Acute lymphoblastic leukemia (ALL) arising in infants less than 1 year of age is characterized by genetic rearrangements of the KMT2A gene (previously MLL) and an exceedingly poor prognosis. We have previously shown that infant KMT2A -rearranged (KMT2A -R) ALL has one of the lowest numbers of somatic mutations of any sequenced cancer with a mean of only 1.3 non-silent mutations being present in all leukemia cells per patient (Andersson et al., Nat Genet, 2017). Despite the paucity of mutations, activating mutations within the PI3K/RAS signaling pathway were present in about half of cases, most of which were subclonal with a mutant allele frequency (MAF) <0.30. In addition, some patients harbored multiple activating mutations at varying MAFs, suggesting the presence of multiple low-frequency leukemia clones. Based on these findings, we hypothesized that the genetic landscape of infant KMT2A -R ALL is more clonally heterogeneous than other pediatric leukemias, which might contribute to its poor prognosis as current treatment regimens may not eliminate all clones.

Fusion genes are potent driver mutations in cancer. In this study, we delineate the fusion gene landscape in a consecutive series of 195 paediatric B-cell precursor acute lymphoblastic leukaemia (BCP ALL). Using RNA sequencing, we find in-frame fusion genes in 127 (65%) cases, including 27 novel fusions. We describe a subtype characterized by recurrent IGH-DUX4 or ERG-DUX4 fusions, representing 4% of cases, leading to overexpression of DUX4 and frequently co-occurring with intragenic ERG deletions. Furthermore, we identify a subtype characterized by an ETV6-RUNX1 -like gene-expression profile and coexisting ETV6 and IKZF1 alterations. Thus, this study provides a detailed overview of fusion genes in paediatric BCP ALL and adds new pathogenetic insights, which may improve risk stratification and provide therapeutic options for this disease.

Acquired activating mutations in kinase/PI3K/RAS signaling pathways occur in about half of pediatric acute leukemia cases with Mixed Lineage Leukemiagene rearrangements (MLL-R; MLL, also known as KMT2A). Mutations resulting in activated signaling cooperate with the MLL-R in mouse leukemia models, however, detailed insight on the effect on the transcriptional and proteomic landscape is lacking. In infant MLL-R acute lymphoblastic leukemia (ALL), a subtype with a very poor prognosis, a majority of the activating mutations are subclonal, but the biological mechanisms by which subclonal mutations affect leukemogenesis remain unclear.

Myelodysplastic/myeloproliferative neoplasms, unclassifiable (MDS/MPN-U) are rare genetically heterogeneous hematologic diseases associated with older age and a poor prognosis. If the disease progresses into acute myeloid leukemia (AML), it is often refractory to treatment. To gain insight into genetic alterations associated with disease progression, whole exome sequencing and single nucleotide polymorphism arrays were used to characterize the bone marrow and blood samples from a 39-year-old woman at MDS/MPN-U diagnosis and at AML progression, in which routine genetic diagnostics had not identified any genetic alterations. The data revealed the presence of a partial tandem duplication of the MLL gene as the only detectable copy number change and 11 non-silent somatic mutations, including DNMT3A R882H and NRAS G13D. All somatic lesions were present both at initial MDS/MPN-U diagnosis and at AML presentation at similar mutant allele frequencies. The patient has since had two extramedullary relapses and is at high risk of a future bone marrow relapse. A directed ex vivo drug sensitivity analysis showed that the patient's AML cells are sensitive to, for example, the MEK inhibitor trametinib and the proteasome inhibitor bortezomib, indicating that she may benefit from treatment with these drugs. © 2016 Wiley Periodicals, Inc.

