Single Nucleotide Polymorphism Array (SNP-A) Genomic Profiling of Mantle Cell Lymphoma (MCL) Against a Large Control Database Reveals Recurring Copy Number Alterations (CNAs) and Copy Neutral Loss of Heterozygosity (CN-LOH)

Abstract Abstract 2001 Background: MCL is characterized by extreme genomic instability. Conventional techniques such as metaphase cytogenetics are unable to detect small deletions, amplifications, or uniparental disomy (UPD) in the MCL tumor genome. SNP-A analysis permits high resolution karyotyping and detection of unbalanced DNA defects, including somatic UPD. We performed SNP-A analysis on MCL tumor samples, excluded CNA present in normal controls, and assessed our results in context of clinical outcome and Ki-67 index. Methods: With IRB approval, available frozen tissue from 18 patients diagnosed with cyclin D1-positive MCL between 1997–2006 was analyzed using high-resolution genome-wide human SNP Array 6.0 (Affymetrix). Signal intensity and SNP calls were determined using the Gene Chip Genotyping Analysis Software Version 4.0 (GTYPE) (Affymetrix). Copy number was also determined. Somatic MCL CNAs and CN-LOH were discerned from germline variants (CNV) by comparing to a database of 1535 normal controls subjected to 250K and/or SNP Array 6.0 analysis. Clinical data was available for all patients, and 15/18 samples were subject to immunohistochemistry (IHC) for Ki67 using an automated immunostainer (Discovery; Ventana Medical Systems). Kaplan-Meier survival analysis was performed. Results: Analysis of 18 MCL patient samples revealed an average 13 CNAs (6.2 gains, 6.4 losses) and 0.4 CN-LOH per patient (Figure 1). Gains were frequently observed in 3q (33%), 8q (22%), 12q (17%), and 18q (11%). A unique 12q micro-gain in one patient narrowed the minimal common region (MCR) to linear region 130.4 – 131.3 Mbp, overlapping with an area of CN-LOH, and including candidate genes MMP17 (upregulated in invasive breast cancer), ULK1 (regulator of autophagy), and EP400 (regulator of chromatin remodeling, proliferation and apoptosis). Recurring deletions were observed at 11q (50%), 1p, 6q (39%), 9p (33%), 13 q (28%), 9q (22%), 7q, and 17p (17%). Similar to prior studies, losses at 1p encompassing CDKN2C and FAF were seen though we further narrowed a common MCR, spanning 93.1–99.7 Mpb and including ARHGAP29 (PARG1)—previously identified in MCL by aCGH/gene expression, whose promoter is a frequent target of MCL methylation. As previously reported, losses in components of the Hippo tumor suppressor pathway were frequently affected by these recurring deletions (6q: LATS1, 9p: MOBKL2B) and by one deletion on 19p (MOBK2LA). Other high-frequency losses encompassed CDKN2A, CDKN2B, and MTAP (on 9p), RB1 and DLEU1/2/miR15a/16-1 (13q), and TP53 (17p). Unique homozygous losses were detected at 9p (3.2-3.3 Mbp; involving only RFX3), 11q (94.5-111.7 Mbp; spanning the ATM region), and 13q (82.7-99.5 Mbp; including the miR17-92 region), and micro-deletions at 6q (121.1-121.9; GJA1/Cx43), 12p (7.5-7.6; CD163), and 13q (73.3-73.4; KLF12, and 75.2–75.3; LMO7). CN-LOH was observed at 6p, similar to prior studies, though we found novel regions of UPD at 4p, 8q, 18q, 19q, and 22q. An overall survival of 3.6 years and relapse-free survival of 1.3 years was observed. Survival was significantly worse among 8 pts with Ki67 >75% (OS 1.4 years, p=.003), but was unaffected by del 11q, del 9p, gain 3q, or gain 8q. Conclusions: SNP-A analysis of 18 primary samples confirms that gains in 3q and 8q and losses in 11q, 6q, and 9p represent common secondary genetic lesions in MCL, and are not frequent in normal controls. We narrowed the MCR of several deletions, potential targets for gene sequencing, and confirm the presence of deletions of potential relevance to the Hippo pathway. Further analysis of our findings in light of tissue micro-array and fluorescence in-situ hybridization studies is underway, to assess pathobiologic consequences of genomic lesions as well as potential therapeutic targets. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v110.11.1585.1585 ◽

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.

