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Clonal hematopoiesis of indeterminate potential (CHIP) is associated with increased risk of cancers and inflammation-related diseases. This phenomenon becomes common in persons aged ≥80 years, in whom the implications of CHIP are not well defined. We performed a mutational screening in 1794 persons aged ≥80 years and investigated the relationships between CHIP and associated pathologies. Mutations were observed in one-third of persons aged ≥80 years and were associated with reduced survival. Mutations in JAK2 and splicing genes, multiple mutations (DNMT3A, TET2, and ASXL1 with additional genetic lesions), and variant allele frequency ≥0.096 had positive predictive value for myeloid neoplasms. Combining mutation profiles with abnormalities in red blood cell indices improved the ability of myeloid neoplasm prediction. On this basis, we defined a predictive model that identifies 3 risk groups with different probabilities of developing myeloid neoplasms. Mutations in DNMT3A, TET2, ASXL1, or JAK2 were associated with coronary heart disease and rheumatoid arthritis. Cytopenia was common in persons aged ≥80 years, with the underlying cause remaining unexplained in 30% of cases. Among individuals with unexplained cytopenia, the presence of highly specific mutation patterns was associated with myelodysplastic-like phenotype and a probability of survival comparable to that of myeloid neoplasms. Accordingly, 7.5% of subjects aged ≥80 years with cytopenia had presumptive evidence of myeloid neoplasm. In summary, specific mutational patterns define different risk of developing myeloid neoplasms vs inflammatory-associated diseases in persons aged ≥80 years. In individuals with unexplained cytopenia, mutational status may identify those subjects with presumptive evidence of myeloid neoplasms.

Introduction:In elderly acute myeloid leukemia (AML) patients (pts) the debate about the use of intensive chemotherapy (iC) as opposed to non-intensive therapy (niT) or best supportive care (BSC) is a hot topic, particularly after the introduction of venetoclax (VEN) use. Age, comorbidities, functional impairment, therapy benefits and risks, pts preferences, and AML features influence this choice. The SIE/SIES/GITMO (“fitness”) criteria (Ferrara, 2013) are a valuable and comprehensive tool, but a revalidation is needed in the light of the new treatment options.

Background. The only curative treatment of Myelodysplastic syndromes (MDS) is represented by allogeneic hematopoietic cell transplantation (HCT). The risk scoring system conventionally used in clinical practice for transplant decision-making is IPSS-R (Greenberg PL et al, 2012), which has been shown to predict the HCT outcome(Della Porta MG et al, 2014). The recent broader application of next generation sequencing (NGS) in clinical practice provides an abundance of molecular data and poses the challenge of better prognostic stratification in MDS patients. Recently, Bernard et al(Elsa Bernard et al, 2022) developed a molecular prognostic score called IPSS-Molecular (IPSS-M) which defines six risk categories based on the patient's molecular profile combined with clinical and cytogenetic data. In the original study, only 9% of patients underwent HSCT, therefore the utility of this score in the transplant decision making needs to be explored.

BAD, the proapoptotic member of the “BH3-only” subfamily of BCL-2 proteins, is inactivated by phosphorylation at serines 112 and 136 and by sequestration in the cytoplasm where it interacts with members of the 14-3-3 family. In BCR/ABL-expressing cells, BAD is constitutively phosphorylated and mainly cytoplasmic, whereas in cells expressing BCR/ABL mutants unable to protect from apoptosis, BAD is nonphosphorylated. We show here that both the wild-type (WT) and the S112A/ S136A double mutant (DM) BAD are more potent inducers of apoptosis in parental than in BCR/ABL-expressing 32D myeloid precursor cells. Stable lines of parental cells expressing DM BAD could not be established and most clones from WT BAD retrovirus-infected parental cells lost BAD expression. On IL-3 withdrawal from parental 32D cells, BAD was rapidly dephosphorylated by the serine-threonine phosphatase 1, and localized in the mitochondria, whereas it remained phosphorylated and did not localize to the mitochondria in the cohort of BCR/ABL-expressing cells escaping apoptosis induced by WT BAD. Moreover, these cells showed high levels of BCL-2 and BCL-XL expression. The cohort of BCR/ABL-expressing cells resistant to apoptosis induced by DM BAD showed only high levels of BCL-2 and BCL-XL. These findings suggest that BCR/ABL-expressing cells are more versatile than normal hematopoietic progenitors in counteracting the apoptotic potential of BAD, and raise the possibility that tumor cells activate multiple antiapoptotic pathways for survival in the face of death-inducing stimuli.

