Background. The Ph chromosome is the most frequent cytogenetic aberration associated with adult ALL and it represents the single most significant adverse prognostic marker. The constitutively active tyrosine kinase encoded by BCR-ABL blocks the B-cell differentiation, prevents apoptosis and also causes genetic instability. Recently, the introduction of array-based analysis of single nucleotide polymorphism (SNP) has allowed the rapid determination of genome-wide allelic information at high density for a DNA sample. Aims. To identify, at submicroscopic level, genetic lesions which can escape standard cytogenetic observations and may provide new insights into the alternative process underlining leukemogenesis and resistance in Ph+ ALL. Methods.We profiled, until now, the genomes of 36 out of 55 Ph+ ALL patients. The median age was 55 years (range, 18-76) and the median blast percentage was 90% (range, 76-100%). 250 ng of genomic DNA were processed on 500K SNP array according to protocols provided by the manufacturer (Affymetrix Inc., USA). Copy number state was calculated with respect to a set of 48 Hapmap normal individuals and a set of samples obtained from acute leukaemia cases in remission using Partek® Genomics Suite. Fluorescence in situ hybridization (FISH), real-time quantitative PCR and western-blot analyses were performed to validate our results. Results. We identified region of high level amplification and homozygous deletion in all patients, with deletions outnumbering amplification almost 3:1. Copy number alterations most frequently involved chromosome 7 and 8. Lesions varied from loss or gain of complete chromosome arms (trisomy 4, monosomy 7, loss of 9p, 10q, 14q, 16q and gain of 1q and 17q) to microdeletions and microduplications targeting genomic intervals. Sub-microscopic lesions encompassing single or only few genes were observed with relative high frequency. Many of these copy number alterations were associated with cellular proliferation and/or apoptosis [e.g. CDKN2A and CDKN2B (n=8), GADD45A (n=2), FAS (n=2), BTG1(n=2)]. We also frequently observed anomalies in genes involved in early B-cell differentiation. These include the paired box gene PAX5 (9p13, n=5) which is a critical determinant of B-lineage commitment; the B-cell adapter containing a SH2 domain protein BLNK (10q23.2-q23.33, n=1); the pre-B lymphocyte gene VPREB1 (22q11.22, n=8) and the transcription factor IKZF1 (7p13-p11.1, n=9), which is required for normal lymphoid development. Reverse transcriptase-PCR and western-blot analysis for IKZF1 showed that in some cases this deletion was associated with the expression of the dominant-negative isoform Ik6 with cytoplasmatic localization. Other recurring copy number abnormalities with relevance to leukemogenesis included deletions of BTLA, TOX, MDS, PBX1, RUNX1, ETV1 and PTEN. It is noteworthy that some lesions felt in regions lacking annotated genes. Conclusions. High-resolution SNP-based genomic profiling is a reliable and valid method for the identification of genomic anomalies and genes relevant for the development of leukaemia. Multiple independent copy number anomalies were frequently observed in genes involved in cell cycle regulation and B-cell differentiation, suggesting that these processes could contribute to the poor prognosis of Ph+ ALL. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Fondazione del Monte di Bologna e Ravenna, PIO project 2007, Strategico di Ateneo.

I. Iacobucci, E. Ottaviani, A. Astolfi, T. Storlazzi, N. Testoni, I. Luciana, et al. (2008). HIGH-RESOLUTION GENOMIC PROFILING OF PH-POSITIVE ACUTE LYMPHOBLASTIC LEUKAEMIA (ALL) IDENTIFIED RECURRENT COPY NUMBER ANOMALIES IN GENES REGULATING THE CELL CYCLE AND THE B-CELL DIFFERENTIATION.

