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Purpose: The 9p21 locus, encoding three important tumor suppressors (p16/CDKN2A, p14/ARF, and p15/CDKN2B), is a major target of inactivation in the pathogenesis of many human tumors.Patients and Methods: To explore, at high resolution, the frequency and size of alterations affecting this locus in adult BCR-ABL1–positive acute lymphoblastic leukemia (ALL) and to investigate their prognostic value, 112 patients (101 de novo and 11 relapsed cases) were analyzed by genome-wide single-nucleotide polymorphism arrays and gene candidate deep exon sequencing. Paired diagnosis–relapse samples were further available and analyzed for 19 (19%) cases.Results:CDKN2A/ARF and CDKN2B genomic alterations were identified in 29% and 25% of newly diagnosed patients, respectively. Deletions were monoallelic in 72% of cases, and in 43% of them, the minimal overlapping region of the lost area spanned only the CDKN2A/B gene locus. An analysis conducted at relapse showed an increase in the detection rate of CDKN2A/ARF loss (47%) compared with the time of diagnosis (P = 0.06). Point mutations within the 9p21 locus were found at very low levels, with only a nonsynonymous substitution in the exon 2 of CDKN2A. Of note, deletions of CDKN2A/B were significantly associated with poor outcomes in terms of overall survival (P = 0.0206), disease free-survival (P = 0.0010), and cumulative incidence of relapse (P = 0.0014).Conclusions: Inactivation of the 9p21 locus by genomic deletion is a frequent event in BCR-ABL1–positive ALL. Deletions are frequently acquired during leukemia progression and are a poor prognostic marker of long-term outcomes. Clin Cancer Res; 17(23); 7413–23. ©2011 AACR.

In adult patients, acute lymphoblastic leukemia (ALL) is a rare hematological cancer with a cure rate below 50% and frequent relapses. With traditional therapies, patients with relapsed or refractory (R/R) ALL have a survival that may be measured in months; in these patients, inotuzumab ozogamicin (IO) is an effective therapy. IO was linked to increased risk of veno‐occlusive disease/sinusoid obstruction syndrome (VOD/SOS), liver injury, and various grade of liver‐related complications during clinical trials and real‐life settings; however, hepatologic monitoring protocol is not established in this population. In our institution, 21 patients who received IO (median of 6 doses of IO administered) for R/R ALL were prospectively followed for hepatologic surveillance, including clinical evaluation, ultrasonography, and liver stiffness measurement (LSM) biochemistry. After a median follow‐up of 17.2 months, two SOS events were reported (both after allogeneic transplant) as IO potentially related clinically relevant adverse event. Mild alterations were reported in almost the totality of patients and moderate‐severe liver biochemical alterations in a quarter of patients. Within biochemicals value, AST and ALP showed an augment related to IO administration. LSM linearly augmented for each IO course administered. Baseline LSM was related to liver‐related changes, especially with the severity of portal hypertension (PH)‐related complications. Pre‐transplant LSM was higher in patients receiving IO when compared with a control cohort. PH‐related complications were discovered in nearly 77% of patients, with clinically significant PH occurrence and development of ascites in 38% and 14%, respectively. This prospective experience constitutes the rationale to design a hepatologic monitoring program in patients receiving IO. LSM may be of pivotal importance in this program, constituting a rapid and effective screening that quantitatively correlates with liver alterations.

Background Despite the recent progress that has been made in the understanding and treatment of acute lymphoblastic leukemia (ALL), the outcome is still dismal in adult ALL cases. Several studies in solid tumors identified high expression of WEE1 kinase as a poor prognostic factor and reported its role as a cancer-conserving oncogene that protects cancer cells from DNA damage. Therefore, the targeted inhibition of WEE1 kinase has emerged as a rational strategy to sensitize cancer cells to antineoplastic compounds, which we evaluate in this study. Methods The effectiveness of the selective WEE1 inhibitor AZD-1775 as a single agent and in combination with different antineoplastic agents in B and T cell precursor ALL (B/T-ALL) was evaluated in vitro and ex vivo studies. The efficacy of the compound in terms of cytotoxicity, induction of apoptosis, and changes in gene and protein expression was assessed using different B/T-ALL cell lines and confirmed in primary ALL blasts. Results We showed that WEE1 was highly expressed in adult primary ALL bone marrow and peripheral blood blasts (n = 58) compared to normal mononuclear cells isolated from the peripheral blood of healthy donors (p = 0.004). Thus, we hypothesized that WEE1 could be a rational target in ALL, and its inhibition could enhance the cytotoxicity of conventional therapies used for ALL. We evaluated the efficacy of AZD-1775 as a single agent and in combination with several antineoplastic agents, and we elucidated its mechanisms of action. AZD-1775 reduced cell viability in B/T-ALL cell lines by disrupting the G2/M checkpoint and inducing apoptosis. These findings were confirmed in human primary ALL bone marrow and peripheral blood blasts (n = 15). In both cell lines and primary leukemic cells, AZD-1775 significantly enhanced the efficacy of several tyrosine kinase inhibitors (TKIs) such as bosutinib, imatinib, and ponatinib, and of chemotherapeutic agents (clofarabine and doxorubicin) in terms of the reduction of cell viability, apoptosis induction, and inhibition of proliferation. Conclusions Our data suggest that WEE1 plays a role in ALL blast’s survival and is a bona fide target for therapeutic intervention. These data support the evaluation of the therapeutic potential of AZD-1775 as chemo-sensitizer agent for the treatment of B/T-ALL. Electronic supplementary material The online version of this article (10.1186/s13045-018-0641-1) contains supplementary material, which is available to authorized users.

BACKGROUND: Many efforts have been made in the attempt to address the conundrum question of fitness definition ad prognosis prediction in elderly acute myeloid leukemia (AML) patients. Parametric definitions are expected to give an advantage in patient stratification; however, clinical examination remain de facto pivotal to formulate therapy decisions and frequently the comorbidities are empirically evaluated. METHODS: We conducted a multicenter study collecting baseline comorbidity, laboratory data, CTCAE 4.0.3 adverse events (AE), and outcome of elderly patients (>65 years old) with new onset AML who received hypomethylating agents as 1st line therapy. We tested the impact on prognosis of baseline clinical and biological risk factors. Furthermore, we evaluated a score - acute myeloid leukemia-composite model, AML-CM (Mukherjee et al, 2017) - developed in chemotherapy-eligible patients, that accounts for baseline comorbidities, laboratory parameters, age and cytogenetic-molecular risk. The study was approved by local Ethical Authority (316/2019/Oss/AOUBo). RESULTS: We collected data from 131 consecutive elderly patients who received 1 st line HMAs between January 2008 and January 2021. Patients had a median age of 76 years (IQR 72 -79). Seventy-seven out of 131 patients (58.8%) had de novo AML, 32/131 (32.8%) had secondary AML, and 11/131 (8.4%) had therapy-related AML. Out of 123 evaluable patients, 43 (34.9%) had complex karyotype, 1 (0.8%) inv(16), 59 (48.4) normal karyotype, 18 (14.7%) other alterations; 8/108 patients harbored FLT3 ITD mutation (7.4%, 23 not tested), 12/101 NPM1 mutation (11.9%, 30 not tested). Based on these data, 111 patients were evaluable for ELN2010 risk stratification; 9 over 111 patients (8.1%) were stratified in the low risk, 42/111 (37.8%) in intermediate-1 risk, 17/111(15.3%) in intermediate-2 risk, and 43/111 (38.7%) in high-risk class. As expected, most of the patients had at least one comorbidity. Particularly, baseline arrhythmia was present in 29/130 (22.1%, 1 no data), cardiovascular comorbidity in 20/130 (15.4%, 1 no data), diabetes in 20/131 (15.3%), cerebrovascular comorbidity in 11/131 (8.4%), kidney disease in 15/130 (11.5%, 1 no data), lung chronic disease 19/130 (14.6% 1 no data), hypoalbuminemia in 25/111 patients (22.5%, 20 no data). With a median follow up of 28.2 months, median overall survival (OS) of the entire cohort was 15.8 months (95% C.I. 11.2-19.4). We confirmed that patient who obtained a response (complete remission, partial response, hematological improvement) after 2 months of therapy had the best OS (figure A, median OS of 21 months for responders vs 7.4 months for non-responders, p To test the impact of comorbidities combined with cytogenetic and molecular risk, AML-CM was used. Our results indicate that AML-CM score was able to stratify prognosis in elderly patients receiving frontline HMAs (figure B, median OS in score group 1: 29.7 months, score group 2: 16.5 months, score group 3: 11.2 months, score group 4: 6.6 months, p=.038). The worse prognosis of patients with higher AML-CM score, which includes patients with increased baseline comorbidities, may be explained with a higher incidence of AEs (84.55, 116.01, 131.45, 229.3 events for 100 patients per year for score group 1,2,3, and 4, respectively) and infections (53.80, 55.10, 85.95, 140.13 events for 100 patients per year for score group 1,2,3, and 4, respectively), in patients with higher baseline comorbidities. CONCLUSION: In this study we found that baseline comorbidities, captured by AML-CM score, may define prognosis of elderly patients receiving 1st line HMAs; parametric comorbidity scores may improve our ability to predict outcome and tailor interventions. The impact of comorbidity on OS may be increased with novel and more aggressive therapy. For this reason, specific studies on functional fitness tests and geriatric assessments are highly warranted in patients receiving HMAs plus venetoclax. This work was supported by Bologna AIL. CP and AC shared last authorship. Figure 1 Figure 1. Disclosures Martinelli: Daichii Sankyo: Consultancy; Pfizer: Consultancy, Speakers Bureau; Astellas: Consultancy, Speakers Bureau; Roche: Consultancy; Abbvie: Consultancy; Stemline Therapeutics: Consultancy; Celgene /BMS: Consultancy, Speakers Bureau; Incyte: Consultancy; Jazz Pharmaceuticals: Consultancy. Cavo: Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Accommodations, Speakers Bureau; Amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: TRAVEL, ACCOMMODATIONS, EXPENSES, Speakers Bureau; Adaptive Biotechnologies: Consultancy, Honoraria; GlaxoSmithKline: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol-Myers Squib: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Papayannidis: Pfizer, Amgen, Novartis: Honoraria. Curti: Abbvie: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; Jazz Pharma: Membership on an entity's Board of Directors or advisory committees.

