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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

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.

Unbiased analysis of CLL genomes using intermediate resolution SNP arrays has identified subtypes of del13q14, del17p and del11q as well as high genomic complexity CLL. Additional information on genomic aberrations and polymorphic copy number variants (CNVs) in CLL may be obtained through application of the latest generation ultra-high-density SNP array technology to CLL genome analysis. We have analyzed 50 paired DNA samples (sorted CD19+ cells versus sorted CD3+ cells) from CLL patients with del13q14 type I and del17p on the Affymetrix SNP 6.0 array platform that were previously analyzed on Affy 50K arrays. We have catalogued all somatically acquired lesions as well as all CNVs. In support of data analysis we have refined the software tools PLUT, LOH tool version 2 and dChipSNP. Results: In this cohort of 50 CLL cases in which we had previously identified a total of 98 subchromosomal losses and gains using the 50K SNP array platform, we have now identified 141 such lesions using the SNP 6.0 arrays. Of these CLL cases, 68% had identical lesion calls, 30% of cases displayed more lesions on the 6.0 arrays than on the 50K arrays and only one case had one lesion not identified using the SNP 6.0 array platform. Thus SNP 6.0 arrays allowed for significantly improved short lesion detection, detection of CNVs and improved measurements of genomic complexity in CLL. The breaks of del13q14 type I and del17p lesions were mapped with unprecedented resolution and in some cases have been localized to genomic regions less than 1 kb in length, thus allowing for PCR-based cloning of breaks. CNVs were identified throughout the genome, and were found in combination with monoallelic, somatically acquired genomic lesions (del17p, del13q14 and others) spanning such CNVs, resulting in patchy biallelic (homozogous) genomic losses within these lesions. Such a mechanism of biallelic gene loss (CNV plus paired, somatically acquired subchromosomal gene loss), occurs in otherwise monoallelic deletions and may contribute to the biology of monoallelic lesions in many cancers. As one example, CNVs were identified in CD3+ DNA from CLL patients that are located at approximately 49.448–49.508 Mb physical position on chromosome 13 that converted to homozygous loss in patients with monoallelic del13q14. These CNVs encompassed parts of DLEU2, TRIM13 and KCNRG but did not include the del13q14 resident miR cluster (miR15a/16-1). Such CNVs within del13q14 may have a role in the biology of CLL as they may result in gene expression changes that mimic somatically acquired del13q14 lesions.

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.