Intrachromosomal Amplification Research Articles

What Is the Initiating Mechanism of iAMP21 in Childhood B Cell Precursor ALL?.

Abstract 581Patients with iAMP21 (intrachromosomal amplification of chromosome 21) represent a distinct cytogenetic subgroup of childhood B cell precursor ALL (BCP-ALL; incidence ∼2%) with a poor prognosis on standard therapy (29% EFS at 5 years). From cytogenetic studies the amplification of chromosome 21 appears to be the primary genetic change, however, whether this or associated abnormalities provide the initiating event of leukaemogenesis remains to be elucidated. Currently fluorescence in situ hybridisation (FISH) with probes directed to the RUNX1 gene provides the only reliable detection method. Knowledge of the underlying genetic mechanism will lead to improved diagnosis and determine those patients at high risk of relapse who require more intensive therapy. Our previous study using genomic arrays (1Mb aCGH, n=10; NimbleGen chromosome 21 custom oligonucleotide array, n=5) identified a common region of amplification (CRA) on 21q of 6.6 Mb and a common region of deletion (CRD) of 3.3 Mb within the telomeric region in 100% and 70% of patients, respectively. However, expression profiling (Affymetrix U133A, n=8) indicated that no important genes within the CRA or CRD were differentially expressed. We now have clinical, cytogenetic and FISH data on 94 iAMP21 patients (44 F, 50 M; median age 9.5y, range 2-30); the largest iAMP21 cohort investigated to date. Higher resolution analysis of 17/94 diagnostic samples by aCGH (Agilent 185K platform) have refined these regions to 5.1 Mb and 2.5 Mb in 100% and 88% of patients, respectively. Recent genome mapping has highlighted the presence of the microRNA miR-802 within the CRA and shown this region overlaps with the Down Syndrome Critical Region at 21q22.3 encompassing the genes DSCR1, DSCR3 and DYRK1. Detailed analysis of chromosome 21 identified recurrent breakpoints centromeric of the CRD within three genes: PDE9A (n=2), COL6A2 (n=2) and DSCAM (n=2). The role of these genes in the initiation of the chromosomal instability associated with iAMP21 is currently under investigation. Global analysis of the iAMP21 genome identified an average of 13 copy number alterations (CNA) per case with recurrent deletions of the leukaemia associated genes PAX5 (n=2), IKZF1 (n=4) and CDKN2A (n=4). To determine the incidences of PAX5, IKZF1 and CDKN2A deletions among the cohort of 94 iAMP21 patients, FISH using probes targeting these genes was carried out on availabel samples. IKZF1 (exons 3-6) was deleted at the highest incidence of 21% (13/62), while CDKN2A and PAX5 were deleted at a lower incidence of 17% (12/71) and 8% (4/48), respectively. The high incidence of IKZF1 deletions found in this patient group correlates with their association with high risk ALL. We have recently reported a deletion within the PAR1 region of the sex chromosomes, centromeric of the CRLF2 gene involving CSF2RA and IL3RA, which leads to deregulated expression of CRLF2. This deletion occurred at an incidence of 23% (17/73), as determined by FISH. Among pairs of matched diagnostic and relapse samples; one had this deletion at both diagnosis and relapse while the second had a deletion in the diagnostic sample only. Gain of an additional × chromosome was identified in 28% (18/65). Of note is that the 3 aberrations: abnormalities of chromosome 21, gain of the × chromosome and high incidence of deletions centromeric of CRLF2 in this patient group mirror the findings in Down Syndrome (DS) ALL, although we have not identified any mutations of the JAK2 kinase domain among this cohort of iAMP21 patients to date. This genome-wide study provides further support for the proposal that amplification of genes within the CRA on chromosome 21 is the primary event driving leukaemogenesis in iAMP21 patients. It has also highlighted that the 3 abnormalities in common with DS ALL and/or deletions of IKZF1, which all occur at a high incidence, contribute towards the progression of this high risk subtype of childhood BCP-ALL. Disclosures:No relevant conflicts of interest to declare.

Met amplification and tumor progression in Cdkn2a‐deficient melanocytes

While many genetic alterations have been identified in melanoma, the relevant molecular events that contribute to disease progression are poorly understood. Most primary human melanomas exhibit loss of expression of the CDKN2A locus in addition to activation of the canonical mitogen-activated protein kinase signaling pathway. In this study, we used a Cdkn2a-deficient mouse melanocyte cell line to screen for secondary genetic events in melanoma tumor progression. Upon investigation, intrachromosomal gene amplification of Met, a receptor tyrosine kinase implicated in melanoma progression, was identified in Cdkn2a-deficient tumors. RNA interference targeting Met in these tumor cells resulted in a significant delay in tumor growth in vivo compared with the control cells. MET expression is rarely detected in primary human melanoma but is frequently observed in metastatic disease. This study validates a role for Met activation in melanoma tumor progression in the context of Cdkn2a deficiency.

