. The average number of colonies was reduced to 44 ± 16% and 4 ± 4% upon treatment with 10 µM and 30 µM GANT61, respectively, while no colony formation was observed with a concentration of 90 µM GANT61 in primary AML cells (n=4 pts). Conclusion In conclusion, Gli2 expression is a negative prognostic factor in AML that mirrors activated Hedgehog signaling induced by paracrine stimulation through BM niche cells. Inhibition of Hedgehog signaling by blocking Gli activity might represent a promising therapeutic approach for AML treatment with special regard to patients carrying a mutated FLT3. Disclosures: Fiedler: Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding.

HOXA9 Gene Expression in Acute Myeloid Leukemia

Homeobox genes encode the class of transcription factors in vertebrates and are found in clusters called A, B, C, and D on four separate chromosomes. HOXA9 gene is part of the cluster A on chromosome 7 and encodes a DNA-binding transcription factor which may regulate gene expression, morphogenesis, and differentiation. The objective of this study was to determine the HOXA9 gene expression in acute myeloid leukemia (AML). For this purpose, semi-quantitative reverse transcriptase-polymerase chain reaction was used to measure HOXA9 gene expression in human erythroleukemia (HEL) cell line and bone marrow mononuclear cells from 54 AML patients and 20 healthy individuals. The data show that HOXA9 mRNA expression was negative in 20 healthy individuals but was positive in HEL cells and in 22 out of 54 (40.74%) AML patients. The complete remission rate (45.45%) of the patients who expressed the gene was significantly lower than that (71.86%) in patients who did not express the gene after chemotherapy. Therefore, it was concluded that HOXA9 gene might be involved in the pathogenesis of AML and played as a worse prognostic factor in AML.

Conventional chemotherapy or hypomethylating agents for older patients with acute myeloid leukaemia?

Acute myeloid leukaemia (AML) is the second more frequent hematologic malignancy in developed countries and primarily affects older adults with a median age at diagnosis of 69 years. Given the progressive ageing of the general population, the incidence of the disease in elderly people is expected to further increase in the years to come. Along with cytogenetics at diagnosis, age represents the most relevant prognostic factor in AML, in that the outcome steadily declines with increasing age. Reasons for poor prognosis include more frequent unfavourable karyotype and other adverse biologic characteristics, such as high rates of expression of genes drug resistance related and high prevalence of secondary AML. Noticeably, as compared with young adults, poorer results in elderly patients have been reported within any cytogenetic and molecular prognostic subgroup, because of frequent comorbid diseases, which render many patients ineligible to intensive chemotherapy. Therefore, predictive models have been developed with the aim of achieving best therapeutic results avoiding unnecessary toxicity. Following conventional induction therapy, older AML patients have complete remission rates in the range of 45-65%, and fewer than 10% of them survive for a minimum of 5 years. On the other hand, hypomethylating agents, such as azacytidine and decitabine offer the possibility of long-term disease control without necessarily achieving complete remission and can represent a reasonable alternative to intensive chemotherapy. Either intensive chemotherapy or hypomethylating agents have lights and shadows, and the therapeutic selection is often influenced by physician's and patient's attitude rather than definite criteria. Research is progress in order to assess predictive biologic factors, which would help clinicians in the selection of patients who can take actual benefit from different therapeutic options.

Upregulation of CD200 is associated with Foxp3+ regulatory T cell expansion and disease progression in acute myeloid leukemia

