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Background: Fusions (Fs) are major molecular biological abnormalities in acute leukemias (ALs), and all well-known Fs in leukemias are founder variations are routinely used as molecular markers for the diagnosis, classification, risk stratification, and targeted therapy but there is a considerable part of ALs that are not screened for other known/unknown transcripts. For example in B-Other B Acute Lymphoblastic Leukemia (B-ALL) [Ph-/-/-; negative for t(9;22); t(1;19); t(4;11); 61% of adult B-ALL], many chimeric genes have been identified leading to a refined classification of B-ALL and to, in some cases, tailored therapies. At this point, a RNA-seq approach is needed but challenging for many aspects, among them a not standardized and very heterogeneous F data analysis. Aims: We developed and validated our integrated pipeline in order to assess targetable biomarkers and to better classify patients (pts). Methods: we performed 1385 gene RNAseq (Illumina) of 224 adult AL samples (112 Ph-/-/-, 41 Ph+, 6 t(1;19), 16 T-LBL/T-ALL and 49 AML). Starting from Ph-/-/-, we developed a combined 4 tool analysis that is further implemented with a filtering strategy with a specific ALs fusion literature filter (Patent PCTEP2021-065692 and 749 ALL Fs database copyright) (Fig.1A). We validate our strategy using: RT-PCR, FISH SNP Arrays; MLPA and total RNA-seq. Results: From 3022 candidate Fs, we retained 160 of them (5.3%; excluding WHO canonical Fs) not otherwise detected, in 120 pts with a high Fs rate in T-LBL/T-ALL, Ph-/-/- and Ph+ (62.5%, 62.2% and 61% respectively) denoting that ALs are not deeply characterized (Fig 1B). The lower rate was found in AML (26.5%), but we were able to identify new ETV6 rearrangement (r) and a FISH cryptic F in a 46, XY pt (TBL1XR1-MECOM). We validated 98 Fs that have been already reported in the literature and 43 novel F transcripts. We obtained a validation rate of 81%. The majority of fused samples (65.2%) had only one detectable F while a smaller group was characterized by multiple Fs. Many of these Fs were previously described in B/T-ALL and AML (e.g. KMT2A-MLLT1, ABL1/2-RCSD1, IGH-MYC, NUP214-SET, ETV6-MECOM). In our bigger sub-cohort (Ph-/-/-), 44 Fs out of 109 (40.3%) were never been reported in Ph- ALL cases. In 15 Ph-like pts, we identified and validate 11 new transcripts. Ph-/-/- F detection help to sub-classify our fused pts in Ph-/-/- subgroups (ZNF384r-11.8%; Ph-like-19.1%; DUX4r- 4.4%; HLFr, MLLr and BCL2/MYC in 2.9%; MEF2Dr-1.5%). Conclusions: we identified pivotal transcripts in all ALs and an unexpected high rate of secondary Fs in adult ALs subgroups (52.4%) that are not characterized with conventional diagnostic methods. The use of an NGS approach and a powerful pipeline permit us to detect Fs useful for a better classification, prognostic identification (e.g. TBL1XR1-MECOM) and in some cases to find targetable Fs (e.g. ABL1-2/RCSD1, NUMA1-CSF1R, ZMYM2-FLT3). Supported by: L3P2505. Citation Format: Anna Ferrari, Silvia Vitali, Eugenio Fonzi, Andrea Ghelli Luserna Di Rora, Chiara Domizio, Cristina Papayannidis, Maria Teresa Bochicchio, Giovanni Marconi, Daniele Dall'Olio, Michela Tebaldi, Michela Rondoni, Barbara Giannini, Fabio Giglio, Crescenza Pasciolla, Monica Fumagalli, Sara Galimberti, Gastone Castellani, Daniel Remondini, Giorgia Simonetti, Giovanni Martinelli. Fusion landscape in acute leukemias: A submerged world of not routinely characterized transcripts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6163.

Currently, the role of autophagy in acute lymphoblastic leukemia (ALL) is not fully understood however it has been linked to drug resistance. Several autophagy modulators have proven their efficacy in single agents or as chemo-sensitizer in ALL. Recently, the 3rd generation BCR-ABL1 inhibitor, ponatinib, has been showed to promote autophagy in chronic myeloid leukemia (CML) cells and the combination between ponatinib and hydroxychloroquine (HCQ) has proven its efficacy in neuroblastoma cells. Here, we tested the efficacy of combining different autophagy modulators (ponatinib, HCQ and rapamycin) against ALL cell lines and primary leukemic ALL cells. Firstly, we established the IC50 values of a panel of Philadelphia (Ph)-positive/negative B-/T-ALL cell lines (n=6) treated with ponatinib, HCQ and rapamycin in single agent. Then, using sub-toxic concentrations, we evaluated the efficacy of the combinations HCQ+Ponatinib and HCQ+Rapamycin in the reduction of the cell viability after 24, 48 and 72 hours of simultaneous treatment. Combination index analyses showed a synergic time and dosage-dependent reduction of the cell viability in all the cell lines treated with HCQ+Ponatinib. The combination HCQ+Rapamycin resulted synergic only in Ph-negative ALL cell lines. These results were confirmed by the significant induction of apoptosis (Annexin V and/or Caspase-3 staining) in the samples treated with HCQ+Ponatinib. The two combinations also significantly reduced the proliferation capacity of ALL cell lines in comparison with controls and single treatments. Moreover, the efficacy of the two combinations was confirmed on primary leukemic blasts isolated form adult ALL patients(n=6). Indeed, a significant induction of apoptosis was seen in samples treated with the two combinations in comparison with single treatments. To deeper understand the mechanism of action of the combinations we evaluated LC3A/B expression by flow cytometry. Interestingly, the expression of LC3A/B was modified heterogeneously among the different ALL cell lines and did not correlate with the response in term of cell viability or induction of apoptosis. Light microscopy analyses showed a significant increment in the number and diameter of autophagy vesicles in both ALL cell lines and primary leukemic ALL cells treated with HCQ+Ponatinib in comparison with single treatments. Interstingly, no significant effect on autophagy vesicles number or diameter was seen in the samples treated with HCQ+Rapamycin in comparison with single treatments. In conclusion, our findings show that the identified combinations effectively inhibit cell viability and induce apoptosis in Ph-positive/negative B-/T-ALL cells, suggesting the fundamental role of autophagy regulation in ALL survival. Citation Format: Andrea Ghelli Luserna Di Rora, Eugenio Fonzi, Lorenzo Ledda, Roberta Napolitano, Anna Ferrari, Antonella Padella, Martina Ghetti, Maria Teresa Bochicchio, Mouna Jandoubi, Cristina Mazzotti, Matteo Paganelli, Claudio Cerchione, Giovanni Marconi, Gerardo Musuraca, Giovanni Martinelli, Giorgia Simonetti. Targeting autophagy in acute lymphoblastic leukemia: Synergism between ponatinib, hydroxychloroquine and rapamycin [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3001.