Emicizumab is a humanized bispecific recombinant monoclonal antibody that binds the enzyme factor IXa and the substrate factor X and mimics the function of FVIII. It solved some unmet needs of haemophilia A (HA) treatment, such as regimen (once weekly up to once monthly infusion) and route of administration (subcutaneous). Most importantly, emicizumab reduce bleeding episodes and improved the quality of life of these patients. Emicizumab received FDA approval for prophylaxis in HA patients with inhibitors in November 2017 and for patients without inhibitors in October 2018.The aim of this study is to provide information on treatment with emicizumab in patients with severe haemophilia A with and without inhibitors in terms of efficacy, safety and quality of life. Here we report our experience with 9 patients with severe haemophilia A (5 with inhibitors and 4 without inhibitors) that were switched from replacement therapy to emicizumab prophylaxis. Median age at initiation of treatment was 26.8 years for patients with inhibitors and 18.7 years for those without inhibitors. Median duration of therapy was 12 months for patients with inhibitors and 8 months for those without inhibitors. All patients were started on emicizumab with a loading dose of 3 mg/kg once weekly for 4 weeks followed by a maintenance dose of 1.5 mg/kg once weekly or every two weeks. The mean annual bleeding rates (ABR) in patients without inhibitor prior to emicizumab was 1.5 compared to 0 while on emicizumab prophylaxis. In subjects with inhibitors ABR was 1.8 compared to 0.4 while on emicizumab prophylaxis. Furthermore, no adverse events including thrombotic events occurred in course of emicizumab prophylaxis. Bleeding events in course of emicizumab prophylaxis did not request any treatment with replacement therapy or bypassing agents. The questionnaire evaluating quality of life (HAL v 2.0 - 2015 ITA; pedHAL v 2.0 - 2015 ITA) was administered to the patients before switch to emicizumab and after a follow up of six months. The scores showed a significant improvement in terms of quality of life in course of emicizumab prophylaxis. In conclusion, our experience suggests that emicizumab prophylaxis is safe and improved protection against bleeding and quality of life compared to replacement therapy in patients with severe A haemophilia.

Introduction.Sickle Cell Disease (SCD) is an evolving public health issue with a significant impact on patient survival, quality of life and costs for health systems also in Italy, a multiethnic country where epidemiology has deeply changed. We present final results from the GREATalyS study that aimed to better understand the SCD burden in Italy in terms of prevalence, clinical features, treatments and resource consumption in the clinical practice setting.

Forni: Bluebirdbio: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees. Castiglioni: Novartis Farma SpA: Current Employment. Condorelli: Novartis Farma SpA: Current Employment. Valsecchi: Novartis Farma SpA: Current Employment. Premoli: Novartis Farma SpA: Current Employment. Fiocchi: Novaris Farma SpA: Current Employment.

Di Raimondo: Amgen: Honoraria; Jazz Pharmaceutical: Honoraria; Pfizer: Honoraria; Janssen Pharmaceuticals: Honoraria; Bristol Myers Squibb: Honoraria; AbbVie: Honoraria.

Gamma-globin chain synthesis has been evaluated in individual bursts and subcolonies that were generated by normal adult blood BFU-Es in methylcellulose cultures containing semipurified erythropoietin (Ep) and then analyzed via either isoelectric focusing (IEF) of globin chains or immunofluorescence techniques. At variance with previously reported results, based on plasma clot culture and immunofluorescence, all bursts and subcolonies analyzed synthesize gamma-globin chains. Identification of gamma-chains has been confirmed by preparative IEF of HbF, followed by either carboxymethylcellulose chromatography or IEF analysis of the resulting globin chains. In all bursts analyzed, the relative synthesis of the two types of gamma-globin chains (G gamma and A gamma) shows an adult ratio (i.e., approximately 1:1). The results obtained via IEF have been confirmed by immunofluorescence studies, which apparently showed presence of at least some HbF-positive cells within all scrutinized bursts or subcolonies. The significance of these studies is discussed in the light of current hypotheses on mechanism(s) underlying HbF synthesis in normal adults.