Somatic mutations of DNMT3A occur in about 20% of acute myeloid leukemia (AML) patients. They mostly consist in heterozygous missense mutations targeting a hotspot site at R882 codon, which exhibit a dominant negative effect and are associated with high myeloblast count, advanced age, and poor prognosis. Other types of mutations such as truncations, insertions, or single-nucleotide deletion also affect the DNMT3A gene, though with lower frequency. The present study aimed to characterize two DNMT3A gene mutations identified by next-generation sequencing (NGS), through analysis of protein stability and DNA methylation status at CpG islands. The first mutation was a single-nucleotide variant of DNMT3A at exon 20 causing a premature STOP codon (c.2385G > A; p.Trp795∗; NM_022552.4). The DNMT3A mutation load increased from 4.5% to 38.2% during guadecitabine treatment, with a dominant negative effect on CpG methylation and on protein expression. The second mutation was a novel insertion of 35 nucleotides in exon 22 of DNMT3A (NM_022552.4) that introduced a STOP codon too, after the amino acid Glu863 caused by a frameshift insertion (c.2586_2587insTCATGAATGAGAAAGAGGACATCTTATGGTGCACT; p. Thr862_Glu863fsins). The mutation, which was associated with reduced DNMT3A expression and CpG methylation, persisted at relapse with minor changes in the methylation profile and at protein level. Our data highlight the need to better understand the consequences of DNMT3A mutations other than R882 substitutions in the leukemogenic process in order to tailor patient treatments, thus avoiding therapeutic resistance and disease relapse.

Background The treatment of Philadelphia chromosome-positive Acute Lymphoblastic Leukemia (Ph+ ALL) patients who harbor the T315I BCR-ABL1 mutation or who have two or more mutations in the same BCR-ABL1 molecule is particularly challenging since first and second-generation Tyrosine Kinase Inhibitors (TKIs) are ineffective. Ponatinib, blinatumomab, chemotherapy and transplant are the currently available options in these cases. Case presentation We here report the case of a young Ph+ ALL patient who relapsed on front-line dasatinib therapy because of two independent T315I-positive subclones, resulting from different nucleotide substitutions -one of whom never reported previously- and where additional mutant clones outgrew and persisted despite ponatinib, transplant, blinatumomab and conventional chemotherapy. Deep Sequencing (DS) was used to dissect the complexity of BCR-ABL1 kinase domain (KD) mutation status and follow the kinetics of different mutant clones across the sequential therapeutic approaches. Conclusions This case presents several peculiar and remarkable aspects: i) distinct clones may acquire the same amino acid substitution via different nucleotide changes; ii) the T315I mutation may arise also from an ‘act’ to ‘atc’ codon change; iii) the strategy of temporarily replacing TKI therapy with chemo or immunotherapy, in order to remove the selective pressure and deselect aggressive mutant clones, cannot always be expected to be effective; iv) BCR-ABL1-mutated sub-clones may persist at very low levels (undetectable even by Deep Sequencing) for long time and then outcompete BCR-ABL1-unmutated ones becoming dominant even in the absence of any TKI selective pressure. Electronic supplementary material The online version of this article (doi:10.1186/s12885-017-3511-2) contains supplementary material, which is available to authorized users.

Context Post-HSCT relapse of B-cell ALL is associated with a dismal outcome. IO allows, in the setting of R/R B-ALL patients, high response rates, but with limited duration. Objective To report the outcome of 8 B-ALL adult patients, relapsed after allogeneic HSCT, treated with IO and DLI. Design Retrospective analysis (first patient treated in April 2015, last patient in October 2019). Setting Multicenter (Italian centers). Patients or other participants Five Ph-negative and 3 Ph-positive B-ALL adult patients, with BM disease (6 morphological relapse and 2 MRD-positivity) were treated; 2 patients had additionally extramedullary (EM) disease. Three patients were in salvage 1, 5 patients in salvage ≥ 2; 50% of the patients had previously received Blinatumomab. Median time from transplant to relapse and from relapse to IO was 11 (range 2-21) and 2.5 (range 0-65) months, respectively. Interventions Patients received a median of 3 (range 2-6) IO courses. Four patients received IO and DLI in an alternate schedule and 4 patients sequentially after IO; a median of 3 (range 2-4) DLI at escalating dose was administered (1st dose ranging from 1 × 104/kg CD3+ to 5 × 106/kg CD3+; following doses from 1 × 105 to 5 × 107/kg CD3+). Main outcome measures CR and MRD rate, EM response, DFS, OS, toxicity. MRD was evaluated with BCR-ABL fusion transcript or V(D)J IgH/TCR disease-specific rearrangement on BM. DFS and OS were calculated from start of IO. Toxicity was graded according to CTCAE version. 4.03. Results All patients achieved CR after 1st IO cycle. Six of 8 patients obtained MRD negativity after 2nd IO cycle (4 of 6 patients after 1st cycle). Both PET+ patients achieved PET-negativity. With a median follow-up of 23.5 (range 3-58) months, 6 of 8 (75%) patients are alive. Four of 8 (50%) patients relapsed, of which 2 with CNS localization. Median DFS is 12 months (range 3-58) and median OS is 23.5 (3-58) months. Four out of 8 patients experienced G4 thrombocytopenia. Two patients experienced grade 1 GvHD and no VOD were reported. Conclusions IO and DLI are a safe combination in the post-HSCT setting that may ameliorate the dismal prognosis of this patient subset.

Background 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. Case presentation 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. Conclusions 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.

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.

7038 Background: Nowadays, science is debating if autophagy in cancer can lead to therapy resistance or it can favor apoptosis. Autophagy pathways are involved pro-apoptotic mechanism, or they can improve stresses survival eliminating damaged mitochondria and proteins. Levels and activity of pro-apoptotic and anti-apoptotic proteins (eg. bcl-2 and p53), high levels of cAMP, and a pink/park complex could play as fulcrum on this lever. Our study aims to define the role of autophagy in AML. Methods: We analyzed 148 consecutive non M3 AML with Affymetrix SNP array. We screened all patients for TP53, FLT3, NMP1 mutations. Patients was treated with intensive induction chemotherapy regimens. Survival data were collected prospectively, with a median follow-up of 18 months. Results: Autophagy alteration (gene group 1: ULK1 CHR11; ULK1 CHR17; BECN1; ATG14; AMBRA1; UVRAG; ATG9A; ATG9B; PIK3C3; PIK3R4) was related to lower Complete Remission rate (CR%) after induction in univariate (p < .001) and multivariable regression model with age, karyotype, secondary AML, TP53 mutation (p = .014); autophagy alteration shown to confer worst Overal Survival (OS) (p < .001) and was significantly associated with complex karyotype and TP53 mutation (p < .001). We detected significant differences in term of survival independently both in gain and loss in group 1 genes (p < .001). Alterations in genes in cAMP pathway (group 2: SESN1; PRKAA1 CHR 3; PRKAB1: PRKAA1 CHR 1: PRKAG1 CHR11; PRKAG1 CHR 7; PRKAG3; PRKAB1) and in genes that could be related to a switch from a physiological role of autophagy to a resiliency mechanism (group 3: CCND1; BCL2; PINK1; PARK2; TP53; MDM1; MDM4) showed to confer worst OS (p < .001 in both groups); Alteration in group 2 and group 3 were related to lower CR% after induction (p < .001 in both groups). Whole Exome Sequencing on 56 patients in our set did not found any significant mutation in genes we analyzed with the exception of TP53. Conclusions: Alterations in autophagy regulator genes are associated with poor prognosis and therapy resistance. A loss in autophagy could block apoptosis, a gain could confer cell resiliency. Acknowledgements: ELN, AIL, AIRC, Progetto Regione-Università 2010-12,FP7 NGS-PTL, HARMONY

