Molecular Characterization of Genomic Structural Alterations through CGH Arrays in Acute Myeloid Leukemia with Normal Cytogenetic Findings.

Abstract Acute myeloid leukemia (AML) is an heterogeneous disease. Cytogenetic findings are one of the main prognosis criteria beside age, leucocytosis, and antecedent of previous hemopathy. More than 50% of AML patients, mainly with normal cytogenetic, belong to the intermediate cytogenetic risk group. Even in this group, cryptic molecular abnormalities as mutation are frequent with prognosis value. In this work we have studied 31 AML patients (Pts) with normal caryotype by high resolution comparative genomic hybridization array CGH using agilent 185k chips to detect gene copy number alterations (CNA). Tumor DNA was obtained from bone marrow diagnosis samples (>60% of blasts) and control DNA from pooled blood samples obtained from 30 healthy donors or from AML patients in CR during the follow up. The design of the study was first to hybridize tumor DNA from the 31 patients against pooled control DNA to detect both copy number polymorphisms (CNP) and acquired CNA in AML and in a second time to hybridize tumor DNA from the 10 pts with the greater number of abnormalities against autologous DNA obtained at CR time to distinguish acquired CNA from CNP. Briefly DNA labelling was performed using Cy3dUTP and Cy5 dUTP respectively for control and tumor samples. Fluorescence ratios were normalized and spots with value that deviated significantly from background were retained as significant evidence of DNA copy number alterations after statistical analysis using Feature Extraction and CGH Analytics AGILENT software. BAC from the Human Genome Center (Wellcome Trust Sanger) were used to obtain FISH probes to confirm abnormalities observed by CGH arrays on metaphase spreads obtained from bone marrow diagnosis sample when available. In each case, two colour FISH was performed by the simultaneous hybridization on a green BAC probe located in the CNA regions and a red control BAC probe located in the vicinity of the CNA regions obtained from the UCSC genome browser data. Results 98 DNA copy number alterations were observed with 56 losses and 42 gains cases. Those CNA were variable in size from 8,6 Mb to <100 Kb. 8 CNA regions were recurrent (5 losses in 7q31 (12pts), 4q13 (3pts), 1q31 (4pts), 2q37 (2pts) et 22q13 (3pts) and 3 gains in 1q23 (3pts), 2p11(2pts) et 17q21(2pts). Non recurrent CNA were located in 1p36.2, 1p34.3, 1p21.1, 1p22, 1q31.2, 3q12.2, 4p13, 5p15.3, 5q14.1, 6p22.1, 7p22.1, 7q11.23, 10q26.3, 11q13.2, 15q11.2, 15q15.3, 15q25.3, 15q26.1, 16q24.2, 17p13.2, 17p11.2, 22q13,33 for gains and in 1p33, 1q32.1, 1q32.2, 3p14.1, 3p13-p12.3, 4q26, 7p11.2, 7p14.3, 7q35, 8p23.2, 8q23.1, 9p24.1, 9p13.1-p12, 9q34.11-q34.12, 12p13.31, 13q14.2, 14q21.2, 14q22.1, 14q23.2, 15q22.31, 16q22.1, 21q21.2, 21q22.12 for losses. No one of the patients had a genome without CNA and 6 patients (20%) had more than 5 CNAs. The CGH data showed clearly that 80% of the CNA observed with pool control DNA were not present when tumor DNA was hybridized against the autologous CR DNA confirming the high frequency of CNP as reported on http://projects.tcag.ca/variation website. All of the acquired CNA regions observed were concordant with the FISH results involving genes known to be crucial in leukemia as RUNX1, Abl, CEBPa but also many other genes. In conclusion cryptic abnormalities are frequent in normal caryotype AML and 20% of the patients studied had more than 5 CNA traducing a great instability of the genome of leukemic cells.

Download Full-text

Oligonucleotide array-CGH reveals cryptic gene copy number alterations in karyotypically normal acute myeloid leukemia

Leukemia ◽

10.1038/sj.leu.2404543 ◽

2007 ◽

Vol 21 (3) ◽

pp. 571-574 ◽

Cited By ~ 38

Author(s):

A Tyybäkinoja ◽

E Elonen ◽

K Piippo ◽

K Porkka ◽

S Knuutila

Keyword(s):

Acute Myeloid Leukemia ◽

Copy Number ◽

Myeloid Leukemia ◽

Array Cgh ◽

Gene Copy Number ◽

Oligonucleotide Array ◽

Gene Copy ◽

Copy Number Alterations ◽

Acute Myeloid

Download Full-text

Genetic changes including gene copy number alterations and their relation to prognosis in childhood acute myeloid leukemia

