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Myelodysplastic neoplasms (MDSs) and chronic myelomonocytic leukemia (CMML) are clonal disorders driven by progressively acquired somatic mutations in hematopoietic stem cells (HSCs). Hypomethylating agents (HMAs) can modify the clinical course of MDS and CMML. Clinical improvement does not require eradication of mutated cells and may be related to improved differentiation capacity of mutated HSCs. However, in patients with established disease it is unclear whether (1) HSCs with multiple mutations progress through differentiation with comparable frequency to their less mutated counterparts or (2) improvements in peripheral blood counts following HMA therapy are driven by residual wild-type HSCs or by clones with particular combinations of mutations. To address these questions, the somatic mutations of individual stem cells, progenitors (common myeloid progenitors, granulocyte monocyte progenitors, and megakaryocyte erythroid progenitors), and matched circulating hematopoietic cells (monocytes, neutrophils, and naïve B cells) in MDS and CMML were characterized via high-throughput single-cell genotyping, followed by bulk analysis in immature and mature cells before and after AZA treatment. The mutational burden was similar throughout differentiation, with even the most mutated stem and progenitor clones maintaining their capacity to differentiate to mature cell types in vivo. Increased contributions from productive mutant progenitors appear to underlie improved hematopoiesis in MDS following HMA therapy.

Lymphangiogenesis is a dynamic process that involves the directed migration of lymphatic endothelial cells (LECs) to form lymphatic vessels. The molecular mechanisms that underpin lymphatic vessel patterning are not fully elucidated and, to date, no global regulator of lymphatic vessel guidance is known. In this study, we identify the transmembrane cell signalling receptor Plexin D1 (Plxnd1) as a negative regulator of both lymphatic vessel guidance and lymphangiogenesis in zebrafish. plxnd1 is expressed in developing lymphatics and is required for the guidance of both the trunk and facial lymphatic networks. Loss of plxnd1 is associated with misguided intersegmental lymphatic vessel growth and aberrant facial lymphatic branches. Lymphatic guidance in the trunk is mediated, at least in part, by the Plxnd1 ligands, Semaphorin 3AA and Semaphorin 3C. Finally, we show that Plxnd1 normally antagonises Vegfr/Erk signalling to ensure the correct number of facial LECs and that loss of plxnd1 results in facial lymphatic hyperplasia. As a global negative regulator of lymphatic vessel development, the Sema/Plxnd1 signalling pathway is a potential therapeutic target for treating diseases associated with dysregulated lymphatic growth.

Progressively acquired somatic mutations in hematopoietic stem cells are central to pathogenesis in myelodysplastic syndromes (MDS) and chronic myelomonocytic leukemia (CMML). They can lead to proliferative advantages, impaired differentiation and progressive cytopenias. MDS or CMML patients with high-risk disease are treated with hypomethylating agents including 5-azacytidine (AZA). Clinical improvement does not require eradication of mutated cells and may be related to improved differentiation capacity of mutated hematopoietic stem and progenitor cells (HSPCs). However, the contribution of mutated HSPCs to steadystate hematopoiesis in MDS and CMML is unclear. To address this, we characterised the somatic mutations of individual stem, progenitor (common myeloid progenitor, granulocyte monocyte progenitor, megakaryocyte erythroid progenitor), and matched circulating (monocyte, neutrophil, naïve B cell) haematopoietic cells in treatment naïve and AZA-treated MDS and CMML via high-throughput single cell genotyping. The mutational burden was similar across multiple hematopoietic cell types, and even the most mutated stem and progenitor clones maintained their capacity to differentiate to mature myeloid and, in some cases, lymphoid cell types in vivo. Our data show that even highly mutated HSPCs contribute significantly to circulating blood cells in MDS and CMML, prior to and following AZA treatment.Key points*Highly mutated HSPCs contribute significantly to circulating blood cells in MDS and CMML, prior to and following AZA treatment.*The mutational burden in matched bone marrow and peripheral blood cells in MDS and CMML was similar throughout myelopoiesis.

Evofosfamide (TH-302) is a hypoxia-activated DNA-crosslinking prodrug currently in clinical development for cancer therapy. Oxygen-sensitive activation of evofosfamide depends on one-electron reduction, yet the reductases that catalyze this process in tumors are unknown. We used RNA sequencing, whole-genome CRISPR knockout, and reductase-focused short hairpin RNA screens to interrogate modifiers of evofosfamide activation in cancer cell lines. Involvement of mitochondrial electron transport in the activation of evofosfamide and the related nitroaromatic compounds EF5 and FSL-61 was investigated using 143B ρ0 (ρ zero) cells devoid of mitochondrial DNA and biochemical assays in UT-SCC-74B cells. The potency of evofosfamide in 30 genetically diverse cancer cell lines correlated with the expression of genes involved in mitochondrial electron transfer. A whole-genome CRISPR screen in KBM-7 cells identified the DNA damage-response factors SLX4IP, C10orf90 (FATS), and SLFN11, in addition to the key regulator of mitochondrial function, YME1L1, and several complex I constituents as modifiers of evofosfamide sensitivity. A reductase-focused shRNA screen in UT-SCC-74B cells similarly identified mitochondrial respiratory chain factors. Surprisingly, 143B ρ0 cells showed enhanced evofosfamide activation and sensitivity but had global transcriptional changes, including increased expression of nonmitochondrial flavoreductases. In UT-SCC-74B cells, evofosfamide oxidized cytochromes a, b, and c and inhibited respiration at complexes I, II, and IV without quenching reactive oxygen species production. Our results suggest that the mitochondrial electron transport chain contributes to evofosfamide activation and that predicting evofosfamide sensitivity in patients by measuring the expression of canonical bioreductive enzymes such as cytochrome P450 oxidoreductase is likely to be futile.

Recent advances in next-generation sequencing have made it possible to perform genome wide identification of somatic mutation in cancers. Most studies focus on identifying somatic mutations in the protein coding portion of the genome using whole exome sequencing (WES). Every human genome has around 4 million single nucleotide polymorphisms (SNPs). A sizeable fraction of these germline SNPs is very rare and will not be found in the databases. Thus, in order to unambiguously identify somatic mutation, it is absolutely necessary to know the germline SNPs of the patient. While a blood sample can serve as source of germline DNA from patients with solid tumours, obtaining germline DNA from patients with haematological malignancies is very difficult. Tumor cells often infiltrate the skin, and their DNA can be found in saliva and buccal swab samples. The DNA in the tips of nails stems from keratinocytes that have undergone keratinization several months ago. DNA was successfully extracted from nail clippings of 5 probands for WES. We were able to identify somatic mutations in one tumor exome by using the nail exome as germline reference. Our results demonstrate that nail DNA is a reliable source of germline DNA in the setting of hematological malignancies.