11611 Background: Necroptosis is a type of necrotic cell death involving several genes transcription and activation of molecular mechanisms as death receptors, interferon, toll-like receptors, intracellular RNA and DNA sensors.The process is leading by the family of receptor-interacting protein kinase ( RIPK3, RIPK2, RIPK1) and the MLKL substrate. Losses of RIPK3 or MLKL, as well as deficiency in apoptosis, could allow tumor cells to escape the immunomediated cells death (ICD). Methods: We performed SNP Arrays (Cytoscan HD and SNP 6.0, Affymetrix) on a cohort of 300 non-M3 AML patients at diagnosis and we analyzed the Overall Survival (OS) of our patients with deficiency on necroptosis pathways. Survival was analyzed with Kaplan-Mayer method and Log-Rank test. We further analyze the relevance of different prognostic factors by the use of COX-Hazard Ratio statistical analysis. Results: We find that 18 patients presented a loss of RIPK1 or MLKL (nobody presented losses in RIPK3/RIPK2) and 13/18 patients were older than 65 years old. The Overall Survival (OS) of patients with alterations in these genes is significantly lower than control group, with a median OS of 3 vs 6 month respectively (p

Monitoring of minimal residual disease (MRD) by quantification of BCR-ABL1 transcript levels has become a main part of the management of patients with BCR-ABL1-positive acute lymphoblastic leukemia (ALL) in treatment with tyrosine kinase inhibitors (TKIs). The failure to achieve molecular negativity shortly after starting TKI has been demonstrated to be predictive of relapse, suggesting that an accurate measurement of low BCR-ABL1 levels may have a role in preventing hematological relapse. Despite the big efforts made by many European laboratories within the European Study Group, at the time of writing a standardized procedure to quantify and express results is still missing for BCR-ABL1-positive ALL. In this study, in order to detect with high sensitivity low levels of BCR-ABL1 transcripts, we used a new technology and a new molecular approach based on microfluidic digital polymerase chain reaction (dPCR) using Taqman chemistry and we compared obtained results with those generated by the conventional method based on reverse transcriptase PCR reaction (RQ-PCR) for BCR-ABL1 and total ABL1, with TaqMan chemistry and with Applied Biosystems instrument. We demonstrated the dPCR is high-sensitive (able to detect a single copy of BCR-ABL1) and reliable (results are comparable to those obtained by BCR-ABL1 quantification with conventional technology), allowing an accurate monitoring of BCR-ABL1-positive ALL patients in complete remission.

Rationale: Inotuzumab ozagomicin (IO) has been linked to an increased incidence of veno-occlusive disease (VOD) and liver alterations. Most VOD events occurred during hematopoietic stem cell (HSCT) transplantation after IO therapy. We have previously described that the measurement of liver stiffness can anticipate the diagnosis of VOD in the context of HSCT. The mechanisms underlying the increased risk of VOD and liver damage in patients receiving IO are not well understood; in the pathogenesis endothelial damage, ozagomicin release and on-target off-tumor effects may be involved. Here, we aimed to assess the effects of IO on the changes of liver, vascular and biochemistry parameters. Methods: Intensive monitoring of the liver was incorporated into the standard of care of patients who received IO for relapsed or refractory (R / R) acute lymphoblastic leukemia (ALL). Upper abdomen ultrasound with Doppler was performed at baseline and at the end of therapy; liver stiffness measurement (LSM) by Fibroscan® (Echosens, Paris, France) at every IO course or at every IO infusion. With the exception of ursodeoxycholic acid, the patients did not receive prophylaxis for VOD. Data was collected after anonymous aggregation, in accordance with GCP and Helsinki declaration. Results are reported as median with interquartile ranges (IQR). Results: At data cut-off, 1st Apr 2019, 16 patient received baseline assessment and at least a post-IO assessment in our monitoring program. In our patent set, median age was 44.5 (IQR 30.7 - 64.0); 12/16 (75 %) patients relapsed after the last treatment and 4/16 (25 %) patients were refractory to the last treatment; patients received a median of 3 (IQR 2 - 3.7) lines before IO; 6/16 (37.5 %) patients undergone HSCT before IO, of which a patient had 1st and 2nd HSCT before IO; 5/16 (31.25 %) undergone HSCT after IO therapy (no patients had second HSCT after IO). Patients received a median of 2 (IQR 2.0 - 3.7) IO administration according to the schedule of the phase 3 trial. The median duration of the therapy was 61.5 days (IQR 43.2 - 114.0) and median progression-free survival in our population was 278.0 days (95% C.I. 264.0 - 292.0). In our patient set, we performed 113 biochemistry determination, 30 liver ultrasounds with Doppler and 116 LSM examination. One patient received a liver biopsy. Among the biochemical exams (AST, ALT, GGT and alkaline phosphatase) only the AST values significantly increased after 1st course of IO (from the median value of 21 U/L to 53 U/L after course 3). Liver ultrasound with Doppler revealed portal hypertension signs in half of the patients during IO monitoring program. Among these patients 7/16 (44%), 3/16 (17%), 5/16 (33.3%) and 3/16 (17%) showed splenomegaly, recanalization of the paraumbilical vein, dilatation of portal vein and ascites, respectively. Median LSM significantly increased from a baseline value of 6 kPa to 7.8 kPa after last post-IO assessment (p-value 7.1 kPa). With a median follow up of 387.5 days (IQR 182.8-524.5) we observed one VOD event (7%); the VOD was graded severe and occurred after HSCT post-IO. Conclusions: Our clinical experience represents the first step to better understand the IO-related liver alterations, as we described the frequency and relevance of quantitative markers. Most of the patients in our set developed ultrasound and/or elastography alteration during IO therapy. Furthermore, these alterations do not seem to correlate with biochemistry. Even if most of the patients had sub-clinical vascular and parenchymal alterations of the liver portal-hypertension related, VOD incidence in our set is comparable with literature. Long-term follow-up results are expected to test whether alterations return or evolve over time. Stratifying the tailored risk liver complications with prospective non-invasive and marker-driven strategies in term of IO dosing and HSCT timing could be a great benefit for patients. * FR and GM contributed to this manuscript equally # AC and CP contributed to this manuscript equally Figure Disclosures Martinelli: Roche: Consultancy; BMS: Consultancy; Novartis: Consultancy; Pfizer: Consultancy; ARIAD: Consultancy. Cavo:janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; bms: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; novartis: Honoraria; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau. Papayannidis:Amgen: Honoraria; Novartis: Honoraria; Teva: Honoraria; Pfizer: Honoraria; Incyte: Honoraria; Shire: Honoraria.

Background Although much efforts have been made to precisely define fitness of AML patients, in patients who are not candidate to chemotherapy, there is no prognostic model and the respective weight of AML biology and patient fitness are not well established. Here we test AML-CM score (Sorror, JAMA 2018), that is validated in fit population, in a set of old AML patients who received HMAs. Methods We retrospectively collected data of consecutive patients who received HMAs in our institution from 1st Jan 2008 with an age > 65 years at AML diagnosis. AML-CM score was applied to all the patients. Patients were divided in 4 groups (score 1-4: group 1, score 5-6: group 2; score 7-9: group 3, score > 9: group 4) and in 2 macro-groups (score 1-6: group A and score > 6 group B) for the analyses. Descriptive data are presented as median with interquartile ranges (IQR). Adverse events are graded according to CTCAE v4.03. Survival analysis was conducted with Kaplan-Meyer and are presented as 95% confidence intervals (C.I.) and differences in overall survival (OS) were tested with 2-side log rank test. Fisher exact test and Person's chi squared test were used whenever appropriate. Results At data cut-off, 1st Jan 2019, 60 consecutive patients received decitabine or azacytidine as 1st line therapy for AML. Median age of the population was 75.94 years (IQR 72.53-80.38). Most of the patients (37/62, 59.7%) had de novo AML, 19/62 (30.6%) had AML secondary to previous myeloid disorders and 6/62 (9.7%) had AML secondary to chemotherapy or radiotherapy. Most of the patients were smokers (19/33, 57.57%, 29 no data), and few were usual drinkers (4/16, 25.00%, 46 no data). In our set, out of 62 patients, 2 patients (3.2%) had inv(3), 1 (1.6%) a translocation involving 11q23, 1 (1.6%) del(5q), 4 (6.4%) mon(7) or del (7q), 1 (1.6%) del(17p), 15 (24.2%) complex karyotype, 27 (43.5%) normal karyotype, 4 (6.5%) other alterations and 5 were not evaluable; 3/17 (17.65%, 45 no data) harbored IDH2 mutation, 1/16 (6.25%) IDH2 mutation, 2/33 FLT3 mutation (6.06%, 29 no data), 1/24 (4.17%, 38 no data), 2/15 (13.33%, 47 no data) TP53 mutation. According to ELN 2017, 3/62 patients (4.83%) had low risk, 34/62 (54.84%) intermediate risk and 23/62 (37.10%) high risk AML. According to AML-CM score, 13/62 patients (20.97%) were in group A, 20/62 (32.36%) in group B, 21/62 (33.87%) in group C, 6/62 (9.68%) in group D, 2/62 (3.23%) were not allocated for incomplete AML-CM score. There was no difference in term of age, ELN risk, secondary AML prevalence, HMA administered, or response to HMA according to ELN criteria between group 1, 2, 3, 4 or between macro-group A and B. Cardiovascular comorbidity, diabetes mellitus, obesity, previous tumor, hypoalbuminemia, elevated LDH were prevalent in higher risk AML-CM groups (3-4) and in macro-group B. Median OS was 658 days (95% C.I. 316-1000) in group 1, 556 days (95% C.I. 463-649 in group 2, 243 days (95% C.I. 153-353) in group 3, 107 days (95% C.I. 47-167) in group 4 (p=.021, figure 1A). Furthermore, we observed a median OS of 589 days (95% C.I. 328-850) in macro-group A and 219 days (95% C.I. 96-342) in macro-group B (p=.003, figure 1B). Reduced survival was correlated with a non-statistical trend toward augmented incidence of infections and adverse events in higher risk AML-CM groups (3-4). Conclusions AML-CM is a useful indicator of prognosis in old patients that receive HMAs. Prognosis in our set is influenced by comorbidity (measured with AML-CM, a quantitative score) more than by disease biology. We identified a group of patients (macro-group A) that has median OS after HMAs outlying OS reported in literature. This brilliant result can be due to lower comorbidity. AML-CM could help in defining candidate patients for therapy intensification and care utilization or for team comorbidity management. GM and RDN equally contributed Figure 1 Disclosures Martinelli: Roche: Consultancy; Novartis: Consultancy; ARIAD: Consultancy; BMS: Consultancy; Pfizer: Consultancy. Baccarani:Novartis: Consultancy, Speakers Bureau; Incyte: Consultancy, Speakers Bureau; Takeda: Consultancy. Papayannidis:Pfizer: Honoraria; Teva: Honoraria; Shire: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Incyte: Honoraria. Cavo:janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; bms: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel accommodations, Speakers Bureau; AbbVie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; novartis: Honoraria; takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; amgen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

