Michaela Feuring-Buske

Age-dependent frequencies of NPM1 mutations and FLT3-ITD in patients with normal karyotype AML (NK-AML)

Prognosis of AML in elderly patients is poor due to adverse patient characteristics and comorbidities. In addition, disease-associated parameters reveal differences between older and younger patients with AML. Survival in normal karyotype AML (NK-AML) is influenced by different clinical and molecular markers. The aim of this work was to investigate the frequencies of molecular markers in patients with NK-AML with a focus on NPM1 mutations and FLT3-ITD in different age groups. In the present study, we analyzed the frequencies of mutations of NPM1 and FLT3-ITD in a cohort of 1,321 adult patients and 148 children with AML treated within the AMLCG99, the AML98, and AML04 trials and their distribution in different age groups. Additionally, the frequencies of mutations in CEBPA genes, FLT3-TKD, and MLL-PTD were analyzed in the cohort with NK-AML (nu2009=u2009729). Our data show that the presence of mutations of NPM1 (from 60% to 40%) and FLT3-ITD (from 50% to 20%) significantly decreased with age in adult AML. Consequently, the proportion of NPM1−/FLT3-ITD− patients increased with age. The decreasing frequency of NPM1 mutations in elderly patients was paralleled by a reduced complete remission (CR) rate in the elderly of 55% compared to 80% in the younger patients. By contrast, the frequencies of other gene mutations, like FLT3-TKD and MLL-PTD, and mutations in CEBPA were not age-dependent. The decreasing frequency of the favorable NPM1 mutations with increasing age may partially explain the worse outcome in the elderly patients. Furthermore, the increasing amount of elderly patients without NPM1 mutations or FLT3-ITD suggests that other molecular and clinical risk factors may influence prognosis in this age group.

An eight-gene expression signature for the prediction of survival and time to treatment in chronic lymphocytic leukemia

An eight-gene expression signature for the prediction of survival and time to treatment in chronic lymphocytic leukemia

The homeobox gene CDX2 is aberrantly expressed and associated with an inferior prognosis in patients with acute lymphoblastic leukemia.

Molecular characterization of acute lymphoblastic leukemia (ALL) has greatly improved the ability to categorize and prognostify patients with this disease. In this study, we show that the proto-oncogene CDX2 is aberrantly expressed in the majority of cases with B-lineage ALL and T-ALL. High expression of CDX2 correlated significantly with the ALL subtype pro-B ALL, cALL, Ph+ ALL and early T-ALL. Furthermore, high expression of CDX2 was associated with inferior overall survival and showed up as a novel and strong risk factor for ALL in bivariate analysis. Functional analyses showed that overexpression of Cdx2 in murine bone marrow progenitors perturbed genes involved in lymphoid development and that depletion of CDX2 in the human ALL cell line Nalm6 inhibited colony formation. These data indicate that aberrant CDX2 expression occurs frequently and has prognostic impact in adult patients with ALL.

The FLT3ITD mRNA level has a high prognostic impact in NPM1 mutated, but not in NPM1 unmutated, AML with a normal karyotype

The impact of a FLT3-internal tandem duplication (FLT3ITD) on prognosis of patients with acute myeloid leukemia (AML) is dependent on the ratio of mutated to wild-type allele. In 648 normal karyotype (NK) AML patients, we found a significant independent effect of the quantitative FLT3ITD mRNA level--measured as (FLT3ITD/wtFLT3)/(FLT3ITD/wtFLT3+1)--on outcome. Moreover, this effect was clearly seen in 329 patients with a mutated NPM1 gene (NPM1+), but not in 319 patients without a NPM1 mutation (wtNPM1). In a multivariate Cox regression model, the quantitative FLT3ITD mRNA level showed an independent prognostic impact on overall survival (OS) and relapse-free survival (RFS) only in the NPM1+ subgroup (OS: hazard ratio, 5.9; [95% confidence interval [CI]: 3.1-11.2]; RFS: hazard ratio, 7.5 [95% CI: 3.4-16.5]). The FLT3ITD mRNA level contributes to relapse risk stratification and might help to guide postremission therapy in NPM1-mutated AML.

Expression analysis of genes located in the minimally deleted regions of 13q14 and 11q22-23 in chronic lymphocytic leukemia-unexpected expression pattern of the RHO GTPase activator ARHGAP20.

