Genome-Wide DNA Copy Number Analysis by SNP Arrays of B-Cell Chronic Lymphocytic Leukemia: Correlation with Known Biological and Molecular Prognostic Markers.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1061-1061
Author(s):  
Laura Mosca ◽  
Sonia Fabris ◽  
Giovanna Cutrona ◽  
Luca Agnelli ◽  
Serena Matis ◽  
...  
Keyword(s):  
High Resolution ◽  
Copy Number ◽  
Prognostic Markers ◽  
Snp Array ◽  
Lymphocytic Leukemia ◽  
Array Analysis ◽  
Genome Wide ◽  
Trisomy 12 ◽  
Snp Array Analysis ◽  
Cell Chronic Lymphocytic Leukemia

Abstract B-cell chronic lymphocytic leukemia (B-CLL) is a genetically heterogeneous disease with a variable clinical course. Chromosomal changes have been identified by FISH in approximately 80% of patients, and the presence of specific lesions, such as trisomy 12 and 13q14, 11q23, 17p13.1 and 6q23 deletions represent prognostic markers for disease progression and survival. In order to characterize further the complexity of B-CLL genomic lesions, we performed high density, single nucleotide polymorphism (SNP) array analysis in highly purified neoplastic cells (>92%) from a panel of 100 untreated, newly diagnosed patients (57 males and 43 females; age, median 63 years, range 30–87) in Binet stage A. All patients were investigated by FISH for the presence of trisomy 12 (21 cases); 13q14 deletion (44 cases, 34 as the sole abnormality); 11q22.3, 17p13.1 and 6q23 (15, 7 and 2 patients, respectively). In addition, ZAP-70 and CD38 expression resulted positive in 42 and 46 patients, whereas IgVH genes were mutated in 45 patients. Genome-wide DNA profiling data were generated on GeneChip® Human Mapping 250K NspI arrays (Affymetrix); copy number alterations (CNA) were calculated using the DNA copy Bioconductor package, which looks for optimal breakpoints using circular binary segmentation (CBS) (Olshen et al, 2004). A total of 782 CNAs (ranging from 1 to 31 per sample, mean and median values 7.82 and 7, respectively) were detected; DNA losses (365/782=46.67% loss; 194/782=24.81% biallelic deletion) were found to be more frequent than gains (148/782=18.93% gain; 75/782=9.59% amplification). The most recurrent alterations detected by FISH were all confirmed by SNP array analysis, strengthening further the good reliability of such high-resolution technology. We identified 12 minimally altered regions (MARs) larger than 100 kb with a frequency higher than 5%. Among well known alterations, the largest was represented by chromosome 12 trisomy, followed by 6q, 17p and 11q23 deletions (32.87, 19.09 and 10.43 Mb, respectively) and 13q14 deletion (635 kb). Gain of 2p25.3 involves a common region of 4.39 Mb region in 7 patients, although it was extended to the whole short arm of chromosome 2 in 3 cases. Among those alterations previously described in B-CLL, we found losses at 14q32.33 (12 pts) and 22q11.2 (5 pts) involving the IGH and IGLλ loci, respectively. With regard to novel regions, we identified losses at 4q35.2 (5 pts) and 11q25 (6 pts). In addition we found a high frequency of losses/gains at 14q11.2 (42 pts) and 15q11.2 (33 pts), two genomic regions reported to be affected by DNA copy number variations in normal individuals. As regards correlations between CNAs and biological markers, we found that the number of CNAs is significantly higher in cases with unmutated IgVH (9.4; range 2–31) as compared with mutated IgVH (6; range 1–13) (p=0.002), while neither CD38 nor ZAP-70 expression showed significant correlation. In addition, a significant higher number of either CNAs (p=0.001), total MARs (p<0.0001) or even only novel MARs (p=0.009) was significantly associated with cases with 17p deletion or multiple cytogenetic aberrations as evaluated by FISH analysis. Our data indicate that genetic abnormalities involving chromosomal gains and losses are very common in early-stage B-CLL and further support the application of high resolution SNP array platforms in the characterization of genetic changes in the disease. In addition, we detected novel altered chromosomal regions that warrant further investigations to better define their pathogenetic and prognostic role in B-CLL.

