Fedor Denkers

Concurrent methylation of promoters from tumor associated genes predicts outcome in acute myeloid leukemia.

By assessment of the methylation status of 25 candidate tumor suppressor genes (TSGs) in 119 acute myeloid leukemia (AML) patients and 5 controls, we aimed to determine whether simultaneous methylation of multiple TSGs exerts prognostic impact. Methylation-specific multiplex ligation probe amplification (MS-MLPA) revealed methylation of at least one TSG in 59/119 patients, while no methylation was found in controls. Methylation of different TSGs within patients was substantially correlated (intra-class correlation; 0.38). ESR1 methylation (34/119) strongly predicted concurrent methylation of other genes, OR 7.33 (95%CI 4.13–12.99). A Cox regression model that included the three most frequently methylated TSGs ESR1, CDKN2B/p15 and IGSF4, showed ESR1 to have opposite effects on overall survival (OS) compared with the other two, HR 0.22 (95% CI 0.09–0.53) and HR 1.66 (95% CI 0.73–3.79), HR 1.61 (95%CI 0.66–3.93). By assessment of CDKN2B/p15 and IGSF4 methylation, patients with methylation at multiple loci can be identified. Accumulation of methylation aberrancies is much more pronounced in ESR1 methylated patients. When combined, the methylation status of ESR1, CDKN2B/p15 and IGSF4 enable identification of patient subgroups with large differences in OS (p <0.0001). This study shows that methylation profiling allows risk stratification in AML. In addition, ESR1 methylation may reflect a biological pathway that leads to hypermethylation of multiple genes, which is reflected by methylation of IGSF4 and/or CDKN2B/p15.

MicroRNA profiling can classify acute leukemias of ambiguous lineage as either acute myeloid leukemia or acute lymphoid leukemia.

Purpose: Classification of acute leukemia is based on the commitment of leukemic cells to the myeloid or the lymphoid lineage. However, a small percentage of acute leukemia cases lack straightforward immunophenotypical lineage commitment. These leukemias of ambiguous lineage represent a heterogeneous category of acute leukemia that cannot be classified as either acute myeloid leukemia (AML) or acute lymphoid leukemia (ALL). The lack of clear classification of acute leukemias of ambiguous lineage as either AML or ALL is a hurdle in treatment choice for these patients. Experimental Design: Here, we compared the microRNA (miRNA) expression profiles of 17 cases with acute leukemia of ambiguous lineage and 16 cases of AML, B-cell acute lymphoid leukemia (B-ALL), and T-cell acute lymphoid leukemia (T-ALL). Results: We show that leukemias of ambiguous lineage do not segregate as a separate entity but exhibit miRNA expression profiles similar to AML, B-ALL, or T-ALL. We show that by using only 5 of the most lineage-discriminative miRNAs, we are able to define acute leukemia of ambiguous lineage as either AML or ALL. Conclusion: Our results indicate the presence of a myeloid or lymphoid lineage-specific genotype, as reflected by miRNA expression, in these acute leukemias despite their ambiguous immunophenotype. miRNA-based classification of acute leukemia of ambiguous lineage might be of additional value in therapeutic decision making. Clin Cancer Res; 19(8); 2187–96. ©2013 AACR.

IGFBP7 induces apoptosis of acute myeloid leukemia cells and synergizes with chemotherapy in suppression of leukemia cell survival

Despite high remission rates after chemotherapy, only 30–40% of acute myeloid leukemia (AML) patients survive 5 years after diagnosis. This extremely poor prognosis of AML is mainly caused by treatment failure due to chemotherapy resistance. Chemotherapy resistance can be caused by various features including activation of alternative signaling pathways, evasion of cell death or activation of receptor tyrosine kinases such as the insulin growth factor-1 receptor (IGF-1R). Here we have studied the role of the insulin-like growth factor-binding protein-7 (IGFBP7), a tumor suppressor and part of the IGF-1R axis, in AML. We report that IGFBP7 sensitizes AML cells to chemotherapy-induced cell death. Moreover, overexpression of IGFBP7 as well as addition of recombinant human IGFBP7 is able to reduce the survival of AML cells by the induction of a G2 cell cycle arrest and apoptosis. This effect is mainly independent from IGF-1R activation, activated Akt and activated Erk. Importantly, AML patients with high IGFBP7 expression have a better outcome than patients with low IGFBP7 expression, indicating a positive role for IGFBP7 in treatment and outcome of AML. Together, this suggests that the combination of IGFBP7 and chemotherapy might potentially overcome conventional AML drug resistance and thus might improve AML patient survival.

Primary acute myeloid leukemia cells with overexpression of EVI-1 are sensitive to all- trans retinoic acid

Enhanced expression of ecotropic viral integration site 1 (EVI-1) occurs in ∼10% of acute myeloid leukemia (AML) patients and is associated with a very poor disease outcome. Patients with EVI-1-positive AML have poor initial responses to chemotherapy and high relapse rates, indicating an urgent need for alternative treatment strategies improving clinical outcome for these patients. Because treatment of acute promyelocytic patients with all-trans retinoic acid (ATRA) has improved the survival of these patients substantially, we investigated whether ATRA might also be effective for the subgroup of AML patients with EVI-1 overexpression. Here, we show that a substantial part of the EVI-1-positive AML cases respond to ATRA by induction of differentiation and decreased clonogenic capacity of myeloid blasts. Most importantly, we demonstrate that in vivo treatment of primary EVI-1-positive AML with ATRA leads to a significant reduction in leukemic engraftment. Altogether, our results show that a considerable part of the EVI-1-positive primary AML cases are sensitive to ATRA, suggesting that combining ATRA with the currently used conventional chemotherapy might be a promising treatment strategy decreasing relapse rates and enhancing complete remissions in this poor prognostic subgroup of AML patients.