Immunosuppression in acute myeloid leukemia (AML) is an important mechanism of tumor escape. CD200, as an immunosuppressive molecule, is overexpressed in some hematological malignancies and it has also been shown to be an independent prognostic factor in AML. In the current study, simultaneous CD200 expression and Foxp3(+) regulatory T cell levels were investigated in Iranian patients with AML by flow cytometry. We also assessed the effect of CD200-CD200R blockade on Th1 and T-reg cytokine production and T cell proliferation in autologous AML- and monocyte-DC mixed lymphocyte reactions (MLRs). ELISA assay was performed to detect IL-2, IL-12, IFN-γ, IL-10, and TGF-β production in MLR supernatants. Expression of Foxp3, IL-10, and TGF-β mRNAs in MLRs were detected by real-time PCR. Our results demonstrated significant overexpression of CD200 (P = 0.001) in association with higher frequencies of Foxp3(+) T cells in AML patients (r = 0.8, P < 0.001). Blocking of CD200-CD200R interaction demonstrated a significant decrease in TGF-β and IL-10 expression in AML-DC MLRs and a significant increase in IL-12 and IFN-γ expression in monocyte-DC MLRs. Elevated T cell levels with lower Foxp3 intensity was also shown in CD200-CD200R-blocked MLRs. Expression of IL-10 mRNA declined significantly only in AML-DC MLRs where CD200-CD200R interaction was blocked and the same result was observed for TGF-β and Foxp3 mRNA in both AML- and monocyte-DC MLRs. These data present a significant role for CD200 in suppressing anti-tumor immune response through stimulation of regulatory mechanisms in AML patients and suggest that CD200 may have a prognostic value in this malignancy and its blockade may be used as a target for AML immunotherapy.

Outcomes in Patients with Acute Myeloid Leukemia Preceded by Breast Cancer

Abstract Abstract 4316 Background: As breast cancer has become a more curable disease, the risk of second malignancy either related to therapy or genetic alterations has become apparent. Recent data from the National Cancer Institute's SEER database showed an increased risk of Acute Myeloid Leukemia (AML) in female patients with Stage III breast cancer. To investigate the outcomes of AML after breast cancer treatment, we reviewed all annotated cases with AML preceded by breast cancer (BrCa) at our institution. Methods: Between 2001 and 2011, the Moffitt Cancer Center Total Cancer Care database was used to identify patients with a diagnosis of AML preceded by breast cancer. Individual charts were subsequently reviewed. Chi square test was used to compare categorical variables in univariate analysis. Kaplan Meier estimates were used to calculate OS. Log rank test was used for comparison between the 2 groups and Cox regression analysis was used for multivariable analysis of survival. All analyses were conducted using SPSS version 19.0 software. Results: We identified 60 female patients with AML preceded by a diagnosis of breast cancer. Median age at AML diagnosis was 65 years (range 43–81). Median time from BrCa diagnosis to AML diagnosis was 7 years. Twenty nine (48%) had stage ≥ 2 disease at time of BrCa diagnosis. Forty nine (82%) of patients had t-AML, 36 (60%) of whom had received chemotherapy, either alone or in combination with radiation, for breast cancer management. Nineteen (32%) had adverse karyotype and 22 (37%) had MDS that preceded the diagnosis of AML. Thirty-six (60%) underwent induction chemotherapy for AML with an cytarabine/anthracycline-based regimen (most commonly “7+3”), and CR/CRiwas achieved in 21 (54%). Ten (17%) patients eventually proceded to allogeneic hematopoietic cell transplant, with a median survival of 20 months (95% C.I. 10 –29.9 mo) Median survival following AML diagnosis was 10.4 mo (95% C.I. 5.4 – 15.4 months). Univariate analysis of prognostic variables associated with overall survival are shown in Table 1. Variables significantly associated with inferior survival included history of MDS preceding AML, adverse karyotype, and no induction therapy for AML. Interestingly, prior BrCa chemotherapy did not adversely impact survival. None of the individual variables retained prognostic significance in multivariate analysis. Conclusions: AML arising in the setting of prior BrCa has a generally poor prognosis that mimics outcomes in older adults with AML. Many traditional adverse prognostic factors in AML are present in patients with AML from BrCa, likely accounting for the poor outcomes. However, many patients are able to receive and benefit from induction chemotherapy and ultimately proceed to allogeneic transplant, indicating that traditional therapy has an important role in the management of AML from BrCa. Larger numbers of patients will be necessary to confirm the importance of varied risk factors and treatment options in this population. A comparison with matched historic control patients will also be presented. Disclosures: No relevant conflicts of interest to declare.

