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 SNP array analysis of tyrosine kinase inhibitor-resistant chronic myeloid leukemia identifies heterogeneous secondary genomic alterations

Blood ◽  
2010 ◽  
Vol 115 (5) ◽  
pp. 1049-1053 ◽  
Author(s):  
Daniel Nowak ◽  
Seishi Ogawa ◽  
Markus Müschen ◽  
Motohiro Kato ◽  
Norihiko Kawamata ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Blast Crisis ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Genomic Alterations ◽  
Tki Resistance

AbstractTo elucidate whether tyrosine kinase inhibitor (TKI) resistance in chronic myeloid leukemia is associated with characteristic genomic alterations, we analyzed DNA samples from 45 TKI-resistant chronic myeloid leukemia patients with 250K single nucleotide polymorphism arrays. From 20 patients, matched serial samples of pretreatment and TKI resistance time points were available. Eleven of the 45 TKI-resistant patients had mutations of BCR-ABL1, including 2 T315I mutations. Besides known TKI resistance-associated genomic lesions, such as duplication of the BCR-ABL1 gene (n = 8) and trisomy 8 (n = 3), recurrent submicroscopic alterations, including acquired uniparental disomy, were detectable on chromosomes 1, 8, 9, 17, 19, and 22. On chromosome 22, newly acquired and recurrent deletions of the IGLC1 locus were detected in 3 patients, who had previously presented with lymphoid or myeloid blast crisis. This may support a hypothesis of TKI-induced selection of subclones differentiating into immature B-cell progenitors as a mechanism of disease progression and evasion of TKI sensitivity.

scholarly journals Metabolic Plasticity Is an Essential Requirement of Acquired Tyrosine Kinase Inhibitor Resistance in Chronic Myeloid Leukemia

Cancers ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 3443
Author(s):  
Miriam G. Contreras Mostazo ◽  
Nina Kurrle ◽  
Marta Casado ◽  
Dominik Fuhrmann ◽  
Islam Alshamleh ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Mitochondrial Respiration ◽  
Myeloid Leukemia ◽  
Chemotherapeutic Agents ◽  
Response To Therapy ◽  
Metabolic Reprogramming ◽  
Metabolic Inhibitors ◽  
Metabolic Plasticity ◽  
Tki Resistance

Tyrosine kinase inhibitors (TKIs) are currently the standard chemotherapeutic agents for the treatment of chronic myeloid leukemia (CML). However, due to TKI resistance acquisition in CML patients, identification of new vulnerabilities is urgently required for a sustained response to therapy. In this study, we have investigated metabolic reprogramming induced by TKIs independent of BCR-ABL1 alterations. Proteomics and metabolomics profiling of imatinib-resistant CML cells (ImaR) was performed. KU812 ImaR cells enhanced pentose phosphate pathway, glycogen synthesis, serine-glycine-one-carbon metabolism, proline synthesis and mitochondrial respiration compared with their respective syngeneic parental counterparts. Moreover, the fact that only 36% of the main carbon sources were utilized for mitochondrial respiration pointed to glycerol-phosphate shuttle as mainly contributors to mitochondrial respiration. In conclusion, CML cells that acquire TKIs resistance present a severe metabolic reprogramming associated with an increase in metabolic plasticity needed to overcome TKI-induced cell death. Moreover, this study unveils that KU812 Parental and ImaR cells viability can be targeted with metabolic inhibitors paving the way to propose novel and promising therapeutic opportunities to overcome TKI resistance in CML.

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.

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 (&gt;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&lt;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.

Expression of CAMKIIγ, HSP70 and HSP90 Genes Are Differentially Associated with the Mutational Status of Tirosine Kinase Domain in Chronic Myeloid Leukemia Resistant Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4877-4877
Author(s):  
Mariana Selena Gonzalez ◽  
Carlos Daniel De Brasi ◽  
Michele Bianchini ◽  
Cristian Ferri ◽  
Raquel Bengió ◽  
...  
Keyword(s):  
Heat Shock ◽  
Chronic Myeloid Leukemia ◽  
Heat Shock Protein ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Kinase Domain ◽  
Chromosome 9 ◽  
Common Mechanism ◽  
Resistance Mutations ◽  
Tki Resistance

