Chromothripsis: A New Mechanism For Tumorigenesis? i Fellow s Conference Cheryl Carlson 6/10/2011

Transcription

1 Chromothripsis: A New Mechanism For Tumorigenesis? i Fellow s Conference Cheryl Carlson 6/10/2011

2 Massive Genomic Rearrangement Acquired in a Single Catastrophic Event during Cancer Development Cell 144, 27 40, January 7, 2011

3 Background: Tumor Evolution Stimulate growth Attenuate / abrogate cell death Destroy checkpoint controls Promote further genetic instability Enable metastatsis s

4 Background: Tumor Evolution Breast Colorectal Esophageal Cervical Anal Multiple Myeloma

5 Background: Colorectal Tumorigenesis

6 Background: Chromothripsis i Mechanisms Copy number changes Deletion of tumor suppressors Amplification of oncogenes Translocations Gene expression dysregulation

7 Methodology Deep sequencing of tumor samples Illumina platform Paired-end protocol 5 ug of tumor DNA SNP array (Affymetrix) Multiplex flourescence in situ hybridization

8 Results: 1/10 CLL patients t 42 genomic rearrangements involving 4q

9 Results: CLL Patient t Despite a separate 13q -, confined to 4q and focal points on 1, 12, and 15.

10 Results: CLL patient t Copy number varies between only 2 states

11 Results: CLL patient t Not just one simple deletion 8 deletions, 9 tandem duplications, 6 head-head inversions, 10 tail-tail inversions

12 Results: CLL patient t Pronounced clustering of breakpoints

13 Results: CLL patient t Equal number of near by and far way junctions

14 CLL Patient s t Clinical i l Course 62 yo previously untreated female Clinical course showed rapid progression Treated with alemtuzumab Quickly relapsed Relapse sample taken 31 months after Relapse sample taken 31 months after initial sample

15 Results: CLL patient t All rearrangements and CNVs were present in relapse sample No new ones found, suggesting underlying mechanism no longer active

16 How Unique Is This Phenomenon? High-resolution i copy number profiles of 746 cancer cell lines (SNP array) 18/746 (2.4%) had similar copy # changes confined to localized genomic regions rapidly alternating between 1,2, or rarely 3 states Seen involving the whole chrom, entire arm, interstitial region, or telomere Multiple tumor types Melanoma (4), SCLC (3), glioma (3), heme (2), NSCLC, sarcoma, esophageal, colorectal, renal, and thyroid

17 Results: Tumor Cell Lines

18 Results: Primary Tumors Examined Segmented SNP array data from 2792 cancers (Beroukim et al, 2010: The landscape of somatic copy-number alteration across human cancers.) 80% were primary tumors Evidence for chromothripsis in a similar proportion of cases Supplemental data missing

19 Next Step Selected 4 cell lines for further genomic analysis Deep sequencing Cytogenetics

20 Results: 4 Tumor Cell Lines Colon Thyroid Renal Lung

21 Results: 4 Tumor Cell Lines

22 Results: 4 Tumor Cell Lines 55

23 The Rearrangements are Limited to a Single Parental Copy of the Chromosome (Renal) Consistent with CNV profile showing 4 with LOH and 6 with heterozygosity

24 The Rearrangements are Limited to a Single Parental Copy of the Chromosome TK10 Renal

25 Additional studies Rearrangement screens in primary tumor samples from 20 patients with bone cancer 5 (25%) showed large numbers of clustered rearrangements c/w chromothripsis 3 osteosarcomas and and 2 chordomas Pt age ranged from 9 to 64 years In 4/5 more than 1 chromosome was affected

26 Bone Cancer Results 147 rearrangements 49 intrachromosomal 98 interchromosomal

27 Bone Cancer Results 88 rearrangements 38 rearrangements

28 Two Models to Explain Their Observations

29 Monte Carlo Simulation of the Progressive Model Rearrangements were randomly sampled from the breakpoints seen in SNU-C1 (colon cell line) with reconstruction of the resulting chromosome structure t Repeated several times to generate different numbers of rearrangements and configurations

30 2nd Argument Against the Progressive Model There was observed retention of heterozygosity in regions with higher copy number Once lost, heterozygosity can t be regained Therefore, deletions could only occur very late in the sequence of rearrangements SNU-C1 had 239 rearrangements on chrom 15, very difficult to imagine a sequence of events that t would spare the heterozygosity observed in >20 separate segments In contrast, it s the natural consequence of the catestophic model

31 3rd Argument Against the Progressive Model B k i t h i ifi tl l t i Breakpoints show significantly more clustering along the chrom than expected by chance

32 Chromothripsis Can Promote Carcinogenesis SCLC-21H Chrom breakpoints 15

33 Chromothripsis Can Promote Carcinogenesis

34 Loss or Disruption of Tumor Suppressors Chordoma

35 Loss or Disruption of Tumor Suppressors SCLC-21H Expression levels decreased in ~5% of genes on chrom 8

36 Creation of Novel Oncogenic Fusion Genes? Found 17 in-frame novel fusions among their rearrangements None were classic like BCR-ABL or EWS-FLI1 Didn t investigate further

37 Conclusions Chromothripsis is real Phenomenon whereby 10s to 100s of rearrangements involving localized genomic regions are acquired during a single catastrophic event Occurs in 2-3% of all malignancies, spanning a wide variety of tumor types May be particularly frequent in bone cancers (25%) Can lead to tumorgenesis Deletion of tumor suppressors Amplification of oncogenes Translocations Gene expression dysregulation

38 Speculations Probably bl occurs during mitosis i when chroms are condensed Sharply circumscribed genomic locations Mechanism of DNA shattering? Pulse of ionizing radiation Telomere attrition (breakage-fusion-bridge cycle) Premature e chromosome oso compaction o Mechanism of repair? Likely by the nonhomologous end-joining DNA repair system

39 Strengthsth Plethora of data Included DNA from primary tumors not just cell lines Verified by multiple methodologies Deep sequencing SNP analysis FISH Protein expression

40 Weaknesses Largely observational / descriptive only Does not explore underlying mechanisms How does the chromosome fragment shatter? How is the repair done? Probable cherry picking of the data to depict a certain point

41 Relevance Chromothripsis i identifies a rare and aggressive entity among newly diagnosed multiple myeloma patients. Magrangeas F, Avet-Loiseau H, Munshi NC, Minvielle S. Blood May 31. [Epub ahead of print] Analysis of high resolution copy number profiles obtained using single-nucleotide polymorphism array data from 764 newly diagnosed MM identified large numbers of genomic rearrangements with the hallmarks of chromothripsis in 1.3% of samples. Moreover this catastrophic event confers a poor outcome. Observed death in 4/9 patients with an average OS of 16 months with a striking example of a 34 yo patient w/ PCL who died 6 months after diagnosis Chromothripsis as a mechanism driving complex de novo structural rearrangements in the germline. Kloosterman WP, Guryev V, van Roosmalen M, Duran KJ, de Bruijn E, Bakker SC, Letteboer T, van Nesselrooij B, Hochstenbach R, Poot M, Cuppen E. Hum Mol Genet May 15;20(10): Epub 2011 Feb 24.