Background: The genetically heterogeneous and poor survival group of Philadelphia negative (Ph-) B-ALL group that doesn’t have the most recurrent adult rearrangements (t(9;22); t(1;19); t(4;11)) is collectively referred to as “triple negative” (Ph-/-/-). 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: Understand the genomic background of all Ph-/-/- ALL and subsequently clustering Ph-/-/- considering CRLF2 overexpression event, in order to define new biomarkers in these subgroups. Pts and Methods: Ph-/-/- pts were sequenced by WES (44pts/93 samples) and 92 B-Other pts were analyzed with NGS target seq (NTS) to validate the 8 most mutated genes (Fig1A). GEP were performed on 55 Ph-/-/-, 29 B-ALL Ph+ and on 7 donors. We cluster triple negative GEP data with our validated pipeline, based on CRLF2 upregulation and in a 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 (Pancancer and/or RNASeq; dMLPA-MRC-Holland; SNP Array; 454 Junior and PCR). Results: WES analysis identified some recurrent mutated genes (NRAS, PAX5, KRAS, PTPN11, EP400, JAK2, TP53, CREBBP) previously reported to be involved in B-ALL, confirming the pivotal roles of these gene in ALL. For the first time we described a little known gene PKHD1L1 as highly mutated (7.2%). TP53 was the most mutated gene (Fig1A) and that between these gene is the only one associated to a worst OS (p=0.004). Combining our new Ph-/-/- GEP clustering, WES, NTS, Fusion and CNA results we identify a defined 2-clusters-subdivision (Gr1 and Gr2). The Gr2 (14.1% of all B-ALL) is characterized by CTGF, CRLF2 and CD200 (Gr2=3C-up; Fig 1B) co-overexpression. The Gr2 GEP is similar to Ph+ one (Fig1C). Gr1 represents 46.9% of all B-ALL. Fusion, CNA and mutational screening done, detected that 3C-Up group has a higher frequency of Ph-like associated lesions (primarily CRLF2, JAK2, IL7R mut or del), 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. We also validated not previously described fusions. Notably p53 pathway is enriched in both groups but with different deregulated genes: CHEK2 is upreg in the group1 and CDK6 in the Gr2. Preliminary data seems to confirm an higher effect on cell viability of a TKI on Gr2 primary cell pt (vs Gr1 pt). Conclusions: we identified a new signature, related to CRLF2 high expression, to classify Ph-/-/- ALL B. This new subclassification identifies new potential therapeutic targets with available drugs (α-CTGF, α-CD200, CDK2, CHK2 and CDK6 inhibitors; TKIs already effective on Ph+ and Ph-like) to test. Citation Format: Anna Ferrari, Silvia Vitali, Valentina Robustelli, Andrea Ghelli Luserna di Rorà, Eugenio Fonzi, Simona Righi, Carmen Baldazzi, Michela Tebaldi, Samanta Salvi, Cristina Papayannidis, Giovanni Marconi, Mariachiara Fontana, Enrica Imbrogno, Antonella Padella, Giorgia Simonetti, Alessandra Santoro, Jesus María Hernández-Rivas, Maria Teresa Bochicchio, Fabiana Mammoli, Benedetta Giannini, Nicoletta Testoni, Daniele Calistri, Massimiliano Bonafè, Gastone Castellani, Elena Sabattini, Daniel Remondini, Giovanni Martinelli. “3c-up” a new adult Philadelphia negative acute lymphoblastic leukemia subgroup: Novel molecular markers [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 2140.

Introduction: PPM1D (wild-type p53-inducible protein phosphatase WIP1) is a member of the PP2C family serine/threonine phosphatase involved in negative regulation of cell stress response pathways, leading to suppression of p53 after stress. To restore p53 function through MDM2 inhibition (Nutlin-3a) is a promising approach in AML. Promising results of the combination of MDM2 inhibitors and WIP1 inhibitor (WIP1i), obtained in many preclinical studies of solid tumors, paved the way for their application in AML. We investigated whether the inhibition of WIP1 (GSK2830371) could sensitize AML cell lines and primary cells to Nutlin-3a (Nut-3a) in order to obtain a novel therapeutic strategy for AML patients, based on restoration of p53 activity. Methods: In vitro viability (by WST-1 reagent) and Annexin-V/PI apoptosis assay were performed on a panel of TP53-wt AML cells (MOLM-13, MV-4-11 and OCI-AML3), on TP53-mutated NOMO-1 AML cells and on primary AML samples. Gene expression profile (GEP) and Western Blot (WB) analyses were performed on MV-4-11 and NOMO-1 after 16h. Results: Combined inhibition of increasing dosage of Nut-3a (0.5 to 5 uM) and WIP1i (5 to 20 uM) synergistically reduces TP53-wt AML cells viability, while NOMO-1 resulted to be insensitive to the combination. The combination index analyses showed a synergistically effect of the combination of both compounds on TP53-wt AML cells. Annexin V/PI staining showed that WIP1i sensitizes TP53-wt AML cell lines and primary samples to Nut-3a-induced apoptosis, when compared with single treatment. No effect was seen in NOMO-1. GEP demonstrated that MV-4-11 cells exhibits a major response to drug combination with an upregulation of cell cycle control genes, a downregulation of DNA repair machinery genes, upregulation of MDM2 and of TP53-downstream genes (eg.CDKN1A), confirming the activation of p53 pathway. NOMO-1 cells showed upregulation of resiliency-mechanism and confirmed insensitivity to both treatment. WB analysis confirmed GEP data showing an increased expression of p53 and p21 in wt-TP53 cell line after 16h of combined-treatment. Conclusions: We identified a novel synergistic drug combination between Nut-3a and WIP1i that induces apoptosis in AML cell lines and primary samples. GEP and WB of TP53-wt MV-4-11 and TP53-mutated NOMO-1 cells showed mechanisms underlying drug sensitivity and resistance giving novel insights on potential markers of response and novel drugable targets. In vivo studies are needed to confirm these preclinical data. Supported by: AIRC, FP7-NGS-PTL, Fondazione del Monte, HARMONY. Citation Format: Maria Chiara Fontana, Jacopo Nanni, Giovanni Marconi, Martina Pazzaglia, Matteo Bocconcelli, Antonella Padella, Simona Soverini, Ilaria Iacobucci, Cristina Papayannidis, Anna Ferrari, Maria Teresa Bochicchio, Enrica Imbrogno, Michele Cavo, Andrea Ghelli Luserna di Rora, Giorgia Simonetti, Giovanni Martinelli. Pharmacological inhibition of WIP1 by GSK2830371 sensitizes AML cells to MDM2 inhibitor Nutlin-3a [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 2964.

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.

