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Single-Photon Avalanche Diodes (SPADs) are p–n junctions operated in Geiger Mode by applying a reverse bias above the breakdown voltage. SPADs have the advantage of featuring single photon sensitivity with timing resolution in the picoseconds range. Nevertheless, their relatively high Dark Count Rate (DCR) is a major issue for charged particle detection, especially when it is much higher than the incoming particle rate. To tackle this issue, we have developed a 3D Silicon Coincidence Avalanche Detector (3D-SiCAD). This novel device implements two vertically aligned SPADs featuring on-chip electronics for the detection of coincident avalanche events occurring on both SPADs. Such a coincidence detection mode allows an efficient discrimination of events related to an incoming charged particle (producing a quasi-simultaneous activation of both SPADs) from dark counts occurring independently on each SPAD. A 3D-SiCAD detector prototype has been fabricated in CMOS technology adopting a 3D flip-chip integration technique, and the main results of its characterization are reported in this work. The particle detection efficiency and noise rejection capability for this novel device have been evaluated by means of a β − strontium-90 radioactive source. Moreover the impact of the main operating parameters (i.e. the hold-off time, the coincidence window duration, the SPAD excess bias voltage) over the particle detection efficiency has been studied. Measurements have been performed with different β − particles rates and show that a 3D-SiCAD device outperforms single SPAD detectors: the former is indeed capable to detect particle rates much lower than the individual DCR observed in a single SPAD-based detectors (i.e. 2 to 3 orders of magnitudes lower).

In this paper, a novel SPAD architecture implemented in a Fully-Depleted Silicon-On-Insulator (SOI) CMOS technology is presented. Thanks to its intrinsic vertical 3D structure, the proposed solution is expected to allow further scaling of the pixel size while ensuring high fill factors. Moreover the pixel and the detector electronics can benefit of the well-known advantages brought by SOI technology with respect to bulk CMOS, such as higher speed and lower power consumption. TCAD simulations based on realistic process parameters and dedicated post-processing analysis are carried out in order to optimize and validate the avalanche diode architecture for an optimal electric field distribution in the device but also to extract the main parameters of the SPAD, such as the breakdown voltage, the avalanche triggering probability, the dark count rate and the photon detection probability. A comparison between the efficiency in back-side and front-side approaches is carried out with a particular focus on time-of-flight applications.

This work describes the development of a novel position sensitive charged particle detector suitable for nuclear physics applications, in the field of High Energy Physics experiments and emerging medical applications such as hadrontherapy and Proton Computed Tomography (pCT). The “3D Silicon Coincidence Avalanche Detector” (3D-SiCAD) pixel consists of a pair of 3D vertically aligned Geiger-mode avalanche diodes (SPAD) working in time-coincidence mode. This novel detector features single charged particle detection capability, compatibility with both standard CMOS technology and commercially available 3D integration techniques, and very low noise. In order to demonstrate this latter point, a first 3D-SiCAD prototype has been designed and fabricated using a standard CMOS technology and a 3D assembly technique based on gold micro-bumps. In a first phase, preliminary measurements over two adjacent “in-plane” avalanche diodes operated in coincidence-mode have demonstrated a very high noise rejection capability, up to 3-4 orders of magnitude lower than the intrinsic dark count rate of each SPAD cell. Afterwards, measurements on a real 3D pixel have been carried out, obtaining however noise rejection capabilities lower than expected. Optical cross-talk occurring between the two sensing level of the 3D-SiCAD pixel has been found to be the phenomenon responsible for this underperformance. Nevertheless this limitation can be easily overcome by interposing a fully absorbing/reflecting material between the two sensing levels. These first results are very encouraging for the realization of a fully CMOS integrated 3D-SiCAD detector, targeting application requiring high-precision tracking, ultra-fast response times and very low noise.

In this paper, a novel SPAD architecture implemented in Silicon-On-Insulator (SOI) CMOS technology is proposed. Thanks to its intrinsic 3D structure, the proposed solution is expected to allow very small pixels while enabling a very high fill factor. Furthermore, the pixel read-out electronics as well as the whole detector electronics can benefit of the well-known advantages brought by SOI technology with respect to bulk CMOS, such as higher speed and lower power consumption. TCAD simulations based on realistic process parameters provided by the foundry are carried out in order to optimize and validate the avalanche diode architecture for an optimal electric field distribution in the device and to obtain a first order estimation of the main parameters of the SPAD, such as the breakdown voltage, the avalanche triggering probability and the dark count rate.

The goal of this paper is to provide some useful design guidelines at the device level regarding the main challenges to be typically faced in the design and integration of Geiger-mode avalanche diodes in a standard CMOS process. Different techniques are found in literature in order to avoid premature edge breakdown with the aim of limiting the electric field at the edges to be weaker than in the multiplication region. In this article, the use of such techniques, the conditions where they can effectively work and above all their limitations are studied by means of TCAD simulations for various diode architectures. Additionally, the noise performance is discussed by focusing on the band-to-band tunneling and shallow trench isolation enhanced dark count rates. Geiger-mode bias techniques as well as a synthesis on the pros and cons of the various avalanche diode architectures are finally presented aiming at facilitating future design choices.

In this paper, a time-integration based passive quenching — active recharge circuit for Geiger-mode avalanche diodes has been proposed with the aim of minimizing the avalanche charge and providing a hold-off time tunable within wide range. These are indeed important features to be taken into account in the design of the avalanche diode quenching — reset electronics. Furthermore a hold-off time tunable within a wide range is typically desirable in every application where a full characterization of the device dark count rate is required. A correct operation of the proposed circuit, designed in a commercial High-Voltage CMOS 0.35um technology, is assessed through circuit simulations as well as Monte Carlo analysis in the Cadence Environment.

International audience; In this paper a preliminary study of coincidence Avalanche Pixel Sensors (APiX) for High Energy Physics (HEP) applications is presented. In this preliminary work, some PEB prevention techniques found in literature have been studied by TCAD simulations adopting 2D Cylindrical geometrical models and 130nm CMOS process technological data.

