1. Yuri E. Nikiforov Department of Pathology University of Cincinnati Genetic Alterations Involved in the Transition from Well Differentiated to Poorly Differentiated and Anaplastic Thyroid Carcinomas Endocrine Pathology Companion Meeting

2. Outline  Genetic events in thyroid WDC, PDC, and AC  Molecular evidence for progression BRAF and RAS mutations RET/PTC and PAX8-PPAR  rearrangements p53 and β-catenin mutations  Evidence from LOH studies  Molecular pathways in progression of thyroid CA: Summary

3. Thyroid follicular cell Papillary Carcinoma AC PDC PAX8-PPAR γ BRAF RET/PTC RAS P53 β-catenin RAS Molecular Alterations in Thyroid Tumors Hurthle Cell Carcinoma Follicular Carcinoma

4. BRAF

5. SOS B-RAF RAS GRB2 MEKERK SOS c-Jun,Fos, c-Myc, Elk-1 Signaling Pathways Activated by BRAF in Thyroid Tumors Y1015 Y1062 PLC γ Enigma SHC FRS2 Y1096 P P P RET P P P

6. RBD CKinase domain Spectrum of BRAF Point Mutations in Various Tumors PheGlyLeu AlaThe Val LysSerPP 594595598601596599 Val Glu Leu 585 Gly 465 Gly 465 Gly 463

7. Prevalence of BRAF Mutations in Thyroid Tumors Nikiforova et al. 2003 Cohen et al., 2003 Xu et al., 2003 Namba et al., 2003 Fukashima et al., 2003 Trovisco et al., 2004

8. BRAF Mutations Present in Both Well Differentiated and Poorly Differentiated Carcinoma Areas Nikiforova et al. (2003) DNA BRAF + PC,WD PDC

9. BRAF Mutations in Poorly Differentiated and Anaplastic Carcinomas Nikiforova et al. (2003)

10. BRAF Mutations: Summary  PC with BRAF are prone to dedifferentiation and transformation to PDC and AC  Other genetic mutations are required to direct this process N BRAF PC PDC AC Additional mutations

11. RAS

12. Point mutations found in many human cancers and in most types of thyroid tumors K-RAS, H-RAS, N-RAS genes may be involved Hot spots - codons 12, 13 and 61 N-RAS codon 61 mutations most common in thyroid tumors RAS Mutations

13. Mechanism of RAS Activation by Point Mutation RAS GTPGDP GAPs H 2 OPi GTPGDP SOS GDSs CDC25 C3G Downstream Effectors Mutations codons12/13 or 61

14. Molecular Pathways Activated by RAS RAS GRB2 SOS PLC Ral/Cdc42 DAGPKCAKTRhoRac B-RAF MEKERK PI3KJNK P70S6KMEKK1 BCL BAD Apoptosis c-Jun,Fos, c-Myc, Elk-1 Y1062 SHC FRS2 RET PPPP

15. Prevalence of RAS Mutations in Thyroid Tumors

16. RAS in Progression of Thyroid Tumors: Case report of AC with areas of FC FC AC RAS codon 61 CAA CGA + + p53 codon 189 GCC GTC - + Asakawa & Kobayashi (2002) Mutations found:

17. Development of nodules, adenomas, and carcinomas in transgenic mice Increased cell proliferation but insufficient for complete transformation of cultured cells Increased chromosome instability, i.e. micronuclei, centrosome amplification, chromosome misalignment during mitosis Consequences of RAS Activation in Thyroid Cells

18. RAS Mutations: Summary  Predispose FC and PC to dedifferentiation, likely by increasing genomic instability  Require additional mutations for dedifferentiation N RAS PC PDC AC Additional mutations FC

19. RET/PTC Rearrangement

20. RET/PTC Rearrangements

21. PLC γ Enigma SHC FRS2 SOS GRB2 RAF RAS MEKERK PPPP Y1015 Y1062 RET/PTC c-Jun,Fos, c-Myc, Elk-1 Molecular Pathways Activated by RET/PTC

22. Prevalence of RET/PTC in Thyroid Tumors

23. RET/PTC Rearrangements: Summary  No RET/PTC in anaplastic carcinomas  Data on PDC not entirely conclusive  Likely - PC with RET/PTC have low potential for dedifferentiation/progression N RET/PTC PC

24. Molecular Pathways in Thyroid Papillary Carcinogenesis N RET-PTC 15% 40% BRAF RAS 20% PC PC PC PDCAC

25. PAX8-PPAR γ Rearrangement

26. Structure of PAX8-PPAR  Fusion Protein Kroll et al. (2000)

27. Results from fusion of PAX8 (2q13) and PPAR γ (3p25) genes PAX8-PPAR γ chimeric protein has dominant negative effect on wild-type PPAR γ Wild-type PPAR γ may inhibit thyroid cell growth (tumor suppressor gene) PAX8-PPAR γ Rearrangement

28. Prevalence of PAX8-PPAR  in Thyroid Tumors

29. PAX8-PPAR  Rearrangements: Summary  No RET/PTC in PDC and AC  Likely - FC with PAX8-PPAR γ lack potential for dedifferentiation/ progression N PAX8-PPAR γ FC

30. Molecular Pathways in Thyroid Follicular Carcinogenesis FC PAX8-PPAR γ 35% FA RAS 45% PDCAC N FC

31. Mutations Directing Progression/ Dedifferentiation of Thyroid Tumors: p53

32. Mutations Directing Progression/ Dedifferentiation of Thyroid Tumors: β-catenin

33. Specific Genetic Events in Thyroid Tumors: Summary

34. Molecular Evidence for Progression/Dedifferentiation: LOH Studies In the same tumor, WDC and AC components have similar patterns of allelic loss Increased LOH rate in AC component J. Hunt et al. (2003)

35. Molecular Pathways in Progression of Thyroid Carcinomas: Summary Studies of gene mutations and LOH supports the following progression: WDC PDC AC WD tumors with BRAF and RAS mutations are prone for dedifferntiation, but require additional mutations p53 and possibly β-catenin directly guide progression

36. Nikiforov Lab Marina Nikiforova Zhaowen Zhu Raffaele Ciampi Christy Caudill Manoj Gandhi Acknowledgements James Fagin University of Cincinnati Todd Kroll Emory University Giovanni Tallini