SNP-A Based Karyotyping Facilitates Improved Mapping of Deletions and Uniparental Disomy within the Long Arm of Chromosome 5 in Myeloid Disorders.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2435-2435 ◽  
Author(s):  
Lukasz P. Gondek ◽  
Andrew Dunbar ◽  
Christine O’Keefe ◽  
Michael A. McDevitt ◽  
Denise Batista ◽  
...  
Keyword(s):  
Copy Number ◽  
Chromosomal Rearrangement ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Chromosome 5 ◽  
Chromosome 5Q ◽  
Complex Chromosomal Rearrangement ◽  
Pathogenic Genes ◽  
Definition Of ◽  
Copy Number Changes

Abstract In MDS, cytogenetics has a major prognostic influence on the phenotype of the malignant clone and specific defects may point towards potential therapeutic targets. However, traditional metaphase cytogenetics (MC) has limited resolution and does not allow for detection of uniparental disomy (UPD). Defects on chromosome 5q have been studied using various methods to identify a minimal commonly deleted region (CDR). SNP-array karyoptyping (SNP-A) allows for precise detection of copy number changes as well as UPD. We hypothesized that SNP-A may reveal new lesions on chromosome 5 and allow for better definition of CDRs and pathogenic genes. Of 512 patients, 15% showed a 5q abnormality as a sole or associated aberration by MC. DNA was available in 189 patients and was subjected to 250K SNP-A. In 7 patients with normal/non-informative MC, a deletion on 5q was clearly detectable by SNP-A; in total, SNP-A identified 5q abnormalities in 14% patients in this group (vs. 11% by MC). UPD 5q was found in one patient with CMML. By SNP-A, 6/27 patients showed an isolated 5q deletion. SNP-A can also be used to construct precise cytogenetic maps. The commonly deleted region (CDR1,5q31.2, 137,472,900–139,451,900) was present in 24/27 patients. Significant overlap occurs with the CDR previously defined by Fairman, Zhao, Horrigan et al. This region includes important genes such as Cdc25C and EGR1. Of 24 patients with a deletion in CDR1, 21 had multilineage dysplasia predominantly in the megakaryocytic line (92%). While elevated platelet counts occurred in 3 patients, increased levels of megakaryocytes were common (83%). Previous studies by Bouldwood/Jaju suggested that the minimal CDR among patients with 5q- syndrome (CDR2, 5q33.1-33.2) differs slightly from that associated with secondary AML/MDS (CDR1). However, when patients (5/27) with classical 5q- syndrome were analyzed, all displayed single deletions spanning both CDR1 and CDR2. Other areas of partial overlaps were also identified (5q12.1; 5q13.3) more centromeric to CDR1 and present in 7/27 patients. 2 cases were particularly interesting: 1 with segmental UPD involving the CDR, the other showing a small deletion defining the CDR itself. In the latter patient, marked thrombocytosis was present and SNP-A demonstrated a complex chromosomal rearrangement. While MC revealed a segmental deletion of 5q and a concomitant duplication of this abnormal homolog, SNP-A showed that while the p arm portion had been duplicated, the q arm, with the exception of two small deletions (1.35 and 1.98Mb in length, confirmed by FISH), had a normal diploid set. SKY clarified that chr. 5 material had indeed been displaced to both chr. 3 and 7 with a reciprocal translocation of chr. 3 material occurring on the abnormal chr. 5. In sum, our studies demonstrate the utility of SNP-A as a karyotyping tool that can detect previously cryptic areas of LOH on chr. 5 and facilitate definition of shared 5q defects. We also show that our patients with 5q- syndrome had lesions spanning both 5q33 and the more proximal 5q31.2 area, making pathogenic distinction based on cytogenetics difficult.