Leukemia & Lymphoma ◽

10.3109/10428190903350397 ◽

2009 ◽

Vol 51 (1) ◽

pp. 114-124 ◽

Cited By ~ 10

Author(s):

Gemma Armengol ◽

Anna Canellas ◽

Yolanda Álvarez ◽

Pilar Bastida ◽

José Sánchez De Toledo ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Copy Number ◽

Myeloid Leukemia ◽

Gene Copy Number ◽

Gene Copy ◽

Copy Number Alterations ◽

Genetic Changes ◽

Childhood Acute Myeloid Leukemia ◽

Acute Myeloid

Download Full-text

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.

Download Full-text

High Concordance between Pharmacogenetic Analysis of Bone Marrow and Buccal Cell Samples in Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v108.11.2364.2364 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2364-2364

Author(s):

Joli R. Weiss ◽

Maria R. Baer ◽

Christine B. Ambrosone ◽

Javier G. Blanco ◽

Alan Hutson ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Excellent Agreement ◽

Myeloid Leukemia ◽

Buccal Cell ◽

Cell Phenotype ◽

Kappa Statistics ◽

Exon 2 ◽

Tumor Dna ◽

Acute Myeloid

Abstract Archived tumor tissue is a useful resource for large-scale pharmacogenetic studies designed to identify associations between genetic polymorphisms and treatment outcomes. In cancer patients, genotypes determined from tumor DNA and from non-diseased tissue DNA may differ due to changes in the tumor’s genome acquired during malignant transformation. Consequently, the assessment of discordances between tumor and non-diseased tissue genotypes is pertinent when identifying pharmacogenetic determinants that may impact drug disposition in both the tumor and the host. Leukemia cells frequently have karyotypic abnormalities; therefore, discordances between host and tumor DNA genotypes are of particular relevance for pharmacogenetic studies on leukemia. In this methodological study, we investigated the concordance between genotypes determined in DNA extracted from paired cryopreserved pre-treatment bone marrow samples and non-diseased tissue (buccal cell samples) from 80 adult patients diagnosed with acute myeloid leukemia (AML) by WHO criteria at Roswell Park Cancer Institute between 2004 and 2006. Samples were genotyped for a panel of polymorphisms (19 SNPs and 2 polymorphic gene deletions) of genes whose products are involved in drug metabolism (CAT, MnSOD, MGMT, GSTP1, GSTT1, GSTM1 CYP3A4, CYP2C8, CDA), drug transport (MDR1, MRP1, BCRP) and DNA repair processes (XPD, XRCC1). All genotypes with the exception of GSTM1 and GSTT1 were determined by using Sequenom’s high-throughput matrix-assisted laser desorption/ionization time of-flight (MALDI-TOF) mass spectrometry. GSTM1 and GSTT1 gene deletion genotypes were determined using multiplex polymerase chain reaction. Kappa statistics were used to determine the concordance rate of genotype calls between the paired tumor and normal tissue DNA samples. Kappa statistics for the paired bone marrow and buccal DNA samples ranged between 0.93 and 1.00, indicating excellent agreement (CAT=0.97; MnSOD=0.98; MGMT=1.00; GSTP1=0.96; GSTT1=1.00; GSTM1=1.00; CYP3A4=0.94; CYP2C8=0.96; CDA=1.00; MDR1-03=1.00; MDR1-05=0.96; MDR1-24=0.98; MRP1(exon 8)=1.00; MRP1 (exon 28)=0.98; MRP1 (exon 9)=0.98; BCRP (exon 5)=1.00; BCRP (exon 2)=1.00; XPD312=0.93; XPD751=0.98; XRCC1=1.00). Significantly, the GSTT1 and GSTM1 genotypes were in perfect concordance for the paired samples. These genes are of particular importance, since there is great potential for misclassification if the null genotype is a result of disease-related loss of heterozygosity. We also observed excellent agreement for those genes on chromosomes that are commonly involved in deletions or translocations as part of the leukemic process, including MDR1 at 7q21.1 and MRP1 at 16p13.1. These data demonstrate that archived bone marrow samples may be used to accurately perform genotyping for the genes that we studied. However, while the data validate the use of bone marrow samples for this panel of genotypes for AML pharmacogenetic studies, it may be important to account for gene amplification or deletion and chromosome gain or loss when using bone marrow DNA to study variation and clinical outcomes in order to account for quantitative differences that may affect the concordance between germline genotype and cancer cell phenotype. Further studies in this area are warranted and are ongoing.