Cytogenetics of paediatric and adolescent acute lymphoblastic leukaemia

Cytogenetics has determined the incidence and prognostic significance of chromosomal abnormalities in acute lymphoblastic leukaemia (ALL). The development of fluorescence in situ hybridization (FISH) and array technologies has led to the discovery of novel aberrations. Five 'hot topics' are presented in which cytogenetics and related techniques have been instrumental in understanding the role of genetics in leukaemogenesis: (i) genetic changes are integral to the biology of T-cell ALL; (ii) intrachromosomal amplification of chromosome 21 is a new recurrent abnormality in precursor-B ALL (BCP-ALL); (iii) the immunoglobulin heavy chain gene (IGH@) is significant in BCP-ALL; (iv) alterations in genes involved in B-cell development and cell cycle control contribute to the pathogenesis of BCP-ALL; (v) age-related cytogenetic profiles define ALL in children and adolescents as distinct biological entities. In this molecular era, cytogenetics continues to be integral to our understanding of the genetics of this disease.

Enhanced gene amplification in human cells knocked down for DNA-PKcs

Gene amplification, a key mechanism for oncogene activation and drug resistance in tumour cells, involves the generation and joining of DNA double-strand breaks. Amplified DNA can be carried either on intra-chromosomal arrays or on extra-chromosomal elements (double minutes). We previously showed that, in rodent cells deficient in DNA-PKcs, intra-chromosomal amplification is significantly enhanced. In the present work, we studied gene amplification in human HeLa cell lines in which the expression of the DNA-PKcs gene was constitutively inhibited by shRNAs. These cell lines showed an increased sensitivity to ionizing radiations, an enhanced frequency of chromosomal aberrations and an increased rate of occurrence of methotrexate resistant colonies compared to the control cell lines (6–18 times). The main mechanism of resistance to methotrexate was extra-chromosomal amplification of the dihydrofolate reductase gene. These results indicate that, in human cells, inhibition of DNA-PKcs gene expression favours gene amplification occurring via the production of double minutes. In addition, they show that cell lines constitutively expressing shRNAs are good model systems to study the role of specific functions in gene amplification.

Heterogeneous patterns of amplification of the NUP214-ABL1 fusion gene in T-cell acute lymphoblastic leukemia

Episomes with the NUP214-ABL1 fusion gene have been observed in 6% of T-ALL. In this multicentric study we collected 27 cases of NUP214-ABL1-positive T-ALL. Median age was 15 years with male predominance. Outcome was poor in 12 patients. An associated abnormality involving TLX1 or TLX3 was found in all investigated cases. Fluorescent in situ hybridization revealed a heterogeneous pattern of NUP214-ABL1 amplification. Multiple episomes carrying the fusion were detected in 24 patients. Episomes were observed in a significant number of nuclei in 18 cases, but in only 1-5% of nuclei in 6. In addition, intrachromosomal amplification (small hsr) was identified either as the only change or in association with episomes in four cases and two T-ALL cell lines (PEER and ALL-SIL). One case showed insertion of apparently non-amplified NUP214-ABL1 sequences at 14q12. The amplified sequences were analyzed using array-based CGH.These findings confirm that the NUP214-ABL1 gene requires amplification for oncogenicity; it is part of a multistep process of leukemogenesis; and it can be a late event present only in subpopulations. Data also provide in vivo evidence for a model of episome formation, amplification and optional reintegration into the genome. Implications for the use of kinase inhibitors are discussed.

Integration of genomic and gene expression data of childhood ALL without known aberrations identifies subgroups with specific genetic hallmarks

Pediatric acute lymphoblastic leukemia (ALL) comprises genetically distinct subtypes. However, 25% of cases still lack defined genetic hallmarks. To identify genomic aberrancies in childhood ALL patients nonclassifiable by conventional methods, we performed a single nucleotide polymorphisms (SNP) array-based genomic analysis of leukemic cells from 29 cases. The vast majority of cases analyzed (19/24, 79%) showed genomic abnormalities; at least one of them affected either genes involved in cell cycle regulation or in B-cell development. The most relevant abnormalities were CDKN2A/9p21 deletions (7/24, 29%), ETV6 (TEL)/12p13 deletions (3/24, 12%), and intrachromosomal amplifications of chromosome 21 (iAMP21) (3/24, 12%). To identify variation in expression of genes directly or indirectly affected by recurrent genomic alterations, we integrated genomic and gene expression data generated by microarray analyses of the same samples. SMAD1 emerged as a down-regulated gene in CDKN2A homozygous deleted cases compared with nondeleted. The JAG1 gene, encoding the Jagged 1 ligand of the Notch receptor, was among a list of differentially expressed (up-regulated) genes in ETV6-deleted cases. Our findings demonstrate that integration of genomic analysis and gene expression profiling can identify genetic lesions undetected by routine methods and potential novel pathways involved in B-progenitor ALL pathogenesis.