The MLL/AF9 fusion is found in approximately 30% of MLL-rearranged leukemias and has an intermediate prognosis. Genomically well-characterized murine leukemia models enable us to understand leukemogenesis. We generated a retroviral transduction murine bone marrow transplantation leukemia model (MBMTLM) using the MLL/AF9 fusion gene. Fifteen of 20 mice transplanted with syngeneic bone marrow transduced with a MLL/AF9 carrying retrovirus developed leukemia after a median latency of 149 days. Half a million leukemic bone marrow (LBM) cells from two of these primary leukemias, MA03-P and MA86-P, were transplanted into irradiated recipient mice to establish secondary leukemias, MA03-S (n=3) and MA86-S (n=4). Half a million LBM cells from these secondary leukemias were further transplanted into irradiated recipient mice to generate tertiary leukemias, MA03-T (n=3) and MA86-T (n=4). The latency of the leukemias shortened from 141 days in MA03-P to 18 and 22 days in MA03-S and MA03-T, respectively. Similarly, MA86-P had a latency of 98 days, and the latency was reduced to about 28 days in MA986-S and MA986-T. We used retroviral insertion sites (RISs) to track leukemia clones during serial transplantation. We identified 5 RISs in MA03-P. One RIS, RIS#1-03 at chromosome 7:4602500-4609499 accounted for 52.5% of the total RIS-related reads in MA03-P, while the other four RISs were each represented by fewer than 5% of the reads. Only RIS#1-03 was detected in all of the MA03 secondary and tertiary leukemias , indicating that the cells with RIS#1-03 were the dominant clone in MA03 leukemias. Two RISs were detected in MA86-P: RIS#1-86 at chromosome 19:41338500-41341999 and RIS#2-86 at chromosome 10:127106000-127109499 at 46.7% and 2.5%, respectively . RIS#1-986 was contained in the dominant clone as only this RIS was subsequently detected in the secondary and tertiary MA86 leukemias. The relatively long latency to leukemia development in our MLL/AF9 model was most likely due to the requirement of cooperating somatic mutations. We performed whole exome sequencing on DNA from LBM (n=15) and DNA from their corresponding germline (n=2). An average of 4.5 of single nucleotide variants (SNVs) and 11.4 indels affecting protein coding sequences were found in the MA03 family of leukemias (n=7) which, among others, mutated genes involved in tyrosine kinase pathways such as Epha5 and Pik3r1. We identified an average of 14.8 (SNVs) and 0.5 indels per exome in the MA86 leukemias (n=8). Transcription regulator (Brd1) and tumor suppressor genes (Stk11 and Trp53) were affected by somatic changes in the MA86 family. RNA sequencing was performed on LBM (n=15) and healthy bone marrow (HBM) (n=8). Principal component analysis (PCA) on the expression profiles showed that LBM samples clustered together. Differential gene expression analysis identified genes such as Six1, Eya1 and Bcor which had been reported in previous studies to be essential for leukemogenesis in MLL/AF9 murine model. We also observed downregulation of genes such as Gata2, Btg1, Ifitm1, which had been implicated in other types of leukemias. We next investigated the effect of the RISs and somatic mutations on gene expression. RIS#1-903 was in intron 1 of Ppp6r1. A reduction in fragments per kilobase of transcript per Million mapped reads (FPKM) of Ppp6r1 was observed in MA03 family leukemias compared to leukemias of the MA86 family which did not have RIS#1-03 and showed no difference to HBM samples (MA03: 87.71±1.5; MA86: 132.1±5.1; HBM: 77.56±1.7, p< 0.001). We then determined the expression of Tm9sf3 as it is located 600bp away from RIS#1-986. The FPKM of Tm9sf3 was significantly higher in LBM (both of MA903 and MA986 leukemias) than in HBM (LBM: 146.0±12.7; HBM: 64.66±2.8, p In conclusion, we have established a MBMTLM driven by the MLL/AF9 fusion gene. This well-characterized model provides insights to further understand leukemia development and drug testing. Moreover, we demonstrated that RISs can have an impact on gene expression. Future work on whether Ppp6r1 and Tm9sf3 identified by our RIS analysis are drivers in MLL/AF9 leukemias is warranted. Disclosures Browett: MSD: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria.

Background Acute Leukaemia (AL) is a genetically heterogeneous disorder caused by somatic mutations and acquired chromosomal translocations. Translocations can lead to the formation of fusion genes such as the MLL/AF9 fusion, which results from the t(9;11)(p22;q23). A better understanding the molecular pathophysiology of AML, of the mechanisms of treatment resistance, disease relapse can be achieved by developing animal models. The MLL/AF9 fusion is frequently used to model AML in mice. However, to date, no MLL/AF9 leukemia models in zebrafish have been reported. Aim Our aim was to establish a transgenic zebrafish leukemia model using the human MLL/AF9 fusion gene. Methods To generate transgenic fish, two constructs (pTol2-Runx1+23: MLL-AF9-IRES-EGFP-cmlc-GFP and pTol2-Runx1+23: MLL-AF9-IRES-mCherry-cmlc-GFP) were injected together with Tol2 transposase mRNA into one-cell stage zebrafish embryos. We used the murine Runx1+23 enhancer to direct MLL/AF9 expression to hematopoietic stem cells and EGFP or mCherry as fluorescent markers. We selected transgenic embryos 24 hours post-fertilization based on the heart marker expression (cmlc). Results 29% (100 of 340 embryos) of the transgenics reached adulthood (6 weeks). After 6 to 24 months, 77% (77) of them developed signs of sickness. They became less active with protruding eyes and hump formation on the nose. Some started bleeding from the gills and/or showed tumor formation around the abdomen and head. Sick fish were euthanized and dissected. The autopsies showed pale and dysmorphic kidneys, pale and enlarged spleens, and in some cases white granular spots on the spleen. Histological sections revealed increased kidney, spleen, and liver cellularity with massive cellular infiltration of cells in these organs. In flow cytometry, kidney marrow cells from the transgenic fish showed a different forward scatter (FSC) and side scatter (SSC) profile compared to that of the normal zebrafish kidney marrow cells. The transgenic F 0 zebrafish showed increased numbers of lymphoid cells (12 fish), precursor cells (9 fish), or myeloid cells (6 fish). Peripheral blood smears showed many intermediate-sized mononuclear cells with an increased nuclear-cytoplasmic ratio, with nuclei containing dipsersed chromatin and inconspicuous nucleoli resembling blasts. There were occasional myelocytes and no mature granulocytes. These data are consistent with the development of acute leukemia in our MLL/AF9 transgenic fish. Whole-mount in situ hybridization (WISH) was performed on F 1 embryos. RNA probes for early hematopoietic markers (gata1, scl, runx1, ikaros, cmyb, mpx, and lyz) were hybridized to 24 and 48 hpf F1 transgenic embryos. There were expression changes of these markers compared to age-matched wild-type larvae, including low expression of gata1, scl, cmyb and high expression of lyz, mpx and ikaros in the caudal hematopoietic organ. We also performed transplantation experiments with the kidney marrow cells from diseased fish to test whether the disease was transplantable. The disease was serially transplantable into secondary and tertiary recipient fish. Transplanted fish had a significantly shorter latency to disease development of only 2 to 6 weeks. The morphological evidence and the serial transplantability of the disease proves that we have in fact succeeded in establishing an MLL/AF9-driven acute leukemia model in zebrafish. The long latency and incomplete penetrance observed in our F 0 MA9 zebrafish, along with a shorter latency in the transplanted fish, suggests that additional somatic mutations are required for leukemogenesis in this model. We performed whole exome sequencing to find cooperating somatic mutations and RNASeq to identify differentially expressed genes in MA9 leukemia fish. Whole exome sequencing on six samples identified putative somatic mutations in genes such as Stat5, Cyp2j20, Ms4a17a.3, Tapbp.1 and Herc5.3, which have been reported to be mutated in human cancer. RNA-seq analysis on seven samples showed 67 differentially expressed genes with a q value < 0.05 (e.g., cxcl32b.1, myof1, ctdsp2, egr3, il2rb) and nine enriched pathways with a P-value of < 0.054 (e.g.: KRAS, TP53, MEK) in our transgenic leukemic MLL/AF9 fish. Conclusion Our MLL/AF9 zebrafish acute leukemia model will be a helpful tool to understand leukemia biology and enable testing of new therapeutic strategies. Disclosures Browett: AbbVie: Honoraria; Janssen: Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees.