Lars Bullinger, Jinghui Zhang, Jeffery Klco, James Downing and colleagues report a detailed genomic analysis of pediatric and adult core-binding factor acute myeloid leukemias (CBF-AMLs). They identify recurrent mutations in CCND2, MGA, DHX15 and ZBTB7A and highlight dramatic differences in the landscape of cooperating mutations between different CBF-AML subtypes. Acute myeloid leukemia (AML) comprises a heterogeneous group of leukemias frequently defined by recurrent cytogenetic abnormalities, including rearrangements involving the core-binding factor (CBF) transcriptional complex. To better understand the genomic landscape of CBF-AMLs, we analyzed both pediatric (n = 87) and adult (n = 78) samples, including cases with RUNX1-RUNX1T1 (n = 85) or CBFB-MYH11 (n = 80) rearrangements, by whole-genome or whole-exome sequencing. In addition to known mutations in the Ras pathway, we identified recurrent stabilizing mutations in CCND2, suggesting a previously unappreciated cooperating pathway in CBF-AML. Outside of signaling alterations, RUNX1-RUNX1T1 and CBFB-MYH11 AMLs demonstrated remarkably different spectra of cooperating mutations, as RUNX1-RUNX1T1 cases harbored recurrent mutations in DHX15 and ZBTB7A, as well as an enrichment of mutations in epigenetic regulators, including ASXL2 and the cohesin complex. This detailed analysis provides insights into the pathogenesis and development of CBF-AML, while highlighting dramatic differences in the landscapes of cooperating mutations for these related AML subtypes.

Anna Andersson, Tanja Gruber, James Downing and colleagues report a genomic analysis of infant acute lymphoblastic leukemias with MLL rearrangements. They identify recurrent activating mutations in tyrosine kinase, phosphatidylinositol 3-kinase and RAS pathway genes but find that these mutations were often present in minor subclones and lost at the time of relapse. Infant acute lymphoblastic leukemia (ALL) with MLL rearrangements (MLL-R) represents a distinct leukemia with a poor prognosis. To define its mutational landscape, we performed whole-genome, exome, RNA and targeted DNA sequencing on 65 infants (47 MLL-R and 18 non–MLL-R cases) and 20 older children (MLL-R cases) with leukemia. Our data show that infant MLL-R ALL has one of the lowest frequencies of somatic mutations of any sequenced cancer, with the predominant leukemic clone carrying a mean of 1.3 non-silent mutations. Despite this paucity of mutations, we detected activating mutations in kinase-PI3K-RAS signaling pathway components in 47% of cases. Surprisingly, these mutations were often subclonal and were frequently lost at relapse. In contrast to infant cases, MLL-R leukemia in older children had more somatic mutations (mean of 6.5 mutations/case versus 1.3 mutations/case, P = 7.15 × 10−5) and had frequent mutations (45%) in epigenetic regulators, a category of genes that, with the exception of MLL, was rarely mutated in infant MLL-R ALL.