Overexpression of Sharp-1 in MLL-AF6-Positive Acute Myeloid Leukemia

Abstract Abstract 3493 Patients with acute myeloid leukemia (AML) carry various chromosomal translocations and/or mutations. Although t(6;11)(q27;q23) is observed in only less than 0.5 % of adult AML patients, this translocation is reported to be one of the independent poor prognostic factors in AML. This translocation induces expression of MLL-AF6 fusion protein, but the precise mechanism of leukemogenesis by MLL-AF6 has not yet been elucidated compared to other MLL fusion proteins. During the analysis of gene expression profiling of &gt;500 AML patients registered in Erasmus University Medical Center in Rotterdam, we happened to find that all of five patients carrying t(6;11) translocation exhibited overexpression of Sharp-1/Dec2, which is involved in the circadian rhythm. AML cell lines carrying this translocation, ML-2 and CTS, also showed more than 10-fold upregulation of Sharp-1 expression. Knockdown of MLL-AF6 expression to 10 % in ML-2 cells by shRNA suppressed expression of Sharp-1 to less than 1 % through the change in the methylation status of histone H3 lysine residues in the Sharp-1 gene promoter region confirmed by chromatin immunoprecipitation. In addition, this knockdown induced growth suppression of ML-2 cells to 40–50 %. Proliferation of ML-2 cells was also suppressed by the knockdown of Sharp-1. Growth suppression of ML-2 cells is neither due to increased apoptosis nor increased differentiation, but due to increased number of cells undergoing cellular senescence together with the upregulation of p21. Furthermore, exogenous expression of Sharp-1 in murine bone marrow cells induced the proliferation of myeloid cells with the reduction of lymphoid cells in the bone marrow transplantation model. These results suggested that Sharp-1 plays a crucial role in the pathogenesis of AML with t(6;11)(q27;q23) and that targeting of Sharp-1 in leukemic stem cells would be a novel therapy for this poor outcome AML. Disclosures: No relevant conflicts of interest to declare.

IS5-1 - Identification and Characterization of Acute Myeloid Leukemia-Stem Cells by Single-Cell Gene-Expression Profile

ABSTRACT While the identification of cancer stem cells as therapeutic targets is now actively being pursued in a variety of human malignancies, leukemic stem cells (LSCs) in acute myeloid leukemia (AML) are a paradigm of such a strategy. However, significant improvements in outcome based on these insights have not been forthcoming, because the molecular basis to distinguish the properties of LSCs from those of normal hematopoietic stem cells (HSCs) remains largely unknown. Meanwhile, current advances in single-cell gene profiling can provide definitive evidence regarding the conversion of one cell type into another at a high level of resolution. To characterize biological subtypes of LSCs in AML, in this study, we systematically analyzed the cDNA profiles of a large number of single LSCs. We first carried out the comparative DNA microarray analysis with primary leukemic cells and reconstituted cells in xenograft, and extracted 22 intrinsic genes from 5,599 genes, of which expression was significantly up-regulated in reconstituted cells, as the markers of high grade, aggressive AML. Among them, Evi1 and MN1, both previously identified potential poor prognostic factors in AML, were included. Next, we isolated BM CD34 + 38- LSCs from refractory AML patients, randomly picked up single cells, and amplified a total of 96 single-cell cDNA samples from each patient, as well as CD34 + 38- cells derived from a healthy volunteer to obtain information from normal HSCs. By analyzing expression-patterning of the 22 identified genes in each cell, we have identified a set of genes, of which expression pattern was significantly correlated with that of Evi1. This correlation was not observed in their normal counterparts, indicating that it was specific for AML LSCs. Furthermore, in one case, who developed AML from myelodysplastic syndrome (MDS), the hierarchical cluster based on the molecular signatures was identified only in LSCs at AML stage, but not at MDS. These results suggest that single-cell gene-expression profiling would allows us to classify AML-LSCs and provide new insights into functional properties of LSCs. Currently, we are comprehensively evaluating the expression of surface molecules on AML LSCs, and would like to disclose the clinical practicality of this methodology in our presentation.

