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  • 1
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    IKZF1 (IKAROS) Deletions Are Independent On BCR-ABL1 Rearrangement and Are Associated with a Peculiar Gene Expression Signature and Poor Prognosis in Adult B-Progenitor Acute Lymphoblastic Leukemia (ALL) Patients.
    American Society of Hematology ; 2009
    In:  Blood Vol. 114, No. 22 ( 2009-11-20), p. 912-912
    Details
    In: Blood, American Society of Hematology, Vol. 114, No. 22 ( 2009-11-20), p. 912-912
    Abstract: Abstract 912 Background: Recent genome-wide analyses in B-precursor acute lymphoblastic leukemia (ALL) demonstrated that deletions of IKZF1, which encodes the transcription factor Ikaros, play an important role in the pathogenesis of BCR-ABL1-positive and BCR-ABL1-like acute leukemias. IKZF1 deletions have been associated with poor outcome in children with ALL but a full understanding of their biological implications and clinical significance has not yet been defined in adult patients. Purpose and Methods: In order to address this issue and to evaluate whether the cases harbouring IKZF1 alterations display a peculiar gene expression profile, a cohort of 144 adult de novo ALL patients (106 BCR-ABL1-postive and 38 B-progenitor ALL negative for known molecular rearrangements) were analyzed with the use of single-nucleotide–polymorphism (SNP) microarrays (Affymetrix 250K NspI and SNP 6.0), FISH for IKZF1 deletions and gene expression profiling (HGU133 Plus 2.0 gene chips, Affymetrix). Patients had a median age of 49 years (range 18-78) and were enrolled into institutional (n = 17) or GIMEMA AL Working Party (n = 121) clinical trials. Results: Deletions of IKZF1 were identified in 75% adult BCR-ABL1-positive and in 58% BCR-ABL1-negative ALL cases, suggesting that IKZF1 deletion is more frequent in the BCR-ABL1-positive ALL subtype (p= 0.04). FISH analysis using a pool of fosmid probes for IKZF1 and genomic quantitative PCR confirmed SNP results. Among 144 patients, the entire IKZF1 locus was deleted in 18 (13%) whereas in 84 (58%) patients only a subgroup of exons or the genomic region immediately upstream of IKZF1 was deleted. In particular, in 46 patients (32%) there was a deletion of the coding exons 4 through 7, which resulted in the expression of a dominant-negative isoform, Ik6, lacking the DNA binding domain. In 24 cases (17%) we identified the loss of exons 2 through 7, producing an Ikaros isoform lacking the translation start site. Using gene-set enrichment analysis to compare the gene-expression data from patients with IKZF1 deletion versus wild-type patients, we identified a peculiar signature irrespective of BCR-ABL1 rearrangement but dependent on IKZF1 genomic status. Indeed, it was characterized by the presence of two subgroups of genes, the expression of which was deregulated in a reciprocal fashion. One subgroup was enriched with up-regulated genes involved in cell-cycle progression (STK17B, SERPINB9, CDKN1A), activation of signalling via JAK-STAT pathway (CISH, SOCS1, SOCS3, STAT3) and DNA damage (GADD45A, GADD45B, NFKBIA, the protoncogene REL). The second subgroup contained down-regulated genes, which are normally expressed during lymphocyte differentiation (e.g. VPREB1, VPREB3, IGLL3, BLK) or are involved in DNA damage repair (MSH2, MSH6) supporting the hypothesis that B-ALL cells with IKZF1 deletions are prone to a block of B-cell differentiation and accumulation of DNA damage events. To investigate whether Ikaros transcription factor is directly involved in the regulation of putative target genes identified in gene expression analysis, cross-linking chromatin immunoprecipitation (ChIP) assay was performed in cell lines and primary ALL cells. We found that the promoters of IGGL1, CD79A, BLK, EBF1, BLC2, MSH2, BUB3, ETV6, YES1, CDKN1A (p21) and CDKN2C (p18) genes, were bound in vivo only by Ikaros full-length protein, but not by Ik6 mutant. These data strongly support a model in which Ikaros deleted isoforms loose the ability to regulate a large set of genes, many of which may play crucial roles in B-ALL development. We next investigated whether the IKZF1 deletions associated with a poor outcome in ALL patients. Univariate analysis showed that the IKZF1 deletion negatively influenced the cumulative incidence of relapse (p=0.02) and disease-free survival (p=0.04, Wilcoxon test) as confirmed by multivariate analysis. Conclusion: In conclusion, our findings shed light on a new subgroup of adult ALL including BCR-ABL1 positive and BCR-ABL1 negative patients and characterized by a unique signature dependent on Ikaros genomic status. Loss of normal Ikaros activity results in the activation of JAK-STAT pathway, DNA repair gene down-regulation and a block of B-cell differentiation. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Strategico di Ateneo, GIMEMA Onlus. Disclosures: No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V114.22.912.912
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2009
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
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  • 2
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    Whole Transcriptome Sequencing of a Philadelphia-Positive Acute Lymphoblastic Leukemia (ALL) with “Next Generation Sequencing” Technology Revealed Novel Point Mutations Associated with Disease-Progression.