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Disruption of the ASXL1 Gene Is Prevalent In PMF: Analysis of Molecular Genetics and Clinical Phenotypes

Blood ◽

10.1182/blood.v116.21.3074.3074 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3074-3074

Author(s):

Brady L Stein ◽

Donna M Williams ◽

Michael A McDevitt ◽

Christine L. O'Keefe ◽

Ophelia Rogers ◽

...

Keyword(s):

Molecular Genetics ◽

De Novo ◽

Conflicts Of Interest ◽

Single Nucleotide Polymorphism Array ◽

Myeloproliferative Neoplasms ◽

Direct Sequencing ◽

Snp Array ◽

Uniparental Disomy ◽

Jak2 V617f ◽

Wild Type

Abstract Abstract 3074 Background: The myeloproliferative neoplasms, PV, ET and PMF, share phenotypic features and molecular lesions, yet PMF distinguishes itself by its unfavorable natural history and rate of leukemic evolution. These distinctions may occur as a result of cooperating genomic lesions specific to PMF compared to PV or ET. We performed single nucleotide polymorphism array (SNP-A)-based karyotyping in 210 MPN patients and identified 20q11 deletions in 10% of PMF cases and in none of the PV or ET cases. The 20q11 deletion region spanned 1,662 KB and encompassed 37 genes, of which ASXL1 was included. To test whether ASXL1 contained lesions in the MPN cohort at large, we directly sequenced key regions of the ASXL1 gene in 65 PMF, 11 PV and 14 ET cases, as well as 7 controls from the SNP-array cohort. Genomic DNA from neutrophils and in select cases, purified CD34+ cells was used for both SNP-A and direct sequencing. Clinical parameters were correlated with genomic findings and the quantitative JAK2 V617F neutrophil allele burden Molecular genetics: 26/65 (40%) of PMF cases had abnormalities in ASXL1 (4 deletions, 22 mutations) whereas none of the 32 PV, ET or control cases had such lesions. The majority of ASXL1 sequence variations were nonsense lesions including the previously reported 1934dupG which comprised 30% of all of the mutations. The residual ASXL1 allele in all 20q11 deletion cases containing the ASXL1 gene was intact. In three PMF cases, more than one distinct ASXL1 mutation was identified, and cloning experiments on two of those cases indicated that the lesions were biallelic. Using banked samples, we observed the acquisition of an ASXL1 lesion over time, and established that ASXL1 lesions detected in 2 post ET-MF cases were also detected at low levels in the ET phase of the MPN. Genotype/Phenotype Correlations: ASXL1 deletions and mutations were prevalent in de novo PMF (37%), post PV-PMF (20%) post ET-PMF (62%) and in PMF/AML (33%). ASXL1 mutations did not associate with chemotherapy exposure as the prevalence of hydroxyurea use was similar in patients with and without mutations, and ASXL1 –mutation positive cases were present in patients who had never received any form of chemotherapy. There was no dependence upon JAK2 status as the presence of ASXL1 mutations were identified in JAK2 V617F-negative cases (9/26); JAK2 V617F-heterozygous cases (10/26); and JAK2 V617F-homozygous cases (7/26). Based on results of SNP-A, patients with ASXL1 mutations were equally as likely to have uniparental disomy (involving 9p or other regions) and loss/gain abnormalities (>1MB) compared to those without ASXL1 mutations. There were no differences in sex, age, or disease duration between PMF patients with and without ASXL1 mutations. In the ASXL1-mutant group, there was a trend toward a lower median white blood cell count (8 vs. 12.5 k/cu mm; p=0.3) and hemoglobin (9.7 vs. 11 g/dl; p=0.3) compared to ASXL1-wild-type patients. Furthermore, those PMF patients with ASXL1 mutations were significantly more likely to have received anemia-directed therapy (transfusion, erythropoietin, immunomodulating agents, steroids) compared to those without mutations (15/26 (58%) vs. 11/39 (23%); p=0.02). Post ET-MF patients comprised 31% (8/26) of ASXL1-mutant cases, compared to only 10% (4/39) ASXL1- wild-type cases (p=0.03). However, the presence of an ASXL1 mutation did not associate with an accelerated transition rate from ET to MF; among the 12 post ET-MF cases in the cohort, the median time of transition from ET to MF was 15.5 years in those with ASXL1 mutations compared to 7 years in those with ASXL1 wild-type status (p=0.02). Conclusion: Disruption of the ASXL1 gene occurs in 40% of PMF cases. The association of ASXL1 lesions, due to either mutation or deletion, suggests that ASXL1 haplo-insufficiency is associated with a PMF phenotype in the context of other known and unknown lesions, and that disruption of ASXL1 function may directly contribute to the pathophysiology and clinical complications of primary and secondary myelofibrosis. These data support the concepts that cooperative lesions in addition to JAK2 V617F are critical in generating PMF, that PMF is molecularly more complex than either PV or ET, and that the transition of PV or ET to PMF is associated with the acquisition of genomic lesions, such as ASXL1, that are present in PMF at large. Disclosures: No relevant conflicts of interest to declare.

