Acute Myeloid Leukemia (AML) is an extremely heterogeneous group of hematological neoplasms, for which allogeneic stem cell transplantation (HSCT) still represents the only potentially curative option in the majority of cases. However, elderly age and clinically severe comorbidities may often exclude a wide amount of patients from this therapeutic approach, underlying the urgent need for alternative strategies. Thanks to the introduction of advanced high-throughput techniques, light is being shed on the pathogenesis of AML, identifying molecular recurrent mutations as responsible for the onset, as well as progression, of disease. As a consequence, and in parallel, many new compounds, including targeted therapies (FMS-like tyrosine kinase 3 (FLT3) and Isocitrate dehydrogenase 1-2 (IDH1-2) inhibitors), have found a wide room of application in this setting, and are now available in daily practice, or in late phases of clinical development. Moreover, several further innovative molecules are currently under investigation, and promising results for many of them have already been reported. In this review, we will present an update on the most relevant molecular alterations of AML, focusing on the most frequent genomic mutations of the disease, for which compounds have been approved or are still currently under investigation.

Extramedullary involvement of B-cell Acute Lymphoblastic Leukemia (EM-ALL) is a rare occurrence, characterized by dismal outcome and the absence of a defined and shared therapeutic approach. In the landscape of innovative compounds, inotuzumab ozogamicin (IO) is a promising drug, whose mechanism of action relies on the killing of CD22 positive leukemic cells, through the delivery, after cell binding, of a molecule of calicheamicin. We report two cases of CD22 positive relapsed EM-ALL treated with IO, obtained as compassionate use. Case 1, a 66 years old woman, affected by Philadelphia (Ph) negative B-ALL, relapsed with extramedullary involvement after 6 standard chemotherapy courses, who reached a complete metabolic response with IO treatment. Case 2, a 67 years old man with Ph positive B-ALL, initially treated with ponatinib, a third generation tyrosine-kinase inhibitor (TKI), obtaining a prolonged deep molecular remission. Nevertheless, for skin relapse during TKI treatment, the patient received local radiotherapy and, shortly after, standard chemotherapy, as multiple abdominal sites of relapse were detected too, with no response. The patient then received IO, obtained as compassionate use, with a good metabolic response. These two cases suggest a possible key role of IO in the setting of advanced CD22 positive ALL, and underline its potential activity also in patients with EM involvement, relapsed after or refractory to conventional chemotherapy. Despite the well known hepatotoxic effect of the compound (Sinusoid Occlusive Syndrome), neither of them had such adverse event, moreover the second patient safely underwent allogeneic bone marrow transplantation.

In acute myeloid leukemia (AML), the restoration of p53 activity through MDM2 inhibition proved efficacy in combinatorial therapies. WIP1, encoded from PPM1D, is a negative regulator of p53. We evaluated PPM1D expression and explored the therapeutic efficacy of WIP1 inhibitor (WIP1i) GSK2830371, in association with the MDM2 inhibitor Nutlin-3a (Nut-3a) in AML cell lines and primary samples. PPM1D transcript levels were higher in young patients compared with older ones and in core-binding-factor AML compared with other cytogenetic subgroups. In contrast, its expression was reduced in NPM1-mutated (mut, irrespective of FLT3-ITD status) or TP53-mut cases compared with wild-type (wt) ones. Either Nut-3a, and moderately WIP1i, as single agent decreased cell viability of TP53-wt cells (MV-4-11, MOLM-13, OCI-AML3) in a time/dosage-dependent manner, but not of TP53-mut cells (HEL, KASUMI-1, NOMO-1). The drug combination synergistically reduced viability and induced apoptosis in TP53-wt AML cell line and primary cells, but not in TP53-mut cells. Gene expression and immunoblotting analyses showed increased p53, MDM2 and p21 levels in treated TP53-wt cells and highlighted the enrichment of MYC, PI3K-AKT-mTOR and inflammation-related signatures upon WIP1i, Nut-3a and their combination, respectively, in the MV-4-11 TP53-wt model. This study demonstrated that WIP1 is a promising therapeutic target to enhance Nut-3a efficacy in TP53-wt AML.

