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Malnutrition is a common problem after allogeneic hematopoietic stem cell transplantation (allo-HSCT) and could impair immune function. Immune dysfunction after allo-HSCT may be linked with infections, GVHD, and relapse and negatively affect the outcome. Aim of this review was to identify malnutrition biomarkers, potentially useful for immune-system monitoring, in the setting of allo-HSCT. After a systematic search, no satisfying biomarker was found, except for citrulline. Citrulline could be useful in monitoring gastrointestinal function after allo-HSCT and its role in the complex relationship with immune-system function ought to be better explored. A multi-omics approach, including biomarkers and PRO (patient reported outcomes) is, in our opinion, the optimal way to study the relationship between malnutrition and transplant outcomes.

Background Myelodysplastic syndromes and acute leukemias after allogeneic stem cell transplantation (allo-SCT) are mainly caused by recurrence of the primitive leukemic clones. More rarely, they originate from donor hematopoietic stem cells, developing the so-called donor cell leukemia (DCL) or myelodysplastic syndromes (DC-MDSs). DCL and DC-MDS can be considered as an in vivo model of leukemogenesis, and even if the pathogenetic mechanisms remain speculative, a genetic predisposition of donor progenitor cells, an altered host microenvironment, and the impairment of immune surveillance are considered the main causes. Case presentation We report a case of DC-MDS diagnosed 5 years after an allo-SCT from a matched related donor (patient's sister) in a patient with Philadelphia chromosome-positive B-cell acute lymphoblastic leukemia (Ph+ B-ALL). The sex-mismatch allowed us to identify the donor cell origin. At the onset, the DC-MDS was characterized by chromosome seven monosomy and NRAS, RUNX1, and BCOR mutations. Because of a familiar history of colorectal neoplasia and the variant allele frequency (VAF) of NRAS mutation at the onset, this mutation was searched on germline DNA in both the donor and the recipient, but the result was negative. Moreover, after transplant (+4 months), the patient developed severe and long-lasting chronic graft-versus-host disease (cGVHD), requiring multiple lines of treatments. Because of the severe immunosuppression, recurrent infections occurred and, lately, the patient died due to septic shock. Conclusion This case report highlights the need, whenever possible, to evaluate the donor origin of the posttransplant myelodysplasia and acute leukemias. The potential key role of the impaired immune surveillance and of long-lasting immunosuppression appears to be emerging in the development of this case of DC-MDS. Finally, this case reminds the importance to investigate the familiar genetic predisposition in donors with a familiar history of neoplasia.

INTRODUCTION Endothelial cells (EC) play an important role in thrombosis and are increased in frequency in Myeloproliferative Neoplasms (MPN). Notably, the JAK2V617F mutation has been identified in micro-laser-dissected EC in peripheral vasculature (Sozer, 2009) and is present in a subset of endothelial progenitor cells (Teofili, 2011). Primary myelofibrosis (PMF) is also characterized by increased bone marrow vascularity. These data suggest that PMF clones could derive from a common precursor shared between CD34-hematopoietic stem cells (HSC) and EC. We explored this hypothesis with a prospective study aimed at comparing the mutational profiles of paired Circulating Endothelial Cells (CEC) and HSC in patients (pts) with PMF. METHODS 14 PMF pts not previously treated with JAK2 inhibitors, along with one healthy control, were enrolled in the MyCEC0617 study beginning in 2018. The study was approved by the local Ethical Committee and all pts gave written informed consent. HSC were selected using CD34+ immunomagnetic bead-column separation. CEC were detected using the CellSearch system, which uses immunomagnetic selection incorporating ferrofluid nanoparticles and fluorophore-labelled antibodies. The markers CD146, CD105, CD45, and DAPI were used to isolate CEC. We identified cells as CEC when we observed characteristic morphological features and a surface immunophenotype of CD146+, CD105 +, DAPI+ and CD45-. Putative CEC were then sorted using the DEPArray system, which uses a combination of di-electrophoresis technology and high-quality image-based cell selection to manipulate individual cells. Sequencing data was then assessed with the MiSeq Illumina NGS platform using a 54-gene custom panel focused on genes mutated in PMF. The cutoffs to confirm the presence of the mutations were identification of mutant alleles in 30 and 50 reads both in forward and reverse, for HSC and CEC, respectively. RESULTS The characteristics of pts are reported in Figure 1. Median age of pts was 69.5 ys (35-85) and male/female ratio was 9:6. Median time from diagnosis to sample collection was 4 (1-211) months. 2/14 pts had had a previous thrombosis, 78% had splenomegaly, and 29% presented with constitutional symptoms. Considering the molecular profile, 9 pts were JAK2V617F mutated, 3 were CALR mutated, and 1 was MPLW515L positive. 2 pts were triple-negative. Most of the pts (7) presented with an Intermediate-1 DIPSS score, while 5 were intermediate-2, and 2 were high risk DIPSS. CEC were collected for 12 of 15 subjects (including the normal control). No significative differences were found between pts from whom we isolated CEC and those we did not successfully recover CEC. A median of 26 (1-122) CEC in 4 ml of peripheral blood were recovered. Notably, no mutations were found in the CEC or HSC from healthy control whereas molecular alterations were discovered both on CEC and HSC in 11 pts (Fig. 1). The previously-identified MPN driver mutation was identified in MF HSC (except for one JAK2-mutated pt and for all CALR-mutated pts, consistent with the limitations of NGS in detecting these lesions). Interestingly, PMF pts demonstrated both shared and different mutations when comparing mutational profiles of HSC and CEC. In particular, 8 of 11 pts shared at least one mutation between EC and HSC. 2 pts harbored the same driver mutation (JAK2V617F), together with ABL1, IDH1, TET2, and ASXL1, respectively. The most frequently mutated genes shared between both CEC and HSC were JAK2, ASXL1, TET2, NOTCH1, and SRSF2. All of these mutations were observed as shared clonal events in at least 2 cases. We also identified individual paired HSC/CEC with shared mutations in TP53, KIT, KMT2A, IDH1, WT1 and ABL1. One pt shared 4 mutations between HSC and CEC, while 4 and 2 pts shared 1 and 2 mutations, respectively. CONCLUSION HSC and EC in PMF have in common one or more gene mutations implicated in the pathogenesis of PMF. For the first time, we show that mutations other than JAK2 can be identified in EC, and in most instances (70% of pts), these mutations are shared between CEC and HSC. Moreover, we present an analysis of mature cells of endothelial lineage including to the CEC. These preliminary findings using primary pts samples support the notion that PMF-initiating cells may be derived from a common HSC/EC precursor. Further data are needed to validate these findings and provide a rationale for additional studies exploring the antecedent cell of origin in MPN. Disclosures D'Adda: Pfizer: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees. Rossi:Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees; Sanofi: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz: Membership on an entity's Board of Directors or advisory committees; Astellas: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Mundipharma: Honoraria; BMS: Honoraria; Sandoz: Honoraria; Daiichi-Sankyo: Consultancy; Roche: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees. Levine:Qiagen: Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy; Loxo: Membership on an entity's Board of Directors or advisory committees; Roche: Consultancy, Research Funding; Prelude Therapeutics: Research Funding; Isoplexis: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Membership on an entity's Board of Directors or advisory committees; Imago Biosciences: Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy; Lilly: Honoraria; Amgen: Honoraria; Celgene: Consultancy, Research Funding.

Introduction. A multidimensional geriatric assessment has been shown to predict therapy-related toxicities in elderly hematology patients. No data are yet available on the use of combined functional and comorbidity scores in the allogeneic stem cell transplant (alloSCT) setting. The Lymphoma Italian Foundation (FIL) score, a multidimensional approach, combines the geriatric comorbidity score using the cumulative illness rating scale-geriatric (CIRS-G) with the activities of daily living (ADL) score, instrumental activity of daily living (IADL) score, and older age. Aims. The aim of the present study was to evaluate the role of the multidimensional FIL score in predicting alloSCT outcome, in terms of transplant-related mortality (TRM), relapse incidence (RI) and overall survival (OS). Patients and methods. Overall, 228 patients older than 60 years were submitted for alloSCT in Italy and France, during the last 10 years (2009-2018). Patients' characteristics are detailed in Table 1. The median age of the study population was 64 years (range, 60-76), and 155 (68%) were male. Acute myeloid leukemia was the most common indication for transplant (53%) and complete remission status was recorded in 54% of cases at transplantation. A matched unrelated donor, sibling donor or alternative donor was utilized in 41%, 31%, and 28% of cases, respectively. Stem cell source was derived from peripheral blood in 82%, bone marrow in 8%, and cord blood in 10% of cases. Utilization of a myeloablative conditioning regimen was reserved for only 20% of patients. Median number of CD34+ x10^6/Kg infused cells was 5 (range, 0,01-12,9), and of CD3+ x10^7/Kg cells 18 (range, 0,2-47,8). The median follow-up period of the cohort was 36 months (range, 12-138). Overall survival was estimated by the Kaplan-Meier method from the date of transplant to the date of last follow-up or death; the log-rank test was used to detect differences between subgroups. The Fine and Gray competing risk regression model was used for calculation of TRM and RI. Univariate and multivariate analyses were carried out using the Cox proportional-hazards regression model. Results. A total of 121 (53%) patients died at last follow-up. The cause of death was related to TRM in 60 (49%) cases comprising infection (31), toxicity (13), acute GVHD (9), and chronic GVHD (7); disease relapse was the ultimate cause of death in 61 (51%) cases . The 2-year expected TRM, RI and OS were 25%, 36% and 49%, respectively. The FIL score was measured in 215 patients and classified patients as 'fit' in 125 (58%) casesa nd 'unfit/frail' in 90 (42%). According to the FIL score, fit patients were more frequently in complete remission (CR) at alloSCT compared with unfit/frail patients (78% vs 21%, p2 (34%). Interestingly, the HCI-CI (HR 1.06, 95%CI 0.96-1.16, p=0.27) failed to predict alloSCT outcome. In contrast , FIL score, predicted fit patients to have a better 2- and 5-year OS of 66% and 59% compared to 32% and 30% in unfit/frail patients (p 80 (0.53, 95%CI 0.36-0.78, p=0.0013) and FIL frail/unfit status (2.83, 95%CI 1.93-4.17, p80 (HR 0,56, 95%CI 0,37-0,85, p=0.0066) and FIL score (HR 2,20, 95%CI 1,27-3,78, p=0.0046) maintained significant associations with OS (Figure 2). Conclusions. In a fairly large cohort of elderly patients (≥60 years) undergoing alloSCT at two European transplant centers, a multidimensional geriatric approach appears to be more accurate compared to a sole comorbidity assessment, in predicting alloSCT outcome and identifying patients at high risk of TRM. This simple tool could easily be applied before transplant for better patient selection and tailoring of treatment. Disclosures Mohty: Jazz Pharmaceuticals: Honoraria, Research Funding.

