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

Abstract Acute myeloid leukemia (AML) is a heterogeneous disease. Risk factors such as karyotype, FAB subtype, FLT3 status and response to induction therapy are determinants of outcome with current therapies. We hypothesize that array comparative genomic hybridization (CGH) will identify gene copy number changes that are determinants of outcome. Array CGH was performed on diagnostic bone marrow samples from patients on the COG study POG #9421. In order to determine regions of altered gene copy number, labeled genomic DNA samples were hybridized together with sex-matching normal human reference DNA to cDNA microarrays with 41,751 features (corresponding to 24,473 unique Unigene cluster IDs), arrays were obtained from the Stanford University Microarray Core Facility. Control hybridizations were performed to assess intra- and inter-experimental variability. We studied 70 samples with adequate high-quality DNA. Circular binary segmentation was used to distinguish discrete gene copy number transition points from chance noise events and to transform primary clone-by-clone data into genomic regions of equal copy number. Using gain/loss threshold, based on two-standard deviation range of control self-to-self distribution, novel gene amplifications and deletions were found in profiled samples. The highest alteration recurrence was observed for gains of chromosome 8 (21%) and losses of chromosome 6 (29%). The area of chromosome 8 which was found to be gained is notable for the presence of potential oncogenes such as ERK8. The deleted area of chromosome 6 is notable for the presence of potential regulators of oncogenesis: MDC1, DDR1, NFKBIL1, TNF, and BRD2. In summary, array CGH has identified novel areas of gene copy number gain and loss in this population of pediatric de novo AML patients. Further studies are needed to assess whether these genes are associated with outcome, known risk factors and whether they will provide insight into the heterogeneity of de novo AML.

Download Full-text

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

Blood ◽

10.1182/blood.v108.11.105.105 ◽

2006 ◽

Vol 108 (11) ◽

pp. 105-105 ◽

Cited By ~ 1

Author(s):

Christophe Roumier ◽

Sandrine Geffroy ◽

Olivier Nibourel ◽

Sabine Quief ◽

Celine Villenet ◽

...

Keyword(s):

Bone Marrow ◽

Acute Myeloid Leukemia ◽

Copy Number ◽

Myeloid Leukemia ◽

Gene Copy ◽

Copy Number Alterations ◽

Dna Copy Number ◽

Dna Copy Number Alterations ◽

Tumor Dna ◽

Acute Myeloid

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

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

TP53 alterations in acute myeloid leukemia with complex karyotype correlate with specific copy number alterations, monosomal karyotype, and dismal outcome

Yearbook of Oncology ◽

10.1016/j.yonc.2012.07.039 ◽

2012 ◽

Vol 2012 ◽

pp. 150-151

Author(s):

R.J. Arceci

Keyword(s):

Acute Myeloid Leukemia ◽

Copy Number ◽

Myeloid Leukemia ◽

Complex Karyotype ◽

Copy Number Alterations ◽

Monosomal Karyotype ◽

Acute Myeloid

Download Full-text

Marker Chromosomes Can Arise from Chromothripsis and Predict Adverse Prognosis in Acute Myeloid Leukemia

Blood ◽

10.1182/blood.v128.22.2869.2869 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2869-2869

Author(s):

Tilmann Bochtler ◽

Martin Granzow ◽

Friedrich Stölzel ◽

Christina Kunz ◽

Brigitte Mohr ◽

...

Keyword(s):

