Constitutional Segmental Uniparental Disomy in a Twin Pair with t(12;21) Positive Acute Lymphoblastic Leukemia Characterized by the Same Prenatal Clone and Divergent Clonal Evolution.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4549-4549
Author(s):  
Giovanni Cazzaniga ◽  
Julie Irving ◽  
Marco Citterio ◽  
Silvia Bungaro ◽  
Roxane Tussiwand ◽  
...  
Keyword(s):  
Germ Line ◽  
Twin Pair ◽  
Rapid Determination ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Monozygotic Twin ◽  
Nucleotide Polymorphisms ◽  
Overt Leukemia

Abstract We previously reported (Tussiwand R. et al., ASH 2002) the case of a monozygotic twin pair with concordant ALL aged 3.0 years at diagnosis, with only 13 days difference in latency. Twin 1 was classified as pre-B ALL and twin 2 as common-ALL, based on standard immunophenotyping criteria. A large screening for TcR and Ig gene rearrangements was performed, resulting in only one common VKII-Kde rearrangement, the others being not related to each others. Highly sensitive RQ-PCR was performed for all markers in both twins. The result of the crossed analysis was consistent with the hypothesis that after a prenatal event resulting in a preleukemic clone, at least a second independent event must have occurred before overt leukemia. To further identify markers of the clonal evolution, high-resolution single nucleotide polymorphisms (SNP) genotype analysis was performed on the DNA using the 10K SNP array (Affymetrix). Remission bone marrow was taken as a germ line samples. Array-based analysis of SNPs allows the rapid determination of genome-wide allelic information at high density, including the identification of submicroscopical copy number changes and/or loss of heterozygosity (LOH). SNP array analysis have been so far successfully applied to demonstrate allelic imbalance in ALL and AML blasts. A 12p12-13 deletion was observed on twin 2. The deletion of the TEL allele not involved in the t(12;21) was confirmed in twin 2 by FISH and microsatellite analyses. The twin 1 did not show any 12p deletion. Similar observations have been made in late-relapses occurring in ALL carrying the t(12;21) translocation: the predominant clone did not correspond to the same clone observed at diagnosis, but represented a second, independent transformation event within the fetal pre-leukemic clone, even when in the presence of the same genetic background. More interestingly, we found by SNP array a 13Mb area of LOH in remission and presentation samples of both twins involving the 2q13-14.3 region. As further confirmed by FISH with 2q probes, LOH was not associated with chromosomal loss, implying a recombination event resulting in Uniparental Isodisomy (UPD). The UPD area includes 57 known genes, several of them implicated in oncogenesis; they include translin, a gene involved in the control of chromosomal translocation and implicated in lymphoid malignancy. UPD of this region has not been reported in other tumors or in remission samples of leukemia; in genetic diseases few cases have been reported with maternal or paternal UPD 2, never associated with haematological disorders. By contrast, it has been shown that a transmitted deletion of 2q13 to 2q14.1 causes no phenotypic abnormalities. This is the first report on constitutional UPD in leukemia patients. Further analyses are necessary to understand the clinical meaning of this chromosomal abnormality; one hypothesis could be that the twins were born with a genetic predisposition to develop leukaemia. In this context, t(12;21) and additional events (i.e. TEL deletion) may be responsible for the overt leukemia.

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

Relapsed Childhood High Hyperdiploid Acute Lymphoblastic Leukemia: Genome-Wide Screening Reveals the Presence of Preleukemic Ancestral Clones and the Secondary Nature of Microdeletions and RTK-RAS Mutations.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2591-2591
Author(s):  
Josef Davidsson ◽  
Kajsa Paulsson ◽  
David Lindgren ◽  
Henrik Lilljebjörn ◽  
Tracy Chaplin ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Structural Changes ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
Snp Array ◽  
Prognostic Impact ◽  
Genetic Changes ◽  
Ras Mutations ◽  
Paired Samples

