Download - Clinical significance of oncogenic KIT and PDGFRA mutations in gastrointestinal stromal tumours.pdf
-
REVIEW
Clinical significance of oncogenic KIT and PDGFRA mutationsin gastrointestinal stromal tumours
J Lasota & M MiettinenDepartment of Soft Tissue Pathology, Armed Forces Institute of Pathology, Washington DC, USA
Lasota J & Miettinen M
(2008) Histopathology 53, 245266
Clinical significance of oncogenic KIT and PDGFRA mutations in gastrointestinal stromaltumours
Gastrointestinal stromal tumours (GISTs) are the mostcommon mesenchymal neoplasms of the gastrointesti-nal tract. Despite clinicopathological differences, GISTsshare oncogenic KIT or platelet-derived growth factor-alpha (PDGFRA) mutations. Imatinib, KIT andPDGFRA inhibitor, has been successfully used in thetreatment of metastatic GISTs. There are primary KITor PDGFRA mutations diagnosed before imatinibtreatment, linked to GIST pathogenesis, and secondarymutations detected during treatment, causing drugresistance. KIT exon 11 mutations are the mostcommon. Gastric GISTs with exon 11 deletions aremore aggressive than those with substitutions. KITexon 11 mutants respond well to imatinib. Lesscommon KIT exon 9 Ala502_Tyr503dup mutants
occur predominantly in intestinal GISTs and are lesssensitive to imatinib. An Asp842Val substitution inexon 18 is the most common PDGFRA mutation. GISTswith such mutation are resistant to imatinib. PDGFRAmutations are associated with gastric GISTs, epithelioidmorphology and a less malignant course of disease.GISTs in neurofibromatosis 1, Carney triad andpaediatric tumours generally lack KIT and PDGFRAmutations. Secondary KIT mutations affect exons1317. GISTs with secondary mutations in exon 13and 14 are sensitive to sunitinib, another tyrosinekinase inhibitor. KIT and PDGFRA genotyping isimportant for GIST diagnosis and assessment ofsensitivity 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-fixed paraffin-embedded; GIST, gastrointestinal stromal tumour; HGVS, HumanGenome Variation Society; HSP, heat-shock protein; ICC, interstitial cells of Cajal; JM, juxtamembrane; KI, kinaseinsert; NF, neurofibromatosis; 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 mostcommon mesenchymal neoplasms of the gastrointesti-nal tract occurring in its different parts. GISTs representa morphological and biological continuum from inci-dentally discovered, 95% of GISTs,including tumours with KIT wild-type (WT) genotypeand most PDGFRA mutant GISTs. However, in thelatter group, KIT expression may be weaker and focal.Contrary to occasional misunderstanding, KIT muta-tion in GIST does not cause KIT expression, butmodifies 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: [email protected]
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 4q12and might have evolved from a common ancestral geneby gene duplication.5,6 Both genes encode highlyhomologous transmembrane glycoproteins that belongto the type III receptor tyrosine kinase family. Thisprotein family is characterized by a specific molecularstructure (Figure 1) consisting of an extracellular (EC)domain with five Ig-like loops and a cytoplasmicdomain with juxtamembrane (JM) region and a splittyrosine kinase (TK) domain. The latter is divided intoan adenosine triphosphate (ATP) binding region (TK1)and a phosphotransferase region (TK2) by a hydrophilickinase insert (KI). The extracellular and cytoplasmicdomains are connected by a transmembrane region.7
Normally KIT and PDGFRA are activated by theirligands, stem cell factor and PDGFs. Ligand binding tothe receptor EC domain results in the dimerization ofreceptors and phosphorylation of tyrosines in their
cytoplasmic TK domains. This leads to a phosphoryla-tion cascade of the tyrosine residues in multipledownstream signalling molecules and activation ofsignal transduction pathways including Ras MAPkinase, Rac Rho-JNK, PI3K AKT and SFK STATsignalling networks.8 KIT-TK activity is regulated byits JM domain, which inhibits KIT kinase activity in theabsence of KIT ligand.9 Activation of KIT regulatesimportant cell functions, including proliferation, apop-tosis, chemotaxis and adhesion, and is critical for thedevelopment 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 tractmobility and regulation of autonomous neural trans-mission.1017
Based on immunophenotypic, ultrastructural and cellsignalling similarities, GISTs are believed to originate
Location of primary and secondary KIT and PDGFRA activating mutations
5 immunoglobulin-like loopsExtracellular (ligand-binding) domain
(EC)
Transmembrane domainJuxtamembrane domain (JM)
First tyrosine kinase domain(TK1)
Second tyrosine kinase domain(TK2)
Kinase insert (KI)
KIT exon 8KIT exon 9
KIT exon 11KIT exon 13
KIT exon 17
L L
PP
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 15KIT exon 16
KIT exon 14
Decreased apoptosisIncreased 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 orPDGFRA mutations, which are believed to be an earlystep in GIST pathogenesis.2,18 However, polyclonal ICChyperplasia might be the precursor lesion, based onstudies of familial GIST syndrome, suggesting thatprogression to GIST probably requires additionalgenetic changes beyond activating KIT or PDGFRAmutations.19 Also, a high incidence of small GISTs butrelative rarity of malignant ones suggest the same.20
Imatinib mesylate, commercially known as Gleevec GlivecTM (http://www.novartis.com) that specificallyinhibits ABL, KIT and PDGFRA receptor TKs,has been successfully used in the treatment ofclinically advanced, unresectable and metastaticGISTs.21,22 Although many patients benefit from suchtreatment, resistance often develops due to secondaryKIT or PDGFRA mutations, genomic amplification ofKIT, and other incompletely defined molecular mech-anisms.2328 In vitro experiments and data fromclinical trials suggest that the type of KIT or PDGFRAmutation may impact on imatinib sensitivity andtherefore should be considered in devising treatmentstrategies.2932
Mutation nomenclature
In this review, mutation nomenclature follows therecommendations of the Human Genome VariationSociety (HGVS) (http://www.hgvs.org). Nucleotidenumbering is based on human KIT (X06182) andPDGFRA (M21574) mRNA sequences from GeneBank(http://www.ncbi.nlm.nih.gov).
