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Whole Exome Sequencing and INTEGRATED GENOMIC ANALYSIS OF ‘WILD-TYPE’ DESMOIDS IDENTIFIES POTENTIAL DRIVERS OF TUMOR INITIATION. Aimee M. Crago, Juliann Chmielecki, Mara Rosenberg, Rachael O’Connor, Caitlin Byrne, Mono Pirun, Nicholas D. Socci, Gouri Nanjangud, Margaret Leversha,

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slide1

Whole Exome Sequencing and INTEGRATED GENOMIC ANALYSIS OF ‘WILD-TYPE’ DESMOIDS IDENTIFIES POTENTIAL DRIVERS OF TUMOR INITIATION

Aimee M. Crago, Juliann Chmielecki,

Mara Rosenberg, Rachael O’Connor,

Caitlin Byrne, Mono Pirun, Nicholas D. Socci,

Gouri Nanjangud, Margaret Leversha,

Meera Hameed, Matthew Meyerson and Samuel Singer

Memorial Sloan Kettering Cancer Center

Connective Tissue Oncology Society

October 31, 2013

desmoid fibromatosis

WES of desmoids

Desmoid fibromatosis
  • Locally aggressive tumor without metastatic potential.
  • Historically treated with surgery though high rates of local recurrence were reported (25-50%).
  • Associated in 85% of patients with mutation in CTNNB1 gene.
  • Nuclear -cateninobserved in >70% of samples by IHC.
  • Component of Gardner’s syndrome in conjunction with familial adenomatosis polyposis and APC mutation.

Aim:

  • Determine molecular events that modulate desmoid initiation in the absence of CTNNB1 mutation.
slide3

WES of desmoids

Sanger sequencing of CTNNB1

  • Frozen samples collected from 2002-2013 (n=110).
  • 77% primary tumors.
  • Median follow-up 43 months.
  • Pathology confirmed by cryomold.
  • Normal tissue macrodissected from specimen.
  • 17/110 (15%) tumors without classic exon 3 mutation .

T41A mutant

AG

T41A

S45

‘wild-type’

S45F mutant

CT

TCT

ACC

T41

S45F

T41

S45

slide4

WES of desmoids

CTNNB1 mutation is not clearly associated with outcome

CTNNB1 mutation and tumor location

S45P

T41A

none

S45F

None vs. T41A – n.s.

None vs. S45F – n.s.

T41A vs. S45F – p<0.05

p=0.001

Multivariate analysis

abdominal wall

intraabdominal

chest

extremity

other

Extremity vs. abdominal wall p<0.05

Extremity vs. intraabdominal p<0.05

slide5

WES of desmoids

  • Unsupervised
  • clustering
  • based on gene expression
  • 47 desmoid tumors
  • Evaluated by U133A 2.0 microarrays
  • S45F – Red
  • T41A – Blue
  • ‘wild-type’ – Black
  • No S45P included

7/36 ‘wild-type’

4/11 ‘wild-type’

slide6

WES of desmoids

Whole exome sequencing of desmoid tumors

  • 17 tumor samples.
  • 9 with no mutation in CTNNB1 – ‘wild-type’ desmoids
  • 8 tumors with CTNNB1 mutation
  • Normal blood from the same patients.
  • Agilent SureSelect v2 Exome bait for capture
  • IlluminaHiSeqflowcells
  • 87% of the exomes were covered >88x

Range 4-29 mutations per sample

29Mb sequenced per tumor

<1 mutation/Mb

slide7

WES of desmoids

Somatic mutation in desmoid tumors

  • 46 significant mutations
    • Called by MuTect
    • Significance by MutSig
  • CTNNB1 mutationin 12/17 tumors (71%) not just 8 as expected based on Sanger.
  • 2 tumors with APC mutation.