Abstract Abstract 2591 Poster Board II-567 Although childhood high hyperdiploid acute lymphoblastic leukemia is associated with a favorable outcome, 20% relapse. This makes it important to identify these patients already at diagnosis to ensure proper risk-stratification. To identify changes associated with relapse and ascertain the genetic evolution patterns, SNP array and mutation analyses of FLT3, KRAS, NRAS, and PTPN11 were performed on 11 paired diagnostic/relapse samples. The “triples trisomies” +4, +10, and +17 were detected in 64%, a frequency similar to the one generally observed at diagnosis, thus questioning their favorable prognostic impact. Structural changes, mainly cryptic hemizygous deletions, were significantly more common at relapse (P<0.05). No single aberration was linked to relapse, but four deletions, involving IKZF1, PAX5, CDKN2A/B or AK3, were recurrent. Based on the genetic relationship between the paired samples, three groups were delineated: 1) identical genetic changes at diagnosis and relapse (18%), 2) clonal evolution with all changes at diagnosis being present at relapse (18%), and 3) clonal evolution with some changes conserved, lost or gained (64%), suggesting the presence of a preleukemic clone. This ancestral clone was characterized by numerical changes only, with structural changes and RTK-RAS mutations being secondary to the high hyperdiploid pattern and perhaps necessary for overt leukemia. Disclosures: No relevant conflicts of interest to declare.

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

Molecular Diversity Detected by Whole Exome Sequencing in Chronic Myelomonocytic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 310-310
Author(s):  
Basel Rouphail ◽  
Kenichi Yoshida ◽  
Holleh D Husseinzadeh ◽  
Edward P Evans ◽  
Satoru Miyano ◽  
...  
Keyword(s):  
Research Funding ◽  
Germ Line ◽  
Target Therapy ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Gene Mutations ◽  
Molecular Pathogenesis ◽  
Common Mutation ◽  
Mutational Status ◽  
Recurrent Mutations