The following mutation types have been identified inKIT and PDGFRA in GISTs: deletions (del), substitutions(often called point mutations), duplications (dup) (oftencalled internal tandem duplications or insertions;however, the latter should not be use to describe thesemutations), insertions (ins) and complex mutationsincluding deletioninsertions (delins) (often calleddeletions and point mutations), duplicationinsertionsand recently reported deletions with insertion ofinverted complementary sequences, designated by onestudy 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 notethat changes described at the DNA and protein levelmight differ in some cases. For example, deletions orinsertions at the DNA level can lead to deletioninsertions at the protein level. Thus, the HGVS recom-mends authors to identify mutations at both DNA andprotein levels.
Overview of KIT and PDGFRA mutationsin GISTs
There are two categories of KIT and PDGFRA muta-tions in GIST: (i) mutations diagnosed in primarytumours before treatment with a TK inhibitor, linked toGIST pathogenesis (primary KIT and PDGFRA muta-tions), and (ii) mutations detected during treatmentcausing resistance to imatinib-based TK inhibition(secondary KIT and PDGFRA mutations). Structuralstudies have shown that both the primary andsecondary KIT mutations affect the same allele.23,35
Oncogenic KIT or PDGFRA mutations activatereceptor TKs by rendering them a constitutive phos-phorylation. Based on the location, these mutationscould be divided into two categories: mutations of thereceptor regulatory domain (EC and JM) and mutationsof 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 JMdomain affect its autoregulatory function and promotespontaneous kinase activation.37,38 Alternatively,mutations in the EC domain may disrupt an antidi-merization motif and lead to spontaneous receptorhomodimerization.29 Also, KIT exon 9 mutants seem tohave more diverse intracellular signalling than KIT-JMmutants.39 A majority of PDGFRA mutations affect theTK2 domain (exon 18). These mutations changethe activation loop, which conformationally regulatesthe ATP-binding pocket and leads to kinase activationas well.40 Continuous ligand-independent activation ofKIT or PDGFRA kinases leads to activation of down-stream signal transduction pathways promoting cellsurvival and proliferation (Figure 1).
An essential role of mutational activation of KIT andPDGFRA kinases in GIST pathogenesis is supported byclinical findings and in vitro studies. Family memberswith germ-line KIT or PDGFRA mutations (heredit-ary mutations) develop, among other symptoms, ICChyperplasia and multiple GISTs.4156 Also, multipleGISTs are found in transgenic mice with inheritablegain-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 mutantKIT in the cell lines elicits transforming ability.2,59
Moreover, inhibition of KIT signalling in vitro stopsgrowth of GIST cell lines and in clinical treatmentreduces tumour growth, confirming that GISTs aredependent on KIT kinase signalling.21,22,6062 Also, thelatter process can be reversed by a new secondary KITmutation that interferes with drug binding.23,26,35,63
In sporadic GISTs, primary KIT mutations have beenidentified in the EC (exon 9), JM (exon 11), TK1 and
Clinical significance of oncogenic KIT and PDGFRA mutations in GISTs 247
2008 The Authors. Journal compilation 2008 Blackwell Publishing Ltd, Histopathology, 53, 245266.
-
TK2 (exon 13, 17) domains, whereas primary PDGFRAmutations occur in the JM (exon 12), TK1 and TK2(exon 14, 18) domains. Secondary mutations havebeen found exclusively in the KIT-TK1 and -TK2 (exon13, 14, 17) domains and KI (exons 15 and 16) andPDGFRA-TK2 (exon 18) domain (Figure 1).4,64
In human familial GIST syndrome, germ-line KITand PDGFRA mutations are mostly similar to thosefound in sporadic GISTs (Figure 1, Table 1). However,two mutations never seen in sporadic GISTs, KITAsp419del (c.1276_1278delGAC) and PDGFRATyr555Cys (c.1803AG) have been reported in twofamilies with GIST syndrome.52,65 Also, a recent studyhas reported a patient with germ-line PDGFRAAsp561Val mutation, who had developed multiplesmall intestinal fibrous polyps, lipomas and GISTs.66
Ten years of studies on KIT and PDGFRA mutationsin GISTs have shown that some mutations could belinked to certain clinicopathological features.4,64,67
Table 2 summarizes the most important of thesefindings.
