Genomic DNA Copy Number Alterations Present in AML Bone Marrow Samples with Normal Cytogenetics.

Abstract Cytogenetics and comparative genomic hybridization (CGH) have been used to identify large genomic amplifications and deletions in all subtypes of acute myeloid leukemia (AML). Up to 15–20% of AML patients have a normal karyotype at diagnosis. While cytogenetic abnormalities confer important prognostic information for patients with AML, there remain differences in the therapeutic response and outcome among patients with the same cytogenetic profile, implying that other, more subtle, genetic abnormalities may exist. We hypothesized that a subset of AML patients with normal cytogenetics may contain genomic DNA copy number changes that are too small to be detected using standard cytogenetic techniques. To address this possibility, we used high-resolution bacterial artificial chromosome (BAC) array CGH technology to examine 31 AML patients with normal cytogenetics. The BAC arrays contain 2,464 BAC clones spotted in triplicate on glass slides, and provide a 1 Mb resolution of the entire human genome. Technical generation of the arrays, hybridization parameters, and analysis were similar to that reported for murine BAC array CGH (Nat Genet. 2001 Dec;29(4):459–64). The 31 AML samples included 4 M0, 8 M1, 10 M2, and 9 M4 patients. Array CGH experiments were performed using 500 nanograms of Cyanine 5 labeled genomic DNA from unmanipulated AML bone marrow, mixed with an equal amount of control DNA (a pool of DNA from 4 cancer-free individuals) labeled with Cyanine 3. Using the human 1 Mb BAC arrays, we identified amplifications and deletions from multiple samples that were confirmed with G-banding cytogenetics [del(7)(q31), del(7)(p11.2), +8, del(11)(q13q23), +21, add(21)(q22), −X, −Y, +Y]. In addition, BAC arrays robustly detected copy number alterations that were identified in as few as 4/21 metaphases. We identified 5/31 (16%) patients with normal cytogenetics that contained altered genomic DNA copy numbers using BAC array CGH. Copy number changes were confirmed for several of these genomic loci using a dye-swap experiment, where the AML DNA was labeled with Cyanine 3, and the control DNA with Cyanine 5. Two of the 5 patients with abnormalities detected using array CGH would be reclassified from “intermediate” to “unfavorable” cytogenetics [del(7)(q31.31q34), add(11)(q23.3qter), and 17(p12pter)]. These results suggest that a subset of AML patients with normal cytogenetics contain genomic copy number alterations that may effect treatment and outcome. Patient # FAB subtype Genomic location Gain or loss Size (Megabase) Dye-Swap confirmed 1 M0 7(q31.31q34) loss 2.0 Not done 1 11(q23.3qter) gain 16.5 Not done 2 M1 11(p14) loss 7.4 Yes 3 M1 11(q13.2q14.1) gain 15.8 Yes 3 19(p) gain 64.0 Yes 4 M2 17(p12pter) gain 8.6 Not Done 5 M2 19(p13.1pter) loss 14.8 Yes 5 12(q13) loss 5.0 Yes

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Novel DNA Copy Number Changes in Hematological Malignancies: A cDNA-Based CGH Microarray Screening of CML, AML and CLL Cases without Chromosomal Imbalances in G-Banding.

Blood ◽

10.1182/blood.v104.11.4418.4418 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4418-4418

Author(s):

Tuija Lundan ◽

Anne Oikarainen ◽

Lena Hafren ◽

Maija Wolf ◽

Erkki Elonen ◽

...

Keyword(s):