Minimal Residual Disease Values Discriminate Between Low and High Relapse Risk in Children With B-Cell Precursor Acute Lymphoblastic Leukemia and an Intrachromosomal Amplification of Chromosome 21: The Austrian and German Acute Lymphoblastic Leukemia Berlin-Frankfurt-Münster (ALL-BFM) Trials

We aimed to identify relapse predictors in children with a B-cell precursor acute lymphoblastic leukemia (ALL) and an intrachromosomal amplification of chromosome 21 (iAMP21), a novel genetic entity associated with poor outcome. We screened 1,625 patients who were enrolled onto the Austrian and German ALL-Berlin-Frankfurt-Münster (ALL-BFM) trials 86, 90, 95, and 2000 with ETV6/RUNX1-specific fluorescent in situ hybridization probes, and we identified 29 patient cases (2%) who had an iAMP21. Minimal residual disease (MRD) was quantified with clone-specific immunoglobulin and T-cell receptor gene rearrangements. Twenty-five patients were good responders to prednisone, and all achieved remission after induction therapy. Eleven patients experienced relapse, which included eight who experienced relapse after cessation of front-line therapy. Six-year event-free and overall survival rates were 37% +/- 14% and 66% +/- 11%, respectively. Results of MRD analysis were available in 24 (83%) of 29 patients: nine (37.5%) belonged to the low-risk, 14 (58.5%) to the intermediate-risk, and one (4%) to the high-risk group. MRD results were available in 8 of 11 patients who experienced a relapse. Seven occurred among the 14 intermediate-risk patients, and one occurred in the high-risk patient. The overall and early relapse rates in the BFM study were lower than that in a previous United Kingdom Medical Research Council/Childhood Leukemia Working Party study (38% v 61% and 27% v 47%, respectively), which might result from more intensive induction and early reintensification therapy in the ALL-BFM protocols. MRD values were the only reliable parameter to discriminate between a low and high risk of relapse (P = .02).

Intrachromosomal amplification of AML1 gene in childhood acute lymphoblastic leukemia

The introduction of routine molecular cytogenetic assays enabled us to reveal hitherto unknown genetic disorders of childhood acute leukemias. Of special interest is the recognition of those rare cytogenetic mutations of negative prognostic value, which are associated with well-known markers of good prognosis. In our present study we review a novel cytogenetic mutation typical for childhood B-cell ALL, the intrachromosomal amplification of chromosome 21, which requires high-risk therapy irrespective of other risk factors, and which is associated with a cryptic 12;21 translocation of good prognostic value.

Array‐CGH reveals hidden gene dose changes in children with acute lymphoblastic leukaemia and a normal or failed karyotype by G‐banding

A tiling path 33K BAC array was used to study 28 children with acute lymphoblastic leukaemia (ALL) who had normal or failed G-banded karyotypes. Twenty-two patients (79%) had a total of 135 copy number alterations (CNA) (69 gains and 66 losses); most of these patients showed CNA that were below the resolution of G-banding. Molecular cytogenetic and array comparative genomic hybridization results enabled the division of B-precursor ALL patients into five groups: high hyperdiploidy, intrachromosomal amplification of 21q, ETV6/RUNX1 rearrangement, others and no CNA. Apart from a shared deletion of 9p21.3, T-ALL patients had additional small CNA, with no region in common.

Cytogenetic-Specific Heterogeneity in the Kinetics of Minimal Residual Disease (MRD) Clearance in Childhood Acute Lymphoblastic Leukaemia (ALL).

