KIT and PDGFRA Mutations in GISTs Guide Treatment

REVIEW
Clinical signicance of oncogenic KIT and PDGFRA mutations

in gastrointestinal stromal tumours
J Lasota & M Miettinen
Department of Soft Tissue Pathology, Armed Forces Institute of Pathology, Washington DC, USA
Lasota J & Miettinen M
(2008) Histopathology 53, 245266
Clinical signicance of oncogenic KIT and PDGFRA mutations in gastrointestinal stromal
tumours
Gastrointestinal stromal tumours (GISTs) are the most
common mesenchymal neoplasms of the gastrointesti-
nal tract. Despite clinicopathological differences, GISTs
share oncogenic KIT or platelet-derived growth factor-
alpha (PDGFRA) mutations. Imatinib, KIT and
PDGFRA inhibitor, has been successfully used in the
treatment of metastatic GISTs. There are primary KIT
or PDGFRA mutations diagnosed before imatinib
treatment, linked to GIST pathogenesis, and secondary
mutations detected during treatment, causing drug
resistance. KIT exon 11 mutations are the most
common. Gastric GISTs with exon 11 deletions are
more aggressive than those with substitutions. KIT
exon 11 mutants respond well to imatinib. Less
common KIT exon 9 Ala502_Tyr503dup mutants
occur predominantly in intestinal GISTs and are less
sensitive to imatinib. An Asp842Val substitution in
exon 18 is the most common PDGFRA mutation. GISTs
with such mutation are resistant to imatinib. PDGFRA
mutations are associated with gastric GISTs, epithelioid
morphology and a less malignant course of disease.
GISTs in neurobromatosis 1, Carney triad and
paediatric tumours generally lack KIT and PDGFRA
mutations. Secondary KIT mutations affect exons
1317. GISTs with secondary mutations in exon 13
and 14 are sensitive to sunitinib, another tyrosine
kinase inhibitor. KIT and PDGFRA genotyping is
important for GIST diagnosis and assessment of
sensitivity to tyrosine kinase inhibitors.
Keywords: gastrointestinal stromal tumours, KIT, mutation, PDGFRA
Abbreviations: ATP, adenosine triphosphate; DHPLC, denaturing high-pressure liquid chromatography;
EC, extracellular; FFPE, formalin-xed parafn-embedded; GIST, gastrointestinal stromal tumour; HGVS, Human
Genome Variation Society; HSP, heat-shock protein; ICC, interstitial cells of Cajal; JM, juxtamembrane; KI, kinase
insert; NF, neurobromatosis; PCR, polymerase chain reaction; PDGFRA, platelet-derived growth factor receptor-
alpha; SNP, single nucleotide polymorphism; TK, tyrosine kinase; WT, wild type
Introduction
Gastrointestinal stromal tumours (GISTs) are the most
common mesenchymal neoplasms of the gastrointesti-
nal tract occurring in its different parts. GISTs represent
a morphological and biological continuum from inci-
dentally discovered, <10-mm benign nodules to large
sarcomas.
1
Despite clinicopathological differences, most
GISTs share a similar genetic prole, including KIT or
platelet-derived growth factor receptor-alpha (PDG-
FRA) gain-of-function mutations (oncogenic muta-
tions).
24
KIT is expressed in >95% of GISTs,
including tumours with KIT wild-type (WT) genotype
and most PDGFRA mutant GISTs. However, in the
latter group, KIT expression may be weaker and focal.
Contrary to occasional misunderstanding, KIT muta-
tion in GIST does not cause KIT expression, but
modies KIT function.
1
Address for correspondence: J Lasota, MD, Department of Soft Tissue
Pathology, Armed Forces Institute of Pathology, 6825 16th Street,
N.W., Bldg. 54, Washington, DC 20306-6000, USA.
e-mail: lasota@ap.osd.mil
2008 The Authors. Journal compilation 2008 Blackwell Publishing Limited.
Histopathology 2008, 53, 245266. DOI: 10.1111/j.1365-2559.2008.02977.x
KIT and PDGFRA genes map to chromosome 4q12
and might have evolved from a common ancestral gene
by gene duplication.
5,6
Both genes encode highly
homologous transmembrane glycoproteins that belong
to the type III receptor tyrosine kinase family. This
protein family is characterized by a specic molecular
structure (Figure 1) consisting of an extracellular (EC)
domain with ve Ig-like loops and a cytoplasmic
domain with juxtamembrane (JM) region and a split
tyrosine kinase (TK) domain. The latter is divided into
an adenosine triphosphate (ATP) binding region (TK1)
and a phosphotransferase region (TK2) by a hydrophilic
kinase insert (KI). The extracellular and cytoplasmic
domains are connected by a transmembrane region.
7
Normally KIT and PDGFRA are activated by their
ligands, stem cell factor and PDGFs. Ligand binding to
the receptor EC domain results in the dimerization of
receptors and phosphorylation of tyrosines in their
cytoplasmic TK domains. This leads to a phosphoryla-
tion cascade of the tyrosine residues in multiple
downstream signalling molecules and activation of
signal transduction pathways including Ras MAP
kinase, Rac Rho-JNK, PI3K AKT and SFK STAT
signalling networks.
8
KIT-TK activity is regulated by
its JM domain, which inhibits KIT kinase activity in the
absence of KIT ligand.
9
Activation of KIT regulates
important cell functions, including proliferation, apop-
tosis, chemotaxis and adhesion, and is critical for the
development and maintenance of different cell types.
These include haematopoietic cells, mast cells, mela-
nocytes, gametocytes and interstitial cells of Cajal
(ICC), pacemaker cells involved in gastrointestinal tract
mobility and regulation of autonomous neural trans-
mission.
1017
Based on immunophenotypic, ultrastructural and cell
signalling similarities, GISTs are believed to originate
Location of primary and secondary
KIT and PDGFRA activating mutations
5 immunoglobulin-like loops
Extracellular (ligand-binding) domain
(EC)
Transmembrane domain
Juxtamembrane domain (JM)
First tyrosine kinase domain
(TK1)
Second tyrosine kinase domain
(TK2)
Kinase insert (KI)
KIT exon 8
KIT exon 9
KIT exon 11
KIT exon 13
KIT exon 17
L
L
P P
P P
Rac/Rho-JNK
Ras/MAP kinase
SFK/STAT
PI3K/AKT
Oncogenic activation of signalling networks
PDGFRA exon 14
PDGFRA exon 18
PDGFRA exon 12
KIT exon 15
KIT exon 16
KIT exon 14
Decreased apoptosis
Increased proliferation
Figure 1. Activation of receptor
by gain-of-function mutation
(blue, yellow, red dots) inde-
pendent of ligand (L) binding
induces dimerization of the
receptor, autophosphorylation
of tyrosines and causes activa-
tion of downstream signalling
pathways. Location of primary
(sporadic and hereditary) and
secondary (detected during
treatment) KIT and platelet-
derived growth factor receptor-
alpha (PDGFRA) mutations is
indicated by blue, yellow and
red dots, respectively.
246 J Lasota and M Miettinen
2008 The Authors. Journal compilation 2008 Blackwell Publishing Ltd, Histopathology, 53, 245266.
from KIT+ ICC progenitor cells through somatic KIT or
PDGFRA mutations, which are believed to be an early
step in GIST pathogenesis.