Abstract Abstract 310 CMML is characterized by monocytic proliferation, cytomorphologic dysplasia, and frequent progression to AML. Heterogeneity in or subtlety of presentation can make diagnosis challenging. Recent advances in molecular technology set the stage for systematic study of genetic and genomic lesions associated with CMML. Initially, RAS and RUNX1 mutations were identified in CMML; subsequently, mutations in TET2, CBL, ASXL1 and EZH2 have been discovered. Most recently, recurrent mutations in various genes of the spliceosomal machinery have been added, with SRSF2 likely the most common mutation in this condition. We hypothesized that more precise analysis of molecular lesions in CMML may allow for better categorization of this condition according to molecular pathogenesis and may provide clues to target therapy of this rather refractory condition. We identified 136 patients with CMML or secondary AML (sAML) with antecedent CMML: 87 CMML-1, 20 CMML-2 and 29 post-CMML sAML. The original cohort has been expanded by an additional 53 patients since first reported. The mean follow up period was 16 months (range, 0–114). Abnormal cytogenetics were found in 50% of the cohort by both metaphase and SNP-array-based karyotyping. In a representative subset of 27 patients, we have applied whole genome sequencing (WES) for which paired tumor/germ line DNA was used. To minimize false positives and focus on the most prevalent/relevant somatic events, we implemented a rational bioanalytic filtering approach and results were aligned using Burrows-Wheeler Aligner and variants detected using the GATK pipeline (Best Practice Variant Detection from Broad Institute). We focused on somatic defects with a frequency of >5% of the cohort. For the most commonly affected genes, results were validated using an expanded panel of 18 genes in 72 additional patients and, thus, for the most relevant genes a cohort of 95 patients was studied. The most frequently mutated genes were TET2 (48%), SRSF2 (35%), ASXL1 (17%) and RUNX1 (17%), whereas CBL (13%), EZH2 (13%), UTX (8%) and U2AF1 (8%), SETPB1 (10%), and RIT1 (9%) were less frequent. We also found TP53 and RUNX1 mutations in 5% and 16% of patients, respectively. A JAK2 V617F mutation was present in one case of seemingly typical CMML. BCOR and STAG2 mutations were found in 13% and 9% of patients, respectively; KRAS/NRAS mutations were in 10%. Spliceosomal gene mutations seem to be mutually exclusive, but were frequently associated with other non-spliceosomal gene mutations examined. Within the cohort of 28 SRSF2 mutant cases, 15 had coexisting TET2 mutations, 22 had ASXL1 mutations, 7 had RUNX1 and 5 had CBL mutations. Among 10 U2AF1 mutant cases, 3, 5 and 2 had TET2, ASXL1, and RUNX1 mutations, respectively. SETBP1 mutations were present in 34% of CMML-1/2 and frequently associated with RUNX1, SRSF2, CBL (approximately 2% each) and ASXL1 (4%) mutations. Cohesin mutations were less frequent (10%) because RAD21 and SMC mutations were absent. Mutations of PTPN11 and NF1 were less frequent in adult CMML than those reported in JMML. We also identified several less-recurrent gene mutations that likely modify pathogenesis or clinical outcomes of specific cases. Serial studies performed on 6 cases showed insight into the clonal architecture, producing a series of putative ancestral and secondary events, including uniparental disomy and acquisition of KRAS/NRAS or SETPB1 mutations. Association between mutational status and overall survival (OS) was assessed using Kaplan-Meier statistics. While all permutations were tested, we highlight here only significant positive and relevant negative results. In the whole cohort, presence of CBL mutations conferred worse OS (p=.018; HR 2.44, 95%CI 1.18–4.69). Median OS was 16 months for CMML-1, 6 months for CMML-2, and 14-months for sAML. In subgroup analyses, CBL mutations were also significant worse prognostic factor in CMML-1 cohort (p=.037; HR 3.23, 95%CI 1.07–8.04). In sum, WES provides intricate information on the molecular pathogenesis of CMML and the wide mutational spectrum correlates with the clinical diversity. Expert-based analysis of the genomic data may be supplanted by unsupervised and unbiased approaches which would cluster patients based on molecular similarities. Disclosures: Maciejewski: NIH: Research Funding; Aplastic Anemia&MDS International Foundation: Research Funding. Makishima:Scott Hamilton CARES Initiative: Research Funding.

Enhancement of Cytogenetic Diagnosis of Myeloid Disorders through Application of SNP-Array-Based Karyotyping.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 108-108 ◽  
Author(s):  
Lukasz P. Gondek ◽  
Ramon Tiu ◽  
Marcin Wlodarski ◽  
Christine O’Keefe ◽  
Michael McDevitt ◽  
...  
Keyword(s):  
Clinical Relevance ◽  
Germ Line ◽  
Copy Number Variants ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Disease Process ◽  
Normal Karyotype ◽  
Diagnostic Value ◽  
Differential Analysis ◽  
The Impact