Although both MRD and karyotype are powerful determinants of outcome in childhood ALL, few studies have examined the kinetics of MRD clearance by cytogenetic subgroup. ALL2003 is an ongoing UK trial open to all patients aged 1–19 years with ALL, except those with mature-B ALL. At diagnosis, patients are stratified by NCI criteria to receive a three drug (Regimen A - age 1–9 years and white cell count (WCC) <50×109/L) or four drug (Regimen B - age 10–18 years or WCC >50×109/L) induction. MRD is assessed at day 29 and week 11 of therapy using a standardised and quality controlled RQ-PCR of at least two patient specific immunoglobulin or T cell receptor rearrangements. MRD risk groups were defined as:1.MRD high risk (HR) MRD >10−4 at day 29;2.MRD low risk (LR) MRD negative or <10−4 at day 29 and negative at week 11;3.MRD indeterminate risk - all other patients.Cytogenetic analysis was performed on pre-treatment samples and six abnormalities were defined as high risk: t(9;22)(q34;q11), near-haploidy (23–29 chromosomes), low hypodiploidy (30–39 chromosomes), 11q23/MLL translocations, t(17;19)(q22;p13) and intrachromosomal amplification of chromosome 21 (iAMP21). Among the first 1000 patients entered into the trial, 979 (98%) patients were eligible for these analyses. A total of 920 (94%) had a successful cytogenetic result and 555 (57%) were assigned to MRD high or low risk groups. Of these 555 patients, 298 (54%) were classified as MRD-HR whereas 257 (45%) were classified as MRD-LR. Collectively, patients with high risk cytogenetics were more likely to be classified as MRD-HR [20/24 (83%) v 261/501 (52%), p=0.003]. Patients with ETV6-RUNX1 (TEL-AML1) fusion were less likely to be classified as MRD-HR [40/145 (28%) v 241/380 (63%), p<0.001]. In contrast, high hyperdiploid patients were more likely to be MRD-HR compared to all other patients [99/155 (64%) v 182/370 (49%), p=0.002]. However, this effect was driven by the low rate of MRD-HR among the ETV6-RUNX1 positive patients and excluding these patients from the analysis revealed that high hyperdiploid patients were as likely to be MRD-HR as other ETV6-RUNX1 negative patients [99/155 (64%) v 142/225 (63%), p=0.880]. There was no difference between high hyperdiploid patients with and without the triple trisomy of +4, +10 and +17 with respect to MRD status. T-ALL patients were also more likely to be classified as MRD-HR compared to B-cell precursor ALL patients [32/46 (70%) v 249/479 (52%), p=0.022]. In particular, 9/10 patients with t(5;14)/TLX3-BCL11B fusion and 6/6 patients with SIL-TAL1 fusion were classified as MRD-HR. In conclusion, we have clearly demonstrated that MRD status varies by cytogenetic subgroup with ETV6-RUNX1 patients having the fastest MRD clearance rate. Despite the good prognosis associated with high hyperdiploidy, these patients were as likely to be MRD-HR as other standard risk patients. Longer follow-up is required to determine the clinical significance of this finding.

Genome-Wide Profiling of High-Risk Pediatric Acute Lymphoblastic Leukemia (ALL): The ALL Pilot Project for the Therapeutically Applicable Research To Generate Effective Treatments (TARGET) Initiative.

Recent studies have identified a high frequency of recurrent acquired DNA copy number abnormalities in pediatric ALL, most commonly involving genes regulating lymphoid development (Nature 2007; 446:758). The majority of B-progenitor ALL cases in this study harbored recurring chromosomal abnormalities such as hyperdiploidy and recurring translocations, many of which are associated with favorable outcome. Data from large cohorts of poor risk B-ALL have not been reported. Here we report genomic profiling data from a cohort of 221 pediatric ALL cases treated on the Children's Oncology Group P9906 study from 2000–2003 with an augmented BFM regimen (N Engl J Med 1998; 338:1663). The study targeted poor prognosis ALL cases, using age/WBC criteria designed to identify a subset of patients with NCI high risk ALL that historically had a very poor outcome. Cases with favorable (trisomy 4 + 10; ETV6-RUNX1) or unfavorable (BCR-ABL1 or hypodiploid) genetic features were excluded. The cohort included 25 TCF3-PBX1 and 19 MLL-rearranged cases. Profiling of DNA copy number abnormalities was performed using Affymetrix 250k Sty and Nsp arrays, reference normalization, dChipSNP, and circular binary segmentation. Germline SNP array data was available for 210 cases. The most frequent abnormalities were deletions of CDKN2A in 101 cases (45.7%), the B-lineage transcription factors PAX5 (N=68, 30.5%) and IKZF1 (Ikaros, N=32, 14.5%), ETV6 (N=29, 13.1%), RB1 (N=25, 11.3%), BTG1 (N=23, 10.4%), the tumor suppressor candidate TSC22D1 (N=20, 9%), the microRNA cluster at 13q14 (N=19, 8.6%), and deletion (N=3) or amplification (N=20, 4 focal, 16 broad) of the retinoic acid pathway gene CCDC26. Other recurring copy number abnormalities with relevance to leukemogenesis included deletions of BTLA, EBF, IL3RA, ERG, TOX, RAG1/2, KRAS, NRAS, NR3C2 and the adenosine deaminase pathway genes ADAR and ADARB2. An unexpected finding was focal deletion involving DMD (dystrophin) at Xp21.1 in 15 (6.8%) cases. Copy number abnormalities were uncommon in MLL rearranged leukemias, suggesting that fewer secondary mutations are needed for leukemogenesis in this genetic subtype of ALL. Clustering of copy number data using non-negative matrix factorization identified 11 clusters of cases, including clusters driven by isochromosome 7q, deletions of 9p, gains of 1q, multiple whole chromosomal gains, and intrachromosomal amplification of chromosome 21 (iamp21). These findings confirm the high frequency of deletions involving genes regulating B cell development and cell cycle such as PAX5 and CDKN2A in both standard and poor risk B-ALL. Furthermore ETV6 (TEL) deletions are shown to be common in B-ALL cases lacking the ETV6-RUNX1 translocation. The identification of novel recurring abnormalities (IL3RA, KRAS, NRAS) emphasizes the importance of high resolution copy number analysis in leukemia. These data are being integrated with gene expression data to select genes for resequencing as part of the TARGET project.