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 >40 mostly small CN alterations that have not been detected by conventional CGH (median size <5MB for losses and <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 >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 Frequency of Loss of Heterozygosity Due to Uniparental Disomy or Allele Deletion of Ocular Adnexal MALT-Type Lymphoma.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2624-2624
Author(s):  
Yoshitaka Asakura ◽  
Seishi Ogawa ◽  
Motohiro Kato ◽  
Go Yamamoto ◽  
Akiko Maeshima ◽  
...  
Keyword(s):  
Loss Of Heterozygosity ◽  
Copy Number ◽  
Clinical Stage ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Tissue Samples ◽  
Conventional Cytogenetic Analysis ◽  
Genetic Abnormalities ◽  
Malt Type Lymphoma ◽  
Copy Number Changes

Abstract Majority of malignant lymphoma arising from the ocular adnexae are ocular adnexal MALT lymphomas (OAL). Several genetic abnormalities, including t(14;18)(q32;q21), trisomy 18 and trisomy 3 have been reported in OAL. However, none of them are found in more than half of cases with OAL by conventional methods. High density Single Nucleotide Polymorphism (SNP) array analysis with CNAG/AsCNAR algorithm allows high-resolution and genome-wide detection of both loss of heterozygosity (LOH) and copy number abnormality, especially uniparental disomy (UPD), without depending on the availability of paired normal DNA (Yamamoto et al, Am J Hum Genet. 2007; 81:114–26). UPD is acquired by somatic recombination and therefore not detected by conventional cytogenetic analysis or array CGH. In this study we analyzed DNA from OAL for the presence of LOH with or without copy number changes. Tissue samples from patients with OAL at our institute between 1995 and 2003 (N=32) were subject to SNP-array (250K NspI) analysis with CNAG/AsCNAR algorithm. The patients included 21 males and 11 females, and the median age of 56.5 years at the time of diagnosis (range, 15–90 years). Clinical stage was I (29 cases), II (1 case), and IV (2 cases). Trisomy of 3, 18, and 21 were found in 9, 7 and 1 cases, respectively. Overall, LOH due to UPD more than one chromosomal band were found in 16 cases (50%). Recurrent one allele deletion was detected at 6q23-24, 12p13 and 17q21 (N=15, 15 and 21, respectively). In total, 28 (82%) among 32 cases showed one or more of the above changes. Characterization of these recurrent genetic abnormalities might reveal the pathogenesis of OAL.

Genome-Wide Micro-Deletions and Amplifications Acquired at Relapse in Acute Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 166-166 ◽  
Author(s):  
Manoj Raghavan ◽  
Manu Gupta ◽  
Tracy Chaplin ◽  
Sabah Khalid ◽  
T. Andrew Lister ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Copy Number ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Normal Karyotype ◽  
P Value ◽  
Copy Number Changes ◽  
Acute Myeloid

Abstract Abstract 166 Recurrence of acute myeloid leukemia AML has a poor prognosis with only 20% of adults surviving to 5 years. Therefore it is of importance to identify molecular changes that explain the pathogenesis of relapsed AML. Previous studies had not identified consistently acquired cytogenetic changes at relapse. Recently, acquired uniparental disomy due to mitotic recombination was described in 40% of relapsed AML (Raghavan et al 2008). Most of the events lead to homozygosity for FLT3 mutations. This study aimed to discover if there are further genetic abnormalities acquired at disease recurrence that cannot be identified by conventional cytogenetics, i.e. microdeletions or gains. Twenty-one presentation and relapse paired AML patient blood and marrow samples were stored with consent at St Bartholomew's Hospital, London. Eleven patient samples had a normal karyotype at diagnosis, two had favourable prognosis cytogenetics (inv(16) and t(8;21)) and others had varying numerical cytogenetic abnormalities and rearrangements associated with an intermediate prognosis. DNA from the samples was analysed by array based high-resolution single nucleotide polymorphism (SNP) genotyping (Affymetrix Human SNP array 6.0). Data was analysed using Partek Genome Browser (Partek, MO). In all cases, the leukemia infiltrate of the marrow or blood was greater than 60% and most cases were greater than 90% allowing accurate identification of DNA copy number changes. Abnormalities of a size that would be identified by cytogenetics were disregarded. Using segmentation analysis using a p-value less than 0.001, over 400 microdeletions and gains were detected that were acquired at relapse in the 21 pairs. Each of the copy number changes was less than 2 megabases in size. One AML sample with a normal karyotype at diagnosis and trisomy 8 and add(9)(q34) at relapse had not acquired any microdeletions or gains. In contrast, in other samples as many as 69 microdeletions/gains were detected. There was no correlation between increased complexity of the karyotype of the leukemia and the number of microdeletions/gains. Several of the acquired microdeletions/gains were in regions containing genes known to be involved in AML, including a deletion of 234Kb at 13q12.2 involving FLT3 and CDX2, and an acquired deletion at 21p11.2 of 150Kb involving exons encoding the runt domain of RUNX1. Another copy number gain was detected at the MLL locus, suggestive of partial tandem duplication. Other detected locations are in Table 1.Table 1Location by cytobandCopy number changeSize / KbP valueGene13q12.2Deletion23410−33FLT3, CDX221q22.12Deletion15010−13RUNX111q23.3Gain5.10.0099MLL11p15.4Gain830.00001NUP9817q21.31Deletion8.00.0007BRCA1The results indicate that recurrent AML may be associated with the deletion or gain of several genes involved in leukaemogenesis. Many other locations are involved throughout the genome, suggesting at least some of these are also involved in the clonal evolution of the leukaemia at recurrence. Further studies should identify novel genes from these regions involved in the pathogenesis of AML. Disclosures: No relevant conflicts of interest to declare.