Myeloid leukemia is caused by acquired genetic changes in haematopoietic stem cells. The combination of stepwise acquisition of genetic changes together with selection of the fittest clones results in great genetic and clonal heterogeneity. We used a CALM-AF10-driven retroviral transduction murine bone marrow transplantation leukemia model (MBMTLM) to study clonal hierarchy and clonal evolution starting with a primary leukaemia (Fig 1: Leu7) which developed after 131 days and had B220 marker expression on 4% of its cells. Limiting dilution assays (LDAs) showed that the leukemia stem cell (LSC) frequency of Leu7 was 1:2339 (95% confidence interval: 1:794-1:6885). Whole exome sequencing (WES) and analysis of the variant allele fraction of somatic mutations revealed that Leu7 was composed of a main clone (Fig 1: grey) with two subclones (blue and red). Half a million leukemic cells from Leu7 were transplanted into 4 sublethally irradiated recipients, which all developed secondary leukemias after a latency of 19 days (Leu7Sec1 to 4). All secondary leukemias showed similar B220 expression levels to Leu7, and all showed an expansion of the blue subclone. When again half a million cells each of one of the secondary leukemias (Leu7Sec2) were transplanted into 4 recipients, the expansion of the blue subclone continued, the red subclone vanished and, surprisingly, the proportion of B220 expressing cells increased to between 16 to 26%. LDAs showed that the LSC frequency of Leu7Sec2 had not changed. However, several of the leukemias from the LDAs had greatly varying latencies (27 to 193 days) and B220 marker expression (2 to 85%). Four of these tertiary LDA leukemias (Leu7Sec2Ter5 to 8), which each arose from a single LSC, were analysed more closely using WES. Leu7Sec2Ter5 showed a similar latency (27 days) and B220 expression levels like Leu7SecTer1 to 4 and also had the expansion of the blue subclone. Leu7Sec2Ter6 had a long latency of 69 days and a very low B220 expression. Leu7Sec2Ter6 was driven by a new, third subclone (pink), and both the blue and the red subclone disappeared. Very interestingly, Leu7Sec2Ter7 and Leu7Sec2Ter8 had a very long latency of 193 days, and showed an expansion of a subclone (green) of the red subclone. The B220 expression was high (37%) to very high (85%) in these two leukemias. Taken together, these observations paint an interesting picture with the blue subclone outcompeting the red subclone, as leukemias arising from the red subclone only appear after a long latency and in leukemias initiated by a single LSC, when there is no blue subclone LSC present. As the four leukemias (Leu7Sec2Ter5 to 8), which each were derived from a single LSC, showed striking differences in latency and surface marker expression, it can be concluded that this variation in phenotype is an intrinsic property of an individual LSCs most likely a consequence of the distinct combination of somatic mutations present in the individual LSCs. These observations also suggest that distinct LSCs with different properties might be present in a single human leukemia. Figure 1 Figure 1. Disclosures Browett: Janssen: Membership on an entity's Board of Directors or advisory committees; MSD: Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria.

In adult acute myeloid leukemia (AML), the karyotype of the leukemic cell is among the strongest prognostic factors. The Medical Research Council (MRC) and the European LeukemiaNet (ELN) classifications distinguish between favorable, intermediate and adverse cytogenetic risk patients who differ in their treatment response and overall survival. Conventional cytogenetic analyses are a mandatory component of AML diagnostics but they are time-consuming; therefore, therapeutic decisions in elderly patients are often delayed. We investigated whether a screening approach using a panel of seven fluorescence in situ hybridization (FISH) probes would allow rapid identification of adverse chromosomal changes. In a cohort of 334 AML patients, our targeted FISH screening approach identified 80% of adverse risk AML patients with a specificity of 99%. Incorporating FISH screening into diagnostic workup has the potential to accelerate risk stratification and treatment selection, particularly in older patients. This approach may allow therapeutic decisions more quickly, which benefits both patients and physicians and might save costs.

Background With the emergence of immunotherapies as a promising cancer treatment, there is now a pressing need for pre-clinical animal models to test immunotherapy strategies for acute myeloid leukemia (AML). While murine xenotransplant models generated by transplanting human AML cells are frequently used to model AML, they require immune-deficient mice. Syngeneic murine bone marrow transplant leukemia models (MBMTLM), which are established in immune competent mice, usually require radiation of the recipient mouse before leukemic cells are transplanted. This radiation suppresses the immune system and makes it difficult to study how the immune system responds to leukemia cells. Aim Our aim was to establish immunocompetent MBMTLMs to understand how the immune system responds to leukemia cells expressing a highly immunogenic antigen. Method We first established MBMTLMs models driven by the CALM/AF10 or MLL/AF9 fusion genes. Then leukemia cells were transduced with a SIINFEKL expressing retrovirus (MSCV-DsRed-SIINFEKL). SIINFEKL is a highly immunogenic eight amino acid peptide from ovalbumin. Results The transduction efficiency of MSCV-DsRed-SIINFEKL was about 12.9% on leukemic cells of MLL/AF9 and 13% on CALM/AF10 cells. All primary MLL/AF9-SIINFEKL (n=3) developed leukemia with a latency of 22 days. SIINFEKL expression was detected on 75.5±3% of the spleen cells of these mice. These spleen cells were transplanted into irradiated (n=6) and non-irradiated (n=8) mice to establish secondary MLL/AF9-SIINFEKL leukemias (Table 1). In the irradiated recipients, five out of six mice developed leukemia within 22-29 days. In non-irradiated recipients, four out of eight developed leukemia with a latency of 29-45 days. Flow cytometry showed that SIINFEKL was expressed on 82.28±6% of the spleen cells in irradiated recipients. In contrast, fewer than 1% of the spleen cells in non-irradiated mice with secondary MLL/AF9-SIINFEKL leukemia expressed SIINFEKL. Similar experiments were performed with the CALM/AF10 model. All primary CALM/AF10-SIINFEKL transplanted mice (n=2) developed leukemia with a latency of 32 and 42 days and showed SIINFEKL positivity on 99% of the leukemia cells. Secondary CALM/AF10-SIINFEKL leukemias were generated by transplanting these AML cells into irradiated (n=6) and non-irradiated (n=8) mice. All six irradiated recipients developed leukemia within 21-40 days. However, only five of the eight non-irradiated recipients developed leukemia with a latency of 35-42 days. Strikingly, about 99% of the AML cells in the irradiated mice were SIINFEKL positive but fewer than 1% of the AML cells in the five non-irradiated mice who developed leukemia were SIINFEKL positive. Discussion We find that for both MLL/AF9 and CALM/AF10 AML an intact immune system (non-irradiated recipients) is able to largely eliminate AML cells which express the SIINFEKL antigen, even when challenged with a large number of AML cells. In the non-irradiated recipients only 50 to 60% developed leukemia, and the leukemias that did develop had hardly any SIINFEKL-positive cells. In contrast, nearly all irradiated mice (11 of 12) developed leukemia with a high percentage of SIINFEKL expressing AML cells. SIINFEKL was presented on the surface of leukemic cells by the murine MHC class I H-2Kb molecule. Our results differ slightly from research reported by Hasegawa et al. 2015, who showed that MLL/AF9 AML cells expressing ovalbumin caused leukemia after transplantation into non-irradiated mice. However, they did not investigate whether ovalbumin was still being expressed on the leukemia cells. Conclusion We have established syngeneic murine AML models in immunocompetent mice and have evidence that an intact immune system has the ability to suppress or even eliminate rapidly proliferating AML cells if they express a strong antigen. These models should be useful for developing immunotherapy strategies for AML. Disclosures Desai: University of Auckland: Current Employment. Kakadia:University of Auckland: Current Employment. Browett:University of Auckland: Current Employment; BeiGene: Research Funding; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES (paid by any for-profit health care company), Research Funding; Shire: Research Funding; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Bohlander:University of Auckland: Current Employment; Family of Marijana Kumerich: Research Funding; Leukaemia and Blood Cancer New Zealand: Research Funding.