Adult B-ALL patients still have a dismal prognosis, due to a high incidence of relapse even after allogenic SCT. Safety and efficacy of Blinatumomab, an anti CD3-CD19 Bite antibody, has been demonstrated both in MRD positive patients and in relapsed/refractory (R/R) setting. To evaluate safety profile and efficacy of Blinatumomab, obtained through a compassionate use, in a cohort of 18 adult patients affected by MRD+ or R/R B-ALL treated at Bologna University. From March 2015 to December 2017, 18 patients received Blinatumomab at the standard dosage (9 mcg/d x 7 days, 28 mcg/d x 21 days) in 28-days courses. All the patients were hospitalized to receive the first course of therapy. The following courses, based on the good safety profile of the compound, were administered in outpatient setting. 18 patients (M/F = 10/8; median age 43, range 18-73) have been treated. Philadelphia (Ph) chromosome was detected in 8/18 patients. 10 patients were MRD+ (5 Ph pos and 5 Ph neg); E2A-PBX1 and MLL-AF4 rearrangements were found in two patients. 8 patients had a R/R disease (3 Ph pos and 5 Ph neg). Median WBC count before starting therapy with Blinatumomab was 5400/mmc (range 500-76500). All the patients had previously received many lines of therapy (median 4, range 1-7). In 4 cases an alloTMO was already performed, and two patients had received two transplants. 12/18 patients were referred to us by other Italian Institutions. All the patients received at least one course of Blinatumomab. In one case three courses were administered; an elderly patient is actually receiving the fifth course. Globally, 32 courses of therapy have been administered (median 2, range 1-5). Bone marrow evaluations, including cytogenetics, molecular biology and immunophenotyping analysis were performed at the beginning of every course of therapy in order to assess patients' disease status. MRD evaluation was assessed through BCR-ABL fusion transcript quantitative analysis in Ph pos ALL patients and Ig rearrangment in Ph negative patients. Monitoring of adverse events was periodically performed. 16/18 patients are evaluable for response, at least to one cycle (one patient died during the first course, one patient is still receiving the first course). 9/16 (56%) patients obtained a CR (7/9 MRD+ and 2/7 R/R). In 7/9 (78%) responders patients a molecular CR was reached, (in 6 patients after the first course, in one case after the second one). 5 responders proceeded to alloBMT and are actually alive in CR (median follow-up after transplant 240 days). In terms of toxicity, one patient developed a grade IV neurological event (mental confusion, tremor), which completely resolved after a transient drug withdrawal. Our results confirm the high rate of response and to Blinatumomab in a poor patients' population, and the good management profile of the compound. Note: This abstract was not presented at the meeting. Citation Format: Giovanni Martinelli, Stefania Paolini, Claudio Cerchione, Alessandra Santoro, Valentina Robustelli, Simona Soverini, Caterina De Benedittis, Enrica Imbrogno, Andrea Ghelli Luserna Di Rora, Sarah Parisi, Chiara Sartor, Giovanni Marconi, Silvia Lo Monaco, Maria Chiara Abbenante, Maria Chiara Fontana, Antonella Padella, Anna Ferrari, Giorgia Simonetti, Elena Tenti, Federica Frabetti, Francesca Volpato, Samantha Bruno, Fabiana Mammoli, Maria Teresa Bocchicchio, Carmen Baldazzi, Cristina Papayannidis. Blinatumomab is safe and effective in relapsed and MRD positive B-ALL CD19+ patients: The bologna compassionate program experience [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3100.

Background. The incorporation of tyrosine kinase inhibitors (TKIs) in treatment schemes of Ph+ ALL has remarkably improved survival. In adult patients with Ph+ ALL, ponatinib in combination with chemotherapy showed a 3-year event-free survival rate of 69%, a 3-year overall survival (OS) of 83%, and a higher rate of response when compared with dasatinib plus chemotherapy. However, in unfit or elderly ALL patients, TKIs combined with chemotherapy are associations with higher toxicity. Therefore, we examined the efficacy and safety of steroids plus ponatinib alone for the treatment of elderly or unfit patients with Ph+ ALL in a multi-center Phase II prospective clinical Italian trial, GIMEMA LAL1811 (EudraCT number 2012-002761-35). Methods. From March 2014 to December 2016, we enrolled 44 patients with untreated Ph+ ALL, ≥ 60 years or unfit (i.e. for intensive chemotherapy and stem cell transplantation). Two out of 44 patients were not elegible for the study. Patients received oral administration of 45 mg/day of ponatinib for 8 consecutive courses of 6 weeks (w). Steroids were administered from day -14 to day 29 during course 1. Intrathecal therapy with methotrexate, cytarabine and dexametasone was performed every 28 days for central nervous system (CNS) disease prophylaxis. In patients with CNS disease at diagnosis, intrathecal therapy was administered twice a week until complete remission. Dose reduction of ponatinib was allowed for adverse events. Patient samples were obtained at diagnosis and at every course, BCR-ABL mutational analisys and BCR-ABL/ABL ratio by quantitative real time PCR was performed. Complete molecular response (CMR) was defined as BCR-ABL/ABL ratio below 0.01 or undetectable, and with a sensitivity of at least 30,000 molecules of ABL. Results. Forty-two patients were eligible for the study. Median age was 68 years (range 27-85). Nine out of 42 patients were At week 24, 15/42 patients still received 45 mg of ponatinib daily, only 4/42 patients permanently withdrew study drug. During the study, 75 adverse events (AE) were reported; 36 of the 75 AEs were considered related to ponatinib. Twenty-six of the 75 AEs were considered serious (SAE); 13/26 SAEs were considered related to ponatinib. A death was suspected to be related to ponatinib. We performed BCR-ABL mutational analysis in 22 patients at diagnosis, and 15 patients at 24w. T315L (abundance 100%) was detected in a patient relapsed during ponatinib therapy. We could not identify the emergency of other mutations. Conclusions. Ponatinib and steroid show a high efficacy in newly diagnosed unfit/elderly Ph+ ALL patients. Toxicities were manageable and cardiovascular AEs were limited. In the small cohort of patients relapsed in the study, relapse mechanisms were unclear; only one patient had evidence of mutations that caused resistance to ponatinib. The fast and deep reduction of the disease burden in the majority of patients, the ability of ponatinib to prevent the emergence of clones harboring BCR-ABL mutations, and the synthetic lethality with steroids on the BCR-ABL, FLT3, HCK, CDK6, MCL1 pathway could explain the therapeutic effectiveness. Acknowledgments. GIMEMA, ELN, AIL, AIRC, Regione-Università 2010-12, FP7 NGS-PTL, HARMONY, Fondazione del Monte BO e RA. Disclosures Soverini: Bristol-Myers Squibb: Consultancy; Incyte Biosciences: Consultancy; Novartis: Consultancy. Bocchia: Novartis: Other: Travel grant; Celgene: Other: Travel grant; Roche: Other: Travel grant; Jansen: Other: Travel grant. Cuneo: Abbvie: Honoraria, Other: Advisory Board; Janssen: Honoraria, Other: Advisory Board; Gilead: Honoraria, Other: Advisory Board; Roche: Honoraria, Other: Advisory Board. Bonifacio: Pfizer: Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Membership on an entity's Board of Directors or advisory committees. Falini: Roche: Research Funding. Galieni: Takeda: Other: Advisory Board; Abbvie: Other: Advisory Board. Foà: Sandoz: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; AbbVie: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; janssen: Consultancy, Speakers Bureau; Gilead: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; Celgene: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau. Baccarani: Pfizer: Honoraria, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Speakers Bureau; Incyte ARIAD: Consultancy, Honoraria, Speakers Bureau; Novartis: Consultancy, Honoraria, Speakers Bureau.