Abstract Acute myeloid leukemia (AML) results from mutations that promote aberrant self-renewal, proliferation and differentiation. The specific combination of mutations not only influences the phenotype of the leukemia but also its response to therapy. The core-binding factor (CBF) leukemias represent one of the most common forms of AML and are defined by genetic lesions that target the RUNX1(AML1)/CBFβ transcription factor complex. These include t(8;21)[RUNX1-ETO], inv(16) and t(16;16)[CBFβ-MYH11], point mutations in RUNX1, and several other rarer translocations that target RUNX1. The AML1/CBFβ transcription factor complex functions as a master transcriptional regulator that is essential for the generation of definitive hematopoietic stem cells during development, and plays an important role in the differentiation of a number of hematopoietic lineages including T cells and megakaryocytes. Using a conditional Runx1-Eto murine knock-in model, we previously demonstrated that the expression of Runx1-Eto was insufficient to induce leukemia. To define the landscape of mutation that cooperate with RUNX1-ETO in leukemogenesis, the St. Jude Children's Research Hospital - Washington University Pediatric Cancer Genome Project performed whole genome DNA Sequencing (WGS) on a cohort of 17 pediatric CBF leukemia [7 t(8;21) and 10 inv(16) cases. Somatic mutations were identified using the PCGP analytical pipeline and all identified mutations were experimentally validated. The frequency of the identified mutations were then assessed by performing exome sequencing on a validation cohort consisting of 151 pediatric and adult CBF leukemias [79 t(8;21) and 72 inv(16)]. The combined analyses identified very few copy number alterations (CNAs) or structural alterations per case, and an average of only 12.5 somatic non-synonymous single nucleotide variations or insertions/deletions per case. Despite the low burden of mutations, recurrent activating mutations in the Kinase/RAS signaling pathway (NRAS, KIT, FLT3, KRAS, and PTPN11, and inactivating mutations in NF1) were collectively identified in >67% of cases An even more striking result was the identification of a high frequency of loss-of-function mutations in genes that encode key epigenetic regulatory proteins (28% including ASXL2, EZH2, UTX, SETD2, MLL2/3), and missense and loss of function mutations in genes that encode components of the cohesin complex (10% including SMC1A, SMC2, RAD21). Moreover, the frequency of both classes of mutations were significantly higher in the t(8;21) versus inv(16) containing cases (epigenetic regulatory mutations 48% vs 6.25% and cohesin mutations 20% vs 0%). No significant difference was noted in the frequency of cohesin mutations between pediatric and adult cases. The identified mutations targeted genes encoding core components of the cohesion complex required to generate the ring shaped DNA binding structure, SMC1A, SMC3, and RAD21, as well as a single case with a mutation in the SMC3 acetyl transferase ESCO2. In addition, loss of the X chromosome is a frequent somatic event in female patients with t(8;21) leukemia and SMC1A is located on Chr. X and is not subjected to X inactivation, suggesting that loss of Chr. X leads to haploinsufficiency. The majority of leukemias with cohesion mutations also contain activating mutation in the RAS/Kinase signaling pathway, suggesting that these mutations cooperate with AML1-ETO to induce leukemia. Collectively, these data provide an unprecedented view of the range of cooperating mutations that occur in the CBF leukemias and highlight fundamental differences between t(8;21) and inv(16) containing cases. Citation Format: Amanda Larson Gedman, Jinjun Cheng, Xiang Chen, Ina Radtke, Anna Andersson, Heather Mulder, Kristy Boggs, Bhavin Vadodaria, Donald Yergeau, Joy Nakitandwe, Lars Bullinger, Michael Kuehn, Hartmut Doehner, Konstanze Doehner, Sheila Shurtleff, John Easton, Richard Wilson, Jinghui Zhang, James R. Downing. The landscape of somatic mutations in the core-binding factor acute myeloid leukemias. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A15.

The genetic basis of hypodiploid acute lymphoblastic leukemia (ALL), a subtype of ALL characterized by aneuploidy and poor outcome, is unknown. Genomic profiling of 124 hypodiploid ALL cases, including whole-genome and exome sequencing of 40 cases, identified two subtypes that differ in the severity of aneuploidy, transcriptional profiles and submicroscopic genetic alterations. Near-haploid ALL with 24-31 chromosomes harbor alterations targeting receptor tyrosine kinase signaling and Ras signaling (71%) and the lymphoid transcription factor gene IKZF3 (encoding AIOLOS; 13%). In contrast, low-hypodiploid ALL with 32-39 chromosomes are characterized by alterations in TP53 (91.2%) that are commonly present in nontumor cells, IKZF2 (encoding HELIOS; 53%) and RB1 (41%). Both near-haploid and low-hypodiploid leukemic cells show activation of Ras-signaling and phosphoinositide 3-kinase (PI3K)-signaling pathways and are sensitive to PI3K inhibitors, indicating that these drugs should be explored as a new therapeutic strategy for this aggressive form of leukemia.