Molecular Involvement and Prognostic Importance of Fms-like Tyrosine Kinase 3 in Acute Myeloid Leukemia

AML (Acute myeloid leukemia) is a form of blood cancer where growth of myeloid cells occurs in the bone marrow. The prognosis is poor in general for many reasons. One is the presence of leukaemia-specific recognition markers such as FLT3 (fms-like tyrosine kinase 3). Another name of FLT3 is stem cell tyrosine kinase-1 (STK1), which is known to take part in proliferation, differentiation and apoptosis of hematopoietic cells, usually being present on haemopoietic progenitor cells in the bone marrow. FLT3 act as an independent prognostic factor for AML. Although a vast literature is available about the association of FLT3 with AML there still is a need of a brief up to date overview which draw a clear picture about this association and their effect on overall survival.

Guest editorial: Introducing progress in hematology in this issue

This issue of IJH contains five serial review articles on acute myeloid leukemia (AML). AML is a molecularly heterogeneous neoplasm, and the understanding of its molecular pathogenesis continues to grow. The development of chemotherapy and hematopoietic stem cell transplantation (HSCT) have increased 5-year survival rate to 40 % in AML patients younger than 60—65 years, whereas outcome in elderly AML patients remains dismal. In the first review, David Grimwade summarizes the molecular heterogeneity of AML and clinical significance of each molecular abnormality. In his work with the UK Medical Research Council (MRC), he has published important articles on prognostic factors in AML and characterization of the mechanisms underlying leukemiaassociated chromosomal translocations. A consensus had emerged that leukemogenesis requires two classes of mutations in AML (WHO classification 2008). The first of these is abnormalities in transcriptional factors that impair hematopoietic differentiation. Rearrangement of genes such as RUNX1, RARA, or MLL belongs to this class of mutations as well as point mutation of CEBPA (these are known as class II mutations). The second class of mutations consists of FLT3, KIT, or NRAS mutations (class I mutations), which are associated with cellular proliferation and/or survival. The discovery of the NPM1 mutation in 2007, however, suggested that the story is not so simple. From 2009, genome sequence analysis revealed novel gene mutations such as TET2, IDH1/2, and DNMT3A especially in AML showing normal karyotype. These gene products are associated with epigenetic modifications, including DNA methylation, and their biological significance in leukemogenesis is under investigation. In the second perspective, Martin Tallman, chairman of the Leukemia Committee for the Eastern Cooperative Oncology Group (ECOG), reviews induction chemotherapy for AML. Although the fundamental drug combination for remission-induction, DNR or IDA, plus Ara-C, has not changed for more than 30 years, there is still no definitive protocol for dose and schedule. Furthermore, many trials are under way seeking to improve CR rate and survival; these experimental approaches include intensification of anthracycline, high-dose of Ara-C, addition of a third drug, priming usage of G-CSF, and combination with molecular targeted drug. Third, co-chairman of the Japan Adult Leukemia Study Group (JALSG) Shuichi Miyawaki was invited to overview Japanese studies on AML. The JALSG has just celebrated its 25th anniversary with the holding of an international symposium at Tokyo Forum on June 23–24, 2012, which this review article commemorates. JALSG has completed six studies of AML to date. As Ryuzo Ohno previously reviewed the first four JALSG studies from 1987 to 1997 (Int J Hematol. 72:272, 2000), Miyawaki focused primarily on the two studies after 1997. Working with Masamitsu Yanada, I co-authored the fourth review, focusing on elderly AML, which is becoming a more important issue in many countries. In the JALSG data, the median age of newly diagnosed patients with AML and high-risk MDS is around 65 years. Accordingly, at least half of AML patients are not included in clinical studies using intensive chemotherapy and HSCT. Since conventional therapy has limitations in improving outcomes in elderly AML, new therapeutic approaches are urgently needed. T. Naoe (&) Department of Hematology and Oncology, Nagoya University Graduate School of Medicine, Nagoya 466-8550, Japan e-mail: tnaoe@med.nagoya-u.ac.jp