Therapy with Tyrosine Kinase Inhibitors (TKIs) changed the fate of Philadelphia-positive Chronic Myeloid Leukemia (CML). At present, the therapeutic strategy aims to improve the management of the disease and the quality of life of the patients. In July 2015, we started a prospective multicentric randomized trial with the aim to validate the policy of the intermittent de-escalation treatment and to explore the impact of this strategy on the Quality of Life. To this purpose, CML patients older than 60 years in stable (≥2 years) MR3.0 or MR4.0 molecular response were randomized to receive a FIXED intermittent TKIs regimen (one month ON and one month OFF), as previously published (Russo D, Blood 2013; Russo D, BCJ 2015), versus a PROGRESSIVE intermittent TKIs regimen (one month ON and one month OFF for the 1st year; one month ON and two months OFF for the 2nd year; one month ON and three months OFF from the 3rd year) (OPTkIMA study, ClinicalTrials.gov: NCT02326311). Molecular monitoring was performed according to the 2015 ELN guidelines, every 3 months by RT-PCR on peripheral blood (Baccarani M,Blood 2013). In case of MR3.0 (MMR) loss, checked in two monthly consecutive RT-PCR analysis, patients were planned to exit the study and to resume TKIs daily. This first interim report have been focused on the patients who, by intention to treat, have completed the first year of the study for an historical comparison with the previous INTERIM trial (Russo D, Blood 2013; Russo D, BCJ 2015). During the first year, both the patients randomized in the FIXED and in the PROGRESSIVE arms were given the TKIs treatment one month ON and one month OFF. Up to June 2018, 177 patients have been enrolled by 26 Italian Hematological Centers (first patient randomized in July 2015) and 121/177 patients (68%) completed the first year of OPTkIMA study. The median age was 71 years (range 60-89) and 64% of the patients were belonging to the Sokal intermediate/high risk goup. 96/121 (79%), 14/121 (12%) and 11/121 (9%) patients were receiving imatinib (IMA), nilotinib (NILO) and dasatinib (DAS), at the time of enrollment. Overall, 59/121 (49%) and 62/121 (51%) patients have been randomized in the FIXED and PROGRESSIVE arm, respectively. 41/62 patients (66%) randomly assigned to the PROGRESSIVE arm have entered the second year of therapy. 34/121 patients (28%) went out of the study during the first year. The reasons for protocol discontinuation were: informed consent withdrawn (2 cases), second cancer (4 cases), loss of MR3.0 (28 cases). (Table 1). Among the 28 patients who lost the MMR, 17 and 11 were in MR4.0 and MR3.0, respectively, when they were enrolled into the study. Thus the probability of loosing the MR3.0 while on OPTkIMA was 21,7% at one year (Figure 1). All the 28 patients resumed TKIs continuously and all obtained at least the MR3.0 response, within 6 months and are currently included in the study follow up. The intermittent treatment was well tolerated, with 4 serious adverse events (1 appendicitis, 1 atrial fibrillation, 1 cardiac failure, 1 hip fracture) and 3 adverse events (1 diarrohea, 1 pruritus and 1 fever), none of which have been considered treatement-related. None of the patients experienced the TKI withdrawn syndrome. According to this first interim report, we found that a policy of intermittent TKIs administration in elderly patients is safe and well tolerated. Analysis of patient-reported QoL outcomes is ongoing and will further add information on the overall treatment effectivness of the new PROGRESSIVE intermittent TKI administration. After the 1st year, 28/121 patients (23%) lost MR3.0 and all of them re-gained the major molecular response within 6 months from resumption of continuous treatment. The probability of MR3.0 loss while on OPTkIMA at 1 year was 21,7% and this is quite comparable with the 20% MR3.0 loss observed in the previous INTERIM trial (Russo D, Blood 2013; Russo D, BCJ 2015). Disclosures Efficace: Bristol Meyers Squibb: Consultancy; Seattle Genetics: Consultancy; Lundbeck: Research Funding; TEVA: Research Funding; AMGEN: Research Funding; Incyte: Consultancy; Amgen: Consultancy; TEVA: Consultancy; Orsenix: Consultancy. Abruzzese:Novartis: Consultancy; BMS: Consultancy; Pfizer: Consultancy; Ariad: Consultancy. Bonifacio:Incyte: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Novartis: Research Funding; Bristol Myers Squibb: Consultancy. Castagnetti:Incyte: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Bristol Meyers Squibb: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Giai:Novartis: Consultancy; Pfizer: Consultancy.

Two different rabbit anti-thymocyte globulin (ATG) formulations, thymoglobulin-ATG (tATG) and fresenius-ATG (fATG) are usually employed as prophylaxis for graft-versus-host-disease (GVHD), one of t...

The outcome of patients underwent to allogeneic stem cell transplantation (allo- SCT) is closely related to graft versus host disease (GvHD) and graft versus leukemia (GvL) effects which can be mediated by mHAgs. 23 mHAgs have been identified and reported to be differently correlated with GVHD or GVL and the aim of this work was develop a method to genotype the mHAgs described so far. For this study we used MALDI-TOF iPLEX Gold Mass Array technology. We tested 46 donor/recipient matched pairs that underwent allo-SCT because of Philadelphia positive (Ph+) chronic myeloid leukemia (n = 29) or Ph+ acute lymphoblastic leukemia (n =17). Our data show that sibling pairs had a lesser number of mHAgs mismatches compared to MUD pairs. Notably, donor/recipient genomic mismatch on DPH1 was correlated with an increased risk of acute GvHD and LB-ADIR-1R mismatch on graft versus host direction was correlated with a better RFS with no increase of GvHD risk. Our work provides a simple, accurate and highly automatable method for mHAgs genotyping and suggest the role of mHAgs in addressing the immune reaction between donor and host.

T and B lymphocyte subsets have been not univocally associated to Graft-versus-host disease (GVHD) and relapse of hematological malignancies after stem cell transplantation (SCT). Their sequential assessment together with B and T cell neogenesis indexes has been not thoroughly analysed in relation to these changing and interrelated immunologic/clinic events yet. Lymphocyte subsets in peripheral blood (PB) and B and T cell neogenesis indexes were analysed together at different time points in a prospective study of 50 patients. Principal component analysis (PCA) was used as first step of multivariate analysis to address issues related to a high number of variables versus a relatively low number of patients. Multivariate analysis was completed by Fine-Gray proportional hazard regression model. PCA identified 3 clusters of variables (PC1-3), which correlated with acute GVHD: PC1 (pre-SCT: KRECs≥6608/ml, unswitched memory B 44%, CD8+TCM cells>4%; HR 1.9, p = 0.01), and PC3 (at aGVHD onset: CD4+TEMRA69%, switched memory CD19+ = 0 cells and KRECs

Background A normal number of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) is considered a biomarker for adequate new T- and B-cell production. In newborns, detection of TRECs and KRECs by real time PCR from dried blood spotted on filter paper is used for the screening of severe immunodeficiency. In adults, elderly and during diseases, where the number of TRECs is lower than in newborns and children, a large amount of DNA and a sensitive method of amplification are necessary to identify newly produced lymphocytes. Methods DNA was prepared from blood of 203 healthy adults (range: 18–91 years old) absorbed for 10 s on flocked swabs and let to dry, or from peripheral blood mononuclear cells. DNA was subjected to digital PCR and to well established conventional real time PCR-based method using TREC- and KREC-specific primers and probes. The number of TRECs and KRECs was expressed per mL of blood. Statistical analysis was performed by nested ANOVA, Pearson coefficient of determination, and by linear regression tests. Results The novel method for the storage of dried blood on nylon flocked swabs and the use of digital PCR allow quantification of TRECs and KRECs with high degree of sensitivity, specificity, accuracy, and precision. TRECs and KRECs were amplified by digital PCR in all tested blood samples, including those obtained from elderly individuals (>70 years old) and that were negative by real time PCR. Furthermore, values of TRECs and KRECs obtained by digital PCR were in the range of those acquired by real time PCR. Conclusions Our findings demonstrate that DNA isolation from dried blood on flocked swabs followed by digital PCR-based analysis represents a useful tool for studying new lymphocyte production in adults and elderly individuals. This suggests the potential use of the methodology when monitoring of clinical variables is limited by the number of molecules that can be amplified and detected, such as in patients with immunodeficiency or under immunosuppressive therapies. Electronic supplementary material The online version of this article (doi:10.1186/s12967-017-1169-9) contains supplementary material, which is available to authorized users.