Accurate classification of acute myeloid leukaemia (AML) has become increasingly reliant on molecular characterisation of this blood cancer. Throughout Australia and New Zealand massively parallel sequencing (MPS) is being adopted by diagnostic laboratories for the routine evaluation of patients with AML. This technology enables the surveying of many genes simultaneously, with many technical advantages over single gene testing approaches. However, there are many variations in wet and dry lab MPS procedures, which raises the prospect of discordant results between laboratories. This study compared the results obtained from MPS testing of ten diagnostic AML bone marrow aspirate samples sent to eight participating laboratories across Australasia. A reassuringly high concordance of 94% was observed with regard to variant detection and characterisation of pathogenicity. The level of discordance observed, although low, demonstrates the need for ongoing assessment of concordance between diagnostic testing laboratories through quality assurance programs. Refereed/Peer-reviewed

We report the case of a 26 year-old patient presenting with a persistent leukocytosis and CML-like marrow but no evidence of a BCR/ABL1 fusion. Molecular cytogenetics revealed that a portion of the ETV6 locus was inserted into the ABL1 locus. An ETV6/ABL1 fusion transcript could subsequently be confirmed. The patient was started on imatinib and went into complete cytomorphological remission. QRT-PCR measurements showed a 4log reduction of the ETV6/ABL1 fusion. 15 months later, the disease transformed into ALL and the patient expired. Thus, an ETV6/ABL1 fusion positive MPN has the potential to transform very rapidly into ALL.

As a result of a whole-exome sequencing study, we report three mutant alleles in SEC24D, a gene encoding a component of the COPII complex involved in protein export from the ER: the truncating mutation c.613C>T (p.Gln205∗) and the missense mutations c.3044C>T (p.Ser1015Phe, located in a cargo-binding pocket) and c.2933A>C (p.Gln978Pro, located in the gelsolin-like domain). Three individuals from two families affected by a similar skeletal phenotype were each compound heterozygous for two of these mutant alleles, with c.3044C>T being embedded in a 14 Mb founder haplotype shared by all three. The affected individuals were a 7-year-old boy with a phenotype most closely resembling Cole-Carpenter syndrome and two fetuses initially suspected to have a severe type of osteogenesis imperfecta. All three displayed a severely disturbed ossification of the skull and multiple fractures with prenatal onset. The 7-year-old boy had short stature and craniofacial malformations including macrocephaly, midface hypoplasia, micrognathia, frontal bossing, and down-slanting palpebral fissures. Electron and immunofluorescence microscopy of skin fibroblasts of this individual revealed that ER export of procollagen was inefficient and that ER tubules were dilated, faithfully reproducing the cellular phenotype of individuals with cranio-lentico-sutural dysplasia (CLSD). CLSD is caused by SEC23A mutations and displays a largely overlapping craniofacial phenotype, but it is not characterized by generalized bone fragility and presented with cataracts in the original family described. The cellular and morphological phenotypes we report are in concordance with the phenotypes described for the Sec24d-deficient fish mutants vbi (medaka) and bulldog (zebrafish).

Evofosfamide (TH-302) is a clinical-stage hypoxia-activated prodrug of a DNA-crosslinking nitrogen mustard that has potential utility for human papillomavirus (HPV) negative head and neck squamous cell carcinoma (HNSCC), in which tumor hypoxia limits treatment outcome. We report the preclinical efficacy, target engagement, preliminary predictive biomarkers and initial clinical activity of evofosfamide for HPV-negative HNSCC. Evofosfamide was assessed in 22 genomically characterized cell lines and 7 cell line–derived xenograft (CDX), patient-derived xenograft (PDX), orthotopic, and syngeneic tumor models. Biomarker analysis used RNA sequencing, whole-exome sequencing, and whole-genome CRISPR knockout screens. Five advanced/metastatic HNSCC patients received evofosfamide monotherapy (480 mg/m2 qw × 3 each month) in a phase 2 study. Evofosfamide was potent and highly selective for hypoxic HNSCC cells. Proliferative rate was a predominant evofosfamide sensitivity determinant and a proliferation metagene correlated with activity in CDX models. Evofosfamide showed efficacy as monotherapy and with radiotherapy in PDX models, augmented CTLA-4 blockade in syngeneic tumors, and reduced hypoxia in nodes disseminated from an orthotopic model. Of 5 advanced HNSCC patients treated with evofosfamide, 2 showed partial responses while 3 had stable disease. In conclusion, evofosfamide shows promising efficacy in aggressive HPV-negative HNSCC, with predictive biomarkers in development to support further clinical evaluation in this indication.

Background:The small molecule BCL-2 inhibitor Venetoclax (VEN) has emerged as a promising frontline therapy in combination with low dose cytarabine (LDAC) or hypomethylating agents in older patients with acute myeloid leukemia (AML)(DiNardo et al, Lancet Oncol 2018, Blood 2019; Wei et al,JCO 2019). The anti-leukemic activity of single agent VEN as monotherapy in newly diagnosed AML is unknown. This information would be useful for studies considering VEN as a maintenance therapy. We have previously presented preliminary outcomes from a phase 1b/2 study: Chemotherapy and Venetoclax in Elderly AML Trial (CAVEAT), which incorporated a 7-day single agent VEN run-in phase prior to combination with chemotherapy (Wei et al, ASH 2018). The study procedures incorporated a bone marrow assessment performed at study screening and on day 8, prior to commencement of chemotherapy (Figure 1A). This allowed us to explore the single agent activity of venetoclax on bone marrow blasts in treatment naïve patients with AML and to assess molecular dynamics of response. Methods:The CAVEAT trial included 51 de novo or secondary patients with AML aged ≥65 years and considered fit for intensive chemotherapy. The study enrolled patients to 5 VEN dose cohorts (50-100-200-400-600mg). VEN was administered on days 1-14 during induction. Chemotherapy commenced on day 8, with a 7-day VEN monotherapy pre-phase. Molecular studies:a 54-gene myeloid NGS panel (Illumina TruSight) and MassARRAY MALDI-TOF mass spectrometry was performed on DNA extracted from bone marrow aspirates. FLT3-ITD testing was by fragment analysis, PCR and capillary electrophoresis. Minimal residual disease (MRD) testing was performed by digital droplet PCR for IDH mutations (assay sensitivity: 10-4-10-5). Results: A total of 46 patients had paired BM assessments at screening and on day 8 for comparison. A ≥50% relative reduction in BM blasts was recorded in 13/46 (28%) patients. Figure 1B shows the relative BM blast reduction stratified by molecular subgroups. Patients with NPM1(n=9) or IDH2 (n=8) mutation achieved greater BM blast reductions (median of 56% and 55%, respectively) than IDH1 (n=6), TP53 (n=10) or FLT3-ITD (n=5) mutant cases (median of 37%, 17% and 7%, respectively). Three NPM1 mutant cases with Clinically, complete remission (CR) and CR with incomplete count recovery (CRi) rates after chemotherapy were seen for the following mutations: NPM1(80%), IDH2 (100%), IDH1 (62%) and TP53 (36%) (Figure 1D). Despite all four response-evaluable patients with FLT3-ITD achieving CR/CRi, 3/4 relapsed with detectable FLT3-ITD (at 0.8, 1.5 and 12.3 months respectively). Median survival for each genomic sub-group were NPM1 (12.5m), IDH2 (not reached), IDH1 (6.1m),TP53 (3.8m), FLT3-ITD (5.5m). CyTOF analyses for changes in BCL-2 family expression are underway and results will be presented at the meeting. Conclusion: Treatment naïve NPM1 and IDH2 mutant AML blasts are highly sensitive to VEN alone and combination with cytarabine and anthracycline chemotherapy results in a high clinical response rate. TP53 and FLT3-ITD mutant cases were more resistant and outcomes were poor despite VEN combined with chemotherapy. Figure 1 Disclosures Reynolds: Alfred Health: Employment, Other: Biostatistician for trials funded by the Australian government and Abbvie, Amgen, Celgene, GSK, Janssen-Cilag, Merck, Novartis, Takeda, but sponsored by Alfred Health.; AUSTRALASIAN LEUKAEMIA & LYMPHOMA GROUP (ALLG): Consultancy; Novartis AG: Equity Ownership; Novartis Australia: Honoraria. Fong:Pfizer: Consultancy, Speakers Bureau; Amgen: Consultancy, Research Funding, Speakers Bureau; Astellas: Consultancy; Novartis: Speakers Bureau. Ting:NHMRC: Research Funding. Fleming:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria; Pfizer: Honoraria; Ariad: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees. Moujalled:Walter and Eliza Hall Institute of Medical Research: Patents & Royalties: Share in venetoclax royalties . Ivey:Novartis: Honoraria. Roberts:AbbVie: Research Funding; Janssen: Research Funding; Walter and Eliza Hall Institute: Employment, Patents & Royalties: receives a fraction of its royalty stream related to venetoclax. Wei:AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties: AHW is a former employee of the Walter and Eliza Hall Institute and receives a fraction of its royalty stream related to venetoclax, Research Funding, Speakers Bureau; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Macrogenics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Astra Zeneca: Honoraria, Research Funding; Janssen: Honoraria. OffLabel Disclosure: Venetoclax is a BCL-2 inhibitor that is FDA-approved in some indications. This presentation will focus on correlative study results in the CAVEAT trial which combines venetoclax with modified intensive chemotherapy in AML, which is not an approved indication.

