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Comparison between mutations detected by conventional Sanger sequencing and Deep sequencing and estimated clonal composition of the samples. Mutation-relative abundance of conventional Sanger Sequencing results was assessed on the basis of variant peak height. In the TKI/treatment column, the TKI or the treatment being administered at the time of analysis is indicated. In sample ALL-8 and 11, “T315?” denotes that 2 overlapping peaks at adjacent positions (c/t at 1091 and t/c at 1092) of codon 315 were identified in the Sanger Sequencing chromatogram and the resulting amino acid substitution(s) could not be resolved. (PDF 209 kb)

This survey was used in the research project that is reported in the following journal article:Rubin. M., Subasic, E., Giacomini, A., & Paolini, S. (2017). An exploratory study of the relations between women miners’ gender-based workplace issues and their mental health and job satisfaction. Journal of Applied Social Psychology. doi: 10.1111/jasp.12448

No abstract available.

The primary goal of the present research was to explore the relationship between adult attachment styles and four different types of identification with social groups. The results confirmed predictions and revealed that particular prototypic attachment styles are associated with an increase in only certain types of ingroup identification. People with secure attachment style had higher social identification than people with a dismissive-avoidant attachment style. Participants with secure attachment style showed higher communal identification than participants who had either a dismissive-avoidant or a fearful-avoidant attachment style. These findings supported the idea that relationship attachment style has an important effect on the way people identify with their social groups and can serve as a predictor of preferred type of ingroup identity.

The present research tested the idea that the ecological impact of intergroup contact on outgroup attitudes can be fully understood only when relative frequency and relative influence of positive and negative contact are considered simultaneously. Participants from five European countries (Austria, the Czech Republic, Germany, Poland and Slovakia; N = 1,276) freely described their contact experiences with people of neighboring nationalities and then reported on their outgroup attitudes. Contact descriptions were coded for positive versus negative valence and for person- versus situation-framing. Consistently across the five participants groups, positive intergroup contact was reported to occur three times more frequently than negative intergroup contact; however positive contact was found to be only weakly related to outgroup attitudes. On the contrary, the less frequent negative (vs. positive) contact was comparatively more influential in shaping outgroup attitudes, especially when negativity was reported around the contact person, rather than the contact situation. This research’s findings reconcile contrasting lines of past research on intergroup contact and suggest that the greater prevalence of positive contact may compensate for the greater prominence of negative contact, thus leading to modest net improvements in outgroup attitudes after intergroup contact.

The present research investigated linguistic description as a moderator of biased evaluations of counterstereotypical individuals. Members of an online participant pool (N = 237) indicated their liking for stereotypical and counterstereotypical individuals who were described using adjectives or behaviors. There was a significant interaction between target typicality and linguistic description: People liked counterstereotypical individuals more than stereotypical individuals when target individuals were described using adjectives. In contrast, they showed no bias or a negative bias against counterstereotypical individuals who were described using behaviors. This interaction effect generalized across gender targets (men/women) and sexuality targets (gay/straight), and it was partially mediated by subjective processing fluency. Implications for the backlash effect and prejudice reduction are discussed.

Table S1. Patient’s characteristic of gene expression cohort. Table S2. Patient’s characteristic ex vivo AZD-1775 treatment in single agent or in combination. Table S3. Quantitative analyses of G2/M checkpoint-related genes. Differential gene expression of 24 genes involved in the regulation of the G2/M checkpoint of primary leukemic cells in comparison to normal mononuclear cells (MNCs). In the table, the primary leukemic samples have been divided into three groups based on the ex vivo sensitivity to AZD-1775. Very good IC50 10 uM. (PDF 253 kb)

Table S1. Patient’s characteristic of gene expression cohort. Table S2. Patient’s characteristic ex vivo AZD-1775 treatment in single agent or in combination. Table S3. Quantitative analyses of G2/M checkpoint-related genes. Differential gene expression of 24 genes involved in the regulation of the G2/M checkpoint of primary leukemic cells in comparison to normal mononuclear cells (MNCs). In the table, the primary leukemic samples have been divided into three groups based on the ex vivo sensitivity to AZD-1775. Very good IC50 10 uM. (PDF 253 kb)