Favourable/intermediate ELN-risk acute myeloid leukemias (AMLs) (e.g. those harboring t(8;21) or inv(16) or NPM1A mutations or CEBP-alpha bi-allelic mutations) account for 30% to 50% of all newly diagnosed AMLs [1,2]. In this setting, conventional induction treatments may induce complete remission (CR) in up to 70% to 80%, but relapses still occur in 40% to 50% of cases and, at the end, no more than 30% to 40% of patients can be cured. Therefore, the optimization of post- remission therapy represents the greatest challenge in the treatment of favourable/intermediate-I ELN-risk AML.

Philadelphia-positive (Ph+), BCR-ABL1, chronic myeloid leukemia (CML) is a model of leukemia driven by a single, specific, chromosome translocation, the t (9;22) (q22;q11). This translocation, leading to a new, hybrid, leukemia-specific gene (BCR-ABL1) encoding for a deregulated tyrosine-kinase protein (p210), drives the leukemic transformation of hematopoietic stem cells [1-6] and induces the progression of the disease from the early chronic phase (CP) to the late blastic phase BP) which close the natural history of the disease. In the 2000s, the introduction of Imatinib, the first tyrosinekinase inhibitor (TKI) able to target the protein p210, significantly changed the fate of CML to fatal disease in real chronic disease.

Background Bacterial, fungal, and viral infections often affect non-relapse mortality after allogeneic stem cell transplantation (alloSCT). Recovery from infections depends on a balanced integration between innate and adaptive immune responses. In this complex interplay, a key role is played by Toll-like receptors (TLRs), which are sensors of pathogen-associated molecular patterns. To our knowledge, no previous study deals with both expression and function of all human TLRs together, in relation to infections in the setting of alloSCT. Methods We prospectively evaluated 9 TLRs by flow cytometry on T lymphocytes and monocytes of 35 patients in relation to infectious events from day +30 to day +120. Tumor necrois factor-alpha, interleukin-4, interferon-gamma, and monocyte chemoattractant protein-1 induction upon TLR activation was assessed by enzyme-linked immunosorbent assay on cell supernatants. Results In multivariate Cox regression analysis, levels of TLR-9 expression on T lymphocytes (P = 0.01) and values of natural killer cells (P = 0.01) correlated negatively with bacterial infections, whereas cytomegalovirus (CMV) infection resulted as a positive predictor. We observed a trend for negative correlation between TLR-7 levels on T lymphocytes and fungal infections (P = 0.07). Values of monocytes were negatively associated with CMV infection (P = 0.03), whereas levels of TLR-5 on T lymphocytes were positive predictors (P = 0.01). Age (P = 0.03) and bacterial infections (P = 0.006) negatively influenced overall survival. Monocyte values were positive predictors of survival (P = 0.003). Conclusions Bacterial, fungal, and CMV infections were associated with a different expression of some TLRs on T lymphocytes. The protective role of TLR-7 and TLR-9 seemed dominant over other TLRs involved in recognizing fungi and bacteria. We also observed an atypical involvement of TLR-5 in CMV infection. The dominant and atypical role of some TLRs could depend on their pleiotropic functions and the changing inflammatory environment of transplanted patients. A specific TLR profile and an adequate count of monocytes could improve survival, promoting an effective control of infections, and balanced immune responses. If our findings will be confirmed by further studies, these immunological variables could be useful as parameters to predict susceptibility to infections.

Monitoring BCR-ABL1 transcript by quantitative PCR (qPCR) is essential for the management of CML patients treated with TKIs. Currently, up to 40-50% of CML patients treated with TKIs can achieve a deep molecular response (DMR = BCR-ABL1 minor or equal to0,01% IS), but only 50% of them are reported to maintain a stable Treatment Free Remission (TFR). Since qPCR has some intrinsic limitations in detection and quantification of BCR-ABL1 transcript, this method does not appear optimal in the selection of patients eligible for TKIs cessation. A precise monitoring of BCR-ABL1 transcript levels can help even better the clinicians in managing CML patients treated with TKIs and in selecting the best candidates for discontinuation of TKIs without relapse. Digital PCR (dPCR) can be more advantageous than qPCR. It gives the absolute quantification of target nucleic acidsby partitioning the PCR reaction mix over a large number of wells, each containing a single copy or no copies of the target region. Based on the assumption of Poisson's distribution, the number of template copies originally presenting in the sample can be calculated from the number of partitions in which amplification has successfully occurred. In that way, standard curves cannot be necessary and data are more accurate. In this study we set a dPCR assay to quantify the BCR-ABL1 transcript in a preliminary cohort of CML patients with major (MMR or MR3.0) or deep molecular response (MR4.0, MR4.5 and MR5.0). The analysis by dPCR were based on a TaqMan-MGB probes targeting the BCR-ABL1 transcript. Custom assay was designed and produced with a FAM-label, basing on the sequence of routinely-used probes. The experiments were performed on a QuantStudio 3D Digital PCR System (Life Technologies). A commercial BCR-ABL1 Mbcr standard dilutions (Qiagen) and 10 replicates of blank samples (DNA-free, RNA-free water) were used to set dPCR assay and to determine the Limit of Detection and the Limit of Quantification of our method. The values of absolute quantities of BCR-ABL1 transcript assessed by dPCR were expressed as number of copies/ul. Samples for dPCR testing were obtained from peripheral blood (10 ml in EDTA tubes) of CML patients treated with TKIs, namely imatinib, nilotinib, or dasatinib, on time-checks planned for monitoring MMR or DMR through the conventional qPCR. To the purpose of the study, we evaluated 10 cases with stable MR3.0, 7 cases with stable MR4.0, 6 cases with stable MR4.5, 7 cases with stable MR5.0. It has been considered as stable any MR3.0, MR4.0, MR4.5, or MR5.0 molecular response measured by conventional qPCR and detected in the last three consecutive checks performed during the last 12 months. Blank samples served as negative controls, while 10 peripheral blood samples of healthy donors served as normal controls. Digital PCR (dPCR) revealed different levels of BCR-ABL1 copies/µl among the CML patients achieving the major (MR3.0) or deep (MR4.0, MR4.5 AND MR5.0) molecular response with TKIs. Moving from MR3.0 to MR5.0 molecular response the median of BCR-ABL1 copies/µl assessed by dPCR were progressively decreasing (Figure 1), with blanks and healthy controls approximately to zero. Medians with ranges were 0.957 (0.472-1.692) for MR3.0; 0.319 (0.072-0.906) for MR4.0; 0.231 (0.063-0.651) for MR4.5; 0.219 (0.074-0.399) for MR5.0; 0.076 (0.000-0.118) for healthy controls, and 0.000 (0.000-0.129) for blanks. Moreover, dPCR revealed different BCR-ABL1 copies/µl among the patients of each class of molecular response. Importantly, in patients with MR4.5, MR5.0 and with undetectable levels of BCR-ABL1 % IS as measured with qPCR, discrete variable levels of BCR-ABL1 copies/µl have been detected by dPCR. These data, revealing different levels of BCR-ABL1 copies/µl beyond the limit of detection and quantification of conventional qPCR, may explain why no correlation was observed between BCR-ABL1 % IS levels measured by qPCR and numbers of BCR-ABL1 copies/µl measured by dPCR. We are screening CML patients with MMR and DMR, but these preliminary results show that dPCR appears to be more accurate than qPCR for detection and quantification of BCR-ABL1 transcript and it should be seen as a useful step forward in order to better manage the TKI therapy and to better select the candidates for TFR. Acknowledgments: Department of Clinical and Experimental Sciences, University of Brescia; PRIN2009; European Leukaemia Net; BCC "Pompiano e Franciacorta". Figure 1. Figure 1. Disclosures Tiribelli: Bristol Myers Squibb: Consultancy, Speakers Bureau; Ariad Pharmaceuticals: Consultancy, Speakers Bureau; Novartis Farma: Consultancy, Speakers Bureau. Rosti:Bristol Myers Squibb: Honoraria, Research Funding, Speakers Bureau; Novartis: Honoraria, Research Funding, Speakers Bureau. Martinelli:Roche: Consultancy; BMS: Speakers Bureau; MSD: Consultancy; ARIAD: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy.

