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

Blood ◽  
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.

SNP Array Analysis in Refractory Anemia with Ringed Sideroblast Allows for Detection of a High Frequency of Previously Cryptic Chromosomal Defects with Possible Clinical Significance.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2352-2352
Author(s):  
Abdo S. Haddad ◽  
Lukasz P. Gondek ◽  
Araf Alkali ◽  
Mikkael A. Sekeres ◽  
Alan Lichtin ◽  
...  
Keyword(s):  
Snp Array ◽  
Uniparental Disomy ◽  
Refractory Anemia ◽  
Array Analysis ◽  
Monosomy 7 ◽  
Ringed Sideroblasts ◽  
Snp Array Analysis ◽  
Pathologic Lesions ◽  
Risk Categories

Abstract Among WHO low-risk categories of MDS, refractory anemia with ringed sideroblasts (RARS) can be more precisely identified based on typical findings in the iron stain preparations of marrow aspirates. Clonal hematopoiesis and chromosomal (chr) abnormalities are features that reveal a clear pathogenetic relationship to MDS. However, despite characteristic morphology, chr defects in RARS are heterogeneous, RARS shows considerable variability in the clinical course even for patients with identical cytogenetic defects. We analyzed the results of metaphase cytogenetics (MC) in a cohort of patients classified as RARS, RCMD-RS, or more advanced forms of MDS associated with ringed sideroblasts. Similar proportions of defects (around 50%) were found in RARS patients (n=68) as in all other remaining MDS forms studied (n=288). The most commonly occurring defects associated with ringed sideroblasts, compared to other forms of MDS, included those of chr 5 (14% vs 31%), 7 (11% vs 14%) and 8 (17% vs 20%), respectively. However, due to the low resolution of MC and its dependence on cell growth in vitro, this test is often negative or non-informative in MDS. High-density SNP arrays (SNP-A) allow for a precise identification of unbalanced genomic lesions. We hypothesize that cryptic chr aberrations do exist in most, if not all, patients with RARS. Their detection may help to improve prognostication, distinguish distinct subgroups of patients and point towards unifying pathogenic defects. Consequently, we have applied 250K SNP-A to analyze the marrow (n=14) or blood (n=15) in RARS. Pathologic lesions were defined based on a study of 36 normal marrow specimens (O’Keefe at al ASH 2006). By traditional MC, a defective karyotype was present in 18 of 29 patients (34%). Monosomy 7/7q- was found in 5 patients; deletions involving chr 5 and 11 in 2 cases; and 5 patients showed a complex karyotypes. However, when SNP-A was applied as a karyotyping tool (copy number and LOH analysis), we were able to detect lesions in 65% of patients (vs 34% by cytogenetics, p<.01). All but 2 aberrations found by MC were confirmed. New, previously cryptic lesions were identified, including losses of a portion of chr 8 (n=4) and deletions (n=2) as well as gains (n=2) within chr 17q. We have also detected segmental uniparental disomy (UPD) in 2 patients. This type of lesion cannot be detected using MC and provides an additional mechanism that can lead to LOH. When comparing MC to SNP-A, 1 lesion was found in 7/29 patients vs 12/29; 2 lesions in 0/29 vs 5/29; and ≥3 lesions in 3/29 vs 2/29 patients, respectively. Most remarkably, we have identified shared lesions, including an invariant deletion involving 4q31-21 (n=3, with 2/3 diagnosed as RARSt), loss of 8q24.23 (n=2); and a UPD involving 1p21.3-22.2 (n=2). These lesions contain important genes, including IL-15 and GAB1 (4p) or DR1 and TGFBR3 (1p). Our results indicate that if more precise techniques are applied, chr abnormalities can be detected in a higher proportion of patients with RARS. Analysis of clinical outcomes associated with defects identified by A-SNP is currently ongoing in our laboratory.

An Increased Frequency of Abnormalities Including Uniparental Disomy Is Detected by SNP Array Compared with Conventional Karyotype Analysis in Acute Myeloid Leukemia.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 97-97 ◽  
Author(s):  
Manoj Raghavan ◽  
Rosemary E. Gale ◽  
Spyros Skoulakis ◽  
Tracy Chaplin ◽  
Gael Y. Molloy ◽  
...  
Keyword(s):  
Copy Number ◽  
Chromosomal Abnormalities ◽  
Germ Line ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Data Set ◽  
Array Data ◽  
The Mean ◽  
Homozygous Mutations ◽  
Acute Myeloid