Figure S1. Efficacy of AZD-1775 used as single agent. A) The graph shows the IC50 values of B/T-ALL cell lines treated with AZD-1775 for 24, 48, and 72 h. B) Cell viability analysis on CCRF-CEM cell lines showing the effect of high doses of AZD-1775. The percentage of viable cells is depicted relative to untreated controls. C) Immunoblot analysis on BV-173 treated with AZD-1775 (IC50) for 12 h. D) Cell cycle analysis in BV-173 and CCRF-CEM cell lines treated with AZD-1775 (IC50) for 12 h. E) Immunofluorescence analysis of BV-173 cells treated with AZD-1775 (IC50) for 12 h and, then, stained with DAPI and phospho-MPM2. In the picture, a cell dying in mitosis is reported with apoptotic bodies strongly positive for phospho-MPM2 antibody. Representative images are shown at × 100 magnification. F) Viability of mononuclear cells isolated from the peripheral blood of 5 healthy donors incubated with increasing concentration of AZD-1775 (2.5, 5, and 10 uM) for 24 h. G) MYT1 transcript levels in samples isolated from adult BCR-ABL1-positive ALL at diagnosis (n = 17), adult BCR-ABL1-negative ALL at diagnosis (n = 27), adult BCR-ABL1-positive ALL at relapse (unpaired, n = 8), adult BCR-ABL1-negative ALL at relapse (unpaired, n = 6), and in MNCs (n = 7) from the peripheral blood of healthy donors. One-way ANOVA test was performed to assess statistical significance. Results are expressed as Log10 2 exp.[−(ΔΔCt). Figure S2. AZD-1775 in combination with chemotherapy agents and tyrosine kinase inhibitors. A) Growth curve of BV-173 and REH cell lines treated for 4 days with AZD-1775 (185 nM) and doxorubicin (25 nM). B) Viability analyses in ALL cell lines incubated for 24 h with Bos or Bos-I (6 to 5000 nM). The percentage of viable cells is depicted relative to untreated controls. C) Cell viability analysis of BV-173 cell line treated with AZD-1775 (6 to 5000 nM, dilution rate 1:3) and with ponatinib (25, 50, 100 nM) or imatinib (250, 500, and 1000 nM) for 24 h. The percentage of viable cells is depicted relative to untreated controls, 0.1%. Figure S3. Wee1 mRNA expression across different cancer types from the Cancer Cell Line Encyclopedia (CCLE) database. A) Box plots showing the level of expression of Wee1 mRNA in different tumor samples, extracted from CCLE [63]. The red arrows point to B/T-ALL samples. Boxes define the 25th and the 75th percentiles, horizontal line within the boxes indicates the median, and whiskers define the 10th and the 90th percentiles. (PDF 1918 kb)

Figure S1. Efficacy of AZD-1775 used as single agent. A) The graph shows the IC50 values of B/T-ALL cell lines treated with AZD-1775 for 24, 48, and 72 h. B) Cell viability analysis on CCRF-CEM cell lines showing the effect of high doses of AZD-1775. The percentage of viable cells is depicted relative to untreated controls. C) Immunoblot analysis on BV-173 treated with AZD-1775 (IC50) for 12 h. D) Cell cycle analysis in BV-173 and CCRF-CEM cell lines treated with AZD-1775 (IC50) for 12 h. E) Immunofluorescence analysis of BV-173 cells treated with AZD-1775 (IC50) for 12 h and, then, stained with DAPI and phospho-MPM2. In the picture, a cell dying in mitosis is reported with apoptotic bodies strongly positive for phospho-MPM2 antibody. Representative images are shown at × 100 magnification. F) Viability of mononuclear cells isolated from the peripheral blood of 5 healthy donors incubated with increasing concentration of AZD-1775 (2.5, 5, and 10 uM) for 24 h. G) MYT1 transcript levels in samples isolated from adult BCR-ABL1-positive ALL at diagnosis (n = 17), adult BCR-ABL1-negative ALL at diagnosis (n = 27), adult BCR-ABL1-positive ALL at relapse (unpaired, n = 8), adult BCR-ABL1-negative ALL at relapse (unpaired, n = 6), and in MNCs (n = 7) from the peripheral blood of healthy donors. One-way ANOVA test was performed to assess statistical significance. Results are expressed as Log10 2 exp.[−(ΔΔCt). Figure S2. AZD-1775 in combination with chemotherapy agents and tyrosine kinase inhibitors. A) Growth curve of BV-173 and REH cell lines treated for 4 days with AZD-1775 (185 nM) and doxorubicin (25 nM). B) Viability analyses in ALL cell lines incubated for 24 h with Bos or Bos-I (6 to 5000 nM). The percentage of viable cells is depicted relative to untreated controls. C) Cell viability analysis of BV-173 cell line treated with AZD-1775 (6 to 5000 nM, dilution rate 1:3) and with ponatinib (25, 50, 100 nM) or imatinib (250, 500, and 1000 nM) for 24 h. The percentage of viable cells is depicted relative to untreated controls, 0.1%. Figure S3. Wee1 mRNA expression across different cancer types from the Cancer Cell Line Encyclopedia (CCLE) database. A) Box plots showing the level of expression of Wee1 mRNA in different tumor samples, extracted from CCLE [63]. The red arrows point to B/T-ALL samples. Boxes define the 25th and the 75th percentiles, horizontal line within the boxes indicates the median, and whiskers define the 10th and the 90th percentiles. (PDF 1918 kb)

Background: 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. Aim: 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. Design: From March 2015 to July 2016, 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. Patients: 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. Results: 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. Conclusions: Our results confirm the high rate of response and to Blinatumomab in a poor patients' population, and the good management profile of the compound. Acknowledgments: Work supported by ALN, AIL, AIRC, PRIN, Progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Soverini: Ariad: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy.

Chromothripsis is a one-step genome-shattering catastrophe resulting from disruption of one or few chromosomes in multiple fragments and consequent random rejoining and repair. This study defines incidence of chromothripsis in 395 newly diagnosed adult acute myeloid leukemia (AML) patients from three institutions, its impact on survival and its genomic background. SNP 6.0 or CytoscanHD Array (Affymetrix??) were performed on all samples. We detected chromothripsis with a custom algorithm in 26/395 patients. Patients harboring chromothripsis had higher age (p???=???0.002), ELN high risk (HR) (p?????0.001), lower white blood cell (WBC) count (p???=???0.040), TP53 loss, and/or mutations (p???