Genomic and functional alterations of enzymatic activity drive cancer metabolic reprogramming along with mutations of tumor suppressors and oncogenes. IDH1/2 lesions represent a paradigmatic example in acute myeloid leukemia (AML). The study aimed to stratify AML patients based on their metabolic landscape and to map potential connections between their metabolic and genomic profile. The genomic landscape of 166 AML patients was obtained by whole exome sequencing. Variants were called by MuTect and Varscan2. Six additional cases were analyzed by targeted sequencing (SOPHiA GENETICS). Metabolites were quantified by mass spectrometry of bone marrow cells (35 CD34+ and 15 CD33+ AML from the above-mentioned cohort) and compared with CD34+ cord blood and CD33+ healthy blood cells (n=21 each, Metabolon) by Welch's t-test. In AML, 17% of somatic variants targeted metabolism-related genes: 38% were enzymes (according to Recon2), including 3% of electron transport chain genes encoded by mitochondrial DNA (COX1-3, ND1-6), 3% were AML-related genes with a known involvement in cell metabolism (e.g. IDH1/2, MYC, KRAS) and 59% were metabolic regulators, defined by gene ontology annotation. Ninety-one% of patients carried at least one mutation in a metabolism-related gene, with 43% of variants rated as damaging. The most represented pathways were lipid, carbohydrate, nucleotide (AK9, H6PD), amino acids (IDO2) and glucose metabolism (PKM, HK3). Principal component analysis of metabolic data showed a distinct profile between AML and healthy cells, with a predictive accuracy of 86% and 95% for CD34+ and CD33+ cells, respectively. Conversely, few differences were observed between CD34+ and CD33+ AML. Unsupervised hierarchical clustering clearly defined 3 AML clusters (C1-3). Moreover, 3 subgroups could be identified in C3 without ambiguous assignments. C1 was enriched for NPM1-mutated (mut) cases (83%, 33%, 27% in C1, C2 and C3, respectively, p=0.03). NPM1-mutated AML were distinguished by a 12-metabolites signature. They showed increased levels of spermidine, cytidine 2′ or 3′-monophosphate (P), thymidine 3′-monoP, uridine-2',3'-cyclic monoP and decrease of inosine 5'-monoP, suggesting altered polyamine, pyrimidine and purine metabolism. Moreover, NPM1-mut AML had reduced levels of intermediates involved in acyl carnitine, lysophospholipid, phosphatidylethanolamine and sphingolipid metabolism. Overall, mutations of metabolism-related genes are common in AML. We defined a metabolic-based classification of AML and identified a new metabolic signature based on 12 metabolites that distinguish NPM1-mut AML from wild-type cases and healthy CD34+/CD33+ cells. Major alterations in the nucleotide and polyamine pathways suggest novel potential therapeutic approaches. Supported by: EHA research fellowship award, AIRC, FP7-NGS-PTL, Fondazione del Monte. Citation Format: Giorgia Simonetti, Antonella Padella, Eugenio Fonzi, Martina Pazzaglia, Margherita Perricone, Maria Chiara Fontana, Samantha Bruno, Maria Teresa Bochicchio, Eugenia Franchini, Jacopo Nanni, Giovanni Marconi, Italo F. do Valle, Rossella De Tommaso, Anna Ferrari, Enrica Imbrogno, Claudio Cerchione, Cristina Papayannidis, Emanuela Ottaviani, Daniel Remondini, Giovanni Martinelli. Metabolic profiling defines a new characterization of acute myeloid leukemia and identifies NPM1-mutated cases as a distinct subgroup [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 5279.

Introduction: The wild-type (wt) p53-inducible protein phosphatase WIP1 (gene PPM1D), is frequently mutated in solid tumors and in blood of healthy-individuals, but its role is still unknown in Acute Myeloid Leukemia (AML). WIP1 suppresses p53 after cell-stress. The aim of this study is to investigate whether the inhibition of WIP1 (GSK2830371) could increase the sensitivity to MDM2 inhibitors (Nutlin-3a) in order to obtain a novel therapeutic strategy for AML patients restoring p53 activity. Methods: Whole Exome Sequencing (WES) and targeted Next Generation Sequencing (NGS) have been performed to investigate the mutational state of PPM1D in newly diagnosed AML patients. We performed in vitro WST-1 cell-viability and Annexin V-PI apoptosis assays in AML cell lines (MOLM-13, MV-4-11) and AML primary cells. Results: WIP1i and Nut-3a reduced the cell viability as single agent in all the treated cells in a time and dosage-dependent manner. The IC50 values in TP53-wt cells showed that MOLM-13 (10 υM) are more sensitive than MV-4-11 (20 υM) to WIP1i. Combination index analyses confirmed that WIP1i and Nut-3a had a synergistic effect (C.I. Conclusions: This pre-clinical study allows us to affirm for the first time that WIP1i potentiates the sensitivity to Nut-3a in AML. Further studies on TP53-mutated and wt cells will help to confirm these preliminary data, Gene Expression Profile and WB will identify the proteins playing a relevant role in the biological alterations induced by WIP1i. 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. Citation Format: Maria Chiara Fontana, Jacopo Nanni, Giorgia Simonetti, Andrea Ghelli Luserna di Rorà, Antonella Padella, Samantha Bruno, Cristina Papayannidis, Giovanni Marconi, Mariachiara Abbenante, Michele Cavo, Giovanni Martinelli. Pharmacological inhibition of WIP1 sensitizes AML cells to MDM2 inhibitors [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 1872.

BACKGROUND: Apoptotic regulation involves several actors which can have a positive and negative effect and may represent a target for personalized therapy. Nowadays, BCL-2 inhibitors have successfully passed preliminary phases of clinical experimentation, however the involvement of other essential proteins could represent a resistance mechanism to BCL-2 inhibitors and reveal novel therapeutic targets in acute myeloid leukemia (AML). Our study aim to investigate the expression of anti-apoptotic genes in AML patients. METHODS: We performed Human Transcriptome Array 2.0 (Affymetrix) in a cohort of 59 newly diagnosed AML. K-means clustering has been used to categorize patients in different cluster. A pool of 7 healthy donors was used to normalize expression data. Survival analysis was conducted with Kaplan-Meyer method and differences in survival were assessed with Log-Rank test. RESULTS: BCL-2, MCL-1 and BCL2L1 gene expression values in 59 AML patients allowed us to establish 5 clusters (Fig. 1a): cluster 1 with the overexpression of MCL1, cluster 2 with low expression of all analyzed genes; cluster 3 with overexpression of BCL2L1, cluster 4 with overexpression of BCL-2 and cluster 5 with high values of expression of all 3 genes. Clusters 2 and 5 included the majority of patients (40/59, 68%). Furthermore, the 3-D Scatter Plot allowed us to observe how patients out of the central cloud (Clusters 2-5) always presented an overexpression of one out of the three anti-apoptotic genes. Moreover, when BCL-2 level is low (Cluster 1-2-3), MCL-1 and BCL2L1 levels are high, respectively. In term of overall survival (OS), there were no statistically significant difference between Cluster 1-3-4, where one different anti-apoptotic gene is always overexpressed. Finally, analyzing the impact of BCL-2 expression on OS in a cohort of 36 young patients treated with chemotherapy, those with high expression of the three antiapoptotic genes had worse outcome (p=0.07). CONCLUSION: BCL-2, MCL-1 and BCL2L1 expressions are quite balanced in AML patients evaluated. One out of the three genes is often overexpressed, confirming the key role of anti-apoptotic genes in leukemogenesis. Even though BCL-2 expression influence patients chemosensitivity, MCL-1 and BCL2L1 overexpression has been documented when BCL-2 is normally or downregulated. In conclusion, we suppose that BCL-2 inhibitors resistance may be caused by MCL-1 and BCL2L1 overexpression. Finally, a combined approach should be realized to switch-off leukemic anti-apoptotic mechanisms. Gene expression profile could provide a synthetic lethal instrument to choose the proper BH3-mimetic drug or combination. 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. Citation Format: Jacopo Nanni, Giorgia Simonetti, Samantha Bruno, Giovanni Marconi, Maria Chiara Fontana, Cristina Papayannidis, Maria Chiara Abbenante, Lorenzo Montanaro, Michele Cavo, Giovanni Martinelli. Antiapoptotic gene expression signature reveals a combined talk to prevent apoptosis: A model to choose the proper BH-3 mimetic drug [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 2539.