Download Full-text

TP53 Alterations in Acute Myeloid Leukemia with Complex Karyotype Correlate with Specific Copy Number Alterations, Monosomal Karyotype, and Dismal Outcome,

Blood ◽

10.1182/blood.v118.21.3558.3558 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3558-3558 ◽

Cited By ~ 3

Author(s):

Frank G. Rücker ◽

Richard F. Schlenk ◽

Lars Bullinger ◽

Sabine Kayser ◽

Veronica Teleanu ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Copy Number ◽

Myeloid Leukemia ◽

Multivariable Analysis ◽

Complex Karyotype ◽

Copy Number Alterations ◽

Free Survival ◽

Genomic Complexity ◽

Monosomal Karyotype ◽

Acute Myeloid

Abstract Abstract 3558 Acute myeloid leukemia with complex karyotype (CK-AML, CK+) is defined as ≥3 acquired chromosome abnormalities in the absence of recurrent genetic abnormalities (WHO 2008). CK-AML account for 10–15% of all AML and are characterized by a dismal outcome. To delineate prognostic markers in this unfavorable subgroup, we performed integrative analysis using genomic profiling (array-comparative genomic hybridization [CGH] and/or single-nucleotide polymorphism [SNP] analysis), as well as TP53 mutation screening in 234 CK-AML. TP53 mutations were found in 141/234 (60%) CK-AML comprising 130 missense, 21 insertion/deletion, nine nonsense, and eight splice site mutations; genomic losses of TP53 were identified in 94/234 (40%). Combining these data, TP53 alterations were detected in 70% of patients, and at least 66% of these exhibited biallelic alterations. TP53 alterations (loss and/or mutation in TP53) were characterized by a higher degree of genomic complexity, as measured by total number of copy number alterations per case (mean±SD 14.30±9.41 versus 6.16±5.53, P <.0001), and by the association with specific genomic alterations, that is, monosomy 3 or losses of 3q (-3/3q-) (P=.002), -5/5q- (P<.0001), -7/7q- (P=.001), -16/16q- (P<.0001), -18/18q- (P=.001), and -20/20q- (P=.004); gains of chromosome 1 or 1p (+1/+1p) (P=.001), +11/+11q (P=.0002), +13/+13q (P =.02), and +19/+19p (P =.04); and amplifications in 11q13∼25 [amp(11)(q13∼25)]. The recently described cytogenetic category “monosomal karyotype” (MK), defined as two or more autosomal monosomies or one single autosomal monosomy in the presence of structural abnormalities, for which a prognostic impact could be demonstrated even in CK-AML, was correlated with TP53 alterations (P <.0001). Clinically, TP53altered CK-AML patients were older (median age, 61 versus 54 years, P =.002), had lower bone marrow (BM) blast counts (median 65% versus 78%, P=. 04), and had lower complete remission (CR) rates (28% versus 50%, P =.01). For multivariable analysis, a conditional model was used with an age cut point at 60 years to address the different treatment intensities applied in the different age cohorts. In this model the only significant factors for CR achievement were TP53altered (OR, 0.55; 95%-CI, 0.30 to 1.00; P =.05) and age (OR for a 10 years difference, 0.67; 95%-CI, 0.52 to 0.87; P =.003). TP53 altered predicted for inferior survival; the 3-year estimated survival rates for CK+/TP53altered and CK+/TP53unaltered patients were as follows: event-free survival (EFS), 1% versus 13% (log-rank, P =.0007); relapse-free survival (RFS), 7% versus 30% (P =.01); and overall survival (OS), 3% versus 28% (P <.0001), respectively. Other variables predicting for inferior OS in univariable analyses were age and MK. Among the cohort of CK+/MK+ AML, TP53altered patients had a significantly worse OS (P =.0004). Multivariable analysis (stratified for age at cut point of 60 years) revealed TP53altered (HR, 2.43; 95%-CI, 1.56 to 3.77; P =.0001), logarithm of WBC (HR, 1.62; 95%-CI 1.17 to 2.26; P =.004), and age (HR for 10 years difference, 1.26; 95%-CI, 1.01 to 1.56, P =.04), but not MK as significant variables for OS. In addition, explorative subset analysis suggested that allogeneic hematopoietic stem-cell transplantation in first CR which was performed in 30 CK-AML did not impact outcome in TP53altered CK-AML. In summary, TP53 is the most frequently known altered gene in CK-AML. TP53 alterations are associated with older age, genomic complexity, specific DNA copy number alterations, MK, and dismal outcome. In multivariable analysis, TP53 alteration is the most important prognostic factor in CK-AML, outweighing all other variables, including the MK category. TP53 mutational status should be assessed in clinical trials investigating novel agents in order to identify compounds that may be effective in this subset of patients. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Abstract 2451: Genomic wide microarray analysis identifies novel copy number alterations in adult acute myeloid leukemia