Outcome for elderly patients with acute myeloid leukemia (AML) is extremely poor. Intensive induction chemotherapy is often unsuitable. Sixty-six newly diagnosed AML patients (median age: 76 years), ineligible for standard therapy, were consecutively treated with low-dose lenalidomide (10 mg/day orally, days 1–21) plus 10 mg/m2low-dose cytarabine, subcutaneously, twice a day (days 1–15) every six weeks, up to 6 cycles. Complete remission (CR) rate was 36.3% according to intention-to-treat. Responding patients had a longer median overall survival than non-responders (517 vs. 70 days, P < 0.001). The achievement of CR was not predicted by bone marrow blast count, cytogenetics, molecular markers, prior MDS, white blood cell count. Conversely, by studying the global gene expression profile, we identified a molecular signature, including 309 genes associated with clinical response (CR versus no CR). Based on the expression of a minimal set of 16 genes, we developed an algorithm to predict treatment response, that was successfully validated by showing an overall accuracy of 88%. We met the primary endpoint of the study, by beating the estimated successful CR rate (P1) fixed at 30%. Moreover, CR induced by this 2-drug combo was efficiently predicted by genetic profiling, identifying a biomarker that warrants validation in independent series.

// 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.

e23207 Background: Nowadays, Acute Promyelocytic Leukemia (APL) is a disease entity with a very high rate of cure and an estimated 2-year overall survival of 97%. Early death, rather than resistant disease so common in all other subtypes of AML, has emerged as the major cause of treatment failure, and relapse is a very rare occurrence. Methods : We collected data of all the APL referred to our institution from 2014. Within 23 patients, we encountered 20 new diagnosis and 2 relapse of APL. We analyzed blasts in samples obtained from Bone Marrow with Single Nucleotide Polymorphisms Array Cytoscan HD. Results: We compared copy number alterations in both relapsed patients with alterations detected in the pool of 20 newly diagnosed APL and we found specific signatures of CNVs for each patient. There were several copy number alterations related to each patient: the first patient presented gain of ROBO2, GRIP1, CTNNB1, SOX6, PBX1, GRIK2, CDKAL1 and loss FAF1, CREBBP, SBF1; the second patient presented gain of ROBO1, MAPK10, CADPS2, APBA1 and loss of GRIP1 and MYB. Subsequently we focused our attention on ROBO and GRIP1genes because they were alterated in both relapsed patients: ROBO proteins are associated to K channels while GRIP1 is involved in various critical functions, for example in androgen receptor binding, beta-catenin binding, glucocorticoid receptor binding, and it is also a regulator of glutamate metabolism, a well-known pathway in Leukemic Stem Cells. Conclusions: APL relapse is a very rare entity, and it is announced to become rarer with the advances in first line therapy. Molecular characteristics are hard to analyze without an effort to collect and bank samples together from multiple institutions. Since relapses, especially relapses out of follow-up period, represent a sudden life-treating condition for patients, to predict patients at higher risk of relapse we selected two candidate genes that could be involved in pathways favoring relapse. By the analysis of ROBO 1-2 and GRIP1 at the diagnosis of APL we could establish a different and strict follow-up program for patients with these alterations. Acknowledgement: ELN,AIL,AIRC,prog. Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project,HARMONY.

e18521Background: AML is a disease with a high number of various mutations and genomic abnormalities; most of them have a proved prognostic value. We focused on FLT3 gene, which shows a high hetero...

e18235Background: Patients affected by oncohaematology pathology follow multiple therapies that can contribute to the drug interactions resulting in change in pharmacokinetics or pharmacodynamics o...

7044Background: Genomic alterations are proved to initiate cancer and related to prognosis in AML. We tested 101 consecutive AML patients (pts) at diagnosis to find prognostic implications of new g...