In chronic lymphocytic leukemia (CLL) patients, disruptions of the TP53 tumor suppressor pathway by 17p13 deletion (del17p), somatic TP53 mutations, or downregulation of microRNA-34a have been associated with a poor prognosis. So far, the impact of the various TP53 defects has not been evaluated in a large cohort of previously treated and relapsed CLL patients. Here, we present the results of TP53 gene sequencing and fluorescence in situ hybridization for del17p in a phase 3 clinical trial (REACH [Rituximab in the Study of Relapsed Chronic Lymphocytic Leukemia]). Of the 457 patients, 52 had TP53 mutations and 37 had del17p. In 24 (46%) of the TP53 mutated patients, no del17p was found and in 9 of the del17p patients, no TP53 mutation was identified. Based on a predicted proportion of TP53 disruption, a complete disruption of TP53 function, either by a combination of point mutations and/or del17p, was associated with a high risk for disease progression. Progression-free survival of patients with a heterozygous TP53 mutation was not significantly different from patients with a completely intact TP53 locus. In addition, only a complete loss of TP53 function correlated with low microRNA-34a expression levels. This trial was registered at www.clinicaltrials.gov as #NCT00090051.

A novel ABL1 fusion to the SH2 containing inositol phosphatase-1 ( SHIP1 ) in acute lymphoblastic leukemia (ALL)

Cytogenetic analysis in peripheral blood lymphocytes of a 50-year-old female with tongue cancer showed the presence of one to three copies of a small supernumerary marker chromosome (sSMC) in a mosaic state. Family studies also revealed the marker in mosaic form in four (age

The balance between DNA damage, especially double strand breaks, and DNA damage repair is a critical determinant of chromosomal translocation frequency. The non-homologous end-joining repair (NHEJ) pathways seem to play the major role in the generation of chromosomal translocations. The "landscape" of chromosomal translocation identified in malignancies is largely due to selection processes which operate on the growth advantages conveyed to the cells by the functional consequences of chromosomal translocations (i.e., oncogenic fusion proteins and overexpression of oncogenes, both compromising tumor suppressor gene functions). Newer studies have shown that there is an abundance of local rearrangements in many tumors, like small deletions and inversions. A better understanding of the interplay between DNA repair mechanisms and the generation of tumorigenic translocations will, among many other things, depend on an improved understanding of DNA repair mechanisms and their interplay with chromatin and the 3D organization of the interphase nucleus.

SummaryA challenge for the development of therapies selectively targeting leukemic stem cells in acute myeloid leukemia (AML) is their similarity to normal hematopoietic stem cells (HSCs). Here we demonstrate that the leukemia-propagating cell in murine CALM/AF10-positive AML differs from normal HSCs by B220 surface expression and immunoglobulin heavy chain rearrangement. Furthermore, depletion of B220+ cells in leukemic transplants impaired development of leukemia in recipients. As in the murine model, human CALM/AF10-positive AML was characterized by CD45RA (B220)-positive, IG DH-JH rearranged leukemic cells. These data demonstrate in a murine leukemia model that AML can be propagated by a transformed progenitor with lymphoid characteristics, which can be targeted by antibodies that do not crossreact with normal HSCs.

In acute myeloid leukemia (AML), assessment of minimal residual disease (MRD) by flow cytometry (flow MRD) after induction and consolidation therapy has been shown to provide independent prognostic information. However, data on the value of earlier flow MRD assessment are lacking. Therefore, the value of flow MRD detection was determined during aplasia in 178 patients achieving complete remission after treatment according to AMLCG (AML Cooperative Group) induction protocols. Flow MRD positivity during aplasia predicted poor outcome (5-year relapse-free survival (RFS) 16% vs 43%, P0.001) independently from age and cytogenetic risk group (hazard ratio for MRD positivity 1.71; P=0.009). Importantly, the prognosis of patients without detectable MRD was neither impacted by morphological blast count during aplasia nor by MRD status postinduction. Early flow MRD was also evaluated in the context of existing risk factors. Flow MRD was prognostic within the intermediate cytogenetic risk group (5-year RFS 15% vs 37%, P=0.016) as well as for patients with normal karyotype and NPM1 mutations (5-year RFS 13% vs 49%, P=0.02) or FLT3-ITD (3-year RFS rates 9% vs 44%, P=0.016). Early flow MRD assessment can improve current risk stratification approaches by prediction of RFS in AML and might facilitate adaptation of postremission therapy for patients at high risk of relapse.

The balance between DNA damage, especially double strand breaks, and DNA damage repair is a critical determinant of chromosomal translocation frequency. The non-homologous end-joining repair (NHEJ) pathways seem to play the major role in the generation of chromosomal translocations. The "landscape" of chromosomal translocation identified in malignancies is largely due to selection processes which operate on the growth advantages conveyed to the cells by the functional consequences of chromosomal translocations (i.e., oncogenic fusion proteins and overexpression of oncogenes, both compromising tumor suppressor gene functions). Newer studies have shown that there is an abundance of local rearrangements in many tumors, like small deletions and inversions. A better understanding of the interplay between DNA repair mechanisms and the generation of tumorigenic translocations will, among many other things, depend on an improved understanding of DNA repair mechanisms and their interplay with chromatin and the 3D organization of the interphase nucleus.

