Search

PDF file - 242K

Purpose: Rhabdomyosarcomas are a major cause of cancer death in children, described with MYCN amplification and, in the alveolar subtype, transcription driven by the PAX3-FOXO1 fusion protein. Our aim was to determine the prevalence of N-Myc protein expression and the potential therapeutic effects of reducing expression in rhabdomyosarcomas, including use of an antigene strategy that inhibits transcription.Experimental Design: Protein expression was assessed by immunohistochemistry. MYCN expression was reduced in representative cell lines by RNA interference and an antigene peptide nucleic acid (PNA) oligonucleotide conjugated to a nuclear localization signal peptide. Associated gene expression changes, cell viability, and apoptosis were analyzed in vitro. As a paradigm for antigene therapy, the effects of systemic treatment of mice with rhabdomyosarcoma cell line xenografts were determined.Results: High N-Myc levels were significantly associated with genomic amplification, presence of the PAX3/7-FOXO1 fusion genes, and proliferative capacity. Sustained reduction of N-Myc levels in all rhabdomyosarcoma cell lines that express the protein decreased cell proliferation and increased apoptosis. Positive feedback was shown to regulate PAX3-FOXO1 and N-Myc levels in the alveolar subtype that critically decrease PAX3-FOXO1 levels on reducing N-Myc. Pharmacologic systemic administration of the antigene PNA can eliminate alveolar rhabdomyosarcoma xenografts in mice, without relapse or toxicity.Conclusion: N-Myc, with its restricted expression in non-fetal tissues, is a therapeutic target to treat rhabdomyosarcomas, and blocking gene transcription using antigene oligonucleotide strategies has therapeutic potential in the treatment of cancer and other diseases that has not been previously realized in vivo. Clin Cancer Res; 18(3); 796–807. ©2011 AACR.

PDF file - 72K

PDF file - 1.1MB

PDF file - 287K

PDF file - 135K

CCR Translation for This Article from Antitumor Activity of Sustained N-Myc Reduction in Rhabdomyosarcomas and Transcriptional Block by Antigene Therapy

PDF file - 750K

PDF file - 62K

PDF file - 399K

PDF file - 347K

PDF file - 104K

Irritable bowel syndrome (IBS) is a psychosomatic gastrointestinal disorder involving the dysfunctional activation of specific brain regions crucial for interoception and disgust processing. Yet, no study has ever investigated the link between this socio-affective/visceral experience and IBS.The present study investigated whether disgust sensitivity and disgust propensity, which can be socially relevant, relate with IBS symptoms in a nonclinical population.105 healthy participants were asked to complete the Disgust Propensity and Sensitivity Scale-Revised (DPSS-R), the Irritable Bowel Syndrome-Quality of Life Measure (IBS-QOL), and the Chronic Urticaria Quality of Life Measure (CU-Q2OL), as control condition.Results showed higher disgust sensitivity scores in individuals with high IBS-QOL score, compared to individuals with low IBS-QOL score. The correlation analysis corroborates this result by showing a positive relationship between disgust sensitivity and respective IBS-QOL scores.This research provides new insights into understanding the etiopathogenesis of IBS, suggesting the relevance of a socially relevant personality trait such as disgust sensitivity as a potential trigger and / or predisposition factor for this chronic inflammatory disease.

Background Multiple Sclerosis (MS) can be associated with personality changes. Personality traits affect the cognitive functions, mood, and psychological well-being of people with MS and their families. Objective The aim of this study was to evaluate whether personality traits might be predictors of cognitive recovery in people with MS. Methods. Thirty-one patients with a diagnosis of MS were enrolled in this study. Each participant was evaluated by a neuropsychologist before (T0) and after the end of the rehabilitation treatment (T1). The Five-Factor Model (FFM) has been used to describe the basic personality structure by highlighting five personality traits: extroversion, neuroticism, conscientiousness, agreeableness, and openness. The rehabilitation program included conventional physiotherapy, speech therapy, psychological support, cognitive rehabilitation, and nutritional therapy as well as robotic rehabilitation, advanced cognitive rehabilitation, and virtual reality. Logistic regressions are carried out to measure the changes score between T0 and T1. Results The results showed that patients had significant improvement in cognitive and behavioral functioning. Here, we considered the relationships between the scores of cognitive and emotional/behavioral domains, the five scales of the BFQ, besides the psychiatric symptoms. In particular, the mental quality of life, visuospatial and verbal long-term memory were positively influenced by the agreeableness trait, while depression was negatively affected by this trait. Conclusion Our data suggest that personality traits could influence cognitive and behavioral recovery in patients with MS.

Aims: To compare the genomic signatures of wild-type (WT) and mutated GISTs and the murine inter-stitial cells of Cajal (ICCs) to find markers of celldifferentiation and other functions that may identifycells that give rise to WT tumours.Methods and results: We analysed the gene expressionprofiles of a total of 30 tumour samples (four WT GISTsand 26 mutated GISTs), selected the genes mostdifferentially expressed (P < 0.001:448 probe sets)and validated these results by quantitative polymerasechain reaction (PCR) and immunohistochemistry. Inaddition, we conducted a meta-analysis merging datafrom human GISTs with a genomic data set frommurine ICCs. The gene expression profiles of WT andmutated GISTs differed profoundly, especially in theexpression of those genes restricted primarily to neuraltissues. We found that mature ICCs are more similar tomutated GISTs than WT GISTs.Conclusions: WT GISTs have different genomic profilesfrom both mutated GISTs and murine mature ICCs.Considering that IGF1R expression is common to bothWT GISTs and putative precursor ICCs, this studysuggests that WT GISTs may derive either from ICCs ata different step of differentiation or from a different cellof origin.Keywords: gene expression profiling, GIST, ICC, ICC mature, ICC precursor, IGF1R, KIT, PDGFRAAbbreviations: CDH2, cadherin-2; DMP, deep muscular plexus; ELAVL3, embryonic lethal, abnormal vision,Drosophila-like 3; GIST, gastrointestinal stromal tumours; ICC, interstitial cells of Cajal; IGF1R, insulin-like growthfactor 1 receptor; IHC, immunohistochemistry; KIRREL3, Kin of IRRE-like protein 3; Lhx2, LIM ⁄ homeobox protein;MY, myenteric plexus; NEFL, neurofilament light polypeptide gene; NRCA, neuronal cell adhesion molecule; NSE,neurone-specific enolase; PCR, polymerase chain reaction; PDGFRA, platelet-derived growth factor receptor, alpha;TKI, tyrosine kinase inhibitor; WT, wild-type

