BAC Array Comparative Genomic Hybridization Research Articles

High‐resolution genomic profiles of breast cancer cell lines assessed by tiling BAC array comparative genomic hybridization

A BAC-array platform for comparative genomic hybridization was constructed from a library of 32,433 clones providing complete genome coverage, and evaluated by screening for DNA copy number changes in 10 breast cancer cell lines (BT474, MCF7, HCC1937, SK-BR-3, L56Br-C1, ZR-75-1, JIMT1, MDA-MB-231, MDA-MB-361, and HCC2218) and one cell line derived from fibrocystic disease of the breast (MCF10A). These were also characterized by gene expression analysis and found to represent all five recently described breast cancer subtypes using the "intrinsic gene set" and centroid correlation. Three cell lines, HCC1937 and L56BrC1 derived from BRCA1 mutation carriers and MDA-MB-231, were of basal-like subtype and characterized by a high frequency of low-level gains and losses of typical pattern, including limited deletions on 5q. Four estrogen receptor positive cell lines were of luminal A subtype and characterized by a different pattern of aberrations and high-level amplifications, including ERBB2 and other 17q amplicons in BT474 and MDA-MB-361. SK-BR-3 cells, characterized by a complex genome including ERBB2 amplification, massive high-level amplifications on 8q and a homozygous deletion of CDH1 at 16q22, had an expression signature closest to luminal B subtype. The effects of gene amplifications were verified by gene expression analysis to distinguish targeted genes from silent amplicon passengers. JIMT1, derived from an ERBB2 amplified trastuzumab resistant tumor, was of the ERBB2 subtype. Homozygous deletions included other known targets such as PTEN (HCC1937) and CDKN2A (MDA-MB-231, MCF10A), but also new candidate suppressor genes such as FUSSEL18 (HCC1937) and WDR11 (L56Br-C1) as well as regions without known genes. The tiling BAC-arrays constitute a powerful tool for high-resolution genomic profiling suitable for cancer research and clinical diagnostics.

Cytogenetically balanced translocations are associated with focal copy number alterations

Current cytogenetic methods (e.g., G-banding and multicolor chromosomal painting) allow detection of translocation events but lack the resolution to (a) locate the breakpoints precisely at the chromosome band level or (b) discriminate balanced translocations from translocations with copy number alterations not previously reported, or imperfectly balanced translocations. In this study, we demonstrate that cytogenetically balanced translocations are in fact frequently associated with segmental gain or loss of DNA. The recent development of a whole genome tiling path BAC array has enabled tiling resolution analysis of genomic segmental copy number status. Combining tiling resolution BAC array comparative genomic hybridization (array CGH) with G-Banding analysis and multicolor chromosomal painting approaches such as spectral karyotyping (SKY) facilitates high-resolution mapping of genomic alterations associated with imperfectly balanced translocations. Using a refined version of our CGH array we have deduced the copy number status throughout the genomes of three cytogenetically well-characterized prostate cancer cell lines (PC3, DU145, LNCaP) to determine whether translocations are associated with focal gains and losses of DNA. At 78 kb tiling resolution we identified the boundaries of 170, 80, and 34 known and novel copy number alterations (CNA) in these cell line genomes, respectively. Thirty-three of the 36 known translocations (92%, P < 0.001) in DU145 were associated with segmental CNA. Likewise, 80% (P < 0.001) of the known translocations showed association in LNCaP. Although many translocation breakpoints exhibit segmental alteration in PC3, the pattern of chromosomal rearrangements is too complex for use in comprehensive association with CNA boundaries. Our results reveal that imperfectly balanced translocations in tumor genomes are a phenomenon that occurs at frequencies much higher than previously demonstrated.

Discovery of previously unidentified genomic disorders from the duplication architecture of the human genome

Genomic disorders are characterized by the presence of flanking segmental duplications that predispose these regions to recurrent rearrangement. Based on the duplication architecture of the genome, we investigated 130 regions that we hypothesized as candidates for previously undescribed genomic disorders. We tested 290 individuals with mental retardation by BAC array comparative genomic hybridization and identified 16 pathogenic rearrangements, including de novo microdeletions of 17q21.31 found in four individuals. Using oligonucleotide arrays, we refined the breakpoints of this microdeletion, defining a 478-kb critical region containing six genes that were deleted in all four individuals. We mapped the breakpoints of this deletion and of four other pathogenic rearrangements in 1q21.1, 15q13, 15q24 and 17q12 to flanking segmental duplications, suggesting that these are also sites of recurrent rearrangement. In common with the 17q21.31 deletion, these breakpoint regions are sites of copy number polymorphism in controls, indicating that these may be inherently unstable genomic regions.

