Yuri E. NikiforovDepartment of PathologyUniversity of Cincinnati

Genetic Alterations Involved in the Transition from Well Differentiated to Poorly Differentiated and Anaplastic

Thyroid Carcinomas

Endocrine Pathology Companion Meeting

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

Thyroid follicular

cell

Papillary Carcinoma

AC PDC

PAX8-PPARγ

BRAFRET/PTCRAS

P53β-catenin

RAS

Molecular Alterations in Thyroid Tumors

Hurthle Cell Carcinoma

Follicular Carcinoma

BRAF

SOS

B-RAF

RASGRB2

MEK

ERK

SOS

c-Jun, Fos,c-Myc, Elk-1

Signaling Pathways Activated by BRAF in Thyroid Tumors

Y1015

Y1062PLCγ

Enigma

SHC

FRS2Y1096

P

P

P

RET

P

P

P

RBD C Kinase domain

Spectrum of BRAF Point Mutations in Various Tumors

Phe Gly Leu Ala The Val Lys Ser

P P

594 595 598 601596 599Val Glu

Leu

585

Gly

465

Gly

465

Gly

463

Prevalence of BRAF Mutations in Thyroid Tumors

PC FC PDC AC

Mean

N of cases39%

227/580

0

0/77

13%

2/16

14%

5/35

Nikiforova et al. 2003

Cohen et al., 2003

Xu et al., 2003

Namba et al., 2003

Fukashima et al., 2003

Trovisco et al., 2004

BRAF Mutations Present in Both Well Differentiated and Poorly Differentiated

Carcinoma Areas

Nikiforova et al. (2003)

DNA DNA

BRAF + BRAF +

PC,WD PDC

Prevalence ofBRAF mutations

PDCwith no WD componentwith PC componentwith FC/HCC component

0/82/7 (29%)

0/1

ACwith no WD componentwith PC componentwith FC/HCC component

0/203/5 (60%)

0/4

BRAF Mutations in Poorly Differentiated and Anaplastic Carcinomas

Nikiforova et al. (2003)

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

NNBRAF

PCPC

PDCPDC

ACAC

Additional mutations

RAS

• 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

Mechanism of RAS Activation by Point Mutation

RAS

RAS

GTP

GDP

GAPs

H2O

Pi GTP

GDP

SOSGDSsCDC25

C3G

DownstreamEffectors

Mutationscodons 12/13 or 61

Molecular Pathways Activated by RAS

RASGRB2

SOS

PLC Ral/Cdc42

DAG

PKC

AKT

Rho

Rac

B-RAF

MEK

ERK

PI3K

JNK

P70S6K

MEKK1

BCL

BAD

Apoptosisc-Jun, Fos,c-Myc, Elk-1

Y1062

SHC

FRS2

RET

P

P

P

P

Prevalence of RAS Mutations in Thyroid Tumors

PC FC PDC AC

Mean

N of cases15%

38/253

45%

21/47

24%

14/58

55%

12/22

RAS in Progression of Thyroid Tumors: Case report of AC with areas of FC

FCAC

RAS codon 61

CAA CGA + +

p53 codon 189

GCC GTC - +

Asakawa & Kobayashi (2002)

Mutations found:

• 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

RAS Mutations: Summary Predispose FC and PC to dedifferentiation,

likely by increasing genomic instability Require additional mutations for

dedifferentiation

NNRAS PCPC PDCPDC

ACAC

Additional mutations

FCFC

RET/PTC Rearrangement

RET/PTC Rearrangements

Wild type RET (10q11.2)

EC TM TK

RET/ PTC1

RET / PTC2 andnovel types

RET / PTC3

H4 (10q21)

CC

TK

TK

TKELE1 (10q11.2)

CC

Gene N

CC

PLCγ

Enigma

SHC

FRS2

SOS

GRB2

RAF

RAS

MEK

ERK

PP

PP

Y1015

Y1062

RET/PTC

c-Jun, Fos ,c-Myc , Elk-1

Molecular Pathways Activated by RET/PTC

PC FC PDC ACSantoro (1992) 19%

33/1770

0/370

0/15Wynford-Thomas(1993)

00/45

00/19

Tallini (1998) 40%81/201

00/22

00/15

00/17

Nikiforov(unpublished)

19%18/93

00/46

00/12

00/11

Santoro (2002) 13%8/62

Prevalence of RET/PTC in Thyroid Tumors

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

NNRET/PTC

PCPC

Molecular Pathways in Thyroid Papillary Carcinogenesis

NNRET-PTC

15%15%

40%40%BRAF

RAS

20%20%

PCPC

PCPC

PCPC

PDCPDC ACAC

PAX8-PPARγ Rearrangement

Structure of PAX8-PPAR Fusion Protein

PPARPAX8

PD HD DA/B C E/F

Kroll et al. (2000)

• 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)

PAX8-PPARγ Rearrangement

Prevalence of PAX8-PPAR in Thyroid Tumors

PC FC PDC AC

Mean

N of cases1%

1/144

36%

45/125

0

0/12

0

0/32

PAX8-PPAR Rearrangements: Summary

No RET/PTC in PDC and AC Likely - FC with PAX8-PPARγ lack

potential for dedifferentiation/ progression

NNPAX8-PPARγ

FCFC

Molecular Pathways in Thyroid Follicular Carcinogenesis

FCFC

PAX8-PPARγ35%35%

FAFA

RAS45%45%

PDCPDC ACAC

NN

FCFC

Mutations Directing Progression/ Dedifferentiation of Thyroid Tumors: p53

PC FC PDC AC

Mean

N of cases1%

1/110

5%

1/21

24%

21/89

74%

14/19

Mutations Directing Progression/ Dedifferentiation of Thyroid Tumors:

β-catenin

PC FC PDC ACGarsia-Rostan(2001)

00/46

00/12

25%7/28

66%19/29

Rosha (2003) 00/17

PC FC PDC AC

BRAF 39% 0 13% 14%

RAS 15% 45% 24% 55%

RET/PTC 35% 0 9% 0

PAX8-PPAR 1% 36% 0 0

P53 1% 5% 24% 74%

β-catenin 0 0 16% 66%

Specific Genetic Events in Thyroid Tumors: Summary

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)

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

Nikiforov Lab

Marina Nikiforova

Zhaowen Zhu

Raffaele Ciampi

Christy Caudill

Manoj Gandhi

Acknowledgements

James FaginUniversity of Cincinnati

Todd Kroll Emory University

Giovanni Tallini