Osteopontin is a prognostic factor for survival of acute myeloid leukemia patients

Osteopontin (OPN) is a glycoprotein that is secreted by osteoblasts and hematopoietic cells. OPN suppresses the proliferation of hematopoietic stem cells in vitro and may regulate the hematopoietic stem cell pool. Increased serum OPN concentrations occur in chronic myeloid leukemia, multiple myeloma, and acute myeloid leukemia (AML). In the present study, we analyzed the prognostic impact of OPN in AML by investigating the expression and relevance of OPN in newly diagnosed AML patients from 2 large study groups (the German AML Cooperative Group and the Dutch-Belgian Hematology Oncology Cooperative group). IHC (n = 84), ELISAs of blood/BM sera (n = 41), and microarray data for mRNA levels (n = 261) were performed. Expression of OPN protein was increased in AML patients both in BM blasts (IHC) and in BM serum (ELISA) compared with healthy controls. Patients expressing high levels of OPN within the BM (IHC) experienced shortened overall survival (OS; P = .025). Multivariate analysis identified karyotype, blast clearance (day 16), and the level of OPN expression as independent prognostic factors for OS. This prompted us to analyze microarray data from 261 patients from a third cohort. The analysis confirmed OPN as a prognostic marker. In summary, high OPN mRNA expression indicated decreased event-free survival (P = .0002) and OS (P = .001). The prognostic role of OPN was most prominent in intermediate-risk AML. These data provide evidence that OPN expression is an independent prognostic factor in AML.

Expression of CD25 on Acute Myeloid Leukemia (AML) Blasts Is An Independent Risk Factor Associated with Refractory Disease, Which May Be Overcome by Stem Cell Transplantation (SCT),

Abstract Abstract 3560 Introduction: Acute myeloid leukemia (AML) originates from rare leukemia stem cells (LSCs). LSCs are chemotherapy resistant and responsible for disease recurrence. AML containing a high percentage of LSCs displays aggressive biology in animal models. Using humanized mice Saito et al (Sci Transl Med 2010; 2: 1–11) have recently shown that xenografted CD25+ LSCs initiate AML and are chemotherapy resistant. Confirmation with clinical data from human AML is needed. Methods: In order to determine the prognostic impact of CD25 expression on AML outcome we have retrospectively investigated CD25 expression in 56 patients (pts) diagnosed and treated for AML at our institution between 02/2008 to 05/2011. 46 pts who had non-APL morphology, were treated with induction chemotherapy and had an adequate specimen for CD25 assessment were included in further analysis. CD25 expression was assessed in each specimen by flow cytometry and immunohistochemistry and correlated with clinical outcome. Patients: Median age was 61 years (22–84), 18 (39%) pts were older than 65; F:M ratio was 19:27, 3 (7%) patients had good risk (core binding factor leukemias), 26 intermediate (diploid karyotype and no good or high risk; 57%) and 17 (37%) high risk cytogenetics (complex, anbormality of 3q26, monosomy 7 and 5). 6 (13%) pts had NPM1mut/FLT3-ITDwt, 6 (13%) pts had NPM1wt/FLT3-ITDmut and 9 (13%) pts had NPM1mut/FLT3-ITDmut. As induction high dose cytarabine/anthracycline based regimen was used in 36 (78%) pts, 7 pts received 7+3 (15%) and 3 (7%) pts received hypomethylating agent. 24 (53%) pts received stem cell transplantation (SCT; 16 [35%] allogeneic and 8 [17%] autologous). The median follow up of the surviving pts was 11.2 months (1.1–38.7). Results: CD25 was detected in 17 pts (37%; 16 at diagnosis and 1 at relapse). Six CD25+ pts experienced relapse (3 pts with 3 or more relapses) heralded by increase in the percentage of CD25+ blasts. 65% of pts with CD25+ AML also had FLT3-ITDmut (p=0,0012). When comparing CD25+ and CD25- pts there was no statistical difference in distribution of the following characteristics: sex, age (65+), cytogenetics risk, presence of NPM1mut, type of induction, SCT. Fifteen (88%) of CD25+ pts experienced relapse compared to 8 (28%) of CD25- pts (p= 0.00007), 8 (47%) CD25+ pts died and 9 (31%) CD25- pts died (p=ns). The median relapse free survival (RFS) of all pts was 10.8 months with the median overall survival (OS) 12.2 months. The estimated 6-month RFS was significantly decreased in CD25+ pts compared to CD25- pts (26% vs 79%, p= 0.0003). This did not translate into a difference in OS between both groups (1-year OS: CD25+ 43% vs CD25- 65%, p=ns). In univariate analysis CD25 positivity was a stronger predictor for relapse (HR 5.28 [2.21–12.62], p=0.0002) than FLT3-ITDmut (HR 4.72 [2.04–10.92]; p= 0.0003). In multivariate analysis CD25 positivity was also a stronger predictor for relapse (HR 6.54 [1.34–9.15], p=0.01) than FLT3-ITDmut (HR 4.72 [2.04–10.92], p= 0.03). Pts undergoing SCT had significantly longer 1-year OS (66%) compared to pts without SCT (21%; p=0.0004). In multivariate analysis SCT was a predictor for improved OS (HR 0.2 [0.07–0.57], p=0.0002). CD25+ pts who received SCT had also significantly longer 1-year OS (63%) compared to CD25+ pts who did not receive SCT (0%; p=0.0098). SCT did not impact RFS in either group. Conclusion: CD25 represents a novel prognostic factor in AML. The increase in CD25+ blasts at relapse is associated with increased relapsed rate and refractory AML supporting the LSCs hypothesis. The detection of CD25 serves not only as a prognostic marker, but may be valuable for minimal residual disease assessment in patients who lack a molecular marker. In our experience treatment inclusive of stem cell transplantation abrogated the negative impact of CD25 expression on OS. Exploration of CD25 as a therapeutic target in AML is warranted. Disclosures: No relevant conflicts of interest to declare.