11622 Background: SNP microarray can detect Copy Number Alterations (CNAs) which could be predictive of response and can help define therapeutic strategies. Our aim is to improve conventional cytogenetic analysis and identify new genetic alterations relevant to leukemogenesis by a SNP array-based genotyping approach. Methods: We performed SNP 6.0/Cytoscan HD (Affymetrix) on 235 Acute Myeloid Leukemia (AML) patients at diagnosis. Seventy-eight/235 samples were also performed by Whole Exome Sequencing, WES (HiSeq,Illumina). SNP Array data were analyzed by Nexus Copy Number (BioDiscovery) and R Core Team. Results: We found several genes preferentially deleted, including MRPS5 (14.8%), PHF6 (9.3%), SCAPER (7.2%), CASK (5.9%), WNK (4.6%), STAG2 (4.2%), LRRK1 (3.4%), PALB2 (3.4%), genes preferentially amplified were RABL2B (16.1%), NF2 (10.2%), NBPF9 (7.6%), JAK2 (6.8%), RB1, NF1 and KMT2A (4.2%), PTEN (3.4%), TP73 and SMAD2 (2.5%). Single-copy losses and deletions were enriched (p < .001) for genes mapping in these pathways: aberrant PD-1 signaling, loss of function of SMAD4 in cancer and SMAD4 MH2 Domain mutants in cancer. The pathways significantly (p < .001) deregulated in our cohort with single copy gain and homozygous amplification were: regulation of transcription and nucleic acid, negative regulation of metabolic processes, constitutive signaling by aberrant PI3K in cancer and PI3K/AKT network. In order to define driver alterations, we correlate deletions and losses with mutational data. We found losses are also targeted by mutations ( BRCA2, LRRK1), while deleted genes, as CASK, CDK6 and MAPT, were involved in pathways affected by genomic mutations ( CASK deletion and MPP6 mutation, CDK6 deletion and PPM1D mutation, MAPT deletion and SPAG5mutation). Conclusions: We have identified new CNAs and pathways involving novel potential leukemia-related genes. The comparison between SNP and WES data could provide important findings on prognosis of AML patients. Minimal deleted regions of genes in deregulated pathways deserve further investigation in order to identify new genes which could be relevant AML biomarkers. Ackn: ELN, AIL, AIRC,prog. Regione-Università 2010-12 (L. Bolondi),FP7 NGS-PTL project,HARMONY.

E23207 Background: Nowadays, Acute Promyelocytic Leukemia (APL) is a disease entity with a very high rate of cure and an estimated 2-year overall survival of 97%. Early death, rather than resistant disease so common in all other subtypes of AML, has emerged as the major cause of treatment failure, and relapse is a very rare occurrence. Methods : We collected data of all the APL referred to our institution from 2014. Within 23 patients, we encountered 20 new diagnosis and 2 relapse of APL. We analyzed blasts in samples obtained from Bone Marrow with Single Nucleotide Polymorphisms Array Cytoscan HD. Results: We compared copy number alterations in both relapsed patients with alterations detected in the pool of 20 newly diagnosed APL and we found specific signatures of CNVs for each patient. There were several copy number alterations related to each patient: the first patient presented gain of ROBO2, GRIP1, CTNNB1, SOX6, PBX1, GRIK2, CDKAL1 and loss FAF1, CREBBP, SBF1; the second patient presented gain of ROBO1, MAPK10, CADPS2, APBA1 and loss of GRIP1 and MYB. Subsequently we focused our attention on ROBO and GRIP1genes because they were alterated in both relapsed patients: ROBO proteins are associated to K channels while GRIP1 is involved in various critical functions, for example in androgen receptor binding, beta-catenin binding, glucocorticoid receptor binding, and it is also a regulator of glutamate metabolism, a well-known pathway in Leukemic Stem Cells. Conclusions: APL relapse is a very rare entity, and it is announced to become rarer with the advances in first line therapy. Molecular characteristics are hard to analyze without an effort to collect and bank samples together from multiple institutions. Since relapses, especially relapses out of follow-up period, represent a sudden life-treating condition for patients, to predict patients at higher risk of relapse we selected two candidate genes that could be involved in pathways favoring relapse. By the analysis of ROBO 1-2 and GRIP1 at the diagnosis of APL we could establish a different and strict follow-up program for patients with these alterations. Acknowledgement: ELN,AIL,AIRC,prog. Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project,HARMONY.