2,18
However, polyclonal ICC
hyperplasia might be the precursor lesion, based on
studies of familial GIST syndrome, suggesting that
progression to GIST probably requires additional
genetic changes beyond activating KIT or PDGFRA
mutations.
19
Also, a high incidence of small GISTs but
relative rarity of malignant ones suggest the same.
20
Imatinib mesylate, commercially known as Gleevec
Glivec
TM
(http://www.novartis.com) that specically
inhibits ABL, KIT and PDGFRA receptor TKs,
has been successfully used in the treatment of
clinically advanced, unresectable and metastatic
GISTs.
21,22
Although many patients benet from such
treatment, resistance often develops due to secondary
KIT or PDGFRA mutations, genomic amplication of
KIT, and other incompletely dened molecular mech-
anisms.
2328
In vitro experiments and data from
clinical trials suggest that the type of KIT or PDGFRA
mutation may impact on imatinib sensitivity and
therefore should be considered in devising treatment
strategies.
2932
Mutation nomenclature
In this review, mutation nomenclature follows the
recommendations of the Human Genome Variation
Society (HGVS) (http://www.hgvs.org). Nucleotide
numbering is based on human KIT (X06182) and
PDGFRA (M21574) mRNA sequences from GeneBank
(http://www.ncbi.nlm.nih.gov).
The following mutation types have been identied in
KIT and PDGFRA in GISTs: deletions (del), substitutions
(often called point mutations), duplications (dup) (often
called internal tandem duplications or insertions;
however, the latter should not be use to describe these
mutations), insertions (ins) and complex mutations
including deletioninsertions (delins) (often called
deletions and point mutations), duplicationinsertions
and recently reported deletions with insertion of
inverted complementary sequences, designated by one
study as deletioninversions (delinv).
33,34
Single nuc-
leotide substitutions can occur in tandem. However,
according to current mutation nomenclature, designa-
tion of such mutations as deletioninsertions is pre-
ferred over two substitutions. It is important to note
that changes described at the DNA and protein level
might differ in some cases. For example, deletions or
insertions at the DNA level can lead to deletion
insertions at the protein level. Thus, the HGVS recom-
mends authors to identify mutations at both DNA and
protein levels.
Overview of KIT and PDGFRA mutations
in GISTs
There are two categories of KIT and PDGFRA muta-
tions in GIST: (i) mutations diagnosed in primary
tumours before treatment with a TK inhibitor, linked to
GIST pathogenesis (primary KIT and PDGFRA muta-
tions), and (ii) mutations detected during treatment
causing resistance to imatinib-based TK inhibition
(secondary KIT and PDGFRA mutations). Structural
studies have shown that both the primary and
secondary KIT mutations affect the same allele.
23,35
Oncogenic KIT or PDGFRA mutations activate
receptor TKs by rendering them a constitutive phos-
phorylation. Based on the location, these mutations
could be divided into two categories: mutations of the
receptor regulatory domain (EC and JM) and mutations
of the enzymatic domain (TK1 and TK2).
36
In GIST,
most KIT mutations occur in the JM domain (exon 11)
followed by EC domain (exon 9). Mutations in the JM
domain affect its autoregulatory function and promote
spontaneous kinase activation.
37,38
Alternatively,
mutations in the EC domain may disrupt an antidi-
merization motif and lead to spontaneous receptor
homodimerization.
29
Also, KIT exon 9 mutants seem to
have more diverse intracellular signalling than KIT-JM
mutants.
39
A majority of PDGFRA mutations affect the
TK2 domain (exon 18). These mutations change
the activation loop, which conformationally regulates
the ATP-binding pocket and leads to kinase activation
as well.
40
Continuous ligand-independent activation of
KIT or PDGFRA kinases leads to activation of down-
stream signal transduction pathways promoting cell
survival and proliferation (Figure 1).
An essential role of mutational activation of KIT and
PDGFRA kinases in GIST pathogenesis is supported by
clinical ndings and in vitro studies. Family members
with germ-line KIT or PDGFRA mutations (heredit-
ary mutations) develop, among other symptoms, ICC
hyperplasia and multiple GISTs.
4156
Also, multiple
GISTs are found in transgenic mice with inheritable
gain-of-function KIT mutations similar to those diag-
nosed in human sporadic and familial GISTs.
57,58
In vitro studies have shown that expression of mutant
KIT in the cell lines elicits transforming ability.
2,59
Moreover, inhibition of KIT signalling in vitro stops
growth of GIST cell lines and in clinical treatment
reduces tumour growth, conrming that GISTs are
dependent on KIT kinase signalling.
21,22,6062
Also, the
latter process can be reversed by a new secondary KIT
mutation that interferes with drug binding.
23,26,35,63
In sporadic GISTs, primary KIT mutations have been
identied in the EC (exon 9), JM (exon 11), TK1 and
Clinical signicance of oncogenic KIT and PDGFRA mutations in GISTs 247
TK2 (exon 13, 17) domains, whereas primary PDGFRA
mutations occur in the JM (exon 12), TK1 and TK2
(exon 14, 18) domains. Secondary mutations have
been found exclusively in the KIT-TK1 and -TK2 (exon
13, 14, 17) domains and KI (exons 15 and 16) and
PDGFRA-TK2 (exon 18) domain (Figure 1).
4,64
In human familial GIST syndrome, germ-line KIT
and PDGFRA mutations are mostly similar to those
found in sporadic GISTs (Figure 1, Table 1). However,
two mutations never seen in sporadic GISTs, KIT
Asp419del (c.1276_1278delGAC) and PDGFRA
Tyr555Cys (c.1803AG) have been reported in two
families with GIST syndrome.
52,65
Also, a recent study
has reported a patient with germ-line PDGFRA
Asp561Val mutation, who had developed multiple
small intestinal brous polyps, lipomas and GISTs.
66
Ten years of studies on KIT and PDGFRA mutations
in GISTs have shown that some mutations could be
linked to certain clinicopathological features.
4,64,67
Table 2 summarizes the most important of these
ndings.
KIT and PDGFRA mutations are believed to be
mutually exclusive, and only one KIT or PDGFRA
mutation should be found in primary GIST and
subsequent metastases.
3
However, several studies have
reported tumours with second primary silent, missense
or nonsense KIT mutations.
4
Although it is not always
stated if such ndings were reproducible, some of them
clearly do not represent polymerase chain reaction
(PCR) artefacts.