HIGH-RESOLUTION GENOMIC PROFILING OF PH-POSITIVE ACUTE LYMPHOBLASTIC LEUKAEMIA (ALL) IDENTIFIED RECURRENT COPY NUMBER ANOMALIES IN GENES REGULATING THE CELL CYCLE AND THE B-CELL DIFFERENTIATION

IACOBUCCI, ILARIA;ASTOLFI, ANNALISA;LONETTI, ANNALISA;SOVERINI, SIMONA;PAOLINI, STEFANIA;PICCALUGA, PIER PAOLO;PAPAYANNIDIS, CRISTINA;MARTINELLI, GIOVANNI
2008

Abstract

Background. The Ph chromosome is the most frequent cytogenetic aberration associated with adult ALL and it represents the single most significant adverse prognostic marker. The constitutively active tyrosine kinase encoded by BCR-ABL blocks the B-cell differentiation, prevents apoptosis and also causes genetic instability. Recently, the introduction of array-based analysis of single nucleotide polymorphism (SNP) has allowed the rapid determination of genome-wide allelic information at high density for a DNA sample. Aims. To identify, at submicroscopic level, genetic lesions which can escape standard cytogenetic observations and may provide new insights into the alternative process underlining leukemogenesis and resistance in Ph+ ALL. Methods.We profiled, until now, the genomes of 36 out of 55 Ph+ ALL patients. The median age was 55 years (range, 18-76) and the median blast percentage was 90% (range, 76-100%). 250 ng of genomic DNA were processed on 500K SNP array according to protocols provided by the manufacturer (Affymetrix Inc., USA). Copy number state was calculated with respect to a set of 48 Hapmap normal individuals and a set of samples obtained from acute leukaemia cases in remission using Partek® Genomics Suite. Fluorescence in situ hybridization (FISH), real-time quantitative PCR and western-blot analyses were performed to validate our results. Results. We identified region of high level amplification and homozygous deletion in all patients, with deletions outnumbering amplification almost 3:1. Copy number alterations most frequently involved chromosome 7 and 8. Lesions varied from loss or gain of complete chromosome arms (trisomy 4, monosomy 7, loss of 9p, 10q, 14q, 16q and gain of 1q and 17q) to microdeletions and microduplications targeting genomic intervals. Sub-microscopic lesions encompassing single or only few genes were observed with relative high frequency. Many of these copy number alterations were associated with cellular proliferation and/or apoptosis [e.g. CDKN2A and CDKN2B (n=8), GADD45A (n=2), FAS (n=2), BTG1(n=2)]. We also frequently observed anomalies in genes involved in early B-cell differentiation. These include the paired box gene PAX5 (9p13, n=5) which is a critical determinant of B-lineage commitment; the B-cell adapter containing a SH2 domain protein BLNK (10q23.2-q23.33, n=1); the pre-B lymphocyte gene VPREB1 (22q11.22, n=8) and the transcription factor IKZF1 (7p13-p11.1, n=9), which is required for normal lymphoid development. Reverse transcriptase-PCR and western-blot analysis for IKZF1 showed that in some cases this deletion was associated with the expression of the dominant-negative isoform Ik6 with cytoplasmatic localization. Other recurring copy number abnormalities with relevance to leukemogenesis included deletions of BTLA, TOX, MDS, PBX1, RUNX1, ETV1 and PTEN. It is noteworthy that some lesions felt in regions lacking annotated genes. Conclusions. High-resolution SNP-based genomic profiling is a reliable and valid method for the identification of genomic anomalies and genes relevant for the development of leukaemia. Multiple independent copy number anomalies were frequently observed in genes involved in cell cycle regulation and B-cell differentiation, suggesting that these processes could contribute to the poor prognosis of Ph+ ALL. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Fondazione del Monte di Bologna e Ravenna, PIO project 2007, Strategico di Ateneo.
2008
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I. Iacobucci, E. Ottaviani, A. Astolfi, T. Storlazzi, N. Testoni, I. Luciana, et al. (2008). HIGH-RESOLUTION GENOMIC PROFILING OF PH-POSITIVE ACUTE LYMPHOBLASTIC LEUKAEMIA (ALL) IDENTIFIED RECURRENT COPY NUMBER ANOMALIES IN GENES REGULATING THE CELL CYCLE AND THE B-CELL DIFFERENTIATION.
I. Iacobucci; E. Ottaviani; A. Astolfi; T. Storlazzi; N. Testoni; I. Luciana; A. Lonetti; S. Soverini; S. Paolini; P.P. Piccaluga; C. Papayannidis; P....espandi
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/154572
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