Genome-Wide 500K SNP Array Analyses Reveal High Frequencies of Cryptic Genetic Abnormalities and Identify Recurrent Microdeletions Targeting Novel Genes in Adult Acute Lymphoblastic Leukemia.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 458-458
Author(s):  
Kajsa Paulsson ◽  
Jean-Baptiste Cazier ◽  
Jane Stevens ◽  
Tracy Chaplin ◽  
Finlay MacDougall ◽  
...  
Keyword(s):  
Copy Number ◽  
Prognostic Markers ◽  
Lymphoblastic Leukemia ◽  
Snp Array ◽  
Array Analysis ◽  
High Frequencies ◽  
Genome Wide ◽  
Genetic Abnormalities ◽  
Adult Acute Lymphoblastic Leukemia ◽  
Snp Array Analysis

Abstract The long-term disease-free survival rate of adult acute lymphoblastic leukemia (ALL) patients remains less than 40%, in contrast to pediatric cases where it approaches 80%. Furthermore, whereas genetic abnormalities are widely used in childhood ALL for assigning patients to prognostic risk groups, their use in adult ALL is generally restricted to the presence or absence of the t(9;22)/[BCR-ABL1]. Novel prognostic markers, allowing better treatment stratification, and new treatment targets are therefore much needed. We have investigated diagnostic samples from 45 adult ALL cases using genome-wide, high-resolution single nucleotide polymorphism (SNP) array analysis with the Affymetrix 10K, 250K Nsp, and 250K Sty chips, in total including more than 500,000 SNPs with a median inter-marker distance of <2.5 kb. This method, which has not previously been applied to adult ALL, enables the detection of genetic copy number abnormalities as well as uniparental disomies (UPDs) with a much higher resolution than cytogenetic and molecular genetic techniques. We detected 367 genetic abnormalities not corresponding to known copy number polymorphisms among the 45 cases. These comprised 94 copy number gains, 211 hemizygous deletions, 47 homozygous deletions, and 15 UPDs. All but three of the patients (93%) displayed one or more anomaly not detectable with standard genetic analyses. Most notably, we found high frequencies of deletions targeting the genes CDKN2A (P16) (21 cases; 47%), PAX5 (15 cases; 33%), IKZF1 (IKAROS) (8 cases; 18%), ETV6 (7 cases; 16%), RB1 (5 cases; 11%), and EBF1 (2 cases; 4.4%). Thirty-two cases (71%) harbored a deletion of at least one of these genes. CDKN2A deletions were homozygous in 17 cases and associated with partial UPDs in 5 cases. PAX5 deletions were always hemizygous and frequently large, including also CDKN2A in all but three patients. Notably, loss of IKZF1 was found in 5 of 10 (50%) t(9;22)/[BCR-ABL1]-positive ALL cases. Reverse transcriptase-PCR for this gene showed that some t(9;22)-positive cases without this deletion expressed a dominant-negative isoform of IKZF1, suggesting that different mechanisms for downregulation of IKZF1 occur and that this gene is frequently targeted in t(9;22)+ ALL. Furthermore, the SNP array analysis revealed novel recurrent deletions targeting the genes DLG2 (4 cases; 8.9%), LDOC1 (3 cases; 6.7%), ERBB4, and CDH13 (2 cases each; 4.4%). In conclusion, using high-resolution SNP arrays we detect a very high frequency of hidden genetic changes in adult ALL cases. Deletions, frequently cryptic, comprised 70% of the found abnormalities, suggesting that microdeletions are a characteristic feature of adult ALL. Most importantly, the analyses revealed recurrent genetic abnormalities in adult ALL targeting novel genes, not previously implicated in leukemogenesis. In addition to giving new insights into the leukemogenic process, these findings are likely to be clinically important, with the possibility of identifying new prognostic markers as well as future treatment targets in adult ALL.