Acute lymphoblastic leukemia (ALL) is the commonest childhood malignancy and is characterized by recurring structural genetic alterations. Previous studies of DNA methylation suggest epigenetic alterations may also be important, but an integrated genome-wide analysis of genetic and epigenetic alterations in ALL has not been performed. We analyzed 137 B-lineage and 30 T-lineage childhood ALL cases using microarray analysis of DNA copy number alterations and gene expression, and genome-wide cytosine methylation profiling using the HpaII tiny fragment enrichment by ligation-mediated PCR (HELP) assay. We found that the different genetic subtypes of ALL are characterized by distinct DNA methylation signatures that exhibit significant correlation with gene expression profiles. We also identified an epigenetic signature common to all cases, with correlation to gene expression in 65% of these genes, suggesting that a core set of epigenetically deregulated genes is central to the initiation or maintenance of lymphoid transformation. Finally, we identified aberrant methylation in multiple genes also targeted by recurring DNA copy number alterations in ALL, suggesting that these genes are inactivated far more frequently than suggested by structural genomic analyses alone. Together, these results demonstrate subtype- and disease-specific alterations in cytosine methylation in ALL that influence transcriptional activity, and are likely to exert a key role in leukemogenesis.

One of the most rapidly evolving genes in humans, PRDM9 , is a key determinant of the distribution of meiotic recombination events. Mutations in this meiotic-specific gene have previously been associated with male infertility in humans and recent studies suggest that PRDM9 may be involved in pathological genomic rearrangements. In studying genomes from families with children affected by B-cell precursor acute lymphoblastic leukemia (B-ALL), we characterized meiotic recombination patterns within a family with two siblings having hyperdiploid childhood B-ALL and observed unusual localization of maternal recombination events. The mother of the family carries a rare PRDM9 allele, potentially explaining the unusual patterns found. From exomes sequenced in 44 additional parents of children affected with B-ALL, we discovered a substantial and significant excess of rare allelic forms of PRDM9 . The rare PRDM9 alleles are transmitted to the affected children in half the cases; nonetheless there remains a significant excess of rare alleles among patients relative to controls. We successfully replicated this latter observation in an independent cohort of 50 children with B-ALL, where we found an excess of rare PRDM9 alleles in aneuploid and infant B-ALL patients. PRDM9 variability in humans is thought to influence genomic instability, and these data support a potential role for PRDM9 variation in risk of acquiring aneuploidies or genomic rearrangements associated with childhood leukemogenesis.

To define the mutation spectrum in non-Down syndrome acute megakaryoblastic leukemia (non-DS-AMKL), we performed transcriptome sequencing on diagnostic blasts from 14 pediatric patients and validated our findings in a recurrency/validation cohort consisting of 34 pediatric and 28 adult AMKL samples. Our analysis identified a cryptic chromosome 16 inversion (inv(16)(p13.3q24.3)) in 27% of pediatric cases, which encodes a CBFA2T3-GLIS2 fusion protein. Expression of CBFA2T3-GLIS2 in Drosophila and murine hematopoietic cells induced bone morphogenic protein (BMP) signaling and resulted in a marked increase in the self-renewal capacity of hematopoietic progenitors. These data suggest that expression of CBFA2T3-GLIS2 directly contributes to leukemogenesis.

