Understanding the S45F and T41A mutations in desmoid tumors
D Braggio PhD, R Pollock MD PhD, D Lev MD
The Ohio State University James Cancer Center
Columbus, Ohio
Tel Aviv University Sheba Medical Center
Tel Aviv, Israel
My Desmoid Heroines
Dina Lev, MD Danielle Braggio, PhD
Support: Desmoid Tumor Research Foundation
- Low-grade; locally invasive
- Local recurrence common
- Lack ability to metastasize
Desmoid key clinical features
bone
desmoid 13 operations
Intra-abdominal desmoid tumor
Extra-abdominal desmoid tumor
Progress will require multidisciplinary/molecular approaches
Study n/total %
Tejpar et al., 1999 42/42 100
Abraham et al., 2002 27/33 82
Montgomery et al., 2002 9/10 90
Bhattacharya et al., 2005 21/21 100
Ng et al., 2005 14/17 82
Rakheja et al., 2005 4/12 33
-catenin is the most commonly over-expressed protein in desmoid fibromatosis
Only three known -catenin gene (CTNNB1) mutations
-catenin: a key component of the WNT pathway
We asked if CTNNB1 mutational status correlated with desmoid recurrence
Three known mutations of CTNNB1, the gene underlying -catenin protein production
138 MDACC desmoid patients
(JCO study); primary and recurrent tumors: 85% mutated
15% wild type
Desmoid prognosis: effect of 45F mutation
Possible impact on clinical decision-making???
Entire series (n=138 pts)
45 F mutation: strongest prognostic factor for recurrence
Cancer 119:3696; 2013
Ann Surg Onc, 22:1464; 2015
British Jour Can, 102:1032; 2010
Confirming the role of CTNNB1 45F mutations
Exploring the biology underlying desmoid tumors
Study hypothesis
Desmoid tumors harboring the 45F CTNNB1
mutation are resistant to apoptosis, possibly
contributing to the observed decreased recurrence
free survival in these patients
Desmoid cell strains isolated
at MDA/OSU
Confirmed as DT cells via CTNNB1
genotyping of cells and original tumors
TUMOR CTNNB1
Des1 T41A
Des2 T41A
Des4 WT
Des6 S45F
Des7 T41A
Des8 WT
Des9 T41A
Des10 T41A
Des11 S45F
Des14 S45F
Des18 S45F
Des19A S45P
Des20 T41A
Des24 T41A
Des27 T41A
Des38 WT
Des39 T41A
Des40 S45F
Des41 T41A
Des42 T41A
Des43 T41A
Des44 S45P
Des45 T41A
Des50 T41A
Des51 T41A
Des39B T41A
Des26 T41A
Des46 T41A
Des47 H36P
Des55 WT
Des65 T41A
Des67 T41A
OSU-Des1 S45F
OSU-Des16 S45F
OSU-Des17 T41A
OSU-Des9 WT
OSU-Des13 S45F
OSU-Des23 S45F
OSU-Des33 S45P
OSU-Des34 T41A
OSU-Des44 T41A
OSU-Des47 S45P
OSU-Des68 S34R
OSU-Des78 T41A
OSU-Des23.2 S45F
OSU-Des76 T41A
OSU-Des91 T41A
OSU-Des93 WT
OSU-Des104 T41A
OSU-Des113 S45P
OSU-Des119 T41A
OSU-Des101 T41A
OSU-Des128 S45P
OSU-Des124 , OSU-Des130 (124 and 130 not yet genotyped)
A powerful research tool: desmoid TMA
• 195 desmoid specimens
115 primary
80 recurrent
25 primary/recurrent pairs
20 scars (control)
• Clinically annotated
• CTNNB1 mut. status known
T41A S45F
; p = 0.3979
CTNNB1 mut T41A vs S45F functional differences not due to differing -catenin protein expression
Apoptotic genes are differentially expressed
in S45F mut desmoid tumors
Initial gene array with 60 desmoid tumor tissues: 13 S45F mut and 12 T41A mut
Apoptotic genes are differentially expressed
in S45F mut desmoid tumors
** = P > 0.001
Apoptosis-promoting genes Apoptosis-inhibiting genes
**
**
N=27 N=27
S45F mut cells: decreased apoptosis activation
1.0
2.9
5.3 5.9
6.7
1.0 1.5 1.8 2.0 1.9
0
2
4
6
8
0 24 48 72 96
T41A
S45F
Cle
av
ed c
asp
ase
(fold
ch
an
ge)
Time (h)
A
T41 S45F
T41
A
S45F
**
S45F mut cells: decreased apoptosis activation
P >0.001
DMSO
Doxorubicin
(0.2mg/mL)
21.5%
41.6%
OSU-Des76 OSU-Des91
23.8%
51.6%
Des67
7.2%
26.5%
Des65
4.9%
16.6%
Des14
6.2%
6.5%
OSU-Des13
5.1%
5.6%
OSU-Des23
6.8%
7.7%
OSU-Des23.2
6.2%
10.1%
No significant induction of apoptosis in S45F mut DT cells after doxirubicin treatment
T41 S45F
Transfection of mut -catenin genes into 293T cells
mirrors biology observed in desmoid cell strains
S45F transfected 293T cells: decreased apoptosis
1.0 3.2
7.0
14.4
18.1
1.0 1.5 3.2 3.0
1.9
0.0
5.0
10.0
15.0
20.0
0h 24h 48h 72h 96h
293T T41A
293T S45F
Cle
av
ed c
asp
ase
(fo
ld c
ha
ng
e)
Time (h)
293T/ T41A 293T/S45F
293T/ T41A 293T/S45F S45F
S45F transfected 293T cells: decreased apoptosis
** = P >0.001
**
-catenin knockdown decreases desmoid growth--new windows for exploration?
CTNNB1 siRNA:
Reduced protein
Reduced proliferation
Reduced migration
Reduced invasion
Ubiquity of -catenin precludes direct targeting
Biopsy proven desmoid tumor
DNA sequencing CTNNB1
pts with 45F mutation
Arm 1: surgery alone Arm 2: pre-op RT followed by surgery or surgery followed by postop RT
Primary endpoint: recurrence status at 36 months Investigative team: Meng Welliver (Radiation Oncology) Raphael Pollock (Surgical Oncology) Xiaokui Mo (Center for Biostatistics)
OSU randomized Phase II clinical trial (concept)
Randomization
Although the pathway to progress in sarcoma is not always clear…
The clarity we seek will come by working together!
So I’m looking forward to our shared next steps!
Many thanks to our sarcoma colleagues!
Pathology Hans Iwenofu, MD Paul Wakely, MD
Radiation Oncology Douglas Martin, MD
Meng Welliver, MD, PhD Karl Haglund, MD
Medical Oncology David Liebner, MD
James Chen, MD, PhD
Orthopedic Oncology Thomas Scharschmidt, MD
Joel Mayerson, MD
P & RS Ian Valerio, MD
Jeffrey Janis, MD
Neurosurgery
Ehud Mendel, MD
Surgical Oncology Harrison Howard, MD
Valerie Grignol, MD
Sarcoma Research Laboratory Gonzalo Lopez, PhD Kate Lynn Bill, PhD Lucia Casadei, PhD
Danielle Braggio, PhD Abby Zewdu, BS Kara Batte, PhD
Anne Strohecker, PhD Dina Lev, MD
Nationwide Childrens Hospital
Timothy Cripe, MD, PhD Ryan Roberts, MD, PhD
Thank you for your attention; questions or comments?
Sunset over Lake Michigan at Beverly Shores, Indiana; 7/23/16