SNP Array Analysis Reveals Copy Number Alterations and Uniparental Disomy in Mantle Cell Lymphomas at High Resolution.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1585-1585
Author(s):  
Elena M. Hartmann ◽  
Itziar Salaverria ◽  
Silvia Bea ◽  
Andreas Zettl ◽  
Pedro Jares ◽  
...  
Keyword(s):  
High Resolution ◽  
Cell Lines ◽  
Copy Number ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Genetic Alterations ◽  
Array Analysis ◽  
Snp Array Analysis ◽  
Copy Number Changes ◽  
500K Array

Abstract Mantle Cell Lymphoma (MCL) is an aggressive B-Cell Non Hodgkin Lymphoma which is genetically characterized by the translocation t(11;14). This translocation leads to juxtaposition of the Cyclin D1 gene and the IgH locus, resulting in constitutive overexpression of Cyclin D1 and consecutive cell cycle dysregulation. Apart from this typical structural genetic alteration, several studies using conventional or array-based comparative genomic hybridization (CGH) reported a high number of secondary numerical genetic alterations contributing to MCL lymphomagenesis and influencing the clinical behavior. Increasingly, there is evidence that loss of heterozygosity (LOH) without copy number changes (e.g. caused by mitotic recombination between the chromosomal homologues, also referred to as acquired (partial) uniparental disomy (a(p)UPD), is an important alternative mechanism for tumor suppressor gene inactivation. However, this phenomenon is undetectable by CGH techniques. Single Nucleotide Polymorphism (SNP) based arrays allow - in addition to high resolution copy number (CN) analyses and SNP genotyping - in the same experiment the analysis of loss of heterozygosity (LOH) events and hereby enable the detection of copy neutral LOH. We analyzed the 3 t(11;14)-positive MCL cell lines Granta 519, HBL-2 and JVM-2 and 5 primary tumor specimens from untreated MCL patients with both the Affymetrix GeneChip®Human Mapping 100K and 500K array sets. In the 3 cell lines, we found an excellent agreement between the copy number changes obtained by SNP array analysis and previously published array CGH results. Extending published results (Nielaender et al., Leukemia 2006), we found regions of pUPD in all 3 MCL cell lines, which often affected regions reported as commonly deleted in MCL. Intriguingly, HBL-2 that is characterized by relatively few chromosomal losses, carries an increased number of large regions showing copy neutral LOH. Furthermore, we compared the results obtained by the 100K and 500K mapping array sets from 5 primary MCL tumor specimens with previously published conventional CGH data. All cases showed genetic alterations in both conventional CGH and SNP array analysis. The total number of copy number alterations detected by conventional CGH was 35, including 23 losses, 10 gains and 2 amplifications. The total number of CN alterations detected by the mapping 100K and 500K array sets was 81 (50 losses, 26 gains and 5 amplifications) and 82 (50 losses, 27 gains and 5 amplifications), respectively. We found an excellent agreement in the large CN alterations detected by conventional CGH and both SNP array platforms. Furthermore, we identified &gt;40 mostly small CN alterations that have not been detected by conventional CGH (median size &lt;5MB for losses and &lt;3Mb for gains). The CN alterations detected by the 100k and the 500K array sets were highly identical. Importantly, we discovered regions of partial UPD in 4 of the 5 MCL cases (size range from around 2Mb up to a single region &gt;40Mb). In conclusion, the results demonstrate the capability of SNP array analysis for identifying CN alterations and partial UPD at high resolution in MCL cell lines as well as in primary tumor samples.

High Resolution Screening of Copy-Number Alterations in Chronic Lymphocytic Leukemia Using Affymetrix 250K SNP-Arrays Reveals a Higher Complexity of Genomic Alterations in Patients with Unmutated IGHV Genes

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3133-3133
Author(s):  
Rebeqa Gunnarsson ◽  
Anders Isaksson ◽  
Hanna Göransson ◽  
Larry Mansouri ◽  
Mattias Jansson ◽  
...  
Keyword(s):  
High Resolution ◽  
Copy Number ◽  
Average Length ◽  
Lymphocytic Leukemia ◽  
Copy Number Alterations ◽  
Snp Arrays ◽  
Genetic Complexity ◽  
Trisomy 12 ◽  
Allelic Deletion ◽  
Gains And Losses