KIT and PDGFRA mutations are believed to bemutually exclusive, and only one KIT or PDGFRAmutation should be found in primary GIST and
subsequent metastases.3 However, several studies havereported tumours with second primary silent, missenseor nonsense KIT mutations.4 Although it is not alwaysstated if such findings were reproducible, some of themclearly do not represent polymerase chain reaction(PCR) artefacts.27,30
Primary KIT mutations
deletions
In-frame deletions are the most common KIT muta-tions in GISTs. They have been identified exclusively(with only one exception) in KIT exon 11 (KIT-JMdomain). Exon 11 deletions consist of losses of threeto 30 or more nucleotides and lead to deletions orin some cases deletioninsertions at the proteinlevel. Although deletions represent a structurallyhighly heterogeneous group of mutations, theytend to cluster in 5KIT exon 11 between 1669_1704 (Lys550_Glu561), with 1690_1695delTGGAAG(Trp557_Lys558del) being the most common. Somedeletions extend from 5 to 3KIT exon 11 andeliminate 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
LocationMutation atprotein 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 fibrouspolyps, lipomas and GISTs (1)
66
PDGFRA-TK2 (exon 18) Asp846Tyr Familial GIST syndrome (1) 46
Total (19)
*Previously diagnosed as intestinal neurofibromatosis.
PDGFRA, Platelet-derived growth factor receptor-alpha; GIST, gastrointestinal stromal tumour.
248 J Lasota and M Miettinen
2008 The Authors. Journal compilation 2008 Blackwell Publishing Ltd, Histopathology, 53, 245266.
-
common 3KIT exon 11 deletion is 1755_1759del-GAT (Asp579del).6873 Different size deletions in 5KITcan 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 toLys550_Lys558del at the protein level.74,75 Such KITexon 11 deletions have been shown to cause constit-utive phosphorylation of KIT and elicit transformingability in murine lymphoblastoid cell lines in vitro.2,59
The involvement of KIT exon 11 codons by deletionsis shown in Figure 2.
A 2131_2136delAAGAAT in exon 14 (KIT-TK1)leading to Lys704_Asn705del at the protein level is theonly one deletion found outside the KIT-JM domain inGISTs.76 The biological potential of such a deletion isunknown. Also, this mutation has been reported in aGIST with another KIT exon 11 deletion and mightrepresent a second random event.
Table 2. Summary of GIST clinicopathological features associated with KIT and PDGFRA mutations
GeneMutation atprotein level Clinicopathological features Tumour type Prognostic value
KIT-EC(exon 9)
Ala502_Tyr503dup Strongly associated withintestinal GISTs (>90% ofthese mutations were identifiedin small intestinal tumours)
Predominantly spindlecell tumours
No prognostic valuein intestinal GISTs
KIT-JM(exon 11)
Trp557_Lys558del Occur in GISTs from differentparts of GI tract
Spectrum of spindle celland epithelioid tumours
May indicate moremalignant behaviour,especially in gastricGISTs
DeletionsDeletioninsertions
Substitutions May indicate lessmalignant behaviourin gastric GISTs
Duplications Associated with gastric GISTs Predominantly spindlecell tumours
May indicate lessmalignant behaviourin gastric GISTs
KIT-TK1(exon 13)
Lys642Glu Occur in GISTs from differentparts of GI tract
May indicate moremalignant behaviourin gastric GISTs
KIT-TK2(exon 17)
Asn822Lys Two times more frequent inintestinal GISTs
No prognostic value
PDGFRA-JM(exon 12)
DeletionsSubstitutions
Strongly associated with gastricGISTs (>95% of such mutationsidentified in tumours fromstomach)
Predominantly epithelioidor mixed epithelioid andspindle cell tumours
May indicate lessmalignant behaviourin gastric GISTs
PDGFRA-TK1(exon 14)
Substitutions
PDGFRA-TK2(exon 18)
DeletionsSubstitutions
KIT PDGFRA Wild-type Occur in GISTs from differentparts of GI tract
Spectrum of spindle celland epithelioid tumours
No prognostic value
GISTs in NF1 (intestinaltumours)
Almost exclusively spindlecell tumours
No prognostic value
GIST in Carney triad andpaediatric GISTs (gastrictumours)
Predominantly epithelioidtumours
PDGFRA, Platelet-derived growth factor receptor-alpha; GIST, gastrointestinal stromal tumour.
Clinical significance of oncogenic KIT and PDGFRA mutations in GISTs 249
2008 The Authors. Journal compilation 2008 Blackwell Publishing Ltd, Histopathology, 53, 245266.
-
s ingle nucleotide substitutions
Single nucleotide substitutions are the second mostcommon KIT mutations in GISTs, and most of themaffect KIT exon 11. Typically, these mutations clusterin four codons, Trp557, Val559 and Val560 (5KITexon 11) and Leu576 (3KIT exon 11). However, afew substitutions have also been identified in otherlocations.4 The most common missense mutationsidentified in GISTs are Val559Asp, Val560Asp,Trp557Arg, Val559Ala, Val559Gly and Leu576-Pro.6873 Some of these mutations have been shownto cause constitutive phosphorylation of KIT and elicittransforming ability in murine lymphoblast cell linesin vitro.2,67 The biological potential of rare KIT exon11 missense mutations is not known. Recently, anin vitro study showed that a rare KIT mutation,Val559Ile, induces, in contrast to common Val559-Asp mutation, imatinib-resistant constitutive KITactivation.77 Thus, the inhibitory effect of imatinibmight 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) andKIT exon 17 (KIT-TK2).4,71,78 The great majority ofmutations identified in KIT exon 13 representa1945AG substitution resulting in Lys642Glu atthe protein level.79 However recent studies havereported six unique KIT exon 13 mutations:Glu635Lys, Leu641Pro, Val643Ala, Leu647Pro,Met651Val and Asn655Lys in the vicinity of codon642.8084 Furthermore, a GIST with double Lys642Gluand Val643Ile mutation has recently been described.79
Lys642Glu and Asn655Lys have been shown to lead toconstitutive KIT TK phosphorylation and to be imatinibsensitive.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 ingonadal germ cell tumours, seminomas and sinonasalnatural 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 59139 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 5913 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 591n =
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.