Adult rats were subjected sequentially to (1) administration of colloidal carbon, zymosan, or gadolinium; (2) subtotal hepatectomy, bilateral nephrectomy, or sham operation; and (3) a 6-hr bout of hypoxia, starting 1 hr or 24 hr after the operation. Control animals received the respective vehicles. The erythropoietin (Ep) activity was assayed in exhypoxic polycythemic mice on the basis of 48-hr per cent RBC-59Fe incorporation values. Ep levels in serum of anephric rats primed with either colloidal carbon or zymosan were considerably more elevated than in control animals. This potentiating effect was observed in rats subjected to hypoxia starting either 1 or 24 hr after nephrectomy. On the other hand, gadolinium did not enhance the extrarenal Ep response to hypoxia. It is emphasized that both colloidal carbon and zymosan induced marked hyperplasia of the hepatic and splenic reticuloendothelial system (RES), while gadolinium did not induce this effect. A strict correlation was thus established between potentiation of extrarenal Ep production and hyperplasia of the RES. It is therefore tentatively concluded that the RES is a source of extrarenal Ep. Additionally, since the liver plays a prominent role in extrarenal Ep production, Kupffer cells may represent a major source for Ep in the anephric rat. Finally, it is of interest that both colloidal carbon and zymosan did not potentiate the Ep response to hypoxia in sham-operated or subtotally hepatectomized rats.

The Id proteins and basic helix-loop-helix (bHLH) proteins play major roles in specifying cell fate decisions in diverse biologic settings. A potential role for Id and TAL1/E2A bHLH genes in hematopoiesis has been suggested by studies on immortalized cell lines. However, it is uncertain whether these observations reflect normal hematopoiesis. We have investigated the expression pattern of Id2 and TAL1/E2A genes in liquid suspension culture of purified hematopoietic progenitor cell (HPCs) undergoing erythroid or granulopoietic differentiation in the first culture week and maturation to terminal cells in the second week. In quiescent, freshly purified HPCs, Id2 mRNA is detected by reverse transcriptase-polymerase chain reaction (RT-PCR), whereas TAL1 and E2A mRNAs are not. At the onset of erythroid differentiation, Id2 mRNA is downregulated, while E2A and TAL1 mRNAs are concomitantly upregulated: their expression is further increased at erythroblast level. Conversely, Id2 is not downmodulated in granulopoietic culture, except for a late decline at day 10 to 12, while TAL1 and E2A are only transiently induced in the first week of granulopoietic differentiation. The expression pattern of the TAL1/E2A heterodimer, as evaluated by mobility shift assay, is consistent with RT-PCR results (except for lower levels of the heterodimer in late erythroid maturation). TAL1 protein level, analyzed by Western blot, shows a pattern consistent with gelshift results. Functional experiments were performed on purified HPCs treated with phosphorothioate antisense oligodeoxynucleotides to Id2 or TAL1 mRNA. The results are strictly consistent with the expression studies: anti-Id2 oligomer (alpha-Id2) causes a significant dose-dependent increase of erythroid colony formation, whereas alpha-TAL1 induces a selective dose-related inhibitory effect on erythroid colonies, as compared with untreated or scrambled oligomer-treated control HPCs. Finally, murine and human glutathione-S-transferase (GST)-Id2 polypeptides compete the TAL1/E2A- specific DNA binding activity when added to the nuclear extracts derived from erythroid culture cells, thus indicating biochemical and suggesting functional interaction of Id2 with the TAL1/E2A complex. These novel observations indicate a coordinate expression and function of an inhibitory Id protein (Id2) and a stimulatory bHLH/bHLH heterodimer (TAL1/E2A) in normal erythroid differentiation.

The Id proteins and basic helix-loop-helix (bHLH) proteins play major roles in specifying cell fate decisions in diverse biologic settings. A potential role for Idand TAL1/E2A bHLHgenes in hematopoiesis has been suggested by studies on immortalized cell lines. However, it is uncertain whether these observations reflect normal hematopoiesis. We have investigated the expression pattern of Id2and TAL1/E2Agenes in liquid suspension culture of purified hematopoietic progenitor cell (HPCs) undergoing erythroid or granulopoietic differentiation in the first culture week and maturation to terminal cells in the second week. In quiescent, freshly purified HPCs, Id2mRNA is detected by reverse transcriptase-polymerase chain reaction (RT-PCR), whereas TAL1and E2AmRNAs are not. At the onset of erythroid differentiation, Id2mRNA is downregulated, while E2Aand TAL1mRNAs are concomitantly upregulated: their expression is further increased at erythroblast level. Conversely, Id2is not downmodulated in granulopoietic culture, except for a late decline at day 10 to 12, while TAL1and E2Aare only transiently induced in the first week of granulopoietic differentiation. The expression pattern of the TAL1/E2A heterodimer, as evaluated by mobility shift assay, is consistent with RT-PCR results (except for lower levels of the heterodimer in late erythroid maturation). TAL1 protein level, analyzed by Western blot, shows a pattern consistent with gel-shift results. Functional experiments were performed on purified HPCs treated with phosphorothioate antisense oligodeoxynucleotides to Id2or TAL1mRNA. The results are strictly consistent with the expression studies: anti-ld2oligomer (α-ld2)causes a significant dose-dependent increase of erythroid colony formation, whereas α-TAL1induces a selective dose-related inhibitory effect on erythroid colonies, as compared with untreated or scrambled oligomer-treated control HPCs. Finally, murine and human glutathione-S-transferase (GST)-ld2 polypeptides compete the TAL1/E2A-specific DNA binding activity when added to the nuclear extracts derived from erythroid culture cells, thus indicating biochemical and suggesting functional interaction of Id2 with the TAL1/E2A complex. These novel observations indicate a coordinate expression and function of an inhibitory Id protein (Id2) and a stimulatory bHLH/bHLH heterodimer (TAL1/E2A) in normal erythroid differentiation.