27,30
Primary KIT mutations
deleti ons
In-frame deletions are the most common KIT muta-
tions in GISTs. They have been identied exclusively
(with only one exception) in KIT exon 11 (KIT-JM
domain). Exon 11 deletions consist of losses of three
to 30 or more nucleotides and lead to deletions or
in some cases deletioninsertions at the protein
level. Although deletions represent a structurally
highly heterogeneous group of mutations, they
tend to cluster in 5KIT exon 11 between 1669_
1704 (Lys550_Glu561), with 1690_1695delTGGAAG
(Trp557_Lys558del) being the most common. Some
deletions extend from 5 to 3KIT exon 11 and
eliminate a large portion of the JM domain. The most
Table 1. Hereditary KIT and PDGFRA mutations associated with familial GIST syndrome and other related genetic syndromes
Location
Mutation at
protein level Genetic syndrome (n) Reference
KIT-JM (exon 8) Asp419del Familial GIST syndrome (1)
52
KIT-JM (exon 11) Trp557Arg Familial GIST syndrome (2)
47,54
KIT-JM (exon 11) Val559Ala Familial GIST syndrome (4)
43,44,49,55
KIT-JM (exon 11) Val560Gly Familial GIST syndrome (1)
55
KIT-JM (exon 11) Val560del Familial GIST syndrome (1)
41
KIT-JM (exon 11) Gln575_Leu576dup Familial GIST syndrome (1)
48
KIT-JM (exon 11) Asp579del Familial GIST syndrome (2)
50,53
KIT-TK1 (exon 13) Lys642Glu Familial GIST syndrome (2)
42,56
KIT-TK2 (exon 17) Asp820Tyr Familial GIST syndrome (2)
45,51
PDGFRA-TK1 (exon 12) Tyr555Cys Familial GIST syndrome* (1)
65
PDGFRA-TK1 (exon 12) Asp561Val Multiple small intestinal brous
polyps, lipomas and GISTs (1)
66
PDGFRA-TK2 (exon 18) Asp846Tyr Familial GIST syndrome (1)
46
Total (19)
*Previously diagnosed as intestinal neurobromatosis.
PDGFRA, Platelet-derived growth factor receptor-alpha; GIST, gastrointestinal stromal tumour.
common 3KIT exon 11 deletion is 1755_1759del-
GAT (Asp579del).
6873
Different size deletions in 5KIT
can affect KIT intron 10-exon 11 splice-acceptor sites.
These deletions always form a novel intraexonic pre-
mRNA 3 splice acceptor site and consistently lead to
Lys550_Lys558del at the protein level.
74,75
Such KIT
exon 11 deletions have been shown to cause constit-
utive phosphorylation of KIT and elicit transforming
ability in murine lymphoblastoid cell lines in vitro.
2,59
The involvement of KIT exon 11 codons by deletions
is shown in Figure 2.
A 2131_2136delAAGAAT in exon 14 (KIT-TK1)
leading to Lys704_Asn705del at the protein level is the
only one deletion found outside the KIT-JM domain in
GISTs.
76
The biological potential of such a deletion is
unknown. Also, this mutation has been reported in a
GIST with another KIT exon 11 deletion and might
represent a second random event.
Table 2. Summary of GIST clinicopathological features associated with KIT and PDGFRA mutations
Gene
Mutation at
protein level Clinicopathological features Tumour type Prognostic value
KIT-EC
(exon 9)
Ala502_Tyr503dup Strongly associated with
intestinal GISTs (>90% of
these mutations were identied
in small intestinal tumours)
Predominantly spindle
cell tumours
No prognostic value
in intestinal GISTs
KIT-JM
(exon 11)
Trp557_Lys558del Occur in GISTs from different
parts of GI tract
Spectrum of spindle cell
and epithelioid tumours
May indicate more
malignant behaviour,
especially in gastric
GISTs
Deletions
Deletioninsertions
Substitutions May indicate less
malignant behaviour
in gastric GISTs
Duplications Associated with gastric GISTs Predominantly spindle
cell tumours
May indicate less
malignant behaviour
in gastric GISTs
KIT-TK1
(exon 13)
Lys642Glu Occur in GISTs from different
parts of GI tract
May indicate more
malignant behaviour
in gastric GISTs
KIT-TK2
(exon 17)
Asn822Lys Two times more frequent in
intestinal GISTs
No prognostic value
PDGFRA-JM
(exon 12)
Deletions
Substitutions
Strongly associated with gastric
GISTs (>95% of such mutations
identied in tumours from
stomach)
Predominantly epithelioid
or mixed epithelioid and
spindle cell tumours
May indicate less
malignant behaviour
in gastric GISTs
PDGFRA-TK1
(exon 14)
Substitutions
PDGFRA-TK2
(exon 18)
Deletions
Substitutions
KIT PDGFRA Wild-type Occur in GISTs from different
parts of GI tract
Spectrum of spindle cell
and epithelioid tumours
No prognostic value
GISTs in NF1 (intestinal
tumours)
Almost exclusively spindle
cell tumours
No prognostic value
GIST in Carney triad and
paediatric GISTs (gastric
tumours)
Predominantly epithelioid
tumours
PDGFRA, Platelet-derived growth factor receptor-alpha; GIST, gastrointestinal stromal tumour.
si ngle nucleoti de substi tuti ons
Single nucleotide substitutions are the second most
common KIT mutations in GISTs, and most of them
affect KIT exon 11. Typically, these mutations cluster
in four codons, Trp557, Val559 and Val560 (5KIT
exon 11) and Leu576 (3KIT exon 11). However, a
few substitutions have also been identied in other
locations.
4
The most common missense mutations
identied in GISTs are Val559Asp, Val560Asp,
Trp557Arg, Val559Ala, Val559Gly and Leu576-
Pro.
6873
Some of these mutations have been shown
to cause constitutive phosphorylation of KIT and elicit
transforming ability in murine lymphoblast cell lines
in vitro.
2,67
The biological potential of rare KIT exon
11 missense mutations is not known. Recently, an
in vitro study showed that a rare KIT mutation,
Val559Ile, induces, in contrast to common Val559-
Asp mutation, imatinib-resistant constitutive KIT
activation.
77
Thus, the inhibitory effect of imatinib
might differ substantially even among mutants involv-
ing the same codon.
In GISTs, single nucleotide substitutions have occa-
sionally been reported in KIT exon 13 (KIT-TK1) and
KIT exon 17 (KIT-TK2).
4,71,78
The great majority of
mutations identied in KIT exon 13 represent
a1945AG substitution resulting in Lys642Glu at
the protein level.
79
However recent studies have
reported six unique KIT exon 13 mutations:
Glu635Lys, Leu641Pro, Val643Ala, Leu647Pro,
Met651Val and Asn655Lys in the vicinity of codon
642.
8084
Furthermore, a GIST with double Lys642Glu
and Val643Ile mutation has recently been described.
79
Lys642Glu and Asn655Lys have been shown to lead to
constitutive KIT TK phosphorylation and to be imatinib
sensitive.
29,78,83
Most KIT exon 17 mutations are 2487TA substi-
tutions leading to Asn822Lys at the protein level.
However, other missense mutations (Asp816Phe,
Asp816Tyr, Asp820Tyr, Asp820Val, Asn822His,
Tyr823Asp) have been reported in a few cases.
71,79
Structurally similar mutations have been found in
gonadal germ cell tumours, seminomas and sinonasal
natural killer T-cell lymphomas.
8588
An Asp816Val
KIT codons 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591
39 87 53 19
KIT codons 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591
3 13 21 24 31 36 39 37 37 31 25 24 22 19 17 15 12 9 6 5 4
KIT codons 550 551 552 553 554 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586 587 588 589 590 591
n =
n =
n =
7 14 36 48 59 70 67 147 141 67 81 53 31 36 37 36 30 32 36 42 46 45 40 36 36 33 32 12 9 23 1
Figure 2. The involvement of KIT exon 11 codons by different mutation types. Deletions, substitutions and duplications are indicated by black,
white and grey colours, respectively. Figure is based on evaluation of 546 KIT exon 11 mutants from Armed Forces Institute of Pathology
collection. n, how many times the codon was deleted.
mutation identied in KIT-associated mastocytosis and
urticaria pigmentosa has been shown to cause ligand-
independent autophosphorylation of KIT.
