Yuri E. Nikiforov Department of Pathology University of Cincinnati

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

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

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

4. BRAF

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

6. P P RBD C Ala The Lys Ser Spectrum of BRAF Point Mutations in Various Tumors Kinase domain Val Glu 463 465 465 585 594 595 596 598 599 601 Gly Gly Gly Leu Phe Gly Leu Val

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 PDC PC,WD DNA DNA BRAF + BRAF + Nikiforova et al. (2003)

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

10. PDC AC N 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 Additional mutations BRAF PC

11. RAS

12. RAS Mutations • 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

13. GDP RAS GTP Pi H O GTP GDP RAS 2 Mechanism of RAS Activation by Point Mutation SOS GAPs GDSs CDC25 Mutations C3G codons 12/13 or 61 Downstream Effectors

14. RET SOS SHC P P GRB2 FRS2 RAS P P PI3K PLC Ral /Cdc42 AKT MEK DAG MEKK1 Rac P70S6K ERK PKC JNK Rho BCL c-Jun, Fos , c- Myc , Elk-1 Molecular Pathways Activated by RAS Y1062 B-RAF BAD Apoptosis

15. Prevalence of RAS Mutations in Thyroid Tumors

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

17. Consequences of RAS Activation in Thyroid Cells 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

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

19. RET/PTC Rearrangement

20. RET/PTC Rearrangements

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

22. Prevalence of RET/PTC in Thyroid Tumors

23. N 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 RET/PTC PC

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

25. PAX8-PPARγ Rearrangement

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

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

28. Prevalence of PAX8-PPARin Thyroid Tumors

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

30. PDC FC AC N Molecular Pathways in Thyroid Follicular Carcinogenesis FC FA RAS 45% PAX8-PPARγ 35%

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

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

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