Defining the Topography of Deletion 5q Using SNP-A Identifies Patients with More Aggressive Disease and Correlates with Additional Lesions

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2795-2795
Author(s):  
Andres Jerez ◽  
Anna M Jankowska ◽  
Hideki Makishima ◽  
Lukasz P Gondek ◽  
Ramon V Tiu ◽  
...  
Keyword(s):  
Conflicts Of Interest ◽  
Single Nucleotide Polymorphism Array ◽  
Clonal Evolution ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Median Number ◽  
Disease Phenotype ◽  
Aggressive Disease ◽  
Chromosome 5Q ◽  
Definition Of

Abstract Abstract 2795 Interstitial deletions of chromosome 5q are common in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), pointing towards the pathogenic role of this region in disease phenotype and clonal evolution. The higher level of resolution of single nucleotide polymorphism array (SNP-A) karyotyping may be used to find cryptic abnormalities, and to precisely define the topographic features of the genomic lesions allowing for more accurate clinical correlations. In order to better address the genetic and genomic complexity of 5q abnormalities in myeloid malignances, we analyzed a large series of 1,155 clinically well-annotated patients with malignant myeloid disorders with SNP-A-based karyotyping to define: i) the extent of the 5q deletion, investigating whether loss of genes is different among 5q disorders; ii) minimally deleted region(s); iii) associated non-5q genomic lesions with 5q abnormalities; and iv) the association of genomic abnormalities with clinical features. We identified chromosome 5q deletions in 142/1155 patients (12%) and uniparental disomy segments (UPD) in 4/1155 patients (0.35%). With increased resolution there was a shift towards more complex karyotypes and increased identification of additional lesions among the patients with 5q aberrations. By SNP-A, previously cryptic lesions were identified in 52% of the patients who otherwise showed a singular del(5q) lesion by metaphase cytogenetics (MC). The presence of chromosome 5q material in all our cases with apparent monosomy 5 (N=11) by conventional MC serves as an illustration for SNP array-based mapping allowing for a more precise definition of the breakpoints; in addition, 48% of MC results localized both the beginning and end of the deletion to a different band than SNP-A, and in only 9% of cases, MC and SNP-A boundaries coincided. The CDR defined in our 5q-syndrome, though with wider limits (145,279,940–153,809,148), encompasses the CDR described by Boultwood et al; the CDR in advanced del(5q) MDS and AML patients is centered on a sub-section of bands 5q31.2 and 5q31.3 (137,528,564–139,451,907) and includes the defect initially mapped by Le Beau et al. Patients with MDS and deletions involving the centromeric and telomeric extremes of 5q have a more aggressive disease phenotype (median overall survival: 32 months, p=0.04, HR 1.9; median number of chromosome lesions: 5.8 vs. 1.1, p<0.001:; median time to progression: 30 months vs not reached, p<0.001). Moreover, lesions not involving the centromeric or telomeric extremes of 5q are not exclusive to 5q- syndrome but can be associated with other less aggressive forms of MDS. In addition, larger 5q deletions are associated with either del(17p) or UPD17p (25% vs 11% of cases, respectively). We closely investigated the outlying cases of aggressive disease and shorter interstitial deletions. We chose to test for 6 possible tumor suppressor genes, located within the extremes of 5q and related to the TP53 pathway that, if defective, might explain a clonal advantage. In our AML cohort only 5 patients showed a deletion not involving the 5q extremes: 4/5 of them displayed either NPM1 and/or MAML1 heterozygous mutations. In summary, the present study of 5q disorders shows that SNP-A can complement traditional MC, not only by finding cryptic abnormalities, but also by precisely defining the extent of the lesions. Moreover, we have perfomed whole exon sequencing but to date the analysis did not identify genes mutated on 5q rather the ones described above. Our results strongly suggest that while genes widely deleted among 5q disorders may be responsible for the characteristics of the dysplastic clone, the loss of an additional gene or genes in the proximal and telomeric extremes of 5q may be responsible of increasing genomic instability, favoring AML transformation. Disclosures: No relevant conflicts of interest to declare.