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SNP Array Karyotyping Improves Detection Rate of Clonal Chromosomal Abnormalities in Refractory Anemia with Ringed Sideroblasts.

Blood ◽

10.1182/blood.v110.11.4132.4132 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4132-4132

Author(s):

Theodore Ghazal ◽

Lukasz P. Gondek ◽

Abdo S. Haddad ◽

Karl S. Theil ◽

Mikkael A. Sekeres ◽

...

Keyword(s):

Copy Number ◽

Chromosomal Abnormalities ◽

Neutral Loss ◽

Snp Array ◽

Uniparental Disomy ◽

Blood Analysis ◽

Refractory Anemia ◽

Ringed Sideroblasts ◽

Normal Copy Number

Abstract Among WHO low-risk categories of MDS, refractory anemia with ringed sideroblasts (RARS) can be more accurately diagnosed by characteristic pathomorphology. Clonal hematopoiesis and chromosomal abnormalities exemplify a close pathogenetic relationship to other forms of MDS. RARS shows considerable clinical variability even for patients (pts) with identical cytogenetic defects. Due to the low resolution of metaphase cytogenetics (MC) and its dependence on cell growth in vitro, this test is often non-informative in MDS. High-density SNP arrays (SNP-A) allow for a precise identification of unbalanced genomic lesions and copy-neutral loss of heterozygozity. We hypothesize that cryptic chromosomal (chr) aberrations exist in most, if not all, pts with RARS. Their detection may help to improve prognostication, distinguish distinct phenotypes and point towards unifying pathogenic defects. Initially, we analyzed the results of MC in pts with MDS and MDS/MPD (N=455) and in a sub-cohort of RARS, RCMD-RS, RARSt and other MDS subtypes with >15% RS. When we compared pts with/without RS, chr defects were found at comparable frequencies (∼50%). The most commonly occurring defects associated with RS, compared to other forms of MDS, included those of chr 5 (9% vs. 16%, 7 (8% vs. 12%) and 20 (3% vs. 8%). DNA was available for 36 pts with RS and was subjected to 250K SNP-A karyotyping. Pathologic lesions were defined upon exclusion of normal copy number polymorphisms identified in 81 controls (O’Keefe at al ASH 2007), as well as the Database of Genomic Variants (http://projects.tcag.ca/variation). By MC, a defective karyotype was present in 16/36 pts (44%). Deletions involving chr 5, 7 and complex MC were found in 3, 5, and 2pts, respectively. However, when SNP-A was applied as a karyotyping tool (copy number and LOH analysis), all aberrations found by MC were confirmed, but also new lesions were detected so that an abnormal karyotype was established in 62% of pts. Several previously cryptic/recurrent lesions included losses of a portion of chr. 2 (N=2; 2p16.2, 2p16.3), and deletions (N=4; 7p11.1–14.1, 7p21.3, 7q11.23–21.11, 7q21.12-qter) as well as gains (N=1; 7q33) on chr 7. We have also detected segmental uniparental disomy (UPD) in chr 1 (N=2; 1p21.3–22.2, 1p). This type of lesion cannot be detected using MC and provides an additional mechanism leading to LOH. When both bone marrow and blood of 5 RARS patient were tested using SNP-A, blood analysis had 100% accuracy rate as compared to marrow; all defects seen in the marrow were also found in blood. We conclude that chromosomal defects are present in a majority of RARS patients and arrays with higher resolution will identify defects in most, if not all of the patients. Our study also demonstrates testing of peripheral blood by SNP-A can complement marrow MC, especially in cases in which marrow is not available. Detection of clonal marker aberrations in blood of RARS patients suggests that mostly clonal dysplastic progenitor cells contribute to blood production rather than residual “normal” progenitors.

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Reconciling Phenotype with Genotype in the MPN: Impact of SNP Array-Based Chromosomal Analysis.

Blood ◽

10.1182/blood.v114.22.1893.1893 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1893-1893

Author(s):

Michael A McDevitt ◽

Donna Williams ◽

Brady L. Stein ◽

Christine O'Keefe ◽

Ramon V. Tiu ◽

...

