Reconstructing the evolution of a
drug-resistant liposarcoma
David Goode
Sarcoma Genetics & Genomics
Peter MacCallum Cance...
Evolution of Drug Resistance in Tumours
“Clonal cell lineage (clone)”
+2.5 μM Nutlin
95 days
in Nutlin
252 days
in Nutlin
778
(liposarcoma)
Experimental Outline
R95
R252
(‘resistant’)
Copy-num...
SNP genotyping arrays
• B Allele Frequency (BAF)
• Relative frequency of the B allele [0 – 1]
• Log R Ratio (LRR)
• Copy-n...
Localised but frequent changes in
CN observed in Nutlin-resistant lines
R252 - 778
R95 - 778
1 2 3 4 5 6 7 8 9 10 11 12 13...
Change in allelic ratios on chr15 in R252
(red) from 778 (black)
Change in allelic ratios on chr15
occurred between R95 & R252
+2.5 μM Nutlin
Loss of one
copy of chr15
778
(‘parental’)
Ordering chromosomal CN changes
R95
R252
(‘resistant’)
Gain of section of chr11q in resistant lines
+2.5 μM Nutlin
Gain of part
of 11q
Gain of one
copy of chr15
778
(‘parental’)
Ordering chromosomal CN changes
R95
R252
(‘r...
Exome sequencing
• 12 clones each from R252 (resistant) and 778 (parental)
• Clones derived from single cells
• Nimblegen ...
BAF pattern on chr11 not present in
any clones from 778
778 Clone 1 (8/12) 778 Clone 2 (4/12) All R252 clones
+2.5 μM Nutlin
Gain of part
of 11q
Gain of one
copy of chr15
778
(‘parental’)
Ordering chromosomal CN changes
R95
R252
(‘r...
BAF pattern on chr5 present in
minority 778 clone
778 Clone 1 (11/12) 778 Clone 2 (1/12) All R252 clones
Most likely ances...
+2.5 μM Nutlin
Gain of part
of 11q
Gain of one
copy of chr15
778
(‘parental’)
Ordering chromosomal CN changes
R95
R252
(‘r...
778
(‘parental’)
Ordering observed copy-number (CN) changes
[relative to 778 SNP arrays]
R95
R252
(‘resistant’)
8 (4, 3, 1...
Phylogeny of 778 and R252 clones
Phylip (DNApars)
0.87
1.93
0.66
2.79
2.99
2.65
2.45
3.31
2.38
2.16
2.45
2.42
2.32
2.12
1.2
0.7
0.99
0.88
0.96
0.94
1.33
1.06
0.95
1.005
Clo...
+2.5 μM Nutlin
Slowing of CN
mutation rate
778
(‘parental’)
R95
R252
(‘resistant’)
Minor clones
potentially
Nutlin resista...
Summary
• BAFs to identify major CN changes occuring during
evolution of Nutlin resistance
• Sequencing individual clones ...
Acknowledgements
• David Thomas
• Arcadi Cipponi
• Tiffany Pang
• Kevin Mills
• Natnicha Inthavong
• Bioinformatics Core
•...
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Reconstructing tumour evolution: reconstructing the evolution of a drug-resistant liposarcoma - David Goode

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Tumours are a collection of genetically distinct cellular lineages related that must compete against each other and the external environment. Many cell lineages die out, while those with phenotypes that are advantageous expand. A major clinical consequence of this evolutionary process is the emergence of drug resistant tumour cells.

We have established an in vitro model system in which we can induce the human well-differentiated liposarcoma (WDLPS) cell line 778 to acquire resistance to the MDM2 inhibitor Nutlin-3a. I will detail we have applied bioinformatics and evolutionary principles to reconstruct major evolutionary events that occurred as this line acquired drug resistance. Integration of SNP array and exome sequencing data from different time points during the evolution of Nutlin resistance allow us to infer the relative order of genetic changes and how the rate of evolution fluctuated during the course of the experiment.