Overall Survival ◽

Acute Myeloid Leukemia ◽

Copy Number ◽

Myeloid Leukemia ◽

Poor Prognosis ◽

Array Cgh ◽

Newly Diagnosed ◽

Single Chromosome ◽

Marker Chromosomes ◽

Acute Myeloid

Abstract Introduction: Cytogenetic testing is routinely performed in newly diagnosed acute myeloid leukemia (AML) for risk stratification. Elaborate risk classifications based on karyotyping are provided by both the European Leukemia Net (ELN) and the Medical Research Council (MRC). Complex aberrant, monosomal and abnl(17p) karyotypes confer a poor prognosis. In cytogenetic studies, chromosome aberrations that cannot be identified due to gross rearrangement, thereby preventing the allocation to a specific chromosome, are designated as "Marker Chromosomes" (MC). The significance of MC as prognostic factor for AML has remained elusive so far. In this study we have assessed frequency, cytogenetic characteristics and prognostic impact of MC as well as their underlying biological origin. Given the gross structural chromosomal damage inherent to MC we speculated that they may arise from chromothripsis, a recently described phenomenon of chromosome fragmentation in a single catastrophic event. Patients and Methods: Patients recruited intwo large consecutive, prospective, randomized, multicenter clinical trials for newly diagnosed non-M3 AML patients from the German Study Alliance Leukemia (SAL) were analyzed (AML96, NCT00180115; AML2003, NCT00180102). All karyotypes were retrospectively screened for MC. For the detection of chromothripsis array-CGH was used. For each sample 50 ng of DNA were hybridized to an Affymetrix® CytoScan HD Oligo/SNP-array and scanned with the Affymetrix GeneChip® Scanner 3000 7G. Chromothripsis was defined according to the criteria of Rausch et al., which require at least 10 switches in segmental copy number involving two or three distinct copy number states on a single chromosome. Results: MC were detectable in 165/1026 (16.1%) of aberrant non-CBF karyotype cases. Adverse-risk karyotypes displayed a higher frequency of MC (40.3% in complex aberrant, 26.5% in adverse-risk as defined by MRC criteria and 41.2% in abnl(17p) karyotypes, p<.001 each). MC were associated with a poorer prognosis compared to other non-CBF aberrant karyotypes as well as with lower remission rates (CR+CRi; 36.0% vs. 55.8% in AML96 ≤60 years, p=0.01; 14.3% vs. 44.1% in AML2003, p<0.001), inferior event-free survival (2.24 vs. 6.54 months, p<0.001; 3.45 vs. 8.03 months, p<0.001) and overall survival (5.72 vs. 11.87 months, p<0.001; 8.68 vs. 20.78, p=0.01). In multivariate analysis with co-variables age, prior MDS, therapy-related AML and adverse-risk cytogenetics according to MRC criteria, MC independently predicted poor prognosis in AML96 ≤60 years but not in AML2003 with its higher allogeneic transplantation rate. As detected by array-CGH, in about one third of MC karyotypes (18/49, 36.7%, including 3 cases with 8 or 9 copy number switches) MC had arisen from chromothripsis, whereas this phenomenon was virtually undetectable in a control group of complex aberrant karyotypes without MC (1/34) (p<0.001). Chromothripsis in MC karyotypes typically involved one single chromosome (n=11), with two or three chromosomes affected in 5 and 2 patients, respectively. There was no predilection for a particular chromosome. MC karyotypes positive for chromothripsis were characterized by a particularly high degree of karyotype complexity as compared to those that were negative for chromothripsis (complex aberrant 100% vs. 64.5% p<0.01; abnl(17p) 50.0% vs. 16.1%, p=0.01). In 12/18 (66.7%) cases, at least one of the chromothriptic chromosomes was reported as loss in the karyotype formula, suggesting that the grouping of a chromothriptic chromosome as a marker is paralleled by a putative loss of the affected chromosome. The chromothripsis positive MC karyotype subgroup had a particularly dismal prognosis with a combined CR+CRi rate of 2/16 vs. 10/31 (p=0.14). It also displayed inferior event-free and overall survival, though statistical significance was not reached for either endpoint, likely due to the already poor prognosis of the entire MC positive group. Conclusion: This is the first study showing that MC are a frequent finding predominantly in adverse-risk AML and associated with particularly poor prognosis. Our data provide evidence that a substantial portion of MC arise from chromothripsis. Disclosures Thiede: AgenDix: Employment, Other: Ownership.

Download Full-text

Acquired copy number alterations in adult acute myeloid leukemia genomes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0903091106 ◽

2009 ◽

Vol 106 (31) ◽

pp. 12950-12955 ◽

Cited By ~ 177

Author(s):

M. J. Walter ◽

J. E. Payton ◽

R. E. Ries ◽

W. D. Shannon ◽

H. Deshmukh ◽

...

Keyword(s):

Acute Myeloid Leukemia ◽

Copy Number ◽

Myeloid Leukemia ◽

Copy Number Alterations ◽

Acute Myeloid

Download Full-text

Detection of copy number alterations in acute myeloid leukemia and myelodysplastic syndromes

Expert Review of Molecular Diagnostics ◽

10.1586/erm.12.18 ◽

2012 ◽

Vol 12 (3) ◽

pp. 253-264 ◽

Cited By ~ 17

Author(s):

Meagan A Jacoby ◽

Matthew J Walter

Keyword(s):

Acute Myeloid Leukemia ◽

Myelodysplastic Syndromes ◽

Copy Number ◽

Myeloid Leukemia ◽

Copy Number Alterations ◽

Acute Myeloid

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-2011-08-375758 ◽

2012 ◽

Vol 119 (9) ◽

pp. 2114-2121 ◽

Cited By ~ 306

Author(s):

Frank G. Rücker ◽

Richard F. Schlenk ◽

Lars Bullinger ◽

Sabine Kayser ◽

Veronica Teleanu ◽

...

Keyword(s):

Overall Survival ◽

Acute Myeloid Leukemia ◽

Copy Number ◽

Myeloid Leukemia ◽

Multivariable Analysis ◽

Complex Karyotype ◽

Copy Number Alterations ◽

Genomic Complexity ◽

Monosomal Karyotype ◽

Acute Myeloid

Abstract To assess the frequency of TP53 alterations and their correlation with other genetic changes and outcome in acute myeloid leukemia with complex karyotype (CK-AML), we performed integrative analysis using TP53 mutational screening and array-based genomic profiling in 234 CK-AMLs. TP53 mutations were found in 141 of 234 (60%) and TP53 losses were identified in 94 of 234 (40%) CK-AMLs; in total, 164 of 234 (70%) cases had TP53 alterations. TP53-altered CK-AML were characterized by a higher degree of genomic complexity (aberrations per case, 14.30 vs 6.16; P < .0001) and by a higher frequency of specific copy number alterations, such as −5/5q−, −7/7q−, −16/16q−, −18/18q−, +1/+1p, and +11/+11q/amp11q13∼25; among CK-AMLs, TP53-altered more frequently exhibited a monosomal karyotype (MK). Patients with TP53 alterations were older and had significantly lower complete remission rates, inferior event-free, relapse-free, and overall survival. In multivariable analysis for overall survival, TP53 alterations, white blood cell counts, and age were the only significant factors. In conclusion, 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.

Download Full-text