Chromosome number alterations, aneuploidy, is a hallmark of cancer. It occurs in about 15% of acute myeloid leukemia (AML) cases, is generally preserved throughout disease progression (Bochtler et al. Leukemia 2015) and correlates with adverse prognosis (Breems et al. JCO 2008, Papaemmanuil et al. NEJM 2016). This evidence highlights the need of understanding the molecular mechanisms that promote and sustain aneuploidy in AML, in order to define novel potential therapeutic targets. In the NGS-PTL project we profiled the genomic landscape of 536 hematological samples by whole exome sequencing (WES, Illumina). Among them, we analyzed 88 and 68 samples from aneuploid (A-) FLT3-wildtype AML (isolated trisomy and monosomy, complex and monosomal karyotype) and euploid (E-) AML (normal and complex karyotype, A-AML showed an increased genomic instability, as confirmed by a higher mutation load compared with E-AML (median number of variants: 22 (range: 2-95) and 11 (range: 3-45), respectively, pA substitutions, compared with the C>T transition-related signature, which is prevalent in AML. A-AML was associated with mutations and/or heterozygous deletion of TP53 (p We show here for the first time the molecular mechanisms promoting and maintaining aneuploidy in AML. Our results indicate that p53 deficiency, either caused by somatic mutations, copy number loss, impaired DNA damage response and enhanced PLK1 signaling synergize with APCgain, RAD50 structural or functional loss and forced progression through mitosis, to override cell cycle and mitotic checkpoints and allow the formation of daughter cells with an aberrant chromosome number. These mechanisms cooperate with recurrent mutations of genes involved in protein ubiquitination and proteasome-mediated protein catabolic process in A-AML, indicative of the attempt of aneuploid cells to override the proteotoxic stress due to the unbalanced protein load generated by the aneuploid condition. This evidence provides the rationale for exploiting proteasome inhibition (Velcade), p53 reactivation (MDM2/4 inhibitor) and targeting of the cell cycle (CHK1/2 inhibitor) downstream to p53 (WEE1 inhibitor) as strategies for novel combination therapies against aggressive aneuploid AML, which are under clinical investigation in our Institution and may serve as a model for aneuploid tumors. GS and AP: equal contribution. Supported by: FP7 NGS-PTL project, ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi). Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Martinelli:Ariad: Consultancy, Speakers Bureau; MSD: Consultancy; Celgene: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Genentech: Consultancy; Novartis: Speakers Bureau; BMS: Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau.

Introduction . Anti-thymocyte globulin (ATG), has been shown to significantly reduce the incidence and severity of acute and chronic graft versus host disease (GVHD) in pts submitted toallogeneic stem cell transplantation (allo-SCT). Two different ATG formulations, derived from rabbit immunization against T antigens,thymoglobuline-ATG (tATG) andfresenius-ATG (fATG) are usually employed. However, only few retrospective studies are available in order to compare the efficacy and safety of these two drugs. Moreover, the differential impact of the two formulations on immune recovery has been poorly studied. Methods. Clinical and laboratory data of 76 pts receivingfATG (30 mg/kg for 3 days beforeallo-SCT) ortATG (10 mg/Kg for 3 days beforeallo-SCT) as GVHD prophylaxis forallo-SCT from matched unrelated donor (MUD) for hematological malignancies at the Unit of Stem Cells Transplantation ofSpedaliCivili of Brescia between 2009 and 2014, were retrospectively collected. Basic phenotype of circulating lymphocytes was assessed by flow-cytometry at 3-months interval fromallo-SCT. The aims of the study were to compare the two ATG formulations in term of incidence of acute and chronic GVHD, infections, non-relapse mortality, relapse-free survival, overall survival and immune recovery. The study was approved by the local Ethic Committee. Results. Overall, 46 (60%) pts receivedfATG and 30 (40%)tATG as GVHD prophylaxis, in association to cyclosporine andmethotrexate. Baseline characteristics were (median): age, 46 years (range, 17-66); male, 68%; acute leukemia, 53%; complete hematological remission, 51%; HLA full-matched (8/8), 40%; source of stem cells, peripheral blood, 87%;myeloablative conditioning regimen, 49%; median follow-up, 22 months (range, 1-88). No significant differences were observed between the two populations according age, sex, hematological disease, disease status atallo-SCT, HLA match, stem cell source, amount of CD3+ and CD34+ cells infused and conditioning regimen. Overall, 26 (57%)fATGand 19 (61%)tATG-exposed pts developed an acute GVHD (p=0.8); among 18 (24%) pts who developedcGVHD, 2 out of 10 (20%)fATGand 6 out of 8 (75%)tATG-receiving pts evolved towards severecGVHD(p=0.05). In particular, the cumulative incidence of severecGVHD, was 5,2% versus 27,6% at 2 years (p=0.03) infATGandtATGgroup, respectively (Figure 1). Bacterial infections were encountered in 42 (91%) and 25 (81%) (p=0.19), fungal infections in 12 (26%) and 6 (19%) (p=0.58)fATGandtATG-treated pts. CMV reactivation was observed in 28 (61%) and 19 (73%) (p=0.43)fATGandtATG-exposed pts, respectively. However, a trend for a higher incidence of early-CMV reactivation was seen in thetATGgroup, with a cumulative incidence of CMV reactivation at 30 days of 30% versus 52% infATGandtATGcohort, respectively (p=0.09). No differences were observed betweenfATGandtATGpopulations in term of relapse-related mortality (24% versus 40%, p=0.25), non-relapse mortality (28% versus 20%, p=0.48) and overall survival (52% versus 60%, p=0.71). Immunologic reconstitution was evaluated in 48 pts (fATG, 64%). As compared totATG,fATGpts showed a significant lower amount of CD3-/CD16+ NK cells (median, 137/mclvs255/mcl, p=0.05) and a trend for lower CD3+CD4+CD25+CD127- T-regcells (5/mclvs11/mcl, p=0.07) and CD4/CD8 ratio (0.26vs0.57, p=0.08) at 3 months fromallo-SCT. At 6 months CD4/CD8 ratio was significantly lower infATGcompared totATGgroup (0.26vs0.42, p=0.01). At 9 months, lymphoid populations did not differ between the two cohorts. Overall, ATG administration was well-tolerated without grade III-IV adverse events, however an increase in infusion-related events (mainly, fever and chills) was recorded infATGgroup (67%vs32%, p=0.004). Conclusion. This retrospective study, reporting a quite large population ofallo-SCT pts from MUD homogeneously followed in a single Hematology Center, shows thatfATG-treated pts have a lower incidence of severecGVHD compared totATG. On the contrary a slight increase of early CMV reactivation has been observed intATG group. These differences do not result in a higher relapse-related or overall mortality. The distinct immune reconstitution as identified by flow-cytometricanalysis, could justify these observations, that need to be validated by prospective studies. Figure 1 Cumulative incidence of severecGVHD according to type of ATG employed Figure 1. Cumulative incidence of severecGVHD according to type of ATG employed Disclosures No relevant conflicts of interest to declare.

Current therapy of CML with tyrosine kinase inhibitors (TKIs) aims to achieve a Major Molecular Response to avoid the disease progression to blastic phase and, possibly, a Deep Molecular Response (DMR), to gain the opportunity for TKIs discontinuation and Treatment Free Remission (TFR). However, TFR strategies are not yet optimized, as well as the criteria for patients (pts) selection to TKI discontinuation are not well defined. The patient's CML prognostic score, the duration of TKI treatment and "stable" DMR before discontinuation and the levels of DMR are still matters of debate. Due to its intrinsic limitations, RT-qPCR cannot be considered as an optimal tool for a precise detection of minimal BCR-ABL1 transcript levels and DMR monitoring. The digital PCR (dPCR) has emerged as a more sensitive and accurate method to detect and monitor the BCR-ABL1 minimal residual disease (MRD). This study focused on the BCR-ABL1 RT-q/dPCR comparative monitoring in CML pts treated with TKIs and with durable DMR (≥ 2 years), as conventionally assessed by RT-qPCR, before the enrollment. The aim was to evaluate the reliability and the efficiency of dPCR, as compared to RT-qPCR, for a better identification of "stable" DMR and for a better selection of the candidates for treatment discontinuation. 118 CML pts with durable DMR were enrolled and 493 peripheral blood samples were comparatively analyzed by both RT-qPCR, according to the last International Guidelines, and dPCR (QS3D Digital PCR System). RT-qPCR results were assessed as BCR-ABL1 %IS. dPCR results were assessed as number of BCR-ABL1 copies/□l of reaction and the molecular response was categorized as □ 0.468 or < 0.468 BCR-ABL1 copies/□l according to the previously reported cut off (Bernardi S. et al, J Mol Biom Diagn, 2017). Starting from the first RTq/dPCR comparative assessment, the 118 pts were monitored every 3/4 months for at least 3 time points. Grouping the 118 pts according to the RT-qPCR molecular results at the enrolment, 50 cases (42%) resulted in the MR4.0group and 68 (58%) in the MR 4.5-MR5.0 group. No significant differences were observed between these 2 groups in terms of clinical and hematological characteristics. At the same time, the 118 pts were divided in 2 groups based on the comparative dPCR molecular results, according to the above mentioned cut-off value: 29 (25%) and 89 (75%) cases had dPCR values □ and □0.468 BCR-ABL1 copies/□l, respectively. No significant clinical differences were observed between these 2 groups. Out of these 118 pts, 87 (74%) discontinued the TKI treatment and 24 (28%) of them lost the DMR after a median time of 3 months (range 1-30). Based on dPCR, 23/87 (26%) and 64/87 (74%) had dPCR □ 0.468 and < 0.468, respectively. Considering the pts who lost DMR, 12/23 (41%) and 12/64 (20%), had dPCR □ 0.468 and < 0.468, respectively (p=0.0021). Therefore, evaluating BCR-ABL1 transcript levels by dPCR before the discontinuation, we could observe a better TFR (positive predictive value of 80%) in those pts with dPCR < 0.468, with respect to the ones with □ 0.468. However, no significant difference was observed between the groups divided according to the evaluation of BCR-ABL1 transcript levels by RT-qPCR before the discontinuation in terms of TFR prediction. In order to reach a better recognition of stable DMR, which is strictly related with the probability to achieve and maintain TFR after TKIs discontinuation, we comparatively analyzed, by RTq/dPCR, the course of MRD during the follow up period. In the case of monitoring by RT-qPCR, a wide oscillation of the molecular responses was observed and they tended to be superimposable both in the group of pts with MR4.0 and in those with MR4.5 and 5.0. In the case of monitoring by dPCR, the values assessed at the enrolment and during the follow up revealed the capability of the dPCR to measure different and heterogeneous numbers of BCR-ABL1 copies in each pts. Moreover, the pts with a dPCR below the cut-off value of 0.468, at enrolment, were maintaining values (median 0,175; range 0-1,863), significantly lower (p This retrospective study show that the dPCR is more accurate and sensitive than conventional RT-qPCR for detecting and monitoring the BCR-ABL1 molecular levels and potentially able to improve the recognition of the stable DMR and selection of the candidates to TKI treatment discontinuation. Figure 1. Figure 1. Disclosures Bonifacio: Incyte: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Novartis: Research Funding; Bristol Myers Squibb: Consultancy. Gaidano:Morphosys: Honoraria; Amgen: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Gilead: Consultancy, Honoraria; Roche: Consultancy, Honoraria; AbbVie: Consultancy, Honoraria. Rossi:Teva: Membership on an entity's Board of Directors or advisory committees; Mundipharma: Honoraria; Sanofi: Membership on an entity's Board of Directors or advisory committees; Janssen: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Abbvie: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; Gilead: Membership on an entity's Board of Directors or advisory committees; Sandoz: Honoraria; Jazz: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria; Amgen: Membership on an entity's Board of Directors or advisory committees. Gobbi:Novartis: Consultancy; Ariad: Membership on an entity's Board of Directors or advisory committees; Pfister: Membership on an entity's Board of Directors or advisory committees; Amgen: Consultancy; Janssen: Consultancy; Celgene: Membership on an entity's Board of Directors or advisory committees. Voso:Celgene: Research Funding, Speakers Bureau. Abruzzese:Ariad: Consultancy; BMS: Consultancy; Novartis: Consultancy; Pfizer: Consultancy.

