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Blood, Vol. 109, Issue 1, 271-280, January 1, 2007
Homozygous deletions localize novel tumor suppressor genes in B-cell lymphomas
Blood Mestre-Escorihuela et al.
109: 271
Supplemental materials for: Mestre-Escorihuela et al, Vol 109, Issue 1, 271-280
Files in this Data Supplement:
- Table S1. Primers for QRT-PCR, sequence analysis, and MSP (PDF, 142 KB) -
Primers and QRT-PCR measurement of CDK6 and NOXA genes in the B-NHL cell lines, and primers for genomic sequencing and methylation analysis using MSP for PIG7/LITAF, BIM, INK4c/P18, and NOXA are shown.
- Table S2. Full karyotype and standard CGH description of 25 B-NHL cell lines (PDF, 41.1 KB) -
We performed genome-wide scanning for DNA copy number changes in 48 B-NHL cell lines using an array CGH method that covers the genome with a 1.4-Mb resolution (Figure 1). To assess sensitivity and resolution, array CGH patterns were compared with those of standard CGH to chromosomes (s-CGH) and to the cytogenetic analysis in 25 selected cell lines. On average, each cell line displayed 20 genomic changes in array CGH analysis (range, 4-35), comprising roughly equal numbers of gains and losses. In the 25 selected cell lines, s-CGH and cytogenetic analysis revealed a lower number of aberrations than in array CGH studies: mean number of abnormalities per case were 13 (range, 1-25) and 12 (range, 2-20) in s-CGH and G-banding studies, respectively. Of the total of 70 genomic amplifications revealed by array CGH in the 25 cell lines, only 22 (31%) were detected by s-CGH. Similarly, among 230 genomic losses detected by array CGH, only 95 (41%) were revealed by s-CGH. Thus, array CGH provided a more accurate quantitative and qualitative portrait of the DNA copy number changes in the B-NHL cell lines. To evaluate reproducibility of array CGH experiments, the same aliquots from SCI1, PR1, OCI-Ly8, and Z138 cell lines were analyzed twice. In the comparative analysis, only 14 clone pairs (range, 4-33) from each of the paired tumors (representing 0.7% of the probes that were successfully hybridized) from the different array CGH experiments showed discordant values (defined as differences in log2 value exceeding 0.3). In summary, array CGH was more sensitive than conventional CGH and was a highly reproducible method for the scanning of lymphoma genomes.
- Table S3. Representation of amplification sites and genes targeted with elevated expression in B-NHL (PDF, 41.1 KB) -
(A) Map positions and cytogenetic locations are based on data available through the University of California Santa Cruz genome browser (May 2004 freeze). (B)For amplicon borders, the more extern positions of amplified clones has been used. (C) Because abnormal clones that delineated the amplicon borders have been excluded in the May 2004 freeze of the UCSC genome browser, the closest positions of neighboring unamplified clones have been used. (D) Candidate genes with elevated expression are mapped to amplified regions delineated by the closest positions of neighboring unamplified clones.
- Figure S1. Genome-wide array CGH and cDNA microarray analyses of B-NHL (PDF, 92.3 KB) -
Genome-wide DNA and RNA copy number variation in the SSK-41 cell line using array CGH (A) and gene expression profiling (B) across the genome; probes were ordered according to their cytogenetic position from 1p telomere to Xq telomere. Individual amplifications of 3q27-q29, 7q21-q22, and 18q21 and genomic deletion of 11p13 were confirmed, as shown, using s-CGH, FISH, and color cytogenetic analyses. The upper inset shows the partial s-CGH and array CGH analyses of the SSK41 cell line, demonstrating amplification of 7q21-q22 involving CDK6 gene, which also showed deregulated expression in gene expression analysis. Cytogenetic analysis showed a der(7)t(7;15)(q21;q15), +der(7)t(7;11;15)(q21;q21-q22;q15), del(10)(p12), +der(10)t(7;10)(q10;p10). FISH studies using a BAC probe containing the gene showed CDK6 amplification in the SSK41 cell line. Western blot analyses revealed CDK6 overexpression in SSK41 cell line as well in Karpas 1718, both derived from patients with marginal zone lymphoma. Other cell lines with gain of 7q derived from other B-NHL subtypes did not show elevated expression of CDK6. We performed genome-wide scanning for DNA copy number changes in 48 B-NHL cell lines using high-resolution array CGH. To assess sensitivity and resolution, array CGH patterns were compared with those of standard CGH (s-CGH) and to the cytogenetic analysis in 25 selected cell lines. On average, each cell line displayed 20 genomic changes in array CGH analysis (range, 4-35), comprising roughly equal numbers of gains and losses. s-CGH and cytogenetic analysis revealed a lower number of aberrations than array CGH: mean number of abnormalities per cell line were 13 (range, 1-25) and 12 (range, 2-20) in s-CGH and G-banding studies, respectively. Of the total of 70 genomic amplifications revealed by array CGH in 25 cell lines, only 22 (31%) were detected by s-CGH. Similarly, among 230 genomic losses detected by array CGH in 25 cell lines, only 95 (41%) were revealed by s-CGH. These data indicate that array CGH provided a more accurate portrait of the DNA copy number changes in the B-NHL cell lines. To evaluate reproducibility of array CGH, the same aliquots from SCI-1, PR1, OCI-Ly8, and Z138 cell lines were analyzed in duplicate. Only 14 clone pairs (range, 4-33) from each of the paired tumors (representing 0.7% of the probes that were successfully hybridized) from the different array CGH experiments showed discordant values (defined as differences in log2 value exceeding 0.3), confirming the reproducibility of the array CGH method.