250 J Lasota and M Miettinen
2008 The Authors. Journal compilation 2008 Blackwell Publishing Ltd, Histopathology, 53, 245266.
-
mutation identified in KIT-associated mastocytosis andurticaria pigmentosa has been shown to cause ligand-independent autophosphorylation of KIT.89
In KIT exon 9, an unsual single amino acidsubstitution Glu490Gly has been reported in theGIST.90 However, its biological potential remainsunknown.90 The involvement of KIT exon 11 codonsby substitutions is shown in Figure 2.
duplications
Duplications are the third most common KIT mutationsin GISTs.4 These KIT mutations have been identified inexon 9 (distal part of KIT-EC domain)78 and in exon 11(KIT-JM domain).91 Structurally, almost all exon 9duplications are identical 1525_1530dupGCCTATleading to Ala502_Tyr503dup at the proteinlevel.9294 However, 1537_1545dupTTTGCATTT lead-ing to Phe506_Phe508dup at the protein level hasbeen reported in three cases.4,29
Duplications in KIT exon 11 are structurally hetero-geneous. Their sizes vary from one to 18 codons andwith one exception they do not involve intronicsequences.7173,90,91,95106 Typically, these duplica-tions cluster in 3KIT exon 11 and only two of >80reported examples affected central or 5 KIT exon11.73,99 The involvement of KIT exon 11 codons byduplications is shown in Figure 2.
Structurally similar duplications reported in caninemastocytoma and paediatric patients with acute mye-
loid leukaemia were associated with KIT constitutivephosphorylation, ligand-independent growth, anddecreased apoptosis.107109
insertions
Insertions in KIT (other than duplications) are veryrare and have been found only in exon 11, specif-ically in codon 558. Almost all exon 11 insertionshave been structurally identical: 1694_1695insTCCleading to Lys558delinsAsnPro at the protein level.4
However, two variants (Lys558delinsGlnPro andLys558delinsAsnGln) have been reported in a fewcases.69,7173,76 The Lys558delinsAsnPro mutationhas been shown to cause constitutive KIT phospho-rylation.60
complex mutations (deletioninsertions,duplicationinsertions and deletioninversions)
Deletioninsertions and duplicationinsertions are rel-atively rare KIT exon 11 mutations. These mutationsconsist of one to several nucleotide deletions orduplications coexisting with small insertions.
More recently, deletions complicated by insertions ofinverted complementary DNA sequences have beenreported in KIT exon 933 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 ThrLys 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 CA 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.
Clinical significance of oncogenic KIT and PDGFRA mutations in GISTs 251
2008 The Authors. Journal compilation 2008 Blackwell Publishing Ltd, Histopathology, 53, 245266.
-
mutations will translate into deletioninsertions at theprotein level. These unique KIT mutations have notbeen previously reported in cancer. Examples of dele-tioninsertions, deletioninversions and duplicationinsertions are shown in Figure 3.
Primary PDGFRA mutations
s ingle nucleotide substitutions
Single nucleotide substitutions are the most commonPDGFRA mutations in GIST. Most of these mutations
have been identified in exon 18 (PDGFRA-TK2).However, PDGFRA exon 12 (PDGFRA-JM) and exon14 (PDGFRA-TK1) can also be mutated.4
In exon 18, the most common is single nucleo-tide substitution 2664AT leading to Asp842Valmutation.3,30,105,106,110115 However, two variants,Asp842Tyr and Asp842Ile, have been reported.3,30,110,116 Other PDGFRA exon 18 single nucleotidesubstitutions affect codons in the vicinity of codon842 and lead to Asp846Tyr and Tyr849Cys muta-tions at the protein level.30,110 An Asp846Tyrmutation 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.