Background. A Deep Molecular Response (DMR), defined as BCR-ABL1 p210 transcript at levels Aim. To compare and examine values of the three different methods of detecting minimal residual disease (MRD) in CML at RNA and LSC levels in patients in TFR or DMR. Patients and Methods. The twenty-seven patients in this study received treatment with either Imatinib (12), Dasatinib (6), Nilotinib (7), Bosutinib (1) or Interferon (1). Twelve patients were in TFR, while the rest were in DMR. TFR patients had stopped therapy for less than 1 year (3), 250,000 ABL copy numbers). D-PCR analysis used TaqMan-MGB probes targeting the BCR-ABL1 transcript. A custom assay was designed and produced with a FAM-label, basing on the sequence of routinely-used probes. BCR-ABL1 quantifications were performed analyzing 50ng of cDNA on a QuantStudio 3D Digital PCR System (ThermoFisher Scientific). BCR-ABL1 transcript values using dPCR were expressed as number of copies/ul. The secondary analysis were carried out by AnalysisSuite Cloud Software (ThermoFisher Scientific). To detect peripheral blood circulating CD34+/CD38-/CD26+ LSCs, cells were incubated with anti-CD45 (BD Biosciences), anti-CD34 (581), anti-CD38 (HIT2) and anti-CD26 (M-A261) (BD Pharmigen). Acquisition and analysis were performed by FACSCanto II flow cytometer (BD Biosciences, NR Nannini). CD45+ cells acquired for each sample ranged from 500,000 to 1,000,000. Isotype controls were included in each staining. Median absolute number of CD26+ cells/μL were calculated as follows: (# WBCs/μL) × (% CD34+/CD38−/CD26+ stained CD45+ cells). Results. TFR status, the type of TKI treatment, QPCR results, dPCR data and LSCs quantification are summarized in Table 1. Both d-PCR and LSCs showed higher sensitivity than QPCR, exhibiting positive results when transcript levels using QPCR were undetectable (16). None of the patients tested negative with d-PCR, however 14/16 were under the threshold of 0.468 copies/uL, corresponding to a stable DMR. LSC levels were negative in 5 patients, 3 of which also tested negative with QPCR. In all patients QPCR ranged from 0-0.0068, d-PCR from 0.073 to 0.943, and LSCs from 0 to 0.156. Results were divided in quartiles, depending on molecular response for QPCR, the 0.468 threshold for dPCR and the distribution for the LSCs. The 2 lowest quartiles defined the lowest detectable DMR. A strong correlation of these data in TFR patients was noted (10/12 concordant) while 8/15 DMR patients were discordant. Conclusions To our knowledge this is the first attempt to analyze and compare DMR in a CML population using standard (QPCR) and highly sensitive (dPCR and LSCs) methods. TFR patients, some lasting up to 17 years, were in the lowest detectable DMR categories. Very little is known about the biology of CML implications of circulating LSCs. Larger studies and dynamic scoring will help define their informative and predictive value. Disclosures Abruzzese: Pfizer: Consultancy; Ariad: Consultancy; Novartis: Research Funding; BMS: Consultancy.

Introduction In AML and MDS cases, the genetic lesions inherited or acquired by the hematopoietic stem cells are considered as starting events. Familial AML and MDS, recently recognized in the revised WHO classification (2016) provide a useful model for investigation of predisposing genetic mutations. Genetic analysis of several pure familial leukemia pedigrees led to the discovery of well defined syndromes associated with inherited de novo mutations on germline DNA. Growing clinical awareness as well as a widespread use of NGS have led to an enlarged description of familial MDS/AML cases, and the number of mutations involved, suggesting they are more frequent than those previously recognized. Despite the recent discovery of well-established causative gene mutations (RUNX1, GATA2, ETV6, TERT, TERC, SRP72, ANKRD26, DDX41, CEBPA), many cases remain unexplained (about 80%), suggesting that other inherited mutations could predispose to MDS/AML. It is expected that new sequencing approaches will help to the identification of more cases, more genes as well as novel syndromes. In 2017, we started a multicentric prospective study (Clinical trial.gov NCT03058588) aiming to look for predisposing mutations in patients and relatives affected by Familial AML and MDS syndromes (FAMS) by NGS and to screen for old and new mutations potentially associated with the disease. Methods At present, 12 AML/MDS patients have been enrolled. Leukemic (bone marrow) and germline (buccal swab) DNA were analyzed by NGS gene panel approach based on a 28 genes associated to myeloid leukemogenesis, including the 9 above mentioned genes associated to FAMS. NGS libraries were performed by a Nimblegen (Roche) custom panel based on gene capture strategy and the sequencing was performed by MiSeq (Illumina). Results Ten patients did not reveal any germline mutations and the candidates are undergoing to whole exome sequencing. One presented a germline mutation on RUNX1, and the analysis of the affected relatives is on going. One revealed a new mutation. She was a 70 years old woman affected by RARS and her pedigree was characterized by 9 relatives affected by hematologic and solid neoplasia and trombocytopenia (fig 1). The NGS analysis revealed the mutation c.*514C>T in 3'UTR of ETV6 with VAF of 50% on tumor DNA. The variant has never been described before, while ETV6 has been already associated with FAMS. Sanger sequencing confirmed the mutation on the germline DNA in heterozygosis. The screening of 2 affected relatives still alive confirmed the presence of the variant in heterozygosis. In silico analysis performed on PolymiRST Database revealed that c.*514C>T in 3'UTR of ETV6 results in a gain of miRNA binding site: hsa-miR- 4717-3p and hsa-miR- 942-3p. Discussion The variant c.*514C>T in 3'UTR of ETV6 seems to repress ETV6 due to RNA interference. The new binding miRNAs have been already described as over-expressed in solid and hematologic tumors. Moreover, the down-regulation of ETV6 is associated with alteration of cell growth and hematopoiesis. Due to these evidences, c.*514C>T in 3'UTR of ETV6 could be considered as a new mutation involved in FAMS predisposition. Disclosures No relevant conflicts of interest to declare.