Introduction: Little is known about alterations of cyclin dependent kinase inhibitors p15INK4B, p16INK4A and p14ARF due to single nucleotide polymorphisms (SNPs) located within the CDKN2A/B genes and/or neighbouring loci. In order to investigate the potential involvement of such common DNA sequence variants in leukemia susceptibility, an association study was performed. Methods: 23 SNPs spanning the MTAP, CDKN2A/B and CDKN2BAS loci, as well as relative intergenic regions were genotyped in a case-control cohort made up of 149 leukemia patients, including Philadelphia positive (Ph+) ALL and acute myeloid leukemia (AML) samples, and 183 healthy controls. 6 SNPs were selected on the basis of their previous association with several diseases, such as coronary artery disease (rs2891168, rs518394, rs564398, rs10757278), type 2 diabetes mellitus (rs564398), frailty (rs2811712). The remaining 17 SNPs were selected to deepen the SNPs coverage for the examined region. Genotyping was performed using iPLEX Gold technology and MassARRAY high-throughput DNA analysis with Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (Sequenom, Inc., San Diego, CA). Results: A total of 17 SNPs, spanning the 9p genomic interval that encompasses the MTAP, CDKN2A/B and CDKN2BAS loci, were successfully genotyped and used for investigating their potential associations with the leukemia phenotypes. Five SNPs (rs1012713, rs10965179, rs34011899, rs3731232, rs3218010) with MAF 20% missing call rates, were instead excluded from the association analysis and potential population stratification affecting the control sample was ruled out as its genotypes distribution satisfies the Hardy-Weinberg equilibrium criterion. Among the 17 SNPs, rs564398, mapping to the CDKN2BAS locus (exon 2) that encodes for ANRIL antisense non-coding RNA, showed a statistically significant correlation with the ALL phenotype, with a risk pattern that was compatible with an overdominant model of disease susceptibility and a OR of 2 (95% CI, 1.20 to 3.33; p= 7.1 x 10-3). Conclusions: Since a co-ordinated regulation of ANRIL and p14/ARF, p16/CDKN2A, p15/CDKN2B transcription has been already observed in both physiologic and pathologic conditions, we hypothesized that rs564398 association reflects a condition of high linkage disequilibrium between such polymorphism and a causative variant that is able to alter CDKN2A/B expression profiles by changing ANRIL dosage, thus leading to abnormal proliferative boosts and consequent increased ALL susceptibility. Recently, the rs564398 has been demonstrated by Cunnington M.S. et al. (2010) to alter ANRIL expression leading in turn to a deregulation of CDKN2A/B genes. Supported by European LeukemiaNet, AIL, AIRC, FIRB 2006, Ateneo RFO grants, Project of integrated program (PIO), PRIN 2008, Programma di Ricerca Regione – Università 2007 – 2009.

Background: Inter-individual variations in genes encoding drug metabolizing enzymes and transporters have been demonstrated to influence the response to therapy. However so far, how these genetic variations interact to produce specific drug related phenotypes in Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) has not yet been investigated. Aim: In order to investigate potential genetic structure and related pharmacogenetic profiles, around 2000 variants in more than 200 genes involved in drug absorption, distribution, metabolism and elimination were genotyped and studied with a population genetics approach in 45 Ph+ ALL patients. Methods: The Drug Metabolizing Enzymes and Transporters (DMET™, Affymetrix) platform, covering more than 90% of the most biologically relevant drug absorption, distribution, metabolism and excretion (ADME) markers was used for successfully genotyping 1931 variants in Ph+ ALL patients treated with the tyrosine kinase inhibitor Dasatinib. A model-based clustering method for inferring population structure using genotype data was applied by means of the Structure software assuming a model in which there are K populations - each one of them being characterized by a set of allele frequencies at each locus - to which individuals are probabilistically assigned according to their genotypes. Distribution of the genetic variance observed among the identified leukemia sub-groups was investigated with a locus by locus Analysis of the Molecular Variance (AMOVA) by means of the Arlequin 3.01 package, exploiting information on genotypes allelic content and frequencies. Results: Three different sub-groups (G1, G2, G3), made up of 2, 12 and 31 patients respectively, were identified in the examined ALL sample, according to their different patterns of allele frequency. A statistical support for this finding was provided by AMOVA results which pointed out a substantial level of genetic differentiation among G1 and the other two sub-groups (Fst = 0.099, p0.3) and significant Fst values; whereas a total of 50 loci, located on the NAT2, VKORC1, CYP4F2, CYP2B6, UGT2B7 and CYP2D6 genes, showed moderate to high (>0.08) significant Fst values in the G2/G3 comparison. Conclusions: Differences of allele frequencies observed among the identified ALL sub-groups prove that an evident genetic structure is detectable in our sample by genotyping loci involved in drug metabolism. Supported by: Fondazione GIMEMA Onlus, European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, Ateneo RFO grants, Project of integreted program (PIO), Programma di Ricerca Regione – Università 2007 – 2009.

