Introduction: Experimental evidence obtained in transgenic mice revealed that PML-RARA is necessary but not sufficient for the development of APL, suggesting that additional genetic mutations are also required for its development. Aim: To define whether additional submicroscopic genomic alterations may characterize APL and may be used to better classify the disease by dissection of genomic subsets. Patients and Methods: 105 adult patients with acute myeloid leukemia were analyzed. These cases included all French-American-British subtypes, miscellaneous cytogenetic abnormalities and normal karyotype subgroups. Among these, the M3 subtype included 28 patients, representing the 33% of the whole study population. Genomic DNA was isolated from blast cells and applied to Genome-Wide Human SNP 6.0 array (Affymetrix, Santa Clara, CA) following the manufacturer’s instructions. Fluorescence in situ hybridization, quantitative PCR and nucleotide sequencing were used to confirm genomic alterations. Results: A wide spectrum of different copy number alterations (CNAs) were identified in all cases and no significant difference in the average number of alterations was detected among different leukemia cytogenetic subgroups except for the complex subgroup, which had an average of 55 CNA/patient. In APL cases an average of 8 CNAs per case (range, 1-24) was found. The macroscopic alterations were rare, confirmed conventional cytogenetics and involved trisomy of chromosome 8 in 3 cases, loss of chromosome 6, loss of chromosome 20 and deletions on chromosome 9 and 7. Microscopic CNAs (< 1.5 Mbps) involved every chromosome at least once and predominantly chromosomes 1, 2, 9, 15 and 17. For each alteration we interrogated a collated library of copy-number variants (CNVs, Database of Genomics Variants and USCS Genome Browser) to assure that these regions were not known as CNVs and therefore to decrease the noise of raw copy number data. Genetic gains were more common than losses and their median size was 300 kb (range 0.2- 1.4 Mb). The majority of lesions were not recurrent, being identified in only a single patient. Focal genetic alterations were detected at the breakpoints of t(15;17)(q22;q21) in PML and RARA genes, in genes involved in activation of transcription (loss of LMX1 on 1q23.3, loss of MLXIPL and BCL7 on 7q11.23), regulation of cell cycle (gain of PVT1 and MYC on 8q24) and cell adhesion (gain of NCAM1 on 11q23). In order to identify potential pathogenetic alterations, all microscopic CNAs were compared with the list of genes from the Cancer genome project (http://www.sanger.ac.uk/genetics/CPG/Census) finding out that six alterations involved a known cancer-related gene. Most of these genes encode tyrosine kinase proteins (ERBB4) or transcription factors (ETV1, ETV6, ERG). Copy neutral loss of heterozygosity events affected 1p34.2-1p32.3, 10p11.2 (MLLT10), 11p11.2 (WT1, CDKN1C, HRAS). Finally, patients with more than 10 CNAs were found to be associated with a worse prognosis. Conclusions: These data demonstrate that different cooperating events may be involved in the generation of APL. Furthermore, these novel findings may be used to stratify patients according to genomic changes. Supported by: European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, Ateneo RFO grants.

HIGH-RESOLUTION MOLECULAR ALLELOKARYOTYPING IDENTIFIES NOVEL UNIPARENTAL DISOMY AND FOCAL COPY NUMBER ALTERATIONS IN ACUTE PROMYELOCYTIC LEUKEMIA (APL)

IACOBUCCI, ILARIA;LONETTI, ANNALISA;PAPAYANNIDIS, CRISTINA;PAOLINI, STEFANIA;A. Ferrari;M. Rondoni;MARTINELLI, GIOVANNI;
2010

Abstract

Introduction: Experimental evidence obtained in transgenic mice revealed that PML-RARA is necessary but not sufficient for the development of APL, suggesting that additional genetic mutations are also required for its development. Aim: To define whether additional submicroscopic genomic alterations may characterize APL and may be used to better classify the disease by dissection of genomic subsets. Patients and Methods: 105 adult patients with acute myeloid leukemia were analyzed. These cases included all French-American-British subtypes, miscellaneous cytogenetic abnormalities and normal karyotype subgroups. Among these, the M3 subtype included 28 patients, representing the 33% of the whole study population. Genomic DNA was isolated from blast cells and applied to Genome-Wide Human SNP 6.0 array (Affymetrix, Santa Clara, CA) following the manufacturer’s instructions. Fluorescence in situ hybridization, quantitative PCR and nucleotide sequencing were used to confirm genomic alterations. Results: A wide spectrum of different copy number alterations (CNAs) were identified in all cases and no significant difference in the average number of alterations was detected among different leukemia cytogenetic subgroups except for the complex subgroup, which had an average of 55 CNA/patient. In APL cases an average of 8 CNAs per case (range, 1-24) was found. The macroscopic alterations were rare, confirmed conventional cytogenetics and involved trisomy of chromosome 8 in 3 cases, loss of chromosome 6, loss of chromosome 20 and deletions on chromosome 9 and 7. Microscopic CNAs (< 1.5 Mbps) involved every chromosome at least once and predominantly chromosomes 1, 2, 9, 15 and 17. For each alteration we interrogated a collated library of copy-number variants (CNVs, Database of Genomics Variants and USCS Genome Browser) to assure that these regions were not known as CNVs and therefore to decrease the noise of raw copy number data. Genetic gains were more common than losses and their median size was 300 kb (range 0.2- 1.4 Mb). The majority of lesions were not recurrent, being identified in only a single patient. Focal genetic alterations were detected at the breakpoints of t(15;17)(q22;q21) in PML and RARA genes, in genes involved in activation of transcription (loss of LMX1 on 1q23.3, loss of MLXIPL and BCL7 on 7q11.23), regulation of cell cycle (gain of PVT1 and MYC on 8q24) and cell adhesion (gain of NCAM1 on 11q23). In order to identify potential pathogenetic alterations, all microscopic CNAs were compared with the list of genes from the Cancer genome project (http://www.sanger.ac.uk/genetics/CPG/Census) finding out that six alterations involved a known cancer-related gene. Most of these genes encode tyrosine kinase proteins (ERBB4) or transcription factors (ETV1, ETV6, ERG). Copy neutral loss of heterozygosity events affected 1p34.2-1p32.3, 10p11.2 (MLLT10), 11p11.2 (WT1, CDKN1C, HRAS). Finally, patients with more than 10 CNAs were found to be associated with a worse prognosis. Conclusions: These data demonstrate that different cooperating events may be involved in the generation of APL. Furthermore, these novel findings may be used to stratify patients according to genomic changes. Supported by: European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, Ateneo RFO grants.
2010
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15
16
I Iacobucci ;E Ottaviani ;V Guadaguolo ;A Lonetti ;N Testoni ;C Papayannidis ;S Paolini ;A Ferrari ;D Cilloni ;A Candoni ;G Saglio ;M Rondoni ;F Pane ;H Khizer ;M Baccarani ;G Martinelli ;F Lo Coco
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11585/154578
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