Copy Number ◽

Myeloid Leukemia ◽

Array Cgh ◽

Reference Sample ◽

Copy Number Alterations ◽

Dna Copy Number ◽

Chromosomal Imbalances ◽

Single Allele ◽

Dna Copy Number Alterations ◽

Copy Number Changes

Abstract Copy number changes, such as small single allele losses and gains, have important roles in the mechanisms of cancer development. These alterations also have often prognostic significance. Genome wide screening of DNA copy number losses previously conducted by extensive LOH analyses can now be performed with array-based comparative genomic hybridization (array CGH). We assessed the utility of array CGH in the detection of single allele deletions and gains in a cohort of seven patients with chronic myeloid leukemia (CML), seven patients with chronic lymphatic leukemia (CLL) and three patients with acute myeloid leukemia (AML). All the CLL and AML patients had a normal karyotype as assessed by standard G-banding. In CML patients the only clonal abnormality detected by cytogenetics was the reciprocal Philadelphia translocation, t(9;22)(q34;q11). The derivative chromosome 9 [der(9)] deletion status of the CML patients was determined using fluorescence in situ hybridization (FISH) analysis. Four patients did not have the deletion, two had a der(9) deletion spanning both 5′ABL and 3′BCR regions and one patient had a deletion of the 5′ABL region alone. The array CGH experiments were performed using Agilent Technologies Human 1 cDNA microarray slides consisting of 13,000 clones. A total of 6 ug of fluorescently labeled DNA extracted from bone marrow samples was hybridized on cDNA array. Normal male or female DNA was used as the reference sample in the hybridization. The slides were scanned with the Agilent fluorescent scanner and intensity ratio data between the tumor and reference sample was processed using Feature Extraction software. The data was filtered and analyzed using SPSS (version 11) and Origin 7.0 softwares. The processed, untransformed red-to-green fluorescence signal ratio was used for evaluating gene dosage. Ratios greater than 1.1 were considered to indicate DNA copy number gains and ratios below 0.9 DNA copy number losses in tumor samples. In two CML patients who had deletions covering both the 5′ABL and 3′BCR regions in the translocation breakpoint of der(9), the deletion was detectable with the array CGH. In four patients with no deletion the red-to-green ratio profile for der(9) was 1. However, in one patient with an isolated 5′ABL deletion, the deletion was not visible in array CGH. No other obvious DNA copy number alterations were seen in CML patients. Array CGH detected deletions in three of the seven CLL patients. Deletions were found in 13q14, 2q32-33 and 14q24. One of the three AML patients studied showed an amplification in chromosome 9p. No alterations were seen in the other two AML patients. The FISH and array studies are being done on larger set of patient samples to confirm the results. We conclude that array CGH provides new information in patients without chromosomal imbalances in standard cytogenetics and enables the detection of novel small submicroscopic copy number alterations. Furthermore, a cDNA-based array platform can be used both for studies of DNA copy number alterations and gene expression analyses.

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DETECTING AND ANALYZING COPY NUMBER ALTERNATIONS IN ARRAY-BASED CGH DATA

Biomedical Engineering Applications Basis and Communications ◽

10.4015/s1016237216500447 ◽

2016 ◽

Vol 28 (06) ◽

pp. 1650044 ◽

Cited By ~ 2

Author(s):

Mariam A. Sheha ◽

Mai S. Mabrouk ◽

Mahmoud Elhefnawi

Keyword(s):

Copy Number ◽

Genomic Dna ◽

Array Cgh ◽

Artificial Chromosome ◽

Gaussian Mixture ◽

Comparative Genomic ◽

Intensity Ratios ◽

Dna Copy Number Alterations ◽

Copy Number Changes ◽

Tissue Specimens

Copy number changes or alterations are a form of genetic variation in the human genome. Genomic DNA copy number alterations (CNAs) are associated with the development and progression of cancers. Array-based comparative genomic hybridization (a-CGH) is a technique used to identify copy number changes in genomic DNA. It yields data consisting of fluorescence intensity ratios of test and reference DNA samples. The intensity ratios provide information about the number of copies in DNA. Practical issues such as the contamination of tumor cells in tissue specimens and normalization errors necessitate the use of automated statistics algorithms for learning about the genomic alterations from array CGH data. Specifically, there is a need for algorithms that can identify gains and losses in the number of copies based on statistical considerations, rather than merely detect trends in the data. For this purpose the proposed study introduces three different approaches; Circular binary segmentation, Bayesian approach, relying on the hidden Markov model and effective Gaussian mixture (GM) clustering for the analysis of array CGH profiles. Publicly available data on pancreatic adenocarcinoma and Coriell cell line bacterial artificial chromosome (BAC) array were used for the analysis to illustrate the reliability and success of the techniques.

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Mixed Gastric Carcinomas Show Similar Chromosomal Aberrations in Both their Diffuse and Glandular Components

Analytical Cellular Pathology ◽

10.1155/2006/650620 ◽

2006 ◽

Vol 28 (5-6) ◽

pp. 283-294

Author(s):

Beatriz Carvalho ◽

Tineke E. Buffart ◽

Rui M. Reis ◽

Thomas Mons ◽

Cátia Moutinho ◽

...