252 J Lasota and M Miettinen
2008 The Authors. Journal compilation 2008 Blackwell Publishing Ltd, Histopathology, 53, 245266.
-
sporadic GISTs.46,110 This mutation is homologous toKIT exon 17 Asp820Tyr mutation reported twice infamilial GISTs.45,51 An Asp842Val mutant has beenshown to activate PDGFRA both in vitro andin vivo.3,117
Almost all single nucleotide substitutions identifiedin PDGFRA exon 12 represented 1821TA resultingin Val561Asp mutation at the protein level. Thismutation is a second most common substitutionfound in PDGFRA in GISTs.3,30,105,110,117 It has beenshown to activate PDGFRA in vitro.3,117 In thevicinity of codon 561, Glu556Lys and Glu563Lyssubstitutions have been reported in two tumours; thebiological potential of these mutants has not beenstudied.81,111
Single nucleotide substitutions in PDGFRA exon 14are rare,118 with only 15 reported cases.30,105,116,118
All these mutations cluster in codon 659, with amajority representing 2125CA and 2125CG sub-stitutions leading to Asn659Lys at the protein level.However, in a few cases a 2123AT substitutionleading to variant Asn659Tyr mutation has beenfound instead.118 An Asn659Lys has been shown toactivate PDGFRA in vitro.30
deletions
In-frame deletions are the second most commonPDGFRA mutations in GISTs. These mutations havebeen identified in PDGFRA exon 18 (PDGFRA-TK2)and exon 12 (PDGFRA-JM). They consist of losses ofthree to several nucleotides and lead to deletion or insome cases deletioninsertions at the protein level.Although PDGFRA deletions represent a structurallyheterogeneous group of mutations, they tend to clusterbetween codons 840_848 in exon 18 and 559_572in exon 12.3,30,105,106,110115 This type of mutationhas been shown to activate PDGFRA in vitro andin vivo.30
duplications
Duplications are rare and only three such mutationshave been identified in PDGFRA exon 12. Two suchmutations have been found in the vicinity of codon561 and one 20 codons 3 to this mutationalhotspot.3,105,114
insertions
Insertions in PDGFRA are extremely rare. Only onesuch mutation, 561_562insER, has been reported inexon 12.3
complex mutations (deletioninsertions)
Several PDGFRA deletioninsertions have beenreported in exon 18. These mutations consist ofdeletion of several nucleotides and insertion of one tofour nucleotides and cluster in the exon 18 region(between codons 840_849) commonly affected bydeletion.30,110 Some of these mutations have beenshown to activate PDGFRA in vitro.30 The involvementof PDGFRA codons by deletions, substitutions andduplications is shown in Figure 4.
Mutations, tumour location anddemographics
Distribution of KIT and PDGFRA mutations amongbenign and malignant GISTs and among GISTs fromdifferent gastrointestinal locations is unequal.4
Although KIT exon 11 deletions, deletioninsertionsand single nucleotide substitutions have been reportedin GISTs from oesophagus to anus,119124 a greatmajority (>80%) of KIT exon 11 duplications havebeen diagnosed in gastric tumours.7173,90,91,95106
No significant correlation between types of KITexon 11 mutations and tumour morphology hasyet been established. However, KIT exon 11mutants show more often spindle cell than epithelioidmorphology.72
Most KIT exon 9 duplications occur inintestinal73,92,125 and very few in gastricGISTs.72,93,94,106,114,126 A recent study has shownthat small intestinal tumours were two times morefrequent than gastric ones among KIT exon 17mutants.79 Similarly, intestinal tumours were slightlyoverrepresented among KIT exon 13 mutants whencompared 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 withsuch mutations.79 Epithelioid morphology in smallintestinal GISTs is believed to represent malignanttransformation and should not be considered equalwith that in gastric tumours.122
PDGFRA mutations occur almost exclusively in GISTof 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 withsuch 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 mutationPDGFRA vs. KIT can not easily be predicted becauseof overlapping morphological features (Figure 5).
Clinical significance of oncogenic KIT and PDGFRA mutations in GISTs 253
2008 The Authors. Journal compilation 2008 Blackwell Publishing Ltd, Histopathology, 53, 245266.
-
Some reports have suggested a link between the typeof KIT mutation and gender or age, but no suchcorrelation has been supported in larger studies.4
However, paediatric and Carney triad GISTs occurpredominantly in female patients and are associatedwith gastric location and epithelioid morphology.These tumours lack KIT and PDGFRA mutations,suggesting that other mechanisms of KIT activationor unrelated oncogenic mechanisms are opera-tional.128133 A Pro456Ser in KIT exon 9 and non-sense mutation in PDGFRA exon 18 have beenreported in two separate paediatric GISTs,134,135 how-ever, these mutations probably represent randommolecular events.
A KIT-WT and PDGFRA-WT genotype has beenfound in most studies of GISTs of neurofibromatosis(NF) 1 patients, who have an increased risk forGIST.136140 In one study, two KIT and two PDGFRA
mutations were reported in separate tumours from twoNF1 patients.138 These mutations do not correspond toGIST-type of KIT or PDGFRA mutations and mightbe random genetic events. In another study, the sameVal559Asp substitution was identified in three separateprimary lesions from a patient with phenotypic featurestypical for NF1.141 Unfortunately, normal tissue wasnot available for testing and the possibility of a geneticsyndrome with a germ-line KIT mutation could there-fore not be excluded in this case. NF1-associated GISTshave predominantly spindle cell morphology and showa strong predilection to intestinal location.1
Multiple GISTs have also been reported in non-NF1patients. Usually they are small lesions with differentKIT PDGFRA genotype.142 However, a few patientswith multiple mini GISTs carrying the same mutationshave 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).
254 J Lasota and M Miettinen
2008 The Authors. Journal compilation 2008 Blackwell Publishing Ltd, Histopathology, 53, 245266.
- metastases might explain the latter finding. KIT-WTand PDGFRA-WT genotype has also been found in
-
trials and other GIST studies, including those based onspecific populations.
