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Abstract Purpose: This study was conducted to identify novel genes with importance to the biology of adult acute myelogenous leukemia (AML). Experimental Design: We analyzed DNA from highly purified AML blasts and paired buccal cells from 95 patients for recurrent genomic microdeletions using ultra-high density Affymetrix single nucleotide polymorphism 6.0 array–based genomic profiling. Results: Through fine mapping of microdeletions on 17q, we derived a minimal deleted region of ∼0.9-Mb length that harbors 11 known genes; this region includes Neurofibromin 1 (NF1). Sequence analysis of all NF1 coding exons in the 11 AML cases with NF1 copy number changes identified acquired truncating frameshift mutations in two patients. These NF1 mutations were already present in the hematopoetic stem cell compartment. Subsequent expression analysis of NF1 mRNA in the entire AML cohort using fluorescence-activated cell sorting sorted blasts as a source of RNA identified six patients (one with a NF1 mutation) with absent NF1 expression. The NF1 null states were associated with increased Ras-bound GTP, and short hairpin RNA–mediated NF1 suppression in primary AML blasts with wild-type NF1 facilitated colony formation in methylcellulose. Primary AML blasts without functional NF1, unlike blasts with functional NF1, displayed sensitivity to rapamycin-induced apoptosis, thus identifying a dependence on mammalian target of rapamycin (mTOR) signaling for survival. Finally, colony formation in methylcellulose ex vivo of NF1 null CD34+/CD38− cells sorted from AML bone marrow samples was inhibited by low-dose rapamycin. Conclusions: NF1 null states are present in 7 of 95 (7%) of adult AML and delineate a disease subset that could be preferentially targeted by Ras or mammalian target of rapamycin–directed therapeutics. Clin Cancer Res; 16(16); 4135–47. ©2010 AACR.

Supplementary Table S9 from NF1 Inactivation in Adult Acute Myelogenous Leukemia

Supplementary Table S8 from NF1 Inactivation in Adult Acute Myelogenous Leukemia

Despite improvements in treatment, the outcome for some adult patients with acute or chronic leukemias remains poor. Clofarabine, a second-generation purine nucleoside analog, received U.S. Food and Drug Administration approval in 2004 for the treatment of pediatric patients with relapsed or refractory acute lymphocytic leukemia after at least two previous regimens. In addition, clinical studies have shown encouraging safety and efficacy results with clofarabine in the treatment of adult patients with various hematologic malignancies. Although most adult patients with leukemia receive the first course of clofarabine while hospitalized, many can be subsequently treated as outpatients with proper monitoring, support, and education. The most frequent side effects associated with clofarabine are gastrointestinal-related, myelosuppression, hepatotoxicity, renal dysfunction, and anorexia. Careful patient monitoring is essential to ensure early identification and prompt intervention. Younger patients and those of any age with no comorbid health issues, good performance status, and an adequate support network are more likely to tolerate outpatient clofarabine administration. Early identification and proactive pharmacologic and nonpharmacologic interventions may reduce the severity of these toxicities and prevent their progression. Patient education about strategies for prevention and management of symptoms also is essential.

Download pose: This study was conducted to identify novel genes with importance to the biology of adult myelogenous leukemia (AML). erimental Design:We analyzed DNA from highly purified AML blasts and paired buccal cells from tients for recurrent genomic microdeletions using ultra-high density Affymetrix single nucleotide orphism 6.0 array–based genomic profiling. ults: Through fine mapping of microdeletions on 17q, we derived a minimal deleted region of Mb length that harbors 11 known genes; this region includes Neurofibromin 1 (NF1). Sequence analf all NF1 coding exons in the 11 AML cases with NF1 copy number changes identified acquired ting frameshift mutations in two patients. These NF1 mutations were already present in the hematic stem cell compartment. Subsequent expression analysis of NF1 mRNA in the entire AML cohort fluorescence-activated cell sorting sorted blasts as a source of RNA identified six patients (one with a utation) with absent NF1 expression. The NF1 null states were associated with increased RasGTP, and short hairpin RNA–mediated NF1 suppression in primary AML blasts with wild-type acilitated colony formation in methylcellulose. Primary AML blasts without functional NF1, unlike with functional NF1, displayed sensitivity to rapamycin-induced apoptosis, thus identifying a dence on mammalian target of rapamycin (mTOR) signaling for survival. Finally, colony formation in lcellulose ex vivo of NF1 null CD34+/CD38− cells sorted from AML bone marrow samples was ind by low-dose rapamycin. clusions: NF1 null states are present in 7 of 95 (7%) of adult AML and delineate a disease subset Con that could be preferentially targeted by Ras or mammalian target of rapamycin–directed therapeutics. Clin Cancer Res; 16(16); 4135–47. ©2010 AACR.