Although improvements in knowledge of genetic basis of ALL have been made and many new drugs are now available, the prognosis for adult patients with ALL is still a challenge. Current therapies based on chemotherapy regimens or on tailored targets do not completely eradicate the leukemia clone and relapse is a predictable event. Here, we explored the in vitro and in vivo activity of PF-00477736 (Pfizer), an ATP-competitive small-molecule inhibitor of checkpoint kinase 1 (Chk1) and with lower efficacy of checkpoint kinase 2 (Chk2). Chk1 and Chk2 are serine/threonine kinases which following activation by a broad spectrum of damaged DNA coordinate the DNA damage response. By quantitative PCR, higher transcript levels of Chk1 were found in 8 leukemia cell lines and 54 newly diagnosed ALL cases compared to normal bone marrow mononuclear cells (p value < 0.001). Immunohistochemistry of formalin-fixed paraffin-embedded tissue samples collected from 60 ALL patients showed a diffuse positivity for Chk1, Chk2 and the phosphorylated forms of Chk1 (Ser345) in 51/54 (96%), 55/57 (94%) and 45/56 (80%) of the cases respectively, whereas 15/55 (27%) of ALLs stained for phosphorylated form of Chk2 (Thr68). Interestingly, in our ALL series, genomic damage was suggested by the nuclear labeling for γ-H2A.X molecule in 40/59 (68%) of samples. In B-/T-ALL cell lines (BV-173, SUP-B15, REH, NALM-6, NALM-19, MOLT-4, RPMI8402 and CCRF-CEM) inhibition of Chk1 resulted in a dose and time-dependent cytotoxicity (IC50 from 57.4 nM to 1423.0 at 24 hours), induction of apoptosis and increased DNA damage as demonstrated by increased levels of phosphorylated Chk1 (Ser317 and Ser345) and H2A.X (Ser139). Moreover, PF-00477736 efficiently triggered the Chk1-Cdc25-Cdc2 pathway with a decreased phosphorylation of Cdc25c (Ser216) and Cdc2 (Tyr15). Gene expression profiling analysis shed light on molecular mechanisms driven by PF-00477736 revealing a deregulation of genes involved in DNA damage checkpoint, cell cycle control and apoptosis. Among these, Gadd45a and Plk3 were confirmed by qPCR analysis up-regulated, whereas Cdk4 and Chk2 resulted down-regulated between treated and untreated samples. The efficacy of PF-00477736 as a single agent was confirmed ex-vivo in primary leukemic blasts separated from 14 ALL patients but not in in primary cultures of normal bone marrow mononuclear cells. Furthermore, in mice transplanted with T-lymphoid leukemia PF-0477736 increased the survival of treated mice compared with mice treated with vehicle (p = 0.0016). In conclusion, in vitro, ex-vivo and in vivo results support the inhibition of Chk1 as a new therapeutic strategy in acute lymphoblastic leukemia and they provide a strong rationale for its future clinical investigation.

// Andrea Ghelli Luserna Di Rora 1 , Ilaria Iacobucci 1, * , Enrica Imbrogno 1 , Cristina Papayannidis 1 , Enrico Derenzini 1 , Anna Ferrari 1 , Viviana Guadagnuolo 1 , Valentina Robustelli 1 , Sarah Parisi 1 , Chiara Sartor 1 , Maria Chiara Abbenante 1 , Stefania Paolini 1 , Giovanni Martinelli 1, * 1 Institute of Hematology “L. e A. Seragnoli”, Department of Experimental, Diagnostic and Specialty Medicine, University of Bologna, Bologna, Italy * Co-corresponding authors Correspondence to: Ilaria Iacobucci, email: ilaria.iacobucci2@unibo.it Giovanni Martinelli, email: giovanni.martinelli2@unibo.it Keywords: CHK1, cell cycle, acute lymphoblastic leukemia, DNA damage response, chemo-sensitizer agent Received: February 12, 2016 Accepted: June 30, 2016 Published: July 11, 2016 ABSTRACT During the last few years many Checkpoint kinase 1/2 (Chk1/Chk2) inhibitors have been developed for the treatment of different type of cancers. In this study we evaluated the efficacy of the Chk 1/2 inhibitor prexasertib mesylate monohydrate in B-/T- cell progenitor acute lymphoblastic leukemia (ALL) as single agent and in combination with other drugs. The prexasertib reduced the cell viability in a dose and time dependent manner in all the treated cell lines. The cytotoxic activity was confirmed by the increment of apoptotic cells (Annexin V/Propidium Iodide staining), by the increase of γH2A.X protein expression and by the activation of different apoptotic markers (Parp-1 and pro-Caspase3 cleavage). Furthermore, the inhibition of Chk1 changed the cell cycle profile. In order to evaluate the chemo-sensitizer activity of the compound, different cell lines were treated for 24 and 48 hours with prexasertib in combination with other drugs (imatinib, dasatinib and clofarabine). The results from cell line models were strengthened in primary leukemic blasts isolated from peripheral blood of adult acute lymphoblastic leukemia patients. In this study we highlighted the mechanism of action and the effectiveness of prexasertib as single agent or in combination with other conventional drugs like imatinib, dasatinib and clofarabine in the treatment of B-/T-ALL.

Next Generation Sequencing (NGS) studies identified 9 functional categories of mutations in acute myeloid leukemia (AML), with >99% of cases having at least one of those mutations (Ley et al. NEJM 2013). However, multiple genetic hits participate to AML pathogenesis, and metabolic dysregulations, as the one induced by IDH1/2 mutations, play oncogenic functions (Ward et al. Cancer Cell 2010). Aim of the study was to define novel functional categories of AML mutations affecting relevant and druggable biological processes, with focus on genetic determinants of metabolic plasticity. Out of 455 whole exome sequencing (WES) cases from onco-hematological patients collected in the NGS-PTL project, we analyzed 37 AML cases, belonging to our cohort of 239 FLT3-WT samples (886 AML total). We performed 100 bp paired-end WES (HiSeq2000, Illumina) and mapped the sequenced reads with Burrows-Wheeler Aligner. Variants where called with MuTect or GATK for single nucleotide variant (SNV) and indels detection, respectively (>90% confidence). Gene expression profiling was performed using HTA2.0 microarray (Affymetrix) on 56 bone marrow samples, including AML (≥80% blasts) and healthy controls. By WES analysis, we detected an average of 26 somatic variants per patient (range, 7 to 65). Gene ontology annotation identified 8 novel relevant functional categories of mutated genes: transcription, translation and post-translational modifications, protein degradation, cytoskeleton, cell cycle, DNA damage, cell survival and metabolism. Since metabolic pathways are promising targets for tailored therapies (e.g. IDH1/2 and glutaminase inhibitors), we focused our analysis on them. We identified 82 variants (74 SNVs, 2 frameshift and 4 nonframeshift deletions, 2 stopgains) targeting 70 genes involved in metabolism, with 78% of patients carrying at least one mutation in a metabolic gene and 35 variants rated as damaging by CONDEL algorithm. Among mutations in metabolic genes, the most represented pathways according to Recon X database were amino acids, lipids, CoA and nucleotides metabolism, transport and bioenergetics pathways. Notably, IMPDH2, a mediator of MYC-induced proliferation involved in nucleotide interconversion, was mutated and overexpressed in our AML cohort (p=0.01), suggesting a potential oncogenic function. Moreover, ALDH2, a regulator of hematopoietic stem cell functions which is involved in multiple metabolic pathways and associates with metabolic remodeling, was mutated and 2-fold downregulated in AML blasts. Seven genes were mutated in 5-8% of samples: RETSAT, HSPG2, CHPF, ABCA2, ND1, APOBR, NAAA. Among them, RETSAT, HSPG2, CHPF mutations were also predicted as “drivers” by DOTS-Finder tool. Bioenergetics pathways were affected by mutations in glycolysis and gluconeogenesis (GPI, ITPA), oxidative phosphorylation (ND1, ND4, ND5, CYTB), pentose phosphate pathway (H6PD, PGLS). Patients carrying mutations in the bioenergetics pathway showed a strong trend towards reduced overall survival, which did not associate with unfavorable molecular mutations. In conclusion, metabolism is the most represented class of mutated genes (8.6% of variants) in our FLT3-WT AML cohort after signaling, leading us to propose a novel functional category. Our data suggest that, along with mutations in established oncogenes and tumor suppressors involved in metabolic control (KRAS, TP53, MYC pathway), a number of genetic determinants participate to leukemia metabolic plasticity and oncogenic mutations of metabolic enzymes may drive leukemogenesis, impact on patient's survival and become novel targets for personalized therapies. Acknowledgements: ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. GS and AP equally contributed to this work. Citation Format: Giorgia Simonetti, Antonella Padella, Ilaria Iacobucci, Italo Do Valle, Gabriele Fontanarosa, Elisa Zago, Francesca Griggio, Marianna Garonzi, Simona Bernardi, Cristina Papayannidis, Maria Chiara Abbenante, Giovanni Marconi, Giorgio Melloni, Laura Riva, Viviana Guadagnuolo, Mariachiara Fontana, Samantha Bruno, Elisa Zuffa, Eugenia Franchini, Annalisa Astolfi, Carmen Baldazzi, Elisa Dan, Barbara Sinigaglia, Michele Cavo, Nicoletta Testoni, Emanuela Ottaviani, Pier Giuseppe Pelicci, Marco Sazzini, Alberto Ferrarini, Massimo Delledonne, Daniel Remondini, Giovanni Martinelli. European Network NGS-PTL preliminary data: Whole exome sequencing identifies mutations of ALDH2, RETSAT, HSPG2, CHPF and other metabolic genes as a novel functional category in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A27.