Abstract Acute Megakaryoblastic Leukemia (AMKL) accounts for ∼10% of childhood acute myeloid leukemia (AML). Although AMKL patients with Down syndrome (DS-AMKL) have an excellent 5 year event-free survival (EFS), non-DS-AMKL patients have an extremely poor outcome with a 3 year EFS < 40%. To define the landscape of mutations that occur in non-DS-AMKL, we performed transcriptome sequencing on diagnostic blasts from 14 cases. Our results identified chromosomal rearrangements resulting in the expression of novel fusion transcripts in 12/14 cases. Remarkably, in 7/14 cases, we detected an inversion on chromosome 16 [inv(16)(p13.3;q24.3)] that resulted in the juxtaposition of CBFA2T3, a member of the ETO family of transcription factors, next to GLIS2 resulting in a CBFA2T3-GLIS2 chimeric gene encoding an in frame fusion protein. GLIS2 is a member of the GLI family of transcription factors that mediate sonic hedgehog (SHH) signaling and has been demonstrated to play a role in regulating expression of GLI target genes. Evaluation of a recurrency cohort of 52 samples including 24 additional pediatric cases and 28 adult cases revealed 6 additional pediatric samples carrying the fusion for an overall frequency of 34% in pediatric AMKL. To gain insight into the mechanism whereby CBFA2T3-GLIS2 promotes leukemogenesis, we introduced the fusion into murine hematopoietic cells and assessed its effect on in vitro colony replating as a surrogate measure of self-renewal. Cells transduced with a mCherry expressing retrovirus failed to form colonies after the 2nd replating. By contrast, expression of either wild-type GLIS2 or CBFA2T3-GLIS2 resulted in a marked increase in the self-renewal capacity, with colony formation persisting through 12 replatings. Immunophenotypic analysis of the CBFA2T3-GLIS2 expressing colonies revealed evidence of megakaryocytic differentiation. GLI transcription factors modulate expression of multiple downstream targets including components of BMP, WNT, and SHH pathways. To interrogate these pathways as potential contributors to the enhanced self-renewal capacity, we conducted luciferase reporter assays and found that CBFA2T3-GLIS2 functioned as a strong activator of the BMP responsive element. Furthermore, expression of CBFA2T3-GLIS2 in Drosophila resulted in ectopic expression of endogenous dpp, the fly homolog of BMP4, and conferred a dpp gain of function phenotype. Taken together these data identify a novel inv(16)-encoded CBFA2T3-GLIS2 fusion protein as a recurrent driver mutation in ∼35% of non-infant pediatric non-DS-AMKLs. The alteration of a key transcriptional regulator within the SHH signaling pathways in a substantial percentage of pediatric AMKL raises the possibility that inhibition of this pathway or downstream activated pathways may have a therapeutic benefit in this aggressive form of AML. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4867. doi:1538-7445.AM2012-4867

Abstract Hypodiploid acute lymphoblastic leukemia (ALL) is an aggressive form of leukemia characterized by multiple whole chromosomal losses and very dismal outcome. Our previous genome wide study of hypodiploid childhood ALL cases treated by the Children's Oncology Group and St Jude, employed interrogation of DNA copy number alterations using Affymetrix SNP 6.0 microarrays, candidate gene resequencing and gene expression profiling using Affymetrix U133 Plus 2.0 microarrays. These analyses showed that this disease can be divided into multiple subtypes characterized by variation in the degree of aneuploidy, distinct submicroscopic deletions, sequence mutations and gene expression profile. Near haploid ALL (24-31 chromosomes) frequently harbors alterations of genes regulating Ras signaling (67.6%; NF1, NRAS, KRAS, PTPN11, FLT3, and PAG1), IKZF3 (encoding the lymphoid transcription factor AIOLOS; 13.2%), and a histone gene cluster at 6p22 (17.6%), while low hypodiploid ALL (32-39 chromosomes) is enriched for IKZF2 (HELIOS; 52.9%), TP53 (70.6%) and RB1 (41.2%) alterations. A striking finding was exclusivity of Ras signaling and IKZF2/3 alterations, and biochemical indications of Ras pathway activation in both near haploid and low hypodiploid ALL. To further interrogate the genomic changes of hypodiploid ALL, we performed next generation sequencing using either Illumina GAIIx or HiSeq3000 sequencers on both tumor and matched remission DNA. Whole genome sequencing to at least 30 fold haploid coverage was performed on 10 near haploid and 8 low hypodiploid cases, and whole exome sequencing (Agilent SureSelect Human All Exon 50Mb) on 5 near haploid and 1 low hypodiploid cases. The burden of single nucleotide variations (SNVs) and insertion/deletion (indel) mutations was in general low in this ALL subtype, with 0-5 indels and 9-95 SNVs in coding regions and untranslated leader regions in the whole genome sequenced cases, where the majority of cases had fewer than 30 SNVs. Further, the number of structural variations, including the ones too small to be identified by SNP microarray analysis, and structural rearrangements, were also low, with less than 25 structural variations identified in the whole genome sequenced cases. For the whole exomes, between 10 and 42 non-silent SNVs and 1-2 indels were identified per case. No recurrent alterations not previously identified in the hypodiploid cohort were found in these 24 cases, indicating that the initial genome wide study of this cohort identified the major recurrent alterations in hypodiploid ALL. However, the recurrence screening including the remaining 78 near haploid and low hypodiploid cases in our cohort on the alterations identified by the whole genome and exome sequencing study is ongoing. Altogether, these findings provide critical new insights into the genetic basis of hypodiploid ALL, and indicate that therapeutic targeting of the Ras pathway should be pursued in this disease. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4870. doi:1538-7445.AM2012-4870

