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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,
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