- Figure S2. Array CGH analysis of the different B-NHL subtypes (PDF, 457 KB) -
(Left) Array CGH profile of different B-NHL subgroups. DLBCL indicates diffuse large B-cell lymphoma; MCL, mantle cell lymphoma; BL, Burkitt lymphoma; MZL, marginal zone lymphoma; PMBCL, primary mediastinal B-cell lymphoma. (Right) Array CGH profiles show genomic alterations that are associated with particular B-NHL subgroups: deletion of chromosome 1p21.2 only in MCL; gain of 1q21.2-q25.1 in BL; gains of 8q13-q21 and 8q24.1 (MYC locus) in DLBCL with the t(14;18)(q32;q21) and deletion of 9p21.3 (INK4/ARF locus) in MCL. Genomic gains are shown in red, genomic losses in green and regions with normal DNA copy number in black. We investigated the association between specific genomic aberrations and the different B-NHL subtypes. The deletion of chromosome 1p21.2 was the only locus observed in only 1 B-NHL category, namely mantle cell lymphoma, but not in other subgroups (42% versus 0%; P|lt|.001) (Figure S2). Some other genomic changes were correlated with the different B-NHL subgroups. These included the deletion of chromosome bands 9p21.3 at INK4/ARF locus (75% versus 25%; P|=|.003) and 2q13-2q21.2 at BIM locus (33% versus 8%; P|=|.05) and the gain of 3q13.33-q21.3 (33% versus 3%; P|=|.01) in mantle cell lymphoma; the gain of 8q24.1 (MYC locus) (73% versus 27%; P|=|.004) and 11p15.2-p15.4 (33% versus 9%; P|=|.05) in diffuse large B-cell lymphoma with the t(14;18)(q32:q21) translocation; the gain of 13q14.11 in diffuse large B-cell lymphoma without the t(14;18)(q32;q21) translocation (50% versus 12%; P|=|.05) and the gain of 1q21.2-q23.3 (64% versus 22%; P|=|.01) and the deletion of 17p13.1 involving P53 (55% versus 22%; P|=|.04) in Burkitt lymphoma. Therefore, regional genomic gains and losses showed a different distribution pattern among the B-NHL subtypes.
- Figure S3. Identification of targeted oncogenes in B-NHL (PDF, 117 KB) -
(A) Amplification and overexpression of CDK6 in marginal zone lymphoma cell line SSK41. Complementary FISH, color cytogenetic, and Western blot analyses are shown. (B) Amplification of different chromosome 13q segments in SSK41 (left) and MD903 (right) cell lines identified numerous genes targeted at the RNA level. (C) Amplification of 6p21.2 involving PIM1 in the JEKO1 mantle cell lymphoma cell line. Overexpresison of PIM1 was detected in Lymphchip microarrays. In addition, we searched for PIM1 expression in 11 mantle cell lymphoma cell lines using Affymetrix oligonucleotide microarrays, being only JEKO1 the one with 6p amplicon. A number of genes across 6p21.2 showed elevated expression in JEKO1 versus the remaining cell lines. Among them, PIM1 presented higher differential expression at the RNA level.
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