Abstract Abstract 69 Infant (< 1 year of age) acute lymphoblastic leukemia (ALL) is a rare disease characterized by rearrangements of the Mixed Lineage Leukemia (MLL) gene at 11q23 and a poor prognosis. In an effort to determine the total complement of somatic mutations occurring in this high risk leukemia, we performed paired-end whole genome sequencing (WGS) on diagnostic leukemia blasts and matched germ line samples from 22 infants with MLL rearranged ALL using the Illumina platform. In addition, we sequenced 2 paired relapse samples. Somatic alterations, including single nucleotide variations (SNV), and structural variations (SV) including insertions, deletions, inversion, and inter- and intra-chromosomal rearrangements were detected using complementary analysis pipelines including Bambino, CREST and CONSERTING. Validation of identified somatic mutations was performed using PCR amplification of the leukemia and germ line DNA followed by Sanger or 454-based sequencing, or by array-based capture followed by Illumina-based sequencing. Analysis of the structure of MLL rearrangements at the base pair level revealed that over half had complex rearrangements that involved either three or more chromosomes, or contained at the breakpoints deletions, amplifications, insertions, or inversion of sequences. In five of the complex cases, chromosomal rearrangements were predicted to generate not only a MLL-partner gene fusion, but also novel in-frame fusions including KRAS-MLL; RAD51B-MLL / AFF1-RAD51B; MLLT10-CTNNAP3B; MLLT10-ATP5L / ATP5L-YPEL4; and CRTAM-GNL3. An analysis of the sequence surrounding the breakpoints of MLL and its partner genes suggest that the predominant mechanism of rearrangement involved non-homologous end joining. An analysis of the total number of non-silent mutations revealed infant ALL to have the lowest frequency of non-silent somatic mutations of any cancer sequenced to date. After removal of SVs and CNAs associated with the MLL rearrangements, a mean of only 2 somatic SVs and 2 SNVs affecting the coding region of annotated genes or regulatory RNAs were detected per case, with a range of non-silent mutation of between 0 and 11 per case (0–7 SV and 0–5 SNV). Despite the paucity of mutations several pathways were recurrently targeted. Mutations leading to activation of signaling through the PI3K/RAS pathway was observed in 45% of the cases with mutation of individual components including KRAS (n=4), NRAS (n=2), and non-recurrent mutations in NF1, PTPN11, PIK3R1, and the GTPase activating protein ARHGAP32 (p200Rho/GAP), which mediates cross-talk between RAS and Rho signaling. Other pathways altered include B cell differentiation, with 23% of cases containing mono-allelic deletion or gains of PAX5, 14% with deletions of the CDKN2A/B, and 2 cases with focal deletions of the non-coding RNA genes DLEU1/2. WGS of two infant ALL relapse samples and comparison with the data from their matched diagnostic samples revealed a marked increase in the number of mutations at relapse with additional SVs, SNVs, and CNAs identified. Moreover, an analysis of the allelic ratios of mutated genes revealed clonal heterogeneity at diagnosis with relapse appearing to arise from a minor diagnostic clone. Because of the exceedingly low frequency of mutations detected in infant ALL, we decided to define the frequency of non-silent SNVs in MLL rearranged leukemia occurring in older children (7–19 years of age). Exome sequencing was performed on 13 MLL leukemias (8 ALLs and 5 AMLs). This analysis revealed that non-infant pediatric MLL rearranged leukemias harbor a significantly higher number of non-silent somatic SNVs than infant ALL (mean 8/case in older patients versus 2/case in infants, p<0.001). Although the increased frequency of mutations may be a reflection of the older age, the low number of cooperating mutations in infants raises the possibility that the target cell of transformation differs between infants and older children, with the cells present during early development requiring fewer cooperating mutations to induce leukemia. In summary our analysis demonstrated an exceedingly small number of mutations required to generate infant MLL rearranged leukemia. The number of detected somatic mutations may represent the lower limit of mutations required to transform a normal human cell into cancer. Disclosures: Fioretos: Cantargia AB: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Qlucore AB: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Winter indices are a well-established tool for calculating the need for winter maintenance activities in relation to the climate. In this paper, data from 654 Road Weather Information System stations in Sweden were used during the five winter seasons, 1998/1999 to 2002/2003. They where used for studying the distribution of slippery conditions throughout Sweden. The number of incidents involving slipperiness during a winter season varies according to the location in Sweden and the manner in which the slippery conditions were formed. Winter indices were used to calculate the total number of slippery conditions. This was later compared to Sweden’s latitudes. It shows a good correlation between the distribution of slipperiness and Sweden latitudes, both for the total slipperiness and the different types of slipperiness. When severe hoarfrost was analysed at the regional level, a high degree of correlation between slipperiness and latitude was found.