A pilot study using Affymetrix Gene Chip(R) Porcine Genome Arrays was set up to evaluate the impact of time lags from death on gene expression profiling of porcine skeletal muscle at four post mortem times (up to 24 h) during the routine processing of fresh thighs. All post chip parameters and data analyses (Average background, Scale Factors, Percent Presence, 3'/5' ratios of beta-actin and glyceraldehyde-3-phosphase dehydrogenase control genes, RNA degradation diagnostics, principal component analysis, hierarchical clustering, mixed regression models with time effects) did not show any effect of post mortem time. Therefore, microarray data obtained from muscle specimens collected in a processing plan over a quite long period have the potential to identify treatments or pre mortem conditions without any potential bias derived from subtle RNA degradation. These results open new perspectives to develop and analyse gene expression biomarkers for pig production and product authentication.

Approximately 10%-15% of gastrointestinal stromal tumors (GISTs) in adults do not harbor any mutation in the KIT or PDGFRA genes (ie, KIT/PDGFRA wild-type GISTs). Recently, mutations in SDHB and SDHC (which encode succinate dehydrogenase subunits B and C, respectively) but not in SDHA and SDHD (which encode subunits A and D, respectively) were identified in KIT/PDGFRA wild-type GISTs. To search for novel pathogenic mutations, we sequenced the tumor transcriptome of two young adult patients who developed sporadic KIT/PDGFRA wild-type GISTs by using a massively parallel sequencing approach. The only variants identified as disease related by computational analysis were in SDHA. One patient carried the homozygous nonsense mutation p.Ser384X, the other patient was a compound heterozygote harboring a p.Arg31X nonsense mutation and a p.Arg589Trp missense mutation. The heterozygous nonsense mutations in both patients were present in germline DNA isolated from peripheral blood. Protein structure analysis indicates that all three mutations lead to functional inactivation of the protein. This is the first report, to our knowle dge, that identifies SDHA inactivation as a common oncogenic event in GISTs that lack a mutation in KIT and PDGFRA.

In the late 1960s, the Australian film industry was in crisis: audiences were turning to television programmes, in part because they offered more Australian content, and the commercially successful screen comedies of the so-called Ocker were perceived to be damaging the image of Australia abroad. In the 1970s, realizing the potential of cinema as a cultural flagship, the government invested heavily in the film industry through the establishment of the Australian Film Commission (AFC) for the purpose of promoting of high-quality productions. This article examines the main polices of the AFC, and argues that its role in transforming the film industry from a ‘cottage industry into a business’ (Murray 1990: 14) has been overestimated by Australian cinema scholars such as Dermody and Jacka, David Stratton and Tom O'Regan. The AFC's role as a government body meant that the Commission was more accountable to the government than to film industry professionals, such as producers and filmmakers. The final s...

In addition to KIT and PDGFRA mutations, sequential accumulation of other genetic events is involved in the development and progression of gastrointestinal stromal tumors (GISTs). Until recently, the significance of these other alterations has not been thoroughly investigated. We report the first study that integrates gene expression profiling and high-resolution genomic copy number analyses in GIST. Fresh tissue specimens from 25 patients with GIST were collected, and gene expression profiling and high-resolution genomic copy number analyses were performed, using Affymetrix U133Plus and SNP array 6.0. We found that all 21 mutant GIST patients showed both macroscopic cytogenetic alterations and cryptic microdeletions or amplifications, whereas 75% (three of four) of wild-type patients with GIST did not show genomic imbalances. The most frequently observed chromosomal alterations in patients with mutant GIST included 14q complete or partial deletion (17 of 25), 1p deletion (14 of 25) and 22q deletion (10 of 25). Genetic targets of the chromosomal aberrations were selected by integrated analysis of copy number and gene expression data. We detected the involvement of known oncogenes and tumor suppressors including KRAS in chr 12p amplification and KIF1B, PPM1A, NF2 in chr 1p, 14q and 22p deletions, respectively. The genomic segment most frequently altered in mutated samples was the 14q23.1 region, which contains potentially novel tumor suppressors, including DAAM1, RTN1 and DACT1. siRNA-mediated RTN1 downregulation showed evidence for the potential role in GIST pathogenesis. The combination of gene expression profiling and high-resolution genomic copy number analysis offers a detailed molecular portrait of GISTs, providing an essential comprehensive knowledge necessary to guide the discovery of novel target genes involved in tumor development and progression.

