The desmoid tumor proteome: The desmoid tumor proteome: identifying molecular markers identifying molecular markers

using a clinically annotated tissue using a clinically annotated tissue microarraymicroarray

Shohrae Hajibashi, Wei-Lien Wang, Alexander J.F. Lazar, Daniel Tuvin, Carla L. Warneke,

Dolores Lopez-Terrada, Raphael E. Pollock, Dina Lev

Sarcoma Research LaboratoryHouston, TX

Unresolved clinical issuesUnresolved clinical issues

• Lack of prognostic markers

• Need for better therapeutics

• Impediments to progress:– Relative rarity– Limited collected human specimens– No cell lines– No animal models

• Molecular determinants not well understood

Bridging the biological gapBridging the biological gap

• Formalin-fixed, paraffin-embedded desmoid tumors (195 specimens from 160 patients; 1985-2005)

• Clinical information including:– Demographic– Therapeutic– Clinical outcome

UTMDACC desmoid tissue UTMDACC desmoid tissue microarray (TMA)microarray (TMA)

• Automated TMA apparatus: 0.6-mm punch samples (2/case) formatted into three recipient blocks

• 195 specimens: 110 primary/ 85 recurrent; 27 autologous pairs; 18 scars

• H&E-staining of 4-µm TMA sections used to verify all samples

UTMDACC desmoid TMAUTMDACC desmoid TMA

Clinical annotationClinical annotation

•Gender: F 62%; M 38%

•Median age: 32 yr old

•Site

Superficial trunk 39%

Extremity 38%

Deep trunk/mesentery 13%

Head and neck 10%

•Median size: 6 cm

•FAP associated: 8%

Desmoid TMA: useful for Desmoid TMA: useful for identifying over-expressed identifying over-expressed

proteinsproteins

Marker % Positive

% Low-Mod/High

-catenin 98 58/40

p53 99 81/18

ER- 100 48/52

PDGFR-α 95 73/22

PDGFR- 98 18/80

c-kit 0 0

PDGF A 22 22/0

PDGF B 92 71/21

Example: high Example: high -catenin TMA -catenin TMA nuclear expression correlates with nuclear expression correlates with

outcomeoutcome

TMA: ~98% of the desmoids showed nuclear reactivity

p=0.0406

High, n=49

Low-moderate, n=40

Recurrence-Free Survival by Mutation

Time (Years)

Pro

po

rtio

n R

ecu

rre

nce

Fre

e

0 5 10 15 20

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0no mutation4145t

p=0.0002

Specific mutations in the -catenin gene (CTNNB1) correlate with local recurrence

in sporadic desmoid tumorsAm J Path. 173(5):1518-27; Nov 2008

15 (17%)

20 (22%)

47 (53%)

Next steps…an algorithm for Next steps…an algorithm for desmoid investigationdesmoid investigation

1. “Up front” identification of potential desmoid-related/specific over-expressed genes

2. Confirm that gene over-expression leads to protein over production (TMA; different samples)

3. Validate these proteins as prognostic markers

4. Examine as possible targets for therapy

1. “Up front” 1. “Up front” identification of identification of potential desmoid-potential desmoid-related/specific related/specific over-expressed over-expressed genesgenes

• Human exonic evidence based oligonucleotide (HEEBOChip) microarray

• 10 desmoid tumor samples (20 additional specimens; van de Rijn/West; Stanford)

1. “Up front” identification of 1. “Up front” identification of potential desmoid-related/specific potential desmoid-related/specific over-expressed genesover-expressed genes

2. Confirm that gene over-2. Confirm that gene over-expression leads to protein over expression leads to protein over production (TMA)production (TMA)

ADAM12 MDK

100% (+) 43% (+)

DesmoidScarDesmoidScar

3 & 4. Examine ADAM12 as a 3 & 4. Examine ADAM12 as a prognostic marker; possible target prognostic marker; possible target for (future) desmoid therapy?for (future) desmoid therapy?• ADAM12 protein: disintegrin and metalloprotease; up-regulated in

many human cancers

• Role in tumor progression– ECM remodeling– resistance to apoptosis– prognostic marker: breast/prostate cancers

3 & 4. Examine ADAM12 as a 3 & 4. Examine ADAM12 as a prognostic marker; possible target prognostic marker; possible target for (future) desmoid therapy?for (future) desmoid therapy?

Des 15NHF Des 12 Des 2

ADAM12

Actin

Anti-ADAM12 agents in pipe line…

ADAM12 over-expressed in desmoid cell culturesADAM12 over-expressed in desmoid cell cultures

Conclusions and implications:Conclusions and implications:

• Needed: comprehensive strategies to identify desmoid-related prognostic markers and therapeutic targets

• High throughput gene/protein expression arrays for target identification: a potential approach

• TMA studies: intense nuclear -catenin expression associates with less aggressive desmoid behavior

• ADAM12: highly expressed in desmoids; currently being studied as a prognostic factor and therapeutic target

AcknowledgementsAcknowledgements

• The Sarcoma Research Laboratory– D. Lev, MD– R. Pollock, MD/PhD– A. Lazar, MD/PhD– Colleagues and staff

• Stanford University Medical Center– M. van de Rijn, MD/PhD– R. West, MD/PhD

• Desmoid Tumor Research Foundation