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 SocietyOctober 31, 2013

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 -catenin observed 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.

WES of desmoids

WES of desmoids

• 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 .

S45F

Sanger sequencing of CTNNB1

CT

T41S45T41

S45F mutant‘wild-type’

T41A mutant

S45T41A

AG

ACCTCT

Univariate Multivariate

HR p HR p

Mutation (vs. T41A)NoneS45F

1.312.66

0.660.025

1.141.68

0.830.29

Extremity vs. Non-extremity 3.31 0.001 2.58 0.029

p=0.001

CTNNB1 mutation and tumor location

None vs. T41A – n.s.None vs. S45F – n.s.

T41A vs. S45F – p<0.05

S45F

T41A

S45P

none

WES of desmoids

Extremity Abdominal wall

Other Total

S45F 19 (59%) 4 (12%) 9 (28%) 32

T41A 9 (19%) 8 (17%) 31 (65%) 48

None 3 (20%) 6 (40%) 6 (40%) 15

S45P 1 (20%) 1 (20%) 3 (60%) 5

Total 32 (32%) 19 (19%) 49 (49%) 100

Multivariate analysis

CTNNB1 mutation is not clearly associated with outcome

extremity other

chest

intraabdominal

abdominal wall

Extremity vs. abdominal wall p<0.05Extremity vs. intraabdominal p<0.05

Unsupervised clustering based on gene expression

47 desmoid tumors

Evaluated by U133A 2.0 microarrays

S45F – RedT41A – Blue‘wild-type’ – BlackNo S45P included

WES of desmoids

7/36 ‘wild-type’ 4/11 ‘wild-type’

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

Illumina HiSeq flowcells

87% of the exomes were covered >88x

Range 4-29 mutations per sample

29Mb sequenced per tumor

<1 mutation/Mb

• 46 significant mutations• Called by MuTect• Significance by MutSig

• CTNNB1 mutation in 12/17 tumors (71%) not just 8 as expected based on Sanger.

• 2 tumors with APC mutation.

Somatic mutation in desmoid tumors

WES of desmoids

* *

Whole exome sequencing of wild-type desmoid tumors

Sample CTNNB1 mutation

mutant allele frequency

DES_1008 S45F 32%

DES_884 T41A 36%

DES_888 T41A 33%

DES_931 S45F 32%

DES_936 T41A 22%

DES_938 S45F 46%

DES_940 T41A 54%

DES_926 S45F 38%

DES_881 H36del 30%

DES_1003 T41A 10%

DES_956 T41A 21%

DES_961 T41A 10%

DES_890 None N/A

DES_975 None N/A

DES_955 None N/A

DES_999 None N/A

DES_1002 None N/A

Sample APC mutation mutant allele frequency

DES_890 I1918fs 61%

DES_975 K1462fs 70%

known CTNNB1 mutation

‘wild-type’ tumors

average 37% reads

average 16% reads

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

WES of desmoids

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

APC

CTNNB1mutations

APC mutation

Wild-type

CTNNB1mutations

APC mutation

Wild-type

Copy number alterations in desmoid fibromatosis

- 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.

WES of desmoids

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

Chromosome 6

CTNNB1mutations

APC mutation

Wild-type

CTNNB1mutations

APC mutation

Wild-type

Chromosome 6 loss in desmoid fibromatosis

- CapSeg algorithm from Broad

24% nuclei with single

chromosome 6 by FISH

56% nuclei with single

chromosome 6 by FISH

CEP6 staining (red)

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.

WES of desmoids

BMI1 mutation in desmoid

CTNNB1 mutations APC

APC

chr 6

del

chr 6

del

?

- BMI1 c.175C>G- 12/142 reads (7.9%)

- Confirmed by orthogonal 454 sequencing

WES of desmoids

Depth Forward Reverse

Reference (Cytosine)

81014(96.7%)

36784 44230

Alternate (Guanine)

2754(3.28%)

1303 1451

Total 83898 38087 45681

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.

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).

WES of desmoids

AcknowledgementsSamuel Singer, M.D., FACSMeera 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.Gouri Nanjangud Ph.D.Mara Rosenberg, Ph.D.Mono Pirun, Ph.D.Rachael O’Connor, B.S.Katherine Thorn, B.A.

Funding:Kristin Ann Carr FoundationCycle for Survival NCI SPORE in Soft Tissue SarcomaAmerican College of SurgeonsSlim Initiative for Genomic Medicine

WES of desmoids

CharacteristicWhole cohort(n=495)

Sequenced samples (n=110)

Age 15-25y.o. 26-45y.o. 46-65y.o. >65y.o.

95 (19%)244 (49%)110 (22%)46 (9.3%)

17 (15%)62 (56%)24 (22%)7 (6.4%)

Location Abdominal wall Chest wall GI/Intraabdominal Extremity Other

88 (18%)76 (15%)

100 (20%)177 (46%)54 (11%)

19 (17%)12 (11%)32 (29%)34 (31%)13 (12%)

Primary 382 (77%) 85 (77%)Margin R0 R1 R2 Unknown

267 (54%)173 (35%)63 (13%)2 (0.40%)

50 (45%)52 (47%)8 (7.2%)0 (0%)

Size <=5cm >5cm, <=10cm >10cm Unknown

148 (30%)200 (40%)125 (25%)22 (4.5%)

20 (18%)47 (43%)42 (38%)1 (0.91%)

FAP 15 (3.0%) 2 (1.8%)

• 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).

Clinical characteristics of patient cohort

WES of desmoids

34 y.o with 10cm rectus sheath tumor.

WES of desmoids

454 sequencing – signal to noise ratio

BMI1 c.175C>G

WES of desmoids