MDM2 is an important negative regulator of p53 tumor suppressor. In this study, we sought to investigate the preclinical activity of the MDM2 antagonist, Nutlin-3a, in Philadelphia positive (Ph+) and negative (Ph-) leukemic cell line models, and primary B-acute lymphoblastic leukemia (ALL) patient samples. We demonstrated that Nutlin-3a treatment reduced viability and induced p53-mediated apoptosis in ALL cells with wild-type p53 protein, in a time and dose-dependent manner, resulting in the increased expression of pro-apoptotic proteins and key regulators of cell cycle arrest. The dose-dependent reduction in cell viability was confirmed in primary blast cells from B-ALL patients, including Ph+ ALL resistant patients carrying the T315I BCR-ABL1 mutation. Our findings provide a strong rational for further clinical investigation of Nutlin-3a in Ph+ and Ph- ALL.

7043Background: Young patients affected by non APL-Acute Myeloid Leukemia (AML) achieve complete remission (CR) using conventional induction chemotherapy in about 55-70%. The addition of Gemtuzumab...

Introduction Indoleamine 2,3-dioxygenase (IDO) is an intracellular heme-containing enzyme that catalyzes the initial rate-limiting step in tryptophan degradation along the kynurenine pathway. IDO is physiologically expressed by a wide variety of human cells in response to several stimuli and it is known to have a crucial role in the induction of immune tolerance during pregnancy, infections, transplantation, autoimmunity and tumors. IDO-mediated tryptophan degradation results in inhibition of T-cell proliferation, increase of T-cell apoptosis and T-reg induction. Several studies demonstrated that IDO production can induce the increase of Regulatory T-cells (Tregs) directly through the conversion of CD25- into CD25+ T cells, even in acute myeloid leukemia (AML) patients. IDO expression can be considered a novel mechanism of leukemia escape from immune control and its inhibition may represent an antileukemia therapeutic strategy. Aim of our work is to analyze IDO mRNA expression in a cohort of AML patients and to investigate the presence of any significant correlation between IDO expression and standard prognostic factors or clinical outcome. Methods We analyzed a cohort of 68 adult patients aged 18 years or older, who were diagnosed with de novo or secondary AML. IDO mRNA expression was evaluated by Real-Time (RT)-PCR in blood bone marrow and peripheral blood samples at diagnosis. Patients were then retrospectively stratified according to standard risk factors at diagnosis and to IDO mRNA expression levels. Results Median age of analyzed patients was 57 years (range 21-76). Fifty-nine out of 68 patients (87%) had de novo AML, whereas 9 out of 68 patients (13%) had secondary AML. A comprehensive risk assessment was available for 61 patients. Among these 61 patients who were evaluable for risk stratification, 13 patients (21%) resulted to have a favorable risk AML, 30 (49%) had an intermediate risk AML and 17 patients (30%) were stratified as high-risk AML. Sixty out of 68 patients received intensive, standard, induction chemotherapy regimens. The remaining 8 patients were not candidate to receive intensive chemotherapy mainly because of comorbidities. Twenty-three out of 68 patients (34%) were considered eligible for allogeneic stem cells transplantation (alloSCT) as consolidation therapy, after obtaining complete remission with standard chemotherapy. IDO expression in peripheral blood (PB) samples was between 0.07 and 4272.26 (median 5.60). Conversely, IDO expression in bone marrow (BM) samples was between 0.17 and 243.16 (median 1.21). Our data did not establish any significant correlation between IDO expression and leukemia risk factors at diagnosis, in particular cytogenetics, de novo or secondary AML, leukocytosis. Among the 60 patients who received induction chemotherapy, 35 achieved morphological complete remission (CR), 24 did not respond and 1 patient was not evaluable for response. Response to induction chemotherapy was not influenced by IDO mRNA expression levels. Interestingly, among patients undergoing alloSCT, high levels of IDO mRNA expression in PB samples negatively correlated with patients' overall survival. In particular, high IDO expression of more than 10 was associated with worse overall survival after alloSCT even when adjusted by patients' age and disease status at transplant (log rank P=0.02) (Fig.1). With the limitations of the low number of patients, these results from the group of transplanted patients were not likely due to differences in the incidence and severity of graft-versus-host-disease, whereas high IDO mRNA expression level was predictive of increased incidence of relapse. Conclusions This work suggests that IDO mRNA expression levels can be considered as predictive of AML outcome, independently from other risk factors at diagnosis. In our set, higher level of IDO mRNA expression at diagnosis was correlated with worse clinical outcome in patients undergoing alloSCT. Larger studies are warranted in order to establish the real predictive role of IDO mRNA expression in influencing AML outcome. Disclosures Cavo: Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Introduction Monosomy 7 (-7) and interstitial deletions of chromosome 7 (7q-) are among the most recurrent chromosomal aberrations found in myeloid neoplasms. Patients carrying these cytogenetical alterations present a poor overall survival (OS), mainly due to a low sensitivity to standard chemotherapy and a high incidence of relapse. In our study, we aimed to disentangle the biology of patients with -7/7q- and find new candidate therapeutic targets for a disease with such a dismal prognosis, by integrating wide genomic approaches. Methods We collected bone marrow samples from 487 adult patients at diagnosis, treated in 3 institutions: Institute L. & A. Seragnoli (Italy, n = 213), CEMM (Austria, n = 160), University of Michigan (US, n = 114, GSE23452). Three hundred ninety-five samples were analyzed by SNP arrays (Affymetrix™), 51 samples by mass spectrometry (Metabolon™) and 57 samples by GEP (Affymetrix™) approaches. Chi-squared, fisher's exact test and ANOVA were used to test differences in proportion and distributions. False discovery rate, Bonferroni correction, and Welch's correction were calculated whenever appropriate. Results Among the 474 patients with evaluable karyotype, 65 (13.7%) had -7/7q-; 47 (9.9%) had -7, while 18 (3.8%) had 7q-. In our sets, the median age at AML diagnosis was 64 years (21-86) and most of the subjects had a de novo AML (65.1%). WBC count at diagnosis was significantly lower in -7/7q- patients (10.4 vs 35.2 k/mm3 p Within patients tested for FLT3, NPM1 and TP53 mutation at diagnosis, 1/50 among -7/7q- patients vs 59/300 controls harbored FLT3 ITD mutation (350 patients tested, 2% vs 19.7%, p In terms of outcome, -7/7q- AML had a median of overall survival of 10.3 months (95% C.I 5.8-14.8), which accounted for 49.5 months (95% C.I. 40.5-58.4) in other AML cases. GEP data of a cohort of 57 patients (8 with -7/7q- and 49 controls) revealed that 24 genes were under-expressed in -7/7q- AML (with e-4 significance threshold, Figure 1A). Twenty-three out of 24 genes mapped on chromosome 7; one gene, COX17, mapped on chromosome 3 (Figure 1A). COX17 plays a role in the recruitment of copper to mitochondria. By metabolomic analyses, we considered quantitative data of 300 different metabolites in 32 patients (4 with -7/7q- vs 28 controls, 19 patients were excluded because samples at diagnosis were not available). In -7/7q- AML, fatty acids (sphingomyelin, 1-linoleoylglycerol), β-cytrilglutamate and the UDP were overrepresented if compared with other AML cases; on the other hand, galactiol and 1-(1-enyl-palmitoyl)-2-linoleoyl-GPC were underrepresented in -7/7q- patients. Furthermore, -7/7q- AML cells seem to accumulate 3-hydroxy-3-methylglutarate and to have lower levels of 2-Hydroxyglutarate (Figure 1C). With SNP arrays, we considered copy number alterations in 395 patients (52 -7/7q- patients vs 343 controls, Figure 1B). 5q was the most recurrent concurrent deletion, with a minimal common deleted region (MCDR) in q31.3-q33.3. Additionally, 17p (MCDR p11.2 - p13.1), 12p (MCDR p12.3-p13.1), 16q (MCDRs q11.2-q12.1, q21-q22.1 and q24.2-q24.3), 16p (MCDR p11.2) and chromosome 4 (MCDRs q34.1 and q35.2) deletions also co-occurred in -7/7q-, listed per frequencies (Figure 1B). These regions are enriched for genes controlling cell signaling, DNA transcription, post-transcriptional modifications (such as SUMOylation), mRNA splicing and cellular senescence. Conclusions SNP, GEP and metabolomic approaches gave new insights on -7/7q- AML biology, identifying 24 new genes differentially expressed in -7/7q-, and 6 MCDR associated with -7/7q- AML. Deletion of chromosome 16q and 4 were never reported in literature associated to AML. Furthermore, for the first time, we described metabolites associated with -7/7q- AML. These data may represent a useful backbone to search for candidate targets in the setting of one of the most aggressive AML subtypes. Supported by:EHA Non-Clinical Junior Research Fellowship,HARMONY,Fondazione del Monte,FP7-NGS-PTL,AIRC. *MCF and MO equally contributed &CP and GS shared the last authorship Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kralovics:MyeloPro Diagnostics and Research GmbH: Equity Ownership. Soverini:Incyte Biosciences: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Consultancy. Martinelli:Ariad/Incyte: Consultancy; Celgene: Consultancy, Speakers Bureau; Novartis: Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Amgen: Consultancy; Janssen: Consultancy; Abbvie: Consultancy; Roche: Consultancy; Jazz Pharmaceuticals: Consultancy. Cavo:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.