Background Graft versus host disease (GVHD) is a common complication of allogeneic stem cell transplantation (allo-SCT), and represents its major cause of morbidity and mortality. Some “classical” patient-, donor- and transplant characteristics, such as age, gender disparity, donor type, HLA-match, and source of stem cells, have been reported as predictors for acute and chronic GVHD. However, no studies analysed these “classical” variables together with parameters of metabolic and endocrine functions, which may potentially influence the immune system. Thus, patient-and transplant variables together with index of liver and thyroid function, and some parameters of protein and lipid metabolism were retrospectively evaluated at different time points after transplantation, in order to identify possible predictors of acute and chronic GVHD and to calculate a risk score. Methods Clinical and transplant characteristics, number and type of infections before and after SCT were analysed in 161 patients. The following variables were also analysed pre-SCT, at day +7,+14,+21,+28, at + 3 and +6 months: LDH, parameters of liver function; parameters of protein and lipid metabolism (serum albumin, urea, total protein, cholesterol, triglycerides); thyroid function tests; autoimmune parameters (anti-nuclear, anti-DNA, anti-cardiolipin antibodies); body mass index. A 2-step multivariate analysis was performed using principal component analysis (PCA) and Cox regression analysis, in order to solve the problem of the high number of variables (metabolic, patients- and transplant related) in comparison with the relatively limited and heterogeneous pool of patients. Based on the regression coefficient of Cox analysis for each significant predictor, a scoring system for acute and chronic GVHD was calculated. Results In multivariate analysis, diagnosis of Myelodisplastic Syndrome or Chronic Myeloid Leukemia (HR 4,9; p=0.0004), conditioning regimen including Total Body Irradiation (HR 3,3; p=0.0003), and pre transplantation urea > 34 mg/dl with +21 day urea > 54 mg/dl (HR 2,6; p=0.0008) were evidenced as predictors for acute GVHD. Score values for each factor are 2, 1, and 1, respectively. Hence, the score, obtained by the sum of the three score values, ranged from 0 to 4. The probability of acute GVHD ranged from 8% (score 0) to 98% (score 4). Female donor (HR 5,1; p=0.0008), pre-SCT TSH values ≥ 2 mU/L with +28 day urea ≥ 39 mg/dl (HR 3,3; p=0.02), +6 month total protein < 5,5 g/dl with gamma-GT ≥ 347 U/L (HR 7,8, p=0.0001) resulted predictors for moderate/severe chronic GVHD, with score values of 1, 1, and 2 respectively. Risk of chronic GVHD at +6,5 month ranged from 3% (score 0) to 97% (score 4). Discussion Our study evidenced that factors other than “classical” ones may be associated to GVHD. Our scoring system includes routine-parameters (urea, total protein, gamma-GT,TSH), which are easily available in clinical practice. Urea levels depend on the balance between protein intake, endogenous catabolism and urinary excretion. The inflammatory microenvironment of GVHD promotes muscle catabolism, hence, urea levels do increase. Increased urea levels could be an indirect index of increased uremic toxins as well, which may stimulate the production of pro-inflammatory cytokines and the activation of leukocytes. On the other hand, increased urea levels and uremic toxins could derive from a dysregulated metabolism of the gut microbiome, which may influence immune system. Our findings, which require a prospective validation, suggest the usefulness a deeper study of the complex network between metabolic/endocrine functions and immune system, in order to develop a holistic approach of the transplant management. Disclosures No relevant conflicts of interest to declare.

Background: Although a complete remission (CR) can be achieved in 70-80% of newly diagnosed acute myeloid leukemia (AML) patients, relapses occur in up to the 50% of cases. Thus, minimal residual disease (MRD) monitoring is a major issue for early detection of patients at high-risk of treatment failure and relapse. Aim: to dynamically evaluate WT1 pan-leukemic molecular marker of MRD in patients with AML. Matherial and methods: 107 newly diagnosed AML patients consecutively treated between 2010 and 2013 were monitored with quantitative WT-1 from bone marrow (BM) and peripheral blood (PB) at baseline, after induction, after the first consolidation course, before allogeneic stem cell transplantation (allo-SCT), at the 3rd and the 6th month after transplantation Results: At diagnosis, 104/107 (97%) had increased PB and BM WT1 levels assessed according to the ELN assay. Eighty-eight out of 107 patients (82%) achieved a complete remission (CR) after induction, 30/88 (34%) relapsed during follow up and 24/107 (22%) were addressed to allogeneic stem cell transplantation (allo-SCT). By univariate analysis, PB-WT > 50x10^4/ABL and BM-WT1 > 250x10^4/ABL after induction (PB: p=0.02; BM: p=0.04), after consolidation (PB: p=0.003), at the end of treatment (PB and BM: p=0.001), at 3rd month of follow up (PB and BM: p=0.005) and at 6th month of follow up (PB: p=0.005) were associated with a reduced overall survival (OS). By multivariate analysis, a BM-WT1 > 250 x 10^4/ABL at the end of treatment was significantly associated with a reduced OS. In order to adapt the cut-off of WT1 in our series of patients, we considered WT1 levels as continuous variables and categorized them at approximately the 25th, 50th, and 75th percentile. A cut-off of PB-WT1 > 25x10^4/ABL and BM-WT1 > 125x10^4/ABL at the end of the treatment program was identified as correlated with reduced leukemia-free survival (LFS) and OS (p=0.001). Similarly, and restricting the analysis on the 24 patients allo-transplanted in CR, 8/11 (73%) with pre-transplant PB-WT1 ≥ 5 and 4/13 (31%) with PB-WT1 < 5 relapsed, respectively (p=0.04). The incidence of relapse was higher in AML patients with PB-WT1 ≥ 5 measured at 3rd (56% vs 38%; p=0.43) and 6th month (71% vs 20%; p=0.03) after allo-SCT. Interestingly, 5/5 (100%) patients with pre-transplant PB-WT1 ≥ 5 who never reduced this level at 3rd or 6th month after allo-SCT experienced a disease recurrence. Conclusions: our data, although retrospectively collected, show that WT1 monitoring may be useful to predict the relapse in AML patients. Acknowledgments: This work was supported in part by Banca di Credito Cooperativo di Pompiano e Franciacorta and Lions Club Bassa Bresciana Association. Disclosures Russo: Celgene: Research Funding; Gilead: Research Funding; Novartis: Consultancy.

Myelodisplastic syndromes (MDS) and Acute Myeloid Leukemia (AML) are a group of diseases of the elderly that initiates in a hematopoietic stem cell and are characterized by clonal hematopoiesis and uncertain prognosis, mostly due to cytogenetic background. In both diseases, 5-Azacitidine (5-Aza) has been successful, inducing prolonged survival and delayed AML evolution. To identify the genes mostly predictive of treatment response, we use high-throughput genomic analysis (SNP arrays and/or NGS-RNA-seq and/or NGS-WES and/or GEP) in azacitidine-sensitive and resistant MDS/AML patients. NGS-WES or RNA seq HiSeq 2000 (Illumina) was positively done in 35/214 AML samples (16%), GEP (GeneChip Human Transcriptome Array 2.0, Affymetrix Inc.) was performed in 65/214 AML samples (30%). SNPs arrays (CytoScan HD Array, Affymetrix Inc.) was done in 125/214 AML samples (58%) and 18/32 MDS samples (56%) at diagnosis, then analyzed by Chromosome Analysis Suite (ChAS) v1.2 (Affymetrix Inc.), Nexus Copy Number™ v7.5 (BioDiscovery) and GeneGo MetaCore™ software. We treated 246 adult patients (pts) with MDS or AML: 214 pts were AML and 32 were MDS with a median age of 59 and 70 years, respectively. Forty-five pts were treated with 5-Aza (32 MDS / 13 AML), while 201 AML were treated with conventional chemotherapy. Forty-five MDS/AML pts were treated with at least one complete cycle of 5-Aza (75 mg/sqm/daily). SNP arrays was done in 22/45 (49%), 13 pts were defined “insensitive/resistant”, ie. never achieving clinical complete remission (CCR) and 9 were defined “sensitive”, ie. all of them obtaining CCR. Copy Number Alterations (CNAs) ranged from loss or gain of complete chromosome (chr) arms to focal deletions and gains targeting one or few genes involving macroscopic (>1.5 Mbps), submicroscopic genomic intervals (50 Kbps - 1.5 Mbps) and LOH (>5 Mbps) events. Macroscopic CNAs affecting a complete chromosome or its arms were detected in 5 of 22 pts (23%), while classical cytogenetic was able to detect only two cases of trisomy 8 (9%), suggesting superiority of SNPs array for CNAs identifications. Microscopic CNAs abnormalities were detected in all of the patients affecting all the chromosomes. Of interest, some of them were located on chr 2, 3, 4, 5, 7, 8, 11, 12, 15, 17, 20, X, and were involved genes such as: NPM1, CTNNA1, IRF1, RPS14, SPARC, CBL, ETV6, EZH2, CUX1, CDC25C, EGR1, RUNX1, BRAF, ASXL1, ZRSR2, PHF6, BCOR, CDC25C, EGR1, IRAK1 in loss; RAD21, JAK2, KIT, ZRSR2, PHF6, BCOR, IRAK1 in gain; SIRPB1 both in loss and gain. Moreover we found in LOH these genes: NF1, GATA2, FADD, IDH2, SF3B1, BCOR, PHF6, ZRSR2, STAG2, KDM6A, ATRX, IRF1, NPM1, CBL, CTNNA1, EGR1, IRAK1. Chromosomic aberrations disease-related are more statistically frequent on pts “insensitive” versus pts. “sensitive” (64% vs. 35%) (p≤0.01). Moreover we found that from the median of chromosomic alterations lenghts (in kbp) the group of “insensitive” MDS/AML patients to 5-Aza therapy present more gains and losses than “sensitive” ones. By Nexus Copy Number software, we identify 137 genes highly differentially gain (SIRPB1 and KIT with p ≤ 0.05) or loss (SIRPB1, LCE1C, BCAS1, EXD3 with p ≤ 0.05) or LOH between “insensitive” versus “sensitive” to 5-Aza (p ≤ 0.05). Among these genes, we focused on SIRPB1 (cytoband 20p13, 56Kbps), since it was loss on 14/22 (64%) “insensitive” pts (p=0,023) and gain on 7/22 (70%) “sensitive” ones with a significantly (p=0,0324), respectively. SIRPB1 common deletion region goes from 1571 to 1598 (27 kbps) and common amplified region goes from 1561 to 1591 (30 kbps). By NGS-WES we analyzed 35/214 (16%) AML samples at diagnosis and we searched for point mutations, insertion/deletion or other abnormalities, involved in biomarkers of sensitivity/refractory to 5-Aza or chemoteraphy. We found mutations in SF3B1, NPM1, CBL, RUNX1, BCOR, KIT, GATA2, IDH2, KDM6A, KIAA1324L, PRIM2, RRN3, APOBR and again in SIRPB1 an heterozigosity frameshift deletion (c. 388delC; p. H130fs) in exone 2 in a AML pts with normal karyotype. By GEP we further analyzed 48/214 (22%) AML samples for SIRPB1 in order to correlate and confirm the expression or loss of expression of these genes, in correlation with 5-Aza response. We conclude that SIRPB1 is a promising marker of response to 5-Aza treatment in MDS and AML. Acknowledgments: ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7NGS-PTL project. Celgene ITA–VZ–MDS–PI-0298 GRANT-ITA-002 Disclosures Martinelli: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