Keyword(s):

Genomic Instability ◽

Copy Number ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Snp Array ◽

Uniparental Disomy ◽

Disease Classification ◽

Single Mutation ◽

Dependent Manner ◽

Disease Evolution

Abstract Abstract 1893 Poster Board I-916 The diagnosis and accurate prognostication of the myeloproliferative neoplasms (MPN) is complicated by phenotypic mimicry and variable rates of disease evolution. The somatic mutation of JAK2 (JAK2V617F), identified in more than 90% of PV and in 50% of ET and PMF patients, is associated with acquired uniparental disomy (aUPD) on chromosome 9p, generating copy number-neutral loss of heterozygosity (CN-LOH) and results in homozygosity of JAK2V617F in roughly one third of JAK2V617F-positive patients. The molecular events upstream of the mitotic recombination that leads to somatic CN-LOH are essentially unknown. Intriguingly, JAK2V617F itself has been implicated in the generation of genomic instability (Plo et al 2008). Although these observations provide a rationale for how a single mutation could give rise to different clinical pathologies and downstream genomic instability in MPN, the JAK2V617F allele burden (AB) has not been systematically correlated with high resolution evaluation of amplifications, deletions, and CN-LOH in a large MPN cohort. We performed single nucleotide polymorphism arrays (SNP-A), a powerful karyotyping tool with the unique ability to detect CN-LOH on neutrophil DNA from 90 MPN patients. Published copy number variants (CNVs) and those identified in our internal cohort of 995 healthy controls were excluded as well as germline, non-clonal CN-LOH regions based on size criteria. Genomic results were correlated with the quantitative JAK2V617F AB, clinical phenotypes within and between disease classes, and in 21 paired longitudinal samples. SNP-A detected aUPD and/or chromosomal gains or losses in 81% of MPN patients. The prevalence of genomic lesions was lowest in ET compared to PV or PMF (40% of ET cases were lesion free), and lower still in JAK2V617F-negative ET (62% lesion free). aUPD was the most common genomic lesion, occurring in 59% of the MPN cases, and involving every chromosome except chromosomes 18 and 23. As expected, aUPD most commonly involved 9p (38 cases), encompassing JAK2. aUPD encompassing the TET2 gene at 4q24, recently implicated in MPN and other myeloid disorders, was seen in 3 of the 90 cases, with deletions spanning this gene in 2 additional cases. Five cases of aUPD at 2q were observed, and other recurrent regions were limited to 3 cases or fewer. The prevalence of chromosomal losses alone was far lower than UPD (22%), most commonly involved chromosome regions 20q, 13q and 17q, and was a feature primarily of PMF. The prevalence of aUPD in our MPN series of 59% was higher than in MDS (20%, p<0.001), MDS/MPN (35%, p=0.0074), and sAML (23%, p=0.0004) using the same arrays and analytical methods. Independent of MPN classification category, JAK2V617F positive patients had a higher prevalence of UPD than did JAK2V617F negative patients, although when excluding 9p UPD (24/59 vs 11/31) this was not significant. However, homozygous JAK2V617F patients had twice the prevalence of aUPD (again excluding 9P UPD) than did the heterozygous patients (8/25 vs 5/33), and twice the prevalence of multiple aUPD within individual cases. Of the 21 patients studied longitudinally over 5 years (range 2–9), 3 patients with chromosomal lesions present on the first sample were not present in the second: 9p UPD resolved in one PV patient who transformed to PMF, 1p UPD resolved in a JAK2V617F-negative ET patient, and a 20q deletion resolved in a heterozygous JAK2V617F-positive ET patient who transitioned to PV. Additional UPD regions occurred in only 1/8 heterozygous JAK2V617F positive MPN patients, while 5/11 JAK2V617F homozygous patients developed additional aUPD. In contrast, the acquisition of deletions occurred primarily in JAK2V617F heterozygous ET patients, all of whom had transitioned to PV or PMF. We conclude that aUPD is increased in the MPN compared to other myeloid disorders, and associates with the presence of JAK2 V617F in a gene dose-dependent manner. We hypothesize that aberrant JAK2 signaling contributes to the development of UPD but not chromosomal losses or gains. Furthermore, comparison of deletion, amplification, and UPD events in ET and PV relative to PMF suggest that PMF bears a closer genomic resemblance to MDS than to ET or PV. Thus SNP-A provides unique insight into MPN disease classification (PMF), disease progression, and genomic instability mechanisms. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v114.22.166.166 ◽

2009 ◽

Vol 114 (22) ◽

pp. 166-166 ◽

Cited By ~ 2

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.

