Project description:Distinct genetic abnormalities such as TP53 deletion at 17p13.1, have been identified as having an adverse prognostic relevance in B-cell chronic lymphocytic leukemia (B-CLL). Conventional cytogenetic studies have shown that TP53 deletion in B-CLL is associated predominantly with 17p loss resulting from complex chromosomal rearrangements. We performed genome-wide DNA (SNPs arrays), fluorescence in situ hybridization (FISH) and gene expression profiling (GEP) analyses to investigate the significance of 17p loss in a panel of 71 genetically well-characterized B-CLLs in Binet stage A, 18 of which carried a TP53 monoallelic deletion. Combined SNP arrays and FISH approaches showed 17p loss in all of the TP53-deleted cases, with breakpoints scattered along the 17p11.2 region. Mutations in exons 5 to 9 of TP53 were found in 9/12 deleted samples. GEP of 60 B-CLLs, including 7 patients with 17p loss, identified 40 differentially expressed genes in 17p- versus 17p normal samples, 35 of which were down-regulated in 17p- tumors. The majority (30/35) of these transcripts, including putative tumor suppressor genes, mapped to 17p. Overall, these data indicate that, beside TP53 deletion, the concomitant loss of 17p arm may contribute to the strong negative prognostic impact known to be associated with this lesion in B-CLL. Keywords: genomic analysis of B-CLL with 17p loss patients This series of microarray experiments contains the genome-wide profiles of purified B-cell chronic lymphocytic leukemia (B-CLL) cells obtained from 12 newly diagnosed patients (Binet stage A). Peripheral blood mononuclear cells from B-CLL patients were isolated by Ficoll-Hypaque density-gradient centrifugation and the proportion of CD5/CD19/CD23 triple positive B cells in the suspension was determined by direct immunofluorescence performed using a FACS-sort flow cytometer with antibodies to: CD19 FITC/PE, CD23 PE and CD5 Cy-Chrome. If B-CLL cells were less than 90%, T cells, NK cells and monocytes were removed by negative selection using CD3, CD56, CD16, and CD14 monoclonal antibody treatment followed by magnetic beads. 250 nanograms of genomic DNA was processed and, in accordance with the manufacturer's protocols, 40 micrograms of fragmented biotin-labelled DNA were hybridized on GeneChip Human Mapping 50K XbaI Arrays (Affymetrix Inc.). The arrays were scanned using the GeneChip Scanner 3000 7G. The images were acquired using Affymetrix GeneChipÂ® Operating Software (GCOS version 1.4). Copy number values for individual SNPs were extracted and converted from CEL files into signal intensities using GTYPE 4.1 and Affymetrix Copy Number Analysis Tool (CNAT 4.0.1) softwares. Genomic Smoothing analysis was performed by using the smoothing window of 1 Mb, and inferred copy number states were derived from a Hidden Markov Model (HMM) based algorithm implemented in CNAT 4.0.1.