Abstract 76 Checkpoint kinase 1 (Chk1) and 2 (Chk2) are serine/threonine kinases regulated by Ataxia-Telangiectasia and Rad3-related (ATR) kinases and involved in the DNA damage response and in the regulation of cell cycle progression at S-G2 phase. Deregulation of these pathways has been previously described in BCR-ABL positive cells and involved in chemoresistance. Based on the potential utility of DNA checkpoint inhibition in enhancing tumor cell death, in this study we aimed to investigate the preclinical activity of PF-0477736 (Pfizer), a potent and selective Chk1/2 inhibitor, in Ph+ ALL and to determine potential biomarkers of functional inhibition. We first examined Chk1 and Chk2 mRNA expression levels in 45 newly diagnosed Ph+ ALL patients, in their paired remission samples, in 14 relapsed cases and in 3 Ph+ cell lines (BV-173, SUPB-15 and K562) by Fluidigm Dynamic Array real-time qPCR assay (Fluidigm Corporation). Higher transcript levels of Chk1 but not Chk2 were found in newly diagnosed patients compared to remission samples (p = 0.0009 for Chk1 and p= 0.8183 for Chk2). Chk1 transcript levels were comparable between diagnosis and relapsed cases (p = 0.5728), suggesting that the ATR-Chk1 pathway is strongly activated in Bcr-Abl-positive cells. This was confirmed by phosphorylation of Chk1 Ser317 by western blotting analysis in Ph+ cell lines. We then evaluated the effect of PF-0477736 as single agent on cell viability using the Cell Proliferation Reagent WST-1 (Roche). BV-173, SUPB-15 and K562 cell lines were incubated with increasing concentration of PF-0477736 (0.005-2 μM) for 24, 48 and 72 hours. PF-0477736 inhibition of Chk1 resulted in dose and time-dependent cytotoxicity with IC50 at 24 hours of 0.1–0.5 μM, with BV-173 being the most sensitive, while K562 the most resistant. These results were confirmed in primary blasts cells from a Ph+ ALL patient with wild-type Bcr-Abl and from 3 cases harboring the T315I Bcr-Abl mutation found to be insensitive to the available TKIs (IC50 ranged from 0.1–0.5 μM at 48 hours). Consistent with the WST-1 results, Annexin V/Propidium Iodide staining analysis showed a significant increase of apoptosis at 24 and 48 hours in both cell lines and primary cells. To test whether increased apoptosis resulted from Chk1 inhibition, we assessed the changes in phosphorylation of Cdc25c phosphatase, which is inactivated by Chk1 to prevent mitotic entry, and of γH2AX, which is increased in response to DNA damage. Western blot analysis showed that PF-0477736 decreases the inhibitory phosphorylation of Cdc25c Ser216 and increases levels of γH2AX. Since multiple studies reported a higher activity of PF-0477736 against p53-defective cancer cells, we performed a mutational screening by amplification and subsequent sequencing of all coding exons of p53. All cell lines except for K562 and primary leukemia cells lacked mutations in the p53 gene, demonstrating that in Ph+ ALL PF-0477736 is highly effective also on p53 wild-type tumor cells. Finally, in order to elucidate the mechanisms of action of PF-0477736 and to determine biomarkers of response, gene expression profiling analysis (Affymetrix GeneChip Human Gene 1.0 ST) was performed on 3 treated Ph+ cell lines and their untreated counterparts. Consistent with a specific Chk1-mechanism of action, treatment resulted in differential expression (p < 0.05) of 211 genes including those involved in apoptosis and cell cycle (CEBPB, CUL1, Histone H1-H2A, 2B family clusters, Histone H4, DHX15, SNCB, FOS) and DNA damage, such as DNA-damage-inducible transcript 3 (DDIT3) and growth-arrest and DNA damage-inducible proteins GADD34 and GADD45a, suggesting that PF-0477736 contributes to a checkpoint abrogation and to an activation of DNA damage response in Ph+ ALL cells. In conclusion, for the first time we demonstrate in a large cohort of Ph+ ALL patients an over-expression of Chk1, providing a strong rational for its inhibition. In vitro treatment of Ph+ ALL cells with PF-0477736 alone resulted in reduction of inhibitory phosphorylation of Cdc25c, inhibition of proliferation and induction of biomarkers of DNA damage and apoptosis, suggesting that single-agent Chk1/2 inhibition may be an effective treatment strategy for Ph+ ALL. Supported by European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2009, PIO program, Programma Ricerca Regione-Università 2007–2009. PF-0477736 provided by Pfizer. Disclosures: Baccarani: Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Meyers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees. Martinelli:Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

Genetic heterogeneity in drug-metabolizing enzyme/transporter (DMET) genes affects specific drug-related cancer phenotypes. To investigate the relationships between genetic variation and response to treatment in acute myeloid leukemia (AML), we genotyped 1931 variants on DMET genes in 94 CD33-positive AML patients enrolled in a phase III multicenter clinical trial combining Gemtuzumab-Ozogamicin (GO) with Fludarabine-Cytarabine-Idarubicin (FLAI) regimen, with the DMET Plus platform. Two ADH1A variants showed statistically significant differences (odds ratio (OR)=5.68, P=0.0006; OR=5.35, P=0.0009) in allele frequencies between patients in complete/partial remission and patients without response, two substitutions on CYP2E1 (OR=0.13, P=0.001; OR=0.09, P=0.003) and one on SLCO1B1 (OR=4.68, P=0.002) were found to differently influence liver toxicity, and two nucleotide changes on SULTB1 and SLC22A12 genes correlated with response to GO (OR=0.24, P=0.0009; OR=2.75, P=0.0029). Genetic variants were thus found for the first time to be potentially associated with differential response and toxicity in AML patients treated with a combination of GO-FLAI regimen.