Introduction. Autologous Bone Marrow Transplantation (Auto-BMT) is currently rarely used in the treatment of Acute Myeloid Leukemia (AML). However, it may represent a good therapeutic option in a specific subset of patients, mainly in consolidation of both low risk (LR) and MRD negative AML without an available HLA matched donor. Aims. To review our database of AML patients who received Auto-BMT from 2005 to 2014 and who were referred to Bologna Institution, in order to assess the efficacy of the procedure in terms of Overall Survival (OS) and Disease Free Survival (DFS). Patients and methods: From 2005 to 2014, 98 AML patients underwent Auto-BMT in several Italian Institutions. 89/98 patients are evaluable for survival and outcome data. The 89 patients considered (42 female, 47 male), had a median age of 49 years (range 15-70). Cytogenetics was performed in all patients by conventional karyotype (22 patients were also analyzed by Single Nucleotide Polymorphisms Array); molecular analysis (FLT3 TKD and ITD, and NPM1 mutational analysis) was available for 51/89 patients. Molecular monitoring by specific fusion transcripts (CBF-MYH11 and AML1-ETO) was performed in CBF positive leukemias (inv(16) and t(8;21)) at the time of diagnosis, after induction, consolidation courses, and every 3 months in the first 2 years of follow-up. Based on this data, and according to ELN guidelines, a risk stratification identified 41 patients with a LR AML (t(8:21), inv(16) or NPM1+/FLT3- with normal karyotype), 4 patients with a high risk (HR) AML (complex karyotype or FLT3 ITD mutated or inv(3) or t(6;9)) and 44 patients with a standard risk (SR) AML (normal karyotype, other alterations). Results. All the patients received an induction chemotherapy treatment, as follows: a "3+7-like" course in 48 cases, a Fludarabine-based regimen in 20 patients and a Gemtuzumab-ozogamicin (GO)-based regimen in 21. 83/89 (93.3%) patients received a median of 2 consolidation courses of chemotherapy (range 1-4) before proceeding to Auto-BMT, performed in 1st CR. 6/89 (6.7%) patients received Auto-BMT in first relapse. 41 patients relapsed after auto-BMT and were treated with a re-induction chemotherapy, or were enrolled in clinical trials. 24 patients reached a 2nd complete remission, and 12 patients underwent an allogeneic BMT in 2nd CR. With a median follow up of 6 years, the median Overall Survival (OS) of the entire population was 64.3 months (range 5.8-294.2 months); the 1 year OS and the 5 years OS were, 97.1%, and 67.9%, respectively. The median Disease Free Survival (DFS) of the 83 patients treated with Auto-BMT in 1st CR was 36 months (range 1.3-293 months). The 1-year DFS and the 5-years DFS were 85% and 56.7%, respectively. Transplant related mortality (TRM, death in 100 days after BMT) was 1.2% for auto-BMT and 6.5% for allogeneic BMT. First, to assess the role of the number of consolidation courses we compared patients who received none or 1 consolidation course with patients who received 2 or more cycles, who showed a better OS (p= 0.0061, Figure 1). There was no statistical difference in terms of OS between young and elderly patients (cut off=65 years). Second, we compared patients who achieved a negative minimal residual disease status before auto-BMT (n=37) with patients who did not (n=9). MRD negativity offered a significantly better outcome in terms of 5-years OS (83.4% and 50% respectively); the median OS of MRD neg was not yet reached; the median OS of MRD pos was 27 months (p= 0.0130) (Figure 2). Conclusions: Auto-BMT offers a chance to achieve long-term DFS and OS if used as a consolidation therapy both in patients with LR and SR AML. The major role could be played in MRD negative patients, offering the best chances to achieve a long-term OS. Auto-BMT can be also a good choice as consolidation therapy for elderly patients, in which allo-BMT could induce high morbidity and mortality rates. The small patients cohort and the retrospective analysis don't allow us to define the best induction therapy to be used before auto-BMT. However, based on our findings we suggest a therapy schedule including two or more consolidation courses in patients who obtain a first CR, and to proceed then to auto-BMT. Acknowledgments: work supported by ELN, AIL, AIRC, Progetto Regione-Università 2010-12 (L.Bolondi), Fondazione del Monte di Bologna e Ravenna, FP7 NGS-PTL project. Figure 1. Figure 1. Figure 2. Figure 2. Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Rodeghiero:Celgene Corporation: Honoraria, Research Funding. Cavo:Janssen-Cilag, Celgene, Amgen, BMS: Honoraria. Martinelli:AMGEN: Consultancy; Novartis: Consultancy, Speakers Bureau; Ariad: Consultancy; BMS: Consultancy, Speakers Bureau; ROCHE: Consultancy; Pfizer: Consultancy; MSD: Consultancy.

Abstract 2497 Introduction: The gene TP53 encoding the tumour suppressor protein p53 is among the most commonly mutated genes in human cancer. TP53 tumour-associated alterations often cause dramatic defects in p53 function and correlate with increased malignancy, dismal survival and resistance to treatment. In contrast, only a small fraction, if any, of the >200 single nucleotide polymorphisms (SNPs) of TP53 in human populations are expected to cause measurable perturbation of p53 function. Aim: Since the pattern, frequency and significance of TP53 aberrations and SNPs in adult BCR-ABL1 positive ALL have still to be investigated, in this study we used a massively parallel pyrosequencing technique to address these issues. Patients and methods: Forty-three adults with BCR-ABL1 positive ALL were analyzed (median age 63 years, range 18–84). Twenty-four cases (56%) were analyzed only at the time of diagnosis, four cases (9%) only at the time of relapse and fifteen cases (35%) at both time points. Massively parallel pyrosequencing in picoliter-sized wells was used to allow highly-sensitive deep-sequencing detecting molecular aberrations at a low burden rate. As part of the IRON (Interlaboratory RObustness of Next generation sequencing) II study network, we applied preconfigured plates including primers for TP53 (exons 4 to 11) and allowing the simultaneous screening of 11 patients, each being recognized by a unique molecular identifier. For each plate, after generating 88 amplicons, 8 per each patient, PCR products were purified using Agencourt AMPure XP beads and Biomek 3000 Laboratory Automation Workstation (Beckam Coulter) and quantified using the Quant-iT PicoGreen kit (Invitrogen). All amplicons were pooled in an equimolar ratio to generate one single library. Subsequent emulsion PCR and amplicon sequencing was performed according to the manufacturer's recommendations on the Genome Sequencer Junior Instrument (Roche Applied Science). Data were analyzed using the GS Amplicon Variant Analyzer software version 2.7 (Roche Applied Science). For the detection of variances, filters were set to display sequence variances occurring in more than 5% of bidirectional reads per amplicon in at least one sample. Results: On average, we generated 63,068 sequencing beads (key pass wells) per plate (range, 50,798-79,486) with a median read length range between 284 and 365 base pairs. The median number of generated reads per case was 5,413 (range, 687-9,604). The median number of reads per amplicon was as follows: exon 4: 275 (range, 0–888), exon 5: 222 (range, 0–1,013); exon 6: 316 (range, 84–854); exon 7: 317 (range, 5–720); exon 8: 313 (range, 0–784); exon 9: 215 (range, 0–785); exon 10: 328 (range, 0–826), exon 11: 447 (range, 0–1,511). Forward and reverse reads were homogeneously distributed allowing a sensitive detection of variances. In total, 8 single nucleotide variations were identified. All variances, except for one nucleotide substitution occurring at position 7576743 (GRCh37/hg19), were found to represent SNPs according to the NCBI dbSNP Build 137. They included: rs1042522 C/G (41/43, 95%) and rs1800370 A/G (1/43, 2%) in exon 4, rs1800372 A/G (2/43, 5%) in exon 6, rs1625895 A/G (42/43, 98%) in intron 6–7, rs12947788 A/G (3/43, 7%) and rs12951053 A/C (4/43, 9%) in intron 7–8 and rs1800899 C/T (1/43, 2%) in intron 9–10. Interestingly, in 2 cases (12%) loss of heterozygosity occurred at the relapse at the C wild-type allele of rs1042522 in leukemia cells. The same mechanism has been identified for one case at the wild-type allele of rs1625895 with the expansion of the variant form at relapse. Both rs1042522 and rs1625895 have been described to alter p53 functionality and increase susceptibility to cancers (Whibley et al.,2009). Although the role of rs12947788 and rs12951053 has not yet deeply investigated, in our study they were found in 3 cases that all relapsed. Conclusion: Comprehensive next generation deep-sequencing of TP53 by a screening assay set up within the IRON II study has demonstrated its ability to efficiently detect TP53 variant. The inactivation of the wild-type allelic forms of rs1042522 and rs1625895, altering the p53 functionality, may serve as an important background for leukemia progression in BCR-ABL1-positive ALL. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment. Luppi:CELGENE CORPORATION: Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Equity Ownership. Baccarani:ARIAD, Novartis, Bristol Myers-Squibb, and Pfizer: Consultancy, Honoraria, Speakers Bureau. Martinelli:NOVARTIS: Consultancy, Honoraria, Speakers Bureau; BMS: Consultancy, Honoraria, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