89
In KIT exon 9, an unsual single amino acid
substitution Glu490Gly has been reported in the
GIST.
90
However, its biological potential remains
unknown.
90
The involvement of KIT exon 11 codons
by substitutions is shown in Figure 2.
dupli cati ons
Duplications are the third most common KIT mutations
in GISTs.
4
These KIT mutations have been identied in
exon 9 (distal part of KIT-EC domain)
78
and in exon 11
(KIT-JM domain).
91
Structurally, almost all exon 9
duplications are identical 1525_1530dupGCCTAT
leading to Ala502_Tyr503dup at the protein
level.
9294
However, 1537_1545dupTTTGCATTT lead-
ing to Phe506_Phe508dup at the protein level has
been reported in three cases.
4,29
Duplications in KIT exon 11 are structurally hetero-
geneous. Their sizes vary from one to 18 codons and
with one exception they do not involve intronic
sequences.
7173,90,91,95106
Typically, these duplica-
tions cluster in 3KIT exon 11 and only two of >80
reported examples affected central or 5 KIT exon
11.
73,99
The involvement of KIT exon 11 codons by
duplications is shown in Figure 2.
Structurally similar duplications reported in canine
mastocytoma and paediatric patients with acute mye-
loid leukaemia were associated with KIT constitutive
phosphorylation, ligand-independent growth, and
decreased apoptosis.
107109
i nserti ons
Insertions in KIT (other than duplications) are very
rare and have been found only in exon 11, specif-
ically in codon 558. Almost all exon 11 insertions
have been structurally identical: 1694_1695insTCC
leading to Lys558delinsAsnPro at the protein level.
4
However, two variants (Lys558delinsGlnPro and
Lys558delinsAsnGln) have been reported in a few
cases.
69,7173,76
The Lys558delinsAsnPro mutation
has been shown to cause constitutive KIT phospho-
rylation.
60
complex mutati ons (deleti oni nserti ons,
dupli cati oni nserti ons and deleti on
i nversi ons)
Deletioninsertions and duplicationinsertions are rel-
atively rare KIT exon 11 mutations. These mutations
consist of one to several nucleotide deletions or
duplications coexisting with small insertions.
More recently, deletions complicated by insertions of
inverted complementary DNA sequences have been
reported in KIT exon 9
33
and exon 11.
34
At DNA level,
the name deletioninversion (delinv) has been pro-
posed for this type of mutation.
34
However, such
A
G A G G A G T T G T T G G A A G G T G A C A T G A A G T A T G T A C C C A A A ' 5
Gln Leu Gly Thr
A C C C A A A ' 5 C T T C T G G T C G A G G A G T T G T T G G A
B 562 561 560 559 558 557 556 555 554 553 552 551 550
562 561 560 559 558 557 556 555 554 553 552 551 550
E Glu Val Val Lys Trp Gln Val Glu Tyr Met Pro Lys
E Glu Val Val Lys Trp Gln Val Glu Tyr Met Pro Lys
G A G G A G T T G T T G G A A G G T G A C A T G A A G T A T G T A C C C A A A ' 5
A C A A C C T T C C A C T G T A
Tyr Gln Lys Lys Asn His
C A T G A A G T A T G T A C C C A A A ' 5 A T G T C A C C T T C C A A C A G A G T
C 586 585 584 583 582 581 580 579 578 577 576 575 573 574 587
Thr Lys Pro Phe Glu Trp Lys His Asp Tyr Pro Leu Gln Thr Pro
A C A A A A C C C T T T G A G G G T A A A C A C T A G T A T T C C T T C A A C A C A A C C
Gln Ile Pro Trp
A C A C A A C G T G A G G G T A A A C A C T A G T A T T C C T T C T A T C G G G C A A A
dup seq
KIT-MT
KIT-MT
KIT-WT
KIT-WT
KIT-MT
KIT-WT
Figure 3. Examples of complex KIT exon 11 mutations: deletioninsertion (A), deletioninversion (B) and duplication with deletioninsertion
(C). Deleted sequences are indicated by clear boxes on KIT-WT (wild-type). Inserted sequences are red in KIT-MT (mutant). Duplication is marked
by a grey box. A silent mutation is indicated by a black box.
mutations will translate into deletioninsertions at the
protein level. These unique KIT mutations have not
been previously reported in cancer. Examples of dele-
tioninsertions, deletioninversions and duplication
insertions are shown in Figure 3.
Primary PDGFRA mutations
si ngle nucleoti de substi tuti ons
Single nucleotide substitutions are the most common
PDGFRA mutations in GIST. Most of these mutations
have been identied in exon 18 (PDGFRA-TK2).
However, PDGFRA exon 12 (PDGFRA-JM) and exon
14 (PDGFRA-TK1) can also be mutated.
4
In exon 18, the most common is single nucleo-
tide substitution 2664AT leading to Asp842Val
mutation.
3,30,105,106,110115
However, two variants,
Asp842Tyr and Asp842Ile, have been reported.
3,30,
110,116
Other PDGFRA exon 18 single nucleotide
substitutions affect codons in the vicinity of codon
842 and lead to Asp846Tyr and Tyr849Cys muta-
tions at the protein level.
30,110
An Asp846Tyr
mutation has been reported in both familial and
PDGFRA codons 555 556 557 558 559 560 561 562 563 564 565 566 567 568 569 570 571 572 581 582 583 584 585 586 587
n = 2 2 2 2 4 31 3 3 3 2 7 7 7 7 7 7 1 1 1 1 1
PDGFRA codons 838 839 841 842 843 844 845 846 847 848 849 850 840 841 842 443 844 845 846 847 848 849 850
n = 4 56 72 70 67 30 7 1 229 2 3
A
B
Figure 4. The involvement of platelet-derived growth factor receptor-alpha (PDGFRA) exon 12 (A) and 18 (B) codons by different mutation
types. Deletions, substitutions and duplications are indicated by black, white and grey colours, respectively. Figure is based on previously
published studies.
3,27,30,96,105,106,110,113116,178,179
n, how many times the codon was deleted.
sporadic GISTs.
46,110
This mutation is homologous to
KIT exon 17 Asp820Tyr mutation reported twice in
familial GISTs.
45,51
An Asp842Val mutant has been
shown to activate PDGFRA both in vitro and
in vivo.
3,117
Almost all single nucleotide substitutions identied
in PDGFRA exon 12 represented 1821TA resulting
in Val561Asp mutation at the protein level. This
mutation is a second most common substitution
found in PDGFRA in GISTs.
3,30,105,110,117
It has been
shown to activate PDGFRA in vitro.
3,117
In the
vicinity of codon 561, Glu556Lys and Glu563Lys
substitutions have been reported in two tumours; the
biological potential of these mutants has not been
studied.
81,111
Single nucleotide substitutions in PDGFRA exon 14
are rare,
118
with only 15 reported cases.