Background: The heterogeneous and poor survival group of Philadelphia negative (Ph-) B-ALL patients (pts) that doesn't have the most recurrent adult rearrangements (BCR-ABL1 t(9;22); TCF3-PBX1 t(1;19); MLL-AF4 t(4;11)) are collectively referred to as "triple negative" (Ph-/-/-) ALL. CRLF2 is frequently altered in adult B-ALL, especially in Ph-like pts (50-75% of cases). Alterations that lead, in the majority of cases, to a CRLF2 overexpression. Adult pts with CRLF2 upregulated have poor outcome and novel strategies are needed to improve it. Aims: Clustering and biological characterization of Ph-/-/- ALL (that represents 61% of adult B-ALL; Roberts KG, J Clin Oncol. 2016), considering CRLF2 overexpression event, in order to define and assess biomarkers in this subgroup to test new drugs. Patients and Methods: Gene Expression Profiling (GEP; HTA 2.0 Affymetrix) were performed on 55 Ph-/-/- ALL, 29 B-ALL Ph+ at different time point of the disease and on 7 mononuclear cell of healthy donors. Data were normalized with the Expression Console Software. Successively we cluster triple negative GEP data with our validated pipeline, based on CRLF2 upregulation and in the top ten-gene list. Ph-/-/- ALL samples were then characterized for the presence of gene fusions, Copy Number Alterations (CNAs) and mutations using different approaches (TruSight Pancancer-Illumina; MLPA and/or dMLPA-MRC-Holland; SNP Array-Affymetrix; 454 Junior-Roche and PCR). Results: Clustering our Ph-/-/- gene expression data using the impact of the 10 single genes in our cohort, we could identify a defined 2-clusters-subdivision (Gr1 and Gr2; Fig 1A). The Gr2 is characterized by CTGF, CRLF2 and CD200 (Gr2=3C-up; Fig 1B) overexpression and it represents 14.1% of all B-ALL. The Gr2 GEP is similar to Ph+ one. Fusion copy number alteration and mutational screening done, detected that 3C-Up group has a higher frequency of Ph-like associated lesions (primarily CRLF2, JAK2, IL7R mutations or deletion), that mainly affect JAK-STAT pathway. Also IKZF1 and EBF1 deletions are significantly associated to Gr2 (p=0.003; p=0.016). RAS pathway genes are highly affected in Gr1. Molecular characterization shed light on a very heterogeneous scenario especially in the group 1, suggesting the need of a more discerning clustering for this group. In spite of the small number of cases is required, preliminary Gr1 subclustering discerns MLLr and ZNF384 gene expression subgroups. Notably p53 pathway is enriched in both groups but with different deregulated genes: CHEK2 is upregulated in the group1 and CDK6 in the Gr2. CRLF2 and CD200 immunoblotting and CD200 immunohistochemistry preliminary analyses suggest that protein expression of CRFL2 and CD200 are higher in Gr2 in comparison to Gr1. Conclusions: we identified a new signature, related to CRLF2 high expression, to classify Ph-/-/- ALL B-based on 10 genes. 3C-up represents 14.1% of all B-ALL and it is characterized by a) high co-expression of three main genes: CRLF2, CTGF and CD200; b) IKZF1 deletion; c) JAK-STAT pathway mutations/fusions/deletions. Gr1 represents 46.9% of all B-ALL. Gr2 GEP similarity to Ph+ one, suggests that this Gr2 could contain Ph-like pts. This new Ph-/-/- subclassification identify new potential therapeutic targets with available drug (α-CTGF, α-CD200, CDK2, CHK2 and CDK6 inhibitors; tyrosine kinase inhibitors already effective on Ph+ and Ph-like) to test. Supported by: ELN, AIL, AIRC, project Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project, HARMONY project, Fondazione del Monte BO e RA project. Figure. Figure. Disclosures Cavo: Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Martinelli:Novartis: Speakers Bureau; Abbvie: Consultancy; Jazz Pharmaceuticals: Consultancy; Janssen: Consultancy; Pfizer: Consultancy, Speakers Bureau; Roche: Consultancy; Celgene: Consultancy, Speakers Bureau; Ariad/Incyte: Consultancy; Amgen: Consultancy.

Introduction Relapsed/refractory (R/R) AML patients continue to be a formidable clinical challenge, mainly in consideration of associated very poor outcome, with a median overall survival (OS) of less than 12 months. SCT represents the only curative option for these patients. Although, there is no standard-of-care approach which may serve as a bridge to SCT. Our study aims to investigate the effectiveness of MEC regimen as a rescue therapy for R/R AML patients by specifically addressing the CR rate, including minimal residual disease (MRD) negativity, the number of patients who subsequently underwent SCT and the presence of predictive factors of response. Methods Fifty-five consecutive adult AML patients were treated with MEC regimen in our Institution. In patients under 66 years old, we administered mitoxantrone 6 mg/sqm/die from day 1 to day 6, etoposide 100 mg/sqm/die from day 1 to day 6 and cytarabine 1000mg/sqm/die from day 1 to day 6, whereas in patients over 66 years old, the treatment schedule was reduced to 4 consecutive days. Data were retrospectively collected by using RedCap in accordance with Helsinki declaration and GCP. We used Kaplan-Meyer to estimate survival, and log rank to test differences in survival. Chi-squared, fisher's exact test and linear-by-linear correlation were used to test differences in proportions and distributions. Response was defined in accordance with 2017 ELN recommendations. CTCAE 4.03 was used to grade adverse events. MRD was assessed with WT1 or specific fusion transcripts. Results Fifty-five patients received MEC from 2008 to 2018. Age at diagnosis ranged from 17 to 72 years, with a median age of 51 years. Our set was enriched for high-risk patients. Interestingly, twenty percent of patients harbored FLT3-ITD at diagnosis (table 1). Two main groups were included: resistant AML, 28/55 patients (50,9%), and relapsed AML, 27/55 patients (49,1%). At induction, almost half of patients received "3+7" (n=25, 45,5%), while fludarabine-based regimens were administered to 14 patients (25,5%). In our set, after MEC median duration of hospitalization was 30 days (14-78); PMN >500/mm3 was reached after 26 days (range 18-67). Fever and febrile neutropenia was the most recurrent adverse events (AE). AEs were low in grade; out of 80 graded AEs, 38 (47,5%) were grade 2, 27 (33,8%) were grade 3, 9 (11,3%) were grade 4 and only 3 events resulted in death (3,8%). E. coli was the most recurrent cause of infection (10 cases). Overall, 25/55 patients (45,5%) achieved a complete remission (CR) after one course of MEC chemotherapy. Twelve patients (21.9%) achieved MRD negativity and 13 patients (23,6%) obtained an MRD+ CR or had no MRD test. Six patients (10,9%) had a partial response (PR) and 1 patient (1,8%)had hematological improvement (HI). Four patients (7.3%) died during post-MEC aplastic phase. Disease risk at diagnosis and R/R status did not influence the chance to obtain CR (figure 1 A). In 12 patients, a second MEC was administered. Four out of 12 patient improved their response with the 2nd MEC (2 patients obtained MRD - from MRD+ CR, 1 patient obtained PR and 1 patients obtained CR from hematological improvement). MEC was an effective bridge to SCT, 32/55 patients (58,2%, figure 1 B), received SCT; 15/32 patients (46,9%) received SCT directly after the 1st course of MEC, 9/32 patients (28,1%) after the 2nd course of MEC and 2 patients (6,3%) after an additional course of post-remission chemotherapy. Of note, only 6 patients (18,8%), who were not responsive to MEC, underwent SCT after an alternative rescue therapy. Median overall survival (OS) from MEC was 455 days (95% C.I. 307-602 days.); 1-year OS, 3-year OS and 5-years OS were 57,9%, 33,2% and 23,1%, respectively (std. error ± 0,067). Patients who responded to MEC (CR MRD+ or CR MRD- after 1 or 2 courses) had better OS than non-responders (median OS 1389 vs 160 days, p=.003). Stepwise multiple logistic regression analysis with COX-HR model established that pre-MEC R/R status, diagnosis class risk, response to one or two courses of MEC, and SCT were independent predictors of survival in the optimal model. Conclusions Taken together, our data indicate that MEC is an effective salvage regimen with affordable toxicity, and gives a high chance to obtain CR. MEC is particularly useful as a bridge to SCT, and has to be considered as a rescue therapy whenever a clinical trial is not available. *GM and AT equally contributed Disclosures Martinelli: Ariad/incyte: Consultancy; Pfizer: Consultancy; Celgene: Consultancy; Amgen: Consultancy; Janssen: Consultancy; Roche: Consultancy. Cavo:Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Adaptive Biotechnologies: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Background: Although there has been remarkable progress, there is a need to improve the molecular dissection of subtypes, identifying genetic alterations that predict the risk of treatment failure and developing novel and targeted therapies. B-ALL patients (pts) that do not have the most recurrent adult rearrangements (BCR-ABL1 t(9;22); TCF3-PBX1 t(1;19); MLL-AF4 t(4;11)) are collectively referred to as “triple negative” (Ph-/-/-) ALL. Aims: Biologic characterization of Ph-/-/- ALL considering CRLF2 overexpression event (that represents near to 57% of B-ALL; Roberts KG, J Clin Oncol 2016), in in order to define and assess biomarkers in this subgroup to test new drugs. Patients and Methods: Gene Expression Profiling (GEP; HTA 2.0 Affymetrix) was performed on 51 Ph-/-/- ALL, 25 B-ALL Ph+ at different time point of the disease and on 7 mononuclear cell of healthy donors. Data were normalized and analyzed with the Expression Console and the Transcriptome Analysis Console (TAC) Software (Affymetrix). Successively we cluster triple-negative GEP data with our validated pipeline, based in a top ten gene list. Results: Comparing GEP of Ph-/-/- and Ph+ to donors we found some shared top upreg genes to focus on (e.g., EBF1, CD19, BLNK, PDLIM1, PXDN, NAV1, CTGF, LEF1, CD200, CRLF2). In triple-negative ALL GEP top upreg gene analysis we identify a well-defined 2-clusters-subdivision (Gr1 and Gr2). Furthermore, a third group , in the Gr1, can be identified by the algorithm without ambiguous assignments. The Gr2 is characterized by CTGF, CRLF2 and CD200 overexpression and it represents 11.3% of all B-ALL. Two groups t-test has been performed between Ph+ and the isolated subgroups of Ph- to determine the similarity of these two groups to Ph+. The Gr2 GEP is similar to Ph+ one. Conclusions: We identified a new signature, related to CRLF2 high expression, to classify Ph-/-/- ALL B-based on 10 genes. Gr2 represents 11.3% of all B-ALL and it is characterized by high expression of three main genes: CRLF2, CTGF and CD200. Gr1 represents 46% of all B-ALL. Gr2 GEP similarity to Ph+ one, suggests that this Gr2 could contain Ph-like pts. This new Ph-/-/- subclassification identifies new potential therapeutic targets to test as a single agent or in combination. ELN, AIL, AIRC, project Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project, HARMONY project, Fondazione del Monte BO e RA project. Citation Format: Anna Ferrari, Silvia Vitali, Valentina Robustelli, Andrea Ghelli Luserna Di Rorà, Simona Righi, Cristina Papayannidis, Giovanni Marconi, Enrica Imbrogno, Alessandra Santoro, J M. Hernández-Rivas, Carmen Baldazzi, Maria Chiara Abbenante, Stefania Paolini, Nicoletta Testoni, Gastone Castellani, Elena Sabattini, Michele Cavo, Daniel Remondini, Giovanni Martinelli. Gene expression profiling identifies new adult "triple-negative" acute lymphoblastic leukemia (ALL) subgroups [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2951.