Background: FLT3 internal tandem duplication (ITD) is frequently detected in AML patients and is an independent predictor of unfavourable outcome, while secondary point mutations in the FLT3 tyrosine kinase domain (KD) are common causes of acquired clinical resistance to FLT3 inhibitors, such as AC220 and Sorafenib. Technologies allowing massively parallel, ultra-deep sequencing (UDS) are currently being evaluated in diagnostic settings since they may conjugate throughputness, sensitivity and accurate quantification of mutated clones. Aim: Since recent whole genome sequencing studies have suggested that FLT3 ITD may evolve from small subclones undetectable at diagnosis by routine PCR, we tested the ability of an UDS strategy for FLT3 mutation screening to highlight small clones harbouring ITD mutations. Furthermore we evaluated if an UDS strategy could highlight in AML patients treated with FLT3 inhibitors emerging clones harbouring critical mutations, anticipating the development of drug-resistance. Methods: 886 AML patients were analyzed in Seràgnoli Institute of Bologna between 2002 and 2013 for a panel of genetic alterations, including FLT3. For our purpose, we retrospectively analyzed five AML (four CN-AML and one t(3;3) AML) who were found negative for FLT3 ITD- at diagnosis by conventional PCR and Sanger Sequencing, but were then found FLT3 ITD+ during follow-up at relapse or disease progression and ten AML (five FLT3+ and five FLT3-) treated with the FLT3 inhibitor AC220. In order to reconstruct the dynamic of mutation emergence, we performed a longitudinal re-analysis of RNA samples with UDS on a Roche GS Junior. UDS achieved a lower detection limit between 0,1% and 1%, depending on the relative number of sequence reads per sample obtained in each run. Results: 886 AML patients were analyzed for a panel of genetic alterations including FLT3 ITD and TKD and 239 of 886 (27%) were FLT3+. In particular 256 were cytogenetically normal (CN) AML and of these 66 (25,8%) were FLT3+ and 192 were FLT3-.UDS strategy revealed that all the CN-AML analyzed already carried at diagnosis a small clone FLT3 ITD+ (allelic ratio 0.2-2%), that increased over time during follow-up, while in the t(3;3) AML the ITD clone emerged only at disease progression. In the three CN patients treated with chemotherapy the ITD+ was a minor clone until complete remission (CR), while after relapse the ITD+ clone expanded; one of these patients carried two rare ITD clones at diagnosis and only one became dominant at relapse, likely through loss of heterozygosity (LOH) of the mutated allele. In one CN patient who was treated only with FLT3 inhibitor AC220 the allelic load of the mutated clone increased over time before treatment and then followed the dynamic of the disease (regression at complete remission). For the five AML FLT3- treated with AC220 we didn’t find any novel FLT3 mutation after treatment, while for the five AML FLT3-ITD+ analyzed, we were able to follow the allelic load of the FLT3-ITD+ clone during treatment and the appearance in two patients of novel TKD mutations after treatment that are able to confer drug resistance (allelic ratio 2-55%). Conclusions: The high sensitivity of UDS technology allows detection of emergence of mutated clones earlier than conventional methods: this is a precious tool to find small clones FLT3 ITD+ that may evolve over time and worsen the prognosis of otherwise good prognosis CN-AML patients and to better calibrate therapy for these patients. Furthermore the prognostic value of determining the presence of FLT3-ITD by UDS is stronger than conventional methods because of the possibility to determine the ratio of mutated versus wild-type allele, the length and the size of insertion within a single analysis. In the setting of FLT3 inhibitors, UDS gives advantage in monitoring MRD by determining exactly the allelic load of mutated FLT3 ITD clones before and after treatment and high sensitivity in highlighting emerging mutated clones during treatment that may confer resistance, giving possibility to switch eventually to other inhibitors before coming out of overt clinical resistance to therapy. For monitoring patients treated with FLT3 inhibitors with UDS we will go on by screening AML treated with Sorafenib, to follow the emergence of any novel critical mutation during treatment. Acknowledgments: ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Martinelli: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