Abstract Chronic lymphocytic leukemia (CLL) is a biologically heterogeneous disease where no common genetic aberration so far has been described. Although recurrent genomic aberrations of prognostic significance are well established (e.g. deletions of 11q, 13q, 17p and trisomy 12), less is known about the overall genetic complexity. Recent development of high-resolution single nucleotide polymorphism (SNP)-arrays will allow screening of genetic complexity, which includes detection of smaller copy-number alterations (CNAs) in addition to the known, recurrent aberrations. We here applied the Affymetrix GeneChip® Mapping 250K Nsp arrays and screened peripheral blood samples from 203 newly diagnosed CLL patients (≥70% tumor cells) from a population-based Scandinavian cohort. The male:female ratio was 2:1, the majority of patients was in stage A (70%) and the median age at diagnosis 61 years. Sixty percentage of cases displayed mutated IGHV genes whereas 35% showed unmutated IGHV genes. The Nexus copy-number software (Biodiscovery, Inc.) was applied and CNAs were identified in all CLL samples investigated, where deletions were more commonly detected than gains (in average, 3.5 vs. 2.2 per sample, respectively). The average length of deletions was 3.5 Mb while gains had an average length of 9.5 Mb (3.6 Mb when excluding trisomy 12). More than 50% of deletions and gains had a size ranging between 0.1–1 Mb, whereas 22% of CNAs were less than 0.1 Mb and 25% larger than 1 Mb. When investigating known, recurrent alterations, 105 samples (52%) carried del(13)(q14); 82 samples displayed a mono-allelic deletion, 15 samples a bi-allelic deletion and 8 samples carried 2 mono-allelic deletions of different sizes. The minimal overlapping region was 0.44 Mb and most of the 13q14 deletions covered the genomic region of miR-15, miR-16 and/or DLEU7. Twenty-one samples (10%) showed trisomy 12 and del(11q) was detected in 27 samples (13%), all covering the ATM gene. del(17p) was detected in 7 patients (3.4%) with one patient also carrying several gains and losses of 17q. Moreover, large alterations involving chromosome arms or entire chromosomes were recurrently observed among the samples, for instance, 3 large 8p losses and 4 large 8q gains. Interestingly, 5 samples, which all carried del(11q), displayed a gain covering whole or large parts of the p-arm of chromosome 2, a region which covers MYCN, REL and BCL11A. Furthermore, comparison of samples with different IGHV mutation status (M vs. UM) illustrated a significantly higher frequency of del(11q) (32% of UM vs. 4% of M), trisomy 12 (22% of UM vs. 5% of M) and gain of 2p (12% vs. 0% in M) in samples with unmutated IGHV genes. As expected, patients with mutated IGHV genes showed a higher frequency of del(13q) (61% of M vs. 31% of UM). In addition, large aberrations (&gt;1Mb) were more common in UM samples which in average displayed 0.57 gains and 1.22 deletions compared to 0.26 gains and 0.67 deletions in M samples. In conclusion, with whole-genome screening using high resolution SNP-arrays, higher complexity was revealed in CLL, including a higher number of gains and losses and a higher frequency of larger aberrations in UM compared to M samples. A novel, recurrent combination of del(11q) and gain of 2p was demonstrated which deserves further investigation.

High Density SNP Array Analysis of Tyrosine Kinase Inhibitor (TKI) Resistant Chronic Myeloid Leukemia (CML) Shows Secondary Genomic Alterations

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3182-3182
Author(s):  
Daniel Nowak ◽  
Norihiko Kawamata ◽  
Tadayuki Akagi ◽  
Ryoko Okamoto ◽  
Nils Thoennissen ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Copy Number ◽  
Myeloid Leukemia ◽  
Snp Array ◽  
High Density ◽  
Array Analysis ◽  
Snp Arrays ◽  
Tki Resistance ◽  
Snp Array Analysis