30,105,116,118
All these mutations cluster in codon 659, with a
majority representing 2125CA and 2125CG sub-
stitutions leading to Asn659Lys at the protein level.
However, in a few cases a 2123AT substitution
leading to variant Asn659Tyr mutation has been
found instead.
118
An Asn659Lys has been shown to
activate PDGFRA in vitro.
30
deleti ons
In-frame deletions are the second most common
PDGFRA mutations in GISTs. These mutations have
been identied in PDGFRA exon 18 (PDGFRA-TK2)
and exon 12 (PDGFRA-JM). They consist of losses of
three to several nucleotides and lead to deletion or in
some cases deletioninsertions at the protein level.
Although PDGFRA deletions represent a structurally
heterogeneous group of mutations, they tend to cluster
between codons 840_848 in exon 18 and 559_572
in exon 12.
3,30,105,106,110115
This type of mutation
has been shown to activate PDGFRA in vitro and
in vivo.
30
dupli cati ons
Duplications are rare and only three such mutations
have been identied in PDGFRA exon 12. Two such
mutations have been found in the vicinity of codon
561 and one 20 codons 3 to this mutational
hotspot.
3,105,114
i nserti ons
Insertions in PDGFRA are extremely rare. Only one
such mutation, 561_562insER, has been reported in
exon 12.
3
complex mutati ons (deleti oni nserti ons)
Several PDGFRA deletioninsertions have been
reported in exon 18. These mutations consist of
deletion of several nucleotides and insertion of one to
four nucleotides and cluster in the exon 18 region
(between codons 840_849) commonly affected by
deletion.
30,110
Some of these mutations have been
shown to activate PDGFRA in vitro.
30
The involvement
of PDGFRA codons by deletions, substitutions and
duplications is shown in Figure 4.
Mutations, tumour location and
demographics
Distribution of KIT and PDGFRA mutations among
benign and malignant GISTs and among GISTs from
different gastrointestinal locations is unequal.
4
Although KIT exon 11 deletions, deletioninsertions
and single nucleotide substitutions have been reported
in GISTs from oesophagus to anus,
119124
a great
majority (>80%) of KIT exon 11 duplications have
been diagnosed in gastric tumours.
7173,90,91,95106
No signicant correlation between types of KIT
exon 11 mutations and tumour morphology has
yet been established. However, KIT exon 11
mutants show more often spindle cell than epithelioid
morphology.
72
Most KIT exon 9 duplications occur in
intestinal
73,92,125
and very few in gastric
GISTs.
72,93,94,106,114,126
A recent study has shown
that small intestinal tumours were two times more
frequent than gastric ones among KIT exon 17
mutants.
79
Similarly, intestinal tumours were slightly
overrepresented among KIT exon 13 mutants when
compared with population-based studies.
79
KIT exon 9,
13 and 17 mutants often have spindle cell morphology.
However epithelioid cell features have been occasion-
ally reported in malignant small intestinal GISTs with
such mutations.
79
Epithelioid morphology in small
intestinal GISTs is believed to represent malignant
transformation and should not be considered equal
with that in gastric tumours.
122
PDGFRA mutations occur almost exclusively in GIST
of stomach and omentum, suggesting that these tum-
ours are interrelated.
30,96,103,105,106,111,112,114,115,127
However, a few intestinal and mesenteric GISTs with
such mutations have also been reported.
84,103,116
Although most PDGFRA mutants have epithe-
lioid or mixed epithelioid spindle cell mor-
phology,
96,103,105,106,112,113
the type of mutation
PDGFRA vs. KIT can not easily be predicted because
of overlapping morphological features (Figure 5).
Some reports have suggested a link between the type
of KIT mutation and gender or age, but no such
correlation has been supported in larger studies.
4
However, paediatric and Carney triad GISTs occur
predominantly in female patients and are associated
with gastric location and epithelioid morphology.
These tumours lack KIT and PDGFRA mutations,
suggesting that other mechanisms of KIT activation
or unrelated oncogenic mechanisms are opera-
tional.
128133
A Pro456Ser in KIT exon 9 and non-
sense mutation in PDGFRA exon 18 have been
reported in two separate paediatric GISTs,
134,135
how-
ever, these mutations probably represent random
molecular events.
A KIT-WT and PDGFRA-WT genotype has been
found in most studies of GISTs of neurobromatosis
(NF) 1 patients, who have an increased risk for
GIST.
136140
In one study, two KIT and two PDGFRA
mutations were reported in separate tumours from two
NF1 patients.
138
These mutations do not correspond to
GIST-type of KIT or PDGFRA mutations and might
be random genetic events. In another study, the same
Val559Asp substitution was identied in three separate
primary lesions from a patient with phenotypic features
typical for NF1.
141
Unfortunately, normal tissue was
not available for testing and the possibility of a genetic
syndrome with a germ-line KIT mutation could there-
fore not be excluded in this case. NF1-associated GISTs
have predominantly spindle cell morphology and show
a strong predilection to intestinal location.
1
Multiple GISTs have also been reported in non-NF1
patients. Usually they are small lesions with different
KIT PDGFRA genotype.
142
However, a few patients
with multiple mini GISTs carrying the same mutations
have also been reported.
55
Although germ-line muta-
tions have been excluded in these cases, multiple local
A
C
B
D
Figure 5. Histological images of gastric gastrointestinal stromal tumours, two spindle cell (A,B) and two epithelioid (C,D) with different KIT
and platelet-derived growth factor receptor-alpha (PDGFRA) mutations show that type of mutation cannot be easily predicted based on
morphological features. KIT mutants, Tyr557_Val559delinsPhe (A) and Tyr557_Lys558del (C); PDGFRA mutants, Asp842Val (B,D).
metastases might explain the latter nding. KIT-WT
and PDGFRA-WT genotype has also been found in
<10% of sporadic GISTs. No distinctive clinicopatho-
logical features have been described in tumours with
such a genotype, except poor response to imatinib.
Ethnic differences
There are no extensive studies comparing KIT and
PDGFRA mutation proles in GIST patients from
different populations. Comparison of data from Asian
and Western GIST study groups indicates possible
differences in the frequency of specic KIT and
PDGFRA mutants. For example, Ala502_Tyr503dup
mutants are rare (6.6%) in gastric GISTs from Euro-
pean and US studies and, in contrast, relatively
frequent (32%) among gastric tumours from combined
Asian studies.
4
Frequency of primary KIT and PDGFRA
mutations
The frequency of KIT and PDGFRA mutations in GISTs
has been a subject of debate because of different
frequences in various studies. Two early published
reports showed almost a fourfold difference in the
frequency of KIT-JM mutations (25% vs. 92%).
71,91
Several factors may have contributed to such discrep-
ancies, including technical problems (see Technical
considerations), and differences in the composition of
analysed cohorts. The studies based on material
from cancer centres and imatinib trials include
more KIT mutants linked to malignant, clinically
aggressive GISTs and fewer PDGFRA mutants, typically
gastric epithelioid GISTs with indolent clinical
behaviour.
24,27,35,84
Also, in cohorts from cancer
centres and imatinib trials, small intestinal GISTs are
overrepresented, because of their higher frequency of
malignancy leading to an apperantly higher frequency
of KIT exon 9 mutants linked to intestinal loca-
tion.