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.

Functional profiling is an emerging trend in precision medicine. Manual laboratory methods proved to be effective in predicting the response to drug treatments both in patients stratified according to genetic profiling and in patients without any specific molecular aberration. In this work, we validated the predictive power of an innovative functional profiling platform, based on lab-on-a-chip technology, which allows testing the response of live tumor cells exposed to anticancer drugs in a fully-automated process, requiring only a Ficoll stratification as a manual step. 15 AML patients (Age: 40% 70 years; Gender: 40% Female; ELN Classification: 20% Favorable, 26% Intermediate-I, 33% Intermediate-II, 14% Adverse; AML stage: 53% De novo, 7% Relapse, 40% Refractory; Treatment: 20% FLAI-3, 40% FLAI-5, 26% Decitabine; 7% Cytarabine, 7% 5-azacitidine) were treated at the Policlinico Sant'Orsola in Bologna. At the same time, bone marrow samples were challenged with the same drugs at scaled concentrations in the functional profiling platform developed by CellPly and an output score was extracted from time-lapse fluorescence image analysis of patient tumor cells exposed to the drug or drug combination for 24 hours. Clinical follow up resulted in the following classification: complete hematologic response (CR) was found in 6/15 patients (6/6 with a De Novo disease), while the remaining 9/15 patients (2/9 de novo; 1/9 Relapse; 6/9 Refractory) were classified as stable disease (SD). In-vitro functional profiling of 14 patients provided a correlation with the clinical outcome (p=0.01) irrespective of the stage of the disease. 100% of the patients identified as responders by functional profiling (n = 5), resulted in a CR outcome. 89% of the patients identified as non-responders (n = 9) resulted in an SD outcome. The same patient samples were also investigated for the molecular assessment of the most relevant genes commonly analyzed in AML patients. FLT3-ITD mutation was found in 2/14 patients, both in SD. Negative FLT3 was not correlated to clinical outcome. FLT3-TKD mutation was found in 3/14 patients, 2/3 resulted in CR, 1/3 in SD. TP53 mutation was found in 1/13 patients, classified as SD. Intronic variant rs1625895 was found in 6/13 patients equally distributed in CR and SD. NPM1 mutation was identified in 4/13 patients, 2/4 resulted in CR, 2/4 in SD indicating no direct correlation. IDH2 was found in 1/11 patients (with R172K mutation) with an SD. As a conclusion, functional profiling proved to be highly correlated with clinical outcome irrespective of patient stage and demonstrated a superior predictive power compared to molecular profiling. Citation Format: Laura Rocchi, Andrea Faenza, Viviana Guadagnuolo, Laura Rambelli, Giovanni Marconi, Maria Chiara Fontana, Martina Pazzaglia, Cristina Papayannidis, Giorgia Simonetti, Nicola Pecorari, Luca Giulianelli, Dario Biscarini, Marco Bettelli, Michele Federici, Rita Ruggiano, Giovanni Martinelli, Roberto Guerrieri, Massimo Bocchi. Lab-on-a-chip-based in-vitro functional profiling proves to be effective in predicting therapy outcome in AML patients [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 3613.

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.

Background: Mutations in TP53 gene predict a poor prognosis in patients with AML. The reported TP53 mutation rate in AML is low (2.1%) by contrast AML with a complex karyotype (CK) is higher (69-78%) and have a poor outcome. Quite common is to found paired TP53 mutation together and a segmental 17p deletion. Aims: To investigate the frequency, the types of mutations, the associated cytogenetic, the correlation with known molecular alterations and the prognostic role TP53 mutations in adult AML pts. Moreover we would identify genes/pathways that are mainly affected in the mutated TP53 group compared to the wt one. Patients and Methods: 258 adult AML pts with miscellaneous cytogenetic abnormalities and normal karyotype were examined in our Institution for TP53 mutations using several methods, including Sanger sequencing, NGS and HiSeq2000 platform and were correlated with cytogenetic analysis. 124/258 samples were genotyped with SNP arrays (Affymetrix, 3 250K, 43 SNP 6.0, 78 CytoScan HD). Copy Number Alterations (CNAs) analyses were performed using Chromosome Analysis Suite (Affymetrix) and Nexus Copy Number (BioDiscovery) software. Results: Mutation analysis detected TP53 mutations on 39 patients with 48 different types of mutations (32 deleterious point mutations; 4 deletions); nine pts have 2 mutations. We found 34/48 (70%) missense mutations, 5 mutations in the splice sites, 4 deletions ,2 intronic and and 3 others mutations. The mutation rate is of 15.1%. Mostly (28/39) of the TP53 mutated pts (71.8%) had CK while only 11/39 (28.2%) mutated pts have “no CK” (P>0.0001). Alterations of TP53 were significantly associated with poor outcome (OS and EFS p Conclusions: Our data demonstrated that TP53 mutations occur in 15.1% of AML with a higher frequency in the subgroup of CK-AML (p Citation Format: Anna Ferrari, Eugenio Fonzi, Maria Chiara Fontana, Andrea Ghelli Luserna Di Rorà, Marco Manfrini, Antonella Padella, Carmen Baldazzi, Cristina Papayannidis, Giovanni Marconi, Stefania Paolini, Viviana Guadagnuolo, Margherita Perricone, Valentina Robustelli, Enrica Imbrogno, Eugenia Franchini, Claudia Venturi, Elisa Zuffa, Maria Chiara Abbenante, Giorgia Simonetti, Nicoletta Testoni, Emanuela Ottaviani, Martinelli Giovanni. Distinct pattern of alterations in tp53 mutated and wild type acute myeloid leukemia (AML) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1766. doi:10.1158/1538-7445.AM2017-1766