Abstract 3890: MicroRNA-551b is highly expressed in hematopoietic stem cells and expression in acute myeloid leukemia is associated with relapse and poor survival

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Despite high remission rates after chemotherapy, only 30-40% of acute myeloid leukemia (AML) patients survive five years after diagnosis. The main cause for this treatment failure is insufficient eradication of a subpopulation of chemotherapy resistant leukemic cells with a stem cell-like character. These so-called leukemic stem cells (LSC) are thought to be responsible for relapse. We hypothesized that success of novel anti-AML therapies relies on functional manipulation of genes including microRNAs, resulting in elimination of leukemic (stem) cells while sparing residual co-existing normal hematopoietic stem cells (HSC). We aimed at identification of microRNAs differentially expressed between HSC, LSC and the AML bulk obtained from the same AML bone marrow, taking into account the effects of the leukemic microenvironment. To that end, we described immunophenotypic markers that can distinguish LSC from HSC. Moreover, we identified that HSC have higher aldehyde dehydrogenase activity than LSC (Schuurhuis et al. Plos One 2013). Comparing the microRNA expression profile of LSC with that of HSC showed that microRNA-551b (miR-551b) is highly expressed in residual HSC in the AML bone marrow. To determine whether miR-551b is a HSC specific microRNA we purified stem and progenitor cell subsets from normal bone marrow and showed that miR-551b is the highest expressed in the two most primitive CD34+CD38- populations i.e. CD90+CD45RA- HSC and CD90-CD45RA- multipotent progenitors. To investigate whether the expression of miR-551b is of clinical importance in AML we determined its expression in AML bone marrow samples (n=154) and showed that high miR-551b is associated with lower complete remission (CR) rates after the first cycle of induction chemotherapy, shorter relapse free survival and a worse overall survival. In line with miR-551b being a stem cell miRNA, high expression in AML was associated with an undifferentiated morphology (FAB M0). To shed more light on the functional role of miR-551b in AML we correlated the expression of miR-551b with overall gene expression in a large panel of AML patients. Many of the genes that highly correlated with miR-551b like; MLLT3, INPP4B, HTR1F, HOPX, PROM1 and others, are also present in published HSC signatures. In conclusion, miR-551b is specifically expressed in normal stem and multipotent progenitor cells and high expression in AML is associated with poor prognosis. Currently, our research focuses on the function of miR-551b in AML. Citation Format: David C. de leeuw, Fedor Denkers, Peter Valk, Iris de Rink, Ron Kerkhoven, Gerrit Jan Schuurhuis, Gert J. Ossenkoppele, Linda Smit. MicroRNA-551b is highly expressed in hematopoietic stem cells and expression in acute myeloid leukemia is associated with relapse and poor survival. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3890. doi:10.1158/1538-7445.AM2014-3890

Abstract LB-246: MicroRNA profiling can classify acute leukemias of ambiguous lineage as either acute myeloid leukemia or acute lymphoid leukemia.

Classification of acute leukemia (AL) is based on commitment of the leukemic cells to either the myeloid or the lymphoid lineage. However, a small percentage of AL cases display features of both hematopoietic lineages and lacks immunophenotypical lineage commitment. These leukemias of ambiguous lineage represent a heterogeneous category of AL that cannot be classified as either myeloid AL (AML) or lymphoid AL (ALL). The lack of a clear classification of acute leukemias of ambiguous lineage as either AML or ALL is a hurdle in treatment choice for these patients. MicroRNAs are small single stranded RNA molecules which regulate gene expression by promoting degradation of mRNAs or repressing their translation. MicroRNA expression profiles have shown to be able to accurately discriminate AL of the lymphoid lineage from those of the myeloid lineage. Here, we compared the microRNA expression profiles of acute leukemias of ambiguous lineage with those of B-ALL, T-ALL and AML. MicroRNA expression profiles of nine patients with leukemia of ambiguous lineage and eleven patients with AML, B-ALL or T-ALL were analyzed. Unsupervised clustering analysis of the AML, B-ALL and T-ALL samples resulted in a clear separation between cases of the myeloid and lymphoid lineages. The top differentially expressed microRNAs between AML and ALL were miR-199b, miR-27a/b, miR-223, miR-23a, miR-221 and miR-150. Subsequently, we compared the general miRNA expression profiles of AL of ambiguous lineage with those of the AML, B-ALL and T-ALL cases thereby showing that these leukemias of ambiguous lineage do not segregate as a separate entity. Moreover, unsupervised clustering of all the AL samples using the top 10 percent of most variable microRNAs resulted in clustering of leukemias of ambiguous lineage with either AML, B-ALL or T-ALL. Furthermor, qRT-PCR expression analysis of the five most discriminative microRNAs between ALL and AML was able to define AL of ambiguous lineage as either AML or ALL. Thus, our results indicate the presence of a lymphoid or myeloid lineage specific microRNA expression profile in AL cases of ambiguous lineage, despite their mixed immunophenotype. At diagnosis, the classification of AL of ambiguous lineage by microRNA expression analysis might be of additional value in therapeutic decision making. Citation Format: Dave C. de Leeuw, Willemijn van den Ancker, Fedor Denkers, Rene X. Menezes, Theresia M. Westers, Gert J. Ossenkoppele, Arjan A. van de Loosdrecht, Linda Smit. MicroRNA profiling can classify acute leukemias of ambiguous lineage as either acute myeloid leukemia or acute lymphoid leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-246. doi:10.1158/1538-7445.AM2013-LB-246