Abstract SNP array technology permits the simultaneous analysis of copy number and allelotype data. This approach has revealed the somatic acquisition of uniparental disomy (UPD) in approximately 20% of acute myeloid leukemias (AMLs) (Raghavan et al. Cancer Res2005;65:375–378). UPD, which mostly appears to be the consequence of mitotic recombination, cannot be detected by conventional analysis. We have conducted a pilot investigation of samples from the UK MRC AML 10 trial in order to confirm these findings in a larger data set from a large clinical trial. The Affymetrix 10K GeneChip Mapping Array was used to type DNA from 100 AML blast samples of which 87 produced arrays with call rates in excess of 90%. Analysis was performed using the genome orientated laboratory file (GOLF) system, an in-house software package designed to interpret SNP array data. Control germline DNA was not available for each AML and GOLF was used to create a control copy number experiment from the mean of 52 array data sets from normal tissue (blood and remission marrow). The copy number ratio was calculated for each SNP for each sample. In 44 samples the karyotype concurred with the SNP array results (excluding balanced translocations which cannot be detected by SNP array). In the other samples, 54 abnormalities were detected that were not seen in the karyotype (6 samples had no karyotype information). Nine were amplifications, 12 were deletions and 31 were UPDs (35%). Of the UPDs, 12 were either whole chromosome or extended to the most telomeric SNP, with the others therefore being interstitial changes. The mean size of an interstitial LOH was 13.3 Mb, with the smallest detected being 3.2 Mb. Recurrent chromosomal abnormalities not detected by giemsa banding are listed in table 1. Twenty samples that were regarded as normal karyotype by gene banding had abnormalities by SNP array. In three examples numerical karyotypic abnormalities were not seen on SNP array analysis (add4q, −8 and a hypodiploid AML). This may be because a minority clone of the AML cells had this karyotype. UPD is known to be associated with homozygous mutations in AML (Fitzgibbon et al. Cancer Res 2005; in press). In this series, two patients had UPD13, both with biallelic FLT3 internal tandem duplication mutations. However, this study has identified several new areas to look for potential homozygous mutations. Given that in this study germ-line comparison has not been made one should not rule out the possibility of some of the smaller abnormalities being copy number polymorphisms (Bailey et al. Science2005;297;1003–1007). However, consanguinity can probably be ruled out given the lack of widespread homozygosity. This study confirms the frequency of UPD and illustrates the potential of SNP arrays for highlighting novel genetic events in AML. Table 1: Recurrent chromosomal abnormalities not detected by gene banding Previously Undetected Abnormality Number UPD1p 2 Amplified 1p 2 UPD2p 2 Del7q 3 UPD8p 2 UPD11p 2 UPD11q 3 Amplified 12 3 Amplified 13 2 UPD16p 4 UPD16q 2 Del20q 2

SNP-Array Based Karyotyping Complements Routine Cytogenetics in Diagnosis and Risk Stratification Schemes of MDS

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 639-639
Author(s):  
Ramon V. Tiu ◽  
Lukasz P Gondek ◽  
Jungwon Huh ◽  
Christine O’Keefe ◽  
Mikkael A. Sekeres ◽  
...  
Keyword(s):  
Risk Stratification ◽  
Chromosomal Abnormalities ◽  
Diagnostic Yield ◽  
Neutral Loss ◽  
Prognostic Significance ◽  
Significant Proportion ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Abnormal Karyotype ◽  
Novel Technologies