The MDR1/ABCB1 regional amplification in large inverted repeats with asymmetric sequences and microhomologies at the junction sites

A multidrug-resistant lung cancer cell line PTX250, established by treatment with the anti-cancer drug paclitaxel, has been demonstrated to have an increased copy number in the 7q21.12 region including the MDR1/ABCB1 gene. The amplicon is 2.7 megabases in size, and the copy number increase is 11-fold compared with the parental cell line. Here, we examined the amplicon structure and determined nucleotide sequences at both junctions of the amplicon. Fluorescence in situ hybridization analysis using an MDR1 probe demonstrated a cluster of fluorescent signals at the chromosomal end, suggesting an intra-chromosomal amplification. DNA fragments of both junctions were cloned and sequenced. The distal junction was a head-to-head fusion with a 4-base pair (bp) overlap separated by an asymmetric sequence of 1,265 bp, and the proximal junction was a tail-to-tail fusion with a 2-bp overlap intervened by an asymmetric sequence of 2,203 bp. These results suggest that the amplicon has a large palindromic structure with an asymmetric sequence and has been amplified through the breakage-fusion-bridge cycle. Specific sequences, which might be related to the occurrence of double-strand-breakages, were found at or near the junctions of the amplicon — an inverted repeat in the distal junction and a highly AT-rich region near the proximal junction.

Bexarotene (LGD1069, Targretin), a Selective Retinoid X Receptor Agonist, Prevents and Reverses Gemcitabine Resistance in NSCLC Cells by Modulating Gene Amplification

Acquired drug resistance is a major obstacle in cancer therapy. As for many other drugs, this is also the case for gemcitabine, a nucleoside analogue with activity against non-small cell lung cancer (NSCLC). Here, we evaluate the ability of bexarotene to modulate the acquisition and maintenance of gemcitabine resistance in Calu3 NSCLC models. In the prevention model, Calu3 cells treated repeatedly with gemcitabine alone gradually developed resistance. However, with inclusion of bexarotene, the cells remained chemosensitive. RNA analysis showed a strong increase of rrm1 (ribonucleotide reductase M1) expression in the resistant cells (Calu3-GemR), a gene known to be involved in gemcitabine resistance. In addition, the expression of genes surrounding the chromosomal location of rrm1 was increased, suggesting that resistance was due to gene amplification at the chr11 p15.5 locus. Analysis of genomic DNA confirmed that the rrm1 gene copy number was increased over 10-fold. Correspondingly, fluorescence in situ hybridization analysis of metaphase chromosomes showed an intrachromosomal amplification of the rrm1 locus. In the therapeutic model, bexarotene gradually resensitized Calu3-GemR cells to gemcitabine, reaching parental drug sensitivity after 10 treatment cycles. This was associated with a loss in rrm1 amplification. Corresponding with the in vitro data, xenograft tumors generated from the resistant cells did not respond to gemcitabine but were growth inhibited when bexarotene was added to the cytotoxic agent. The data indicate that bexarotene can resensitize gemcitabine-resistant tumor cells by reversing gene amplification. This suggests that bexarotene may have clinical utility in cancers where drug resistance by gene amplification is a major obstacle to successful therapy.

Interconversion of intra‐ and extra‐chromosomal sites of gene amplification by modulation of gene expression and DNA methylation

We previously showed that plasmids containing a mammalian replication initiation region and a matrix attachment region were efficiently amplified to few thousand copies per cell, and that they formed extrachromosomal double minutes (DMs) or chromosomal homogeneously staining regions (HSRs). In these structures, the plasmid sequence was arranged as a tandem repeats, and we suggested a mechanism of plasmid amplification. Since amplification was very efficient, easy, and convenient, it might be adapted to a novel method for protein production. In the current study, we found that gene expression from the tandem plasmid repeat was suppressed. We identified several strategies to overcome this suppression, including: (1) use of higher concentrations of antibiotic during cell selection; (2) treatment of cells with agents that influence DNA methylation (5-azacytidine) or histone acetylation (butyrate); (3) co-amplification of an insulator sequence; and (4) co-amplification of sequences that encode a transcriptional activator. Expression from the plasmid repeat was always higher at DMs compared to HSRs. We found that continuous activation of a plasmid-encoded inducible promoter prevented the generation of long HSRs, and favored amplification at DMs. Consistent with this finding, there was a synergistic effect of transcriptional activation and inhibition of DNA methylation on the fragmentation of long HSRs and the generation of DMs and short HSRs. Our results indicate that both transcriptional activation and DNA methylation regulate the interconversion between extra- and intra-chromosomal gene amplification. These results have important implications for both protein production technology, and the generation of chromosomal abnormalities found in human cancer cells.