Diagnostic and prognostic value of primaryKIT or PDGFRA mutations
Most GISTs independent of mutation status are KIT+,including KIT-WT GISTs, such as those in NF1 patientsand children. However, some GISTs (
-
progression, being present in metastases, but not inprimary tumours.24,81,148 A recent study has shownthat 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 similarmolecular mechanism has been shown for homozygousKIT exon 13 mutations.78 Gastric and small intestinalGISTs with homozygous KIT exon 11 mutations arealmost invariably associated with malignant tumourbehaviour.149
Initially, GISTs with KIT exon 9 duplications wereassociated with malignant outcome.73,125 However, arecent study of 145 small intestinal GISTs with KIT exon9 mutations did not show significant differences inclinical outcome between KIT exon 9 and KIT exon 11mutants. Thus, the previously reported findings wereprobably related to the higher mortality of patients withsmall intestinal vs. gastric tumours.120,122
Although KIT exon 13 and KIT exon 17 mutationsare 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 previouslypublished studies from US and European clinical trials
Gene Exon
Primary KIT and PDGFRAmutations identified in GISTsfrom imatinib clinical trials (n) Sensitivity to imatinib mesylate
KIT 9 Ala502_Tyr503dup Sensitive to imatinib in vitro29
Complete remission in 5%, partial response in 29%, stabledisease in 47%, progressive disease in 17% as reported byEORTC phase III trial84
A high-dose regimen increased progression-free survival84
11 Deletion deletioninsertionSubstitutionDuplication
Most common mutants sensitive to imatinib in vitro29
Rare Val559Ile mutant resistant to imatinib in vitro77
Complete remission in 6%, partial response in 61%, stabledisease in 25%, progressive disease in 3% as reported byEORTC phase III trial84
13 Lys642Glu (8)Glu635Lys (1)
Sensitive to imatinib in vitro29
Partial response or stable disease reported in all ninecases29,35,84
17 Asp820Tyr (1)Asn822Lys (2)Asn822His (2)
Asn822Lys and Asn822His sensitive to imatinib in vitro29
Partial response reported in four mutants includingAsn820Tyr, Asn822Lys, Asn822His29,84 Primary resistancereported in Asn822Lys mutant27
PDGFRA 12 Asp561Val (4)Deletion deletioninsertionDuplication, insertion
Asp561Val and some other exon 12 mutants testedsensitive to imatinib in vitro30,117 Objective responsereported in the majority of a few cases treated withimatinib29,84
14 Asn659Lys This mutant tested sensitive to imatinib in vitro30
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 toimatinib in vitro30,117 Objective response reported in themajority of a few cases treated with imatinib29,84
Asp842Val resistant to imatinib in vitro29,30,117
Resistance reported in seven cases includingAsp846Val29,35,84; stable disease in one case after 5 monthsof imatinib treatment 35
KITPDGFRA
9, 11, 13, 1712, 14, 18
Wild-typeWild-type
Partial response in 23%, stable disease in 50%, andprogressive disease in 19% as reported by EORTC phase IIItrial84
GIST, Gastrointestinal stromal tumour; PDGFRA, platelet-derived growth factor-alpha.
Clinical significance of oncogenic KIT and PDGFRA mutations in GISTs 257
2008 The Authors. Journal compilation 2008 Blackwell Publishing Ltd, Histopathology, 53, 245266.
-
shown that KIT exon 13 mutants tend to be signifi-cantly more aggressive than gastric GISTs on average,whereas gastric GISTs with KIT exon 17 mutationsshow no such tendency. Furthermore, the behaviour ofsmall intestinal GISTs with KIT exon 13 or KIT exon 17mutations did not differ from that of other smallintestinal GISTs.79
In general, PDGFRA mutants have low mitotic rateand good prognosis; most of them represent gastricGISTs, many of which would previously have beendiagnosed as leiomyoblastomas.1,4
Primary and secondary KIT and PDGFRAmutations and TK inhibitor treatment
Since the first patient with GIST was successfullytreated in 2000 with KIT and PDGFRA TK inhibitor,imatinib mesylate [STI571, commercially known asGleevc GlivecTM (http://www.novartis.com)], manypatients have benefited from this targeted treat-ment.21,22,150 However, the response to imatinibtreatment to some extent depends on the tumour KIT
or PDGFRA mutation status. Table 5 summarizes dataon in vitro sensitivity to imatinib and response toimatinib treatment of different KIT and PDGFRAmutants.
Clinical observations have shown that KIT exon 11mutants in general respond better to imatinib mesylatetreatment than KIT exon 9 mutants and KIT-WTtumours.29,84 Thus, to achieve similar therapeuticresults, patients with KIT exon 9 mutant GISTs mightrequire a higher dosage of imatinib mesylate.84
GISTs with PDGFRA Asp842Val substitutions areresistant to imatinib treatment.29,35,84 This mutationcorresponds to imatinib-resistant KIT Asp816Val muta-tion reported in human mastocytosis.29
During imatinib mesylate treatment, resistance oftendevelops due to detected secondary KIT or PDGFRAmutations.2327 Almost all such mutations reported inGISTs affect KIT. The only exceptions are two GISTs withprimary KIT and secondary PDGFRA, Asp842Valmutations.24,27 Structurally, most secondary KIT muta-tions represent single nucleotide substitutions affectingspecific codons in KIT exon 13 and 14 (TK1), exon 15
Glu
TyrTyr n = 2
Deletion n = 5His
Ile Glyn = 7 His n = 3
n = 4Glyn = 2 Glu
Ala Glu Gly Glu n = 4 Lys AspKIT-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 823KIT-WT Val Thr Ser Asp Leu Cys Arg Asp Lys Asp Asn Tyr
Ex 13 Ex 14 Ex 15 Ex 16 Ex 17TK1 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, Asn822Lys8. 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
identified in 10 patients. Figure
is based on previously pub-
lished studies.2327,32,35,151160
258 J Lasota and M Miettinen
2008 The Authors. Journal compilation 2008 Blackwell Publishing Ltd, Histopathology, 53, 245266.
-
and 16 (KI) and 17 (TK2).2328,151160 KIT-TK1 domainmutations affect codons never mutated in primarytumours, whereas some of the secondary KIT-TK2mutations, Asn822Lys and Tyr823Asp and PDGFRA-TK2 mutation Asp842Val, have been reported asprimary KIT mutations also.79 In some cases, differentmutations in different lesions, or simultaneous evolu-tion of multiple clones in one lesion, have beenreported.27,35,151153,155 Figure 7 shows the frequencyand distribution of secondary KIT mutations in GISTs.