Abstract Abstract 165 Genomic aberrations are of dominant importance to the biology and clinical outcome of patients with acute myelogenous leukemia (AML). To further our understanding of such aberrations in AML, we analyzed DNA from highly purified AML blasts and paired buccal cells from 95 patients for subchromosomal copy number changes and allele identities using ultra-high-density Affymetrix SNP 6.0 array-based genomic profiling. A total of 358 somatically acquired copy number changes were detected in 95 AML genomes. We detected 16 losses and 22 gains of entire chromosomes, 285 subchromosomal losses and 35 subchromosomal gains. No recurrent high-level amplifications or recurrent homozygous deletions were identified. Eight of the 34 AML cases (24%) with normal karyotype each had one lesion detected through 6.0 array profiling, all but one of which was less than 4Mb in length. Focusing on microdeletions as potential indicators of the locations of novel tumor suppressor genes or genes with importance to AML biology, we identified 60 deletions that were less than 1 Mb in length and 158 deletions of less than 5 Mb, the vast majority of which were undetectable by conventional cytogenetics. Through fine mapping of microdeletions on 17q, we identified Neurofibromin 1 (NF1) null states due to mutations or absent expression in ∼7% of AML. NF1 mutations were present in the hematopoetic stem cell compartment (CD34+/CD38- cell population) and siRNA-mediated NF1 suppression using recombinant lentiviruses significantly increased colony formation of primary AML blasts in methylcellulose. Further, AML blasts without functional NF1 displayed sensitivity to rapamycin-induced apoptosis, thus identifying a dependence on mTOR signaling for survival. As an additional validation of using microdeletions to guide pathogenetic gene discovery, we identified deletions involving RUNX1, IRF8, Core Binding Factor Beta (CBFB) and Casitas B-cell lymphoma B (CBLB), genes known to be altered in AML. IRF8 expression was found to be absent in ∼30% of all AML but sequencing of all coding exons of IRF8 of 48 AML cases did not disclose somatically acquired mutations. In summary, this comprehensive description of subchromosomal copy number changes and microdeletions in adult AML substantially adds to our knowledge of the pathological anatomy of the AML genome and should inform future searches for novel genes with importance to AML biology. Disclosures: Malek: Cephalon: Honoraria, Speakers Bureau; Celgene: Honoraria, Speakers Bureau; Affymetrix: Research Funding. Erba:Lilly: Research Funding; Antisoma: Research Funding; Wyeth: Research Funding; Cephalon: Honoraria, Research Funding; MGI Pharma: Honoraria; Pharmion: Honoraria; Celgene: Honoraria; BMS: Honoraria; Novartis: Honoraria, Research Funding; Genzyme: Consultancy, Honoraria, Research Funding; Gemin-X: Research Funding; Kanisa: Research Funding.

13536 Background: Multidrug resistance (MDR) associated with Pgp overexpression in blasts is common in high-risk AML and is associated with poor outcomes. Erba et al (JCO 2007) reported a 42% complete remission (CR) rate in patients (pts) with sAML [prior MDS or treatment-related AML, (tAML)] (N = 88) treated with amonafide and std dose cytarabine. In laboratory studies of Pgp+ human leukemia cells, amonafide was neither a substrate nor an inhibitor of Pgp-mediated efflux, in contrast to daunorubicin (DNR). We sought to correlate the lack of Pgp effect with treatment outcome. Methods: AML blasts from 15 pts from the Phase 2 trial cited above were retrospectively assessed for Pgp expression and function as well as amonafide and DNR uptake and retention in the presence and absence of the Pgp inhibitor cyclosporin A. Pts: median age, 62 yrs (range 51–87); 7 with prior MDS; 8 had tAML; 10 with unfavorable cytogenetics. Pgp-mediated efflux was assessed by comparing uptake of the Pgp substrate DiOC2(3) in the presence and absence of the Pgp inhibitor PSC-833. Pgp- mediated transport (effluxapp) was calculated as differential uptake and retention of amonafide and DNR with (a) and without (b) PSC-833, normalized to apparent influx, using the formula [(a-b)/a]×100, reported as the mean±s.e.m. Results: The 15 sAML samples showed significantly less effluxapp of amonafide (5.2%±3.2) than of DNR (16%±2.1; p=0.0083). The unfavorable cytogenetic subset showed much less effluxapp of amonafide (0.13%±3.7) compared to DNR (16%±2.1; p=0.0015). CR pts also showed less effluxapp of amonafide (4.0%±6.7) than of DNR (21%±2.9; p=0.035). Conclusions: The relative lack of Pgp-mediated efflux of amonafide, compared to DNR, from sAML blasts provides a rationale for its observed clinical efficacy in the Phase 2 trial. Prospective assessment of these MDR parameters is underway in a randomized Phase 3 clinical trial in sAML comparing amonafide to DNR in combination with std dose cytarabine for remission induction. Author Disclosure Employment or Leadership Consultant or Advisory Role Stock Ownership Honoraria Research Expert Testimony Other Remuneration Xanthus Pharmaceuticals Xanthus Pharmaceuticals Xanthus Pharmaceuticals