Purpose: Rhabdomyosarcomas are a major cause of cancer death in children, described with MYCN amplification and, in the alveolar subtype, transcription driven by the PAX3-FOXO1 fusion protein. Our aim was to determine the prevalence of N-Myc protein expression and the potential therapeutic effects of reducing expression in rhabdomyosarcomas, including use of an antigene strategy that inhibits transcription. Experimental Design: Protein expression was assessed by immunohistochemistry. MYCN expression was reduced in representative cell lines by RNA interference and an antigene peptide nucleic acid (PNA) oligonucleotide conjugated to a nuclear localization signal peptide. Associated gene expression changes, cell viability, and apoptosis were analyzed in vitro. As a paradigm for antigene therapy, the effects of systemic treatment of mice with rhabdomyosarcoma cell line xenografts were determined. Results: High N-Myc levels were significantly associated with genomic amplification, presence of the PAX3/7-FOXO1 fusion genes, and proliferative capacity. Sustained reduction of N-Myc levels in all rhabdomyosarcoma cell lines that express the protein decreased cell proliferation and increased apoptosis. Positive feedback was shown to regulate PAX3-FOXO1 and N-Myc levels in the alveolar subtype that critically decrease PAX3-FOXO1 levels on reducing N-Myc. Pharmacologic systemic administration of the antigene PNA can eliminate alveolar rhabdomyosarcoma xenografts in mice, without relapse or toxicity. Conclusion: N-Myc, with its restricted expression in non-fetal tissues, is a therapeutic target to treat rhabdomyosarcomas, and blocking gene transcription using antigene oligonucleotide strategies has therapeutic potential in the treatment of cancer and other diseases that has not been previously realized in vivo. Translational Relevance Rhabdomyosarcomas are the most common pediatric soft tissue sarcoma. They are a leading cause of cancer death in children with few therapeutic options in highrisk categories. N-Myc is a transcription factor belonging to the MYC protein family that has a highly restricted expression pattern in non-fetal tissues. N-Myc protein expression in patient samples of rhabdomyosarcoma and an apoptotic response to sustained reduction of expression in rhabdomyosarcoma cell lines imply that N-Myc represents a tumor-specific therapeutic target in these tumors. Systemic treatment of mice with rhabdomyosarcoma cell line xenografts using an antigene peptide nucleic acid (PNA) oligonucleotide conjugated to a nuclear localization signal peptide that inhibits transcription of MYCN shows promise by eliminating tumors. This direct antigene approach may have therapeutic potential in the treatment of other cancers and diseases.

BackgroundGenome-wide association studies of pooled DNA samples were shown to be a valuable tool to identify candidate SNPs associated to a phenotype. No such study was up to now applied to childhood allergic asthma, even if the very high complexity of asthma genetics is an appropriate field to explore the potential of pooled GWAS approach.Methodology/principal findingsWe performed a pooled GWAS and individual genotyping in 269 children with allergic respiratory diseases comparing allergic children with and without asthma. We used a modular approach to identify the most significant loci associated with asthma by combining silhouette statistics and physical distance method with cluster-adapted thresholding. We found 97% concordance between pooled GWAS and individual genotyping, with 36 out of 37 top-scoring SNPs significant at individual genotyping level. The most significant SNP is located inside the coding sequence of C5, an already identified asthma susceptibility gene, while the other loci regulate functions that are relevant to bronchial physiopathology, as immune- or inflammation-mediated mechanisms and airway smooth muscle contraction. Integration with gene expression data showed that almost half of the putative susceptibility genes are differentially expressed in experimental asthma mouse models.Conclusion/significanceCombined silhouette statistics and cluster-adapted physical distance threshold analysis of pooled GWAS data is an efficient method to identify candidate SNP associated to asthma development in an allergic pediatric population.

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.

Background: About 10%-15% of GISTs do not harbour KIT/PDGFRA mutations (wild-type, WT). We analyzed the tumor transcriptome of two sporadic young adults WT GISTs patients using massively parallel sequencing approach in order to identify novel genetic mutations. Methods: Patients were a 28 years-old female (GIST_07) and a 30 years-old male (GIST_10). Primary tumors were in the stomach, multi-centric in GIST_07 and uni-centric in GIST_10, and presented metastases at diagnosis. RNA-Seq paired-end libraries were sequenced on the Illumina GAIIx system using a paired-end strategy at a read length of 75bp. Bioinformatic analysis highlighted 9 candidate genes common to both patients. Mutations in these genes were then filtered with SNP&GO, a tool suited to compute the likelihood of a mutation to be disease-related or not, depending on the protein sequence and its functional annotation. The identified candidate gene was analyzed by Sanger sequencing on tumor (T) and peripheral blood (PB) DNA from GIST_07 and GIST_10 patients, and on additional 8 tumor and 3 PB DNA from 8 sporadic WT GISTs patients. Immunohistochemistry (IHC) was also performed. Results: Only mutations in the sequence of SDHA were identified as disease-related with a high reliability index. GIST_07 tumor DNA was homozygous for an exon 9 p.S384X nonsense mutation; GIST_10 patient showed compound heterozygosity for a germinal exon 2 p.R31X nonsense mutation and a somatic exon 13 p.R589W missense mutation.Heterozygous nonsense mutations were already present in germline DNA in both patients. All mutations lead to functional inactivation of the protein. IHC revealed that both patients showed the down regulation of SDHA. The expanded study showed that 2/10 carried SDHA mutations in the tumor: compound heterozygosity for an exon 9 p.G419R and exon 13 p.E564K mutations in one patient, and a somatic heterozygous exon 5 p.R171C mutation in the other. Conclusions: Recently, SDHB and SDHC mutations were also reported in these patients. This the first study that identifies mutations on SDHA, in 4/10 WT GIST patients. This gene should be evaluated in larger seriesand be added in the sequencing screening of WT GISTs.

10056 Background: Gastrointestinal stromal tumors (GISTs) are characterized by oncogenic mutations of KIT and PDGFRA receptors. However about 10%-15% of adult GISTs do not harbor any KIT or PDGFRA ...