Introduction PI3P is a molecule that regulate cell growth and mediates cell proliferation via PI3K/AKT/mTOR in response to various growth signals. Abnormal activation of genes in its pathway is associated to oncogenic activity and poor Overall Survival (OS). AMPK plays a role as a regulator of cellular energy homeostasis. Aims The aim of the this study is to define the role of PI3P pathways and AMPK pathway in AML. Methods In this work we analyzed 208 consecutive newly diagnosed non M3 AML patients, screened for TP53, FLT3, NMP1, IDH1, IDH2, and DNMT3A mutations. Remission status was assessed with bone marrow biopsy. We performed Microarray-based Comparative Genomic Hybridization with Affymetrix SNP array 6.0 or Cytoscan HD in all the patients; we performed Whole Exome Sequencing (WES)in 80/208 patients. Survival data were collected prospectively, with a median follow-up of 18 months. Survival analysis was performed with Kaplan Meyer method using log rank test. Univariate and multivariable regression and Cox Hazard Ratio(HR) model was performed. Correlation between variables was assessed with Fisher’s exact test. Results We selected genes in pathways basing on literature and GO data. Alterations in these pathways involved 103/209 patients (48%). We analyzed the gene in two different pathways. PI3K/AKT/mTOR pathway includes the following genes: pik3ca, cdkn1a, akt1, akt3, mtor and pten, pdk1,pik3r1 and irs1. The second one is AMPK pathway and it include: sesn, prkaa1, prkab1, prkag1, prkag3. Alterations in PI3K/AKT/mTOR pathway confer worst OS (p = .035) when compared with unaltered patient, but events in these pathways did not affect therapy response. Alterations in AMPK pathway confer worst OS (p Conclusions Our work investigates the role of PI3P and cAMP pathways in AML. Surprisingly, it showed that alterations in these pathways are associated with poor prognosis. Significantly, alterations in cAMP pathways were associated with therapy resistance. Acknowledgement: ELN, AIL, AIRC, PRIN, Progetto Regione-Università 2010-12, FP7 NGS-PTL project, HARMONY Citation Format: Mariachiara Abbenante, Mariachiara Fontana, Giovanni Marconi, Giorgia Simonetti, Antonella Padella, Elena Tenti, Eugenia Franchini, Anna Ferrari, Sarah Parisi, Emanuela Ottaviani, Nicoletta Testoni, Viviana Guadagnuolo, Chiara Sartor, Silvia Lo Monaco, Cristina Papayannidis, Giovanni Martinelli. Alterations in phosphatidylinositol 3-phosphate (PI3P) pathway and cAMP pathway confirm poor prognosis and reduced overall survival (OS) in a series of 209 acute myeloid leukemia patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 515. doi:10.1158/1538-7445.AM2017-515

The endopeptidase Separase, encoded by the ESPL1 gene, plays a key role in faithful segregation of sister chromatids by cleaving the cohesin complex at the metaphase to anaphase transition. Its overexpression associates with aneuploidy and bad prognosis in solid tumors. Little is known in Acute Myeloid Leukemia (AML). We profiled the genomic landscape of 405 and 78 AML cases by SNP array (SNP 6.0 and Cytoscan HD, Affymetrix) and whole exome sequencing (100 bp, paired-end, Illumina), respectively. Bone marrow blasts from 61 patients were analyzed by gene expression profiling (HTA 2.0, Affymetrix). Separase expression was determined by Immunohistochemistry (1:600 antibody dilution Abnova, clone 6H6) in 44 AML and 4 control bone marrow specimens. One patient exhibited a nonsynonimous mutation in ESPL1 (1.3%), which was predicted to alter the protein function. Moreover, ESPL1 copy number gain was observed in 5/405 cases (1.2%): 2 hyperdiploid AML, one trisomy 12 and 2 cases with a short gain at 12q. Notably, protein level detection in one of the 12q-gain cases confirmed Separase overexpression. To determine the incidence of Separase overexpression, we performed Immunohistochemistry on additional 43 AML. Separase was overexpressed in 29/44 AML (66%, Separase-high), being comparable to control marrow biopsies in the remaining 15 samples (Separase-low). Sixty-two percent of Separase-high AML were aneuploid. However, no significative association was observed, as previously reported for mutations in the cohesin genes in AML. Separase overexpression correlated with increased patients’ age (median age 64 vs. 57 years, p=.01), 17-fold upregulation of CD34 (p=.004) and a trend towards reduced overall survival (6-years follow-up). Separase overexpression was not mutually esclusive with cohesin gene mutations, it co-occurred with NPM1 and FLT3 lesions and frequent mutations in genes involved in protein post-translational modification and ubiquitination (p=.04). Separase-low cases were enriched for mutations in RAS signaling pathway (NRAS, KRAS, NF1, RIT1, GRAP2, RALGDS; p=4.5x10-5) and in cell migration-related genes (LIMS2, S1PR1, PPIA, PLXNB1, FAT1). Separase-high cases also showed a defined transcriptomic profile, characterized by reduced expression of HOXA/B family genes, the DNA damage repair gene ATM, the p53 regulator MDM2 and forced expression of the cell cycle markers CDC20, AURKB, NUSAP1 and of MYC, independently of chromosome 8 gain. Taken together, our data suggest that genomic lesions targeting ESPL1 are a rare event in AML. However, Separase overexpression is a common feature and defines a new subset of AML cases with a distinct gene expression profile, which may benefit of innovative targeted therapies including CDC20 and bromodomain inhibitors. Supported by: ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Citation Format: Giorgia Simonetti, Antonella Padella, Simona Righi, Maria Chiara Fontana, Marco Manfrini, Cristina Papayannidis, Giovanni Marconi, Carmen Baldazzi, Marianna Garonzi, Alberto Ferrarini, Massimo Delledonne, Nicoletta Testoni, Elena Sabattini, Giovanni Martinelli. Separase overexpression defines a new subset of acute myeloma leukemia patients characterized by high CD34 and MYC levels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3472. doi:10.1158/1538-7445.AM2017-3472

Introduction There are no clear evidences if autophagy can lead to therapy resistance or favor apoptosis in cancer; it can function as a pro-apoptotic mechanism, or it can improve stresses survival clearing damaged mitochondria and proteins accumulation. Levels and activity of pro-apoptotic and anti-apoptotic proteins, particularly bcl-2 and p53, high levels of cAMP, and a complex made by pink/park could play as fulcrum of this yin and yang effect of autophagy. Our study aims to define the role of autophagy in AML, and to establish if gain or loss in autophagy could reduce the patients’ chance to respond to induction, and to worsen OS. Methods We analyzed 148 consecutive newly diagnosed non M3 AML patients treated with induction chemotherapy regimens containing at least one dose of anthracycline. We screened all patients for TP53, FLT3, NMP1 mutations; we performed Affymetrix SNP array 6.0 or Cytoscan HD. 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) were related to lower Complete Remission rate (CR%) after induction in univariate (p Conclusions Our work investigates for the first time with a genomic approach the role of autophagy in AML. We found that both gain and loss in autophagy key regulator genes are associated with poor prognosis and therapy resistance. A loss in autophagy could enhance proliferation and block apoptosis; a gain could give cell resiliency, favoring cytoplasm turnover, damaged mitochondria elimination, and neutralizing oxidative damages. Acknowledgements: ELN, AIL, AIRC, PRIN, Progetto Regione-Università 2010-12, FP7 NGS-PTL project. Citation Format: Giovanni Marconi, Cristina Papayannidis, Maria Chiara Fontana, Antonella Padella, Anna Ferrari, Eugenia Franchini, Stefania Paolini, Maria Chiara Abbenante, Chiara Sartor, Francesca Volpato, Viviana Guadagnuolo, Silvia Lo Monaco, Elena Tenti, Andrea Ghelli Luserna Di Rora, Valentina Robustelli, Nicoletta Testoni, Giorgia Simonetti, Emanuela Ottaviani, Giovanni Martinelli. The alteration in key regulator genes of autophagy is mainstream mechanism of therapy resistance and impact prognosis of acute myelogenous leukemia (AML): results from diagnosis genomic analysis on 148 consecutive patients treated with intensive chemotherapy and long-term survival follow-up [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3311. doi:10.1158/1538-7445.AM2017-3311