Introduction. The BCR-ABL1 fusion gene defines the subgroup of acute lymphoblastic leukaemia (ALL) with the worst clinical prognosis. Aim and Methods. To identify oncogenic lesions that combine with BCRABL1 to cause ALL, we used Affymetrix Genome-Wide Human SNP arrays (250K NspI and SNP 6.0) and genomic PCR to study 106 cases of adult BCR-ABL1-positive ALL. Results. The most frequent somatic copy number alteration was a deletion on 7p12 of IKZF1, which encodes the transcription factor Ikaros and was identified in 80 of 106 patients (75%). Two major deletions occurred: the first one (D4-7) was characterised by a loss of exons 4 through 7 (44/106, 42%), and the second one (D2-7) that we cloned was characterised by removal of exons 2 through 7 (20/106, 19%). A variable number of nucleotides (patient-specific) were inserted at the conjunction and maintained with fidelity at the time of relapse. The extent of the D4-7 deletion correlated with the expression of a dominant-negative isoform with cytoplasmic localisation and oncogenic activity, while the D2-7 deletion resulted in a transcript lacking the translation start site. The IKZF1 deletion was also identified in the progression of chronic myeloid leukaemia (CML) to lymphoid blast crisis (66%), but never in myeloid blast crisis or chronic phase CML, or in acute myeloid leukaemia patients. Known DNA sequence and structural features were mapped along the breakpoint cluster regions including heptamer recombination signal sequences (RSS) recognised by RAG enzymes during V(D)J recombination, suggesting that IKZF1 deletions could arise from aberrant RAG-mediated recombination. Using gene-set enrichment analysis to compare the gene-expression signatures of patients with IKZF1 deletion versus not-deleted patients, we identified a unique signatures characterized by down-regulation of genes involved in pre-B-cell differentiation (e.g. VPREB1, VPREB3, IGLL3, BLK) and upregulation of genes involved in cell-cycle progression (STK17B, SERPINB9, CDKN1A). These findings suggest that genetic alterations of IKZF1 influence the transcriptome and contribute to an impaired B-cell differentiation. We next investigated whether the IKZF1 deletions associated with a poor outcome. Univariate analysis showed that the IKZF1 deletion is a negative prognostic marker influencing the cumulative incidence of relapse (10.1 months for pts with deletion vs 56.1 months for wild-type pts, p=0.0103) and disease-free survival (DFS) (10.1 months vs 32.1 months, respectively, p=0.0229). The negative prognostic impact of the IKZF1 deletion on DFS was also confirmed by multivariate analysis (p=0.0445). Conclusions. Deletion of IKZF1 is an important event in the development of BCR-ABL1 B-progenitor ALL which significantly influences clinical outcome. It is likely that Ikaros loss combines with BCRABL1 to induce lymphoblastic leukaemia, arresting B-lymphoid maturation. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Strategico di Ateneo, GIMEMA Onlus, Fondazione del Monte di Bologna e Ravenna, PIO project 2007.

Several trials have shown the superior impact of high-dose melphalan (usually 200 mg/m(2), MEL200) vs standard therapy in myeloma patients. Intermediate-dose melphalan (100 mg/m(2), MEL100) is also superior to the standard dose, but has not been clinically compared with MEL200. A total of 90 patients at diagnosis were treated with two MEL100 courses. Their clinical outcome was compared with that of a control group of 90 pair mates matched for serum beta2-microglobulin levels and Durie and Salmon clinical stage. These patients were treated at diagnosis with two MEL200 courses. Patient characteristics were similar in both groups except that the median age of the MEL100 group was significantly higher (P0.0001). Complete remission was 35% after MEL100 and 48% after MEL200 (P=0.08). Median event-free survival (EFS) was 32 months in the MEL100 group and 42 months in the MEL200 group (P0.005), but overall survival (OS) was not different. Transplant-related mortality was not significantly different. Haematological and extra-haematological toxicity was significantly reduced after MEL100. Despite the significant age difference, tandem MEL100 was less toxic than tandem MEL200, and MEL100 was inferior to MEL200 in terms of EFS but not in terms of OS. The intensified nonmyeloablative MEL100 regimen is an effective first-line treatment.

The clinical guideline used in daily medical practice is the result of a complex process based on the definition, enactment and evaluation of a clinical trial. The paper illustrates the life cycle of a clinical trial, the process of its definition, the interrelation of descriptive elements using a logical-semantic and temporal relationship, as well as the integrated schema of a data dictionary.

The potential role of autologous stem cell transplantation (ASCT) as an alternative therapeutic strategy in chronic myelogenous leukemia (CML) has been widely explored in pilot studies, but the clinical results in terms of survival have so far been evaluated only retrospectively and in heterogeneous groups of patients. The goal of our prospective study was to evaluate the feasibility and long-term efficacy of unmanipulated ASCT followed by low-dose interferon-alpha in a homogeneous group of patients affected by CML in a very early phase of disease.Twenty-six unselected consecutive patients with CML in chronic phase underwent stem cell collection at diagnosis, then received cytoreductive treatment with hydroxyurea and, subsequently, a busulphan-melphalan myeloablative regimen followed by unmanipulated stem cell graft within one year of diagnosis. Interferon was given a median of 6.5 months after transplant at escalating doses, starting from 0.5 x 10(6) IU 3 times/week, on the basis of the clinical and hematologic tolerance.Median chronic phase duration from diagnosis is 9 years. The ten-year projected probability of overall survival from diagnosis is 55% with a median follow-up of surviving patients of 9.5 years (8-10.5); median survival has not been reached after ten years.Our experience suggests that high-dose therapy followed by unmanipulated peripheral blood stem cell transplantation and low-dose interferon-alpha is a feasible approach, which results in long-term survival in newly diagnosed CML patients. These data need to be confirmed in controlled trials comparing ASCT with other therapeutic approaches, such as the use of interferon-alpha alone or in combination with other agents.

The clinical trial has to be rigidly followed because it identifies a uniform clinical behaviour, which has to be adopted by the different physicians carrying out the test. The life cycle of the clinical trial is therefore based on a planning and definition phase, a next experimental phase connected with its diffusion to the involved centres and finally an evaluation phase of the results. An information system, which supports the users in the different phases of the clinical trial life cycle, has to take into account the different characteristics of each phase. The aim of this paper is to illustrate the role of WF technology as a component of an information system which supports the life cycle of a clinical trial also on the basis of the experience of the Italian Group for Haematological Disease of Adults (GIMEMA).

BACKGROUND AND METHODS. The usefulness of hematological parameters provided by the Technicon H*1 was investigated for the diagnosis of acute lymphoblastic leukemias (ALL) and for the identification of blast cells in peripheral blood samples.When automated blast detection was compared with microscopic examination in 62 samples with 4-98% morphologically recognizable lymphoblasts on peripheral blood smears, a false-negative result occurred in 28% (17/62) of the ALL cases. However, in 14 of these 17 false-negative cases the H*1 generated an output listing that would require microscopic evaluation in any event. The H*1 output listing interpretation (based on abnormal leukograms, blast flags and bicytopenia) led us to suspect the presence of ALL in 76% (47/62) of the cases. Three ANLL were recognized as ALL. In three out the 62 ALL, the H*1 output listing interpretation missed identify a leukemic process. In most cases (77%) we noted a characteristic deformation of the mononuclear population profile in the baso/lobularity display, and the cloud of analytical points was shifted toward to the left, in keeping with the presence of a large number of hypodense nuclei.