Expression of Gli2, a Downstream Mediator of the Activated Hedgehog Pathway, Represents a Negative Prognostic Marker in AML,

Abstract Abstract 3535 The cancer and leukaemia stem cell hypothesis was first proposed over 40 years ago. This hypothesis suggests that malignant stem cells serve as reservoir for leukaemia cell renewal. Stem cell activators such as Wnt/β-catenin, transforming growth factor β (TGF-β), Notch and Hedgehog signaling pathways have been implicated into stem cell capacity for self-renewal, production of heterogeneous progeny and resistance to chemotherapy. Until now, few data on the contribution of the Hedgehog pathway in acute myeloid leukaemia (AML) exist. Therefore, the aim of this study was to investigate the expression of members of the Hedgehog pathway in primary AML samples and to correlate expression with clinical outcome. Pre-treatment samples from 95 patients with newly diagnosed AML who were entered into the AMLSG 07–04 trial were analyzed. The trial was conducted between August 2004 and August 2009 (ClinicalTrials.gov Identifier: NCT00151242). In brief, the trial design was as follows: Patients between 18 and 60 years of age with de novo or secondary AML [excluding patients with t(15;17)] were entered into the study. All patients received two induction courses with cytarabine, idarubicin and etoposide. Patients with complete remission (CR) obtained high dose cytarabine consolidation. At study entry patients were randomized between additional therapy with valproic acid, ATRA, both or none. After an interim analysis the randomization to the valproic acid and the combination arm was suspended. Mean follow-up of the analyzed patients was 580 days. Expression of ligands Sonic, Indian and Dessert Hedgehog, receptors Patched and Smoothened as well as downstream transcription factors Gli1-3 were analyzed by qPCR. No appreciable expression of Sonic, Indian or Desert Hedgehog was found in 30 patients each, and thus no further samples were analyzed. Expression of Smoothened and Patched was found in 73% and 41% of cases, respectively. Mutations within exon 9 or 10 of the Smoothened gene represent a frequent genetic alteration in basal cell carcinomas and medulloblastomas. Therefore, both Smoothened exons have been analyzed by direct sequencing in AML samples. No mutations in Smoothened exon 9 or 10 were found in 30 AML patients. Measurable expression of Gli1, Gli2 and Gli3 was detected in 73%, 22% and 26% of cases, respectively. Patient characteristics including gender, age, categorized karyotype, FLT3 mutation, NPM-1 mutation or MLL status were correlated with the expression of the various members of the Hedgehog pathway. In this analysis, a significant association between FLT3 mutation and Gli2 expression could be established (p&amp;lt;0.001). Clinical outcome of patients was also correlated to Hedgehog expression variables. The CR rate was 57% for Gli2 expressors compared to 71% in Gli2 negative patients. Furthermore, Gli2 expression had a significant influence on relapse free survival and overall survival (p=0.026 and p=0.008, respectively). In conclusion, Gli2 expression in contrast to the more frequently expressed Gli1 is a negative prognostic factor in AML. Blockade of Smoothened activity by specific inhibitors may result in repression of the Gli2 gene and may therefore represent a new therapeutic approach in AML. Acknowledgment: Supported by a grant from Pfizer Disclosures: Fiedler: Pfizer: Research Funding.

