ALBERT

Description: Pediatric mixed-lineage leukemia (MLL)–rearranged acute monoblastic leukemia with t(9;11)(p22;q23) has a favorable outcome compared with other MLL-rearranged AML. The biologic background for this difference remains unknown. Therefore, we compared gene expression profiles (GEPs; Affymetrix HGU133 + 2.0) of 26 t(9;11)(p22;q23) patients with 42 other MLL-rearranged AML patients to identify differentially expressed genes. IGSF4, a cell-cell adhesion molecule, was found to be highly expressed in t(9;11)(p22;q23) patients, which was confirmed by real-time quantitative polymerase chain reaction and Western blot. IGSF4 expression within t(9;11)(p22;q23) patients was 4.9 times greater in French-American-British morphology classification (FAB)–M5 versus other FAB-types (P = .001). Methylation status investigation showed that high IGSF4-expressing t(9;11)(p22;q23) patients with FAB-M5 have no promoter hypermethylation, whereas all other cases do. Cell-line incubation with demethylating agent decitabine resulted in promoter demethylation and increased expression of IGSF4. Down-regulation of IGSF4 by siRNA did not affect proliferation or drug sensitivity. In a cohort of 79 MLL-rearranged AML cases, we show significant better overall survival for cases with high IGSF4 expression (5-year overall survival 0.70 vs 0.37, P = .03) In conclusion, we identified IGSF4 overexpression to be discriminative for t(9;11)(p22;q23) patients with FAB-M5, regulated partially by promoter methylation and resulting in survival benefit.

Description: Abstract 3963 Poster Board III-899 Children with Down Syndrome (DS) have an increased risk of developing acute lymphoblastic leukemia (ALL). DS ALL patients differ in presenting characteristics from ALL patients without DS (non-DS ALL). Recent evidence suggests that a unique genetic event may characterize DS ALL, i.e. an activating mutation localized at R683 in the Janus Kinase 2 (JAK2) gene, which occurs in 18% of DS ALL cases, and differs from the JAK2 mutations that are typically found in Myeloproliferatieve diseases (Bercovich et al, The Lancet 2008). However, Mullighan et al. recently reported also JAK R683 mutations in non-DS high risk ALL (PNAS, 2009). Furthermore, we and others described deletions in B-cell development genes in high-risk ALL (Den Boer et al, Lancet Oncology 2009, and Mullighan, NEJM 2009). One of these genes, IKZF1, which encodes the lymphoid transcription factor IKAROS, was found to be an indicator of poor prognosis in this high-risk group. In the present study, we studied deletions in B-cell development genes in DS ALL, utilizing array-Comparative Genomic Hybridisation (array-CGH, 105K Agilent). Moreover, genomic DNA was PCR-amplified with specific primers to detect the isoform 6 of IKAROS, which consists of a deletion of exon 3-6 resulting in expression of a dominant-negative form of IKAROS, with intact homodimerization but reduced DNA-binding capacity. We used direct sequencing for mutation screening of the pseudo-kinase and kinase domains of JAK2. Of 34 DS ALL patients treated according to the DCOG treatment protocols samples were available. All 34 patients had B-cell precursor ALL. Median follow up time was 5.7 years (range 1.2 – 15.4 years).In total, 19/34 (56%) DS ALL patients had one or more deletions in B-cell development genes (Table 1). Affected genes included the transcription factors IKZF1 (41%, n=14), PAX5 (12%, n=4) and VPREB1 (18%, n=6). These aberrations were not mutually exclusive. No deletions were found in EBF1 and TCF3 (E2A). Deletions in the PAX5 gene were part of larger deletions (≥0.5 million bases), whereas the other genes were mainly affected by focal deletions (

Description: Abstract 2390 Poster Board II-367 In recent years miRNA expression patterns have been related to tumor (sub)type and disease outcome in various types of cancer, including acute myeloid leukemia (AML). Therefore, miRNA profiling may provide information for better classification and risk stratification of AML subtypes, and in addition may shed light on the underlying disease biology. To date large scale miRNA profiling has only been performed in adult AML, which differs from childhood AML in many ways, reflected in differences in cytogenetic subgroups, response to therapy and prognosis. However, knowledge on the role of miRNAs in childhood AML is limited. To answer the question if differential expression of miRNAs can be observed in subtypes of pediatric AML, we used quantitative RT-PCR to determine the expression levels of miR-29a, -155, -196a and -196b in a selection of de novo pediatric AML patients (n=49-84) representing the different cytogenetic subtypes. These miRNAs have been reported to be differentially expressed in cytogenetic and morphological subtypes of adult AML. In AML with t(10;11) MLL-rearrangements (n=5) versus all other AML samples expression of miR-29a was 2.4-fold lower (p=0.005). However, differences in expression of miR-29a in all MLL-rearranged AML compared to other AML patients were small and not significant, in contrast to what was found in adults. MiR-155 was upregulated 2.3-fold (p=0.0003) in FLT3-ITD-mutated AML (n=8) compared to all other AML samples, which is consistent to what has been reported for adult AML. The expression of both miRNA-196a and –196b differed extremely between patients. High expression of both miRNAs was observed in patients carrying MLL-gene rearrangements, NPM1 mutations, or FLT3-ITD mutations in a normal karyotype background. Low expression was found in t(8;21), inv(16) and t(15;17) subtypes (including those with FLT3-ITD mutations), and in patients with mutated CEBPA. The median difference between these groups of patients was 147-fold for miR-196a (n=73, p