A neurological and electroencephalographic (EEG) prospective study was performed in 34 leukemic patients receiving busulfan (BU) plus cyclophosphamide (CY) before bone marrow autologous transplant. During BU treatment and briefly thereafter, all patients were given anticonvulsant prophylaxis with phenobarbital. Neurological evaluations were performed daily and EEGs, recorded one week before and soon after the end of the BU regimen, were re-evaluated two months later. Basal EEGs were normal in 23 of the 34 patients, focal abnormalities were detected in 9 cases, and generalized epileptic discharges were present in one (one subject was not studied). EEGs performed at the end of BU administration showed generalized spike/polyspike-and-wave discharges in 21 of the 34 patients, with associated myoclonic epilepsy in 10 subjects appearing on the 3rd or 4th day of treatment. Focal abnormalities were present in 6 patients and constant EEG normality in 7. It is known that myoclonic epilepsy can be induced by drugs. Oral administration of high-dose BU is followed by a high-dose cerebrospinal fluid concentration of the drug. Therefore, myoclonic epilepsy and/or paroxysmal epileptiform EEG discharges may be observed in leukemic patients undergoing high-dose BU therapy.

The authors have evaluated the influence of some obstetric events (type and duration of labour and method of delivery) and some pharmacological factors (oxytocic drugs during labour, local infiltration, duration of general anaesthesia, oxytocic drugs and/or ergot derivatives during puerperium) on frequency of breast-feeding at the time of discharge from hospital. About 57% of 356 newborns were breast-fed (wholly or prevalently) at discharge from the "A. Gemelli" University Hospital in Rome, during two months period (march-april 1981). The results showed that all those conditions requiring medical and paramedical action, either instrumental or pharmacological (such as: induced labour, prolonged labour, operative delivery either vaginal or abdominal, particularly in emergency; use of local infiltration; use of oxytocic drugs during labour, particularly for induction; prolonged general anaesthesia in caesarean section) may have an unfavorable effect, nearly always significant, on breast-feeding. According with these results, the Authors suggest the following measures for breast-feeding promotional programme: modification of neonatal routine care to promote an earlier mother-infant interaction during lying-in; limitation of avoidable obstetric procedures and of drugs giving to mothers; a better understanding of pharmacokinetics, both in mothers (placentar passage of drugs and through milk) and in newborns during labour, delivery and puerperium.

We performed 56 HDIVG administrations in adults (26) and children (30) affected by idiopathic thrombocytopenic purpura with two different schedules: a five-day course at 400 mg/kg/d and a "bolus" 1 gr/kg. Forty-five cases were treated in preparation for surgical operations and eleven for bleeding episodes. Response to the five-day course in adults was good in 3/24, fair in 13/24, poor in 8/24, while in children it was good in 14/26, fair in 9/26, poor in 3/26. The difference between adults and children is statistically significant. None of the responder patients submitted to surgical operation needed platelet infusions during surgery. Response to the "bolus" schedule (6 cases) in adults was poor in 1 case and good in the other one, and in children, fair in 3 cases and good in the other one. In responder patients treated for bleeding episodes we obtained clinical improvement with hemorrhage arrest. In adults the treatment was well-tolerated, while in children we observed 3/30 mild side effects and 1/30 important side effect.

Abstract 2574 Recently, using genome-wide single nucleotide polymorphism arrays and gene candidate deep exon sequencing, we identified lesions in CDKN2A gene, encoding p16/INK4A and p14/ARF tumor suppressors, in 27% (32/117) adult newly diagnosed Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) patients and in 47% (14/30) relapsed cases. Clinically, in our cohort CDKN2A deletions were associated by univariate analysis to a worse outcome in terms of overall survival (OS), disease-free survival (DFS) and cumulative incidence of relapse (CIR) (OS: 27.7 vs 38.2 months, p = 0.0206; DFS: 10.1 vs. 56.1 months, p = 0.0010; CIR: 73.3 vs 38.1, p = 0.0014). Noteworthy, the negative prognostic impact of CDKN2A deletion on DFS was also confirmed by the multivariate analysis (p = 0.0051). These results showed that there are genetically distinct Ph+ ALL patients with a different risk of leukemia relapse and that testing for CDKN2A alterations at diagnosis may help in risk stratification. Furthermore, since the loss of CDKN2A eliminates the critical tumor surveillance mechanism and allows proliferation and tumor cell growth by the action of MDM2, a negative regulator of p53, we investigated the preclinical activity of the MDM2 antagonist RG7112 in primary B-ALL patient samples and leukemic cell line models. BV-173, SUPB-15 and K562 Ph+ cell lines were incubated with increasing concentration of RG7112 (0.5–10 μM) and its inactive enantiomer for 24, 48 and 72 hours (hrs). MDM2 inhibition by RG7112 resulted in a dose and time-dependent cytotoxicity with IC50 (24 hrs) of 2 μM for BV-173 and SUPB-15 which harbor homozygous deletion of CDKN2A but wild-type p53. No significant changes in cell viability were observed in K562 p53-null cell line after incubation with RG7112. The time and dose-dependent reduction in cell viability were confirmed in primary blast cells from a Ph+ ALL patient with the T315I Bcr-Abl kinase domain mutation found to be insensitive to the available tyrosine kinase inhibitors and from a t(4;11)-positive ALL patient (IC50 at 24 hrs equal to 2 μM). Consistent with the results of cell viability, Annexin V/Propidium Iodide analysis showed a significant increase in apoptosis after 24 hrs in BV-173, SUPB-15 and in primary leukemia blasts, whereas no apoptosis was observed in K562 cells. To examine the possible mechanisms underlying RG7112-mediated cell death, western blot analysis was performed. Protein levels of p53, p21 (an important mediator of p53-dependent cell cycle arrest), cleaved caspase-3 and caspase-9 proteins increased upon treatment with RG7112 after 24 hrs of incubation with concentrations equal to the IC50. These data demonstrate the ability of RG7112 to activate the intrinsic apoptotic pathway by a p53-dependent mechanism. In order to better elucidate the implications of p53 activation and to identify biomarkers of clinical activity, gene expression profiling analysis (Affymetrix GeneChip Human Gene 1.0 ST) was next performed, comparing sensitive cell lines (BV-173 and SUPB-15) after 24 hrs exposure to 2 μM RG7112 and their untreated counterparts (DMSO 0.1%). A total of 621 genes (48% down-regulated vs 52% up-regulated) were differentially expressed (p < 0.05). They include genes involved in cell cycle and apoptosis control (e.g. Histone H1, TOP2, GAS41, H2AFZ) and in the down-regulation of the Hedgehog signaling (e.g. BMI1, BMP7, CDKN1C, POU3F1, CTNNB1, PTCH2) with a strong repression of stemness genes and re-activation of INK4/ARF as illustrated in Figure 1. Actually, both GAS41 (growth-arrest specific 1 gene) and BMI1 (a polycomb ring-finger oncogene) are repressors of INK4/ARF and p21 and their aberrant expression has found to contribute to stem cell state in tumor cells. In our data they were strongly down-regulated (fold-change −1.35 and −1.11, respectively; p-value 0.02 and 0.03, respectively) after in vitro treatment as compared to control cells, suggesting that these genes have a potential as new biomarkers of activity. In conclusion, inhibition of the p53–MDM2 interaction by RG7112 can activate the p53 pathway, resulting in apoptosis and inhibition of stemness genes in B-ALL with wild-type p53. Our findings provide a strong rational for further clinical investigation of RG7112 in Ph+ ALL. Supported by: ELN, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, Ateneo RFO grants, Project of integrated program, Programma di Ricerca Regione–Università 2007–2009. Disclosures: Baccarani: Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Novartis: Honoraria; Bristol Myers Squibb: Honoraria; Pfizer: Honoraria; Ariad: Honoraria. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