*

*

whole exome sequencing of wild type desmoid tumors

WES of desmoids

Whole exome sequencing of wild-type desmoid tumors

known CTNNB1 mutation

average 37% reads

average 16% reads

‘wild-type’ tumors

Only 3 of nine ‘wild-type’ tumors without CTNNB1 or APC mutation.

slide9

WES of desmoids

Copy number alterations in desmoid fibromatosis

Chromosome 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y

CTNNB1

mutations

APC mutation

Wild-type

APC

CTNNB1

mutations

APC mutation

Wild-type

  • CapSeg algorithm from Broad Institute (McKenna et al., in preparation)
  • Few copy number events are observed in desmoid fibromatosis.
  • APC mutants with likely heterozygous deletion of the gene.
slide10

WES of desmoids

Chromosome 6 loss in desmoid fibromatosis

Chromosome 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y

CTNNB1

mutations

APC mutation

Wild-type

CEP6 staining (red)

Chromosome 6

24% nuclei with single chromosome 6 by FISH

CTNNB1

mutations

APC mutation

Wild-type

56% nuclei with single chromosome 6 by FISH

  • - CapSeg algorithm from Broad
chromosome 6 deletions

WES of desmoids

Chromosome 6 deletions
  • Copy number events affecting chromosome 6 are uncommon in oncogenesis.
  • High incidence in HNPCC families without aberrations in the microsatellite instability pathway.
  • Identified in a subset of medulloblastoma patients with Wnt pathway activation
  • Potential tumor suppressors on chromosome 6 known to downregulate Wnt signaling:
    • QKI, DACT2, BMP6, LATS1

Clifford et al. (2006) Cell Cycle 5(22): 2666-70.

Blӓker et al. (2008) Genes Chrom. Cancer 47(2): 159-64.

slide12

WES of desmoids

BMI1 mutation in desmoid

APC

chr 6 del

chr 6 del

APC

?

CTNNB1 mutations

- BMI1 c.175C>G

- 12/142 reads (7.9%)

- Confirmed by

orthogonal 454 sequencing

slide13

WES of desmoids

Whole exome sequencing identifies BMI1 mutation in a ‘wild-type’ desmoid

  • Stem cell marker
  • Positively regulated by Wnt
  • Positive regulator of Wnt
  • BMI1 mutation a rare event –
  • Not detected by Sanger sequencing in any additional (n=96) desmoids

Cho et al. (2013) J. Biol. Chem. 288(5): 3406-18.

conclusions

WES of desmoids

Conclusions
  • Patient outcomes and gene expression profiles associated with ‘wild-type’ desmoids do not differ significantly from those associated with CTNNB1 mutated tumors.
  • Next generation sequencing identifies CTNNB1 mutation in a significant number of ‘wild-type’ desmoids
    • ~95% of tumors can now be associated with mutated CTNNB1 or APC.
  • Genomic events that have potential to regulate Wnt signaling are identified in all desmoids without mutation in CTNNB1 or APC (e.g., chromosome 6 loss, BMI1 mutation).
acknowledgements

WES of desmoids

Acknowledgements

Samuel Singer, M.D., FACS

Meera Hameed, M.D.

Matthew Meyerson, M.D., Ph.D.

Juliann Chmielecki, Ph.D.

Nicholas Socci, Ph.D.

Li-Xuan Qin, Ph.D.

Raya Khanin, Ph.D.

Caitlyn Byrne, Ph.D.

Margaret Leversha, Ph.D.

GouriNanjangud Ph.D.

Mara Rosenberg, Ph.D.

Mono Pirun, Ph.D.

Rachael O’Connor, B.S.

Katherine Thorn, B.A.

Funding:

Kristin Ann Carr Foundation

Cycle for Survival

NCI SPORE in Soft Tissue Sarcoma

American College of Surgeons

Slim Initiative for Genomic Medicine

slide16

WES of desmoids

Clinical characteristics

of patient cohort

  • Frozen samples collected from 2002-2013 (n=110).
  • Clinical characteristics as compared to all surgically resected tumors between 1982 and 2011.
  • Median follow-up 43 months (range 0 -126 months).
slide17

WES of desmoids

34 y.o with 10cm rectus sheath tumor.