Introduction: Novel array-based technique as SNP microarray can detect losses or gains of chromosomic material, which could be predictive of response and can help define therapeutic strategies. The aim of this study is to improve conventional cytogenetic analysis and identify new genetic alterations relevant to leukemogenesis, by a SNP array-based genotyping approach. Materials and Methods: We performed SNP 6.0 or Cytoscan HD (Affymetrix) in 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 v8.0 (BioDiscovery) and R Core Team. Results: Copy Number Alterations (CNAs) were scattered across all chromosomes and all pts showed CNA events. SNP array analysis showed that several genes were 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%), while the 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 Conclusion: We have identified new CNAs and pathways involving novel potential leukemia-related genes. Our results suggest that the comparison between SNP and WES data could provide important findings on prognosis of AML patients. Minimal deleted regions of genes implicated in deregulated pathways deserve further investigation in order to identify new candidate genes which could be relevant AML biomarkers. Acknowledgements: ELN,AIL,AIRC,progetto Regione-Università 2010-12 (L. Bolondi),FP7 NGS-PTL project,HARMONY. Citation Format: Maria Chiara Fontana, Giovanni Marconi, Cristina Papayannidis, Eugenio Fonzi, Giorgia Simonetti, Antonella Padella, Anna Ferrari, Emanuela Ottaviani, Silvia Lo Monaco, Stefania Paolini, Simona Soverini, Giovanni Martinelli. Genomic wide microarray analysis identifies novel copy number alterations in adult acute myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2451. doi:10.1158/1538-7445.AM2017-2451

Partner and localizer of BRCA2 (PALB2) plays a key role in the DNA damage repair (DDR) by recruiting BRCA1, BRCA2 and RAD51. Alterations in PALB2 were described in hereditary breast cancer and Fanconi Anemia (FA). Little is known in Acute Myeloid Leukemia (AML). Aim of the study is to define the frequency and interplay of PALB2 alterations with patterns of somatic mutations and copy number variants (CNVs) in AML. We genotyped 233 AML samples by Single Nucleotide Polymorphism array (SNP 6.0 and Cytoscan HD, Affymetrix) to detect CNVs. We also performed Whole Exome Sequencing (WES, Illumina) of 56 cases to detect single nucleotide variants and small indels (MuTect and Varscan 2.0). Differences in survival were assessed by Kaplan-Meier survival analysis and Breslow test. We detected PALB2 loss in 12/233 patients (5%), with a minimal common region of deletion of 6.6 Kb including exon 12, which encodes for the domain of interaction with RAD51, BRCA2 and POLH. PALB2 deletion were correlated with CN loss of MYH11, CREBBP, PLK1 and FANCA on chromosome (chr) 16 (p≤.005), deletions of TP53, NF1 and BRCA1 on chr 17 (p≤.004), 5q deletions (p≤.001) and loss of XPO1 on chr 2 (p≤.006). PALB2-loss patients were enriched for alterations in genes involved in the protein kinase pathway (p=5×10-4), JUN kinase activity (p=.0006) and, notably, in genes belonging to the chromosome breakage pathway (p=.001; TP53, BRCA1 and BRCA2). To identify mutations that co-operates with CN alterations, we integrated WES data of 56 AML patients. No mutations in PALB2 were detected. However, we identified mutations in other DDR genes, including FANCE, BRCA2, TP53 and BRCA1. Of note, alterations in the DDR genes frequently co-occurred with both mutations and CN loss of TP53, leading to homozygous loss of function of TP53. In addition, KRAS, IDH1, TET2 and BCOR mutations were mutually exclusive with PALB2 loss. Preliminary gene expression profiling data revealed a 2-fold upregulation of FANCA in samples with DDR genes alterations compared with patients with no alterations (p=.04), while the other genes of the pathway were not affected. Finally, patients harboring PALB2 loss had a poorer prognosis compared complex karyotype cases (p=.021). We here define a new subset of AML patients, characterized by the synergistic loss of key DDR genes, which may in turn result in FANCA over-expression as a mechanism to overcome the DDR deficit. Our data suggest that the leukemogenic process associated PALB2 loss integrates defects in the DDR together with enforced proliferative stimuli leading to uncontrolled proliferation of cells, which potentially accumulate genetic lesions. These results may help improve patient stratification and define ad hoc therapeutic strategies for this aggressive leukemia type. Supported by: ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Citation Format: Antonella Padella, Giorgia Simonetti, Maria Chiara Fontana, Marco Manfrini, Giovanni Marconi, Anna Ferrari, Italo Faria do Valle, Marianna Garonzi, Cristina Papayannidis, Eugenia Franchini, Elisa Zuffa, Viviana Guadagnuolo, Samantha Bruno, Andrea Ghelli Luserna di Rorà, Emanuela Ottaviani, Daniel Remondini, Massimo Delledonne, Giovanni Martinelli. Co-occurrence of alterations in the DNA damage repair genes synergize with uncontrolled proliferation and associate with very-poor prognosis in acute myeloid leukemia patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4671. doi:10.1158/1538-7445.AM2017-4671