Abstract 871 Since 1993, Italian pediatric patients (age The 6-year Kaplan-Meier estimates of overall survival (OS) and disease-free survival (DFS) are 89.7% (CI 95%: 84.7–95) vs 96% (CI 95%: 91.7–100), (p 0.05) and 73.1% (CI 95%: 66.7–80.2) vs 82.5% (CI 95%: 75.9–89.8), (p 0.28) in the AIDA 0493 and 2000 protocols, respectively. For low/intermediate risk children, OS and DFS at 6 years are 94.2% (CI 95%: 89.1–99.5) and 76.7% (CI 95%: 68.9–85.4) in the AIDA 0493 vs 95.6% (CI 95%: 90.0–100) and 82.7% (CI 95% 74.9–91.3) in the AIDA 2000 trial, respectively (p 0.57 and 0.73); considering high risk patients, OS and DFS at 6 years are 81.6% (CI 95%: 72.1–92.3) and 65.2% (CI 95%: 54.7–77.6) in the AIDA 0493 vs 96.8% (CI 95%: 90.9–100) and 82.3% (CI 95%: 70.1–96.5) in the AIDA 2000 trial (p 0.05 and 0.20). These results confirm the high anti-leukemic efficacy of the ATRA + idarubicin induction combination. For low/intermediate risk children, the anthracycline-based plus ATRA consolidation is equally effective as the previous cytarabine-containing regimen. The risk-adapted strategy including ATRA for consolidation resulted into a significant improvement in OS for all children. Furthermore, our results highlight the role of cytarabine coupled to anthracyclines and ATRA during consolidation in the high-risk group. Disclosures: Foa: Roche: Consultancy, Speakers Bureau.

Abstract 12 Background: Recently, in genome-wide analyses of DNA copy number abnormalities using single nucleotide polymorphism (SNP) microarrays, genetic alterations targeting PAX5 were identified in over 30% of pediatric acute lymphoblastic leukemia cases (Mullighan et al. Nature 2008). However, so far, the occurrence of PAX5 alterations and their clinical correlation has not been investigated in adults with BCR-ABL1-positive ALL. Aim: To characterize the rearrangements on 9p involving PAX5 and their clinical significance in adult BCR-ABL1-positive ALL. Patients and Methods: Eighty-nine patients with de novo adult BCR-ABL1-positive ALL were enrolled into institutional (n = 15) or Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto Acute Leukemia Working Party (n = 74) clinical trials and after obtaining informed consent their genome was analyzed by SNP arrays (Affymetrix 250K NspI and SNP 6.0), FISH, genomic PCR and resequencing. Results: By SNP array analysis we identified a loss of PAX5 in 29 patients (33%). In all cases the deletion was hemizygous. Four patterns of PAX5 deletion were observed: 1) focal deletion involving only the PAX5 gene in one case (3%); 2) deletions involving only a portion of PAX5 and both telomeric and centromeric genes in 11 cases (38%) with a median size of 310 kb (range: 101 kb-16,395 kb); 3) broader deletions involving the entire PAX5 locus and a variable number of flanking genes in 10 patients (34%) with a median size of 1,999 kb (range: 567 kb-1,208 kb); 4) deletion of all chromosome 9 or 9p in 7 patients (24%). These deletions may result in either PAX5 haploinsufficiency or expression of PAX5 alleles with impaired DNA-binding or transcriptional activation activity. In one patient the deletion of PAX5 involved only a subset of exons (exons 2-6) resulting in an alternative transcript encoding a prematurely truncated protein lacking key PAX5 functional domains. To investigate the consequences of genomic PAX5 alteration, quantitative PCR (q-PCR) was used, demonstrating that genomic alterations on 9p13.2 lead to a significant down-modulation at the transcript level of Pax5. Furthermore, both normal and deleted PAX5 subgroups were investigated for point mutations by sequencing analysis but we did not found nucleotide substitutions, suggesting that deletions are the main mechanism of inactivation of PAX5 in BCR-ABL1 positive ALL. However, when we investigated the association of PAX5 deletions with clinical outcome, no association with PAX5 status was detected (overall survival: p=0.3294; disease free-survival,DFS: p=0.9249 and cumulative incidence of relapse, CIR: p=0.945), suggesting that PAX5 deletions are not associated with outcome. Twenty-three patients (26%) had deletion of both PAX5 and IKZF1, encoding for the transcription factor Ikaros. We assessed the contribution of both PAX5 and IKZF1 alterations on clinical outcome finding out that the occurrence of both normal PAX5 and IKZF1 loss is associated with a shorter DFS (p=0.04) and higher CIR cumulative incidence of relapse in comparison to patients with both normal IKZF1 and PAX5 (p = 0.009). These results were confirmed by multivariate analysis. Conclusion: PAX5 deletions are frequent events in adult BCR-ABL1 positive ALL and are often associated with IKZF1 loss. The genomic status of both PAX5 and IKZF1 can be useful to identify at diagnosis groups of patients with worse prognosis. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Strategico di Ateneo, GIMEMA Onlus. Disclosures: No relevant conflicts of interest to declare.