Uniparental Disomy May Be Associated with NPM Mutations in AML with a Normal Karyotype.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2310-2310
Author(s):  
Elena Serrano ◽  
Vanesa Orantes ◽  
Camino Estivill ◽  
Adriana Lasa ◽  
Salut Brunet ◽  
...  
Keyword(s):  
Complete Remission ◽  
Copy Number ◽  
Germ Line ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Normal Karyotype ◽  
Copy Number Analysis ◽  
Complex Germ ◽  
Flt3 Mutations ◽  
Tandem Duplications

Abstract Acute myeloid leukemia (AML) is a heterogeneous group of neoplastic disorders characterized by an abnormal proliferation of the myeloid precursors and a maturation block. A large proportion of AML cases have either a normal karyotype or non-recurrent chromosomal alterations. Underlying genetic lesions of some of these cases have been characterized with the discovery of MLL-internal tandem duplications, activating FLT3 mutations and NPM mutations. Loss of heterozygosity (LOH) derives from the loss of one of the two alleles at a given locus and can be a sign of inactivation of tumor-suppressor genes. We performed a high-resolution genotype analysis on DNA obtained from 19 AML patients with a normal karyotype, both at diagnosis and in samples obtained in complete remission(assessed by multiparametric flow cytometry) using the 10K SNP Array (Affymetrix). Both LOH and copy number analysis, as well as visualization of these analysis were performed by means of the dChip software (M. Lin et al., Bioinformatics (2004), 20:1233–40). A mean call rate of 96.8%. SNP array-based LOH analysis revealed that 4 patients presented large regions of homozygosity at diagnosis which were absent from samples in complete remission. In all four patients copy number analysis indicated no gross chromosomal losses or gains, as was confirmed by conventional cytogenetic analysis. Therefore, it can concluded that the LOH observed in these four patients was due to the presence of uniparental disomy. Simultaneous analysis of FLT-3 internal tandem duplications (FLT-3/ITD), FLT3- D835 mutations, NPM mutations and MLL rearrangements was performed using conventional molecular methods. Two of these patients (UPN2 and UPN12) had FLT-3/ITD in association with NPM mutations. UPN4 had a mutated form of NPM whereas in patient UPN16 FLT-3 and NPM genes were in the germ line configuration. All four cases were negative for MLL rearrangements and FLT-3-D835 mutations. These results suggest that NPM and FLT3 mutations may be associated with acquired somatic recombinations. It remains to be investigated whether there are loci preferentially involved by these events. Uniparental disomy and genetic lesions in normal karyotype AML Patient LOH FLT3 NPM D835 MLL UPN2 13q Mutated Mutated Germ line Germ line UPN4 6pter-p12.212q13.12-qter Germ line Mutated Germ line Germ line UPN12 2p Mutated Mutated Germ line Germ line UPN16 complex Germ line Germ line Germ line Germ line