Isolated trisomy 13 (AML+13) is a rare chromosomal abnormality in acute myeloid leukemia (AML), and its prognostic relevance is poorly characterized. We analyzed the clinical course of 34 AML+13 patients enrolled in the German AMLCG-1999 and SAL trials and studied their biological characteristics by exome sequencing, targeted sequencing of candidate genes and gene expression profiling. Relapse-free (RFS) and overall survival (OS) of AML+13 patients were inferior compared to other ELN Intermediate-II patients (n=855) (median RFS, 7.8 vs 14.1 months, p=0.006; median OS 9.3 vs. 14.8 months, p=0.004). Besides the known high frequency of RUNX1 mutations (75%), we identified mutations in spliceosome components in 88%, including SRSF2 codon 95 mutations in 81%, of AML+13 patients. Moreover, recurring mutations were detected in ASXL1 (44%) and BCOR (25%). Two patients carried mutations in CEBPZ, suggesting that CEBPZ is a novel recurrently mutated gene in AML. Gene expression analysis revealed a homogenous expression profile including upregulation of FOXO1 and FLT3 and downregulation of SPRY2. This is the most comprehensive clinical and biological characterization of AML+13 to date, and reveals a striking clustering of lesions in a few genes, defining AML+13 as a genetically homogenous leukemia subgroup with alterations in a few critical cellular pathways. These studies were registered at clinicaltrials.gov, identifiers: AMLCG-1999: NCT00266136; AML96: NCT00180115; AML2003: NCT00180102; and AML60+: NCT00893373.

Purpose Cytogenetically normal (CN) acute myeloid leukemia (AML) is the largest and most heterogeneous cytogenetic AML subgroup. For the practicing clinician, it is difficult to summarize the prognostic information of the growing number of clinical and molecular markers. Our purpose was to develop a widely applicable prognostic model by combining well-established pretreatment patient and disease characteristics. Patients and Methods Two prognostic indices for CN-AML (PINA), one regarding overall survival (OS; PINAOS) and the other regarding relapse-free survival (RFS; PINARFS), were derived from data of 572 patients with CN-AML treated within the AML Cooperative Group 99 study ( www.aml-score.org ). Results On the basis of age (median, 60 years; range, 17 to 85 years), performance status, WBC count, and mutation status of NPM1, CEBPA, and FLT3-internal tandem duplication, patients were classified into the following three risk groups according to PINAOS and PINARFS: 29% of all patients and 32% of 381 responding patients had low-risk disease (5-year OS, 74%; 5-year RFS, 55%); 56% of all patients and 39% of responding patients had intermediate-risk disease (5-year OS, 28%; 5-year RFS, 27%), and 15% of all patients and 29% of responding patients had high-risk disease (5-year OS, 3%; 5-year RFS, 5%), respectively. PINAOS and PINARFS stratified outcome within European LeukemiaNet genetic groups. Both indices were confirmed on independent data from Cancer and Leukemia Group B/Alliance trials. Conclusion We have developed and validated, to our knowledge, the first prognostic indices specifically designed for adult patients of all ages with CN-AML that combine well-established molecular and clinical variables and that are easily applicable in routine clinical care. The integration of both clinical and molecular markers could provide a basis for individualized patient care through risk-adapted therapy of CN-AML.

The t(8;21) and inv(16)/t(16;16) rearrangements affecting the core-binding factors RUNX1 and CBFB, respectively, are found in 15% to 20% of adult de novo acute myeloid leukemia (AML) cases and are associated with a favorable prognosis. Since the expression of the fusion genes CBFB / MYH11 or RUNX1 / RUNX1T1 alone is not sufficient to cause leukemia, we performed exome sequencing of an AML sample with an inv(16) to identify mutations, which may collaborate with the CBFB/MYH11 fusion during leukemogenesis. We discovered an N676K mutation in the adenosine triphosphate (ATP)-binding domain (tyrosine kinase domain 1 [TKD1]) of the fms-related tyrosine kinase 3 ( FLT3 ) gene. In a cohort of 84 de novo AML patients with a CBFB/MYH11 rearrangement and in 36 patients with a RUNX1/RUNX1T1 rearrangement, the FLT3 N676K mutation was identified in 5 and 1 patients, respectively (5 [6%] of 84; 1 [3%] of 36). The FLT3-N676K mutant alone leads to factor-independent growth in Ba/F3 cells and, together with a concurrent FLT3-ITD (internal tandem duplication), confers resistance to the FLT3 protein tyrosine kinase inhibitors (PTKIs) PKC412 and AC220. Gene expression analysis of AML patients with CBFB / MYH11 rearrangement and FLT3 N676K mutation showed a trend toward a specific expression profile. Ours is the first report of recurring FLT3 N676 mutations in core-binding factor (CBF) leukemias and suggests a defined subgroup of CBF leukemias. This trial was registered at www.clinicaltrials.gov as #NCT00266136.

We recently identified the marginal zone B and B1 cell-specific protein (MZB1) as part of a gene expression signature associated with outcomes in chronic lymphocytic leukemia (CLL). MZB1 is important for B cell function as a key regulator of antibody secretion, calcium homeostasis and adhesion. Therefore, we analyzed the role of MZB1 expression levels in 139 patients with CLL using quantitative real-time polymerase chain reaction (qRT-PCR) and microarray data sets in CLL, follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), multiple myeloma (MM) and acute myeloid leukemia (AML). High MZB1 expression was associated with inferior survival in CLL (hazard ratio [HR]: 1.63 [confidence interval (CI): 1.14-2.33], p = 0.007), FL (221286_s_at HR: 1.16 [CI: 0.98-1.37], p = 0.086; 223565_at: HR: 1.3 [CI: 1.1-1.61], p = 0.015) and DLBCL (221286_s_at: HR: 1.17 [CI: 1.06-1.3], p = 0.003; 223565_at: HR: 1.21 [CI: 1.08-1.35], p = 0.001). In DLBCL MZB1 expression was an additive prognostic marker in a multivariate model including activated B-cell like (ABC) versus germinal center (GCB) subtype. Additionally, MZB1 expression correlated with a unique gene expression pattern. This study is the first to show that the expression level of a single gene has prognostic significance in different lymphoma subtypes. Due to its biological function, MZB1 may play a central role in B cell neoplasms and is a potential target for future therapeutic interventions.

Cytogenetically normal acute myeloid leukemia (CN-AML) with biallelic CEBPA gene mutations (biCEPBA) represents a distinct disease entity with a favorable clinical outcome. So far, it is not known whether other genetic alterations cooperate with biCEBPA mutations during leukemogenesis. To identify additional mutations, we performed whole exome sequencing of 5 biCEBPA patients and detected somatic GATA2 zinc finger 1 (ZF1) mutations in 2 of 5 cases. Both GATA2 and CEBPA are transcription factors crucial for hematopoietic development. Inherited or acquired mutations in both genes have been associated with leukemogenesis. Further mutational screening detected novel GATA2 ZF1 mutations in 13 of 33 biCEBPA-positive CN-AML patients (13/33, 39.4%). No GATA2 mutations were found in 38 CN-AML patients with a monoallelic CEBPA mutation and in 89 CN-AML patients with wild-type CEBPA status. The presence of additional GATA2 mutations (n=10) did not significantly influence the clinical outcome of 26 biCEBPA-positive patients. In reporter gene assays, all tested GATA2 ZF1 mutants showed reduced capacity to enhance CEBPA-mediated activation of transcription, suggesting that the GATA2 ZF1 mutations may collaborate with biCEPBA mutations to deregulate target genes during malignant transformation. We thus provide evidence for a genetically distinct subgroup of CN-AML. The German AML cooperative group trials 1999 and 2008 are registered with the identifiers NCT00266136 and NCT01382147 at www.clinicaltrials.gov.

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.

Background The RUNX1 ( AML1 ) gene is a frequent mutational target in myelodysplastic syndromes and acute myeloid leukemia. Previous studies suggested that RUNX1 mutations may have pathological and prognostic implications. Design and Methods We screened 93 patients with cytogenetically normal acute myeloid leukemia for RUNX1 mutations by capillary sequencing of genomic DNA. Mutation status was then correlated with clinical data and gene expression profiles. Results We found that 15 out of 93 (16.1%) patients with cytogenetically normal acute myeloid leukemia had RUNX1 mutations. Seventy-three patients were enrolled in the AMLCG-99 trial and carried ten RUNX1 mutations (13.7%). Among these 73 patients RUNX1 mutations were significantly associated with older age, male sex, absence of NPM1 mutations and presence of MLL -partial tandem duplications. Moreover, RUNX1 -mutated patients had a lower complete remission rate (30% versus 73% P =0.01), lower relapse-free survival rate (3-year relapse-free survival 0% versus 30.4%; P =0.002) and lower overall survival rate (3-year overall survival 0% versus 34.4%; P

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 (n = 729). 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.