Among all cytogenetic subtypes of ALL, the BCR-ABL1 fusion gene, which is causative of chronic myeloid leukemia (CML), defines the subgroup of ALL with the worst prognosis. The reasons of the aggressive nature of BCR-ABL1-positive ALL have not yet been completely understood. To identify, at the submicroscopic level, oncogenic lesions that cooperate with BCR-ABL1 to induce ALL and blastic transformation of CML, we used an integrated molecular approach including high resolution genomic analysis of copy number alterations by single nucleotide polymorphism (SNP) arrays (500K, Affymetrix Inc., Santa Clara, CA, USA), genomic amplification, cloning, deep sequencing, gene expression profiling (GEP) and analysis of epigenetic changes to study 97 de novo BCR-ABL1- positive ALL adult patients (pts). High level amplification and homozygous deletions were identified in all pts, with deletions outnumbering amplification almost 3:1. The most frequent somatic copy number alteration was deletion on 7p12 of IKZF1, which encodes the transcription factor Ikaros identified in 59/97 pts (61%). Ikaros is required for the earliest stages of lymphoid lineage commitment and acts as tumor suppressor in mice. The IKZF1 deletions were predominantly mono-allelic (64% vs 36%) and were limited to the gene in all cases, identifying IKZF1 as the genetic target. Real-time quantitative PCR and FISH analysis confirmed SNP results. We characterized and mapped all breakpoints to recognize that two major deletions occur in BCR-ABL1-positive ALL: type A characterized by loss of exons 4–7 (37/97, 38%) and due to breakpoints occurring in the region spanning from 50380371 to 50380388 and from 50431123 to 50431167 in introns 3 and 7, respectively; type B characterized by removal of exons 2–7 (18/97, 19%) and due to a variable pattern of breakpoints in introns 1 (from 50317112 to 50317936) and 7. A variable number of nucleotides (patient-specific) were inserted at the conjunction and maintained with fidelity at the time of relapse. The extent of both deletions correlated with the expression of a dominant-negative isoform Ik6 with cytoplasmic localization and oncogenic activity, and of an aberrant transcript containing only exons 1 and 8, respectively. Interestingly, 2 pts with a homozygosis deletion showed simultaneously both type A and B deletions. In other 2 pts, the deletion involved the whole IKZF1 gene. The IKZF1 alteration was not a frequent event across different leukemias, since it was identified also at the progression of CML to lymphoid blast crisis (66%), but never in myeloid blast crisis CML (n=10), chronic phase CML (n=30) and acute myeloid leukemia (n=28). Known DNA sequences and structural features were mapped along the breakpoint cluster regions including heptamer recombination signal sequences recognized by the RAG enzymes during V(D)J recombination and activation-induced cytidine deaminase (AID) consensus motifs (DGYW/WRCH), suggesting that IKZF1 deletions could arise from aberrant RAG and/or AID-mediated recombination. GEP analysis of pts with IKZF1 deletion vs wild-type pts identified a unique signature characterized by a down-regulation of genes involved in pre-B-cell differentiation (e.g. VPREB1, VPREB3, IGLL3), demonstrating that genomic IKZF1 alterations have a strong impact on trascriptoma and contribute to a block of B-cell differentiation. For 83 out of 97 BCR-ABL1 ALL pts correlation between molecular data and clinical outcome was available. Univariate analysis showed that the IKZF1 deletion is a negative prognostic marker influencing the cumulative incidence of relapse (10.1 months for pts with deletion vs 56.1 months for wild-type pts, p=0.0103) and disease-free survival (DFS) (10.1 months vs 32.1 months, respectively, p=0.0229). The negative prognostic impact of the IKZF1 deletion on DFS was also confirmed by multivariate analysis (p=0.0445). In conclusion, deletion of IKZF1 resulting in either haploinsuffciency and in the expression of the dominant negative isoforms is an important event in the development of BCR-ABL1 B-progenitor ALL which significantly influences clinical outcome.