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High-Resolution Molecular Allelokaryotyping of Chronic Myeloid Leukemia Patients in Blast Crisis by 6.0 SNP-Arrays Shows a High-Frequency of Uniparental Disomy and Focal Copy Number Alterations Affecting the Whole Sequence or Specific Exons of Oncogenes and Tumor Suppressor Genes.

Blood ◽

10.1182/blood.v114.22.2176.2176 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2176-2176

Author(s):

Simona Soverini ◽

Sabrina Colarossi ◽

Alessandra Gnani ◽

Fausto Castagnetti ◽

Annalisa Astolfi ◽

...

Keyword(s):

High Resolution ◽

Tumor Suppressors ◽

Copy Number ◽

Myeloid Leukemia ◽

Blast Crisis ◽

Snp Array ◽

Uniparental Disomy ◽

Copy Number Alterations ◽

Snp Arrays ◽

Array Technology

Abstract Abstract 2176 Poster Board II-153 Progression from chronic phase to blast crisis (BC) remains a major hurdle on the road to effective treatment of chronic myeloid leukemia (CML). BC is known to be associated with accumulation of additional genetic alterations, but these alterations have so far been only partially characterized. The development of SNP-arrays as a tool for high-resolution karyotyping now allows to perform high-throughput genome-wide screens for submicroscopic genomic alterations with unprecedented informativity and resolution and to precisely map all the genes involved in these alterations. We have used Human 6.0 SNP Arrays (Affymetrix) to perform high-resolution molecular allelokaryotyping of 25 DNA samples from BC (myeloid, n=16; lymphoid, n=9) CML patients (pts). The 6.0 SNP Array technology relies on 1.8 million markers evenly spaced across the genome, with a median inter-marker distance <700 bp. Loss of Heterozygosity (LOH) analysis identified several recurrent regions of uniparental disomy (UPD) ranging from 970Kb to 2.4Mb: 3p21.31-3p21.2 (19 pts); 4p15.1 (n=18 pts); 14q23.3 (n=18 pts); 8q22.2 (n=15 pts); 7q31.31 (n=14 pts); 3q11.2 (13 pts); 17q23.2 (n=13 pts); 12q24.11-12q24.13 (n=12 pts); 15q15.2-15q15.3 (n=12 pts); 16q22.1 (n=12 pts); 10q22.1-10q22.2 (n=11 pts); 1p34.3 (n=10pts); 7q11.22 (n=10 pts); 8p11.12 (n=10 pts); 15q23-15q24.1 (n=9 pts); 20q11.22-20q11.23 (n=7 pts); 16q11.2 (n=6 pts); 17q11.2 (n=5 pts). Three pts had evidence of UPD involving the whole long arm of chromosomes 5, 14 and 19, respectively. Macroscopic copy number alterations (CNAs) (+8, +19, +14q; +21q; -7; -18, -16q; -17p; -6p; -6q; -9q) were frequent and easily detected. A variety of submicroscopic CNAs were also detected. However, we decided to exploit the unprecedented resolution power of Human 6.0 SNP Arrays and the ability of Genotyping Console 3.0.2 (Affymetrix) software to precisely pinpoint the borders of these CNAs. We thus aimed our analysis to the identification of very small CNAs that may have been missed by previous studies - all using less sensitive assays. This approach revealed a high number of focal gains or losses ranging from 4 to 47Kb, affecting single genes or even some exons only. Genes involved in >2 pts are listed in the Table below. Gains/losses mapping to known regions of copy number variation (CNV) were excluded. All the genes found to harbor CNAs were transcription factors, adaptor proteins, receptor and non-receptor kinases involved in cell proliferation and apoptosis - with a known role as oncogenes or tumor suppressors or oncogene/tumor suppressor interactors. Although these results confirm a high degrees of heterogeneity in the alterations detectable in BC CML pts, members of the RAS pathway (indicated with an asterisk) were the most frequently altered genes. Further characterization by polymerase chain reaction and sequencing is ongoing. In conclusion, the power of 6.0 SNP Array technology allowed us to detect previously unidentified alterations targeting whole or part of key oncogenes or tumor suppressors whose deregulation may play a role in determining the aggressive phenotype of BC CML, and which may represent potential therapeutic targets. Supported by European LeukemiaNet, AIL, AIRC, PRIN, Fondazione del Monte di Bologna e Ravenna. Disclosures: No relevant conflicts of interest to declare.

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

Blood ◽

10.1182/blood.v118.21.2795.2795 ◽

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.