Aberrations of the Insulin-like Growth Factor (IGF) system have been found in association with a variety of cancer types. The potential role of IGF1R has been postulated in a small subset of GISTs, but until now the implications of its aberrations have not been defined. The aim of the study was to examine the IGF1R status in patients with gastric GIST in regard to KIT and PDGFRA genotype. Fresh resection specimens were collected from 8 primary tumours [2 wild-type (WT) and 6 mutant cases]. IGF1R was studied as gene expression profiling with Affymetrix GeneChip HG-U133Plus 2.0 arrays and as genomic copy number with SNP array analysis Affymetrix Genome Wide Human SNP 6.0 arrays, and at protein level with western blotting (WB) and immunohistochemistry (IHC). The unsupervised analysis of gene expression profiling of our patients merged with a data set from gastric GISTs identified 2 patients out of 8 with different expression of IGF1R. The data were confirmed by WB and IHC. In particular, IGF1R was upregulated in 2 young patients (

Nowadays molecular biology represents one of the most interesting topics in medical oncology, because it provides a global and detailed view on the molecular changes involved in tumour progression, leading to a better understanding of the carcinogenesis process, to discovering new prognostic markers and novel therapeutic targets. The gene expression profiling analysis with microarray technology has shown a great potential in cancer research and in medical oncology, mapping simultaneously the expression of thousands of genes in a single tumour sample and giving a measurement of articulated genes expression patterns. Colorectal cancer represents a wide and exciting area of research for molecular biology, due to the growing need of a molecular classification as well as prognostic and predictive molecular factors that may guide oncologists in patient's clinical management. The aim of this review is to analyze the state of art of gene expression profile in colorectal cancer using microarrays technologies and to explore some perspectives in this research field.

Acute Myeloid Leukemia (AML) is a heterogeneous malignancy characterized by the expansion of myeloid precursor cells with limited or abnormal differentiation capacity. A relatively common event in AML is represented by chromosome gain or loss. Numerical chromosome abnormalities, which define the aneuploid condition, have a detrimental effect in primary non-malignant cells, since they dramatically reduce cellular fitness. However evidence suggests that they have a causative role in tumorigenesis and that they are well tolerated in transformed cells belonging to the myeloid lineage. Aim of the study is to elucidate the pathogenic mechanisms that sustain and contribute to aneuploidy in AML. We have performed gene expression profile (GEP) analysis of bone marrow cells from 42 AML patients, including 19 aneuploid cases and 23 cases with normal karyotype. All samples contained more than 80% blast cells. The aneuploid cohort included AML cases carrying one (or more) monosomy, trisomy or a monosomal karyotype. Our analysis covered more than 245,000 and 40,000 coding and non-coding transcripts, respectively (the latter comprising microRNAs), and a significant number of exon-exon junctions, which allow the analysis of multiple splicing isoforms. Quality controls confirmed that the data show comparable signal values. The gene expression signature of aneuploid samples have been compared to normal karyotype ones. We have identified a set of coding and non-coding transcripts which are differentially expressed between the two groups (p≤0.05, including more than 20 genes with a fold difference ≥2) and defined a gene signature that allows the discrimination between aneuploid and euploid samples in our dataset. The analysis of an increased number of cases will confirm the results and allow the sub-stratification of aneuploid samples according to their GEP. Our data will be further validated by comparing them with published GEP datasets and the gene signature will be characterized by pathway analysis. This study provides novel insights into the molecular mechanism that sustain aneuploidy in AML. The biological validation of genes which are commonly and specifically deregulated in aneuploid AML patients will guide the design of future therapeutic strategies targeting key players in the disease. Acknowledgements: European LeukemiaNet, AIRC, AIL, Prin 2010-2011, FP7 NGS-PTL project. Citation Format: Giorgia Simonetti, Antonella Padella, Viviana Guadagnuolo, Cristina Papayannidis, Francesca Volpato, Emanuela Ottaviani, Serena Formica, Annalisa Astolfi, Ilaria Iacobucci, Giovanni Capranico, Daniel Remondini, Giovanni Martinelli. Gene expression signature of aneuploidy in acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2243. doi:10.1158/1538-7445.AM2014-2243

e20527 Background: Liver and peritoneum are the most common site of recurrences from gastrointestinal stromal tumors (GISTs). Bone metastases represent a rare finding in clinical practice and their...

10046 Background: Recently SDH variations were identified in KIT and PDGFRA wild type GISTs. However the causative genomic alterations of these GISTs still remain unknown. Massively parallel sequen...

Despite the striking efficacy of targeted therapy for chronic myeloid leukemia (CML), a proportion of patients (pts) still experience progression from the initial chronic phase to an acute phase (blast crisis; BC) characterized by high disease aggressiveness and poor prognosis. BC is known to be associated with accumulation of additional genetic alterations, but these alterations have so far been only partially characterized. We used Human 6.0 SNP Arrays (Affymetrix) to perform high-resolution molecular karyotyping of 29 DNA samples from BC (myeloid, n=17 lymphoid, n=11) CML pts. The 6.0 SNP Array technology relies on 1.8 million markers evenly spaced across the genome, with a median inter-marker distance Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2143.