24,27,35,84,143
Similarly, GIST cohorts from aca-
demic centres and consulting institutions might be
enriched with diagnostically difcult tumours, includ-
ing those with epithelioid morphology.
106,120
Thus, the
overall frequency of KIT and PDGFRA mutations can
only be established based on population studies free of
selection bias.
97,144
However, in one such study KIT
immunoreactivity was an inclusion criterion in the
selection of cases, so that the reported frequency of
PDGFRA mutations (5.1% versus 14.3% in another
population-based study) was relatively low compared
with other population-based studies.
97,144
Tables 3 and
4 compare differences in the frequencies of KIT and
PDGFRA mutations in GISTs in cohorts from clinical
Table 3. Frequency of KIT exon 9 and exon 11 and PDGFRA mutants among gastric and intestinal GISTs analysed in clinical
trials, and studies based on a population, or academic institution
Location Mutations
GISTs from EORTC
phase III trial*
(n = 124 S; n = 93 Sb)
GISTs from two
population-based studies
(n = 148 S; n = 89)
GISTs diagnosed in
one institution
(n = 45 S; n = 24 Sb)
Stomach KIT 106 (85.5) 95 (64.2) 27 (60)
PDGFRA 3 (2.4) 13 (8.8) 11 (24.4)
KIT exon 11 99 (79.8) 92 (62.1) 25 (55.6)
KIT exon 9 4 (3.2) 1 (0.7) 1 (2.2)
Small bowel KIT 75 (80.6) 59 (66.3) 21 (87.5)
PDGFRA 4 (4.3) 2 (2.3) 0
KIT exon 11 47 (50.5) 47 (52.8) 14 (58.3)
KIT exon 9 25 (26.9) 10 (11.2) 7 (29.2)
Values in parenthesis are presented as percentage.
*EORTC clinical trial.
84
Swedish and Norwegian population-based studies.
97,144
Consecutively collected GISTs at the Institute of Pathology, Heidelberg University, Germany between 1996 and 2003.
106
5.1% and 14.3% in Swedish and Norwegian population-based study, respectively.
n, Number of GISTs reported; S, stomach; Sb, small bowel; GIST, gastrointestinal stromal tumour.
trials and other GIST studies, including those based on
specic populations.
Diagnostic and prognostic value of primary
KIT or PDGFRA mutations
Most GISTs independent of mutation status are KIT+,
including KIT-WT GISTs, such as those in NF1 patients
and children. However, some GISTs (<5%), especially
gastric PDGFRA mutant GISTs, can be KIT) or only
weakly positive.
1,110,116
In these cases, mutation
analysis can help to establish the diagnosis of GIST.
However, the potential of nding GIST-specic KIT
mutations in KIT) tumours is extremely small.
There are controversies regarding the prognostic
value of KIT exon 11 mutations in GISTs. Some early
studies showed that KIT exon 11 mutations are more
common in large, mitotically active tumours or corre-
late with malignant disease outcome.
68,69,145
However,
others have suggested that KIT mutations are a
ubiquitous feature of GISTs and can be found in
malignant and clinically indolent, benign tumours
also.
71,146
More recent studies have shown that the type of KIT
exon 11 mutation may correlate with clinical out-
come.
4
Pre-imatinib gastric GISTs with KIT exon 11
deletions follow a more malignant course of disease
than those with single nucleotide substitutions.
120
Also, the presence of specic deletion Tyr557_Lys558-
del has been linked to malignant behaviour.
90,97,147
However, small intestinal KIT exon 11 deletion
mutants were not more aggressive than those with
single nucleotide substitutions.
118,122
Gastric GISTs
with duplication in 3KIT have been linked by some
studies to benign clinical behaviour.
73,103
However, a
few malignant, advanced GISTs with such mutations
have been reported in series from imatinib trials.
24,35
Homozygous KIT exon 11 mutations have been
occasionally diagnosed by direct sequencing in primary
GISTs (Figure 6). Also, a shift from heterozygosity to
homozygosity has been documented during tumour
Table 4. Frequency of deletions, substitutions, and duplications among KIT exon 11 mutations diagnosed in gastric and
intestinal GISTs from clinical trials and studies based on a population, and an academic institution, and a consultation centre
Location
KIT exon 11
mutation
Malignant GISTs
treated with imatinib*
(n = 25 S; n = 41 Sb)
GISTs from a
population study
(n = 60 S; n = 35 Sb)
GISTs diagnosed
in one institution
(n = 25 S; n = 14 Sb)
GISTs submitted
to AFIP
(n = 114 S; n = 88 Sb)
Stomach Deletions 21 (84) 33 (55) 12 (48) 69 (60.5)
Substitutions 1 (4) 10 (16.7) 7 (28) 34 (29.8)
Duplications 1 (4) 17 (28.3) 5 (20) 9 (7.9)
Small bowel Deletions 34 (82.9) 25 (71.4) 10 (71.4) 60 (68.2)
Substitutions 7 (17.1) 10 (28.6) 4 (28.6) 28 (31.8)
Duplications 0 0 0 0
Values in parenthesis are presented as percentage.
*Combined data from imatinib treatment clinical trial studies
27,35,152
include 10 GISTs treated paliatively with imatinib.
143
Swedish population-based study.
97
Consecutively collected GISTs at the Institute of Pathology, Heidelberg University, Germany between 1996 and 2003.
106
Tumours submitted from the USA to the Armed Forces Institute of Pathology (AFIP) between 1970 and 1996.
120,122
n, Number of GISTs reported; S, stomach; Sb, small bowel; GIST, gastrointestinal stromal tumour.
A
B
Figure 6. Heterozygous (A) and homozygous (B) KIT exon 11
mutation 1690_1695delTGGAAG (Trp557_Lys558del) diagnosed by
polymerase chain reaction amplication and direct sequencing. Note
shift of KIT-WT (wild-type) sequence (A) and lack of KIT-WT
sequence (B).
progression, being present in metastases, but not in
primary tumours.
24,81,148
A recent study has shown
that the loss of KIT-WT allele and subsequent duplica-
tion of KIT-MT allele lead to the shift from hetero-
zygosity to homozygosity in GISTs.
149
A similar
molecular mechanism has been shown for homozygous
KIT exon 13 mutations.
78
Gastric and small intestinal
GISTs with homozygous KIT exon 11 mutations are
almost invariably associated with malignant tumour
behaviour.
149
Initially, GISTs with KIT exon 9 duplications were
associated with malignant outcome.
73,125
However, a
recent study of 145 small intestinal GISTs with KIT exon
9 mutations did not show signicant differences in
clinical outcome between KIT exon 9 and KIT exon 11
mutants. Thus, the previously reported ndings were
probably related to the higher mortality of patients with
small intestinal vs. gastric tumours.
