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

**

Therapeutic implications???

-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