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SNP-Based Mapping Arrays Reveal High Genomic Complexity in Monoclonal Gammopathies: From the MGUS to Myeloma Status

Blood ◽

10.1182/blood.v118.21.295.295 ◽

2011 ◽

Vol 118 (21) ◽

pp. 295-295

Author(s):

Lucía López-Corral ◽

Maria Eugenia Sarasquete ◽

Sílvia Beà ◽

Ramón García-Sanz ◽

Maria Victoria Mateos ◽

...

Keyword(s):

Copy Number ◽

Plasma Cells ◽

Conflicts Of Interest ◽

Neutral Loss ◽

Strong Association ◽

Snp Array ◽

Fragile Sites ◽

Symptomatic Disease ◽

Monoclonal Gammopathies ◽

Genetic Events

Abstract Abstract 295 Genetic events mediating transformation from the pre-malignant monoclonal gammopathies (MG) to myeloma (MM) are unknown. Previous FISH analyses have highlighted that most genetic lesions typical of MM are already present in MGUS. However, the genetic abnormalities characteristic of each evolving stage of MG have not been elucidated. To obtain a comprehensive genomic profile of MG cases from the early to the late stages we performed for the first time high resolution analysis on purified plasma cells from 20 MGUS, 20 Smoldering MM (SMM) and 34 MM by high density 6.0 SNP-array. Ten matched non-tumor DNA samples were also included in the analysis. We examined DNA copy number alterations (CNA), copy number neutral loss of heterozygosity (CNN-LOH) and the spectrum of minimally altered regions which could contain relevant genes. Moreover, visual inspection allowed us to detect intermediate situations which corresponded to imbalances present in minor populations (less than 50%) coexisting with the major diploid population. CNA were identified in 69 (93%) of the 74 patients analyzed with a median of 8 imbalances per abnormal case. The only 5 cases with no CNA corresponded to asymptomatic entities. We observed a progressive increase in the incidence of genomic imbalances from MGUS (median, 5/case) to SMM (media, 7.5/case) to MM (median, 12/case) (P=0.006; MGUS vs MM). In particular, gains on 1q, 3p, 6p, 9p, 11q, 19, 21q together with losses on 1p, 16q and 22q may be important genetic events associated with MGUS-MM transition, as they were less frequent in MGUS than in MM (P<0.038). Otherwise, 11p+ and 4q- would be implicated in the transition from SMM to symptomatic disease, as they were lower in SMM compared to MM (P<0.038). Chromosomal gains were usually associated with gains and losses with losses with the exception of 1q gains that were significantly associated with losses. The subclones analysis allowed us to illustrate that PC bearing CNA increased from MGUS to SMM and to MM. Thus, we observed at least one minor subclone (<50% of PC) carrying a CNA more frequently in MGUS (75% of cases with a median of 3.5 of subclones/case, range 1–12) than in SMM (30% of cases with a median of 1, range 1–19) and MM (56% of cases with a median of 1/case, range 1–16) (p=0.017, MGUS vs SMM+MM comparison). Moreover, the four abnormalities apparently exclusive of MM disease (+11q, +21q, -16q y -22q) were also found as minor subclone in MGUS samples, supporting the previous notion that karyotypic instability is initiated in MGUS (Figure 1).Figure 1Percentage of cases with CNAs as major and minor subclonesFigure 1. Percentage of cases with CNAs as major and minor subclones Overall, a total of 65 CNN-LOH were detected and 7 locus had a uniparental trisomy with a median number of genes of 188 per CNN-LOH. The frequency of CNN-LOH was higher in active MM as compared to the asymptomatic entities (MM, 53% vs SMM, 25% vs MGUS 25%; p=0.047). Most of the cases showed more than one region affected by this phenomenom. Of note, there were two identical interstitial CNN-LOH in one MGUS and one MM at 8q11.21-q11.23. We identified 12 homozygous deletions (HZD) corresponding to 5 MGUS (25%), 1 SMM (5%) and 3 MM (9%) patients, some of them containing relevant genes such as PRAME, BIRC2, BIRC3, MMPs 7 and 20. Interestingly, two couples of MGUS patients showed the same HZD at 2p22.3 and 8p11.23-p11.22. The latest contained only the gene ADAM 3A, whose function remains unknown, but its HZD has been recently reported in gliomas (Figure 2).Figure 2HZD at 8p11.23-p11.22 in two MGUS (CHAS software).Figure 2. HZD at 8p11.23-p11.22 in two MGUS (CHAS software). A strong association between genetic lesions and fragile sites (FRA) was also detected as more than one third of the focal-recurrent CNA and HZD, and more than a half of the minimal common regions and interstitial CNN-LOH overlapped with known FRA. In summary, our study shows an evolving cytogenetic profile of increasing complexity from MGUS to SMM and to MM. However, although MM display more CNA and CNN-LOH than early steps, MGUS are as genetically aberrant as MM, and the transition from MGUS to MM is not associated to a particular chromosomal imbalance but rather to an expansion of altered clones already present in MGUS. The analysis of sequential samples from the same individual evolving from MGUS and SMM to active MM is essential to confirm these results. In addition, our study reveals new chromosomal regions involved in CNA, HZD and CNN-LOH. A comprehensive investigation of the genes contained in these regions may provide new insights in MM pathogenesis. Disclosures: No relevant conflicts of interest to declare.