Keyword(s):

Gastric Cancer ◽

Chromosomal Aberrations ◽

Copy Number ◽

Array Cgh ◽

Comparative Genomic Hybridisation ◽

Comparative Genomic ◽

Dna Copy Number ◽

Gastric Carcinomas ◽

Phenotypic Divergence ◽

Copy Number Changes

Gastric cancer is one of the most frequent malignancies in the world. Nonetheless, the knowledge of the molecular events involved in the development of gastric carcinoma is far from complete. One of the hallmarks of gastric cancer is chromosomal instability resulting in abnormal DNA copy number changes throughout the genome. Mixed gastric carcinomas constitute a rare histological entity, containing the two main histological phenotypes (diffuse and intestinal). Very little is known about the underlying mechanisms of phenotypic divergence in these mixed tumours. To the best of our knowledge only E-Cadherin mutations were implicated so far in the divergence of these tumours and nothing is known about the involvement of chromosome copy number changes in the two divergent histological components. In this study, we compared the DNA copy number changes, in the two different components (diffuse and intestinal) of mixed gastric carcinomas by microarray – comparative genomic hybridisation (array CGH). The analysis of 12 mixed gastric carcinomas showed no significant differences in array CGH profiles between the diffuse and intestinal components of mixed carcinomas. This supports the idea that the phenotypic divergence within mixed gastric carcinomas is not caused by DNA chromosomal aberrations.

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Distinguishing melanocytic nevi from melanoma by DNA copy number changes: comparative genomic hybridization as a research and diagnostic tool

Dermatologic Therapy ◽

10.1111/j.1529-8019.2005.00055.x ◽

2006 ◽

Vol 19 (1) ◽

pp. 40-49 ◽

Cited By ~ 147

Author(s):

Jurgen Bauer ◽

Boris C. Bastian

Keyword(s):

Comparative Genomic Hybridization ◽

Diagnostic Tool ◽

Copy Number ◽

Comparative Genomic ◽

Genomic Hybridization ◽

Dna Copy Number ◽

Melanocytic Nevi ◽

Copy Number Changes

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High-resolution array CGH of metanephric adenomas: lack of DNA copy number changes

Histopathology ◽

10.1111/j.1365-2559.2009.03473.x ◽

2010 ◽

Vol 56 (2) ◽

pp. 212-216 ◽

Cited By ~ 12

Author(s):

Adrianna Szponar ◽

Maria V Yusenko ◽

Gyula Kovacs

Keyword(s):

High Resolution ◽

Copy Number ◽

Array Cgh ◽

Dna Copy Number ◽

Copy Number Changes

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High Density SNP Array Analysis of Acute Promyelocytic Leukemia (APL) Detects New Common Genomic Copy Number Alterations as Possible Cooperating Lesions

Blood ◽

10.1182/blood.v116.21.2721.2721 ◽

2010 ◽

Vol 116 (21) ◽

pp. 2721-2721

Author(s):

Daniel Nowak ◽

Marion Klaumuenzer ◽

Benjamin Hanfstein ◽

Maximilian Mossner ◽

Florian Nolte ◽

...

Keyword(s):