Chromosome gain or loss, which is the hallmark of aneuploidy, occurs in about 10% of adult Acute Myeloid Leukemia (AML) cases (Farag et al. IJO 2002, Breems et al. JCO 2008), despite inducing a dramatic reduction of cellular fitness in non-malignant cells (Torres et al. Science 2007). The study aimed to identify AML-specific molecular mechanisms having a causative and/or tolerogenic role towards aneuploidy. We performed 100 bp paired-end whole exome sequencing (WES, Illumina Hiseq2000) of 38 aneuploid (A) and 34 euploid (E) AML cases, identified according to cytogenetic analysis and SNP array (CytoScan HD, Affymetrix). Variants were called with GATK, MuTect and VarScan. We also compared the transcriptomic profile of leukemic bone marrow cells from 21 A-AML and 28 E-AML cases (HTA 2.0, Affymetrix). A-AML showed a significantly higher mutation load compared with E-AML (median number of variants: 25 and 15, respectively, p Our data show a link between aneuploidy and genomic instability in AML and highlight novel molecular mechanisms for the acquisition and/or maintenance of the aneuploid phenotype. Deregulation of the cell cycle machinery and DNA damage/repair checkpoints, either through mutations, copy number and transcriptomic alterations, cooperate with leukemogenic pathways, as KRAS signaling, to develop A-AML and overcome the unfitness barrier. This evidence suggests that a number of A-AML patients may benefit from pharmacological reactivation of TP53 and inhibition of KRAS pathway. Supported by: FP7 NGS-PTL project, ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi). Citation Format: Giorgia Simonetti, Antonella Padella, Marco Manfrini, ĺtalo Faria do Valle, Cristina Papayannidis, Carmen Baldazzi, Maria Chiara Fontana, Viviana Guadagnuolo, Anna Ferrari, Elisa Zago, Marianna Garonzi, Simona Bernardi, Annalisa Astolfi, Maria Chiara Abbenante, Giovanni Marconi, Elisa Zuffa, Eugenia Franchini, Ilaria Iacobucci, Michele Cavo, Emanuela Ottaviani, Nicoletta Testoni, Alberto Ferrarini, Massimo Delledonne, Torsten Haferlach, Daniel Remondini, Giovanni Martinelli. A cell cycle-related genomic and transcriptomic signature distinguish aneuploid and euploid acute myeloid leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 90.

Introduction. Intensive induction chemotherapy in non-M3 young Acute Myeloid Leukemia (AML) patients is represented by the association of an antracycline and Cytarabine. Some treatment regimens including fludarabine or the addition of Gemtuzumab Ozogamicin (GO) as a third or fourth drug, proved to give a benefit in terms of CR rates. Aims of the study. In a group of 49 patients treated with intensive chemotherapy, we evaluated chromosomal abnormalities with SNP 6.0 and Cytoscan HD (Affymetrix) in order to improve conventional cytogenetic analysis and discover novel chromosomic aberrations related to clinical data and therapy response. Patients and Methods. From 2001 to 2014, 489 patients were treated in our Institution. Among those, in 49 newly diagnosed AML patients (median age 54 (range 19-71)), SNP microarray based-genotyping were performed and then analyzed by Nexus Copy Number™ v7.5 (BioDiscovery) and R Development Core Team. According to karyotype, FLT3 and NPM1 mutational status, 55.9% of the patients were considered at High Risk (HR) and 4.1% at low risk (LR). Ten patients had secondary AML. Patients were treated with induction schemes including MyFLAI, MyAIE, FLAI, FLAN, FLAG, 3+7 or DAE. Results. The CR rate after induction was 87.8% (43/49 patients). Deaths during induction (DDI), occurring in the first 50 days from 1st line therapy, were 1/49. The median OS was 135 months, the 5-years OS in our patients was 55,1%. Patients treated with GO showed a non-statistical trend toward a better OS than patients treated with other regimens (median OS not reached vs 133 months, respectively). We explored the alterations found by SNP array in our patients searching for novel markers of therapy resistance. We found a median of 192,5 total copy number aberrations (range 72- 1071): a median of 145,5 total copy number aberrations in responding patients group (RPG), and a median of 361 total copy number aberrations (p = ns) in non-responding patients group (NRPG). We compared the frequency of detected aberrations in RPG and in NRPG with Fisher's exact test. We found that PIK3CA, Gain chr3:178,927,088-178,929,550 (p = 0,0016), SMAD4, Gain chr18:48,573,154-48,573,255 (p = 0,0166) and several other gene's loci (CASC18, TCF12, UTY, GRB10, ZFY) are significant aberrations in NRPG compared with RPG. Conclusions. We identified a number of genes with significant aberrations in NRPG, particularly PIK3CA, a protein-coding gene involved in cell proliferation and metabolic pathway with interaction with HRAS/KRAS and EGF, and SMAD4, a transcription factor activated by TGF-beta. Those 2 genes were found overexpressed in other solid tumors. We suppose that those genes may be involved in a hyper-proliferative pathway that underlies a mechanism of chemo-resistance. Acknowledgments Work supported by ELN, AIL, AIRC, Progetto Regione-Universit⁁ 2010-12 (L.Bolondi), FP7 NGS-PTL project. Citation Format: Cristina Papayannidis, Maria Chiara Fontana, Giovanni Marconi, Viviana Guadagnuolo, Giorgia Simonetti, Antonella Padella, Simona Soverini, Stefania Paolini, Maria Chiara Abbenante, Sarah Parisi, Chiara Sartor, Silvia Lo Monaco, Marco Manfrini, Elisa Zuffa, Eugenia Franchini, Claudia Venturi, Maria Teresa Bochicchio, Andrea Ghelli Luserna di Rorà, Emanuela Ottaviani, Giovanni Martinelli. Specific chromosomic alterations confer therapy resistance in a cohort of 49 patients with newly diagnosed acute myeloid leukemia treated with intensive chemotherapy. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 368.

Acute Myeloid Leukemia (AML) is a clonal hematopoietic disorder characterized by an abnormal proliferation and differentiation of immature blast cells in the bone marrow. SNP microarray approach has resulted in genome-wide screening for genomic alterations with information not previously achievable and also it allows to map all the genes involved in these alterations which may plays a role in oncogenesis. Our objective is to evaluate the prognostic impact of these genetic alterations on clinical outcome. We analyzed 285 AML samples at diagnosis of which 221 by SNP Array 6.0 (Affymetrix) (Affymetrix), while 64 by by CytoScan HD Array (Affymetrix). The results obtained were analyzed by Chromosome Analysis Suite (ChAS) v1.2 (Affymetrix Inc.) and Nexus Copy Number™ v7.5 softwares (BioDiscovery). The 285 AML patients (pts) analyzed are equally distributed between both sexes, they have a median age of 60 years, include miscellaneous cytogenetic abnormalities and normal karyotype. The 44% of pts are treated with chemotherapy (CHT), the 16% of pts with low-dose cytosine arabinoside, the 20% of pts with 5-azacytidine and the 19% of pts are not treated or had only supportive therapy. The ratio between responders and non responders pts was 57/43. Each pt presented an average of 1448 events so distributed: 31% of homozygous deletions, 32% of homozygous amplifications, while irrelevant events are related to heterozygous deletions and amplifications. We also found 36% of uniparental disomy (UPD)events. Among all these macroscopic and submicroscopic alterations, we focused on JAK2 gene, which is located on human chromosome 9 at p24.1 locus. It is composed of 25 exons and encodes a 1132 amino-acid protein of 130.7 kDa. This gene was amplified in 12/285 pts (4%) and deleted in 13/285 (5%). All these pts are treated with CHT but only those characterized by JAK2 deletion are responsive in first line. We showed that the group of pts which present this deletion had a better overall survival rate than the group with the amplification of this gene (p-value < 0,01). The deletion event of JAK2 gene involved two different regions: the first region had a length of 7.429 bp involving the intron 4 and was found in the database genome variant (DGV); the second deletion presented a variable length between different pts: the smallest deleted region had a length of 3342 bp and included the exons 17, 18, 19, while the largest one had a length of 76903 bp which included the portion from exon 9 to exon 19. This second deletion has not been described in DGV and it involved the pseudokinase and kinase domain of JAK2 protein. We have identified Copy Number Variation involving important cancer genes in AML. We have identified a new JAK2 deletion involving its Pseudokinase and Kinase domain and we observed that this deletion of JAK2 correleted with overall survival. ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project Citation Format: Viviana Guadagnuolo, Maria Chiara Fontana, Cristina Papayannidis, Marco Manfrini, Antonella Padella, Giorgia Simonetti, Anna Ferrari, Giovanni Marconi, Andrea Ghelli Luserna di Rorà, Stefania Paolini, MariaChiara Abbenante, Sarah Parisi, Chiara Sartor, Emanuela Ottaviani, Giovanni Martinelli. New JAK2 heterozygous loss: A role in overall survival in acute myeloid leukemia patients. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4507.