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The Identification of Further Minimal Regions of Overlap in Chronic Lymphocytic Leukemia Using High-Resolution SNP Arrays

Blood ◽

10.1182/blood.v124.21.3315.3315 ◽

2014 ◽

Vol 124 (21) ◽

pp. 3315-3315

Author(s):

Samantha JL Knight ◽

Ruth Clifford ◽

Pauline Robbe ◽

Sara DC Ramos ◽

Adam Burns ◽

...

Keyword(s):

High Resolution ◽

Chronic Lymphocytic Leukemia ◽

Candidate Genes ◽

Copy Number ◽

Conflicts Of Interest ◽

Neutral Loss ◽

Lymphocytic Leukemia ◽

Targeted Sequencing ◽

Bioinformatics Pipeline ◽

Large Patient

Abstract Background:Historically, the identification of minimal deleted regions (MDRs) has been a useful approach for pinpointing genes involved in the pathogenesis of human malignancies and constitutional disorders. Microarray technology has offered increased capability for newly identifying or refining existing MDRs and minimal overlapping regions (MORs) in cancer. Despite this, in chronic lymphocytic leukemia (CLL), published MORs that pinpoint only a few candidate genes have been limited and with the advent of NGS, the utility of high resolution array work as a discovery tool has become uncertain. Here, we show that profiling copy number abnormalities (CNAs) and cnLOH using arrays in a large patient series can still be a valuable approach for the identification of genes that are disrupted or mutated in CLL and have a role in CLL development and/or progression. Methods: 250 CLL patient DNAs from individuals enrolled in two UK-based Phase II randomised controlled trials (AdMIRe and ARCTIC trials) were tested using Infinium HumanOmni2.5-8 v1.1 according to manufacturer’s guidelines (Illumina Inc, San Diego, CA). Data were processed using GenomeStudioV2009.2 (Illumina Inc.) and analysed using Nexus Discovery Edition v6.1 (BioDiscovery, Hawthorne, CA). All Nexus plots were inspected visually to verify calls made, identify uncalled events and exclude likely false positives. To exclude common germline CNVs, the Database of Genomic Variants (DGV), a comprehensive catalog of structural variation in control data, was used. Copy number (CN) changes that encompassed fully changes noted in the DGV were excluded from further analysis. Regions of copy neutral loss of heterozygosity (cnLOH) were recorded if >1Mb in size, but were not used to define or refine MORs. Data from 1275 age-appropriate control samples minimised the reporting of common cnLOH events. All genomic coordinates were noted with reference to the GRCh37, hg19 assembly. MORs were investigated using Microsoft Excel filtering functions. A subset of genes (n=91) selected from MORs mainly on the basis of event frequency and/or number of genes within the MOR and/or literature interest were taken forward for targeted sequencing (exons only) of appropriate samples with/without CN Losses or cnLOH (Set 1 n=124; Set 2 n=126). These were tested using custom designed TruSeq Custom Amplicon panels (Illumina Inc) and processed according to manufacturer’s instructions. SAMHD1 was excluded from these panels since it had been studied separately within our laboratory. The data were analysed using an in-house bioinformatics pipeline that uses the sequence aligners MSR and Stampy and the variant callers GATK and Platypus, followed by stringent filtering. Results: Using our datasets we have identified >50 MORs previously unreported in the literature. Six of these showed copy number (CN) losses in >3% of patients studied. Furthermore, we have refined 14 MORs that overlapped with regions described previously and that had also a CN loss frequency of >3%. Thirteen MORs involved only a single reference gene, often a gene implicated previously in cancer (eg. SAMHD1, MTSS1, DCC and RFC1). Of the 91 genes taken forward for targeted sequencing, stringent data filtering led to a subset of 19 genes of interest harbouring exonic mutations. Genes with mutations identified include DCC, BAP1 and FBXW7, also implicated previously in cancer. Conclusion: We have generated high resolution CNA and cnLOH profiles for 250 first-line chemo-immunotherapy treated CLL patients and used this information to document newly identified MORs, to refine MORs reported previously and to identify mutation harbouring genes using targeted NGS. Functional knowledge supports our hypothesis that these genes may have a contributory role in CLL. For two genes, SAMHD1 and FBXW7, relevance in CLL has been established already. Taken together, our data validate the utility of high resolution arrays studies for the identification of candidate genes that may be involved in CLL development or progression when disrupted. Further studies are required to confirm a role for these genes in CLL and to elucidate the nature of the underlying biological mechanisms. Disclosures No relevant conflicts of interest to declare.