Introduction High-resolution genome-wide profiling analysis of B-cell precursor acute lymphoblastic leukemia (BCP-ALL) samples has identified many novel somatic genetic alterations, several of which have clear implications for risk stratification or future therapeutic targeting. However, most of the studies focused on children and therefore a deep molecular characterization of adults is still challenging, especially for those cases lacking recurrent fusion genes. Subjects and Methods In order to shed light on the molecular features of this ALL subgroup, we retrospectively analyzed 28 newly diagnosed BCR-ABL1-negative BCP-ALL subjects (19 males/9 females; median age 41.5 years; negative for known fusion genes) and 28 BCR-ABL1-positive BCP-ALL subjects as a comparison group, since it represents the most frequent genetic subgroup in adults with ALL. In BCR-ABL1-negative ALL karyotype was normal in 10/28 (36%), showed abnormalities in 5/28 (18%) and failed or was not available in 13/28 (46%) cases. The overall survival rate was very poor with a median of 14 months (range, 1-75). We analyzed copy number alterations (CNA) of IKZF1, CDKN2A/B, PAX5, EBF1, ETV6, BTG1, RB1, and genes within PAR1: CRLF2, CSF2RA, IL3RA by the SALSA MLPA kit P335 IKZF1 (MRC Holland). In addition, mutation status was assessed for TP53, CRLF2, JAK2, LEF1, PAX5 and IL7R by next-generation deep-sequencing (NGS) (Roche Applied Science; IRON-II study oligonucleotide primer plates). Positivity for newly described BCR-JAK2, PAX5-JAK2, ETV6-ABL1, EBF1-PDGFRB, NUP-ABL1 gene fusions occurring in BCR-ABL1-like ALL (Roberts KG et al., Cancer Cell. 2012) was assessed by PCR amplification and sequencing. Finally, SNP arrays (SNP 6.0, Affymetrix) and gene expression profile analyses (GeneChip® Human Transcriptome Array 2.0) were performed to more fully assess genomic complexity. Results Overall, 76% of BCR-ABL1-negative subjects showed an abnormality of at least one of the analyzed genes: 7 (25%) had one, 4 (14%) had two, 6 (21%) had three, and 6 (21%) had four or more alterations. In subjects showing no abnormalities, SNP arrays analysis revealed amplifications of chromosome 1q in 2/6 cases (33%). Deletions of CDKN2A/B were the most frequent (39%) and in 73%, they occurred together with other abnormalities, suggesting that multiple events are needed to induce the full leukemia phenotype. Other common CNA included: deletions of IKZF1 (25%), ETV6 (25%), PAX5 (14%), EBF1 (11%), PAR1 region (11%) and RB1 (7%). NGS showed mutations of TP53 in 18% of cases (W147*, V172L/G, G245C, Del244-246, D259Y), while JAK2 and CRLF2 were mutated in 7% (R683S/G) and 4% (F232C), respectively. No positivity for newly described fusion genes activating tyrosine kinase was confirmed. Importantly, subjects with no abnormalities showed better survival rates compared to those with one or more molecular alterations (p < 0.01). The BCR-ABL1-positive subgroup shared the same CNA of BCR-ABL1-negative cases, such as deletions of IKZF1 (71%), CDKN2A/B (21%), PAX5 (14%), BTG1 (11%), EBF1 (11%), and ETV6 (4%), but they did not show mutations in the analyzed genes. Conclusions BCP-ALL lacking recurrent fusion genes is a highly heterogeneous and complex disease. Current diagnostic procedures need to be revised to improve risk assessment and to guide therapeutic decisions. Supported by AIL, AIRC, PRIN 2010-2011, Programma Ricerca Regione-Università 2010-2012, FP7 “NGS-PTL” project. Disclosures: Soverini: Bristol-Myers Squibb: Consultancy; Novartis: Consultancy; ARIAD: Consultancy. Chiaretti:Roche Diagnostics: Research Support Other. Kohlmann:MLL Munich Leukemia Laboratory: Employment; Roche Diagnostics: Honoraria. Martinelli:Novartis: Consultancy, Speaker fees Other; Bristol-Myers Squibb: Consultancy, Speaker fees, Speaker fees Other; Pfizer: Consultancy, Speaker fees, Speaker fees Other; Ariad: Consultancy, Speaker fees, Speaker fees Other.