Bone Marrow ◽

Acute Promyelocytic Leukemia ◽

Copy Number ◽

Genomic Dna ◽

Snp Array ◽

Promyelocytic Leukemia ◽

High Density ◽

Copy Number Alterations ◽

Array Analysis ◽

Snp Array Analysis

Abstract Abstract 2721 Introduction: Acute Promyelocytic Leukemia (APL) is characterized by the typical chromosomal translocation t(15;17)(q22;q21) leading to the fusion product PML-RARA, which blocks granulocytic differentiation in the promyelocyte stage. Several experimental in vitro and in vivo studies have demonstrated that PML-RARA is necessary but not sufficient for the generation of APL. This circumstance has motivated the search for additional leukemogenic and cooperating molecular lesions. Patients and Methods: We have analyzed 101 APL patient bone marrow samples with high density Genome-Wide Human SNP 6.0 arrays, which interrogate >900.000 SNPs and >900.000 non-polymorphic copy number markers throughout the genome (Affymetrix, Santa Clara, CA, USA) in search for copy number alterations (CNAs) potentially relevant in the pathogenesis of APL. Genomic DNA from samples at initial diagnosis of 94 patients was analyzed. Furthermore, DNA from 11 samples at relapse was available, whereby 4 of these relapse samples also had paired DNA from initial diagnosis. Data analysis was carried out with the CNAG 3.3 software using anonymous references. For exclusion of copy number polymorphisms, all detected CNAs were compared with the databases of known copy number polymorphisms in the UCSC genome browser. For data validation, putatively acquired CNAs and regions of copy number neutral loss of heterozygosity (CNLOH) were confirmed by hybridization of DNA from paired normal samples when the patients were in remission, by quantitative real time PCR of genomic DNA and by direct sequencing of informative SNPs. Results: The high density SNP array analysis detected a total of 120 heterozygous deletions, 97 duplications or amplifications and 7 regions of telomeric CNLOH leading to an average of 2.3 CNAs per sample (range 0–30). The most common numerical and large structural aberrations were found on chromosome (chr.) 8 with either trisomy 8 (n=11) or duplication of regions on chr. 8q (n=10) followed by heterozygous deletions of chr. 7q (n=5) and chr. 16q (n=5). Furthermore, unbalanced translocations of chr. 15 and 17 involving PML and RARalpha were detected in five cases leading to duplication of the PML-RARA fusion or deletion of genomic regions flanking either PML or RARalpha. Recurrent microlesions (<1Mbp) were found in several regions as heterozygous deletions on chr. 1q31.3 containing the micro RNAs MIR181B1 and MIR181A1 (n=5), on chr. 2q32.3 containing serine/threonine kinase 17b (STK17B) (n=5) or chr. 3p24.3 containing ankyrin repeat domain 28 (ANKRD28) (n=5). One recurrent region of telomeric CNLOH was found on chr. 19q in two samples. Of note, besides the few regions of telomeric CNLOH a large number of intrachromosomal CNLOH regions (n=265) was identified, with recurrent regions on chr. 6p21.1 (n=10) or chr. 5q23.3-5q31.1 (n=6) containing genes relevant in hematopoiesis such as IL3, CSF2 or DNA damage repair such as RAD50. Although these CNLOH regions were not somatically acquired they may possibly harbor genetic predispositions for disease. Conclusions: We describe a detailed high density SNP array genomic profiling of bone marrow DNA from patients with APL, which has led to the identification of several new cryptic recurrent genomic lesions. These genomic alterations point to candidate genes, which could be cooperating factors in addition to PML-RARA. Therefore, our data helps to provide a better understanding of the molecular mechanisms underlying the development of APL. Disclosures: Kohlmann: MLL Munich Leukemia Laboratory: Employment. Lengfelder:Cephalon: Research Funding.

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Targeted Oligonucleotide Array Assessment of Genomic Copy Number Alterations for Risk Stratification in Chronic Lymphocytic Leukemia

Blood ◽

10.1182/blood.v118.21.1773.1773 ◽

2011 ◽

Vol 118 (21) ◽

pp. 1773-1773

Author(s):

Jane Houldsworth ◽

Asha Guttapalli ◽

Xiao J. Yan ◽

Charles Ma ◽

Weiyi Chen ◽

...

Keyword(s):