Abstract Cytogenetic testing improves diagnosis in myeloid disorders; chromosomal (chr) aberrations have important clinical implications. SNP arrays (SNP-A) can be applied for karyotyping with a superb resolution of unbalanced defects and detection of uniparental disomy (UPD). We stipulated that SNP-A will enhance diagnostic value of metaphase cytogenetics (MC) and uncover new random/recurrent lesions. We applied 250K SNP-A to analysis of 76 controls and 318 patients, including 95 MDS, 64 AA, 20 PNH, 48 MDS/MPD, and AML both as primary (N=32) and secondary (N=59). Multiple samples were obtained in 13 patients. Minimal clonal size detectable by SNP-A was 25–50% by dilution studies. Repetitive testing resulted in congruent results; analysis of chr X in males showed >99% fidelity. To obtain reference, deletions and duplications seen in controls were analyzed. These abnormalities correspond to germ line encoded copy number variants (CNV). In patients such CNV were not deemed pathogenic. SNP-A confirmed 82% of unbalanced chr lesions detected by MC; discordant cases included defects involving smaller clones (<8/20 metaphases) and aberrations of Y. SNP-A allowed for detection of defects in 63% vs. 37% by MC, including 77% vs. 58% in MDS, 75% vs. 37% in MDS/MPD, 33% vs. 0% in AA, 30% vs. 0% in PNH, 59% vs. 31% in AML and 76% vs. 53% in sAML. New lesions were confirmed by paired SNP-A and microsatellite analysis. Concurrent analysis of blood and marrow showed concordant results suggesting utility of SNP-A performed on blood. Serially followed patients N=6, showed occurrence of new lesions (del(4)(q) and del(7)(q)) and earlier detection of the chr aberrations. In sAML, differential analysis of blasts and granulatocytes revealed occurrence of new lesions e.g., UPD6 or 7. In both MDS and AML, UPD of various chrs was present in 20% of patients and found in up to 35% of MDS/MPD (in addition to 9p involving also chrs 6,7,11 & 14). Other newly detected lesions included isolated/recurrent microdeletions and duplications involving genes such as AML1 or Ftl3 among others. Clinical utility of SNP-A depends on whether SNP-A karyotypig will have impact on disease parameters. In all groups tested the newly detected lesions showed impact on overall survival. While the detailed results will be a subject of our presentation, survival analysis in AML can illustrate our point; cases with a normal karyotype showed superior OS to those with newly detected defects (21 vs. 6 mo, p=.05). Similarly, new additional lesions worsen the survival as compared to those with confirmed MC (3 vs. 10 mo, p=.004). The impact on OS was also established for some of the new recurrent lesions such as UPD7q (3 vs. 39 mo, p=.002). Clinical relevance of SNP-A karyotyping is also demonstrated in AA; it may help to distinguish AA from hypocellular MDS (clonal chr. defects, including UPD, occur in 33% of AA patients), AA with normal SNP-A testing showed superior response to immunosuppression as compared to patients with a totally normal karyotype. Aside of the clinical relevance, new overlapping/recurrent lesions point towards genes involved in the disease process. We conclude that SNP-A karyotyping may enhance MC in diagnosis of chr. defects and allow for a better clinical correlations of the defects with the phnenotypic and clinical features.

Sequence of Genetic Events in ETV6-RUNX1 Positive B Precursor ALL: Insights from Identical Twins with Concordant Leukaemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2-2 ◽  
Author(s):  
Caroline M Bateman ◽  
Sharon W. Horsley ◽  
Tracy Chaplin ◽  
Bryan D Young ◽  
Anthony M Ford ◽  
...  
Keyword(s):  
Gene Fusion ◽  
Twin Pair ◽  
Snp Array ◽  
Monozygotic Twin ◽  
Cell Receptor ◽  
Identical Twins ◽  
Childhood Leukaemia ◽  
Genomic Changes ◽  
Genomic Deletions ◽  
Genetic Events