120,122
Although KIT exon 13 and KIT exon 17 mutations
are rare in GISTs, a recent multicentre study has
Table 5. KIT and PDGFRA genotypes, in vitro sensitivity to imatinib and response to imatinib treatment based on previously
published studies from US and European clinical trials
Gene Exon
Primary KIT and PDGFRA
mutations identied in GISTs
from imatinib clinical trials (n) Sensitivity to imatinib mesylate
KIT 9 Ala502_Tyr503dup Sensitive to imatinib in vitro
29
Complete remission in 5%, partial response in 29%, stable
disease in 47%, progressive disease in 17% as reported by
EORTC phase III trial
84
A high-dose regimen increased progression-free survival
84
11 Deletion deletioninsertion
Substitution
Duplication
Most common mutants sensitive to imatinib in vitro
29
Rare Val559Ile mutant resistant to imatinib in vitro
77
Complete remission in 6%, partial response in 61%, stable
disease in 25%, progressive disease in 3% as reported by
EORTC phase III trial
84
13 Lys642Glu (8)
Glu635Lys (1)
Sensitive to imatinib in vitro
29
Partial response or stable disease reported in all nine
cases
29,35,84
17 Asp820Tyr (1)
Asn822Lys (2)
Asn822His (2)
Asn822Lys and Asn822His sensitive to imatinib in vitro
29
Partial response reported in four mutants including
Asn820Tyr, Asn822Lys, Asn822His
29,84
Primary resistance
reported in Asn822Lys mutant
27
PDGFRA 12 Asp561Val (4)
Deletion deletioninsertion
Duplication, insertion
Asp561Val and some other exon 12 mutants tested
sensitive to imatinib in vitro
30,117
Objective response
reported in the majority of a few cases treated with
imatinib
29,84
14 Asn659Lys This mutant tested sensitive to imatinib in vitro
30
No clinical experience
18 Asp842_His845del (2)
Asp842_Met844del (1)
Ile843del (1)
Ile843_His845del (1)
Asp842Val (7)
Asp846Val (1)
Some of these and similar mutants tested sensitive to
imatinib in vitro
30,117
Objective response reported in the
majority of a few cases treated with imatinib
29,84
Asp842Val resistant to imatinib in vitro
29,30,117
Resistance reported in seven cases including
Asp846Val
29,35,84
; stable disease in one case after 5 months
of imatinib treatment
35
KIT
PDGFRA
9, 11, 13, 17
12, 14, 18
Wild-type
Wild-type
Partial response in 23%, stable disease in 50%, and
progressive disease in 19% as reported by EORTC phase III
trial
84
GIST, Gastrointestinal stromal tumour; PDGFRA, platelet-derived growth factor-alpha.
shown that KIT exon 13 mutants tend to be signi-
cantly more aggressive than gastric GISTs on average,
whereas gastric GISTs with KIT exon 17 mutations
show no such tendency. Furthermore, the behaviour of
small intestinal GISTs with KIT exon 13 or KIT exon 17
mutations did not differ from that of other small
intestinal GISTs.
79
In general, PDGFRA mutants have low mitotic rate
and good prognosis; most of them represent gastric
GISTs, many of which would previously have been
diagnosed as leiomyoblastomas.
1,4
Primary and secondary KIT and PDGFRA
mutations and TK inhibitor treatment
Since the rst patient with GIST was successfully
treated in 2000 with KIT and PDGFRA TK inhibitor,
imatinib mesylate [STI571, commercially known as
Gleevc Glivec
TM
(http://www.novartis.com)], many
patients have beneted from this targeted treat-
ment.
21,22,150
However, the response to imatinib
treatment to some extent depends on the tumour KIT
or PDGFRA mutation status. Table 5 summarizes data
on in vitro sensitivity to imatinib and response to
imatinib treatment of different KIT and PDGFRA
mutants.
Clinical observations have shown that KIT exon 11
mutants in general respond better to imatinib mesylate
treatment than KIT exon 9 mutants and KIT-WT
tumours.
29,84
Thus, to achieve similar therapeutic
results, patients with KIT exon 9 mutant GISTs might
require a higher dosage of imatinib mesylate.
84
GISTs with PDGFRA Asp842Val substitutions are
resistant to imatinib treatment.
29,35,84
This mutation
corresponds to imatinib-resistant KIT Asp816Val muta-
tion reported in human mastocytosis.
29
During imatinib mesylate treatment, resistance often
develops due to detected secondary KIT or PDGFRA
mutations.
2327
Almost all such mutations reported in
GISTs affect KIT. The only exceptions are two GISTs with
primary KIT and secondary PDGFRA, Asp842Val
mutations.
24,27
Structurally, most secondary KIT muta-
tions represent single nucleotide substitutions affecting
specic codons in KIT exon 13 and 14 (TK1), exon 15
Glu
Tyr
Tyr n = 2
Deletion n = 5
His
Ile Gly
n = 7 His n = 3
n = 4
Gly
n = 2 Glu
Ala Glu Gly Glu n = 4 Lys Asp
KIT-MT n = 30 n = 5 Phe Asn Val n = 2 n = 2 Arg Ala n = 11 n = 11
654 670 709 716 783 809 815 816 818 820 822 823
KIT-WT Val Thr Ser Asp Leu Cys Arg Asp Lys Asp Asn Tyr
Ex 13 Ex 14 Ex 15 Ex 16 Ex 17
TK1 TK1 KI KI TK2
Cases with multiple KIT mutations
1. Val654Ala, Thr670Ile
2. Val654Ala, Asp816His (n = 2)
3. Val654Ala, Asp820G
4. Val654Ala, Asn822Lys
5. Val654Ala, Thr670Glu, Tyr823Asp
6. Asn818Lys, Asn822Lys, Tyr823Asp
7. Asp816Glu, Asp820Val, Asp820Glu, Asn822Lys
8. Asp820Glu, Asn822Lys, Asn822Tyr
9. Asn822Tyr, Cys809Glyn
Figure 7. Frequency and
distribution of 95 recently
reported secondary KIT
mutations. Window above
shows a summary of oligo-
clonal KIT mutation genotypes
identied in 10 patients. Figure
is based on previously pub-
lished studies.
2327,32,35,151160
and 16 (KI) and 17 (TK2).
2328,151160
KIT-TK1 domain
mutations affect codons never mutated in primary
tumours, whereas some of the secondary KIT-TK2
mutations, Asn822Lys and Tyr823Asp and PDGFRA-
TK2 mutation Asp842Val, have been reported as
primary KIT mutations also.
79
In some cases, different
mutations in different lesions, or simultaneous evolu-
tion of multiple clones in one lesion, have been
reported.
27,35,151153,155
Figure 7 shows the frequency
and distribution of secondary KIT mutations in GISTs.
More recently, sunitinib malate, also known as
SU11248 (http://www.pzer.com), a multitargeted
inhibitor of KIT, PDGFRs, vascular endothelial growth
factor receptors, FLT3 and RET receptor TKs, has been
used for treatment of imatinib-resistant GISTs as the
rst second-generation TK inhibitor.
161163
In vitro
and in vivo studies have shown that the clinical benet
of sunitinib is signicantly inuenced by KIT and
PDGFRA mutation status.
31,32,164
Although clinical
benet was observed in all major mutant types, the
primary response rate was signicantly higher for KIT
exon 9 mutants. The inhibitory effect of sunitinib on
KIT kinase activity was not substantially affected by
secondary KIT mutations in TK1. However, GISTs with
KIT-TK2 secondary mutations were resistant to suni-
tinib treatment.