Adult rats were subjected sequentially to (1) administration of colloidal carbon, zymosan, or gadolinium; (2) subtotal hepatectomy, bilateral nephrectomy, or sham operation; and (3) a 6-hr bout of hypoxia, starting 1 hr or 24 hr after the operation. Control animals received the respective vehicles. The erythropoietin (Ep) activity was assayed in exhypoxic polycythemic mice on the basis of 48-hr per cent RBC(-59) Fe incorporation values. Ep levels in serum of anephric rats primed with either colloidal carbon or zymosan were considerably more elevated than in control animals. This potentiating effect was observed in rats subjected to hypoxia starting either 1 or 24 hr after nephrectomy. On the other hand, gadolinium did not enhance the extrarenal Ep response to hypoxia. It is emphasized that both colloidal carbon and zymosan induced marked hyperplasia of the hepatic and splenic reticuloendothelial system (RES), while gadolinium did not induce this effect. A strict correlation was thus established between potentiation of extrarenal Ep production and hyperplasia of the RES. It is therefore tentatively concluded that the RES is a source of extrarenal Ep. Additionally, since the liver plays a prominent role in extrarenal Ep production, Kupffer cells may represent a major source for Ep in the anephric rat. Finally, it is of interest that both colloidal carbon and zymosan did not potentiate the Ep response to hypoxia in sham-operated or subtotally hepatectomized rats.

Three cases of adult pure red cell aplasia (PRCA) are reported. All patients proved refractory to various combinations of androgens and corticosteroids. The first case, harboring a thymoma, showed a complete clinical remission following cyclophosphamide therapy. The second and third responded similarly to either a combined cyclophosphamide + antilymphocyte globulin (ALG) treatment or to ALG administration preceded by a small dosage of cyclophosphamide, which had proved ineffective when administered alone. Serum IgG inhibitors to erythropoiesis were demonstrated in all cases by means of in vivo and/or in vitro techniques. The inhibitors), although directed against the erythroid marrow in both the first and third patients (PRCA type A), apparently functioned as an antibody to circulating erythropoietin (Ep) in the second case (PRCA type B). The inhibitor(s) was always absent in postremission samples. Additionally, experimental models for both types of human PRCA were established in normal rodents. The present studies support the contention that adult PRCA is an autoimmune disease. The therapeutic role of cytotoxic-immunodepressive agents in PRCA patients is confirmed. It is emphasized that ALG may represent an additional therapeutic tool in cases resistant to cyclophosphamide and/or steroids. In addition, cyclophosphamide proved effective in a patient harboring a thymoma not amenable to surgery. Finally, it is postulated that IgG serum auto-antibodies, directed against either an early erythroid precursor (PRCA type A) or, more rarely, circulating Ep (PRCA type B), play a major role in the pathogenesis of the disease.

Gamma-globin chain synthesis has been evaluated in individual bursts and subcolonies that were generated by normal adult blood BFU-Es in methylcellulose cultures containing semipurified erythropoietin (Ep) and then analyzed via either isoelectric focusing (IEF) of globin chains or immunofluorescence techniques. At variance with previously reported results, based on plasma clot culture and immunofluorescence, all bursts and subcolonies analyzed synthesize γ-globin chains. Identification of γ-chains has been confirmed by preparative IEF of HbF, followed by either carboxymethylcellulose chromatography or IEF analysis of the resulting globin chains. In all bursts analyzed, the relative synthesis of the two types of γ-globin chains (Gγ and Aγ) shows an adult ratio (i.e., approximately 1:1). The results obtained via IEF have been confirmed by immunofluorescence studies, which apparently showed presence of at least some HbF-positive cells within all scrutinized bursts or subcolonies. The significance of these studies is discussed in the light of current hypotheses on mechanism(s) underlying HbF synthesis in normal adults.