Identification of Novel Genetic Lesions in Myelodysplastic Syndromes Using High Density SNP-Arrays.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2430-2430
Author(s):  
Saskia Langemeijer ◽  
Roland Kuiper ◽  
Peter Vandenberghe ◽  
Estelle Verburgh ◽  
Jan Boezeman ◽  
...  
Keyword(s):  
Copy Number ◽  
Single Gene ◽  
Snp Array ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Trisomy 8 ◽  
Entire Genome ◽  
Monosomy 7 ◽  
Snp Arrays ◽  
Copy Number Changes

Abstract Conventional cytogenetics and FISH reveal chromosomal defects in approximately 50% of MDS patients. These mostly consist of gross gains and losses of specific chromosomal regions or entire chromosomes like 5q-, monosomy 7 and trisomy 8. Currently, the genes that are critical for MDS development remain largely unknown, which hampers both a proper diagnosis of clonal disease as well as development of targeted therapy. To identify the affected genetic loci and to map the critical regions and genes in MDS, we performed high-resolution (250k) SNP-based CGH. So far, 231 controls and 87 MDS patients from various subclasses were analyzed. In all patients and controls, loss of heterozygosity (LOH) without copy number changes was observed at multiple loci across the entire genome. Although large areas of LOH encompassing the main part of the p- or q-arm of chromosomes were only seen in MDS patients, no genomic regions were identified that were statistically more often affected in patients compared to control DNA. Copy number changes (excluding known regions of normal variation) were seen in 53% of patients with a normal karyotype (n=54). In 231 controls and in non-malignant T cells of a subset of patients, these areas were not affected, indicating that they were disease-specific. The number of affected regions per patient ranged from 0–7. The majority (82%) of karyotypic aberrations were confirmed using SNP-arrays. Only balanced translocations and some subclonal aberrations could not be detected. Importantly, SNP-array analysis revealed additional copy number changes in 70% of patients with an abnormal karyotype. Copy number changes that were observed in only one patient might reflect general genomic instability in the tumor cells and may not represent genes that are implicated in the pathogenesis of MDS. Therefore, we selected areas that were affected in at least two patients. In total, we found 51 different recurrent genomic loci. This indicates that MDS is genetically diverse, which is in agreement with its diverse clinical and morphological presentation. Among the 51 recurrent loci, 15 contained only a single gene (Table). Among these genes, there were several known to be implicated in MDS (e.g. ETV6 and RUNX1), whereas others represent novel genes that are potentially implicated in the pathogenesis of MDS. For several of these, a biological function has been described that may be linked to control of differentiation and proliferation, like the transcription- and proliferation-regulating gene JARID2 and the transcription factor DMTF1. Currently, we are performing a high thoughput mutation- and expression-analysis of these genes in a larger group of patients. Single gene copy number changes in MDS Chr Cytoband Loss/Gain Cases Size (Mb) Gene 1 p35.1 loss 2 0.01 CSMD2 3 p24.2 loss 2 0.07 LRRC3B 6 p22.3 loss 3 0.02 JARID2 8 p23.2-1 gain 2 0.14 MCPH1 9 p13.2 gain 2 0.23 MELK 9 p24.3 gain 2 1.14 SMARCA2 11 q22.3 gain 2 0.05 SLC35F2 12 p12.1 loss 3 0.08 ST8SIA1 12 p13.2 loss 4 0.08 ETV6 12 q23.2 loss 2 0.03 IGF1 16 q23.3 loss 2 0.06 MPHOSPH6 21 q22.12 loss 3 0.07 RUNX1 21 q22.2 gain 2 0.62 DSCAM 22 q12.2 gain 2 0.00 PES1 X q13.1 loss 2 0.17 EDA

scholarly journals Genetic Analysis Using a High Density SNP Array in Myelodysplastic Syndrome: Clinical Utility and Comparative Analysis Study Compared to Metaphase Chromosome Analysis

