Benjamin Misemer, Amy Skubitz, Carlos Manivel,

1. Identification of Heterogeneity in Aggressive Fibromatosis: Validation by immunohistochemistry of markers identified by differential gene expression analyses Benjamin Misemer, Amy Skubitz, Carlos Manivel, Stephen Schmechel, Edward Cheng, Jonathan Henriksen, Joseph Koopmeiners, and Keith Skubitz Masonic Cancer Center University of Minnesota Minneapolis

2. Aggressive Fibromatosis (AF) • Characterized by a monoclonal proliferation of myofibroblasts • AF has a variable clinical course • Optimal treatment is unclear • WNT pathway, or beta-catenin pathway, has been implicated in pathogenesis

3. AF – earlier studies Four genes (ADAM12, FAP-alpha, WISP-1, and Sox-11) were uniquely over-expressed in AF compared with 448 samples comprising 16 different normal tissues Skubitz, et al J Lab Clin Med 144:193-200, 2004.

4. FAP-alpha • Integral membrane serine protease expressed in reactive stromal fibroblasts • FAP inhibition inhibits tumor growth in some mouse models

5. WISP-1 (WNT-inducible signaling protein-1) • Growth factor that regulates diverse cellular functions • Enhances ECM deposition in fibroblasts • Up-regulated in Idiopathic Pulmonary Fibrosis • Up-regulated in a mouse model of bleomycin-induced lung fibrosis, where anti-WISP-1 attenuates fibrosis

6. SOX-11 • Transcripton factor • Accelerates in vivo nerve regeneration

7. ADAM-12 • A disintegrin and metalloprotease implicated in cell-cell and cell-matrix interactions

8. ADAM-12 • Dulauroy et al recently identified ADAM12+ perivascular cells as a major source of profibrotic cells during acute tissue injury. These were a subset of PDGFR-alpha+ cells.

9. AF – earlier studies Gene expression patterns suggested two distinct subgroups of AF; this may reflect disease biology Skubitz, et al J Lab Clin Med 144:193-200, 2004.

11. Methods • To confirm protein expression in AF, immunohistochemistry (IHC) was performed on 29 AF cases

14. Results • IHC revealed protein expression of each of the four probes. • ADAM12 staining in AF has been reported (Hajibashi et al, and Colombo et al.)

15. FAP-alpha

16. Methods • Automated imaging analysis was performed to quantify: • IHC staining • Nuclear size • Nuclear chromatin density

17. ROI = Region of interest

18. Methods • A nuclear chromatin density ratio (CDR) was defined to serve as a surrogate for nuclear activity = Nuclear area of light hematoxylin staining Nuclear area of dark staining

20. Results • Nuclear size and chromatin density ratio (CDR) were strongly correlated (p<0.001). • Thus, CDR is a reasonable surrogate for nuclear size.

21. Methods • A morphologic tumor activity score (MTA) was determined on 322 regions of interest (ROI) and correlated with nuclear size and CDR

22. MTA Score

23. Results • Both average nuclear size and CDR correlated with MTA score (p<0.001).

24. Methods • The relationship between CDR and IHC staining intensity was assessed for each of the 4 proteins on ~26,000 ROIs from the 29 AF samples

25. Results • Image analysis revealed moderate correlations between intensity of staining and CDR • ADAM12 r = 0.44, p<0.001 • FAP-alpha r = 0.39, p<0.001 • WISP-1 r = 0.27, p<0.001 • No significant correlation for SOX-11

26. Conclusions • Confirmed protein expression of ADAM12, FAP, WISP-1, and SOX-11 in AF, found in our earlier gene expression studies • Prominent regional variation of morphologic tumor activity is present within and among AF cases

27. Conclusions • These four proteins were more highly expressed in areas where cellular morphology suggested more active tumor cells. • These proteins may be involved in AF pathogenesis

28. University of Minnesota • Sarah Bowell • Denis Clohisy • Stefan Pambuccian • Roby Thompson