In chronic lymphocytic leukemia (CLL), 13q14 and 11q22‐23 deletions are found in 2/3 of the cases. 11q22‐23 deletions are associated with poor survival, whereas 13q14 deletions as single abnormality are often found in indolent disease forms. The molecular basis for this difference in prognosis is not known. We examined the 13q14 and 11q22‐23 minimally deleted regions (MDRs) for differentially expressed genes by analyzing 154 microarray CLL gene expression data sets. We were able to generate a detailed gene expression map of the MDRs demonstrating a gene dosage effect. Surprisingly, ARHGAP20 encoding the RHO GTPase activating protein 20, which is located in the 11q22‐23 MDR, showed—counterintuitively—a significantly higher expression in cases with 11q22‐23 deletions compared with cases with no detectable genetic lesion or trisomy 12. Interestingly, cases with 13q14 deletions also had higher ARHGAP20 expression. These expression level changes were confirmed by quantitative PCR in 110 additional CLL samples. The ARHGAP20 gene encodes an evolutionarily conserved protein. In the zebra fish (Danio rerio) genome the syntenic regions of human chromosomal bands 13q14 and 11q22‐23 are juxtaposed. The similar expression profiles of ARHGAP20 in 13q14 and 11q22‐23 deleted CLL cases suggest a molecular connection and an intriguing mechanism of regulation.

AML1–ETO meets JAK2: clinical evidence for the two hit model of leukemogenesis from a myeloproliferative syndrome progressing to acute myeloid leukemia

AML1–ETO meets JAK2: clinical evidence for the two hit model of leukemogenesis from a myeloproliferative syndrome progressing to acute myeloid leukemia

The leukemogenicity of Hoxa9 depends on alternative splicing.

Although the transforming potential of Hox genes is known for a long time, it is not precisely understood to which extent splicing is important for the leukemogenicity of this gene family. To test this for Hoxa9, we compared the leukemogenic potential of the wild-type Hoxa9, which undergoes natural splicing, with a full-length Hoxa9 construct, which was engineered to prevent natural splicing (Hoxa9FLim). Inability to undergo splicing significantly reduced in vivo leukemogenicity compared to Hoxa9-wild-typed. Importantly, Hoxa9FLim could compensate for the reduced oncogenicity by collaborating with the natural splice variant Hoxa9T, as co-expression of Hoxa9T and Hoxa9FLim induced acute myeloid leukemia (AML) after a comparable latency time as wild-type Hoxa9. Hoxa9T on its own induced AML after a similar latency as Hoxa9FLim, despite its inability to bind DNA. These data assign splicing a central task in Hox gene mediated leukemogenesis and suggest an important role of homeodomain-less splice variants in hematological neoplasms.

GPR56 contributes to the development of acute myeloid leukemia in mice

The G protein-coupled receptor 56 (GPR56) was identified as part of the molecular signature of functionally validated leukemic stem cells isolated from patients with acute myeloid leukemia (AML). This report now demonstrates particularly high expression of GPR56 in patients with mutant NPM1 and FLT3-length mutation and association of high GPR56 expression with inferior prognosis in a large patient cohort treated in two independent multicenter phase III trials. Functional relevance of GPR56 expression was validated in mice, in which co-expression of Gpr56 significantly accelerated HOXA9-induced leukemogenesis and vice versa knockdown of Gpr56 delayed onset of HOXA9/MEIS1-induced AML. Overexpression of Gpr56 grossly changed the molecular phenotype of Hoxa9-transduced cells affecting pathways involved in G protein-coupled receptors (GPRCs) and associated intracellular signaling. Blockage of surface GPR56 by an anti-GPR56 antibody successfully impaired engraftment of primary human AML cells. In summary, these data demonstrate that high expression of GPR56 is able to contribute to AML development and characterize the GPR56 as a potential novel target for antibody-mediated antileukemic strategies.

Leukemic progenitor cells are susceptible to targeting by stimulated cytotoxic T cells against immunogenic leukemia-associated antigens

Leukemic stem cells (LSC) might be the source for leukemic disease self‐renewal and account for disease relapse after treatment, which makes them a critical target for further therapeutic options. We investigated the role of cytotoxic T‐lymphocytes (CTL) counteracting and recognizing LSC. Leukemia‐associated antigens (LAA) represent immunogenic structures to target LSC. We enriched the LSC‐containing fraction of 20 AML patients and hematopoietic stem cells (HSC) of healthy volunteers. Using microarray analysis and qRT‐PCR we detected high expression of several LAA in AML cells but also in LSC. PRAME (pu2009=u20090.0085), RHAMM (pu2009=u20090.03), WT1 (pu2009=u20090.04) and Proteinase 3 (pu2009=u20090.04) showed significant differential expression in LSC compared with HSC. PRAME, RHAMM and WT1 are furthermore also lower expressed on leukemic bulk. In contrast, Proteinase 3 indicates a higher expression on leukemic bulk than on LSC. In colony forming unit (CFU) immunoassays, T cells stimulated against various LAA indicated a significant inhibition of CFUs in AML patient samples. The LAA PRAME, RHAMM and WT1 showed highest immunogenic responses with a range up to 58‐83%. In a proof of principle xenotransplant mouse model, PRAME‐stimulated CTL targeted AML stem cells, reflected by a delayed engraftment of leukemia (pu2009=u20090.0159). Taken together, we demonstrated the expression of several LAA in LSC. LAA‐specific T cells are able to hamper LSC in immunoassays and in a mouse model, which suggests that immunotherapeutic approaches have the potential to target malignant stem cells.