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High-Resolution SNP Array Profiling of Relapsed Acute Leukemia Identifies Genomic Abnormalities Distinct from Those Present at Diagnosis.

Blood ◽

10.1182/blood.v110.11.234.234 ◽

2007 ◽

Vol 110 (11) ◽

pp. 234-234 ◽

Cited By ~ 1

Author(s):

Charles G. Mullighan ◽

Ina Radtke ◽

Jing Ma ◽

Sheila A. Shurtleff ◽

James R. Downing

Keyword(s):

High Resolution ◽

Acute Leukemia ◽

Loss Of Heterozygosity ◽

Copy Number ◽

Neutral Loss ◽

Clonal Evolution ◽

Snp Array ◽

Remission Induction ◽

Dna Copy Number ◽

Leukemic Clone

Abstract Failure of initial remission-induction therapy and disease recurrence remains a major problem in the management of acute leukemia, however the nature of the biologic factors promoting relapse are incompletely understood. To identify genomic abnormalities associated with relapse, we performed high-resolution, genome-wide analysis of DNA copy number abnormalities and loss-of heterozygosity using Affymetrix single nucleotide polymorphism (SNP) microarrays in 33 cases of relapsed acute leukemia. Sixteen ALL (2 ETV6-RUNX1, 2 MLL rearranged, 6 pseudodiploid or cytogenetically normal B-progenitor ALL, and 6 T-lineage) and 17 AML (two RUNX1-RUNX1T1 [AML1-ETO], two MLL-rearranged, one M7, and 12 with normal karyotype or miscellaneous cytogenetic abnormalities) were studied. Samples with less than 80% blasts were flow sorted to at least 90% purity prior to DNA extraction. Diagnostic samples were available for all cases, and germline samples for 22. DNA copy number and LOH analysis was performed using Affymetrix 250k Nsp and Sty arrays was performed for all samples. Data were analysed using a karyotype-guided normalization algorithm, dChipSNP, and circular binary segmentation. In a detailed comparative analysis of paired diagnostic and relapse samples, changes in DNA copy number abnormalities were identified in the relapse sample in 14 of 16 (87.5%) ALL cases. A striking finding was loss of copy number lesions present at diagnosis in 8/16 ALL relapse samples, and the acquisition of new copy number lesions in 4 of these 8 samples. In each case, the pattern of deletions at antigen receptor loci was comparable between relapse and diagnosis, suggesting the emergence of a related leukemic clone, rather than the development of a distinct second leukemia. An additional 8 ALL relapse samples retained the copy number lesions present at diagnosis, but 6 of these acquired additional copy number abnormalities at relapse, a finding further suggestive of significant clonal evolution. Of the newly acquired copy number abnormalities in the relapse samples, deletions (62.5% of cases) were more common than gains (12.5%). In constrast to ALL a more restricted range of copy number abnormalities was seen in AML, with new abnormalities at relapse seen in 5/17 (29.4%) cases, and deletions (29.4%) outnumbering gains (17.6%). The loss of lesions present at diagnosis was only observed in two AML relapse samples. Examining the entire cohort, the CDKN2A/B locus was most commonly involved (N=5), gains of 1q were noted in two cases, otherwise all observed copy number changes were noted in single cases only, and included focal deletions of ERG and RUNX1. Copy neutral loss of heterozygosity was uncommon, with the exception of three AML cases with UPD of the entire chromosome 13. These observations indicate that relapse is frequently the result of the emergence of a leukemic clone that shows significant genetic differences from the diagnostic clone. Whether these represent rare clones present at the time of diagnosis or are the emergence of new clones as the result of ongoing genomic abnormalities can now be determined using genomic probes specific for the newly acquired deletions.

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