1. 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

2. Desmoid fibromatosis Aim:
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 -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.

3. Sanger sequencing of CTNNB1
WES of desmoids
Sanger sequencing of CTNNB1
Frozen samples collected from (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
S45
T41
S45F

4. CTNNB1 mutation is not clearly associated with outcome
WES of desmoids
CTNNB1 mutation is not clearly associated with outcome
CTNNB1 mutation and tumor location
S45P
T41A
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%) 15
S45P
1 (20%)
1 (20%)
3 (60%) 5
32 (32%)
19 (19%)
49 (49%)
100
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
Univariate
Multivariate HR p
Mutation (vs. T41A)
None
S45F
1.31
2.66
0.66
0.025
1.14
1.68
0.83
0.29
Extremity vs.
Non-extremity
3.31
0.001
2.58
0.029
intraabdominal
chest
extremity
other
Extremity vs. abdominal wall p<0.05
Extremity vs. intraabdominal p<0.05

5. based on gene expression
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’

6. Whole exome sequencing of desmoid tumors
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

7. Somatic mutation in desmoid tumors
WES of desmoids
Somatic mutation in desmoid tumors
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. * *

8. Whole exome sequencing of wild-type 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
33%
DES_931
DES_936
22%
DES_938
46%
DES_940
54%
DES_926
38%
DES_881
H36del
30%
DES_1003
10%
DES_956
21%
DES_961
DES_890
None
N/A
DES_975
DES_955
DES_999
DES_1002
APC mutation
I1918fs
61%
K1462fs
70%
known CTNNB1 mutation
average 37% reads
average 16% reads
‘wild-type’ tumors
We were able to detect b-catenin mutations in 12/17 desmoid tumors, including 3 mutations not detected by clinical testing.
We also found an additional 2 APC mutations (b-catenin pathway) in 2/17 tumors; mutually exclusive with b-cat mutations.
Therefore, 14/17 tumors show b-catenin activation. RNA-seq confirms that b-catenin is highly expressed in both mutant and WT tumors, suggesting that they all rely on this pathway.
Only 3 of nine ‘wild-type’ tumors without CTNNB1 or APC mutation.

9. Copy number alterations in desmoid fibromatosis
WES of desmoids
Copy number alterations in desmoid fibromatosis
Chromosome 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.

10. Chromosome 6 loss in desmoid fibromatosis
WES of desmoids
Chromosome 6 loss in desmoid fibromatosis
Chromosome X Y
CTNNB1
mutations
APC mutation
Wild-type
Chromosome 6
CEP6 staining (red)
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

11. 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):
Blӓker et al. (2008) Genes Chrom. Cancer 47(2):

12. BMI1 mutation in desmoid
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
Depth
Forward
Reverse
Reference (Cytosine)
81014
(96.7%)
36784
44230
Alternate (Guanine)
2754
(3.28%)
1303
1451
Total
83898
38087
45681

13. 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):

14. 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).

15. Acknowledgements Funding: Samuel Singer, M.D., FACS Meera Hameed, M.D.
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.
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 Foundation
Cycle for Survival
NCI SPORE in Soft Tissue Sarcoma
American College of Surgeons
Slim Initiative for Genomic Medicine

16. Sequenced samples (n=110)
WES of desmoids
Clinical characteristics
of patient cohort
Characteristic
Whole 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
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 (n=110).
Clinical characteristics as compared to all surgically resected tumors between 1982 and 2011.
Median follow-up 43 months (range months).

17. 34 y.o with 10cm rectus sheath tumor.
WES of desmoids
34 y.o with 10cm rectus sheath tumor.

18. 454 sequencing – signal to noise ratio
WES of desmoids
454 sequencing – signal to noise ratio
BMI1 c.175C>G