More recently, sunitinib malate, also known asSU11248 (http://www.pfizer.com), a multitargetedinhibitor of KIT, PDGFRs, vascular endothelial growthfactor receptors, FLT3 and RET receptor TKs, has beenused for treatment of imatinib-resistant GISTs as thefirst second-generation TK inhibitor.161163 In vitroand in vivo studies have shown that the clinical benefitof sunitinib is significantly influenced by KIT andPDGFRA mutation status.31,32,164 Although clinical
benefit was observed in all major mutant types, theprimary response rate was significantly higher for KITexon 9 mutants. The inhibitory effect of sunitinib onKIT kinase activity was not substantially affected bysecondary KIT mutations in TK1. However, GISTs withKIT-TK2 secondary mutations were resistant to suni-tinib treatment.32 Thus, the search for other second-line drugs inhibiting KIT and PDGFRA TK activitymust continue.165,166
Also, inhibition of alternative targets, such asdownstream components of KIT and PDGFR pathwaysincluding AKT and mTOR proteins in the AKT path-way, has been tested in vitro and in clinical trials. Thebest known example of these is everolimus.167 Theseagents can be used alone or in combination with TKinhibitors.
In addition to the inhibition of KIT and PDGFRA andtheir signalling pathways, an antitumour effect can beachieved 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 Pfizer
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 Infinity 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.
Clinical significance of oncogenic KIT and PDGFRA mutations in GISTs 259
2008 The Authors. Journal compilation 2008 Blackwell Publishing Ltd, Histopathology, 53, 245266.
-
Several anti-angiogenesis agents including sunitinib,already approved for GIST treatment, have beendeveloped and their therapeutic potential tested inclinical trials.
More recently, strategies to inhibit KIT signalling byabolishing KIT or diminishing KIT expression havebeen developed. One of these is based on inhibitionof heat-shock protein (HSP)-90, a member of thechaperone family of proteins, which plays a role inprotein folding into three-dimensional shapes andstabilizes and protects KIT from degradation. Inhibitionof HSP90 prevents protective interaction betweenHSP90 and KIT and leads to KIT degradation andtumour cell apoptosis.167 Another, very new strategyto inhibit KIT signalling is to abrogate KIT mRNAexpression with a transcriptional inhibitor, such asflavopiridol.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 mayhave an impact on planning the targeted treatment,genotyping of GISTs should be considered a standardclinical test in all primary tumours with a significantrisk of metastasis, especially to rule out mutantsprimarily resistant to imatinib. Also, testing for sec-ondary KIT and PDGFRA mutations in GISTs underimatinib treatment could be valuable in monitoringdrug resistance.
Technical considerations
Contamination of tumour samples with non-tumourcells, such as lymphocytes, other inflammatory cellsand entrapped smooth muscle cells can lead to relativedecrease of tumour DNA in the analysed sample andcause false-negative results in PCR-based mutationanalysis by elevating PCR amplification of KIT-wildtype versus KIT-mutant allele. Thus, histopathologicalevaluation of the sample and enrichment of tumourtissue for DNA extraction are necessary.
Detection of KIT and PDGFRA mutations informalin-fixed paraffin-embedded (FFPE) GISTs ap-pears to be lower than expected in some studies.Three recent studies performed independently ondifferent material by two different groups have shownthat a detection rate of KIT and PDGFRA tendsto decrease with increasing age of paraffinblocks.97,120,144 A possible explanation for this phe-nomenon is ongoing degradation of tumour DNA inarchival paraffin blocks.
Most KIT and PDGFRA mutation studies in GISTshave 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 fractioncollector, substantially increases the detection of muta-tions compared with standard direct sequencing of PCRproducts.169,170 However, one study has shown that alarge duplication may not be easy amplifiable frompartially degraded DNA obtained from FFPE tissuesand missed by both DHPLC screening and directsequencing of PCR products. Thus, obtaining relativelysmall amplicons by design of primer systems is help-ful in PCR-based detection of duplications in FFPEGISTs.171 According to another recent study, screeningof PCR amplification products for KIT and PDGFRAmutations using high-resolution melting ampliconanalysis might be slightly more sensitive thanDHPLC.115
In general, multiple primary KIT mutations affectingthe same or different exons have rarely been reported.However, one study identified multiple KIT exon 11mutations in as many as 9% (seven of 78) primaryGISTs.172
Also, KIT STOP codon frame-shift mutations havebeen reported occasionally in primary and metastaticGISTs.27,90,135,173 These mutations might reflect sec-ondary changes related to tumour progression. It seemsthat they might involve KIT-WT allele rather than theprimarily mutated allele and, in fact, lead to functionalKIT homozygosity.27
STOP
Gln828
A
B
Figure 8. Example of an artefact created by polymerase chain
reaction (PCR) amplification. 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 amplification
from the same DNA sample (B).