Background: AML is a heterogeneous disease. The karyotype provides important prognostic information that influences therapy and outcome. Identification of AML patients (pts) with poor prognosis such as those with complex karyotype (CK) has great interest and impact on therapeutic strategies. TP53 is the most frequently mutated gene in human tumours. TP53 mutation rate in AML was reported to be low (2.1%), but the incidence of TP53 mutations in AML with a complex aberrant karyotype is still debated. Aims: To investigate the frequency of TP53 mutations in adult AML pts, the types of mutations, the associations with recurrent cytogenetic abnormalities and their relationship with response to therapy, clinical outcome and finally their prognostic role. To this aim, we focused on a subgroup of TOT/886 AML pts treated at the Serˆgnoli Institute of Bologna between 2002 and 2013. Patients and Methods: 886 AML patients were analysed for morphology, immunophenotype, cytogenetic and for a panel of genetic alterations (FLT3, NPM1, DNMT3A, IDH1, IDH2 mutations, WT-1 expression, CBF fusion transcripts). Of these, 172 adult AML pts were also examined for TP53 mutations using several methods, including Sanger sequencing, Next-Generation Deep-Sequencing (Roche) and HiSeq 2000 (Illumina) platform. 40 samples were genotyped with Genome-Wide Human SNP 6.0 arrays or with CytoScan HD Array (Affymetrix) and analysed by Nexus Copy Numberª v7.5 (BioDiscovery). Results: Of the 886 AML patients, 172 pts were screened for TP53 mutations. Sanger sequencing analysis detected TP53 mutations in 29/172 AML patients with 36 different types of mutations; seven pts (4%) had 2 mutations. At diagnosis, the median age of TP53 mutated and wild type patients was 68 years (range 42-86), and 65 years (range 22-97) respectively. Median WBC count was 8955/mmc (range 580-74360/mmc) and 1240/mmc (range 400-238000/mmc). Conventional cytogenetics showed that: a) 52 pts (30,2%) had 3 or more chromosome abnormalities, i.e. complex karyotype; b) 71 (41,3%) presented with one or two cytogenetic abnormalities (other-AML); c) 34 pts (19,8%) had normal karyotype. Most of the TP53 mutated pts (23/29, 79.3%) had complex karyiotype, whereas only 6/29 mutated pts had “no complex Karyotype” (21% and 3% of the entire screened population, respectively). Overall, TP53 frequency was 44.2% in the complex karyotype group, suggesting a pathogenetic role of TP53 mutations in this subgroup of leukemias. As far as the types of TP53 alterations regards, the majority of mutations (32) were deleterious.. Copy Number Alterations (CNAs) analysis performed on 40 cases by Affymetrix SNP arrays showed the presence of several CNAs in all cases: they ranged from loss or gain of the full chromosome (chr) arm to focal deletions and gains targeting one or few genes involving macroscopic (>1.5 Mbps), submicroscopic genomic intervals (50 Kbps - 1.5 Mbps) and LOH (>5 Mbps) events. Of relevance, gains located on chr 8 were statistically associated with TP53 mutations (p = 0.001). In addition to the trisomy of the chr 8, others CNAs, located on chromosomes 5q, 3, 12, 17 are significantly associated (p = 0.05) with TP53 mutations. WES analysis was performed in 37 pts: 32 TP53 were wt while 5 pts were TP53 mutated. Interestingly, TP53 mutated patients had more incidence of complex karyotype, more aneuploidy state, more number of somatic mutations (median mutation rate 30/case vs 10/case, respectively). Regarding the clinical outcome, as previously reported (Grossmann V. et Al. Blood 2013), alterations of TP53 were significantly associated with poor outcome in terms of both overall survival (median survival: 4 and 31 months in TP53 mutated and wild type patients, respectively; p Figure 1: Overall Survival curve of 172 AML patients with (red) or without (blue) TP53 mutations (p< 0.0001). Conclusions: Our data demonstrated that TP53 mutations are more frequent at diagnosis in the subgroup of complex karyotype AML (16.86%) (p< 0.0001–Fisher's exact test). They are mostly deleterious mutations and are significantly correlated with worst prognosis, fail to respond to therapy and rapidly progress. We recommend TP53 mutation screening at least in AML pts carrying either complex karyotype or chr. 8 gain. Supported by: ELN, AIL, AIRC, PRIN, progetto Regione-Universitˆ 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures No relevant conflicts of interest to declare.

Introduction PI3P is a key regulator of 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). PI3P is also a core activator of autophagy. Which role autophagy plays in cancer is not well established; it can function as a pro-apoptotic mechanism, or it can improve survive to stresses clearing damaged mitochondria and proteins accumulation, preventing apoptosis. Levels and activity of pro-apoptotic and anti-apoptotic proteins, particularly bcl-2 and p53, membrane signaling via mTOR, high levels of cAMP, a complex made by pink/park, promote a switch from apoptotic autophagy toward a mechanism that augment cell resiliency. Our study aims to define the role of PI3P pathways in AML, and to establish if autophagy could reduce the patients' chance to respond to induction, and to worsen OS. Methods 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. In all the patients, we perform Microarray-based Comparative Genomic Hybridization with Affymetrix SNP array 6.0 or Cytoscan HD; we perform 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 analyzed 4 pathways (Table 1); we selected genes in pathways basing on literature and GO data. Alterations in these pathways involved 103/209 patients (48%). PI3K/AKT/mTOR pathway alterations (both gains or losses) were shown to confer worst OS (p = .035, Figure 1a) when compared with unaltered patients; events in these pathways did not affect therapy response. Autophagy pathway alterations were shown to confer worst OS (p AMPK pathway alterations were shown to confer worst OS (p Autophagy switch pathway confer worst OS to patients(p Having at least an altered pathway is associated with worst prognosis (p WES in a sub-cohort of patients did not found any significant mutation in genes we analyzed. This data is consistent with literature. Conclusions Our work investigates for the first time the role of PI3P pathways and autophagy in AML. Surprisingly, it showed that both positive and negative alterations in these pathways are associated with poor prognosis. Significantly, alterations in cAMP and autophagy pathways were associated with therapy resistance. These results point out that both positive and negative regulation of autophagy could worsen patients OS; a diminished autophagy could be linked to a hyper-proliferative state via activation of AKT/mTOR but an augmented autophagy could give cell resiliency, favoring cytoplasm turnover, damaged mitochondria elimination, and neutralizing oxidative damages to proteins. A pan-PI3K inhibitor could target these mechanisms and improve chemo-sensitivity in high risk AML. Acknowledgment: ELN, AIL, AIRC, PRIN, Progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Guadagnuolo: CellPly S.r.l.: Employment. Soverini:Ariad: Consultancy; Bristol-Myers Squibb: Consultancy; Novartis: Consultancy. Martinelli:Novartis: Speakers Bureau; MSD: Consultancy; Ariad: Consultancy, Speakers Bureau; BMS: Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Genentech: Consultancy; Roche: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau.