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Reconstructing tumour evolution: reconstructing the evolution of a drug-resistant liposarcoma - David Goode

  1. 1. Reconstructing the evolution of a drug-resistant liposarcoma David Goode Sarcoma Genetics & Genomics Peter MacCallum Cancer Centre March 28, 2014
  2. 2. Evolution of Drug Resistance in Tumours “Clonal cell lineage (clone)”
  3. 3. +2.5 μM Nutlin 95 days in Nutlin 252 days in Nutlin 778 (liposarcoma) Experimental Outline R95 R252 (‘resistant’) Copy-number (CN) profiling using SNP genotyping arrays 778 (‘parental’)
  4. 4. SNP genotyping arrays • B Allele Frequency (BAF) • Relative frequency of the B allele [0 – 1] • Log R Ratio (LRR) • Copy-number relative to normal diploid genomes A/B BA
  5. 5. Localised but frequent changes in CN observed in Nutlin-resistant lines R252 - 778 R95 - 778 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y
  6. 6. Change in allelic ratios on chr15 in R252 (red) from 778 (black)
  7. 7. Change in allelic ratios on chr15 occurred between R95 & R252
  8. 8. +2.5 μM Nutlin Loss of one copy of chr15 778 (‘parental’) Ordering chromosomal CN changes R95 R252 (‘resistant’)
  9. 9. Gain of section of chr11q in resistant lines
  10. 10. +2.5 μM Nutlin Gain of part of 11q Gain of one copy of chr15 778 (‘parental’) Ordering chromosomal CN changes R95 R252 (‘resistant’)
  11. 11. Exome sequencing • 12 clones each from R252 (resistant) and 778 (parental) • Clones derived from single cells • Nimblegen exome capture • Illumina Hi-Seq (100X+ coverage) • SNVs called with GATK’s Unified Genotyper • Use frequency of alternate (non-reference) allele to identify CN polymorphisms
  12. 12. BAF pattern on chr11 not present in any clones from 778 778 Clone 1 (8/12) 778 Clone 2 (4/12) All R252 clones
  13. 13. +2.5 μM Nutlin Gain of part of 11q Gain of one copy of chr15 778 (‘parental’) Ordering chromosomal CN changes R95 R252 (‘resistant’)
  14. 14. BAF pattern on chr5 present in minority 778 clone 778 Clone 1 (11/12) 778 Clone 2 (1/12) All R252 clones Most likely ancestor of resistant clones
  15. 15. +2.5 μM Nutlin Gain of part of 11q Gain of one copy of chr15 778 (‘parental’) Ordering chromosomal CN changes R95 R252 (‘resistant’) Gain one copy of chr5
  16. 16. 778 (‘parental’) Ordering observed copy-number (CN) changes [relative to 778 SNP arrays] R95 R252 (‘resistant’) 8 (4, 3, 1) 3 (2, 1, 0) 6 (3, 2, 1) # of CN changes (Loss, Gain, Other) +2.5 μM Nutlin
  17. 17. Phylogeny of 778 and R252 clones Phylip (DNApars)
  18. 18. 0.87 1.93 0.66 2.79 2.99 2.65 2.45 3.31 2.38 2.16 2.45 2.42 2.32 2.12 1.2 0.7 0.99 0.88 0.96 0.94 1.33 1.06 0.95 1.005 Clone phylogeny labeled with Nutlin IC50 values *share a non-syn TP53 SNV (C238F) Arcadi Cipponi
  19. 19. +2.5 μM Nutlin Slowing of CN mutation rate 778 (‘parental’) R95 R252 (‘resistant’) Minor clones potentially Nutlin resistant Evolutionary history of R252 Clones Acquired null TP53 mutationMany CN changes in quick succession
  20. 20. Summary • BAFs to identify major CN changes occuring during evolution of Nutlin resistance • Sequencing individual clones improves resolution of evolutionary events and reveals population substructure • Selection for CN changes early in evolution • Point mutations in clonal subpopulations later on • Next: WGS on experimental replicates and on other cell line/drug combinations
  21. 21. Acknowledgements • David Thomas • Arcadi Cipponi • Tiffany Pang • Kevin Mills • Natnicha Inthavong • Bioinformatics Core • Maria Doyle • Joshy George • Jason Li • Jason Ellul • Molecular Genomics Core • LSCC/VLSCI • Gayle Philip • Jessica Chung • Andrew Lonie • Leonardo Meza-Zapeda (Oslo) • microarrays

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