Whole exome and transcriptome sequencing (WES and RNAseq) technologies are able to provide a comprehensive analysis of the genomic aberrations acquired by malignant cells, of their synergistic effects and functional consequences. In particular, RNAseq enables the detection of gene fusions originating from rare chromosomal translocations that have been involved in the pathogenesis of Acute Myeloid Leukemia (AML). We performed WES and RNAseq of AML patients to identify novel genetic abnormalities playing a causative role in leukemia development. We collected bone marrow or peripheral blood samples of 31 patients. Sequencing was performed using the Illumina Hiseq2000 platform. WES raw data were analysed with Whole-Exome sequencing Pipeline web tool for variants detection (WEP). The presence of gene fusions was assessed in RNAseq data with deFuse and Chimerascan. Selected genes fusions and variants were validated by Sanger sequencing. By RNAseq we identified a rare gene fusion transcript involving the BCL11B gene, which been previously suggested to play an oncogenic role in AML. The gene encodes for a zinc-finger protein participating to chromatin remodelling and regulating the differentiation and apoptosis of hematopoietic cells. The fusion was identified in a patient with poorly differentiated leukemia phenotype and unfavourable karyotypic abnormalities: 46,XX, t(2;14)(q21;q32), t(11;12)(p15;q22), who received standard chemotherapy, underwent allogeneic bone marrow transplantation and is currently in complete remission. Differently from previous data, the BCL11B translocation was associated neither with FLT3-ITD nor DNMT3A mutations. WES analysis revealed mutations in the TET2 and WTAP genes, which are known to act as co-players in the leukemic transformation. The exome data of our AML cohort identified neither INDELs nor nonsynonymous mutations in the BCL11Bgene, suggesting that the oncogenic function of BCL11B is activated by chromosomal translocations. Gene expression profiling showed a 4-fold upregulation of BCL11B transcript in the patient’s blasts, compared to 53 AML samples with no chromosomal aberrations in the 14q32 region, according to cytogenetic analysis. The increased expression of BCL11B was associated with an upregulation of potential targets including the antiapoptotic protein SPP1. Our data suggest that chromosomal translocations involving the BCL11B gene are rare events in AML and associate with somatic mutations in the malignant transformation of myeloid lineage cells, potentially by altering the differentiation and apoptotic processes. Future studies will investigate putative fusion partners of BCL11Band elucidate the biological consequences of its upregulation in AML pathogenesis. The results highlight the molecular heterogeneity of AML and the need for high-resolution sequencing analysis of leukemic samples at diagnosis in order to tailor personalized therapies. Supported by: FP7 NGS-PTL project, ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi). Disclosures Martinelli: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

AMLs are clonal disorders characterized by high genomic heterogeneity and several chromosomal and molecular alterations affecting patients' outcome. In about 40% of AML patients who do not show any citogenetic alteration, sequencing analysis identified different gene mutations which play a pivotal role in leukemogenesis and have a negative prognostic impact: FLT3, ASXL1, TET2, IDH1, IDH2, RUNX1, CBL, CEBPα, DNMT3A and TP53. Conventional Sanger sequencing may detect clones representing more than 20% of the total tumor population, whereas Next Generation Sequencing (NGS) can identify mutations in less than 1% of leukemic cell burden. The detection of these variants is relevant because they can play an important role in driving drug resistance and disease relapse and for biologic risk assessment. To that purpose, we designed a 23-genes panel including: FLT3, DNMT3A, RUNX1, ASXL1, IDH1, IDH2, BCOR, NRAS, KRAS, TET2, TP53, U2AF1, ZRSR2, SF3B1, SRSF2, CBL, CEBPα, EZH2, NPM1, TERT, TERC, ETV6, GATA2. By using a 454 GS Junior by Roche Diagnostics with an amplicon-based sequencing approach and performing three sequencing runs per sample in order to reach a sensitivity of at least 1%, we settled an investigative strategy in order to assess the genomic profile of AMLs at diagnosis and possibly at the time of relapse or resistance. By this approach, we were able to confirm all the variants (i.e. FLT3, NPM1) previously documented with conventional tests; to reveal the presence of variants under the Sanger threshold of 20% in the genes resulted as wild type with routinely analysis; to evaluate the AML clonal heterogeneity, by assessing the coexistence of several mutated clones which have been described to be related to drug resistance, poor prognosis or to prior myelodysplastic syndrome. This gene panel NGS-based strategy may be proposed as a highly accurate and sensitive approach for genomic characterization of acute myeloid leukemias and as an useful tool for planning a target and personalized AML therapy. Disclosures No relevant conflicts of interest to declare.

Introduction The long-term efficacy of allogeneic haematopoietic stem cell transplantation (SCT) relies primarily on the Graft-versus-tumor (GVT) effect, which partially overlaps with Graft versus Host disease (GvHD), the most common cause of morbidity and mortality in SCT. Researches on GVHD-biomarkers are still ongoing and a set of validate markers are still lacking, especially for chronic GVHD. Furthermore, immune parameters that univocally associate with GVHD or GVT have not been identified yet. In this study, lymphocyte subsets together with TCR-repertoire analysis, and index of thymic and bone marrow output were evaluated at different time points, in order to identify possible predictors of GVHD and ineffective GVT. Methods Prospective evaluations of lymphocyte subsets, thymic and bone marrow output were performed in 40 patients before SCT, at 30, 90, 180 days and 1 year after SCT. CD4+/CD8+ naïve, central memory, effector memory, terminally differentiated effector memory (TEMRA) cells, subsets of regulatory T-lymphocytes, immature B cells, naïve, switched and unswitched memory B cells, memory double negative (IgD-CD27-) B cells were analysed by flow cytometry. Analysis of thymic and bone marrow output was performed by detection of T cell receptor excision circles (TRECs) and kappa-deleting recombination circles (KRECs). TRECs and KRECs were simultaneously quantified by a duplex quantitative Real-Time PCR. Heteroduplex assay was used to perform TCR-repertoire analysis. A 2-step multivariate analysis was performed using principal component analysis (PCA) and Cox regression analysis, to solve the problem of the high number of variables (immunological, patients- and transplant related) in comparison with the relatively limited and heterogeneous pool of patients. Results Twenty patients developed acute GVHD (median time: 28 days, range 19-120). Chronic GVHD was observed in 9 patients (median time: 6 months, range 4-10). In multivariate analysis, acute GVHD correlated positively with pre-transplant percentage of CD4+ central memory cells, and with values of regulatory effector memory T-cells and CD4+TEMRA cell at day +30 (p=0,0006). Pre-transplant percentage of unswitched memory B cells was also associated with acute GVHD, whereas pre-transplant levels of KRECs were inversely correlated (p=0,0005). Chronic GVHD was associated with matched unrelated donor and with (p -values of regulatory effector memory T-cells at +30, percentage of CD8+TEMRA cells at +90, values of immature B cells and levels of KRECs at +180 (positive correlation) -percentage of CD4+ central memory and CD8+ effector memory cells at +90 (negative correlation). The relapse rate (27%; median time: 5,5 months, range 3-12) was used as clinical index of ineffective GVT. The following cluster of immunological parameters at day +90 correlated positively with relapse: CD8+ effector memory cells, immature B cells, naïve, switched memory B cells, memory double negative (IgD-CD27-) B cells (p=0,006). Discussion Different clusters of immunological parameters at different time points were evidenced as predictors of GVHD and ineffective GVT, allowing a clear-cut distinction between these immunological reactions. Changes in pre- and post-transplant B-lymphopoietic microenvironment and specific imbalances in the subset of B-cells may be involved in acute and chronic GVHD development. The atypical association of regulatory T-cells with GVHD may be explained by the relative efficiency of different subsets of regulatory T-cells (naïve>effector memory), as shown in some experimental models. Increased values of CD8+ effector memory cells could be an early sign of ineffective GVL. Imbalance toward a lymphocyte B-response, and especially toward "senescent" memory (IgD-CD27-) B cells, could promote tolerance to tumor cells. The validation of these clusters of immunological parameters as specific early predictors of GVHD or GVT, even before SCT, could potentially allow the development of pre-emptive and targeted therapies. Disclosures No relevant conflicts of interest to declare.