Abstract 4259 Philadelphia-chromosome positive (Ph+) CML is generally regarded as a quite genetically heterogeneous disease and therapy with Bcr-Abl tyrosine kinase inhibitors results in high response rates. Nevertheless, resistance may develop, especially in high Sokal risk patients, and progression to blast crisis still represents a major concern. The biological bases underlying Sokal risk, as well as the determinants of disease progression remain largely unknown. Several high-throughput technologies have been recently developed that allow to perform genome-wide studies with unprecedented informativity and resolution. For the first time in CML, we have integrated three such technologies – massively-parallel sequencing, gene expression profiling (GEP) by microarrays and high-resolution karyotyping by SNP-arrays – for a deeper characterization of a tyrosine kinase inhibitor-resistant patient at diagnosis and at the time of progression to BC. A 62-year-old female was diagnosed with Philadelphia-chromosome positive (Ph+), BCR-ABL (p210)-positive CML, high risk according to both Sokal (2.4) and Euro (1704.39) scores. At presentation, no additional chromosomal abnormalities (ACA) were detected. The patient received first-line therapy with nilotinib 800 mg/d, achieved a major molecular response (MMR; Bcr-Abl transcript levels, 0.1% according to the International Scale as assessed by real-time quantitative PCR) after 3 months, but suddenly progressed to lymphoid BC after 6 months from diagnosis and died of her disease one month thereafter. At the time of progression, a T315I mutation was identified by conventional direct sequencing of the BCR-ABL kinase domain (KD); no ACAs were detected by chromosome banding analysis. After having obtained written informed consent from her next of kin, the samples collected at diagnosis, at the time of remission (MMR) and at the time of progression to BC were used for RNA and DNA extraction. Poly(A) RNA was then obtained and used to prepare double-stranded cDNA libraries for paired-end sequencing on an Illumina/Solexa Genome Analyzer. The number of 75bp-long sequence reads obtained was 40,193,384 (corresponding to 3.01 billion bases), 35,592,588 (2.7 billion bases), and 32,867,700 (2.5 billion bases) for diagnosis, remission and progression samples, respectively. The open-source software MAQ (http://maq.sourceforge.net) was used for read alignment and mapping against the human reference genome (hg18, NCBI build 36.1) and for subsequent single nucleotide variant (SNV) calling. Aligned reads were 31,886,732 (16,566,482 with no mismatches and 15,320,250 with at least one mismatch), 27,722,967 (16,199,728 with no mismatches and 11,523,239 with at least one mismatch) and 25,563,423 (14,315,149 with no mismatches and 11,248,274 with at least one mismatch) for diagnosis, remission and relapse, respectively. Comparison of the SNVs identified in the diagnosis and relapse samples with the SNVs identified in the remission sample was crucial to rule out all the inherited sequence variants non-specific of Ph+ cells. Nonaligned reads were further analyzed for the discovery of small insertions/deletions (indels) and novel or aberrantly spliced transcripts. In parallel, high-resolution ( Although this study focuses - at present - on a single CML case, it first offers a comprehensive overview of the complexity of the Ph+ cell genome and transcriptome of a high-risk patient both at the time of diagnosis and at the time of TKI-resistance and progression to BC. Genome-wide integrated approaches like this might provide novel insights and fill the gaps in our knowledge of the pathogenesis of CML and of the mechanisms of disease progression and Abl KD mutation outgrowth. For this reason, whole-transcriptome sequencing of additional CML cases has already been planned. Supported by European LeukemiaNet, AIL, AIRC, PRIN, Fondazione del Monte di Bologna e Ravenna. Disclosures: No relevant conflicts of interest to declare.

10559 Background: Besides mutually exclusive cKIT or PDGFRA mutations, sequential accumulation of other genetic events may be involved in GISTs development and progression, but very few data is still available. Methods: Fresh tissues specimens of GISTs from 10 patients (9 gastric and 1 intestinal) were collected and used for RNA and DNA extraction, labeled and hybridized to HG-U133Plus 2.0 and SNP array 6.0, respectively (Affymetrix). Six patients had exon 9 or exon 11 c-KIT mutation, two PDGFRA mutation, and other two wild-type disease. Gene expression data were quantified by the RMA algorithm, filtered and analysed with supervised techniques (SAM algorithm). Genomic copy number data were analysed with Partek Genomic Suite software against a reference set of 90 Ceu HapMap individuals using a segmentation algorithm with stringent p-value cutoff. Results: Almost all patients exhibited both macroscopic cytogenetic alterations and cryptic microdeletions or amplifications by SNP-array copy number data analysis. The most frequent chromosomal alterations were: 14q complete or partial deletion (7/10), chromosome 19 monosomy (3/10), 22q and 1p deletion (2/10), chromosome 5 trisomy (2/10). The minimal overlapping region ranged from 14q22.3 to 14q32.33, covering a region including 320 genes. The integration of copy number and gene expression data showed that at least 40% of the genes inside the 14q deleted region were significantly downregulated (FDR No significant financial relationships to disclose.

10087 Background: KIT/PDGFRA wild-type (WT) GISTs harbour mutations on SDHB and SDHC and, more recently, we described mutations on SDHA using massively parallel sequencing approach. We sequenced SDHA and SDHB genes in a larger series in order to validate the data. Methods: SDHA gene (1-15 exons) and SDHB gene (1-8 exons) (even not all exons in all samples) were sequenced on tumor (T) and/or peripheral blood (PB) of WT GIST patients by Sanger Sequencing method. DNA was extracted from tumor specimens by the QIAmp DNA Mini kit (Qiagen, Milan, Italy) and amplified with specific primer pairs designed to amplify exons but not SDHA pseudo-genes located on chromosomes 3 and 5. Then, PCR products were purified with the Qiaquick PCR purification kit (Qiagen, Milan, Italy) and sequenced on both strands using the Big Dye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems). Sanger sequencing was performed on ABI 3730 Genetic Analyzer (Applied Biosystems). Results: SDHA gene exons were sequenced on a total of 27 WT GIST patients, in particular on T, PB and both from 12, 6 and 9 patients respectively. SDHB gene exons were sequenced on a total of 18 out of 27 patients, in particular on T, PB and both from 7, 8 and 3 patients respectively. 8 SDHA mutations were found in 5 samples (18.5%). Besides those previously identified, 5 new SDHA mutations were found in other 3 samples: one sample harboured R171C and R589Q heterozygous missense mutation in exons 5 and 13 respectively. The other one harboured G419R and E564K heterozygous missense mutations in exons 9 and 13 respectively. The third sample harboured a delCAG immediately upstream of exon 5, in heterozygosis on PB and in homozygosis on T. A SDHB heterozygous mutation (301delT) in exon 4 was found on 1 PB sample. Conclusions: the presence of SDHA mutations has been confirmed in a subgroup of WT GIST patients. All subunits of SDH complex should be sequenced on WT GIST patients in order to explore the frequency and any linkage between each other and the pathogenetic and clinical significance.