Abstract Abstract 757 Acute Megakaryoblastic Leukemia (AMKL) accounts for ∼10% of childhood acute myeloid leukemia (AML). Although AMKL patients with down syndrome (DS-AMKL) have an excellent 5 year event-free survival (EFS), non-DS-AMKL patients have an extremely poor outcome with a 3 year EFS of less than 40%. With the exception of the t(1;22) translocation seen in infant non-DS-AMKL, little is known about the molecular genetic lesions that underlie this leukemia subtype. To define the landscape of mutations that occur in non-DS-AMKL, we performed transcriptome sequencing on diagnostic blasts from 14 cases (discovery cohort) using the illumina platform. Our results identified chromosomal rearrangements resulting in the expression of novel fusion transcripts in 12/14 cases. Remarkably, in 7/14 cases we detected an inversion on chromosome 16 [inv(16)(p13.3;q24.3)] that resulted in the juxtaposition of the CBFA2T3, a member of the ETO family of transcription factors, next to GLIS2 resulting in a CBFA2T3-GLIS2 chimeric gene encoding an in frame fusion protein. 6 cases in the discovery cohort fused exon 10 of CBFA2T3 to exon 3 of GLIS2, while 1 case carried a larger product that fused exon 11 of CBFA2T3 to exon 1 of GLIS2. Both products retain the 3 CBFA2T3 N-terminal nervy homology regions that mediate protein interactions, and the 5 GLIS2 C-terminal zinc finger domains that bind the Glis DNA consensus sequence, along with one of its N-terminal transcriptional regulatory domains. GLIS2 is a member of the GLI super family of transcription factors and has been demonstrated to play a role in regulating expression of GLI target genes as well as inhibiting WNT signaling through the binding of beta catenin. Although GLIS2 is not normally expressed in hematopoietic cells, the translocation results in high level expression of the CBFA2T3-GLIS2 fusion protein. In addition to CBFA2T3-GLIS2, chimeric transcripts were detected in 6/7 cases that lacked evidence of the inv(16)(p13.3;q24.3). Specifically, we detected GATA2-HOXA9, MN1-FLI1, NIPBL-HOXB9, NUP98-KDM5A, GRB10-SDK1 and C8orf76-HOXA11AS, each in an individual case. Importantly, several of the genes involved in these translocations either play a direct role in normal megakaryocytic differentiation (GATA2 and FLI1), or have been previously shown to be involved in leukemogenesis (HOXA9, MN1, HOXB9). Evaluation of a recurrency cohort of 42 samples including 14 additional pediatric cases and 28 adult cases by RT-PCR revealed 4 additional pediatric samples carrying CBFA2T3-GLIS2 for an overall frequency of 39% in pediatric AMKL. In addition to these somatic structural variations, we also identified mutations in genes previously shown to play a role in megakaryoblastic leukemia including activating mutations in JAK2 and MPL (36%). To gain insight into the mechanism whereby CBFA2T3-GLIS2 promotes leukemogenesis, we introduced the fusion into murine hematopoietic cells and assessed its effect on in vitro colony replating as a surrogate measure of self-renewal. Hematopoietic cells transduced with a mCherry expressing retroviral vector failed to form colonies after the second replating. By contrast, expression of either wild-type GLIS2 or the CBFA2T3-GLIS2 fusion resulted in a marked increase in the self-renewal capacity, with colony formation persisting through eight replatings. Immunophenotypic analysis of the CBFA2T3-GLIS2 expressing colonies revealed evidence of megakaryocytic differentiation. Importantly, the CBFA2T3-GLIS2 cells remained growth factor dependent suggesting that cooperating mutations in growth factor signaling pathways are required for full leukemic transformation. Taken together these data identify a novel cryptic inv(16)-encoded CBFA2T3-GLIS2 fusion protein as a recurrent driver mutation in approximately 40% of non-infant pediatric non-DS-AMKLs. Moreover, the majority of pediatric cases that lacked this lesion were shown by transcriptome sequence analysis to contain other chromosomal rearrangements that encoded fusion proteins that directly alter megakaryocytic differentiation and/or myeloid cell growth. The alteration of a key transcriptional regulator within the hedgehog signaling pathways in a substantial percentage of pediatric AMKL raises the possibility that inhibition of this pathway may have a therapeutic benefit in this aggressive form of AML. *TAG and ALG contributed equally to this work. Disclosures: Biondi: BMS, Novartis, Micromed: Consultancy, Membership on an entity's Board of Directors or advisory committees. Ravandi:Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria. Kantarjian:Novartis: Consultancy, Research Funding; Pfizer: Research Funding; BMS: Research Funding. Doehner:Hoffmann La Roche: Honoraria.