Genomic profiles of colorectal cancers differ based on patient smoking status

Human sporadic colorectal cancer is the result of a lengthy somatic evolutionary process facilitated by various forms of genomic instability. Such instability arises endogenously from mutations in genes whose role is to preserve genomic integrity, and exogenously from environmental agents that generate genomic damage. We have found that cigarette smoking shifts the genomic profiles and genomic instability patterns of colorectal carcinomas. The genomic profiles of 57 consecutive cancers were examined; 31 cases were current or former smokers and 26 were nonsmokers. Genome-wide allelotypes of 348 markers were examined, along with comparative genomic hybridization (CGH) on ordered BAC microarrays, microsatellite instability, and inter-(simple sequence repeat) polymerase chain reaction instability. Tumors from nonsmokers exhibited losses of heterozygosity, particularly on chromosomes 14 and 18, whereas tumors from smokers exhibited a more diffuse pattern of allelic losses. Tumors from smokers exhibited higher overall rates of loss of heterozygosity, but showed lower rates of background microsatellite instability (MSI-L). On BAC array CGH, higher levels of generalized amplifications and deletions were observed in tumors from smokers, differentially affecting male smokers. In the transforming growth factor-β signaling pathway, MADH4 mutations were more common in tumors from smokers, whereas transforming growth factor-β RII mutations were more common among nonsmokers.

An Isothermal Method for Whole Genome Amplification of Fresh and Degraded DNA for Comparative Genomic Hybridization, Genotyping and Mutation Detection

Molecular genotyping has important biomedical and forensic applications. However, limiting amounts of human biological material often yield genomic DNA (gDNA) in insufficient quantity and of poor quality for a reliable analysis. This motivated the development of an efficient whole genome amplification method with quantitatively unbiased representation usable on fresh and degraded gDNA. Amplification of fresh frozen, formalin-fixed paraffin-embedded (FFPE) and DNase-degraded DNA using degenerate oligonucleotide-primed PCR or primer extension amplification using a short primer sequence bioinformatically optimized for coverage of the human genome was compared with amplification using current primers by chromosome-based and BAC-array comparative genomic hybridization (CGH), genotyping at short tandem repeats (STRs) and single base mutation detection. Compared with current primers, genome amplification using the bioinformatically optimized primer was significantly less biased on CGH in self-self hybridizations, and replicated tumour genome copy number aberrations, even from FFPE tissue. STR genotyping could be performed on degraded gDNA amplified using our technique but failed with multiple displacement amplification. Of the 18 different single base mutations 16 (89.5%) were correctly identified by sequencing gDNA amplified from clinical samples using our technique. This simple and efficient isothermal method should be helpful for genetic research and clinical and forensic applications.

Genomic profiling of breast cancer

Cancer and other genetic diseases are characterized by genome alterations, including DNA copy number changes. Comparative genomic hybridization (CGH) represents a powerful technique to detect and map these aberrations, and recent improvements in resolution and sensitivity have been possible through implementation of microarray-based platforms. Germline mutations in the two major breast cancer susceptibility genes, BRCA1 and BRCA2, account for a significant proportion of all hereditary breast cancers. Earlier studies have shown that inherited and sporadic tumors progress along different somatic genetic pathways and that global gene expression profiles distinguish between these groups. Using 1 Mbp resolution BAC-array CGH analysis, we now show that genomic copy number profiles similarly discriminate between BRCA1/BRCA2-related tumors and sporadic tumors. Overall, BRCA1 tumors had a higher frequency of copy number alterations than sporadic breast cancers. In particular, frequent losses on 4p, 4q and 5q in BRCA1 tumors and frequent gains on 7p and 17q24 in BRCA2 tumors distinguish these from sporadic breast cancer. Distinct amplicons at 3q27.1-q27.3 were identified in BRCA1 tumors, and amplicons at 17q23.3-q24.2 in BRCA2 tumors. Moreover, evidence of a homozygous deletion in a BRCA1 tumor on 5q12.1 was obtained. Using a set of 169 BAC clones that detect significantly different frequencies of copy number changes in inherited and sporadic tumors, these subsets could be discriminated into separate groups using hierarchical clustering. Further validation may prove this tumor classifier to be useful for selecting familial breast cancer cases, likely to carry BRCA1 or BRCA2 germline mutations, for further mutation screening, particularly as these data can be obtained using DNA prepared from archival tumor tissue. Further improved genomic profiling was obtained by construction of microarrays comprising 32,433 BAC clones, offering complete genome coverage at single gene resolution, on average <50 kbp. These new tiling 32k-arrays were evaluated on breast cancer cell lines (BT-474, MCF7, HCC1937, SK-BR-3, L56Br, ZR-75-1), validated by FISH and gene expression analysis. Known amplicons were resolved and found to include complex patterns of narrow peaks, occasionally including a few or even single genes. Several amplified regions and genes on 17q and 20q were depicted and confirmed by demonstrating strong correlations between gene copy numbers and expression. Previously described as well as novel homozygous deletions, ranging from a few BAC clones (<300 kb) to several Mbp, were observed, including PTEN and other regions on 10q, CDH1/CDH3 on 16q22, and new regions on 4q34 and 19p12, emphasizing the power of array CGH in pinpointing genes of importance in tumor development. Array CGH is a promising diagnostic tool in profiling of somatic and constitutional genomic alterations.