The regulation of iron absorption is only partially understood. A cause and effect relationship between the erythropoietic and the absorption rates has been postulated, but not firmly established. Experiments in polycythemic mice were undertaken in an attempt to separate the effect of hypoxia on iron absorption from that on the erythropoietic activity. The latter parameter was evaluated on the basis of per cent values of peripheral reticulocytes, nucleated red cells in red pulp of spleen, and/or RBC-59Fe incorporation. In the first series of experiments the erythroid response to an 18- or 96-hr period of hypoxia was selectively suppressed by administration of antierythropoietin (anti-Ep) serum. However, the enhancement of iron absorption induced by an equivalent hypoxic stimulus was not significantly modified by anti-Ep. In additional studies, polycythemic mice were subjected to either fractionated Ep administration or hypoxic stimuli, lasting 24, 48, or 72 hr. The stimulatory effect on erythropoiesis induced by Ep was either greater or equivalent to that of hypoxia. However, although these hypoxic stimuli induced a four- to sixfold increase in iron absorption, Ep administration evoked either little or no enhancement of iron absorption. These experiments provide evidence against concepts linking the primary regulation of iron absorption to the erythropoietic rate.

Three cases of adult pure red cell aplasia (PRCA) ARE REPORTED. All patients proved refractory to various combinations of androgens and corticosteroids. The first case, harboring a thymoma, showed a complete clinical remission following cyclophosphamide therapy. The second and third responded similarly to either a combined cyclophosphamide + antilymphocyte globulin (ALG) treatment or to ALG administration preceded by a small dosage of cyclophosphamide, which had proved ineffective when administered alone. Serum IgG inhibitors to erythropoiesis were demonstrated in all cases by means of in vivo and/or in vitro techniques. The inhibitor(s), although directed against the erythroid marrow in both the first and third patients (PRCA type A), apparently functioned as an antibody to circulating erythropoientin (Ep) in the second case (PRCA type B). The inhibitor(s) was always absent in postremission samples. Additionally, experimental models for both types of human PRCA were established in normal rodents. The present studies support the contention that adult PRCA is an autoimmune disease. The therapeutic role of cytotoxic-immunodepressive agents in PRCA patients is confirmed. It is emphasized that ALG may represent an additional therapeutic tool in cases resistant to cyclophosphamide and/or steroids. In addition, cyclophosphamide proved effective in a patient harboring a thymoma not amenable to surgery. Finally, it is postulated that IgG serum autoantibodies, directed against either an early erythroid precursor (PRCA type A) or, more rarely, circulating Ep (PRCA type B), play a major role in the pathogenesis of the disease.

The regulation of iron absorption is only partially understood. A cause and effect relationship between the erythropoietic and the absorption rates has been postulated, but not firmly established. Experiments in polycythemic mice were undertaken in an attempt to separate the effect of hypoxia on iron absorption from that on the erythropoietic activity. The latter parameter was evaluated on the basis of per cent values of peripheral reticulocytes, nucleated red cells in red pulp of spleen, and/or RBC-59Fe incorporation. In the first series of experiments the erythroid response to an 18- or 96-hr period of hypoxia was selectively suppressed by administration of antierythropoietin (anti-Ep) serum. However, the enhancement of iron absorption induced by an equivalent hypoxic stimulus was not significantly modified by anti-Ep. In additional studies, polycythemic mice were subjected to either fractionated Ep administration or hypoxic stimuli, lasting 24, 48, or 72 hr. The stimulatory effect on erythropoiesis induced by Ep was either greater or equivalent to that of hypoxia. However, although these hypoxic stimuli induced a four- to sixfold increase in iron absorption, Ep administration evoked either little or no enhancement of iron absorption. These experiments provide evidence against concepts linking the primary regulation of iron absorption to the erythropoietic rate.

Clonal hematopoiesis of indeterminate potential (CHIP) is associated with increased risk of cancers and inflammation-related diseases. This phenomenon becomes very common in oldest-old individuals, in whom the implications of CHIP are not well defined. We performed a mutational screening in 1794 oldest-old individuals enrolled in two population-based studies and investigate the relationships between CHIP and associated pathologies. Clonal mutations were observed in one third of oldest-old individuals and were associated with reduced survival. Mutations in JAK2and splicing genes, multiple mutations (DNMT3A, TET2, ASXL1with additional genetic lesions) and variant allele frequency ≥0.096 had positive predictive value for myeloid neoplasms. Combining mutation profiles with abnormalities in red blood cell indices improved the ability of myeloid neoplasm prediction. On this basis, we defined a predictive model that identifies 3 risk groups with different probabilities of developing myeloid neoplasms. Mutations in DNMT3A, TET2, ASXL1or JAK2(most occurring as single lesion) were associated with coronary heart disease and rheumatoid arthritis. Cytopenia was a common finding in oldest-old population, the underlying cause remaining unexplained in 30% of cases. Among individuals with unexplained cytopenia, the presence of highly-specific mutation patterns was associated with myelodysplastic-like phenotype and a probability of survival comparable to that of myeloid neoplasms. Accordingly, 7.5% of oldest-old subjects with cytopenia had presumptive evidence of myeloid neoplasm. In conclusion, specific mutational patterns define different risk of developing myeloid neoplasms vs.inflammatory-associated diseases in oldest-old population. In individuals with unexplained cytopenia, mutational status may identify those subjects with presumptive evidence of myeloid neoplasms.