Intrachromosomal amplification of chromosome 21 (iAMP21) may arise from a breakage–fusion–bridge cycle

Intrachromosomal amplification of chromosome 21 (iAMP21), involving amplification of the RUNX1 gene and duplication of chromosome 21, dup(21q), defines a new cytogenetic subgroup in B-lineage acute lymphoblastic leukemia (ALL) with a poor prognosis. Characterization of this abnormality has become vital to ensure that the most accurate detection method is used. We have previously defined common regions of amplification and deletion of chromosome 21 in these patients, although the level and extent of amplification within the amplicon was highly variable. This study, using interphase fluorescence in situ hybridization (FISH) with chromosome 21 locus specific probes, substantiated these findings in a large series of patients and confirmed that the amplicon always included RUNX1. Thus, FISH with probes directed to the RUNX1 gene remains the most reliable detection method. Metaphase FISH, supported by G- and multiple color chromosomal banding (mBAND) revealed the patient specific morphology and genetic profile of the dup(21q) chromosomes, as well as the complexity of the intrachromosomal changes giving rise to them. These findings suggested that iAMP21 had arisen from a breakage-fusion-bridge cycle: a mechanism previously described in tumors, which we report for the first time in ALL.

Characterization of 14 NUP214-ABL1 Fusions in T-Cell Acute Lymphoblastic Leukaemia (T-ALL) Exhibits a Genomic Heterogeneity.

The recently described episomal amplification of the NUP214-ABL1 fusion in 6% of T-ALL leads to the expression of a constitutively activated chimeric tyrosine kinase sensitive to imatinib. We collected additional cases in order to better characterize this new entity with respect to genetic presentations and clinical course.We collected 14 new NUP214-ABL1 positive cases by FISH (LSI-BCR/ABL1 ES, Vysis and ABL1 break-apart home-made probes) or RT-PCR (fusion transcript) screening.FISH analysis detected episomal amplification of NUP214-ABL1 in 11 patients with a highly variable number of nuclei with amplification (<1% to 90%). Interestingly, one case showed a higher percentage of nuclei with amplification when using frozen non cultured cells rather than cultured fixed cells (10% vs 1%) suggesting loss of episomes during culture. FISH showed also intrachromosomal amplification of NUP214-ABL1 in two cases: one presented a HSR at the original 9q34 site without detectable episomes, the other associated HSR (probably on a chromosome 10), episomes (<1% of nuclei) and 9q34 chromosomal insertions including NUP214 and ABL3′ that encode the tyrosine kinase domain but not ABL5′, on variable chromosomes including 14p (33%). One NUP214-ABL1 RT-PCR positive case did not show any FISH aberration. Median age: 16 y (3–45) with a male predominance (10:4). There were no T-cell lymphoblastic lymphoma. Immunophenotype (EGIL): mature (n=2), cortical (n=6) or pre-T (n=4). Karyotype: structural chromosomal alterations in 8 patients (including 4 with 10q24/HOX11 rearrangements), only numerical chromosomal alterations in 4 (including 2 with + 8), normal in 1, failed in 1. All samples with available information (10/14) showed a HOX11 or HOX11L2 abnormality. Among the 13 cases with available outcome data, we observed 5 early relapses, including both patients with NUP214-ABL1 HSR, and 1 refractory ALL.These observationsemphasize the interest of combining both (quantitative) RT-PCR and FISH for the screening and characterisation of NUP214-ABL1 fusion and amplification,demonstrate the coexistence of different NUP214-ABL1 genomic presentations in one patient (episomal amplification, 9q34 insertions, HSR) compatible with the model in which genomic amplification start with episome formation in order to create the NUP214-ABL1 fusion followed by their amplification and optional secondary reintegration,confirm occurrence of NUP214-ABL1 in T-ALL with HOX11 and HOX11L2 involvement,raise the question of the rather worse prognosis for cases with intrachromosomal amplification as previously suggested,raise the signification of minor NUP214-ABL1 clones and variable genomic presentations in the leukemogenesis of this subgroup of T-ALL that could potentially benefit from imatimib.° on behalf of the GFCH (Groupe Francophone de Cytogénétique Hématologique) and the BCGHO (Belgian Cytogenetic Group for Hematology and Oncology).

Identification of New Genomic Lesions in Childhood ALL without Known Genetic Aberrations: A Microarray Study of Gene Expression and Genotype Data.