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5259-5259 ◽  
Author(s):  
Sally Jeffries ◽  
Nicola Trim ◽  
Emma Huxley ◽  
Laura Ford ◽  
Manoj Raghavan ◽  
...  
Keyword(s):  
Genetic Analysis ◽  
Metaphase Chromosome ◽  
Copy Number ◽  
Clinical Utility ◽  
Snp Array ◽  
Gene Mutations ◽  
Chromosome Analysis ◽  
Genetic Aberrations ◽  
Allelic Gene ◽  
Copy Number Changes

Abstract There is no single technology capable of detecting the various genetic and genomic aberrations observed in patients with neoplasia. Patients with myelodysplastic syndrome (MDS) may present with chromosomal copy number changes (duplication, deletion, and amplification), balanced chromosome rearrangements, copy neutral loss of heterozygosity (CN-LOH) and/or gene mutations. Currently only microscopic chromosomal changes, as dictated by the international prognostic scoring system (IPSS-R), are used to determine the genetic risk in MDS. However, different genetic aberrations, particularly gene mutations are anticipated to be incorporated into the IPSS-R in the near future. The Affymetrix CytoScan® HD Array is a high definition array with over 2.6 million markers (both copy number and SNP) allowing resolution capabilities way beyond that of metaphase chromosome analysis. The incorporation of 750,000 SNPs also allows for detection of CN-LOH, regions known to harbour bi-allelic gene mutations. A real-time comparative study using the Affymetrix CytoScan® HD Array against traditional metaphase chromosome analysis is being performed on patients with confirmed or highly suspected MDS referred for genetic analysis at the West Midlands Regional Genetics Laboratory, UK. The study is expected to utilise 600 arrays over two years at presentation and on serial surveillance samples. The preliminary results available after the first 100 patients are presented with examples demonstrating the capabilities and clinical utility of SNP array genetic analysis. The study so far, in patients with MDS at presentation, has demonstrated: An increased number of genetic aberrations (CN changes and CN-LOH) detected by SNP array (38/105 (36%) by metaphase analysis and 62/105 (60%) by SNP array analysis).The ability to reliably detect deletions at the single gene level including CUX1, TET2 and RUNX1, and rarely copy number changes within genes including duplication within KMT2A, consistent with partial tandem duplication.The detection of CN-LOH regions, which may contain bi-allelic gene mutations, in 20/105 (19%) of cases, including chromosomal regions 1p (MPL, NRAS), 4q (TET2), 7q (CUX1), 11q (CBL), 17p (TP53). Confirmatory studies are on-going.A lower failure rate for SNP array compared to metaphase analysis (1% v 4%). Of the four failed metaphase cases, three showed recurrent genetic aberrations observed in MDS including del(5q), del(20q) with CN-LOH 17p, and trisomy 8. The failed SNP array had a normal karyotype.Confirmed balanced rearrangements (without gain or loss of genetic material) are not detected.The ability to detect abnormal clones with copy number changes (deletion or gain at one or more loci) at sensitivities as low as between 5 and 20% dependent on aberration size.Twenty-two cases with a normal karyotype had abnormal genetic aberrations detected by SNP array. Two cases with an abnormal karyotype had a normal SNP array profile due to a balanced rearrangement and a low level abnormal clone (4%) beyond the sensitivity of the test.The ability to utilise peripheral blood instead of marrow as no dividing cells are required, thereby allowing genetic analysis in patients too frail or otherwise unsuitable for marrow aspirationThe ability to detect deletion or CN-LOH of the HLA gene region, which is critical in patients being considered for stem cell transplant as HLA typing may be inaccurate or ambiguous.The ability when performed as a complementary tool to metaphase analysis to detect sub-clones or concurrent clonal neoplasia with different copy number changes. Patient benefits are expected to be the potential to improve patient outcomes through improved confidence in diagnosis, prognosis and monitoring. Disclosures Jeffries: Affymetrix: Research Funding.

scholarly journals Genomic Analysis of Myelodysplastic Syndromes Among Nagasaki Atomic Bomb Survivors