Abstract Despite the success story of tyrosine kinase inhibitors (TKIs) for the treatment of Chronic Myeloid Leukemia (CML), patients can develop resistances against the drugs. The main known causes for resistance are mutations or over-expression of the BCR/ABL fusion protein, reduced bioavailability of the drugs and activation of compensatory molecular pathways. It is hypothesized that during disease progression, genomic instability of CML cells increases, which may lead to new genomic lesions harboring additional mechanisms of resistance. In this context, we studied genomic DNA profiles of 32 Imatinib resistant CML patients with high density 250K SNP arrays (Affymetrix). Molecular allelokaryotyping for allele specific copy number and loss of heterozygosity analysis was performed with the CNAG software. Single DNA samples from 27 patients were extracted after they had acquired resistance to Imatinib or alternative TKIs such as Nilotinib or Dasatinib. DNA from 12 patients could be analyzed in sequential samples from the initial diagnosis timepoint and a second timepoint upon the emergence of TKI resistance. All patients were positive for BCR/ABL by PCR and FISH. 10 relapse patient samples had known BCR/ABL mutations of which two were T315I mutations. High density allelokaryotyping confirmed pre-existent data on unbalanced translocations, amplifications and deletions from routine cytogenetics: 5 samples displayed a genomic duplication of the BCR/ABL fusion gene, 4 samples had trisomy 8, 1 sample showed deletion of chromosome 17p, 1 sample had heterozygous deletion of chromosome 9. Apart from this, SNP array analysis revealed numerous new submicroscopic genomic lesions. After exclusion of genomic copy number polymorphisms (CNPs) by comparison to recorded CNPs in the UCSC Genome Browser (http://genome.ucsc.edu/) the following results were obtained: Two patients displayed common heterozygous microdeletions of the reciprocal ABL/BCR fusion product. Furthermore, single samples displayed heterozygous micro-deletions on chromosomes 1, 2, 10, 12, 15, 17, and 22 or microduplications on chromosomes 2,3,6, 8, 9, 11, 12, 14, 15, 22. The affected regions contained potentially interesting genes in respect to resistance to therapy such as tumor suppressor candidate MBP-1, apoptosis related protein RERE, metastasis associated gene MTA3, nuclear body associated gene SP100, alpha-T-catenin (CTNNA3), Cbl-interacting protein Sts-1 and the DNA repair associated gene RAD51. As a new genomic alteration in CML, we detected acquired uniparental disomy (UPD) in 5 samples with a common site of UPD on chromosome 19q in 2 patients. In conclusion, in 14 out of 39 TKI resistant cases, high density SNP arrays enabled us to identify submicroscopic copy number lesions and regions of UPD containing promising candidate genes, which merit further research as sites conferring TKI resistance.

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

Blood ◽  
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.

scholarly journals Erratum to: high-resolution SNP array analysis of patients with developmental disorder and normal array CGH result

2014 ◽  
Vol 15 (1) ◽  
Author(s):  
Linda Siggberg ◽  
Sirpa Ala-Mello ◽  
Tarja Linnankivi ◽  
Kristiina Avela ◽  
Ilari Scheinin ◽  
...  
Keyword(s):  
High Resolution ◽  
Developmental Disorder ◽  
Array Cgh ◽  
Snp Array ◽  
Array Analysis ◽  
Snp Array Analysis

Dissection of Submicroscopical Aberrations and Clonal Evolution from Birth To Diagnosis and Relapse in a Childhood AML FLT3-ITD Positive Patient by RQ-PCR and SNP Array Analyses.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4337-4337
Author(s):  
Giovanni Cazzaniga ◽  
Silvia Bungaro ◽  
Manoj Raghavan ◽  
Chiara Beretta ◽  
Maria G. Dell’Oro ◽  
...  
Keyword(s):  
Disease Progression ◽  
Copy Number ◽  
Clonal Evolution ◽  
Snp Array ◽  
Array Analysis ◽  
Highly Sensitive ◽  
Snp Array Analysis ◽  
Childhood Aml ◽  
Copy Number Changes ◽  
Array Analyses

Abstract We have performed a combined Real Time Quantitative-PCR and single nucleotide polymorphisms array analyses for dissecting the clonal evolution in a childhood AML patient who experienced two relapses and for whom we had the availability of the cord blood (CB) sample. The patient was diagnosed at 6 years of age with an AML-M1 and showed normal karyotype and a FLT3-ITD mutation as a sole abnormality. She underwent autologous-BMT; however, 3.5 months later the patient relapsed, and 4 months after an allogeneic-BMT she suffered from a second relapse and died for disease progression. Highly sensitive (10−4) monitoring of FLT3-ITD was performed by patient-specific RQ-PCR, and showed a progressive decrease of minimal residual disease (MRD) during induction therapy. MRD was below the detection limit before auto-BMT, but the same FLT3-ITD clone re-emerged three months after auto-BMT, and preceded the clinical relapse. Thus, the same FLT3-ITD mutation was detected at the time of relapse, suggesting that the leukemic clone responsible for the first diagnosis was still present and could be potentially used as a marker for backtracking the leukemia into the CB. When tested by highly sensitive RQ-PCR, the DNA from CB resulted negative for the FLT3-ITD mutation. Although the relatively limited sensitivity of the technique might impair the interpretation, the FLT3-ITD negative result in CB is consistent with the hypothesis that FLT3-ITD mutations are secondary events, not sufficient by themselves to induce leukemia transformation in hematopoietic stem cells without a necessary primary event. With the aim to find additional submicroscopical genetic changes associated to the highly aggressive nature of the patient disease, we performed a genome wide SNP array analysis on the patient DNA through the clinical evolution of the disease, from birth to relapse. This new SNP array strategy is emerging as a powerful method to detect loss of heterozygosity (LOH) and/or copy number changes in a DNA sample with high resolution. The Affymetrix GeneChip® Mapping 10K platform has been used, which allows the scanning of more than 10.000 SNPs. SNP array analysis on DNA from the first relapse showed the deletion of the long arm of chromosome 9, a recurring chromosomal aberration in AML, and LOH on the whole chromosome 13 not associated with copy number changes. This latter has been confirmed by FISH, and it is consistent with uniparental isodisomy (UPD) as a responsible mechanism for the somatically acquired homozygosity of FLT3-ITD at 13q14. This mechanism is emerging as a frequent way of disease progression and represents a subsequent event to FLT3-ITD heterozygous mutation. The deletion of the wild type FLT3 allele has been confirmed by PCR. 10K SNP array analysis failed to reveal LOH or copy number changes in the diagnostic and in CB samples. These findings are compatible with a somatic post-natal origin of the FLT3-ITD positive AML subtype. Additional abnormalities can be responsible for the disease progression, via different mechanisms, including UPD. Other methods must be applied to find the primary event(s) giving rise to leukemia in association with FLT3-ITD mutation.

