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. 2008;9(3):R59.
doi: 10.1186/gb-2008-9-3-r59. Epub 2008 Mar 25.

A sequence-based survey of the complex structural organization of tumor genomes

Benjamin J Raphael  1 , Stanislav Volik, Peng Yu, Chunxiao Wu, Guiqing Huang, Elena V Linardopoulou, Barbara J Trask, Frederic Waldman, Joseph Costello, Kenneth J Pienta, Gordon B Mills, Krystyna Bajsarowicz, Yasuko Kobayashi, Shivaranjani Sridharan, Pamela L Paris, Quanzhou Tao, Sarah J Aerni, Raymond P Brown, Ali Bashir, Joe W Gray, Jan-Fang Cheng, Pieter de Jong, Mikhail Nefedov, Thomas Ried, Hesed M Padilla-Nash, Colin C Collins
Affiliations

A sequence-based survey of the complex structural organization of tumor genomes

Benjamin J Raphael et al. Genome Biol. 2008.

Abstract

Background: The genomes of many epithelial tumors exhibit extensive chromosomal rearrangements. All classes of genome rearrangements can be identified using end sequencing profiling, which relies on paired-end sequencing of cloned tumor genomes.

Results: In the present study brain, breast, ovary, and prostate tumors, along with three breast cancer cell lines, were surveyed using end sequencing profiling, yielding the largest available collection of sequence-ready tumor genome breakpoints and providing evidence that some rearrangements may be recurrent. Sequencing and fluorescence in situ hybridization confirmed translocations and complex tumor genome structures that include co-amplification and packaging of disparate genomic loci with associated molecular heterogeneity. Comparison of the tumor genomes suggests recurrent rearrangements. Some are likely to be novel structural polymorphisms, whereas others may be bona fide somatic rearrangements. A recurrent fusion transcript in breast tumors and a constitutional fusion transcript resulting from a segmental duplication were identified. Analysis of end sequences for single nucleotide polymorphisms revealed candidate somatic mutations and an elevated rate of novel single nucleotide polymorphisms in an ovarian tumor.

Conclusion: These results suggest that the genomes of many epithelial tumors may be far more dynamic and complex than was previously appreciated and that genomic fusions, including fusion transcripts and proteins, may be common, possibly yielding tumor-specific biomarkers and therapeutic targets.