Abstract Monozygotic twin pairs with concordant ALL have provided unique insights into the molecular pathogenesis and natural history of childhood leukaemia. Data from twin pair studies and neonatal blood spot screening indicate that ETV6-RUNX1 usually arises as an early or initiating pre-natal event. Its consequence appears to be the generation of a clinically silent or covert but persistent pre-leukaemic clone. Conversion to overt, clinical ALL then requires the acquisition of one or more additional genetic lesions that functionally complement ETV6-RUNX1, often including deletions of the non-rearranged ETV6 allele. Recent genome wide single nucleotide polymorphism (SNP) array based studies have revealed considerably more genetic complexity than previously suspected, with ETV6-RUNX1 cases having an average of 6 (range 1–21) genomic losses or gains (Mullighan et al., Nature2007, 446: 758). It is however unclear from these descriptive screens or audits when these multiple changes arise in relation to the presumed initiating gene fusion and what functional contribution they make. We have used a series of identical twin pairs with ETV6-RUNX1 positive B precursor ALL to test the proposition that, as we reported previously for ETV6 deletion (Maia et al., Blood2001, 98: 478), all presumed functional or ‘driver’ genomic changes are post-natal in origin and therefore secondary to ETV6-RUNX1 fusion. If this were to be correct then we anticipated that genomic deletions and gains should be different or distinct within each twin pair. We used 250K Sty and 250K Nsp Affymetrix SNP mapping arrays on 5 pairs of identical twins concordant for ETV6-RUNX1 gene fusion positive ALL. We identified copy number variation using the “in-house” Genome Orientated Laboratory File v2.2.9 software package. The SNP array was performed using leukaemic DNA compared to matched remission DNA for 4 out of 5 cases. The fifth case was compared to a pool of remission DNA. The total number of genetic aberrations found was 51 (excluding T cell receptor and immunoglobulin rearrangements): 36 of these lesions were deletions (mean = 7.2) and 15 amplifications. The commonest aberration, found in 8 out of the 10 children, was a deletion on 12p13.2 involving the ETV6 gene. This was discordant in all cases, consistent with our previous reports using microsatellite markers. Other aberrations included deletions of PAX5, CDKN1B, CDKN2A and CD200/BTLA. The status of these, and other, presumed ‘drivers’ of leukaemogenesis were always different when diagnostic DNA of twins, within a pair, were compared i.e. either the genetic change was absent in one but present in the other, or the alteration was present in both but had distinct genomic boundaries. However in 2 of 5 twin pairs concordant, identical lesions were detected. These were idiosyncratic or very rare genomic changes in ALL and were either in gene sparse regions or involved loci with no known or likely contribution to B cell regulation or leukaemogenesis (e.g. CRYGD). We consider the most likely explanation for these shared genetic events in twin cases is that they arise simultaneously with (or immediately prior to) ETV6-RUNX1 fusion, and in the same incipient pre-ALL stem cell, as collateral damage or ‘passenger’ mutations. These data indicate that the common and presumed ‘driver’ genetic changes that accompany ETV6-RUNX1 in ALL are all secondary to gene fusion and most probably post-natal in origin. It remains to be established whether they contribute at all to the sustained pre-leukaemic state and whether they arise independently of each other and sequentially or as a timed suite or bolus perhaps proximate to diagnosis.

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.

Susceptibility to Philadelphia-Positive acute Lymphoblastic Leukemia (ALL) Is Associated with a Germline Polymorphism In the ANRIL (CDKN2BAS) Locus.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1670-1670
Author(s):  
Ilaria Iacobucci ◽  
Marco Sazzini ◽  
Anna Ferrari ◽  
Annalisa Lonetti ◽  
Alessio Boattini ◽  
...  
Keyword(s):  
Dna Analysis ◽  
Kinase Inhibitors ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Snp Array ◽  
Control Sample ◽  
Normal Karyotype ◽  
Nucleotide Polymorphisms ◽  
B Locus ◽  
Genome Wide