Abstract While current pathomorphologic criteria distinguish MDS, MDS/MPD and MDSderived AML (sAML), these conditions share common unbalanced chromosomal abnormalities highly predictive of prognosis. Reflecting their clinical importance, IPSS is highly influenced by metaphase cytogenetics (MC), the standard for detection of chromosomal abnormalities. Due to its low resolution and need for cell divisions, MC can only detect abnormalities in 50% of patients with MDS, novel technologies have been developed that could improve the diagnostic yield. Among these methods, single nucleotide polymorphism arrays (SNP-A) have been adopted as a cytogenetic platform. SNP-A karyotyping can be performed on interphase cells and allows for detection of smaller lesions as well as for copy-neutral loss of heterozygosity (LOH). As a result, some reference laboratories offer this test for routine cytogenetic diagnostics. However, the clinical relevance of lesions detected by SNP-A remains unclear and, consequently, we designed this comprehensive study to clarify the clinical impact of cytogenetic results generated through a combined application of SNP-A and MC. A cohort of 352 patients (218 MDS, 59 MDS/MPD, 75 sAML) was studied using Affymetrix 250K (N=160), Affymetrix 6.0 (N=190) and both platforms (N=95). Controls included 362 and 118 samples analyzed by 250k and 6.0 arrays, respectively. CNAGv3 or GCv2.1 software was used for analysis. To avoid biological and technical artifacts, known copy number variants as well as areas of LOH&lt;2.5Mba were excluded. All other lesions, if not present on MC, were confirmed in germ line DNA if possible. The median follow-up time was 29 months (95% CI; 21-38 mo) and for many patients serial samples were studied, showing sequential acquisition of chromosomal defects. Overall, the detection rate of chromosomal abnormalities was improved compared to MC (57% vs. 44%; p=.0096), MDS/MPD (66% vs. 39%, p=.0055) and sAML (63% vs. 45%, p=.048). Somatic uniparental disomy (UPD) accounted for a significant proportion of newly detected defects (26% of new lesions). Overall, UPD was detected in 100 patients (78 as the sole and 22 as an additional lesion). Chromosomal lesions were identified in 98/180 with normal MC. In 61% of patients with non-informative MC (N=18), SNP-A demonstrated an abnormal karyotype; those with newly detected lesions showed inferior outcomes in overall survival (OS) [16 vs. 36 mo, p=.03]. Analysis of OS showed that patients with new defects detected by SNP-A and previously normal MC have worse outcomes compared to those in whom normal chromosomes were confirmed (39 vs. 73 mo, p=.03). Moreover, additional lesions also conferred worse prognosis for patients with abnormal karyotype by MC (17 vs. 38 mo, p=.01). This trend was even more pronounced when specific subgroups were analyzed based on OS and event free survival (EFS). For example, detection of new lesions in MDS (OS p=.02), MDS/MPD (OS p=.009, EFS p=.01) or sAML (OS p=.02, EFS p=.008) results in significant worsening of OS and EFS as compared to patients with truly normal cytogenetics. Newly detected lesions allowed for more precise prognostication within established IPSS strata of MDS and MDS/MPD patients. The detection of SNP-A defects negatively impacted OS/EFS in patients with low IPSS (NR vs. 69 mo, p=&lt;.0001; 42 vs. 112 mo, p=&lt;.0001), decreased EFS in Int-1/2 (24 vs. 29 mo; p=&lt;.0001) and decreased OS/ EFS in high IPSS categories (11 vs. 26 mo, p=&lt;.0001; 7 vs. 14 mo, p=&lt;.0001). In sAML, patients with SNP-A lesions, regardless of MC, have worse OS (6 vs. 21 mo, p=.009) and EFS (5 vs. 13 mo, p=.01). In summary, SNP-A karyotyping facilitates detection of lesions which can complement MC and contribute to a better molecular diagnosis. SNPdetected lesions have impact on prognostic parameters which will likely affect future risk stratification schemes and have prognostic significance regardless of MC in sAML. As a result, new molecular subtypes of MDS may be identified and better-defined recurrent lesions will contribute to identification of causative genes.

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)

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2001-2001
Author(s):  
Michael J. Rauh ◽  
Juraj Bodo ◽  
Eric Hsi ◽  
Bill Richendollar ◽  
Yuka Sugimoto ◽  
...  
Keyword(s):  
High Resolution ◽  
Copy Number ◽  
Conflicts Of Interest ◽  
Single Nucleotide Polymorphism Array ◽  
Neutral Loss ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Tissue Micro Array ◽  
Ki 67 ◽  
Normal Controls

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.

scholarly journals Hidden abnormalities and novel classification of t(15;17) acute promyelocytic leukemia (APL) based on genomic alterations

Blood ◽  
2009 ◽  
Vol 113 (8) ◽  
pp. 1741-1748 ◽  
Author(s):  
Tadayuki Akagi ◽  
Lee-Yung Shih ◽  
Motohiro Kato ◽  
Norihiko Kawamata ◽  
Go Yamamoto ◽  
...  
Keyword(s):  
Acute Promyelocytic Leukemia ◽  
Copy Number ◽  
Chromosomal Abnormalities ◽  
Neutral Loss ◽  
Chromosomal Translocation ◽  
Promyelocytic Leukemia ◽  
Genomic Alteration ◽  
Trisomy 8 ◽  
Genomic Changes ◽  
Normal Copy Number