Project description:Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease characterized by chromosomal aberrations of prognostic significance. Recent studies showed that gain of chromosome 2p is a recurrent lesion in CLL. We investigated the 2p gain and its relationship with prognostic biomarkers in a prospective series of 287 early-stage CLLs (Binet A). The 2p gain was detected by FISH in 17 patients (6%) and further characterized by single nucleotide polymorphism-array. Overall, unfavorable cytogenetic deletions, i.e. del(11)(q23) and del(17)(p13) (P=0.002) as well as unmutated (UM) status of IGHV (P<1M-CM-^W10-4) and CD38 (P<1M-CM-^W10-4) and ZAP-70 positive expression (P=0.003) were significantly more prevalent in 2p gain cases. Furthermore, 2p gained patients showed a significantly higher occurrence of stereotyped HCDR3 sequences compared to 2p normal CLLs (P=0.009). Among the stereotyped subsets, the incidence of subset #1 in 2p positive patients was significantly higher than that found in the remaining CLLs (P=0.031). Finally, gene expression profiling analysis identified a number of genes significantly upregulated in 2p gain CLLs. Among those located at 2p, NCOA1 and ROCK2 are known for their involvement in tumor progression in several human cancers, whereas among those located in different chromosomes, CAV1 at 7q31.1 has been recently identified to play a critical role in CLL progression. Our study indicates that 2p gain is a recurrent lesion in early CLL, correlated with the major biological and cytogenetic risk markers of the disease. Moreover, we provide insights to define novel candidate genes that may play additional pathogenetic roles in CLL. This series of microarray experiments contains the genome-wide profiles of purified B-cell chronic lymphocytic leukemia (B-CLL) cells obtained from 10 patients (Binet stage A) showing 2p gain alteration. Peripheral blood mononuclear cells from B-CLL patients were isolated by Ficoll-Hypaque density-gradient centrifugation and the proportion of CD5/CD19/CD23 triple positive B cells in the suspension was determined by direct immunofluorescence performed using a FACS-sort flow cytometer with antibodies to: CD19 FITC/PE, CD23 PE and CD5 Cy-Chrome. If B-CLL cells were less than 90%, T cells, NK cells and monocytes were removed by negative selection using CD3, CD56, CD16, and CD14 monoclonal antibody treatment followed by magnetic beads. 250 nanograms of genomic DNA was processed and, in accordance with the manufacturer's protocols, 90 micrograms of fragmented biotin-labeled DNA were hybridized on GeneChip Human Mapping 250K NspI Arrays (Affymetrix Inc.). The arrays were scanned using the GeneChip Scanner 3000 7G. The images were acquired using Affymetrix GeneChipM-BM-. Operating Software (GCOS version 1.4). Copy number values for individual SNPs were extracted and converted from CEL files into signal intensities using GTYPE 4.1 and Affymetrix Copy Number Analysis Tool (CNAT 4.0.1) softwares. The raw data for individual SNPs were extracted from CEL files and converted into signal intensities using GTYPE 4.1 and Affymetrix Copy Number Analysis Tool (CNAT 4.0.1) softwares using the Hidden Markov Model algorithm with a genomic smoothing window set to 0. After the pre-processing, piecewise constant estimates of the underlying local DNA copy number (CN) variation was calculated using the DNAcopy Bioconductor package, which looks for optimal breakpoints using circular binary segmentation (CBS).

Project description:Distinct genetic abnormalities such as TP53 deletion at 17p13.1, have been identified as having an adverse prognostic relevance in B-cell chronic lymphocytic leukemia (B-CLL). Conventional cytogenetic studies have shown that TP53 deletion in B-CLL is associated predominantly with 17p loss resulting from complex chromosomal rearrangements. We performed genome-wide DNA (SNPs arrays), fluorescence in situ hybridization (FISH) and gene expression profiling (GEP) analyses to investigate the significance of 17p loss in a panel of 71 genetically well-characterized B-CLLs in Binet stage A, 18 of which carried a TP53 monoallelic deletion. Combined SNP arrays and FISH approaches showed 17p loss in all of the TP53-deleted cases, with breakpoints scattered along the 17p11.2 region. Mutations in exons 5 to 9 of TP53 were found in 9/12 deleted samples. GEP of 60 B-CLLs, including 7 patients with 17p loss, identified 40 differentially expressed genes in 17p- versus 17p normal samples, 35 of which were down-regulated in 17p- tumors. The majority (30/35) of these transcripts, including putative tumor suppressor genes, mapped to 17p. Overall, these data indicate that, beside TP53 deletion, the concomitant loss of 17p arm may contribute to the strong negative prognostic impact known to be associated with this lesion in B-CLL. Keywords: transcriptional analysis of B-CLL with 17p loss patients This series of microarray experiments contains the gene expression profiles of purified B-cell chronic lymphocytic leukemia (B-CLL) cells obtained from 60 newly diagnosed patients (Binet stage A). Peripheral blood mononuclear cells from B-CLL patients were isolated by Ficoll-Hypaque density-gradient centrifugation and the proportion of CD5/CD19/CD23 triple positive B cells in the suspension was determined by direct immunofluorescence performed using a FACS-sort flow cytometer with antibodies to: CD19 FITC/PE, CD23 PE and CD5 Cy-Chrome. If B-CLL cells were less than 90%, T cells, NK cells and monocytes were removed by negative selection using CD3, CD56, CD16, and CD14 monoclonal antibody treatment followed by magnetic beads. 3 micrograms of total RNA was processed and, in accordance with the manufacturer's protocols, 15 micrograms of fragmented biotin-labelled cRNA were hybridized on GeneChip Human Genome U133A Arrays (Affymetrix Inc.). The arrays were scanned using the GeneChip Scanner 3000 7G. The images were acquired using Affymetrix GeneChipÂ® Operating Software (GCOS version 1.4) and the probe level data converted to expression values using the Bioconductor function for the Robust Multi-Array average (RMA) procedure (Irizarry et al, 2003), in which perfect match intensities are background adjusted and quantile-quantile normalised.

