Diapositiva 1

1
Molecular Biology of GIST
Elena Tamborini Laboratory of Experimental
Molecular Pathology, Fondazione IRCCS Istituto
dei Tumori, Milan, Italy.
2
The molecular characteristic (signature) of
GISTs is the presence of activating mutations
in
KIT PDGFRA
KIT mutations are present in 75-85 of all
GISTs PDGFRA mutations are present in
5-10 10-15 of GISTs lack detectable mutations
in KIT or PDGFRA.
3
Human receptor tyrosine kinases
out
in
Blume-Jensen P, Hunter T Nature 2001
INT MILANO
4
NH2
KIT PDGFRA
SCF PDGFA/B
Ligand binding domain
Extracellular region
KIT PDGFRA
Dimerization domain
Exon 9 10
Exon 11 12
Juxtamembrane domain
TK 1
Exon 13 14
Cytoplasmic region
Kinase domain
TK 2
Exon 17 18
COOH
5
PDGFRA Kinase domain
KIT Kinase domain
Red exon 11 wt juxtamembran domain Blue
exon 13 TK1 C-Helix Yellow
exon 17 TK2 Activation
Loop
Red exon 12 wt Blue exon 14 Yellow exon 18
Pricl S, Tamborini E, Submitted
6
How ATP is located into the pocket
KIT
PDGFRA
It binds the ACTIVE conformation of the kinase
Pricl S, Tamborini E, Submitted
7
HOT-SPOTs of mutations in GISTs

• Exon 11 KIT ( juxtmembrane domain) exon 12
  PDGFRA
• Exon 9 KIT (extracellular domain)
• Exon 13 KIT (I part of TK domain)
  exon 14 PDGFRA
• Exon 17 KIT (II part of TK domain) exon
  18 PDGFRA

frequency
frequency




E12
E13 E14
E14
E15
E16
E17 E18
E18
E6
E7
E8
E9 E10
E10
E11 E12


extracellular
tyrosin kinase
juxtamembrane
8
1. Classification of the mutations KIT and
PDGFRA
Primary mutations before the
treatment Secondary mutations after the
treatment they affect exons 13, 14 and 17 of
KIT 15 and 16 exon 18
of PDGFRA
9
2. Classification of the mutations KIT and PDGFRA
Depending on the altered domain
Receptor regulatory domain dimerization
(extracellular) domain and JM domain Enzymatic
domain TK1 and TK2
10

• Which type of mutations are detected
• 1. Deletions
• 2. Substitutions / point mutations
• 3. Duplications / internal tandem duplication /
  insertions
• 4. Insertions
• 5. Complex mutations
• Deletions insertions / deletions/point mutations
• Duplications/insertions
• Single nucleotide substitutions can occur in
  tandem

11
KIT Regulatory domain
1. In frame deletions. KIT They represent a
very heterogeneous group of mutations They
cluster at 5 end of exon 11 of KIT, between 1669
and 1704 bp. Other mutations (less frequent)
affect the 3 end of th exons and frequently
they eliminate a consistent part of JM domain. .
In this group, the most frequent mutation is
represented by Del 557-558.
The most common is the Del of Asp579
Lasota, Miettinen, 2008
KIT exon 11
3
5
12
2. Single nucleotide substitutions/point
mutations. KIT Exon 11 They cluster in 4
codons Try 557 versus Arg Val 559 versus
Asp, Ala and Gly Val 560 versus Asp Leu 576
versus Pro.
KIT Regulatory domain
Hirota (Lab Invest. 2007 Apr87(4)365-71)
reported that the mutation Val 559 Ile,
detected in one case of aggressive systemic
mastocytosis, was Imatinib resistant
13
2. Single nucleotide substitutions/point
mutations. KIT Exon 13 and exon 17 Lys 642 Glu
Glu 635 Lys Leu 641 Pro Val 643 Ala Leu 647
Pro Met 651 Val Asn 655 Lys Asn 822 Lys Asp
816 Phe, Tyr Asp 820 Tyr, Val Asn 822 His Tyr 823
Asp
KIT Enzymatic domain
13
17
KIT regulatory domain
Glu 490 Gly
9
14
KIT Regulatory domain
3. Duplications . KIT Exon 9 Dupl Ala 502- Tyr
503 Exon 11 structurally heterogeneous and
cluster in 3 end of the exon
4. Insertions. KIT Not duplications are very
rare and affect only exon 11 at codon 558 Asn,
Pro and Glu Pro 5. Complex mutations.
KIT Very rare and always detected in exon 11
15

• Single nucleotide substitutions. PDGFRA
• Most of these mutations cluster in exon 18, but
  can also
• affect exon 12 and 14
• Asp 842 Val in exon 18 is the most frequent
  mutated codon
• Asp 842 Tyr
• Asp 842 Ile
• Asp 846 Tyr
• Tyr 849 Cys
• Val 561 Asp second as frequency
• Glu 556 Lys
• Glu 563 Lys

PDGFRA Enzymatic domain
18
PDGFRA regulatory domain
12
PDGFRA Enzymatic domain
14
16
2. In frame deletions. PDGFRA All these
mutations involve exon 18 Generally they
interest codons 840-848 exon 12 Codons
559-572 3. Duplication. PDGFRA Only exon
12 4. Insertions. PDGFRA 5. Complex
mutations. PDGFRA Exon 12
exon 18 insertion between
cds 840-849
PDGFRA Enzymatic domain
17
Correlation between prognosis and type of
mutations Deletion in KIT exon 11 , high
metastatic risk Del 557-558, poor
prognosis KIT single substitutions longer
PFS better OS Duplications in KIT exon 11
(3 end) less aggressive behaviour PDGFRA
mutations low malignant behaviour
Correlation between predictivity and type of
mutations GISTs carrying KIT exon 11 mutations
respond better to Imatinib treatment PDGFRA
mutation Asp 842 Val is insensitive to the
treatment
18
PROGNOSIS Unfavorable exon11 mutated GISTs
PREDICTIVITY Favorable exon11 mutated GISTs
19
Gleevec/Imatinib/STI571
2-phenylaminopyrimidine derivative
20
Pricl S, Tamborini E, Submitted
21
How Imatinib is located into the pocket
KIT
PDGFRA
Activation loop
Activation loop
It binds the INACTIVE conformation of the kinase
hampering the entrance of ATP
Pricl S, Tamborini E, Submitted
22
KIT receptor
wt exon 11
mutated exon 11
Sterical hindrance
Pricl S, Tamborini E, Submitted
23
Summarizing.
Primary or intrinsic resistance is due to a
conformation of the ATP pocket which does not
fit with the Imatinib entrance.
Mutated exon 11 KIT fit better than wt KIT
24
Experimental Molelcular Pathology S. Pilotti E.
Tamborini S. Brich E. Conca F. Bozzi T. Negri
M. Orsenigo M. Virdis S. Arisi Rota

• Department of Experimental Oncology
• Greco
• MA. Pierotti

INT Clinical Staff PG. Casali A.
Gronchi R. Bertulli M.
Fiore E.Fumagalli F. Grosso S. Stacchiotti
Molecular Simulation Engineering (MOSE)
Laboratory, University of Trieste S.
Pricl G.M.Panemi