Transcript Whole

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
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.
WES of desmoids
Sanger sequencing of CTNNB1
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Frozen samples collected from 20022013 (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 .
‘wild-type’
ACC
T41
T41A mutant
AG
T41A
S45
S45F mutant
CT
TCT
S45
T41
S45F
WES of desmoids
CTNNB1 mutation is not clearly associated with
outcome
CTNNB1 mutation and tumor location
S45P
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
T41A
none
S45F
None vs. T41A – n.s.
None vs. S45F – n.s.
T41A vs. S45F – p<0.05
p=0.001
abdominal wall
Multivariate analysis
intraabdominal
chest
extremity
other
Extremity vs. abdominal wall p<0.05
Extremity vs. intraabdominal p<0.05
Mutation (vs. T41A)
None
S45F
Extremity vs.
Non-extremity
Univariate
Multivariate
HR
p
HR
p
1.31
2.66
0.66
0.025
1.14
1.68
0.83
0.29
3.31
0.001
2.58
0.029
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’
WES of desmoids
Whole exome sequencing of desmoid tumors
17 tumor samples.
• 9 with no mutation
in CTNNB1 – ‘wildtype’ 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
WES of desmoids
Somatic mutation
in desmoid tumors
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46 significant mutations
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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.
*
*
WES of desmoids
Whole exome sequencing of wild-type desmoid tumors
known
CTNNB1
mutation
‘wild-type’
tumors
Sample
CTNNB1
mutation
mutant allele
frequency
DES_1008
DES_884
DES_888
DES_931
DES_936
DES_938
DES_940
DES_926
DES_881
DES_1003
DES_956
DES_961
DES_890
DES_975
DES_955
DES_999
DES_1002
S45F
T41A
T41A
S45F
T41A
S45F
T41A
S45F
H36del
T41A
T41A
T41A
None
None
None
None
None
32%
36%
33%
32%
22%
46%
54%
38%
30%
10%
21%
10%
N/A
N/A
N/A
N/A
N/A
Sample
APC mutation
mutant allele
frequency
DES_890
DES_975
I1918fs
K1462fs
61%
70%
average 37% reads
average 16% reads
Only 3 of nine ‘wild-type’ tumors without CTNNB1 or APC mutation.
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
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.
X
Y
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
CTNNB1
mutations
APC mutation
Wild-type
CEP6 staining (red)
Chromosome 6
CTNNB1
mutations
24% nuclei
with single
chromosome 6
by FISH
APC mutation
Wild-type
- CapSeg algorithm from Broad
56% nuclei
with single
chromosome 6
by FISH
X
Y
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
?
chr 6 del
APC
CTNNB1 mutations
APC
chr 6 del
BMI1 mutation in desmoid
- 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
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.
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
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
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).
Characteristic
Age
15-25y.o.
26-45y.o.
46-65y.o.
>65y.o.
Location
Abdominal wall
Chest wall
GI/Intraabdominal
Extremity
Other
Primary
Margin
R0
R1
R2
Unknown
Size
<=5cm
>5cm, <=10cm
>10cm
Unknown
FAP
Whole
cohort
(n=495)
Sequenced
samples
(n=110)
95 (19%)
244 (49%)
110 (22%)
46 (9.3%)
17 (15%)
62 (56%)
24 (22%)
7 (6.4%)
88 (18%)
76 (15%)
100 (20%)
177 (46%)
54 (11%)
382 (77%)
19 (17%)
12 (11%)
32 (29%)
34 (31%)
13 (12%)
85 (77%)
267 (54%)
173 (35%)
63 (13%)
2 (0.40%)
50 (45%)
52 (47%)
8 (7.2%)
0 (0%)
148 (30%)
200 (40%)
125 (25%)
22 (4.5%)
15 (3.0%)
20 (18%)
47 (43%)
42 (38%)
1 (0.91%)
2 (1.8%)
WES of desmoids
34 y.o with 10cm rectus sheath tumor.
WES of desmoids
454 sequencing – signal to noise ratio
BMI1 c.175C>G