BRAF mutation

Molecular genetic analysis
and diagnostics
of thyroid cancer
Bela Bendlova, Sarka Dvorakova, Vlasta Sykorova,
Eliska Vaclavikova, Tereza Halkova, Josef Vcelak
Dept. of Molecular Endocrinology, Institute of Endocrinology, Prague
Thyroid carcinomas
Parafollicular cell
Medullary
MTC
< 10%
Follicular cell
Papillary
PTC
> 80%
Follicular
FTC
10-20%
Anaplastic
ATC
2-5%
Incidence
1986
Chernobyl
26. 4. 1986
Causes of increasing incidence of thyroid cancer, especially PTC:

Improvement of diagnosis, microcarcinoma

Changes in diagnosis

Over-supply iodine

Chernobyl accident (26 April 1986)
Genetics of thyroid carcinomas

Proto-oncogenes and tumor-suppresor genes
 Chromosomal rearrangements:
 RET/PTC, NTRK1, PAX8/PPARγ,
BRAF/AKAP9
 Point mutations:
 Germ-line mutations in the RET
gene in FMTC and MEN2 syndromes
 Somatic mutations in the BRAF
and RAS genes, in PDTC and ATC
also TP53, CTNNB1, PIK3CA, PTEN
and AKT1
Factors in the development of DTC
Landa I , Robledo M J Mol Endocrinal 2011;47:R43-R58
Genetic markers

Diagnosis of disease

More accurate prognosis

Prediction of treatment reaction

Target gene therapy
Target therapy – inhibitors of tyrosine kinases
VANDETANIB
Keefe S M et al. Clin Cancer Res 2010;16:778-783
Genetics of PTC
Extracellular
space
Cytoplasm RET/PTC
Growth
factor
Tyrosine kinase
receptor
PTEN mutation
TP
NTRK1 rearrangement
rearrangement
RAS mutation
Ras
met amplification
BRAF mutation
Raf
AKAP9/BRAF rearrangement
MAPK
Nucleus
TP53 mutation
Transcription
factor
Sample cohorts

Fresh frozen thyroid samples
Department of Otorhinolaryngology and Head and Neck Surgery, 1st Faculty
of Medicine and Faculty Hospital Motol, Charles University, Prague
Department of Surgery, 2nd Faculty of Medicine, Charles University, Prague
Department of Otorhinolaryngology, Na Homolce Hospital, Prague

Paraffin embedded formalin-fixed samples
The Fingerland Department of Pathology, Hradec Kralove
Institute of Pathology, 1st Faculty of Medicine, Charles University, Prague
Institute of Pathology and Laboratory of Molecular Pathology, Faculty
of Medicine, Palacky University, Olomouc

Fine needle aspiration biopsies from Institute of Endocrinology

Peripheral blood samples of healthy controls
Molecular-genetic analysis:
 234 PTC, 6 FTC, 1 FTA, 8 PDTC, 3 ATC, 23 MTC
 172 healthy controls
Clinicopathological characteristics
 sex
 age
 tumor size
 histological variant of PTC
 tumor invasion
 nodal metastasis
 distant metastasis
 multifocality of tumor
 encapsulation
 angioinvasion
 TNM stage
 recurrence
Papillary structures
Methods
Izolation of NK
RNA
Trizol
DNA
Trizol, kit
RNA → cDNA
Real Time PCR
PCR
PCR
Genotyping
Sequencing
Elektroforesis
GAPDH
PAX8
EC RET
TK RET
RET/PTC1
RET/PTC3
RET polymorphisms
A432A – exon 7
G691S – exon 11
L769L – exon 13
S836S – exon 14
S904S – exon 15
RET
BRAF – exon 15
H-RAS – exon 1 and 2
K-RAS – exon 1 and 2
N-RAS – exon 1 and 2
Results – RET/PTC
RET/PTC rearrangements
•
26 RET/PTC from 101 PTC (25.5 %):
2 RET/PTC1, 2 RET/PTC3 and 22 RET/PTCX
RET/PTC1
•
Male, 19 years, T1N0M0, multifocal PTC without a capsule and invasion,
diffuse sclerosing variant, 17 months without recurrence
•
Female, 33 years, T1N1M0, unifocal encapsulated PTC without invasion,
classic variant, 18 months without recurrence
RET/PTC3
•
Female, 12 years, T3N1M1, unifocal PTC without a capsule, with invasion
into adjacent tissue and vessels, mixed follicular/papillary type, 39 months
without recurrence
•
Female, 43 year, T3N0M0, multifocal PTC, follicular variant, 20 months
without recurrence
Results: PTC – RET polymorphisms
Polymorphisms of RET gene
 234 PTC vs. 172 controls
p = 0.02
 101 PTC – differences in exon
7 and 13 in relation to
RET/PTC and sex of patients
 risk alelles – major alelle G
(exon 7) and minor alelle G
(exon 13) carriers of the risk
haplotypes (GGGCC and
GGGTC) – 2 times higher risk
of RET/PTC rearrangements
p = 0.04
Male
Female
Results – BRAF mutation
 detected in 81 of 242 PTC patients (33.5 %), in one of 6 poorly
differentiated carcinomas (16.7 %), in one anaplastic carcinoma and
in none of 23 MTC
 the impact of Chernobyl accident – re trospective study (47 years)
17
100
80
22
10
5
60
4
20
1991-1995
60
50
7
11
40
53
58
31
23
1
BRAF (%)
Prevalence (%)
70
1986-1990
2001-2005
40
2006-2007
30
1996-2000
20
20
06
-2
00
7
20
01
-2
00
5
19
96
-2
00
0
19
91
-1
99
5
19
86
-1
99
0
Be
fo
r
e
19
86
0
10
Before 1986
0
30
35
40
45
50
Age (median of years)
55
60
65
Results – BRAF mutation
BRAF mutation is associated with
 a higher age at diagnosis (p = 0.049)
 a histologic variant (p = 0.001)
(46.4 % classic vs. 13.2 % follicular variant)
 a greater tumor size (p = 0.041)
 with more agressive behavior of tumor
 presence of nodal metastasis (p = 0.029)
 more advanced TNM stage (p = 0.014)
 recurrence of disease (p = 0.008)
Results – RAS mutations
Gene
Exon
Alteration
Codon
Patient
H-RAS
2
V44M
GTG-ATG
A
C51Y
TGC-TAC
B
L79L
CTG-TTG
B
1
E3K
GAA-AAA
A
2
Y40Y
TAC-TAT
C
T50I
ACC-ATC
D
L56L
CTC-CTT
D
G60G
GGT-GGA
E
G60G
GGT-GGC
F
Q61K
CAA-AAA
E, F
Q22Q
CAG-CAA
G
Q22X
CAG-TAG
H
Q61R
CAA-CGA
I, J, K, L
A66T
GCC-ACC
C
D69D
GAC-GAT
G
E76K
GAA-AAA
C
K-RAS
N-RAS
1
2