Description: Abstract 5035 Children with Down Syndrome (DS) have an increased risk of developing leukemia, including both acute myeloid (ML-DS), as well as acute lymphoblastic leukemia (DS-ALL). ML-DS can be preceded by a pre-leukemic clone in newborns (transient leukemia-TL), which in most cases resolves spontaneously. Janus Kinase (JAK) 1-3 belongs to a family of intracellular non-receptor protein tyrosine kinases that transduce cytokine-mediated signals via the JAK-STAT pathway. JAK plays an important role in regulating the processes of cell proliferation, differentiation and apoptosis in response to cytokines and growth factors. Mullighan et al. described JAK 1-3 mutations in non-DS high-risk childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL; PNAS, 2009). In T-ALL, JAK-1 mutations are a frequent event (∼25%) as reported among others by Jeong et al (Clinical Cancer Research, 2008). Mutations in JAK-2 and JAK-3 have been described in TL and ML-DS. Bercovich et al. recently reported mutations within the pseudokinase domain of JAK-2 in DS-ALL patients (Lancet 2008). This activating JAK-2 mutation differs from the V617F exon 14 mutation found in myeloproliferative diseases. However, JAK-1 has never been investigated in Down syndrome leukemias. Therefore we performed mutational analysis of the pseudokinase and kinase domains of JAK-1, 2 and 3 by direct sequencing in 8 TL, 16 ML-DS and 35 DS-ALL samples taken at initial diagnosis. The TL and ML-DS samples were unpaired. In the ML-DS group, 12 patients were classified as FAB M7, 3 as FAB M0 and 1 as FAB M6; all 35 DS-ALL patients were classified as BCP-ALL. Mutations in JAK-1 were found in 1 ML-DS patient (D625R) and in 1 DS-ALL patient (V651M). These mutations were localised in the same region of the pseudokinase domain, but not identical to the activating mutations in JAK1 described in high-risk ALL (Mullighan et al., PNAS 2009). The JAK-1 mutated ML-DS patient had a complex karyogram, and the DS ALL patient a normal karyotype. No events occurred in either of the patients with a follow-up of 2.4 and 3.1 years, respectively. JAK-2 activating mutations at position R683 were found in 5/35 (14.3%) of the DS-ALL patients. These patients had diverse cytogenetic aberrations, and had no events at a median follow up of 4.4 years. In the TL and ML-DS patients no mutations were identified in JAK-2. For JAK-3, 1 TL-patient (13%) and 1 ML-DS patient (6.3%) harboured the A573V-mutation. This activating mutation is previously described in ML-DS patients and the megakaroyblastic cell line CMY ((Kiyoi et al, Leukemia 2007). Because the mutations occur in both TL and ML-DS, this suggests that they do not play a role in the clonal progression model from TL to ML-DS. A mutation at JAK3 R1092C, which to our knowledge has never been reported before, was found in 1 DS-ALL patient. This patient had a deletion on chromosome 12 (p11p13), and was in CCR with a follow up of 5 years. In conclusion, JAK-mutations are rare in DS-leukemias, except for JAK-2 mutations in DS-ALL, which occur in approximately 15% of cases. The rarity of JAK-1 mutations in DS is in accordance with the rarity of T-ALL in DS. Of interest, none of the DS ALL cases with a JAK-2 mutation relapsed so far, which differs from the patients with JAK-2 mutations that were recently in high-risk BCP-ALL. Hence, JAK-2 may be an interesting novel therapeutic target. Disclosures No relevant conflicts of interest to declare.

Description: Hematopoiesis is tightly controlled by transcription regulatory networks, but how and when specific transcription factors control lineage commitment are still largely unknown. Within the hematopoietic stem cell (Lin−Sca-1+c-Kit+) compartment these lineage-specific transcription factors are expressed at low levels but are up-regulated with the process of lineage specification. CCAAT/enhancer binding protein α (C/EBPα) represents one of these factors and is involved in myeloid development and indispensable for formation of granulocytes. To track the cellular fate of stem and progenitor cells, which express C/EBPα, we developed a mouse model expressing Cre recombinase from the Cebpa promoter and a conditional EYFP allele. We show that Cebpa/EYFP+ cells represent a significant subset of multipotent hematopoietic progenitors, which predominantly give rise to myeloid cells in steady-state hematopoiesis. C/EBPα induced a strong myeloid gene expression signature and down-regulated E2A-induced regulators of early lymphoid development. In addition, Cebpa/EYFP+ cells compose a fraction of early thymic progenitors with robust myeloid potential. However, Cebpa/EYFP+ multipotent hematopoietic progenitors and early thymic progenitors retained the ability to develop into erythroid and T-lymphoid lineages, respectively. These findings support an instructive but argue against a lineage-restrictive role of C/EBPα in multipotent hematopoietic and thymic progenitors.