Despite the risk stratification based on the prognostic relevant translocations, approximately 20–25% of childhood ALL patients cannot be classified according to genetic hallmarks. Recent studies based on single nucleotide polymorphism (SNP) analyses showed that Loss of Heterozygosity (LOH), with or without loss of genetic material, is frequently related to childhood ALL and AML. Moreover, there is an increasing interest in integrating the gene expression profiles with chromosomal localizations to identify new genetic subgroups. We applied an integrated approach composed of DNA index estimation, PCR (and/or RT-PCR) and cytogenetics, to exclude patients with known molecular and cytogenetics aberrations. Aim of the study was to identify criptic abnormalities in childhood ALL patients by performing an integrative analysis of gene expression and copy number changes (CNC) data. The 30 patients included in our study met the following inclusion criteria: a) B-cell precursor childhood ALL b) DNA index 1; c) negativity at t(4;11), t(12;21), t(9;22), t(1;19) RT-PCR screening; d) cytogenetics revealing normal karyotype or not technically feasible. Patients genomic DNA has been analyzed by the Affymetrix GeneChip Mapping 100K SNP arrays to identify genomic regions of LOH and CNC. In parallel, biotin-labeled cRNA has been synthesized and hybridized on HG-U133Plus2 Probe Arrays in accordance with the MILE (mircroarray innovation in leukemia) protocols; all samples were part of the B-ALL class without known genetic aberrations.The presence of del(9)(p21) was found in 9/30 (30%) patients, with an homozygous commonly deleted region involving CDKN2A and CDKN2B genes in 7/30. Hemizygous losses on 9p13 were found in two cases. Four patients (13,3%) showed the hemizygous deletion of chromosome 12p13.2, with a commonly deleted region involving the ETV6 gene. In one ETV6 deleted patient FISH analysis identified a translocation involving the first ETV6 exon and an unknown partner gene. Four patients, including three ETV6 deleted, showed the presence of the intrachromosomal amplification of chromosome 21 (iAMP21) with a common region of amplification between 31.3–43.5 Mb and a common region of deletion in 2/4 patients between 43.5–47 Mb. Four patients showed large regions of LOH not associated with CNC. Three patients did not show CNC and random microdeletions were found in single cases. Remission samples were analyzed for 5 patients carrying different aberrations, showing small regions of LOH in both the diagnosis and remission samples in 4 cases. No CNC was found in the remission samples. We found a statistically significant reduction for the expression of the CDKN2A transcript located within the (9)(p21) region. We are applying a locally adaptive statistical procedure to identify genes whose expression signals are specifically modulated by CNC. The integration of genomic variation and gene expression profiling might may be useful to identify new hidden genetic lesions, and to learn how critical regulators of tumor are linked to genomic alterations in cancer cells.

Genome Complexity in Patients with Acute Lymphoblastic Leukaemia Revealed by Array-Based Comparative Genomic Hybridization.

Chromosomal abnormalities are important for the classification and risk stratification in acute lymphoblastic leukaemia (ALL). However, approximately 30% of childhood and 50% of adults lack abnormalities of clinical relevance. Here, we describe the use of array-based comparative genomic hybridization (aCGH) to determine the genetic profile of these patients (n=96) and examine the secondary changes in those with established abnormalities (n=12). DNA from diagnostic bone marrow was obtained from 108 patients with ALL (88 children and 20 adults). Analysis was performed with either a 1Mb BAC- (n=58) or a 185/244K oligonucleotide-array (n=50). DNA copy number alterations (CNA) were validated with FISH. CNA were common, with a combination of simple and complex patterns of imbalance involving chromosomes 6, 9, 12, 15 and 21. Results were concordant with conventional cytogenetics (CC) and FISH in the majority of cases. Deletions of 6q (n=17) were heterogeneous, ranging from 34.5 to 91.1Mb in size. They shared a common deleted region (CRD) of 1.8Mb (104.2–106Mb), which concurs with previous data and demonstrates the sensitivity of aCGH in measuring CNA in ALL. Six patients with 6q loss harbored a complex series of gains and losses, which in five cases corresponded to the presence of a ring chromosome by CC. CNA involving chromosome 9 were deletions of the short (p) arm (n=36), ranging from 0.7 to 36.7Mb in size. All 9p deletions encompassed the CDKN2A locus, and showed considerable heterogeneity in both the extent and location of the deletion. Hence, the association with prognosis may be dependent on the size of the deletion and the loss of other genes in co-operation with CDKN2A. Twelve patients showed loss of 12p, ranging from 0.7 to 20.9Mb in size, all with a CRD between 10.9 and 11.6Mb, encompassing amongst others, the ETV6 gene. In approximately half the patients, the 12p loss accompanied the ETV6-RUNX1 fusion. CNA involving chromosome 21 occurred in patients with intrachromosomal amplification of chromosome 21 (iAMP21, n=27). Each patient harbored at least one CNA, while several patients had complex patterns of four or more distinct regions of gain or loss. This study corroborated our earlier findings (Strefford et al 2006, PNAS 103, 8167–8172) in a larger series and confirmed the variable nature of this abnormality. Five patients showed CNA of chromosome 21 in the absence of iAMP21, suggesting that a genomic rather than a cytogenetic definition may be more appropriate. Chromosome 15 CNA (n=6) were complex, involving several regions, but all with a 6.4Mb CRD. Other recurrent findings included gain of an 11.2Mb region on 17q (n=9) and deletions of 13q between 48.8 and 56.7Mb (n=6). Compared to CC, aCGH showed a three- and four-fold increase in CNA detection per patient (for BAC- and oligo-array, respectively). This is the first report of genome wide detection of CNA in ALL patients using aCGH. It has provided further insight into the size, genomic position, gene content and high-resolution characterization of the breakpoints producing these abnormalities, demonstrating that these changes are often more complex than anticipated. Such precise descriptions of the genetic alterations in ALL may lead to improved molecular diagnosis and prediction of outcome. The sub-microscopic alterations described here may facilitate the identification of novel molecular targets implicated in the pathogenesis of ALL.