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Figures

Figure 1
Figure 1
Schematic of ESP. End sequencing and mapping of tumor genome fragments to the human genome provides information about structural rearrangements in tumors. A bacterial artificial chromosome (BAC) end sequence (BES) pair is a valid pair if distance between ends mapped on the normal human genome sequence and the orientation of these ends and are consistent with those for a BAC clone insert; otherwise, the BES pair is invalid. bp, base pairs; ESP, end sequencing profiling.
Figure 2
Figure 2
PCR validation of breakpoints in MCF7. (a) MCF7 clone 69F1 was sequenced and contained a small piece of chromosome 1 (purple rectangle) to chromosome 17 (yellow rectangle). Arrows on each rectangle indicate whether the fragment is oriented as in the reference genome (pointing to right) or inverted (pointing to left). PCR primers were designed to amplify the breakpoint and these primers were used to assay the other clones in the BES cluster with 69F1. Each of the other clones in the cluster are indicated as lines below 69F1, with the end-points of the lines indicating the locations of the mapped ends relative to the ends of 69F1. The heterogeneous PCR results might result from heterogeneity of the MCF7 cells, or the existence of multiple versions of this breakpoint in MCF7 genome. (b) PCR results for the clones presented in panel a. The expected size of the PCR fragment is 600 base pairs. (c) PCR validation of breakpoints in sequenced clone 37E22 from MCF7 and three additional clones in bacterial artificial chromosome end sequence (BES) cluster all fusing nearby locations from chromosomes 1, 3, and 20. Two other clones have the same complex internal organization as 37E22 with four rearrangement breakpoints. However, clone 34J23 contains only one of these breakpoints, suggesting that the rearrangement history of this clone is different from that of the others in the cluster.
Figure 3
Figure 3
Use of dual-color FISH to validate a BT474 genomic breakpoint. End sequences from clone CHORI518_014-E04 were mapped to chromosomes 1 and 4. Clones RP11-692N22 and RP11-1095F2 were selected from the human RPCI11 library because their sequences map to just outside of tumor bacterial artificial chromosome (BAC) end sequence (BES) locations. These BACs were labeled with fluorescein and Texas red, respectively. Top: two chromosomes containing a merged yellow signal indicating juxtaposition of both probes are indicated with white arrows (and labeled A and B). Bottom: each labeled chromosome is shown with corresponding inverted-DAPI banded chromosome, and red and green image layers. Black arrows identify the region where the red and green probes are juxtaposed to one another. FISH, fluorescence in situ hybridization.
Figure 4
Figure 4
Recurrent rearrangement loci in the three breast cancer cell lines. (a,b) Four loci on 20q13.2-13.3 shared by MCF7 and BT474 and (c) a locus near to the ERBB2 amplicon shared by BT474 and SKBR3. Colored boxes indicate the breakpoint regions for different bacterial artificial chromosome (BAC) clones from MCF7 (blue), BT474 (red), and SKBR3 (green) as a custom track on the University of California, San Francisco (UCSC) genome browser. A breakpoint region is defined as the possible locations of a breakpoint that are consistent with all the BAC end sequence (BES) in the cluster; thus, shorter boxes indicate more precise breakpoint localization. Arrows give the strand of the mapped BES and thus point away from the fused region.
Figure 5
Figure 5
RT-PCR assays of fusion transcripts on a panel of breast cancer cell lines and normal tissues. HMEC-P1 stands for normal human mammary epithelial cells (passage 1), and HMEC-P4 stands for HMEC passage 4 (higher passage). (a) RT-PCR reveals expression of DR00074 (HYDIN gene fusion) in 16 out of 21 tested breast cancer cell lines, normal cultured human breast epithelial cells, and a wide range of normal human tissues. (b) RT-PCR validation of CN272097 a cDNA produced by a complex rearrangement on chromosome 5 fusing the SLC12A2 gene and expressed sequence tag (EST) AK090949. The results provide evidence for expression of the fused transcript in 5 out of 21 breast cancer cell lines and in higher passage but not lower passage human mammary epithelial cells (HMECs). Note that MDAMB435 was recently demonstrated to be derivative of the M14 melanoma cell line and not from breast [62], and the absence of the SLC12A2 fusion is this cell line is consistent with its absence in other nonbreast tissues.
Figure 6
Figure 6
Results of SNP identification in BAC end sequences. (a) The number of high quality isolated single nucleotide polymorphisms (SNPs) in uniquely mapped bacterial artificial chromosome (BAC) end sequences expressed per kilobase (blue). Each tumor sample has a significantly higher rate of SNPs compared with the normal library, whereas the ovarian library exhibits a rate significantly higher than the other tumor samples. Also shown is the fraction of SNPs not found in dbSNP124 (red). The ovarian library shows a significantly higher rate of these novel SNPs. (b) Mutational spectrum of SNPs for each of the samples. For C:G → T:A transitions and C:G → G:C transversions, the fraction at CpG dinucleotides is indicated in red and yellow, respectively.
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References

    1. Pinkel D, Albertson DG. Array comparative genomic hybridization and its applications in cancer. Nat Genet. 2005;37(suppl):S11–S17. doi: 10.1038/ng1569. - DOI - PubMed
    1. Nahta R, Esteva FJ. HER2 therapy: molecular mechanisms of trastuzumab resistance. Breast Cancer Res. 2006;8:215. doi: 10.1186/bcr1612. - DOI - PMC - PubMed
    1. Tomlins SA, Rhodes DR, Perner S, Dhanasekaran SM, Mehra R, Sun XW, Varambally S, Cao X, Tchinda J, Kuefer R, Lee C, Montie JE, Shah RB, Pienta KJ, Rubin MA, Chinnaiyan AM. Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer. Science. 2005;310:644–648. doi: 10.1126/science.1117679. - DOI - PubMed
    1. Raphael BJ, Volik S, Collins C, Pevzner PA. Reconstructing tumor genome architectures. Bioinformatics. 2003;19(suppl 2):II162–II171. - PubMed
    1. Raphael BJ, Pevzner PA. Reconstructing tumor amplisomes. Bioinformatics. 2004;20(suppl 1):I265–I273. doi: 10.1093/bioinformatics/bth931. - DOI - PubMed

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