Abstract 575 Incorporation of the tyrosine kinase inhibitor (TKI) imatinib in the frontline treatment of Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) patients (pts) has significantly improved the anti-leukemic efficacy of induction therapy. In contrast to chronic myeloid leukemia (CML), however, responses are short-lived and relapse is frequently associated with the selection of Bcr-Abl kinase domain (KD) mutations, fostered by the high genetic instability of Ph+ ALL cells. The advent of the 2nd-generation TKIs dasatinib and nilotinib has brought additional treatment options both for newly diagnosed and for imatinib-resistant pts. To analyze the changes they have brought in mutation frequency and type, we have reviewed the database recording the results of BCR-ABL KD mutation analyses done in our laboratory from January 2004 to June 2011. Overall, 781 tests on 258 Ph+ ALL pts (number of tests per pt, range: 1–15) were performed by denaturing high-performance liquid chromatography (D-HPLC) followed by direct sequencing of D-HPLC-positive cases. One hundred and fourty-three pts were analyzed because of imatinib resistance. One hundred and one out of 143 (71%) pts scored positive for one or more KD mutations. Similarly to what is know to occur in CML, hematologic and cytogenetic resistance were by far more frequently associated with mutations than molecular resistance (Bcr-Abl transcript increase as assessed by RT-Q-PCR). Overall, mutations at thirteen residues were detected. In contrast to what can be observed in CML, three mutations were by far the most frequent, accounting for almost 75% of the mutated cases: T315I (n=38 pts, 37%), E255K/V (n=19 pts, 18%) and Y253H (n=19 pts, 18%). The other mutations were, in order of frequency: F359V/I, M244V, M351T, F317L, G250E, Q252H, L387M, D276G, L248R, E279K. Nine out of 103 (9%) pts had two mutations, in the same (2 pts) or in different (7 pts) subclones. In 84 pts who were analyzed because they were reported to have developed resistance to dasatinib (n=72) or nilotinib (n=12) as 2nd- or 3rd-line TKIs, 65 (77%) had newly acquired mutations (57/72 dasatinib-resistant pts and 8/12 nilotinib-resistant pts). The most frequent newly acquired mutation in this setting was the T315I, detected in 35/57 (61%) cases acquiring mutations on dasatinib and in 2/8 cases acquiring mutations on nilotinib. Other recurrent newly acquired mutations were F317L, V299L, T315A in dasatinib-resistant pts and Y253H and E255K in nilotinib-resistant pts. Thirty out of 65 pts (46%) were positive for multiple mutations (2 to 4 mutations, in the same or in different subclones or both) that emerged under the same TKI in 11 cases (37%) and accumulated as a consequence of multiple lines of TKI therapy in the remaining 19 (63%) cases. Mutation analysis was also performed in 15 resistant pts enrolled in a clinical trial of dasatinib as first-line treatment for Ph+ ALL. Twelve pts were positive for mutations; 11/12 had a T315I. Sixty-one pts were analyzed at the time of diagnosis in order to assess whether TKI-resistant mutations could already be detectable. Only two pts (3%) were positive for mutations: one patient had an F311L that disappeared after one month of nilotinib treatment; an additional patient was positive only by D-HPLC, but not by the less sensitive direct sequencing – most likely for the T315I mutation that shortly after the start of dasatinib treatment outgrew and led to resistance. Taking advantage of the recent availability of a next-generation sequencing platform (Roche 454), allowing high sensitivity (0.01%) mutation scanning of the KD, samples collected at the time of diagnosis and during follow-up from selected Ph+ ALL cases who developed mutations and resistance to TKI therapy were retrospectively analyzed – but the mutations were not always already detectable at diagnosis. In conclusion: a) although 2nd generation TKIs may ensure a more rapid debulking of the neoplastic clone and have much fewer insensitive mutations, long-term disease control remains a problem and the T315I becomes an even tougher enemy; b) the clinical usefulness of mutation screening of Ph+ ALL pts at diagnosis before TKI start, even with highly sensitive approaches is low – not all mutations pre-exist since genetic instability remains high and fosters mutational events anytime during treatment. Supported by PRIN, FIRB, AIL and AIRC. Disclosures: Soverini: ARIAD: Consultancy; Novartis: Consultancy; Bristol-Myers Squibb: Consultancy. Luppi:CELGENE CORPORATION: Research Funding. Foà:Bristol-Myers Squibb: Consultancy, Membership on an entity's Board of Directors or advisory committees. Baccarani:Bristol-Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

Abstract 1670 Among genes that are supposed to be involved in altered molecular pathways leading to leukemogenesis, the CDKN2A/B locus is particularly noteworthy since it encodes for the INK4-class cyclin dependent kinase inhibitors p15 INK4B, p16 INK4A and p14 ARF, which act as tumor suppressors. The CDKN2A/B locus has been found to be inactivated in several haeamatologic malignancies mainly due to deletion, aberrant repression or epigenetic silencing, whereas little is known about the potential role of its single nucleotide polymorphisms (SNPs) in leukemia susceptibility. To investigate whether polymorphisms within this broad genomic region can correlate with increased susceptibility to haeamatologic malignancies, an association study was performed by genotyping 23 SNPs spanning the MTAP, CDKN2A/B and CDKN2BAS loci, as well as relative intergenic regions, in a case-control cohort made up of 332 samples: 149 leukemia patients, including Philadelphia positive (Ph+) ALL (n=92) and acute myeloid leukemia (AML) samples (n=57), and 183 unrelated healthy controls. Ph+ ALL patients showed a median age of 52 years (18-78 years); 24 cases (26%) expressed the p210 oncoprotein, 62 (67%) the p190 and 6 (7%) both the proteins. AML patients showed a median age of 53 years (21-71 years) and they included FAB-M0, M1, M2, M3, M4, M5, miscellaneous cytogenetic abnormalities and normal karyotype subtypes. 6 SNPs were selected on the basis of their previous association with several diseases, such as coronary artery disease (rs2891168, rs518394, rs564398, rs10757278), type 2 diabetes mellitus (rs564398), frailty (rs2811712). The remaining 17 SNPs were selected among those included in the Affymetrix Genome-Wide Human SNP Array 6.0 to deepen the SNPs coverage for the examined region. Genotyping was performed using iPLEX Gold technology and MassARRAY high-throughput DNA analysis with Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (Sequenom, Inc., San Diego, CA). Furthermore, all leukemia samples were also previously genotyped using the GeneChip® Human Mapping 250K NspI and Genome-Wide Human SNP 6.0 (Affymetrix). A total of 17 SNPs, spanning the 9p genomic interval that encompasses the MTAP, CDKN2A/B and CDKN2BAS loci, were successfully genotyped and used for investigating their potential associations with the leukemia phenotypes. Five SNPs (rs1012713, rs10965179, rs34011899, rs3731232, rs3218010) with Minor Allele Frequency (MAF) 20% missing call rates, were instead excluded from the association analysis. Potential population stratification affecting the control sample was ruled out as its genotypes distribution satisfies the Hardy-Weinberg equilibrium criterion. Among the 17 SNPs, rs564398, mapping to the CDKN2BAS locus that encodes for ANRIL antisense non-coding RNA, showed a statistically significant correlation with the ALL phenotype, with a risk pattern that was compatible with an overdominant model of disease susceptibility and an Odd Ratio (OR) of 2 (95% CI, 1.20 to 3.33; p= 7.1 × 10-3). Since a co-ordinated regulation of ANRIL and p14/ARF, p16/CDKN2A, p15/CDKN2B transcription has been already observed in both physiologic and pathologic conditions, we hypothesized that rs564398 association reflects a condition of high linkage disequilibrium between such polymorphism and a causative variant that is able to alter CDKN2A/B expression profiles by changing ANRIL dosage, thus leading to abnormal proliferative boosts and consequent increased ALL susceptibility. Supported by European LeukemiaNet, AIL, AIRC, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integrated program (PIO), Programma di Ricerca Regione – Università 2007 – 2009. Disclosures: Baccarani: Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Martinelli:Novartis: Consultancy, Honoraria; BMS: Honoraria; Pfizer: Consultancy.