Complex genomic alterations and gene expression in acute lymphoblastic leukemia with intrachromosomal amplification of chromosome 21

We have previously identified a unique subtype of acute lymphoblastic leukemia (ALL) associated with a poor outcome and characterized by intrachromosomal amplification of chromosome 21 including the RUNX1 gene (iAMP21). In this study, array-based comparative genomic hybridization (aCGH) (n = 10) detected a common region of amplification (CRA) between 33.192 and 39.796 Mb and a common region of deletion (CRD) between 43.7 and 47 Mb in 100% and 70% of iAMP21 patients, respectively. High-resolution genotypic analysis (n = 3) identified allelic imbalances in the CRA. Supervised gene expression analysis showed a distinct signature for eight patients with iAMP21, with 10% of overexpressed genes located within the CRA. The mean expression of these genes was significantly higher in iAMP21 when compared to other ALL samples (n = 45). Although genomic copy number correlated with overall gene expression levels within areas of loss or gain, there was considerable individual variation. A unique subset of differentially expressed genes, outside the CRA and CRD, were identified when gene expression signatures of iAMP21 were compared to ALL samples with ETV6-RUNX1 fusion (n = 21) or high hyperdiploidy with additional chromosomes 21 (n = 23). From this analysis, LGMN was shown to be overexpressed in patients with iAMP21 (P = 0.0012). Genomic and expression data has further characterized this ALL subtype, demonstrating high levels of 21q instability in these patients leading to proposals for mechanisms underlying this clinical phenotype and plausible alternative treatments.

A Breakage-Fusion-Bridge Cycle Generated by Telomeric Loss May Underlie the dup(21q) of Acute Lymphoblastic Leukemia.

A highly complex, rearranged chromosome 21, arising from duplication of 21q and associated with amplification of RUNX1 [dup(21q)], is linked to a poor prognosis in acute lymphoblastic leukemia (ALL). Using BAC array-based CGH (Spectral Genomics 1Mb, USA) (aCGH), we originally identified a characteristic pattern of imbalance, with common regions of amplification (CRA) and deletion (CRD) along 21q, in a series of 10 patients. The extent of these regions was refined to 6.6Mb (between 33.192 and 39.796Mb) and 3.3Mb (between 43.7 and 47Mb, which included sub-telomeric sequences), respectively, by tiling-path oligonucleotide-based aCGH (NimbleGen Inc., USA) (n=15). Six BAC clones (including a sub-telomeric one) from the 1Mb arrays, corresponding to the variable regions of amplification along 21q were used as probes for interphase FISH (iFISH) in 48 patients. The same CRA was confirmed in all, while the CRD was observed in 34/46 (77%) of them. iFISH showed that the degree of amplification corresponding to each chromosomal region differed between patients. Although these techniques, which measure genomic copy number changes, showed consistent patterns of amplification and deletion along 21q, it was intriguing as to why, at the cytogenetic level, the abnormal chromosome 21 had many different forms. The same locus-specific probes, applied to metaphases from 10 patients, revealed complex signal patterns, unique to each patient, in which the signals from each probe were distributed along 21q in unexpected positions relative to each other. Often signals from the same probe were located to more than one region of the abnormal chromosome 21. These findings were indicative of duplications of the chromosomal regions to which the probes were located, in combination with intricate intrachromosomal rearrangements, including inversions. We were able to illustrate these unique rearrangements by multicolor FISH banding (XCyte21, Metasystems, Germany) in two cases. Supervised gene expression analysis (HG-U133A arrays, Affymetrix, USA) showed a distinct signature for eight patients with dup(21q). Genomic copy number correlated with overall gene expression levels within areas of gain or loss. However, there was considerable inter-genic differences and variation between individuals. Of the 40 genes contained within the amplicon, no promising targets were upregulated when compared to patients with high hyperdiploidy, comprising at least one additional copy of chromosome 21. Collectively, our results have provided evidence for extensive intrachromosomal rearrangements and instabilityn of 21q in these patients. Although there was no evidence of other established chromosomal changes, the clonal heterogeneity we observed in the karyotypes was indicative of genome-wide chromosomal instability. These types of genomic alterations, arising from a series of chromatid breaks and reunions, which lead to intrachromosomal amplifications and deletions, are the hallmark of the breakage-fusion-bridge (BFB) cycle in solid tumors. This is the first time that cytogenetic features linked to the BFB mechanism have been described in ALL. We hypothesize that the loss or abnormal functioning of telomeric sequences may be the causal event behind this poor-risk 21q abnormality.