10.1158/1538-7445.am2017-2451 ◽

2017 ◽

Author(s):

Maria Chiara Fontana ◽

Giovanni Marconi ◽

Cristina Papayannidis ◽

Eugenio Fonzi ◽

Giorgia Simonetti ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Microarray Analysis ◽

Copy Number ◽

Myeloid Leukemia ◽

Copy Number Alterations ◽

Acute Myeloid

Download Full-text

Chromosome Instability in Acute Myeloid Leukemia Is Associated to increased spontaneous breakage and Copy Number Variations (CNVs).

Blood ◽

10.1182/blood.v114.22.4240.4240 ◽

2009 ◽

Vol 114 (22) ◽

pp. 4240-4240

Author(s):

Ariela Freya Fundia ◽

Irene Beatriz Larripa ◽

Susana Hayde Acevedo

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Copy Number ◽

Myeloid Leukemia ◽

Conflicts Of Interest ◽

Copy Number Variations ◽

Fragile Sites ◽

Bone Marrow Cultures ◽

Acute Myeloid ◽

Marrow Cultures

Abstract Abstract 4240 Acute Myeloid Leukemia (AML) is a genetically pleiomorphic disease characterized by multiple genetic lesions including structural and numerical chromosome aberrations, gene mutations and changes in gene expression. The underlying mechanism behind the acquisition of these genetic abnormalities is not known, but it is likely that are facilitated by factors that increase chromosomal and genetic instability. The aim of this work was to explore chromosomal instability (CIN) in de novo AML patients by evaluating chromosome fragility and acquired DNA copy number variations (CNVs). Leukemic karyotypes were scored on bone marrow cultures (24-48 hs, without mitogens) from 24 AML patients using conventional cytogenetic and FISH. Spontaneous and FUdR -induced chromosomal fragility was studied on PHA-stimulated lymphocytes cultures (72 hs) from 10 patients and 10 healthy individuals, with and without FUdR (10mg/ml). One hundred metaphases were analyzed blind using conventional cytogenetic with sequential GTG banding. CNVs were scored on bone marrow cultures from 14 AML patients using fluorescence in situ hybridization (FISH) with two probes targeting specific regions on chromosomes 5q31 (LSI EGR/D5S721:D5S23) and 7q31 (LSI D7S522/CEP7), loci reported to be critical hotspots involved in AML. The cut-off value for FISH scoring was calculated after analyzing 250 cells from each of 10 normal bone marrow samples. The cutoff for allelic losses was equal to 0.8% for either 5q or 7q probe. Nine out of 24 AML cases presented normal karyotypes in bone marrow samples while the following abnormalities: t(3;8), t(3;11), t(4;11), t(15;17), t(9;22) and inv(16) were detected in the remaining patients. Significantly increased frequencies of spontaneous chromosome breakage, scored on untreated cultures, were detected in patients (0.22±0.03) respect to controls (0.07±0.03) (p<0.05), showing a random pattern of distribution. No differences were observed between patients and controls with FUdR treatment. Statistical analysis with Ch2 test considering data of FUdR breakpoint distribution over all individuals, identified 21 common fragile sites (c-fra) in AML cases (p<0.005). The most common sites were located at 1p32, 1p22, 1q21, 3p14, 3q27, 4q31, 5q31, 6p21 and 9q13. A high inter-individual variation in the pattern of expression was observed. Using FISH, we obtained the DNA copy numbers at 5q and 7q in all AML samples. Only losses of DNA were found with both probes. The 5q signal count in patients with normal karyotypes was on average 6.4% (range 0.5-30%), while it was 24.9% (range 0.8-90%) for patients with abnormal cytogenetic. The 7q signal count was on average 3.9% (range 0.5-10.3%) and 20.6% (range 2-63%) for cases with normal and abnormal karyotypes, respectively. Particularly, we showed that 12/14 (85.7%) AML cases, either with normal or abnormal karyotypes, exhibited copy number losses at both regions, showing different values between each patient. These findings showed that leukemic patients exhibit a CIN phenotype, providing an unstable background and facilitating the acquisition of additional genetic changes, such as CNVs, which could play an important role in disease progression. Disclosures: No relevant conflicts of interest to declare.

Download Full-text