    American Society of Hematology ; 2009
    In:  Blood Vol. 114, No. 22 ( 2009-11-20), p. 3074-3074
    Details
    In: Blood, American Society of Hematology, Vol. 114, No. 22 ( 2009-11-20), p. 3074-3074
    Abstract: Abstract 3074 Poster Board III-11 Background BCR-ABL1-positve Acute Lymphoblastic Leukemia (ALL) is the most common ALL subtype in adults and is associated with poor prognosis. The pathogenesis of this leukemia is related to the expression of the BCR-ABL1 fusion transcript, but additional recurrent genetic lesions are suspected to be involved in its development and progression. Aim A Next-Generation Sequencing Technology was used to sequence the whole transcriptome of leukemia cells from a BCR-ABL1-positive ALL patient at diagnosis and at relapse following tyrosine kinase inhibitor (TKI) therapy with the aim to detect acquired mutations cooperating with BCR-ABL1 in leukemia manifestation and drug-resistance. Methods Poly(A) RNA was extracted from leukemia cells and used to prepare double-stranded cDNA libraries for Illumina/Solexa Genome Analyzer. Obtained 36 base-pair (bp) sequence reads were mapped to the reference sequence of the human genome (UCSC hg18, NCBI build 36.1) to identify single nucleotide variants (SNVs) and to estimate reads density corresponding to RNA from each known exon, canonical splice event or new candidate gene. This approach allowed us to define a detailed Digital Gene Expression (DGE) profile. Reads that showed no match to the reference genome were subsequently mapped to a dataset of all possible splice junctions created by in silico pairwise combination of the exons of all annotated genes (UCSC knownGene file) to identify alternative splicing (AS) events. Results Whole Transcriptome Shotgun Sequencing (RNA-seq) analysis generated 13.9 and 15.8 million reads from de novo and relapsed ALL samples respectively, achieving approximately 90% diploid coverage and detecting transcripts from 62% and 64% of human annotated genes. The great majority of these active genes (78% at diagnosis and 73% at relapse) showed very low expression levels, with a number of reads per kilobase of exon model per million mapped reads (RPKM value) from 0.01 to 10, whereas 20% and 24% showed moderate expression levels (RPKM 10-100), as well as only 2% and 3% resulted highly expressed (RPKM 100-8000). Moreover, 6,390 and 4,671 AS events were also identified within 4,334 diagnosis and 3,651 relapse annotated transcripts, with the already described ALL-related Ik6 Ikaros isoform observed in both samples. Finally, 2,011 and 2,103 single nucleotide variants (SNVs) were found at diagnosis and relapse respectively, about 94% of which have been already reported in the dbSNP. Of greater interest as potential ALL-related mutations, 124 and 115 non annotated SNVs were also found at diagnosis and relapse, respectively. Of these, 43 affected both samples, while 81 and 72 resulted diagnosis and relapse private variants. In particular, the analysis was focused to the coding sequences of annotated genes, finding that non-synonymous changes were one out of the 19 shared between the two samples and affected a transmembrane receptor gene (PLXNB2). Six out of the 12 diagnosis private variants, affecting genes involved in metabolic process (PDE4DIP, EIF2S3, DPEP1, ZC3H12D, TMEM46) or transport (MVP) and 5 out of the 30 relapse private variants, affecting genes involved in cell cycle regulation (ABL1, CDC2L1), catalytic activity (CTSZ, CXorf21) or with unknown function (FAM116B). Most of these diagnosis and relapse non-synonymous private mutations resulted highly expressed, showing frequencies of mutated unique reads higher than 50%. According to this pattern, diagnosis private mutations may be carried by primary leukemic clones that did not develop again at relapse, whereas relapse private mutations have greater probability to be variants acquired during the disease progression. Interestingly, the T315I point mutation in the Abl kinase domain, that confers resistance to many TKIs, was found at relapse but not at diagnosis. Conclusions An accurate expression profile was obtained for the leukemia cells of the examined ALL patient, as well as the discovery of several new non-synonymous mutations affecting genes from different pathways and for which no correlation was previously found with ALL pathogenesis. These findings demonstrate that RNA-Seq represents a suitable and cost-efficient approach for identifying new genes potentially involved in ALL development and progression. Acknowledgments AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, Ateneo 60% grants, European LeukemiaNet. Disclosures No relevant conflicts of interest to declare.
    Type of Medium: Online Resource
    ISSN: 0006-4971 , 1528-0020
    URL: Article
    DOI: 10.1182/blood.V114.22.3074.3074
    RVK:
    XA 33000
    RVK:
    XA 10000
    Language: English
    Publisher: American Society of Hematology
    Publication Date: 2009
    detail.hit.zdb_id: 1468538-3
    detail.hit.zdb_id: 80069-7
    Library Location Call Number Volume/Issue/Year Availability
    BibTip Others were also interested in ...
    Online Resource
    Open Access Request
    Availability (electronic / print)
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