Castiglioni: Novartis Farma SpA: Employment. Condorelli:Novartis Farma SpA: Employment. Amore:Novartis Farma SpA: Employment. Perrone:CliCon srl: Employment. Alessandrini:CliCon srl: Employment. Veronesi:CliCon srl: Employment. Degli Esposti:CliCon srl: Employment. Premoli:Novartis Farma SpA: Employment. Fiocchi:Novartis Farma SpA: Employment.

Background:

No relevant conflicts of interest to declare.

Background.Acute Myeloid Leukemia is a heterogeneous disease, characterized by the uncontrolled proliferation of hematopoietic precursor cells. AML accounts for 15% of acute leukemias in children, with a cure rate of 55-60% as overall survival (Pession A Blood 2013). Complete remission (CR) is achieved in 80-85% of children with AML. Current protocol defines CR by morphological evaluation (blasts <5%) and cytofluorimetric analyses (blasts <0,1%), after two cycles of high-dose chemotherapy. Around 10-15% of children and 30% of adults do not achieve remission and are defined as Primary Induction Failure (PIF). The majority of these cases are addressed to other form of high-dose chemotherapy blocks, experimental therapies or directly to hematopoietic stem cell transplantation (HSCT) as salvage treatment, showing a very poor prognosis (Quarello P et al. British Journal of Haematology 2015). Strong efforts to generate biological data in order to define this subtype of AML are needed for early identification and for target therapy design.

Conventional cytogenetics is the major prognostic factor in predicting outcome of myelodysplasia (MDS), but at least 50% of patients have a normal karyotype. 5-azacytidine (5-AZA) is a DNA methyltransferase inhibitor used in treatment of high-risk MDS, which is able to delay the progression to AML and improve clinical outcomes, but targets of the methylation status are poor known.To evaluate molecular changes, at genomic or proteomic level, which can be identified as additional targets of 5-azacytidine in MDS patients with normal conventional cytogenetics.By reverse-phase protein microarray (RPMA), we analyzed bone marrow mononuclear cells from 19 patients affected of high risk MDS, treated with 5-azacytidine (median age 71 years, M/F=12/5). Treatment consisted of 4 cycles of 100mg flat dose for 7 days+21 days of wash-out. In 7 cases the sample after 4 cycles of therapy was matched to the sample collected before starting treatment. RPMA was used to quantitatively map 45 cell signaling pathway endpoints, including survival, proliferation, drug resistance, apoptosis, and autophagy. For the first 4 patients we used also a genome-wide approach based on SNP (single nucleotide polymorphism) array (6.0 Affymetrix platform) to detect copy number and allelotype data in order to identify new genetic markers in terms of SNP, CNV and LOH.All patients were evaluable for response one month after the 4th cycle. Three patients were refractory and progressed to AML and 1 was a late responder (after 7 cycles). All other patients experienced hematologic improvement. After 4 cycles of 5-AZA, at proteomic level we found three main signaling pathways were increased and did not correlate with the response: 1) pro-survival signaling: PLC-y-1-Tyr783 (p=0.0017), and its upstream regulators Src-Tyr416, c-Abl-Tyr735 (p=0.002) and downstream target STAT5Tyr694 (p=0.0017) were increased, without affecting proliferative pathways, such as AKT activation status on Ser473 and Thr308 or mTORSer2448. 2) Autophagy: ATG5, Beclin 1 and LC3B were significantly elevated after treatment (p values respectively <0.0001, 0.0056 and 0.0124). Activation of the autophagy pathway occurred downstream of mTOR, since mTORSer2448, AktSer473, AktThr308, ERKThr202Tyr204 (and in general proliferation markers) were not affected. 3) Restore of cell cycle G1-S checkpoint: c-Abl-Tyr735, Rb-Ser608, Rb-Ser780Chk1-Ser345, p53, p53Ser15, and in particular HDAC1, HDAC3 were significantly elevated (p<0.001).At genomic level, we identified 46 losses and 25 gains. In three cases we found a deletion on chromosome 20q13.12 (from 46125 kb to 46178 kb) at level of NCOA3 gene, a transcriptional coactivator protein that contains several nuclear receptor interacting domains and an intrinsic histone acetyltransferase activity. In 2 cases we found a loss in the region 22q13 (from 40871 kb to 40910 kb), at level of MKL1 gene, encoding a nuclear protein involved in transduction signals from the cytoskeleton to the nucleus. MKL1 is involved in a specific translocation event associated with acute megakaryocytic leukemia. Moreover in one case we found a deletion on chromosome 4q24 (from 105800 kb to 107200 kb) in a region involving TET2 gene. Loss of heterozygosity 4q24 and TET2 mutations have been implicated in the pathogenesis of myelodysplastic syndromes. Another patient showed autozigosity regions with an extensive CN-LOH on chromosomes 7 and 11, involving the centromers and recently their neoplastic potential in colon cancer have been reported and never, before, observed in MDS.More informative findings can be detected using a proteomic approach in combination with the genomic one. Autophagy activation can be considered an escape pathway promoting survival in neoplastic cells and the observation that 5-AZA does not affect proliferative pathways suggests the possibility to combine it with anti-proliferative agents. The increase of HDAC1 and HDAC3 can provide the molecular rationale for the development of a combination of 5-azacytidine with HDAC inhibitors.No relevant conflicts of interest to declare.