We and others have shown that cytogenetically normal (CN)-AML patients with biallelic CEBPA gene mutations (biCEBPA) represent a molecularly distinct group with a favorable prognosis. Patients carrying a monoallelic CEBPA mutation (moCEBPA), however, show no different outcome compared to patients with wildtype CEBPA, and these mutations are frequently associated with mutated NPM1 or FLT3-ITD. So far, no molecular or clinical hallmark has been identified to prognostically distinguish moCEBPA patients from patients with wildtype CEBPA. Therefore, we used the data of 663 CN-AML patients treated within the AMLCG 1999 trial to explore the prognostic value of moCEBPA in the context of concomitant clinical and molecular markers (mutated NPM1, FLT3-ITD). Multiple Cox regression in 515 patients adjusting for all available potential confounders revealed that the NPM1 mutation modified the prognostic value of moCEBPA with respect to overall survival (OS, p = 0.017) and event-free survival (EFS, p = 0.011). MoCEBPA was beneficial in NPM1 mutated patients: adjusted OS–hazard ratio (HR) 0.09, 95% confidence interval (CI) 0.01–0.63, p = 0.016; EFS–HR (95% CI) 0.16 (0.04–0.65), p = 0.010. In contrast, moCEBPA had no prognostic impact in patients with wildtype NPM1: OS–HR (95% CI) 1.08 (0.59–1.97), p = 0.804; EFS–HR (95% CI) 1.12 (0.64–1.96), p = 0.682. We found no prognostic effect modification for moCEBPA by FLT3-ITD. The presence of a moCEBPA mutation was shown to be associated with prolonged survival in NPM1 mutated CN-AML patients. Confirmation of these results in larger studies will clarify whether an additional moCEBPA mutation influences the risk stratification of patients with an NPM1 mutated/FLT3-ITD positive genotype.

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

Genomic 5-hydroxymethylcytosine levels correlate with TET2 mutations and a distinct global gene expression pattern in secondary acute myeloid leukemia

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.

Chromosomal translocations generating fusion proteins are frequently found in human leukemias. The fusion proteins play an important role in leukemogenesis by subverting the function of one or both partner proteins. The leukemogenic CALM-AF10 fusion protein is capable of interacting with the histone H3 lysine 79 (H3K79)–specific methyltransferase hDOT1L through the fused AF10 moiety. This interaction leads to local H3K79 hypermethylation on Hoxa5 loci, which up-regulates the expression of Hoxa5 and contributes to leukemogenesis. However, the long latency of leukemogenesis of CALM-AF10 transgenic mice suggests that the direct effects of fusion oncogene are not sufficient for the induction of leukemia. In this study, we show that the CALM-AF10 fusion protein can also greatly reduce global H3K79 methylation in both human and murine leukemic cells by disrupting the AF10-mediated association of hDOT1L with chromatin. Cells with reduced H3K79 methylation are more sensitive to γ-irradiation and display increased chromosomal instability. Consistently, leukemia patients harboring CALM-AF10 fusion have more secondary chromosomal aberrations. These findings suggest that chromosomal instability associated with global epigenetic alteration contributes to malignant transformation in certain leukemias, and that leukemias with this type of epigenetic alteration might benefit from treatment regimens containing DNA-damaging agents. This study is registered with [www.clinicaltrials.gov][1] as [NCT00266136][2]. [1]: http://www.clinicaltrials.gov [2]: /lookup/external-ref?link_type=CLINTRIALGOV&access_num=NCT00266136&atom=%2Fbloodjournal%2F114%2F3%2F651.atom

The presence of CCAAT/enhancer binding protein alpha (CEBPA) gene mutations in patients with cytogenetically normal acute myeloid leukaemia (CN-AML) confers a favourable prognosis. Routine screening of all CN-AML patients for CEBPA mutations is therefore important for individual risk-adapted post-remission therapy and requires a fast and easy screening method. CEBPA mutations are distributed over the entire CEBPA gene and the functional and clinical consequences of the different mutations are still largely unknown. Therefore, we developed a multiplex polymerase chain reaction-based fragment length analysis mutation screening method for the entire CEBPA coding region. We initially evaluated our method by analysing 120 CN-AML samples both by fragment analysis and nucleotide sequencing and reached a sensitivity of 100% and a specificity of 90%. 349 CN-AML samples were subsequently screened for CEBPA mutations by fragment length analysis. Among a total of 469 CN-AML patient samples, 58 CEBPA mutations were detected in 38 CN-AML patients (8.1%). In conclusion, we established a fast and sensitive CEBPA mutation screening method suitable for inclusion in routine AML diagnostics.

Acute myeloid leukemia (AML) with isolated trisomy 13 (AML+13) is rare and frequently associated with FAB M0 morphology. The clinical course is not well characterized but according to the ELN classification of intermediate prognosis. Eighty to one-hundred percent of patients (pts) with AML+13 carry mutations in the RUNX1 gene. Over-expression of FLT3 (located on chromosome 13 [chr 13]) due to the additional gene copy on the third chr 13 was proposed as a mechanism of leukemogenesis in AML+13 (gene dosage hypothesis). We set out to characterize the clinical course of AML+13 pts and elucidate their molecular background using whole exome sequencing, targeted resequencing and gene expression profiling. We identified 23 pts with AML+13 enrolled in a multicenter trial of the German AML Cooperative Group (AMLCG-1999) and compared this group to 386 pts without +13 who were classified in the ELN Intermediate-II genetic category. All pts received intensive induction chemotherapy. There was no significant difference in age, white blood cell or platelet count between the two groups. However, LDH levels were significantly (p=.01) lower in the AML+13 group while bone marrow blast percentage was significantly higher (p=.04). Twelve AML+13 pts (52%) reached complete remission, but all relapsed. Relapse-free and overall survival were inferior in the AML+13 group compared to other ELN Intermediate-II pts (median RFS, 9 vs 15 months, p=.01; median OS, 7 vs. 13 months, p=.03). Remission samples from two AML+13 pts were available as normal control for exome sequencing. Using SureSelect human all exon target enrichment (Agilent) followed by 80bp paired-end sequencing on an Illumina GAIIx platform, we were able to identify non-synonymous leukemia-specific mutations affecting, among others, RUNX1, ASXL1, PTPN11 and CEBPZ. Genes identified by exome sequencing and a panel of genes recurringly mutated in AML were studied by targeted amplicon resequencing in all AML+13 pts with available material (16/23; Figure). As described before, a high incidence of RUNX1 mutations (75%) was identified. In addition, we detected mutations in spliceosome components in 14/16 (88%) of AML+13 pts, including SRSF2 codon 95 mutations in 13/16 pts (81%). One patient without SRSF2 mutation showed a mutation in SF3B1. Moreover, recurring mutations were found in ASXL1 (44%) and BCOR (25%), and were associated with RUNX1 and SRSF2 mutations. Interestingly, both pts without mutations in the splicing machinery had mutations in DNMT3A, which were also mutually exclusive with mutations in RUNX1 or ASXL1. Two pts carried mutations in CEBPZ suggesting that CEBPZ is a novel recurringly mutated gene in AML.FigureMutation frequencies in 16 patients with AML+13Figure. Mutation frequencies in 16 patients with AML+13 To further characterize this genetically homogenous subgroup, we compared gene expression profiles of 9 pts with AML+13 with 509 AML pts without +13. We identified 678 (up-regulated 492; down-regulated 186) probe sets as significantly deregulated. Only 59 (8.7%) of these probe sets were localized on chr 13, but of those, 55 were up-regulated and only 4 were down-regulated. Up-regulated probe sets on chr 13 included FOXO1, FLT3 and RB1. The strongest down-regulated probe set on chr 13 belonged to the tumor suppressor gene SPRY2, which is a negative regulator of receptor tyrosine kinases. Gene set enrichment analysis showed significant deregulation of gene sets associated with regulation of transcription and nuclear transport. In summary, our study is the first to show that AML+13 is significantly associated with inferior OS and RFS compared to other intermediate-risk cytogenetic abnormalities in a homogeneously treated cohort. Furthermore, we present evidence that AML+13 leukemias are a genetically quite homogenous subgroup. AML+13 is not only associated with a high rate of RUNX1 mutations but also with mutations in SRFS2, ASXL1 and BCOR. The incidence of mutations in SRSF2 in AML+13 is the highest of any AML subgroup reported so far. In addition, our gene expression data show a homogenous expression profile associated with AML+13. The striking association of a few recurring mutations in AML+13 suggests a biological relationship with synergistic lesions during leukemogenesis. While mutations in RUNX1, ASXL1 and up-regulation of FLT3 were previously reported as markers of poor prognosis in AML, the combination of these lesions might be responsible for the extremely poor outcome of AML+13. Disclosures: Krebs: Illumina: Honoraria. Greif:Illumina: Honoraria.