Background: The reported TP53 mutation rate in AML is relatively low (7.5-9%, TCGA) and predict a poor prognosis. In 2017 European LeukemiaNet recommended for AML to add TP53 mutations (muts) in the risk stratification. Specific chromosomal aneuploidies are closely correlated with each other and with presence of TP53 muts. Aims: Considering that TP53 mut AML pts have HRisk and no target therapy, we would identify genes/pathways that are mainly CNA-affected (Copy Number Alteration) in the mut TP53 group compared to the wt one. Patients and Methods: 358 adult AML pts were screened for TP53 muts. 219/358 samples were genotyped with SNP arrays. CNA analyses were performed using two software to confirm or integrate karyotype data. Fisher's exact test and pathway enrichment analyses were performed. Results: We detected TP53 muts in 52/358 (14.5%) pts. Mostly (34/52) of the TP53 mut pts (65.4%) had complex karyotype. TP53 alterations were significantly associated with poor outcome (OS and EFS p Citation Format: Anna Ferrari, Eugenio Fonzi, Andrea Ghelli Luserna Di Rorà, Maria Chiara Fontana, Marco Manfrini, Carmen Baldazzi, Cristina Papayannidis, Vincenza Solli, Antonella Padella, Giovanni Marconi, Stefania Paolini, Valentina Robustelli, Enrica Imbrogno, Eugenia Franchini, Perricone Margherita, Maria Chiara Abbenante, Giorgia Simonetti, Nicoletta Testoni, Emanuela Ottaviani, Michele Cavo, Giovanni Martinelli. Distinct pattern of alterations in TP53 mutated/deleted and wild-type high risk acute myeloid leukemia (AML) patients: Identification of new "targetable" genes/pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 656.

Background: Antigen presentation is crucial for tumor immune response and MHC-I plays a key role. Solid and hematological tumors with high load of newly tumor associated antigens develop evading immune surveillance. A subset of patients with acute myeloid leukemia (AML), precisely ones with complex karyotype and TP53 alterations have a high load of mutation or genomic aberrations. In our study, we screened the entire genome of a large set of newly diagnosed AML to discover if MHC I antigen presentation is altered in the set of AML with a high neo-antigen load. Methods: We screened 300 samples of AML bone marrow at diagnosis from 3 hematological Institutions. We analyzed 636 single nucleotide polymorphism (SNP) arrays to detect karyotype aberration at genomic level; germline SNP arrays were performed in 114/300 patients, the remaining 186/300 were normalized with a reference pool of 222 healthy donors. Of the 300 patients, we collected clinical data and we tested TP53, IDH1, IDH2, FLT3, NPM1, DNMT3A and RUNX1 mutations according ordinary practice. Genes in MHC class I antigen presentation pathway were selected basing on Reactome. We considered MHC-I antigen presentation altered if pathway's alteration rate (altered genes in a pathway/gene in a pathway ratio) scored in the top 25% between all pathways. Results: Twenty out of 300 patients (6.7%) had loss of genes in MHC I antigen presentation (group A). Patients had alterations in one or more genes; overall, 53 genes were founded altered in our set. Patients in group A did not have differences in age or in white blood count at diagnosis compared with other patients in our set (group B). Group A was enriched for patients with of complex karyotype (14/20 patients, p Conclusion: We demonstrated that patients with MHCI alterations have a high chance to be TP53 mutated or to have complex karyotype; patients with complex karyotype have a high chance to harbor concomitant alterations in MHC I antigen presentation. In the set of AML with high burden of aberrations, MHC I alterations could be permissive for leukemia development toward immune escape. Our approach is a good candidate for further testing inside clinical trials to identify a cohort of patients that are unlikely to respond to immune check-point inhibitors within patients with larger sets of patients with high burden of alterations. Acknowledgements: ELN, AIL, AIRC, Regione-Università 2010-12, FP7 NGS-PTL, HARMONY, Fondazione del Monte BO e RA. Citation Format: Giovanni Marconi, Maria Chiara Fontana, Cristina Papayannidis, Mariachiara Abbenante, Antonella Padella, Giorgia Simonetti, Michele Cavo, Giovanni Martinelli. Antigen presentation by MHC-I molecule and immune escape in acute myeloid leukemia with high burden of genomic aberrations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3788.

We report a case of a 65-year old patient affected by neurofibromatosis 1, documented by the presence of germ-line mutation on the NF1 gene, who developed various hyperproliferative malignant and benign diseases. He was brought to our attention for the diagnosis of acute myeloid leukemia revealed by major fatigue and dyspnea. The disease characteristics at diagnosis were hyperleukocytosis and complex karyotype with the inversion of the chromosome 16, classifying as a high-risk leukemia. The association between leukemia and neurofibromatosis 1 is controversial and needs to be further investigated. Nevertheless, such patients present a wide number of comorbidities that make therapeutic strategies most difficult.

// Elisa Zuffa 1 , Eugenia Franchini 1 , Cristina Papayannidis 1 , Carmen Baldazzi 1 , Giorgia Simonetti 1 , Nicoletta Testoni 1 , Maria Chiara Abbenante 1 , Stefania Paolini 1 , Chiara Sartor 1 , Sarah Parisi 1 , Giovanni Marconi 1 , Federica Cattina 2 , Maria Teresa Bochicchio 1 , Claudia Venturi 1 , Emanuela Ottaviani 1 , Michele Cavo 1 , Giovanni Martinelli 1 1 “Seragnoli” Institute of Hematology, Sant'Orsola-Malpighi University Hospital, Bologna, Italy 2 Bone Marrow Transplant Unit, University of Brescia, Brescia, Italy Correspondence to: Cristina Papayannidis, e-mail: cristina.papayannidis@unibo.it Keywords: AML, FLT3, ultra-deep sequencing, clonal evolution, minimal residual disease Received: April 21, 2015 Accepted: August 24, 2015 Published: September 05, 2015 ABSTRACT FLT3 internal tandem duplication (ITD), one of the most frequent mutations in Acute Myeloid Leukemia (AML), is reported to be an unstable marker, as it can evolve from FLT3 ITD- to ITD+ during the disease course. A single-gene sensitive mutational screening approach may be helpful for better clarifying the exact timing of mutation occurrence, especially when FLT3 ITD appears to occur late, at disease progression. We developed an amplicon-based ultra-deep-sequencing (UDS) approach for FLT3 mutational screening. We exploited this highly sensitive technology for the retrospective screening of diagnosis, relapse and follow-up samples of 5 out of 256 cytogenetically normal (CN-) AML who were FLT3 wild-type at presentation, but tested ITD+ at relapse or disease progression. Our study revealed that all patients carried a small ITD+ clone at diagnosis, which was undetectable by routine analysis (0,2–2% abundance). The dynamics of ITD+ clones from diagnosis to disease progression, assessed by UDS, reflected clonal evolution under treatment pressure. UDS appears as a valuable tool for FLT3 mutational screening and for the assessment of minimal residual disease (MRD) during follow-up, by detecting small ITD+ clones that may survive chemotherapy, evolve over time and definitely worsen the prognosis of CN-AML patients.