Project description:Chronic lymphocytic leukemia (CLL) is a clinically heterogeneous disease characterized by chromosomal aberrations of prognostic significance. Recent studies showed that gain of chromosome 2p is a recurrent lesion in CLL. We investigated the 2p gain and its relationship with prognostic biomarkers in a prospective series of 287 early-stage CLLs (Binet A). The 2p gain was detected by FISH in 17 patients (6%) and further characterized by single nucleotide polymorphism-array. Overall, unfavorable cytogenetic deletions, i.e. del(11)(q23) and del(17)(p13) (P=0.002) as well as unmutated (UM) status of IGHV (P<1M-CM-^W10-4) and CD38 (P<1M-CM-^W10-4) and ZAP-70 positive expression (P=0.003) were significantly more prevalent in 2p gain cases. Furthermore, 2p gained patients showed a significantly higher occurrence of stereotyped HCDR3 sequences compared to 2p normal CLLs (P=0.009). Among the stereotyped subsets, the incidence of subset #1 in 2p positive patients was significantly higher than that found in the remaining CLLs (P=0.031). Finally, gene expression profiling analysis identified a number of genes significantly upregulated in 2p gain CLLs. Among those located at 2p, NCOA1 and ROCK2 are known for their involvement in tumor progression in several human cancers, whereas among those located in different chromosomes, CAV1 at 7q31.1 has been recently identified to play a critical role in CLL progression. Our study indicates that 2p gain is a recurrent lesion in early CLL, correlated with the major biological and cytogenetic risk markers of the disease. Moreover, we provide insights to define novel candidate genes that may play additional pathogenetic roles in CLL. This series of microarray experiments contains the gene expression profiles of purified B-cell chronic lymphocytic leukemia (B-CLL) cells obtained from 136 patients (Binet stage A) showing 2p gain alteration. Peripheral blood mononuclear cells from B-CLL patients were isolated by Ficoll-Hypaque density-gradient centrifugation and the proportion of CD5/CD19/CD23 triple positive B cells in the suspension was determined by direct immunofluorescence performed using a FACS-sort flow cytometer with antibodies to: CD19 FITC/PE, CD23 PE and CD5 Cy-Chrome. If B-CLL cells were less than 90%, T cells, NK cells and monocytes were removed by negative selection using CD3, CD56, CD16, and CD14 monoclonal antibody treatment followed by magnetic beads. 5.5 micrograms of single-stranded DNA target obtained from 100 ng of purified total RNA was fragmented and then labeled using the WT Terminal Labeling Kit according to the standard Affymetrix protocol (GeneChipM-BM-. Whole Transcript (WT) Sense Target Labeling Assay Manual). The fragmented labeled single-stranded DNA target was hybridized for 16 hours and 30 minutes at 45M-BM-0C on GeneChipM-BM-. Gene 1.0 ST array according to the standard Affymetrix protocol. Washing and scanning were performed using GeneChip System of Affymetrix (GeneChip Hybridization Oven 640, GeneChip Fluidics Station 450 and GeneChip Scanner 7G). Log2-transformed expression values were extracted from CEL files and normalized using NetAffx Transcript Cluster Annotations, Release 31 and robust multi-array average (RMA) procedure in Expression Console software (Affymetrix Inc.). The expression values of transcript cluster ID specific for loci representing naturally occurring read-through transcriptions were summarized as median value for each sample.

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MiRNA and mRNA expression profile of CLL cells

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