1. Cytogenetic Insights in Mesenchymal Tumors Jonathan A. Fletcher, M.D. Pathology & Pediatrics Brigham & Women’s Hospital Dana-Farber Cancer Institute Harvard Medical School Boston, MA

2. How it is done!

3. Mince, then disaggregate cells by overnight treatment with collagenase

4. Disaggregated cells are plated as monolayer cultures on glass slides or in plastic flasks

5. All cultures are inspected daily, to determine whether tumor cells are growing, and when metaphase harvests should be performed

6. Leiomyoma: simple karyotype with t(12;14) HMGA2 (HMGIC)

7. Leiomyosarcoma: G-banded karyotype Complex! Clonal (arrows) and nonclonal aberrations

8. Example 1 Novel biologic mechanisms revealed through indentification of recurrent cytogenetic abnormalities in mesenchymal tumors

9. Aneurysmal Bone Cyst and the fusion fusion oncogene

11. Aneurysmal Bone Cyst Patients are generally < 20 years old Can recur locally, but do not become malignant “Primary ABC” have been generally regarded as nonneoplastic “Secondary ABC” associated with –osteoblastoma –chondroblastoma –giant cell tumor –osteosarcoma

12. Aneurysmal Bone Cyst 1999: Panoutsakopoulos et al. reported translocation t(16;17) in two ABC –neoplastic basis –recurrent oncogenic mechanism

13. Aneurysmal Bone Cyst 17p13 rearrangements in: –“solid variants” of ABC –soft-tissue ABC 25% of ABC have t(16;17) >25% of ABC have alternate translocations, involving 17p13, but not 16q22

14. Aneurysmal Bone Cyst t(16;17) –17p13 gene = USP6 (Ewing’s oncogene) –16q22 gene = CDH11 (aka “osteoblastic cadherin”) –promoter swapping between CDH11 and USP6 fusion of highly active CDH11 promoter to the 5’ UTR of USP6

15. Aneurysmal Bone Cyst Corroboration of “promoter swapping” mechanism in USP6 fusions TranslocationGene Promoter Swapping? t(9;17)osteomodulinYES t(17;17)COL1A1YES t(1;17)TRAP150YES t(3;17)ZNF9YES

16. Aneurysmal Bone Cyst What is the neoplastic cell? Mechanisms in secondary ABC?

17. USP6 oncogene in ABC spindle-cells

18. USP6 oncogene is not found in “secondary ABC” Secondary ABC Associated Chondroblastoma

19. USP6 or CDH11 Rearrangement in ABC 36 of 52 (69%) primary ABC 0 of 17 secondary ABC –giant cell tumor –osteoblastoma –chondroblastoma –fibrous dysplasia

20. USP6 is an evolutionarily-recent fusion of the PRC17 and USP32 genes (hominoid specific) USP6 PRC17USP32 TBC (rabGAP)UBP TBC (rabGAP)UBP >95% identity

21. USP6 Function: regulates endocytosis/destruction of activated proteins TBC (rabGAP)UBP - inactivates rab family members -rab function required for endocytosis of activated EGFR -UBP protease reverses ubiquitination - ?synergize with rabGAP function to inhibit endocytosis/proteolysis CDH 11

22. USP6 Expression Normal expression restricted to embryonic tissues and testis Neoplastic expression restricted to mesenchymal tumors: –2 of 2 osteoblastomas –1 of 4 myofibromas –1 of 3 Ewing’s sarcomas

23. Conclusions USP6 is overexpressed due to promoter swapping mechanisms in most primary ABC USP6 overexpression may stabilize oncogenic proteins USP6 is an evolutionarily recent gene, with likely relevance in sarcoma Useful models of mesenchymal tumor biology can come from unlikely places

24. Example 2 Smooth Muscle Tumors Use of cytogenetic clues to identify clinically-relevant biologic pathways in a genetically complex disease

25. Leiomyoma: simple karyotype with t(12;14) HMGA2 (HMGIC)

26. Leiomyosarcoma: G-banded karyotype

27. How do leiomyomas progress to malignancy? ???

28. Intravenous Leiomyomatosis Typical uterine leiomyoma Balanced t(12;14) Intravenous leiomyomatosis Unbalanced t(12;14) Partial trisomy 12q Paola Dal Cin Brad Quade Cynthia Morton t(12;14) with: partial trisomy 12q partial deletion 14q

29. Cytogenetic correlates for leiomyoma progression Vascular invasion –intravenous leiomyomatosis –unbalanced t(12;14) Increased proliferation –cellular leiomyoma –deletion 1p (also common in lms) Distant metastases –“benign metastasizing leiomyoma” –deletions of 19q and 22q

30. Pulmonary Chondroid Hamartoma (HMAG2 & HMGA1 oncogenes)

31. PCH: primitive mesenchymal, fat, chondroid

32. PCH: primitive mesenchymal, fat, chondroid, smooth muscle

33. Leiomyosarcoma/Leiomyoma: where do they start?

34. Is there proof that any sarcoma arises from a differentiated mesenchymal cell? osteo – bone chondro – cartilage lipo – fat leiomyo – smooth muscle rhabdo – skeletal muscle fibro – myofibroblast “GIST” – interstitial cell of Cajal NO

35. Andre Oliveira Paola Dal Cin Cynthia Morton Marisa Nucci Anette Duensing Chang-Jie Chen Nora Joseph Bryna Mcconarty Felicity Smith Lynn Yu Christopher Hubert Maureen Thyne Vicki Derr Stana Weremowicz George Demetri Christopher Fletcher Sam Singer Antonio Perez-Atayde Mark Gebhardt Andrew Rosenberg Julia Bridge

36. THANK YOU!!!