98 samples – 83 PTC (56 FV PTC,
11 CV PTC, 14 mixed, 2 rare
variants), 7 FTC, 1 FTA, 3 ATC, 4
PDTC

In six patients detected mutation
in codon 61 of N-RAS gene and
K-RAS gene (aktivating domain)

Found other silent, nonsense and
missense genetic changes

All genetic changes detected in
follicular variant of PTC

In silico analysis shows possible
pathogenicity of some missense
mutations (p.Cys51Tyr,
p.Glu3Lys), that needs to be
confirmed
MTC and RET proto-oncogene
• located on chromosome 10q11.2
1 2
3
4
5
6
7
Cadherin-like
domain
8 9
10 11 12
Cysteine-rich
region
13
14
15
16
17 18
19
Tyrosine kinase
domains
Extracellular domain
Intracellular domain
Transmembrane region
Activating germ-line mutations FMTC, MEN 2A, MEN 2B
Activating somatic mutations
Sporadic MTC
Inactivating mutations
Hirschsprung disease
20 21
Genetic screening of the RET gene
Patient with MTC
DNA analysis (blood, tumor)
Mutation detected
only in a tumor
Mutation in blood
and a tumor
MTC
sporadic
MTC
familial
DNA analysis in all
at-risk family members
Prophylactic total
thyroidectomy
Mutation
detected
No mutation
detected
Biochemical
screening
Excluded from
screening
RET germ-line mutations in MTC patients
1 2
3
4
Arg321Gly 1x
5
6
7
8
Cys609Tyr 1x
Cys611Arg 1x
Cys611Tyr 1x
9
10
11 12
14
15
16
17
Cys634Ser 1x
Cys634Phe 1x
Leu790Phe 1x
Tyr791Phe 3x
Val804Leu 1x
Val804Met 4x
Cys634Trp 1x
Glu768Asp 1x
Tyr791Phe 2x
Val804Met 1x
Cys618Arg 1x
Cys620Phe 1x
Cys620Arg 1x
13
Cys634Arg 8x
Cys634Tyr 1x
Cys634Ser 2x
18 19 20
21
Met918Thr 9x
Tyr791Phe 1x
Tyr791Phe 1x
spor. MTC
FMTC
MEN2A
MEN2B
Total
Tested index patients
340
12
14
9
375
Mutation detected
14 (4%)
7 (58%)
13 (93%)
9 (100%)
Tested family members
202
61
69
20
352
Mutation detected
13
19
25
3
60
Excluded from screening
20
20
43
17
100