Background Cytogenetically normal acute myeloid leukemia (CN-AML) is the largest and most heterogeneous cytogenetic AML subgroup. For the practicing clinician it is difficult to know how to use the prognostic information of the growing number of clinical and molecular markers. Our purpose was to develop a widely applicable prognostic model by combining well-established pre-treatment patient and molecular characteristics. Patients and methods Two prognostic indices for CN-AML, one with regard to overall survival (PINAOS) and the other regarding relapse-free survival (PINARFS) were derived based on a cohort of 669 CN-AML patients treated within the AML Cooperative Group 99 (AMLCG99) study. Results Based on age (median: 60 years [range: 17-85 years]), performance status, white blood count, and presence or absence of NPM1 mutation, biallelic CEBPA mutation, and FLT3-ITD, patients were classified into three risk groups according to PINAOS and PINARFS: 29% of all and 32% of responding patients had low risk (5-year OS 72%; 5-year RFS 55%), 56% and 39% intermediate risk (5-year OS 28%; 5-year RFS 27%), and 15% and 29% high risk disease (5-year OS 3%; 5-year RFS 8%) (Figure 1). PINAOS and PINARFS further subdivided the European LeukemiaNet (ELN) favorable-genetic group as well as the ELN intermediate-I-genetic group. Both, PINAOS and PINARFS were confirmed in a large, independent, and comparable CN-AML cohort of 529 patients from the Cancer and Leukemia Group B (CALGB/Alliance) trials (Figure 2). Conclusions We have developed and validated the first prognostic indices specifically designed for CN-AML patients of all ages combining well-established molecular and clinical variables easily applicable in routine clinical care. The integration of both clinical and molecular markers could provide a basis for individualized patient care by risk-adapted therapy of CN-AML. Disclosures: No relevant conflicts of interest to declare.

Abstract 1379 Background: A large number of gene mutations have been recently detected in AML using novel sequencing technologies. We established a rapid, amplicon-based resequencing assay that allows efficient analysis of 16 of the most commonly mutated genes in AML and used it to study a cohort of AML patients (pts) carrying a translocation t(9;11)(p22;q23) (MLLT3-MLL; MLL-AF9). This genetic subgroup, accounting for ∼1% of adult AML, is associated with young age, treatment-related disease, FAB M4/M5 morphology, and an intermediate prognosis. There is limited information on the cooperating genetic lesions in adult AML with t(9;11). Importantly, several widely used murine AML models are based on MLLT3-MLL fusion transcript expression. Thus, a better understanding of the genetic basis of human MLLT3-MLL-rearranged AML is necessary to understand how well these animal models reflect their human counterpart and whether findings from MLLT3-MLL-induced disease are generalizable to other genetic subsets. Patients and Methods: We studied 33 bone marrow samples from adult AML pts with t(9;11)(p22;q23) (age range, 20–71 years; median, 44 years; 21 de novo and 12 therapy-related AML). Mutations in ASXL1, CBL, DNMT3A, FLT3, IDH1, IDH2, KIT, KRAS, NRAS, NPM1, RUNX1, SF3B1, SRSF2, TET2, U2AF1 and WT1 were analyzed from 250ng of genomic DNA using a multiplexed, amplicon-based next-generation sequencing approach (Illumina TruSeq Custom Amplicon assay and MiSeq sequencer). KRAS mutations were independently verified using PCR followed by 454 sequencing (Roche), and NRAS and FLT3 mutations by PCR and melting curve analysis or Sanger sequencing. Results: Per patient, we obtained between 96k and 235k paired-end reads (2×150bp) mapping to the regions of interest, resulting in median coverage depths of the target genes ranging from 180-fold (SRSF2) to >2500-fold (KRAS). Overall, mutations affecting growth factor signalling pathways were detected in 73% of MLLT3-MLL rearranged AML (24/33; Figure): Fourteen pts (42%) carried KRAS mutations mostly affecting the known hotspot codons 12, 13 and 61, 6 pts (18%) had NRAS mutations (mainly at codons 12 or 13), 5 pts had FLT3 mutations (4 tyrosine kinase domain mutations and 1 internal tandem duplication), and 2 pts had mutated CBL. The frequency of RAS gene mutations did not differ significantly between de novo AML and pts with treatment-related disease (P=.26). More than one RAS mutation was found in 7 pts, including pts with 2 (n=3) or 3 (n=1) distinct KRAS mutations, 2 pts with mutations in both NRAS and KRAS, and one patient with 2 NRAS mutations. Interestingly, in some of these pts, one mutation was present in a relatively large proportion of sequencing reads (e.g., patient UPN12 showing a KRAS p.Q61H mutation in 36% of reads, consistent with a heterozygous mutation present in the majority of cells in the specimen), while other coexisting mutations affected a much smaller proportion of reads (in patient UPN12, two different KRAS exon 2 mutations in 5% and 2% of reads, respectively). These results suggest the presence of different subclones within the AML blast population, each carrying a different KRAS mutation. Analyses of follow-up samples are underway to assess changes of clonal architecture over time. Other gene mutations were rarely found in this cytogenetic subgroup of AML: In our 33 pts, we detected 2 ASXL1 mutations, 1 mutation each in TET2, SRSF2 and U2AF1, and no mutations in the other 8 genes we studied. Conclusion: Targeted resequencing using a multiplexed amplicon-based assay is a sensitive and rapid method to screen for mutations in a panel of genes commonly involved in AML pathogenesis. To our knowledge, our report is the first comprehensive analysis of cooperating gene mutations in adult AML with t(9;11)(p22;q23). We demonstrate that MLLT3-MLL-rearranged AML is characterized by frequent mutations in genes involved in growth factor signalling (particularly KRAS and NRAS, mutated in 40% and 18%, respectively, of our MLL-MLLT3 AML cohort compared to only about 5% of unselected AML pts), in the absence of other common AML-related gene mutations. Our results complement recent studies reporting RAS mutations in 45% of infant MLL-rearranged ALL, and functional data from mouse models showing that RAS mutations cooperate with the MLLT3-MLL fusion during leukemogenesis. Finally, our results provide evidence for clonal heterogeneity within MLLT3-MLL rearranged human AML. Disclosures: No relevant conflicts of interest to declare.