2176 Poster Board II-153 Progression from chronic phase to blast crisis (BC) remains a major hurdle on the road to effective treatment of chronic myeloid leukemia (CML). BC is known to be associated with accumulation of additional genetic alterations, but these alterations have so far been only partially characterized. The development of SNP-arrays as a tool for high-resolution karyotyping now allows to perform high-throughput genome-wide screens for submicroscopic genomic alterations with unprecedented informativity and resolution and to precisely map all the genes involved in these alterations. We have used Human 6.0 SNP Arrays (Affymetrix) to perform high-resolution molecular allelokaryotyping of 25 DNA samples from BC (myeloid, n=16; lymphoid, n=9) CML patients (pts). The 6.0 SNP Array technology relies on 1.8 million markers evenly spaced across the genome, with a median inter-marker distance 2 pts are listed in the [Table][1] below. Gains/losses mapping to known regions of copy number variation (CNV) were excluded. | Gene Symbol | Type of CNA | Exons involved | Chromosomal location | No of pts harboring CNAs | |:-----------:| ----------- | -------------- | -------------------- | ------------------------ | | MDM4 | a | All | 7q22.11 | 5 pts | | HGF | a | All | 1q32.1 | 4 pts | | NRAS* | a | All | 1p13.2 | 3 pts | | HRAS* | a | All | 11p15.5 | 3 pts | | KRAS* | a | All | 12p12.1 | 5 pts | | AKT3* | md | One or more | 1q43 | 13 pts | | CDC73 | md | One or more | 1q31.2 | 8 pts | | RB1 | md+a | One or more | 13q14.2 | 8 pts | | ERG | md/bd | One or more | 21q22.3 | 8 pts | | ETS1 | a | One or more | 11q24.3 | 8 pts | | ETV1 | a | One or more | 7p21.2 | 7 pts | | SMAD2 | ma+d | One or more | 18q21.1 | 7 pts | | PIK3CA* | ma+d | One or more | 3p26.32 | 6 pts | | SKAP2 | a | One or more | 7p15.2 | 5 pts | | EPHA3 | a | One or more | 3p11.2 | 5 pts | | KALRN* | md | One or more | 3q21.2 | 5 pts | | FHIT | md | One or more | 3p14.2 | 5 pts | | JAK1 | d | One or more | 1p32.3 | 5 pts | | JAK2 | ma+d | One or more | 9p24.1 | 4 pts | | AKT2* | md | One or more | 19p13.2 | 4 pts | | SOS1* | md | One or more | 2p22.1 | 4 pts | | PTEN* | ma+d | One or more | 10q23.31 | 4 pts | | FGFR2 | a | One or more | 10q26.3 | 4 pts | | ETV5 | a | One or more | 3q27.2 | 4 pts | | MET | a | One or more | 7q31.2 | 4 pts | | PBX4 | md | One or more | 19p13.11 | 4 pts | | NRG1 | md | One or more | 8p12 | 4 pts | | FAF1 | md | One or more | 1p33 | 4 pts | | GAS2 | md | One or more | 11p14.3 | 3 pts | | TEC | a | One or more | 4p12 | 3 pts | | PTCH1 | ma+d | One or more | 9q22.32 | 3 pts | | PIK3R1* | a | One or more | 5q13.1 | 3 pts | | GRB2* | a | One or more | 17q24 | 3 pts | | PAK2* | md | One or more | 3q29 | 3 pts | | RUNX1T1 | a | One or more | 8q21.3 | 3 pts | * Abbreviations: md, monoallelic deletion; bd, biallelic deletion; a, amplification; ma+d, complex pattern of monoallelic deletion of some exons and amplification of other exons All the genes found to harbor CNAs were transcription factors, adaptor proteins, receptor and non-receptor kinases involved in cell proliferation and apoptosis - with a known role as oncogenes or tumor suppressors or oncogene/tumor suppressor interactors. Although these results confirm a high degrees of heterogeneity in the alterations detectable in BC CML pts, members of the RAS pathway (indicated with an asterisk) were the most frequently altered genes. Further characterization by polymerase chain reaction and sequencing is ongoing. In conclusion, the power of 6.0 SNP Array technology allowed us to detect previously unidentified alterations targeting whole or part of key oncogenes or tumor suppressors whose deregulation may play a role in determining the aggressive phenotype of BC CML, and which may represent potential therapeutic targets. Supported by European LeukemiaNet, AIL, AIRC, PRIN, Fondazione del Monte di Bologna e Ravenna. Disclosures: No relevant conflicts of interest to declare. [1]: #T1