Abstract Abstract 4877 Chronic Myeloid Leukemia (CML) is a clonal myeloproliferative disorder characterized by the reciprocal chromosomal translocation between chromosome 9 and 22, t(9;22)(q34;q11), known as Philadelphia chromosome. At molecular level, this cytogenetic aberration associates with a chimeric BCR-ABL fusion gene responsible for the pathogenesis of the disease. Targeting BCR-ABL with tyrosine kinase inhibitors (TKIs) has led to a rapid clinical response in most CML cases; however, harboring of point mutations within the kinase domain (KD) of BCR-ABL is the most common mechanism responsible for acquired resistance to therapy. The objective of this work was to evaluate the expression profiles of 4 different genes involved in proliferation, differentiation or cell survival, in order to study their association with acquired missense mutations within the KD of BCR-ABL. Due to its clear involvement in cell proliferation and apotosis the following genes were analyzed. CAMKIIγ (Ca2+ /calmodulin dependent protein kinase IIγ) is a critical regulator of multiple signalling networks regulating the proliferation in myeloid leukaemia. HSP70 (Heat Shock protein 70) is a chaperone protein that protects cells from apoptotic and necrotic stimuli. HSP90 (Heat Shock protein 90) is a chaperone that facilitates folding of client proteins such as BCR-ABL. Ki-67 is a nuclear antigen associated with cellular proliferation and ribosomal RNA transcription. Total RNA was extracted from 50 TKI-resistant CML-patient's PBMC samples; by quantitative real time-PCR (QRT-PCR) (SybrGreen method, melting analysis and β2-microglobulin as an endogenous control reference gene) we measured the gene expression of CaMKIIγ, Ki67, HSP70, HSP90 and BCR-ABL. All patients were treated with TKIs (26 imatinib, 13 nilotinib, 11 dasatinib) and showed primary lack or loss of haematological and/or cytogenetic response according to the ELN (European Leukaemia Net) criteria. To characterize TKI-resistant mutations within the KD of the chimeric BCR-ABL gene, ABL exons 4, 5, 6 and 7 were subjected to automated DNA sequencing following a nested- BCR-ABL-specific PCR. Mutations were observed in 27/50 cases at 15 different residues: 1 L248V, 1 G250E, 1 Y253H, 5 E255K/V, 1 E279K, 1 V289F, 4 T315I, 2 F317L, 1 L348M, 3 M351T, 1 E355G, 1 N358S, 2 F359V/C, 2 L387M and 1 L389V. Remaining 23 CML cases did not show mutations (detection limit 10–20%). Our results showed a significant increase in the expression of CaMKIIγ and HSP70 and decrease of HSP90 in mutated patients (MT) with respect to cases without mutations (WT) (p<0.01). On the contrary, transcript levels of Ki67 and BCR-ABL did not show significant differences between MT and WT, likely due to the resistant status of both groups. Taking into account these results we design a score (TKI-MT) to estimate the likelihoods for a patient to harbor TKI-resistance mutations using the transcript normalised expression of CaMKIIγ, HSP70 and HSP90 as follows: [Log10(CaMKIIγ) + Log10(HSP70) – Log10(HSP90)]. TKI-MT scores from 27 and 23 patients from the MT and WT groups respectively were analysed by use of ROC curves in order to find an optimal cut-off value to classify new unstudied cases. We found that patients with TKI-MT scores over a cut-off value of −0.79 showed 4.8 times more probabilities to present TKI-resistance mutations than those below −0.79 (OR 4.8, CI95% 1.3–17.6, P<0.02). We concluded that the expression of CaMKIIγ, HSP70 and HSP90 allowed prediction of mutations in the ABL tyrosine kinase domain with 82% of specificity in CML patients treated with TKIs and associated with lack or loss of response. Disclosures: No relevant conflicts of interest to declare.

Use of high-density SNP-array analysis to identify novel chromosomal abnormalities that predict survival in multiple myeloma

2008 ◽  
Vol 26 (15_suppl) ◽  
pp. 8522-8522 ◽  
Author(s):  
H. Avet-Loiseau ◽  
N. Munshi ◽  
C. LI ◽  
F. Magrangeas ◽  
W. Gouraud ◽  
...  
Keyword(s):  
Multiple Myeloma ◽  
Chromosomal Abnormalities ◽  
Snp Array ◽  
High Density ◽  
Array Analysis ◽  
Snp Array Analysis
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