Low expression of PP2A regulatory subunit B55α is associated with T308 phosphorylation of AKT and shorter complete remission duration in acute myeloid leukemia patients

The regulation of Protein Kinase B (AKT) is a dynamic process that depends on the balance between phosphorylation by upstream kinases for activation and inactivation by dephosphorylation by protein phosphatases. Phosphorylated AKT is commonly found in acute myeloid leukemia (AML) and confers an unfavorable prognosis. Understanding the relative importance of upstream kinases and AKT phosphatase in the activation of AKT is relevant for the therapeutic targeting of this signaling axis in AML. The B55α subunit of Protein Phosphatase 2A (PP2A) has been implicated in AKT dephosphorylation but its role in regulating AKT in AML is unknown. We examined B55α protein expression in blast cells derived from 511 AML patients using Reverse Phase Protein Analysis (RPPA). B55α protein expression was lower in AML cells compared to normal CD34+ cells. B55α protein levels negatively correlated with T308 phosphorylation levels. Low levels of B55α were associated with shorter complete remission duration demonstrating that decreased expression is an adverse prognostic factor in AML. These findings suggest that decreased B55α expression in AML is at least partially responsible for increased AKT signaling in AML and suggests that therapeutic targeting of PP2A could counteract this.

Clinical results and issues of acute myeloid leukemia in elderly patients aged 75 years and older

Clinical outcomes of acute myeloid leukemia (AML) in elderly patients still remain unsatisfactory and the optimal treatment has yet to be clearly established. This report describes the results of a retrospective study of clinical outcomes and prognostic factors of AML in patients aged 75 years and older. In addition, we aimed to elucidate the situation of patients with AML accompanied by dementia, which has been largely ignored in previous studies. The subjects consisted of 31 patients with untreated AML (including previous myelodysplastic syndrome: AML/MDS). All patients underwent chemotherapy, with 25 undergoing conventional therapy and six undergoing low-intensity therapy. Complete remission was obtained in 16 of the 31 cases (51.6%), with a 3-year survival rate of 11.5%. However, in seven cases, Alzheimer's disease (AD) was observed. Although we were able to perform induction therapy in each of these cases, consolidation therapy was difficult in cases of moderate AD. The results of this study suggest that even very elderly patients can benefit from chemotherapy. However, it is thought that the treatment selection for cases which are complicated by moderate to severe dementia should be determined carefully while considering the patient's quality of life.