RET germ-line mutations in HSCR patients
1 2
3
4
5
6
7
8
9
10
Pro566Leu 1x
Ala373Val 1x
11 12
13
del603(A)
14
15
16
Ser649Leu 2x
Cys609Tyr 1x
Cys620Arg 2x
17
18 19 20
Tyr791Phe 8x
Gly798Ser 1x
Exon
Mutation
Detected
HSCR phenotype
Dual phenotypic
RET mutations
Exon 10
Cys609Tyr
1x
TCA
Exon 10
Cys620Arg
2x
TCA
RET mutations
typical for MTC
Exon 11
Ser649Leu
2x
1x TCA, 1x RS
Exon 13
Tyr791Phe
8x
2x TCA, 6x RS
Novel
RET mutations
with unknown
function
Exon 6
Ala373Val
1x
RS
Exon 9
Pro566Leu
1x
TCA
Exon 10
del603(A)
1x
TCA
Exon 13
Gly798Ser
1x
TCA
17x
9x TCA, 8x RS
Total 156 patients
21
Haplotypes - MTC
1 – A45A (G/A)
2 – A432A (G/A)
3 – G691S (G/A)
4 – L769L (T/G)
5 – S836S (C/T)
6 – IVS14-24G>A
7 – S904S (C/G)
Control
cohort
p = 0.003
Patients
Publications

Dvorakova S, Vaclavikova E, Sykorova V, Duskova J, Vlcek P, Ryska A, Novak Z, Bendlova B 2006 New
multiple somatic mutations in the RET proto-oncogene associated with a sporadic medullary thyroid
carcinoma. Thyroid 16:311–316.

Bendlová B, Dvořáková S, Václavíková E, Sýkorová V, Vlček P, Škába R 2006 Nádory štítné žlázy a
Hirschsprungova choroba – 10leté zkušenosti s molekulárně-genetickým testováním RET protookogenu. Vnitř Lek 52:926–934.

Sýkorová V, Dvořáková Š, Kodetová D, Astl J, Ryška A, Dušková J, Vlček P, Novák Z, Bendlová B 2007
Genetické příčiny vzniku papilárního karcinomu štítné žlázy Diab Metab Endokrinol Výživa
1:30–35.

Dvorakova S, Vaclavikova E, Sykorova V, Vcelak J, Novak Z, Duskova J, Ryska A, Laco J, Cap J,
Kodetova D, Kodet R, Krskova L, Vlcek P, Astl J, Vesely D, Bendlova B 2008 Somatic mutations in the
RET proto-oncogene in sporadic medullary thyroid carcinomas. Mol Cell Endocrinol, 284(1–2):21–27.

Vaclavikova E, Dvorakova S, Sykorova V, Bilek R, Dvorakova K, Vlcek P, Skaba R, Zelinka T, Bendlova
B 2009 RET mutation – Tyr791Phe – the genetic cause of different diseases derived from neural crest.
Endocrine, 36(3):419–424.

Sykorova V, Dvorakova S, Ryska A, Vcelak J, Vaclavikova E, Laco J, Kodetova D, Kodet R, Cibula A,
Duskova J, Hlobilkova A, Astl J, Vesely D, Betka J, Hoch J, Smutny S, Cap J, Vlcek P, Novak Z,
Bendlova B 2010 BRAFV600E Mutation in the Pathogenesis of a Large Series of Papillary Thyroid
Carcinoma in Czech Republic. J Endocrinol Invest, 33:318-324.
Acknowledgement
Institute of Endocrinology
RNDr. Š. Dvořáková, Ph.D.
Mgr. E. Václavíková
Mgr. Tereza Hálková
Mgr. Josef Včelák
MUDr. Z. Novák, CSc.
Doc. MUDr. J. Vrbíková, CSc.
Department of Nuclear Medicine and Endocrinology, 2nd Faculty
of Medicine and Faculty Hospital Motol, Prague
Doc. MUDr. P. Vlček, CSc.
Department of Otorhinolaryngology and Head and Neck Surgery,
1st Faculty of Medicine and Faculty Hospital Motol, Prague
Doc. MUDr. J. Astl, CSc., MUDr. J. Veselý, Prof. MUDr. J. Betka, DrSc.
Department of Surgery, 2nd Faculty of Medicine and Faculty Hospital Motol, Charles University, Prague
Prof. MUDr. J. Hoch, CSc., MUDr. S. Smutný
Institute of Pathology, 1st Faculty of Medicine and Faculty Hospital Motol, Charles University, Prague
Prim. MUDr. D. Kodetová, Prof. MUDr. R. Kodet, CSc., Mgr. A. Cibula
The Fingerland Department of Pathology, Faculty Hospital Hradec Kralove
Prof. MUDr. A. Ryška, CSc., MUDr. J. Laco
Institute of Pathology and IPVZ, 1st Faculty of Medicine, Charles University, Prague
Doc. MUDr. J. Dušková, CSc.
Institute of Pathology and Department of Molecular Pathology, Faculty of Medicine, Palacky University, Olomouc
MUDr. A. Hlobilková, Ph.D.
Department of Endocrinology, Faculty Hospital Hradec Kralove
Prof. MUDr. J. Čáp, CSc.
Department of Otorhinolaryngology, Na Homolce Hospital, Prague
MUDr. J. Lukáš
Supported by grant project IGA MH CR NR/9165-3
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