Risk Stratification ◽

Copy Number ◽

Array Cgh ◽

Lymphocytic Leukemia ◽

P Value ◽

Copy Number Alterations ◽

Mutation Status ◽

Copy Number Changes ◽

Gain Loss ◽

Genomic Regions

Abstract Abstract 1773 Risk stratification in chronic lymphocytic leukemia (CLL) is highly desirable and should comprise not only evaluation of clinical features but also molecular prognostic markers. Currently such molecular markers include loss of 17p13, 11q22, 13q14, 6q22, and gain of chromosome 12 as assessed by fluorescence in situ hybridization (FISH) and mutation status of the variable region of the IGH gene (IGHV) by sequencing. In recent years, genome-wide scanning technologies such as array-comparative genomic hybridization (array-CGH) have revealed novel and refined known copy number alterations (CNAs) in the CLL genome. In order to evaluate the potential of array-CGH in prognostication in mature B-cell neoplasms, including CLL, and implement array-CGH in a clinical diagnostic laboratory, a targeted oligonucleotide-based microarray was custom designed to represent genomic regions exhibiting gain/loss in these lymphoid neoplasms. The 4 × 44K formatted array included 2 × 17,348 probes for the 80 selected genomic regions (average resolution of 34kbp), and recommended controls including a 1Mbp genome backbone. DNA extracted from two CLL datasets were submitted to array-CGH using an equimixture of commercially available male/female DNA as a reference. CNAs were detected using Genomics Workbench Lite (Agilent Technologies, Inc.) with the ADM2 algorithm. Analytical sensitivity was assessed by cell line DNA dilution and by FISH (116 specimens) and was 30–40% and 20–25%, respectively. Recurrent CNAs in previously untreated patients, greater than 1.5Mbp in size, were analyzed for association with time to first treatment (TTFT) and overall survival (OS) by the log rank test. Association with IGHV mutation status was tested using the Fisher's two-sided exact test. In both datasets for untreated specimens, unmutated IGHV negatively correlated with both TTFT and OS significantly (p < 0.05). Gain of chromosome 12 was detected in 11–12% of untreated specimens in both datasets and as expected did not associate with outcome. Loss of 13q14 as a sole abnormality (excluding copy number changes arising at known sites of normal variation) was associated with an overall favorable outcome, but specimens with loss of both loci (MIR15A/16-1 and RB1) versus one locus (MIR15A/16-1) did not display significantly different outcomes. As expected loss of 17p13 associated with shorter TTFT and OS, and was observed at higher levels in treated specimens. A similar result was observed for 11q22 loss but not in the second dataset, perhaps due to the relatively short follow-up time. Importantly, four additional copy number changes (gain of 2p, 3q, and 8q, and loss of 8p) were found to associate with shorter TTFT and/or OS, and also occurred at higher frequency in treated specimens. Notably, all but one specimen exhibiting two of these CNAs, were Rai Stage 0-II. After multiple comparisons correction, gain of 2p and 3q, and loss of 8p remained significantly associated with an unfavorable outcome. Gain of 2p25.3-p15 was observed exclusively in unmutated IGHV specimens. Loss of 18p and gain of 17q24 were not considered further for testing due to low frequency or lower frequency in treated specimens (data not shown). Uniquely, these data demonstrate in low-intermediate risk CLL cohorts the prognostic value of genomic gain/loss at multiple sites and support implementation of array-CGH into a clinical setting for risk stratification in CLL where genomic gain or loss of multiple clinically relevant genomic regions can be assessed simultaneously. Dataset 1 Untreated n = 81 TTFT p-value OS p-value Treated n = 38 Dataset 2 n = 169 TTFT p-value OS p-value Treated n = 28 Median TTFT 87.6 mo 24.1 mo Median OS 117.7 mo 37.2 mo Rai Stage 0 25 77 I-II 42 48 III-IV 5 1 na 9 43 Unmutated IGHV 46% (n=80) 0.0003 0.0004 38% (n=163) 0.002 0.044 13q14 loss (sole abnormality) 52.5% 0.038‡ 0.087‡ 33.7% 0.144‡ 0.008‡ MIR15A/16-1, RB1 27.5% 0.77 0.337 11.2% 0.011 1 MIR15A/16-1 25.0% 22.5% 11q22 loss (ATM) 12.3% 0.125 0.009 23.7% 8.3% 0.393 0.977 14.3% 17p13 loss (TP53) 2.5% 0.010 0.012 15.8% 4.7% 0.006 <.0001 10.7% 2p25.3-p15 gain 6.2% 0.002 <.0001 10.5% 3.0% 0.702 0.025 10.7% 8q24 gain 2.5% 0.238 0.014 7.9% 4.1% 0.564 0.007 0.0% 3q26-q27 gain 2.5% <.0001 <.0001 5.3% 3.0% 0.850 <.0001 7.1% 8p23-p21 loss 2.5% 0.002 0.016 10.5% 1.2% 1 <.0001 7.1% Unless otherwise noted, all values associated with shorter times ‡ Associated with longer time na not available Disclosures: Houldsworth: Cancer Genetics, Inc.: Employment. Guttapalli:Cancer Genetics, Inc.: Employment. Ma:Cancer Genetics, Inc.: Employment. Chen:Cancer Genetics, Inc.: Employment. Patil:Cancer Genetics, Inc.: Consultancy.

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