The Impact of FLT3-ITD Mutation on the Expression of Multidrug Resistance Genes MDR-1, MRP-1, BCRP and LRP In Adult Acute Myeloid Leukemia

Abstract Abstract 1808 Background Multidrug resistance (MDR) is a clinically relevant problem in the treatment of acute myeloid leukaemia (AML). From 20–50% of patients with AML are primarily resistant to induction chemotherapy and in the great majority of those who respond well to initial therapy the disease relapses. While some mechanisms of MDR have been well characterized, this phenomenon still remains incompletely understood. Three ATP-dependent drug transporting proteins: p-glycoprotein (Pgp; MDR-1), multidrug resistant protein-1 (MRP-1), breast cancer resistance protein (BCRP) and one major vault protein-lung resistance-related protein (LRP) and the internal tandem duplication (ITD) in the receptor tyrosine kinase FLT3 have been found to have a strong correlation with MDR and poor outcome in AML. About 30% of AML cases harbor FLT3-ITD mutation. Mainly MDR-1 protein and FLT3-ITD mutation are targets for inhibition by numerous agents in clinical practice. Objectives In this retrospective study we estimated expression of MDR genes encoding MDR-1, MRP-1, BCRP, LRP proteins and detected FLT3-ITD mutation in patients with AML. We divided all patients into two groups according to FLT3-ITD mutation and we compared expression of MDR genes and other prognostic factors in AML: age (&lt;60 vs ≥60 years), cytogenetic/molecular aberrations (good, intermediate, poor), de novo/secondary AML, white blood cell count (WBC), extramedullary involvement, type of AML according to the FAB classification, CD34 expression on blast cells and myeloperoxidase activity (&lt;50% of blast cells, ≥50%) between groups Moreover the rate of complete remissions (CR) and relapses and disease free survival (DFS) and overall survival (OS) in both groups were estimated. Materials and Methods A total of 126 adult patients at median age of 53 years (range 21–87) with newly diagnosed between June 2005 and March 2010, previously untreated AML and 40 healthy donors were included in this study. Diagnosis of AML was based on standard morphological and immunophenotypical criteria according to the WHO classification. Most of the patients were treated with anthracycline and cytarabine during the induction regime. There were 39 FLT3-ITD positive (30,4%) and 87 FLT3-ITD negative (69,6%) patients. DNA was extracted from bone marrow (90 patients) or peripheral blood (36 patients) samples and used to detect FLT3-ITD with PCR method. RNA was extracted from the same samples and following reverse transcription RQ-PCR method was performed for assessment of expression of MDR-1, MRP-1, BCRP and LRP genes. Parameters of RQ-PCR reaction were established based on the Europe Against Cancer protocol. Every tested sample was amplified simultaneously for the presence of control ABL gene for standardization of mRNA level for tested genes. Results of the expression of tested genes were presented as coefficients calculated with an intermediate method according to Pfaffl's rule. Results To our surprise, significant higher expression of MDR-1 gene was found either in bone marrow or peripheral blood samples of the patients who do not harbor FLT3-ITD in contrast to the patients harboring this mutation (median 0,3025 vs 0,0756, p=0,003 total; median 0,2690 vs 0,0583, p= 0,002 bone marrow samples; median 0,5775 vs 0,0920, p=0,001 peripheral blood samples). For MRP-1, BCRP and LRP genes we did not find significant differences in their expression between two groups. When adjusted for other prognostic factors, only WBC was significantly higher in FLT3-ITD positive group of patients: 1–294×109/l, median 74 vs 0–286×109/l, median 6,5 in FLT3-ITD negative group of patients (p=0,0001). We did not observe significant differences in CR and relapses rates, DFS and OS between groups. Conclusions It seems that FLT3-ITD mutation may in some mechanisms influence MDR-1 gene expression We observed significant inhibition of MDR-1 expression in FLT3-ITD positive patients. Because risk factors for AML and outcome of AML therapy did not differ between groups it seems that FLT3-ITD mutation and high expression of MDR-1 gene are independent high risk factors which negatively influence results of AML therapy. These results might provide a powerful risk classification and useful information for treatment strategies including inhibitors of tyrosine kinase or MDR-1 protein. This project was supported by grant of The Ministry of Science (N N402 208935). Disclosures: No relevant conflicts of interest to declare.