Abstract Abstract 1670 Among genes that are supposed to be involved in altered molecular pathways leading to leukemogenesis, the CDKN2A/B locus is particularly noteworthy since it encodes for the INK4-class cyclin dependent kinase inhibitors p15 INK4B, p16 INK4A and p14 ARF, which act as tumor suppressors. The CDKN2A/B locus has been found to be inactivated in several haeamatologic malignancies mainly due to deletion, aberrant repression or epigenetic silencing, whereas little is known about the potential role of its single nucleotide polymorphisms (SNPs) in leukemia susceptibility. To investigate whether polymorphisms within this broad genomic region can correlate with increased susceptibility to haeamatologic malignancies, an association study was performed by genotyping 23 SNPs spanning the MTAP, CDKN2A/B and CDKN2BAS loci, as well as relative intergenic regions, in a case-control cohort made up of 332 samples: 149 leukemia patients, including Philadelphia positive (Ph+) ALL (n=92) and acute myeloid leukemia (AML) samples (n=57), and 183 unrelated healthy controls. Ph+ ALL patients showed a median age of 52 years (18-78 years); 24 cases (26%) expressed the p210 oncoprotein, 62 (67%) the p190 and 6 (7%) both the proteins. AML patients showed a median age of 53 years (21-71 years) and they included FAB-M0, M1, M2, M3, M4, M5, miscellaneous cytogenetic abnormalities and normal karyotype subtypes. 6 SNPs were selected on the basis of their previous association with several diseases, such as coronary artery disease (rs2891168, rs518394, rs564398, rs10757278), type 2 diabetes mellitus (rs564398), frailty (rs2811712). The remaining 17 SNPs were selected among those included in the Affymetrix Genome-Wide Human SNP Array 6.0 to deepen the SNPs coverage for the examined region. Genotyping was performed using iPLEX Gold technology and MassARRAY high-throughput DNA analysis with Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (Sequenom, Inc., San Diego, CA). Furthermore, all leukemia samples were also previously genotyped using the GeneChip® Human Mapping 250K NspI and Genome-Wide Human SNP 6.0 (Affymetrix). A total of 17 SNPs, spanning the 9p genomic interval that encompasses the MTAP, CDKN2A/B and CDKN2BAS loci, were successfully genotyped and used for investigating their potential associations with the leukemia phenotypes. Five SNPs (rs1012713, rs10965179, rs34011899, rs3731232, rs3218010) with Minor Allele Frequency (MAF) <0.05 in cases and controls, as well as one SNP (rs3931609) showing >20% missing call rates, were instead excluded from the association analysis. Potential population stratification affecting the control sample was ruled out as its genotypes distribution satisfies the Hardy-Weinberg equilibrium criterion. Among the 17 SNPs, rs564398, mapping to the CDKN2BAS locus that encodes for ANRIL antisense non-coding RNA, showed a statistically significant correlation with the ALL phenotype, with a risk pattern that was compatible with an overdominant model of disease susceptibility and an Odd Ratio (OR) of 2 (95% CI, 1.20 to 3.33; p= 7.1 × 10-3). Since a co-ordinated regulation of ANRIL and p14/ARF, p16/CDKN2A, p15/CDKN2B transcription has been already observed in both physiologic and pathologic conditions, we hypothesized that rs564398 association reflects a condition of high linkage disequilibrium between such polymorphism and a causative variant that is able to alter CDKN2A/B expression profiles by changing ANRIL dosage, thus leading to abnormal proliferative boosts and consequent increased ALL susceptibility. Supported by European LeukemiaNet, AIL, AIRC, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integrated program (PIO), Programma di Ricerca Regione – Università 2007 – 2009. Disclosures: Baccarani: Novartis: Consultancy, Honoraria; BMS: Consultancy, Honoraria. Martinelli:Novartis: Consultancy, Honoraria; BMS: Honoraria; Pfizer: Consultancy.

Exposure to Inflammatory Immune Responses As Driver of Clonal Evolution in Childhood Acute Lymphoblastic Leukemia

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 166-166
Author(s):  
Lars Klemm ◽  
Srividya Swaminathan ◽  
Elli Papaemmanuil ◽  
Anthony M Ford ◽  
Mel Greaves ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cells ◽  
Immune Responses ◽  
B Cell ◽  
Cell Cultures ◽  
Lymphoblastic Leukemia ◽  
Clonal Evolution ◽  
B Cell Development ◽  
Inflammatory Stimuli ◽  
Overt Leukemia