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4341-4341
Author(s):  
Masataka Taguchi ◽  
Hiroyuki Mishima ◽  
Yusuke Shiozawa ◽  
Chisa Hayashida ◽  
Akira Kinoshita ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Copy Number ◽  
Myeloid Leukemia ◽  
Atomic Bomb ◽  
Snp Array ◽  
Exposed Group ◽  
Chromosome 7 ◽  
Chromosome 5Q ◽  
Methylation Pathway

Abstract Exposure to ionizing radiation is a well-established risk of cancer development. Atomic bomb (A-bomb) survivors were exposed to A-bomb radiation, then developed acute myeloid leukemia (AML), acute lymphoblastic leukemia, and chronic myeloid leukemia with the highest incidence around five to ten years after the bombing. On the other hand, the risk of myelodysplastic syndromes (MDS) were significantly high among the survivors even after 40 to 60 years from the bombing (Iwanaga et al. J Clin Oncol 2011), indicating a long-lasting influence of A-bomb radiation on human hematopoiesis. In general, genetic mutations are found in almost all MDS, however, the landscape of genetic alterations in MDS among A-bomb survivors remains to be elucidated. To study the role of A-bomb radiation on the pathogenesis of MDS and related conditions, we analyzed 32 patients with MDS and 3 cases diagnosed as idiopathic cytopenia of undetermined significance (ICUS) among A-bomb survivors using next generation sequencing technologies and SNP array karyotyping. Patients were categorized into two groups by the exposure distance; proximally exposed group (PE-group, 18 patients, < 2.7 km from the hypocenter) and distally exposed group (DE-group, 17 patients, directly exposed in 2.7-10 km, or those who entered the bombing area [inside of 2 km from the hypocenter] within two weeks after the bombing [no acute radiation syndromes]). Median exposure distance was 1.1 and 3.4 km in PE- and DE-group, respectively (P < .001). There was no significant difference in sex, subtype of MDS, and ages at bombing and the diagnosis, between the two groups, whereas frequencies of abnormal karyotype (78% vs. 59%, P = .29) and complex karyotype (33% vs. 12%, P = .23) were higher in PE-group without statistical significance. Whole genome sequencing of three patients in PE-group revealed that most frequent nucleotide substitution was cytosine-to-thymine (C to T), which is a hallmark of age-related mutational changes. Using whole exome sequencing and 154 genes-targeted capture sequencing (T-S), we identified significant differences in mutated genes between the two groups. No patients of PE-group had mutations in TET2 although it was most frequently affected in DE-group (29% [5 out of 17 patients], P = .019), and the frequency of gene mutations in DNA methylation pathway was 5.6% (1 out of 18 patients) and 41% (7 out of 17) in PE- and DE-group, respectively (P = .018). TP53 and the genes coding RNA splicing factors (SFs) were mutated equally in both groups (TP53, 11% and 12% [P = 1]; SFs, 33% and 35% [P = 1] in PE- and DE-group, respectively). Copy number alterations were analyzed using SNP-array and T-S data. We found that loss of long arm of chromosome 11 (11q loss) was significantly accumulated in PE-group (33% [6 out of 18 patients] vs. 0%, P = .019), whereas loss of chromosome 5q and chromosome 7 were almost equally identified in both groups (chromosome 5q, 22% and 12% [P = .66]; chromosome 7, 22% and 29% [P = .71] in PE- and DE-group, respectively). On the commonly affected region of 11q in this study, there were three genes recurrently altered in MDS and AML; ATM, KMT2A, and CBL. Copy number loss was found in these three genes, and the alteration of ATM was significantly increased in PE-group (28% [5 out of 18 patients] vs. 0%, P = .046) than DE-group. The remaining allele of ATM also possessed a mutation in two out of five patients in PE-group, indicating a deleterious effect on the function of ATM. In summary, we revealed two unique genetic features in MDS among A-bomb survivors who were proximally exposed to A-bomb radiation; significantly less frequent mutations in DNA methylation pathway and significant accumulation of 11q loss, suggesting a long-lasting effect of ionizing radiation and an important role of 11q loss in the initiation of MDS. Disclosures No relevant conflicts of interest to declare.

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.

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