Aneuploidy causes a proliferative disadvantage, mitotic and proteotoxic stress in non-malignant cells ( Torres et al. Science 2007). Chromosome gain or loss, which is the hallmark of aneuploidy, is a relatively common event in Acute Myeloid Leukemia (AML). About 10% of adult AML display isolated trisomy 8, 11, 13, 21 (Farag et al. IJO 2002), or either an isolated autosomal monosomy or monosomal karyotype (Breems et al. JCO 2008). This evidence suggests that tumor-specific mechanisms cooperate to overcome the unfitness barrier and maintain aneuploidy. However, the molecular bases of aneuploid AML are incompletely understood. We analyzed a cohort of 166 cytogenetically-characterized AML patients (80 aneuploid (A-) and 86 euploid (E-)) treated at Seràgnoli Institute (Bologna). Aneuploidy was significantly associated with poor overall survival (median survival: 13 and 26 months in A-AML and E-AML respectively; p=.006, Fig.1). To identify AML-specific alterations having a causative and/or tolerogenic role towards aneuploidy, we integrated high-throughput genomic and transcriptomic analyses. We performed 100 bp paired-end whole exome sequencing (WES, Illumina Hiseq2000) of 70 samples from our A-AML and E-AML cohort of 166 patients. Variants where called with MuTect or GATK for single nucleotide variant and indels detection, respectively. AML samples were genotyped by CytoScan HD Array (Affymetrix). Gene expression profiling (GEP) was also conducted on bone marrow cells from 24 A-AML, 33 E-AML (≥80% blasts) and 7 healthy controls (HTA 2.0, Affymetrix). We detected a significantly higher mutation load in A-AML compared with E-AML (median number of variants: 31 and 15, p=.04) which was interestingly unrelated to patients' age (median age: 63.5 years in A-AML and 62 years in E-AML, Xie et al, Nat. Med. 2014). C>A and C>T substitutions, which are likely mediated by endogenous 5mCdeamination, were the most frequent alterations (Alexandrov et al. Nat. 2013). However, aneuploidy associated with an increased variability in terms of mutational signatures, with the majority of A-AML displaying 3 or more signatures compared to few E-AML cases (p=.04). WES analysis also revealed a specific pattern of somatic mutations in A-AML. A-AML had a lower number of mutations in signaling genes (p=.04), while being enriched for alterations in cell cycle genes (p=.01) compared with E-AML. The mutated genes were involved in different cell cycle phases, including DNA replication (MCM6, PURB, SSRP1), centrosome dynamics (CEP250, SAC3D1, HEPACAM2, CCP110), chromosome segregation (NUSAP1, ESPL1, TRIOBP), mitotic checkpoint (ANAPC7, FAM64A) and regulation (CDK9, MELK, ZBTB17, FOXN3, PPM1D, USP2). Moreover, genomic deletion of cell cycle-related genes was frequently detected in A-AML. Notably, ESPL1 which associated with aneuploidy, chromosome instability and DNA damage in mammary tumors (Mukherjee et al. Oncogene 2014) was mutated and also upregulated in A-AML compared with E-AML (p=.01), the latter showing expression levels comparable to controls. Among the top-ranked genes differentially expressed between A-AML and E-AML, we identified a specific signature characterized by increased CDC20 and UBE2C and reduced RAD50 and ATR in A-AML (p Our data show a link between aneuploidy and genomic instability in AML. Deregulation of the cell cycle machinery, DNA damage and repair checkpoints either through mutations, copy number and transcriptomic alterations is a hallmark of A-AML. The results define specific genomic and transcriptomic signatures that cooperate with leukemogenic pathways, as KRAS signaling, to the development of the aggressive phenotype of A-AML and suggest that a number of A-AML patients may benefit frompharmacological reactivation of TP53pathway (e.g. MDM2 inhibitor, clinical trial NP28679). Supported by: FP7 NGS-PTL project, ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 GS & AP: equal contribution Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Cavo:JANSSEN, CELGENE, AMGEN: Consultancy. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Martinelli:MSD: Consultancy; BMS: Speakers Bureau; Roche: Consultancy; ARIAD: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy.

Alagille syndrome (ALGS), or arteriohepatic dysplasia, is a congenital multisystem disease due to Notch signalling pathway mutations, most commonly affecting JAGGED-1 (ALGS type 1), and more rarely NOTCH-2 (ALGS type 2), leading to hepatic, lung, renal and ocular dysfunction (chronic cholestasis, peripheral pulmonary artery stenosis, dysplastic kidneys pigmentary retinopathy), and skeletal abnormalies (minor vertebral segmentation, characteristic facies, posterior embryotoxon/anterior segment defects). ALGS is an autosomal dominant disease, but it is characterized also by variable penetrance and clinical expression and somatic/germline mosaicism. A 20-year-old man with ALGS was admitted to the University Hospital of Verona because of pancytopenia. Following analyses led to the diagnosis of Philadelphia chromosome/bcr-abl-negative, CD10-positive, B-lineage acute lymphoblastic leukemia (common B-ALL). In order to identify the genetic components involved in this complex phenotype, we sequenced the exome of a bone marrow sample collected from the patient. By genome interpretation with Knome pipeline applied to the reference genome UCSC hg19, we found missense variants both in NOTCH-2 (E38K) and JAGGED-1 (P871R) genes that are mainly involved in the syndrome, although their effect on protein function was predicted not to be deleterious. However, we detected putative damaging mutations in genes such as PAX5 (R38H) and NOTCH-1 (K1821N), which might be strongly related to the observed disease. In fact, PAX5 is a member of PAX protein family of transcription factors implicated into regulation of early development that binds NOTCH-2 and likely altering its functionality. On the other hand, NOTCH-1 is involved in cell growth and proliferation and thus the predicted alteration of function of the corresponding protein may have an important role in neoplastic transformation. Disclosures Martinelli: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

Alagille syndrome (ALGS), or arteriohepatic dysplasia, is a congenital multisystem disease due to Notch signalling pathway mutations, most commonly affecting JAG1 (ALGS type 1), and more rarely NOTCH2 (ALGS type 2), leading to hepatic, lung, renal and ocular dysfunction (chronic cholestasis, peripheral pulmonary artery stenosis, dysplastic kidneys pigmentary retinopathy), and skeletal abnormalies (minor vertebral segmentation, characteristic facies, posterior embryotoxon/anterior segment defects). ALGS is an autosomal dominant disease, but it is characterized also by variable penetrance and clinical expression and somatic/germline mosaicism. A 20-year-old man with ALGS was admitted to the University Hospital of Verona because of pancytopenia. Following analyses led to the diagnosis of Philadelphia chromosome/bcr-abl-negative, CD10-positive, B-lineage acute lymphoblastic leukemia (common B-ALL). In order to identify the genetic components involved in this complex phenotype, we sequenced the exome of a bone marrow sample collected from the patient. By genome interpretation with Knome pipeline applied to the reference genome UCSC hg19, we found missense variants both in NOTCH2 (E38K) and JAG1 (P871R) genes that are mainly involved in the syndrome, although their effect on protein function was predicted not to be deleterious. However, we detected putative damaging mutations in genes such as PAX5 (R38H) and NOTCH1 (K1821N) which might be strongly related to the observed disease. In fact, PAX5 is a member of PAX protein family of transcription factors implicated into regulation of early development, that binds NOTCH2 and likely altering its functionality. On the other hand, NOTCH1 is involved in cell growth and proliferation and thus the predicted alteration of function of the corresponding protein may have an important role in neoplastic transformation. Disclosures: No relevant conflicts of interest to declare.

Abstract 4676 Introduction Myeloproliferative diseases (MPDs) are a group of haematological disorders characterized by the hyper proliferation of different blood cells in peripheral blood and other hematopoietic organs. The clinical heterogenity of these neoplasms reflects the different gene pathways involved, most of which are only partially known. Given the genetic homology and the physiological similarity to mammals, zebrafish has emerged as an ideal model to study human normal and malignant haematopoiesis. In the last decade several oncogenes involved in the development of hematopoietic neoplasms have been used to model leukemia in zebrafish with the aim to discovery new molecular pathways involved in malignant transformation. Despite the first encouraging results these experimental models failed to fully recapitulate human myeloproliferative disorders. Methods We took advantage of the Gal4/UAS binary system to induce the expression of human oncogenic HRASV12G in the zebrafish hematopoietic compartment. We used a specific transgenic line that drives oncogene expression in zebrafish early hematopoietic progenitors under control of the FLI.1 (Friend Leukemia virus Integration 1) promoter. Results We observed the development of a myelo-erythroid proliferative disease in few days in zebrafish transgenic larva. The pathological phenotype is characterized by the expansion of the hematopoietic tissue, an increased expression of myelo-erythroid specific genes (PU.1, gata1, mpx, c-mpl) associated with a slight increase of staminality markers (lmo2, scl, c-myb, runx.1), and a higher number of l-plastin expressing cells. Moreover blood smear of pathological larva displayed leukemic blasts and the arrest of erythrocyte differentiation whereas kidney marrow of juvenile fish displayed abnormal myelopoiesis characterized by the increase of erythro-myeloid progenitors. We found that the pathological phenotype is associated with a down regulation of the Notch pathway as shown by the decreased gene expression of notch pathways target genes (notch1, notch3, her6). Furthermore we discovered a novel set of genes involved in neoplastic transformation induced by HRASV12 expression through RNA-Seq analysis of pathological larva. Conclusions The expansion of the zebrafish hematopoietic compartment characterized by the hyper-proliferation of the myelo-erythroid progenitors that we found in this model reproduces some of the pathological features of human myeloproliferative disorders. This study showed that forcing oncogene expression in the hemogenic endothelial cells induces the transdifferentiation of the early hemogenic pluripotent stem cells into abnormal myeloerythoid progenitors by repressing the Notch pathways. Transcriptome analysis identified a number of potential effectors of this transformation. Disclosures: No relevant conflicts of interest to declare.