Abstract Background: Pediatric pre-B acute lymphoblastic leukemia (ALL) may develop from prenatal chromosomal translocations acquired in utero. For instance, the ETV6-RUNX1 gene rearrangement (~25% of childhood ALL) is found in the umbilical cord blood and Guthrie blood spots of 1 in 100 healthy newborns, however, only 1 in 14,000 carriers develop overt leukemia. The molecular mechanisms driving clonal evolution towards overt leukemia were not clear. Rationale: Activation Induced Cytidine Deaminase (AID) and Recombination Activation Genes 1 and 2 (RAG1-RAG2) are genetic modifiers of the immunoglobulin (Ig) genes that are expressed during normal B cell development. Although AID and RAG1/RAG2 are thought to be segregated to early (RAG1/RAG2) and late (AID) stages of B cell development, respectively, we found that the two enzymes can be concurrently expressed during early B-lymphopoiesis in the context of repeated inflammatory stimuli. Results: Our experiments identified transitional pre-B cells as the subset that is particularly vulnerable to concomitant expression of AID and RAG1-RAG2 with earlier B cells being protected by IL7 signaling.Human B cells from children lacking a functional IL-7 receptor (IL-7R) displayed both increased expression and activity of AID concurrently with RAG1-RAG2. These results demonstrated that AID activation in both mouse and human early B cell compartments increases genetic instability. Although concurrent activation of AID and RAG1-RAG2 in patient samples implicated a correlation between the two enzymes in the pathogenesis of leukemia, this as such did not prove that the enzymes causally induce overt leukemogenesis. Therefore, we next evaluated the requirement of AID and RAG1-RAG2 in leukemogenic transformation, and identified a condition that leads to massive activation of these enzymes in a pre-leukemic B cell. Importantly, AID and RAG1-RAG2 expression increased dramatically during inflammatory immune responses (e.g. infection), where both these enzymes diversify the antibody repertoire and improve its affinity to antigens from infectious pathogens. We therefore tested whether the pre-B cell subset that concurrently expresses AID and RAG1-RAG2 can respond to an inflammatory stimulus, such as LPS. We observed that pre-B cells require protection from IL7, which prevents aberrant activation of AID. In the absence of protective IL-7, these pre-B cells acquired responsiveness to LPS and strongly activated AID concurrently with RAG1-RAG2 enzymes. We developed IL7-dependent pre-B cell cultures as a disease model for ETV6-RUNX1 pre-leukemia and tested the role of AID and RAG1 in the progression of pre-leukemic clones. To this end, we expanded ETV6-RUNX1 pre-B cells from wildtype (AID and RAG1 expressing) mice, or from mice lacking AID (Aid-/-Rag1+/+) or RAG1 (Aid+/+Rag1-/-). We then challenged pre-B cell cultures by withdrawal of IL7 (loss of protection) and inflammatory stimuli (LPS) and transplanted pre-B cells that had undergone five cycles of -IL7/LPS challenge. Upon transplanting -IL7/LPS-treated Aid+/+Rag1+/+ or Aid-/-Rag1+/+ or Aid+/+Rag1-/- pre-B cells containing ETV6-RUNX1 into NOD-SCID recipient mice, we observed that loss of either Aid or Rag1 dramatically prolonged the latency and reduced the penetrance of leukemia in transplant recipients. This proved that AID and RAG1-RAG2 causally accelerate clonal evolution of a pre-leukemic B cell towards leukemia. Our findings provide a mechanism by which pre-leukemic clones carrying a prenatal genetic lesion such as ETV6-RUNX1 can evolve through infectious and inflammatory stimuli ultimately leading to full blown leukemia. Conclusion: The impact of inflammatory stimuli on leukemogenesis has been previously implicated in multiple epidemiological studies. For instance, day-care attendance primed the immune system during early childhood and is thought to protect against exacerbation of B cell responses and to prevent collateral damage driving clonal evolution towards leukemia. Although inflammation (LPS stimulation) seems to play a role in accelerating pre-B leukemogenesis in our model, further experiments testing actual infectious pathogens are needed to corroborate this concept. Moreover, it is crucial to test whether leukemogenesis is accelerated in individuals infected with restricted classes of pathogens, not all of which may activate AID in pre-B cells. Disclosures No relevant conflicts of interest to declare.

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