Winter weather can be a significant cause of road traffic accidents. This paper uses UKCIP climate change scenarios and a temporal analogue to investigate the relationship between temperature and severe road accidents in the West Midlands, UK. This approach also allows quantification of the changes in the severity of the winter season over the next century in the region. It is demonstrated that the predicted reduction in the number of frost days should in turn reduce the number of road accidents caused due to slipperiness by approximately 50%. However, the paper concludes by warning against complacency in winter maintenance regimes. A warmer climate may result in budget cuts for highway maintenance which in turn may well reverse declining accident trends.

Slippery roads due to ice and snow are a major cause of road traffic accidents in Sweden during the winter. This paper investigates the hypothesis that as the climate becomes increasingly milder there will be a reduction in the number of accidents in winter. Two months are compared in this analysis; one colder and drier than average, the other warmer and wetter than average. Despite the differences in weather between the 2 years, there was approximately the same number of accidents in both cases, although the exact cause of these accidents varied. It is concluded that using the warmer month as a temporal analogue, the accident rate in Sweden will not fall under current climate change scenarios. This result is attributed to the fact that drivers become more complacent in milder weather conditions where the risk of slippery roads is reduced. Copyright. (C) 2010 Royal Meteorological Society

Childhood leukemia is characterized by the presence of balanced chromosomal translocations or by other structural or numerical chromosomal changes. It is well know that leukemias with specific molecular abnormalities display profoundly different global gene expression profiles. However, it is largely unknown whether such subtype-specific leukemic signatures are unique or if they are active also in non-hematopoietic normal tissues or in other human cancer types.

The dynamic mechanical properties and acoustic damping of polyurethane (PU) foams loaded with a hyperbranched polymer (HBP) have been studied. The results show a significant modulus enhancement parallel to an increased vibration damping in the low frequency range of ~1 Hz with increased amount of HBP. Furthermore, an increased acoustic damping in the frequency interval of 500—1600 Hz is observed with increasing amount of HBP. The origin of these property enhancements are sought in the morphology and the chemical composition of the PU foam. It has been found that no visible differences in cell sizes and strut thicknesses exist among the foams. However, FTIR reveals significant differences in chemical composition between the foams of different HBP content, indicating increased crosslinking.