In Philadelphia-positive (Ph+) acute lymphoblastic leukemia (ALL) patients (pts) treated with tyrosine kinase inhibitors (TKIs), responses are generally short-lived and relapse is most often accompanied by selection of point mutations in the Bcr-Abl kinase domain (KD). In order to assess whether mutations are already present at the time of diagnosis, we analyzed RNA samples from 13 pts with newly diagnosed Ph+ ALL enrolled on the GIMEMA LAL1205 protocol – designed to assess activity, safety and tolerability of dasatinib monotherapy as first-line treatment for adult Ph+ ALL. The pts investigated (males/ females: 6/7; median age: 56 years, range 30–73) included 6 pts who subsequently relapsed with dasatinib-resistant mutations, 2 pts who relapsed without evidence of mutations as assessed by direct sequencing and 5 pts in persistent remission. Screening for low level mutations was performed by cloning the entire Bcr-Abl KD (amino acid 206 through 524) in a bacterial vector and sequencing 150 to 200 independent clones for each pt. All pts, irrespective of their subsequent response to dasatinib, had evidence of aberrant KD sequences due to point mutations (13/13 pts), small insertions (2/13 pts) or small deletions (1/13 pts). Two to five point mutations were detectable for each pt. In some cases, multiple mutations could be found to co-exist in the same cloned fragment. A total of 45 different point mutations (including 18 silent mutations, 4 nonsense mutations and 23 missense mutations) were observed. Base substitutions were scattered all over the KD and there were no mutation hotspots. Thirty-nine out of 45 (87%) mutations were transitions: G>A (n=14), A>G (n=10), C>T (n=9), T>C (n=6). Such a high prevalence of transitions (which normally occur 1.4 times more frequently than transversions) suggests that a specific mechanism generating mutations is predominant. Activation-induced cytidine deaminase (AID) has been recently shown to be aberrantly expressed in a proportion of Ph+ ALLs and has been implicated in the development of at least some Bcr-Abl mutations; correlation with AID expression will be presented. Mutations were detected in no more than three independent clones, suggesting that they were present at low levels. Mutations detected in less than two clones were considered only if independently validated by a specifically designed ASO-PCR assay run on the original cDNA sample. The majority of point mutations detected have never been reported in association with TKI resistance. However, three pts were found to harbor known imatinib- or dasatinib-resistant mutations, including two T315I mutations that were later on selected and led to relapse. This supports the theory that mutations randomly arise as a consequence of a high genetic instability and very few of them will confer a growth advantage under the selective pressure of TKIs. Six samples from newly diagnosed chronic phase CML (CP-CML) pts were screened for comparison with the same cloning approach, including 2 pts who later progressed with evidence of KD mutations after 3 and 6 months from the start of imatinib, respectively, and 4 pts who achieved a stable molecular response on imatinib. Both pts who experienced early relapse on imatinib had low level mutations detectable at the time of diagnosis, whereas none of the clones from CP-CML imatinib responders had evidence of KD mutations. The results herein presented support the concept that Ph+ ALL cells have a particularly high genomic instability that allows early escape from TKI inhibitor therapy. Only a fraction of CML cases share this feature. Our data highlight the importance of understanding the mechanism(s) responsible for this ‘mutator’ phenotype, as well as how to interfere therapeutically with it.

Background: BCR-ABL1-positive ALL is the most frequent and prognostically most unfavorable subtype of adult ALL. The main reason for the poor clinical outcome of BCR-ABL1-positive ALL is genetic instability. However, how normal B-cell precursor cells acquire the genetic changes that lead to transformation and progression has not been completely defined. Activation-induced cytidine deaminase (AID) produces immunediversity by inducing somatic hypermutations and class-switch recombinations in human immunoglobulin genes (Ig). Aim: Since at much lower frequency AID can also target non-Ig genes and may even act as a genome-wide mutator, we investigated whether AID was expressed in BCR-ABL1-positive ALL and in chronic myeloid leukemia (CML) at the time of progression to blast crisis. Patients and methods: We analyzed 61 adult de novo Ph+ ALL patients (pts) and 60 CML pts (chronic phase and myeloid/lymphoid blast crisis). AID cDNA, obtained from bone marrow or peripheral blood, was amplified with two pairs of oligonucleotides, the forward primer of each couple conjugated with a fluorescent dye (fluorescein) at its 5′ end. PCR products were then loaded on the ABI Prism 3730 DNA Analyzer for automated capillary gel electrophoresis and the results were plotted with the AbiPrism GeneMapper v3.5 software (Applied Biosystems). Results: On the 61 de novo adult BCR-ABL1-positive ALL pts, AID mRNA and protein were detected in 41 (67%). AID expression correlated with the BCR-ABL1 transcript levels and disappeared after treatment with tyrosine kinase inhibitors at the time of remission. Moreover, AID expression was also found in lymphoid blast crisis CML (60%), but not in myeloid lineage or in chronic phase CML. Different isoforms of AID were expressed. In 10/61 (16%) BCR-ABL1-positive ALL pts the full-length isoform (GeneBank accession number NM_020661) was identified, in 16/61 (26%) the co-expression of the wild-type isoform and of different AID splice variants was found and in 15/61 (25%) only the expression of splice variants was found. These can result from retention of intron 4 (Variant A), omission of exon 4 (Variant B) and 3 (Variant C), and from a deletion of 30 bp in the initial portion of exon 4 (Variant D). In the wild-type mRNA, codon 148 spans exons 3 and 4. In both variants A and B, mRNA splicing disrupts this codon and causes a frameshift, which results in a premature stop codon. If translated, the splice variants produce truncated proteins of 187 and 145 amino acids, respectively. However, the putative deaminase active site, encoded by exon 3, is preserved in both splice variants, but is lacking in the variant C. Since enforced expression of Pax5 induces endogenous AID gene expression, we analyzed the expression levels of Pax5 in all pts (ΔΔCt method, GAPDH as control gene). As expected, we found a very strong difference (p Conclusions: Our findings show that BCR-ABL1-positive ALL cells aberrantly express different isoforms of AID that can act as a mutator outside the Ig gene loci in promoting genetic instability in leukemia cells.