Screening for Copy Number Alterations and Loss of Heterozygosity in Chronic Lymphocytic Leukemia - A Comparative Study of Four Differently Designed, High Resolution Microarray Platforms.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2084-2084
Author(s):  
Rebeqa Gunnarsson ◽  
Johan Staaf ◽  
Mattias Jansson ◽  
Anne Marie Ottesen ◽  
Hanna Göransson ◽  
...  
Keyword(s):  
High Resolution ◽  
Chronic Lymphocytic Leukemia ◽  
Loss Of Heterozygosity ◽  
Copy Number ◽  
Lymphocytic Leukemia ◽  
Allelic Loss ◽  
Copy Number Alterations ◽  
Oligonucleotide Arrays ◽  
Snp Arrays ◽  
Trisomy 12

Abstract Screening for copy number alterations (CNA) has improved by applying genome wide microarrays, where SNP-arrays also allow analysis of loss of heterozygosity (LOH). Currently, comparisons of high resolution microarray platforms are few, thus we performed a study to evaluate the power of differently designed microarrays for copy number analysis and LOH. We here analyzed 10 diagnostic chronic lymphocytic leukemia (CLL) samples (five IGVH mutated and five IGVH unmutated) using four different high-resolution platforms: BAC-arrays (32K), oligonucleotide-arrays (185K, Agilent), and two SNP-arrays (250K, Affymetrix and 317K, Illumina). Comparison of copy number data showed that the platforms are concordant in terms of detecting large CNA, including the known recurrent alterations. Mono-allelic and bi-allelic loss of 13q14 (3 and 1 sample, respectively), mono-allelic loss of 11q (1 sample), trisomy 12 (2 samples) and mono-allelic loss of 17p (2 samples) were concordant in all platforms. These aberrations were validated with FISH, which in addition identified subclones with mono-allelic loss of 13q14 in two cases, only detected with the BAC platform, rendering a cut-off for the power of detecting subclones to approximately 25% of investigated cells. As expected, all poor prognostic aberrations were detected in patients carrying unmutated IGHV genes whereas four of five mutated samples were detected with mono-allelic loss of 13q14, as the only recurrent alteration. Furthermore, detection of small CNA were in many cases discordant between platforms. Therefore, we defined alterations identified by at least two platforms and identified 47 losses and 31 gains using this criterion. We are currently validating the presence of a number of these alterations using other techniques. Evaluation of LOH showed concordance for 86 regions between the Illumina and Affymetrix platforms. Of these regions 12 LOH coincided with CNA, leaving the remaining 74 as copy-neutral LOH. In conclusion, all platforms investigated are powerful tools for screening of CNA, however, since non-overlapping CNA were detected by individual platforms, we emphasize the importance of validating findings. Also, there is a cut-off for detecting subclones, here estimated to 25%. Genomic arrays will improve the detection of new recurrent aberrations, which may potentially refine the prognostic hierarchy established by FISH.

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