Karyotypic abnormalities are of dominant importance in AML risk prognostication and therapy selection. A comprehensive description of subchromosomal genomic copy number changes and allele status together with gene mutation analysis and identification of chromosomal translocations is needed to fully harvest the prognostic and biological power of genomic changes in AML. We have analyzed DNA from 96 AML-derived pure blast populations (purified using column-based multi parameter negative selection followed by multi-gated FACS sorting) compared with paired buccal DNA using the Affymetrix 6.0 SNP platform. To support data analysis and display, we have developed the software tools PLUT and LOH tool version 2 and have refined dChipSNP. Data were supplemented with blast karyotypes and mutation status of Flt3, NPM1, p53, N-ras and K-ras. Results: AML cases carried between zero and thirty-four subchromosomal losses and gains. Approximately 23% of all cases had ≥3 subchromosomal lesions and approximately 50% of all cases had no such lesions. Of the 22 cases with complex and hypercomplex genomic changes by SNP profiling, ~50% had mutations in p53 exons 5–9 and ~50% were p53 wild-type by sequence analysis. Monoallelic deletion of p53 as part of various deletions at 17p was found in 7 of 96 (7%) cases. Importantly, 7/96=7% additional cases demonstrated UPD at 17p which spanned the p53 locus. Of all 14/96=15% of cases with LOH at 17p (with and without copy loss) 9/14=64% carried p53 mutations and 11/14=79% had complex karyotypes. Of the AML cases with NPM1 mutations (19 of 96 cases or 20%), 75% carried no detectable subchromosomal lesions, while 25% carried between 1 and 4 such lesions. Previously unidentified microdeletions resulting in monoallelic NF1 loss were identified on 17p in a total of 10 of 96 (10%) cases, thus suggesting activation of the Ras pathway independent of Ras mutations (all affected cases had wild-type N-ras and K-ras) in a substantial subset of AML cases. Mutational analysis of all coding exons of NF1 on the retained allele in the affected cases is ongoing. Additional recurrent microdeletions were identified on chromosomes 3p and 3q as well as 12p, the latter encompassing the genes ETV6 and p27, as previously reported. Finally, analysis of the prognostically and therapeutically important deletions 5q and 7q identified large deletions without recurrent microdeletions. Regions of minimal loss on 5q have been delineated.

BACKGROUND:Peripheral blood lymphocyte apoptosis is a recognized feature of serious infection and sepsis and can be easily quantified by flow cytometric measurement of annexin V binding to the cell surface. Use of apoptosis as a biomarker in emergency department (ED) studies of sepsis is potentially difficult because of sample processing requirements and limited availability of a research cytometer with which to measure patient samples.OBJECTIVES:To assess, in vitro and in simulation, the relationship between sample stability, timing of patient enrollment, and diagnostic performance of a flow cytometric assay for sepsis in patients evaluated in EDs.METHODS:Assuming any clinical trial would require daily sample batching, the authors measured the stability of lymphocyte samples over time, noting the rate at which annexin V-negative cells became positive as ED processing delays increased. With these data, they then optimized a study design that could evaluate lymphocyte apoptosis as a sepsis biomarker by using a series of Monte Carlo-based simulated clinical trials.RESULTS:The authors found that annexin V-negative lymphocytes become positive during storage delays that would be encountered in an ED sepsis trial. The extent of this deterioration was least among cells left as whole blood at room temperature until just before analysis or when lymphocytes were isolated early and stored in culture media at 4 degrees C until analysis. When the expected rate of sample deterioration was considered in simulated clinical trials, an inverse relationship was found between the rate at which patients are enrolled and the best achievable receiver operating characteristic curve a study could produce.CONCLUSIONS:Peripheral blood samples being analyzed for lymphocyte apoptosis degrade at a rate relevant to the design of ED trials of sepsis. Because of sample processing delays inherent in studying unscheduled septic patients, the performance of annexin V binding as a biomarker for sepsis can approach, but not be expected to exceed, its performance in a comparable intensive care unit-based study.

CLL is the most common leukemia in the Western world and displays considerable clinical heterogeneity. Genetic approaches to elucidate defects in CLL genomes have resulted in the identification of recurrent chromosomal abnormalities with prognostic importance. A genome-wide high-density unbiased view of chromosomal copy number changes or loss of heterozygosity (LOH) may allow for further refinements of genomics-based risk prognostication. We have conducted a genomic profiling study using 50KSNP-oligonucleotide arrays (Affymetrix) on FACS-sorted CD19+ cells and paired buccal DNA from 120 patients with typical CLL (CD5+/CD23+) at various stages of presentation enrolled in a clinical translational protocol. We have found novel genetic subtypes, including novel regions of recurrent LOH and chromosomal copy loss on chromosomes 3p, 14q and 18p at frequencies of 3–6%. Fine mapping of the clinically important regions del(6q), del(11q), del(13q14) and del(17p) suggests molecular heterogeneity of these lesions. Anatomic criteria suggest existence of multiple distinct subtypes of del13q14. Furthermore, the clinically important del(17p) is associated with almost uniform deletion of most of 17p, occasionally displays copy-neutral LOH (undetectable by FISH) with p53 mutations and occurs without p53 mutations in a subset of patients as previously reported. Finally, using a genome-wide composite genomic instability score of copy gains and losses we found a subset of CLL cases (18%) with high genomic instability values (greater than 2 lesions per genome). These findings may have implications for CLL risk-adapted therapies.