Introduction: Genomic rearrangements can drive the development of cancer through different mechanisms: chromothripsis, a catastrophic mechanism of genomic instability, could be relevant for hematological disease. Aim: To discover the mechanisms underlying the pathogenesis of Acute Myeloid Leukemia (AML), we studied chromothripsis in our cohort of patients (pts). Methods: We perform SNP Array 6.0 or Cytoscan HD Array (Affymetrix) in a cohort of 104 AML pts at diagnosis. SNP Array data were analyzed by Nexus Copy Number™ v7.5 (BioDiscovery). Results: Seven/104 pts (6.7%) showed chromothripsis events involving different chromosomes (8, 17, 11, 5 and 16). These pts had median age of 68.5 years (range 56-76), complex karyotype and high risk disease according to ELN definition. Among the pts showing chromothripsis, we compared chromosomic abnormalities of pts with de novo (5/7) and secondary (2/7) AML. De novo AML showed a prevalence of trisomy of chromosome 8 in a non-statistical way (4/5 vs 0/2), due to the low number of cases. However, we identified significant differences in the pattern of genes altered in the two groups. De novo AML had copy number gain of PEX1, ANK1, NCOA2, ESRP1, TPD52, ESRP1, ZFPM2 and MITF (p Conclusion: Our data suggest that different pathways and genomic alterations are involved in chromothripsis events in de novo and secondary AML, which could be explained by the repeated rounds of stress underwent by leukemic cells in secondary AML cases. Cytoskeleton and microtubules formation pathways appear to be the main cellular processes implicated in chromothripsis genesis, while alterations of histone acetyltransferase, immune response and antigen presentation pathways could sustain leukemic cells after chromothripsis. Acknowledgement: ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12(L. Bolondi), FP7 NGS-PTL project. Citation Format: Maria Chiara Fontana, Viviana Guadagnuolo, Cristina Papayannidis, Giovanni Marconi, Giorgia Simonetti, Antonella Padella, Marco Manfrini, Barbara Santacroce, Margherita Perricone, Silvia Lo Monaco, Emanuela Ottaviani, Simona Soverini, Michele Cavo, Giovanni Martinelli. Chromothripsis in AML patients: A new mechanism of cancer initiation and progression. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3582.

Introduction: In Acute Myeloid Leukemia (AML) there is a strong need to develop new diagnostic and therapeutic options: to identify the genes mostly predictive of treatment response, we use Single Nucleotide Polymorphism (SNP) Arrays and Whole Exome Sequencing (WES) in AML patients with heterogeneous karyotypes and different subgroups. Materials and Methods: SNP arrays (CytoScan HD Array, Affymetrix Inc.) was positively done in 58 AML samples and all of them were analyzed by Chromosome Analysis Suite (ChAS) v1.2 (Affymetrix Inc.) and Nexus Copy Number™ v7.5 software (BioDiscovery), while Next Generation Sequencing (NGS)-WES HiSeq 2000 (Illumina) was done in 35 AML patients. Results: We treated 58 AML patients (pts) with a median age of 52 years, of which 9 were insensitive pts and 49 sensitive to therapies, all obtaining complete clinical remission. Copy Number Alterations (CNAs) were detected in all patients affecting all the chromosomes, in particular our cohort of pts present a percentage of CNA, divided as follow: 58% of LOH, 15% of gain, 16% of loss, 3% of high copy gain and 4% of homozygous copy loss. We found that several cancer genes were preferentially amplified: IGH@, KIT, IGL@, TSC1, NOTCH2, SETD2, EZH2, while in high copy gain we found TSC1, PTEN, RB1, IKZF1, ZRSR2, IGH@, NF1, MYC, KRAS. Then several genes were preferentially deleted: CRLF2, ATRX, JAK2, BCOR, PHF6, GATA1, KDM6A and in homozygous copy loss: JAK2, CRLF2, RB1, PDGFRA, RUNX1. Moreover we genes in LOH: DDX5, MTCP1, HOOK3, ZRSR2, GATA1, KDM6A, BCOR, NF1, BRAF, ATRX. We focused on two losses of JAK2: the first deletion, detected in 18/58 (31%) pts, goes from 5030 to 5038 Kbp (7,43 Kbps) including intron 4-5; the second minimal common region of loss, detected in 5/58 (8,6%) pts, goes from 5083 to 5098 Kpb (15 Kbps) including intron 19-20 and exons 20, 21 and 22, suggesting a defective transduction, in fact we showed that the overall survival rate is better for the group of pts which present a deletion of JAK2 rather than the group with an amplification of this gene (p-value < 0,01). We have also found three other genes which are preferentially lost: SIRPB1, ADAM3A and STAG2 with a percentage of 50%, 43% and 69% respectively. By NGS-WES we analyzed 35 AML samples at diagnosis and we searched for point mutations, insertion/deletion or other abnormalities, involved in biomarkers of response to treatment. We found mutations in SF3B1, NPM1, CBL, RUNX1, BCOR, KIT, GATA2, IDH2, KDM6A, KIAA1324L, PRIM2, RRN3, APOBR. Conclusion: By SNP arrays we have identified Copy Number Alterations involving important cancer genes AML and we showed that a new deletion in JAK2 may have a role in overall survival rate. Future prospective will be to correlate the cancer genes alteration and mutations with the prognosis of AML, in order to identify new biomarkers relevant for the disease. Acknowledgement: ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7NGS-PTL project Citation Format: Viviana Guadagnuolo, Maria Chiara Fontana, Antonella Padella, Ilaria Iacobucci, Cristina Papayannidis, Giorgia Simonetti, Anna Ferrari, Giovanni Marconi, Stefania Paolini, Maria Chiara Abbenante, Sarah Parisi, Francesca Volpato, Chiara Sartor, Emanuela Ottaviani, Massimo Delledonne, Michele Cavo, Guido Biasco, Giovanni Martinelli. SNP array reveals a new deletion of JAK2 in AML patients. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4848. doi:10.1158/1538-7445.AM2015-4848