Introduction. Conventional induction treatment in young non APL-Acute Myeloid Leukemia (AML) patients is still represented by the association of an antracycline and Cytarabine, which offers a complete remission (CR) rate not inferior to 60%. The addition of Gemtuzumab Ozogamicin (GO) as a third or fourth drug, already demonstrated to improve clinical outcome, in terms of CR rates. Aims of the study. We retrospectively evaluated and compared the efficacy of different induction schedules, in terms of CR rates and Overall Survival (OS), administered to two groups of AML patients. Group 1 (n=139) was treated with a GO containing course (MyFLAI or MyAIE schedules); Group 2 (n=270) received a non-GO based regimen including or not Fludarabine (FLAI, FLAN, FLAG, 3+7 or DAE). Patients and Methods. From 1997 to 2014,409 newly diagnosed AML patients were treated in 3 Italian Institutions. Their median age was 53 (range 19-74) and 52 (range 17-75) years, respectively. According to karyotype (performed in 392/409 patients), FLT3 (available for 244/409 patients), and NPM1 mutational status (available for 157/409 patients), based on the NCCN-2013 risk stratification criteria, 35.2% of the patients were considered at High Risk (HR) (31.6% and 36.4% in the two groups, respectively) and 7.6% at low risk (LR) (7.8% and 7.0%, respectively). Results. The complete remission (CR) rate after induction was 81.4% and 70.4% for Group 1 and 2, respectively (p=0.01). Deaths during induction (DDI), occurring in the first 50 days from 1st line therapy, were 4/139 (2.9%) in Group 1 and 22/270 (8.1%) in Group 2 (p=0.003). Patients treated with GO showed a better OS than patients of Group 2 (Figure 1); the 5-years OS in the two groups was 54.01% and 34.9%, respectively, and different according to age (54.0% and 34.9% respectively (p=0.0003) in patients Then, with a logistic univariate regression analysis.,we explored the impact of GO therapy in terms of CR rate. The use of GO was identified as a strong predictor of the achievement of a 1st CR (p=0.008). Moreover, a logistic multivariate regression analysis (risk and age) confirmed the role of the compound as a predictor of higher CR rate (p=0.013). We also performed a Cox Regression analysis, to investigate the impact of GO therapy, age and risk on OS: the use of GO was confirmed to be an independent predictor of better OS (p In a sub-analysis performed on HR patients, we observed a significantly better outcome in Group 1 than in Group 2 in terms of OS (p=0.0074, 5-year OS 47.7%; in group 2 OS 21.0% respectively, Figure 3) and EFS (p=0.001). However, there was no difference in terms of CR rate (p=ns). In particular, HR patients with adverse karyotype, may benefit from GO induction therapy in terms of OS (5-years OS 42.9% and 12.8% in Group 1 and 2, respectively, p=0.02); nevertheless FLT3 mutations negative impact canÕt be overcome by the drug administration (5-years OS 66.6% and 61.3% in Group 1 and 2, respectively). In SR AML, GO offered a better OS (p=0.036, 5-years OS 51.4% and 41.9% respectively). Comparing with Fisher's exact test the rate of increment in 5-years OS between Group1 and Group2 in HR and SR AML, we demonstrate that treatment with GO gave the higher benefit in HR AML (p=0.0005). Conclusions. These data showed that a four-drugs intensified induction therapy is a feasible approach in AML patients: adding GO at any induction regimen is an independent and strong predictor of better OS and higher CR rates. In our population, GO was not associated with a higher incidence of DDI. This approach, could be strongly recommended in SR and HR AML patients, due to karyotype abnormalities, in which showed an advantage in term of OS if compared with other standard regimens. On the contrary, we donÕt suggest the same schedule in FLT3 mutated patients, in which no benefits have been observed. Acknowledgments Work supported by ELN, AIL, AIRC, Progetto Regione-Universitˆ 2010-12 (L.Bolondi), FP7 NGS-PTL project. Figure 1. Figure 1. Figure 2. Figure 2. Figure 3. Figure 3. Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Fanin:Novartis Farma: Speakers Bureau. Cavo:BMS: Honoraria; Millenium Pharmaceuticals: Honoraria; Jansenn: Consultancy, Honoraria; Sanofi: Consultancy, Honoraria; Onyx: Honoraria; Celgene: Consultancy, Honoraria; Novartis: Consultancy, Honoraria. Martinelli:Pfizer: Consultancy; BMS: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau; ROCHE: Consultancy; MSD: Consultancy; AMGEN: Consultancy; Ariad: Consultancy.