Abstract 3818

In acute myeloid leukemias (AML) chromosomal aberrations, detectable by conventional cytogenetics or targeted molecular techniques, provide the basis for a classification with prognostic relevance. However, cases with normal cytogenetics and undefined prognosis still constitute the single largest group. Recent advances in genome-wide analysis of submicroscopic DNA segment copy number variations (CNVs) may allow the identification of novel molecular tumor-associated abnormalities in the normal cytogenetics group (somatic CNVs). However, CNVs are also present physiologically in the normal population (germline CNVs) (Redon et al., 2006) and can represent potential predisposition factors in disease. Indeed, CNVs can have dramatic phenotypic consequences as a result of altering gene dosage, disrupting coding sequences, or perturbing long-range gene regulation. We used the last generation of Affymetrix single nucleotide polymorphism (SNP)/CNV microarrays (SNP Array 6.0) containing probes for the detection of CNVs and SNPs, with an inter-marker distance of 680 bases and a resolution power of 100 kb. SNP Array 6.0 Assay kit (Affymetrix, Santa Clara, CA) is able to assess copy number changes (CNVs) at a resolution comparable with data obtained using oligonucleotide-array-comparative genomic hybridization (aCGH) and provides also information on loss of heterozygosity (LOH) of the allelic imbalance and copy number neutral type. In the present communication we report preliminary results of a study aimed to test the ability of such arrays to distinguish tumor-associated somatic CNVs and LOHs from germ-line ones by comparing bone marrow samples from AML patients at diagnosis (>90% blasts) and at the remission phase. So far, 8 M4–M5 FAB subtype AML patients have been studied, 6 females, 2 males (median age 38 years, range 25–51). At diagnosis 4 cases with normal karyotype, 2 cases with trisomies (respectively trisomy 13 in 25% and trisomy 22 in 80% of 20 metaphases), 1 inversion (inv (16) (p13q22)) and 1 balanced translocation (t (6;14) (q27;q23)) were detected by conventional cytogenetic analysis. We obtained arrays with quality control (QC) call rates in excess of 90% in all cases (>95% in 8/13 cases) and MAPD <0.4 (<0.35 in 7/13 arrays), using Genotyping Console Version 2.1 for signal intensity analysis, as recommended by Affymetrix. To obtain copy number and LOH calls we used a predefined reference model file, obtained from 270 healthy individuals (HapMap collection). All samples that were regarded as normal karyotype by chromosomal banding had detectable submicroscopic abnormalities by the SNP/CNV array assay. Results obtained are reported in table 1. We found 13 somatic gains not in overlap with known CNVs deposited in the Toronto Database of Genomic Variants. The only recurrent somatic CNV(2/8 patients) was a gain of 109kb in 7q22.1, where genes MGC57359 and GATS map. Five recurrent germline CNVshave been detected, both at diagnosis and remission samples, which could represent regions determining susceptibility to AML. The trisomy 13 case showed a whole chromosome somatic LOH at chromosome 21. 3/8 patients had an interstitial somatic LOH in 19q13.12 in correspondence with adhesion molecules genes (CEACAM1, MEGF8, PSG 1-6-7, ZNF 526). Finally, we detected an interstitial germline LOH, common to all samples, in 16q22.1, where CBFB (core binding factor beta) gene maps, involved in FAB subtypes evaluated in this study. Although this is an ongoing study, with preliminary results, we think that such genome-wide characterization of sub-microscopic DNA alterations might contribute to the discovery of new markers and target genes, with diagnostic, prognostic or therapeutic relevance.