Abstract Acute promyelocytic leukemia (APL) is a hematopoietic malignant disease characterized by the chromosomal translocation t(15;17), resulting in the formation of the PML-RARA gene. Here, 47 t(15;17) APL samples were analyzed with high-density single-nucleotide polymorphism microarray (50-K and 250-K SNP-chips) using the new algorithm AsCNAR (allele-specific copy-number analysis using anonymous references). Copy-number-neutral loss of heterozygosity (CNN-LOH) was identified at chromosomes 10q (3 cases), 11p (3 cases), and 19q (1 case). Twenty-eight samples (60%) did not have an obvious alteration (normal-copy-number [NC] group). Nineteen samples (40%) showed either one or more genomic abnormalities: 8 samples (17%) had trisomy 8 either with or without an additional duplication, deletion, or CNN-LOH (+8 group); and 11 samples (23%) had genomic abnormalities without trisomy 8 (other abnormalities group). These chromosomal abnormalities were acquired somatic mutations. Interestingly, FLT3-ITD mutations (11/47 cases) occurred only in the group with no genomic alteration (NC group). Taken together, these results suggest that the pathway of development of APL differs in each group: FLT3-ITD, trisomy 8, and other genomic changes. Here, we showed for the first time hidden abnormalities and novel disease-related genomic changes in t(15;17) APL.

Acquired 1p Uniparental Disomy Is Associated with Biallelic MPL W515L Mutation in RARS-T.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1643-1643
Author(s):  
Hadrian Szpurka ◽  
Lukasz P Gondek ◽  
Sanjay R Mohan ◽  
Eric D Hsi ◽  
Karl S Theil ◽  
...  
Keyword(s):  
Clonal Selection ◽  
Neutral Loss ◽  
Significant Proportion ◽  
Uniparental Disomy ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Refractory Anemia ◽  
Molecular Defect ◽  
Ringed Sideroblasts ◽  
V617f Mutation

Abstract Refractory anemia with ringed sideroblasts and thrombocytosis (RARS-T) has been considered a provisional subtype within the diagnostic entity of myelodysplastic/myeloproliferative diseases (MDS/MPD). Since JAK2 V617F mutation is present in a significant proportion of RARS-T patients (Szpurka et al. Blood, 2006), many investigators consider this entity to be more closely related to classical MPD. However, a significant minority of patients with RARS-T do not display a JAK2 V617F mutation. We have studied a cohort of patients with RARS-T (N=18) characterized by the presence of ringed sideroblasts, reticulin fibrosis and thrombocytosis (&gt;600×109/L), that lack obvious causes of secondary thrombocytosis. While 8/18 patients harbored a JAK2 V617F mutation, a molecular pathogenesis for the remaining patients was unexplained. The successful application of SNP-A to characterize the genomic lesions in MDS prompted us to use this technology to study RARS-T. SNP-A allows detection of copy neutral loss of heterozygosity such as UPD9p which is associated with JAK2 V617F mutation. SNP-A facilitated detection of previously cryptic lesions; 9/18 patients showed an abnormal SNP-A-based karyotype often involving multiple lesions (only 5 of these defects were detected by metaphase cytogenetics). The new lesions seen by SNP-A included gains of chromosome 11p, 20q and 21q; deletion of 2p and various areas of UPD including 1p, 9p, 6p, 2p and 8p. SNP-A allowed identification of seemingly invariant UPD1p in 4/18 patients. As this region includes the Mpl gene, we analyzed patients for the presence of MPL W515L/K mutations which have been described in MPD. We did not find any patients with MPL W515K, however MPL W515L mutation was present in 2/4 RARS-T patients with UPD1p; another patient showed monoallelic MPL W515L variant. In addition, 1 patient with UPD1p harbored both JAK2 V617F and MPL W515L mutations. To further delineate the molecular lesion we analyzed all patients for the presence of abnormal STAT5 activation. An aberrant phospho-STAT5 staining pattern was present in all cases that were positive for either JAK2 V617F or MPL W515L mutations (N=10); unexplained STAT5 activation was found in only 4 cases, pointing towards a molecular defect involving this pathway. In these 4 patients, and in 1 additional with UPD1p who did not harbor MPL W515L mutation, we searched for other genes which might explain the pathogenesis of this disease by potentially causing aberrant activation of STAT5. We sequenced Jak1T478S, Jak1V623A and Ntrk1S677N as well as the transmembrane, juxtamembrane and kinase domain of Tie1, Epha2 and Ephb2 genes, but no mutation was found. In addition, we found a group of phospho-STAT5-negative patients (N=4) that showed typical genetic features of myelodysplasia e.g. del(5), +8 and partial loss of chromosome X; these cases are probably best considered to be of MDS origin rather than MPD. To our knowledge, our work is the first description of biallelic MPL W515L mutation and UPD1p found in RARS-T patients. This data is important for understanding the clonal selection process and pathophysiology of activating mutations in MDS/MPD. Overall, our studies demonstrate that somatic UPD1p is associated with homozygous MPL W515L mutation in MDS/MPD cases. Localizing areas of somatic UPD by SNP-A may help identify candidate genes within the shared regions that are likely targets for mutations.

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

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.

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