The desmoid tumor proteome: identifying molecular markers using a clinically annotated tissue microarray Shohrae Hajibashi, Wei-Lien Wang, Alexander J.F. Lazar, Daniel Tuvin, Carla L. Warneke, Dolores Lopez-Terrada, Raphael E. Pollock, Dina Lev Sarcoma Research Laboratory Houston, TX

Unresolved 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 gap

Formalin-fixed, paraffin-embedded desmoid tumors (195 specimens from 160 patients; ) Clinical information including: – Demographic – Therapeutic – Clinical outcome UTMDACC desmoid tissue 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 TMA

Clinical 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 identifying over-expressed proteins Marker% Positive% Low-Mod/High  -catenin 9858/40 p539981/18 ER-  10048/52 PDGFR-α9573/22 PDGFR-  9818/80 c-kit00 PDGF A2222/0 PDGF B9271/21

Example: high  -catenin TMA nuclear expression correlates with outcome TMA: ~98% of the desmoids showed nuclear reactivity p= High, n=49 Low-moderate, n=40

p= Specific mutations in the  -catenin gene ( CTNNB1 ) correlate with local recurrence in sporadic desmoid tumors Am J Path. 173(5): ; Nov (17%) 20 (22%) 47 (53%)

Next steps…an algorithm for desmoid 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” identification of potential desmoid- related/specific over- expressed genes Human exonic evidence based oligonucleotide (HEEBOChip) microarray 10 desmoid tumor samples (20 additional specimens; van de Rijn/West; Stanford)

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

2. Confirm that gene over-expression leads to protein over production (TMA) ADAM12MDK 100% (+)43% (+) DesmoidScarDesmoidScar

3 & 4. Examine ADAM12 as a prognostic marker; possible target 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 prognostic marker; possible target for (future) desmoid therapy? Des 15NHFDes 12Des 2 ADAM12 Actin Anti-ADAM12 agents in pipe line… ADAM12 over-expressed in desmoid cell cultures

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

Acknowledgements 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