Abstract 967 Human genome sequence variation, in the form of single nucleotide polymorphisms (SNPs) as well as more complex structural variation (such as insertions, duplications and deletions) in drug-metabolizing enzyme and transporter genes (DMETs), affects each individual's response to anticancer agents and thus the likelihood of experiencing an adverse drug reaction. Therefore, understanding relationships between genetic variation and differential drug responses is a challenge to improve both the safety and efficacy of drugs. Accordingly, in order to investigate a potential association between SNPs and clinical response and toxicity, we comprehensively assessed the allele frequencies of 1931 genetic variations of 225 absorption, distribution, metabolism and excretion genes in 59 AML patients younger than 65 years, using the new Affymetrix drug-metabolizing enzyme and transport (DMET Plus) genotyping platform (Affymetrix, Inc. Santa Clara, CA, USA). All patients were enrolled in a phase III multicenter clinical trial combining low dose of Gemtuzumab-ozogamicin (GO) with FLAI regimen (Fludarabine, Cytarabine, Idarubicin) as Induction chemotherapy i (eudract: 2007–005248-26; ClinicalTrials.gov NCT00909168). The induction regimen (GO-FLAI) included fludarabine (25 mg/sqm) and Ara-C (2 g/sqm) on days 1–5, idarubicin (10 mg/sqm) on days 1, 3, and 5 and GO (3 mg/sqm) on day 6. Hematopoietic stem cell transplant was planned for all high risk AML patients in first complete remission (CR) after consolidation with intermediate doses of Ara-C and idarubicin (ID-AC and IDA). Cytogenetic, multidrug-resistance phenotype, FLT3 and NPM mutation status, WT1 quantitative expression analyses, were performed at diagnosis in all patients. Furthermore, high-resolution SNP array analysis by GeneChip® Human Mapping 250K NspI and Genome-Wide Human SNP 6.0 (Affymetrix) was also performed. DNA processing and genotype identifications were performed according to the Affymetrix DMET platform's instructions. All statistical analyses were performed using the R package 2.11.1. Allele frequencies estimation and principal component analyses (PCAs) were performed using the adegenet library, whereas an ad hoc R function was developed to compute two-side Fisher's exact test p-values for each SNP in order to test their association with clinical response and toxicity.Results of potential interest were limited to those in which the p-value was 6). In conclusion, we performed an exploratory pharmacogenomic study that related SNPs in multidrug enzymes and transporters genes with the efficacy and toxicity of a combination of Gemtuzumab-ozogamicin (GO) with FLAI regimen used in a clinical trial of AML patients younger than 65 years. We identified for the first time a pharmacogenomic panel made up of 1 gene (ADH1A) associated with clinical outcome and 4 genes associated with toxicity. Findings obtained from this exploratory study are being tested in a larger clinical sample sets to confirm the predictive value of the SNP panel. Supported by European LeukemiaNet, AIL, AIRC, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integreted program (PIO), Programma di Ricerca Regione – Università 2007 – 2009. Disclosures: Martinelli: Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria; Pfizer: Consultancy.

Abstract 3262 Poster Board III-1 CML pts display a certain degree of clinical heterogeneity that is documented by the varying levels of response to tyrosine kinase inhibitor therapy and is best reflected by the Sokal risk score. Clinical differences must be a sign of some biological heterogeneity the basis of which, however, are still poorly understood. Today many high-throughput assays are available that allow to unravel the complexity of cancer cells in a genome-wide fashion. We have used Human 6.0 SNP Arrays (Affymetrix) to perform high-resolution ( The pathways and genes most frequently affected by CNAs or UPD are listed in the Table below: Base Excision Repair (BER) MUTYH DNA glycosilase 1p34.1 loss/upd 10 pts PNKP Polynucleotide kinase 19q13.33 loss 10 pts NEIL1 DNA glycosilase 15q24.2 loss 7 pts POLB DNA polymerase beta 8p11.21 loss/upd 6 pts PCNA Sliding clamp for DNA polymerases 20p12.3 loss 6 pts Mismatch Repair (MMR) PMS2L5 Mut L homolog - mismatch and loop recognition 7q11.23 loss/upd 11 pts MSH2 Mut S homolog - mismatch and loop recognition 2p21 loss 8 pts POLD1 DNA polymerase delta 19q13.33 loss 8 pts POLA2 DNA polymerase alpha, subunit 2 2p16.3 loss/upd 6 pts POLE2 DNA polymerase epsilon, subunit 2 14q21–22 loss 5 pts Nucleotide Excision Repair (NER) ERCC1 5’ incision subunit of TFIIH complex 19q13.32 loss 11 pts ERCC2 5’ to 3’DNA helicase of TFIIH complex 19q13.32 loss 10 pts XAB2 Transcription-coupled NER factor 19p13.2 loss/upd 9 pts CDK7 Kinase subunit of TFIIH complex 5q13.2 loss 7 pts RPA4 Binds damaged DNA in preincision complexes Xp21.33 loss/upd 7 pts RPA2 Binds damaged DNA in preincision complexes 12q24.31 loss/upd 6 pts Homologous Recombination (HR) RAD51C Homologous pairing 17q23.2 loss/upd 7 pts RAD52 Accessory factor for recombination 12p13.33 loss 7 pts XRCC2 DNA break and crosslink repair 7q36.1 loss 5 pts Non-Homologous End Joining (NHEJ) PRKDC DNA-dependent protein kinase, catalytic subunit 8q11.21 loss/upd 5 pts DCLRE1C Artemis nuclease 10p13 loss 5 pts REV7 DNA polymerase zeta, subunit 1p36.22 gain 5 pts Other genes involved in DNA replication/repair/modification or chromatin remodeling CHAF1A Chromatin assembly factor 19p13.3 loss 15 pts RECQL5 DNA helicase 17q25.1 loss/upd 11 pts RAD9B PCNA-like DNA damage sensor 12q24.11 loss/upd 10 pts CHEK2 DNA-damage checkpoint 22q12.1 loss/upd 8 pts RAD17 RFC-like DNA damage sensor 5q13.2 loss 6 pts TREX1 DNAase III exonuclease 3p12.31 loss/upd 6 pts For some genes (e.g., RAD52), the monoallelic deletion we detected was found to translate into reduced mRNA expression, observation that was also independently confirmed in an additional group of high/intermediate versus low Sokal risk pts. In all the 44 pts, multiple pathways and multiple genes within the same pathway were affected, supporting the hypothesis that the lesions we detected might actually have consequences on DNA integrity despite the known partial functional redundancy of pathways and effectors. For many of the genes identified in this screen, activating or inactivating mutations are known to occur, and together with overexpression or haploinsufficiency, have been linked to a mutator phenotype in several malignant conditions. We are currently investigating whether this may be the case also in CML. Supported by European LeukemiaNet, AIL, AIRC, PRIN, Fondazione del Monte di Bologna e Ravenna. Disclosures: Baccarani: Novartis: Honoraria, Research Funding, Speakers Bureau; Bristol-Myers Squibb: Honoraria, Research Funding, Speakers Bureau.