32
Thus, the search for other second-
line drugs inhibiting KIT and PDGFRA TK activity
must continue.
165,166
Also, inhibition of alternative targets, such as
downstream components of KIT and PDGFR pathways
including AKT and mTOR proteins in the AKT path-
way, has been tested in vitro and in clinical trials. The
best known example of these is everolimus.
167
These
agents can be used alone or in combination with TK
inhibitors.
In addition to the inhibition of KIT and PDGFRA and
their signalling pathways, an antitumour effect can be
achieved in GISTs by blocking tumour angiogenesis.
Table 6. Second-generation agents drugs developed for GIST treatment
Agent drug Molecular target of inhibition Developer producer
Sunitib malate, SU11248
(Sugen)
KIT, PDGFR,VEGFRs, FLT3 Pzer
AMN107 (Nilotinib) KIT, PDGFRs, BCR-ABL Novartis
AZD2171 VEGFR, KIT, PDGFRs AstraZeneca
OSI-930 VEGFR, KIT OSI pharmaceuticals
MP-470 KIT, PDGFRs, MET, RET, AXL SuperGen Pharmaceuticals
BMS-354825 (Dasatinib) SRC-family kinase inhibitor, ABL, KIT, PDGFRs Bristol-Myers Squibb
PTK787 ZK22584 VEGFR, KIT, PDGFRs Novartis and Schering AG
XL820 KIT, PDGFRB, VEGFR Exelixis
PKC412 KIT, PDGFRs, VEGFR-2, Protein kinase C
(PKC)
Novartis
AMG 706 VEGFR, KIT, PDGFRs, RET Amgen
Everolimus (RAD001) mTOR in the AKT pathway Novartis
CCI-779 (Temsirolimus) mTOR in the AKT pathway Wyeth Pharmaceuticals
KRX-0401 (Perifosine) AKT KERYX Biopharmaceuticals
BAY 43-9006 (Nexavar) RAF kinases inhibitor in the MAPK pathway, KIT,
PDGRFB, VEGFR-2, VEGFR-3, FLT3, RET
Bayer Pharmaceuticals Corp.
Onyx Pharmaceuticals Inc.
IPI-504 Heat Shock Protein 90 (HSP90) inhibitor Innity Pharmaceuticals
Flavopiridol Suppressor of KIT expression, induces apoptosis.
Also CDK inhibitor
National Institutes of Health,
Bethesda, MD, USA
GIST, Gastrointestinal stromal tumour; PDGFRA, platelet-derived growth factor-alpha.
Several anti-angiogenesis agents including sunitinib,
already approved for GIST treatment, have been
developed and their therapeutic potential tested in
clinical trials.
More recently, strategies to inhibit KIT signalling by
abolishing KIT or diminishing KIT expression have
been developed. One of these is based on inhibition
of heat-shock protein (HSP)-90, a member of the
chaperone family of proteins, which plays a role in
protein folding into three-dimensional shapes and
stabilizes and protects KIT from degradation. Inhibition
of HSP90 prevents protective interaction between
HSP90 and KIT and leads to KIT degradation and
tumour cell apoptosis.
167
Another, very new strategy
to inhibit KIT signalling is to abrogate KIT mRNA
expression with a transcriptional inhibitor, such as
avopiridol.
168
Examples of second-generation agents
drugs for GIST treatment are listed in Table 6 (
167,168
,
http://www.liferaftgroup.org/treat_trials.html).
Because the type of KIT or PDGFRA mutation may
have an impact on planning the targeted treatment,
genotyping of GISTs should be considered a standard
clinical test in all primary tumours with a signicant
risk of metastasis, especially to rule out mutants
primarily resistant to imatinib. Also, testing for sec-
ondary KIT and PDGFRA mutations in GISTs under
imatinib treatment could be valuable in monitoring
drug resistance.
Technical considerations
Contamination of tumour samples with non-tumour
cells, such as lymphocytes, other inammatory cells
and entrapped smooth muscle cells can lead to relative
decrease of tumour DNA in the analysed sample and
cause false-negative results in PCR-based mutation
analysis by elevating PCR amplication of KIT-wild
type versus KIT-mutant allele. Thus, histopathological
evaluation of the sample and enrichment of tumour
tissue for DNA extraction are necessary.
Detection of KIT and PDGFRA mutations in
formalin-xed parafn-embedded (FFPE) GISTs ap-
pears to be lower than expected in some studies.
Three recent studies performed independently on
different material by two different groups have shown
that a detection rate of KIT and PDGFRA tends
to decrease with increasing age of parafn
blocks.
97,120,144
A possible explanation for this phe-
nomenon is ongoing degradation of tumour DNA in
archival parafn blocks.
Most KIT and PDGFRA mutation studies in GISTs
have been based on direct sequencing of PCR products.
A number of recent studies have shown that employ-
ing denaturing high-pressure liquid chromatography
(DHPLC), especially when empowered by fraction
collector, substantially increases the detection of muta-
tions compared with standard direct sequencing of PCR
products.
169,170
However, one study has shown that a
large duplication may not be easy ampliable from
partially degraded DNA obtained from FFPE tissues
and missed by both DHPLC screening and direct
sequencing of PCR products. Thus, obtaining relatively
small amplicons by design of primer systems is help-
ful in PCR-based detection of duplications in FFPE
GISTs.
171
According to another recent study, screening
of PCR amplication products for KIT and PDGFRA
mutations using high-resolution melting amplicon
analysis might be slightly more sensitive than
DHPLC.
115
In general, multiple primary KIT mutations affecting
the same or different exons have rarely been reported.
However, one study identied multiple KIT exon 11
mutations in as many as 9% (seven of 78) primary
GISTs.
172
Also, KIT STOP codon frame-shift mutations have
been reported occasionally in primary and metastatic
GISTs.
27,90,135,173
These mutations might reect sec-
ondary changes related to tumour progression. It seems
that they might involve KIT-WT allele rather than the
primarily mutated allele and, in fact, lead to functional
KIT homozygosity.
27
STOP
Gln828
A
B
Figure 8. Example of an artefact created by polymerase chain
reaction (PCR) amplication. Platelet-derived growth factor receptor-
alpha (PDGFRA) exon 18 sequence with STOP codon mutation (A),
and lack of STOP codon mutation in the second PCR amplication
from the same DNA sample (B).
In some studies it has not been clearly stated whether
additional primary KIT or PDGFRA mutations were
detected by sequencing of PCR amplication products
from at least two independent PCRs. Thus, PCR
amplication sequencing artefacts can not be com-
pletely excluded. Increased frequency of PCR amplica-
tion artefacts has been reported in analysis of DNA from
FFPE tissues.
174
Figure 8 shows an example of PCR
amplication artefact in PDGFRA exon 18.
Several single nucleotide polymorphisms (SNP) in
KIT and PDGFRA coding sequences (SNP database at
http://www.ncbi.nlm.nih.gov) and two alternative
splicing sites in KIT have been reported.
175,176
KIT
and PDGFRA polymorphisms and alternatively spliced
variants of KIT mRNA should not be confused with
activating oncogenic KIT mutations.
177
Disclaimer
The opinions and assertions contained herein are the
expressed views of the authors and are not to be
construed as ofcial or reecting the views of the
Departments of the Army or Defense.
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