In acute myeloid leukemias (AML) chromosomal aberrations, detectable by conventional cytogenetics or targeted molecular techniques, provide the basis for a classification with prognostic relevance. However, cases with normal cytogenetics and undefined prognosis still constitute the single largest group. Recent advances in genome-wide analysis of submicroscopic DNA segment copy number variations (CNVs) may allow the identification of novel molecular tumor-associated abnormalities in the normal cytogenetics group (somatic CNVs). However, CNVs are also present physiologically in the normal population (germline CNVs) (Redon et al., 2006) and can represent potential predisposition factors in disease. Indeed, CNVs can have dramatic phenotypic consequences as a result of altering gene dosage, disrupting coding sequences, or perturbing long-range gene regulation. We used the last generation of Affymetrix single nucleotide polymorphism (SNP)/CNV microarrays (SNP Array 6.0) containing probes for the detection of CNVs and SNPs, with an inter-marker distance of 680 bases and a resolution power of 100 kb. SNP Array 6.0 Assay kit (Affymetrix, Santa Clara, CA) is able to assess copy number changes (CNVs) at a resolution comparable with data obtained using oligonucleotide-array-comparative genomic hybridization (aCGH) and provides also information on loss of heterozygosity (LOH) of the allelic imbalance and copy number neutral type. In the present communication we report preliminary results of a study aimed to test the ability of such arrays to distinguish tumor-associated somatic CNVs and LOHs from germ-line ones by comparing bone marrow samples from AML patients at diagnosis (>90% blasts) and at the remission phase. So far, 8 M4–M5 FAB subtype AML patients have been studied, 6 females, 2 males (median age 38 years, range 25–51). At diagnosis 4 cases with normal karyotype, 2 cases with trisomies (respectively trisomy 13 in 25% and trisomy 22 in 80% of 20 metaphases), 1 inversion (inv (16) (p13q22)) and 1 balanced translocation (t (6;14) (q27;q23)) were detected by conventional cytogenetic analysis. We obtained arrays with quality control (QC) call rates in excess of 90% in all cases (>95% in 8/13 cases) and MAPD <0.4 (<0.35 in 7/13 arrays), using Genotyping Console Version 2.1 for signal intensity analysis, as recommended by Affymetrix. To obtain copy number and LOH calls we used a predefined reference model file, obtained from 270 healthy individuals (HapMap collection). All samples that were regarded as normal karyotype by chromosomal banding had detectable submicroscopic abnormalities by the SNP/CNV array assay. Results obtained are reported in table 1. We found 13 somatic gains not in overlap with known CNVs deposited in the Toronto Database of Genomic Variants. The only recurrent somatic CNV (2/8 patients) was a gain of 109kb in 7q22.1, where genes MGC57359 and GATS map. Five recurrent germline CNVs have been detected, both at diagnosis and remission samples, which could represent regions determining susceptibility to AML. The trisomy 13 case showed a whole chromosome somatic LOH at chromosome 21. 3/8 patients had an interstitial somatic LOH in 19q13.12 in correspondence with adhesion molecules genes (CEACAM1, MEGF8, PSG 1-6-7, ZNF 526). Finally, we detected an interstitial germline LOH, common to all samples, in 16q22.1, where CBFB (core binding factor beta) gene maps, involved in FAB subtypes evaluated in this study. Although this is an ongoing study, with preliminary results, we think that such genome-wide characterization of sub-microscopic DNA alterations might contribute to the discovery of new markers and target genes, with diagnostic, prognostic or therapeutic relevance. CNV or LOH per sample Normal karyotype Abnormal karyotype median range median range CNV (diagnosis) 38 16–52 79 18–163 ratio gain/loss at diagnosis 7 6–7 15 4–22 CNV (remission) 24 16–32 49 20–167 ratio gain/loss at remission 1 1–2 9 1–20 germline CNV 18 14–21 33 7–77 somatic CNV 16 2–30 51 3–86 LOH (diagnosis) 299 285–314 316 307–317 LOH (remission) 291 285–297 295 282–318 germline LOH 283 278–289 287 287–308 somatic LOH 16 12–19 20 8–30 Table 1