260 J Lasota and M Miettinen
2008 The Authors. Journal compilation 2008 Blackwell Publishing Ltd, Histopathology, 53, 245266.
-
In some studies it has not been clearly stated whetheradditional primary KIT or PDGFRA mutations weredetected by sequencing of PCR amplification productsfrom at least two independent PCRs. Thus, PCRamplification sequencing artefacts can not be com-pletely excluded. Increased frequency of PCR amplifica-tion artefacts has been reported in analysis of DNA fromFFPE tissues.174 Figure 8 shows an example of PCRamplification artefact in PDGFRA exon 18.
Several single nucleotide polymorphisms (SNP) inKIT and PDGFRA coding sequences (SNP database athttp://www.ncbi.nlm.nih.gov) and two alternativesplicing sites in KIT have been reported.175,176 KITand PDGFRA polymorphisms and alternatively splicedvariants of KIT mRNA should not be confused withactivating oncogenic KIT mutations.177
Disclaimer
The opinions and assertions contained herein are theexpressed views of the authors and are not to beconstrued as official or reflecting the views of theDepartments of the Army or Defense.
References
1. Miettinen M, Lasota J. Gastrointestinal stromal tumors: pathol-
ogy and prognosis at different sites. Semin. Diagn. Pathol. 2006;
23; 7083.
2. Hirota S, Isozaki K, Moriyama Y et al. Gain-of-function
mutations of c-kit in human gastrointestinal stromal tumors.
Science 1998; 279; 577580.
3. Heinrich MC, Corless CL, Duensing A et al. PDGFRA activating
mutations in gastrointestinal stromal tumors. Science 2003;
299; 708710.
4. Lasota J, Miettinen M. KIT and PDGFRA mutations in
gastrointestinal stromal tumors (GISTs). Semin. Diagn. Pathol.
2006; 23; 91102.
5. Roberts WM, Look AT, Ruossel MF et al. Tandem linkage of
human CSF-1 receptor (c-fms) and PDGF receptor genes. Cell
1989; 55; 655661.
6. Stenman G, Eriksson A, Claesson-Welsh L. Human PDGFA
receptor gene maps to the same region on chromosome 4 as the
KIT oncogene. Genes Chromosomes Cancer 1989; 1; 155158.
7. Pawson T. Regulation and targets of receptor tyrosine kinases.
Eur. J. Cancer 2002; 38; S3S10.
8. Fletcher JA. Role of KIT and platelet-derived growth factor
receptors as oncoproteins. Semin. Oncol. 2004; 31 (Suppl. 6);
411.
9. Mol CD, Dougan DR, Schneider TR et al. Structural basis for the
autoinhibition and STI-571 inhibition of c-Kit tyrosine kinase.
J. Biol. Chem. 2004; 279; 3165531663.
10. Yarden Y, Kuang WJ, Yang Feng T et al. Human proto-
oncogene c-kit: a new cell surface receptor tyrosine kinase for
an unidentified ligand. EMBO J. 1987; 6; 33413351.
11. Chabot B, Stephenson DA, Chapman VM et al. The proto-
oncogene c-kit encoding a transmembrane tyrosine kinase
receptor maps to the mouse W locus. Nature 1988; 335; 8889.
12. Williams DE, Eisenman J, Baird A et al. Identification of a
ligand for the c-kit protooncogene. Cell 1990; 63; 167174.
13. Zsebo KM, Williams DA, Geissler EN et al. Stem cell factor is
encoded at the S1 locus of the mouse and is the ligand for the
c-kit tyrosine kinase receptor. Cell 1990; 63; 213224.
14. Blume-Jensen P, Claesson-Welsh L, Siegbahn A et al. Activa-
tion of the human c-kit product by ligand-induced dimerization
mediates circular actin reorganization and chemotaxis. EMBO
J. 1991; 10; 41214128.
15. Maeda H, Yamagata A, Nishikawa S et al. Requirement of c-kit
for development of intestinal pacemaker system. Development
1992; 116; 369375.
16. Lev S, Blechman J, Nishikawa S et al. Interspecies molecular
chimeras of kit helps define the binding site of the stem cell
factor. Mol. Cell. Biol. 1993; 13; 22242234.
17. Huizinga JD. Gastrointestinal peristalsis: joint action of enteric
nerves, smooth muscle, and interstitial cells of Cajal. Microsc.
Res. Tech. 1999; 47; 239247.
18. Kindblom LG, Remotti HE, Aldenborg F, Meis-Kindblom JM.
Gastrointestinal pacemaker cell tumor (GIPACT): gastrointes-
tinal stromal tumors show phenotypic characteristics of the
interstitial cells of Cajal. Am. J. Pathol. 1998; 152; 12591269.
19. Chen H, Hirota S, Isozaki K et al. Polyclonal nature of diffuse
proliferation of interstitial cells of Cajal in patients with familial
and multiple gastrointestinal stromal tumours. Gut 2002; 51;
793796.
20. Agaimy A, Wunsch PH, Hofstaedter F et al. Minute gastric
sclerosing stromal tumors (GIST tumorlets) are common in
adults and frequently show c-KIT mutations. Am. J. Surg.
Pathol. 2007; 31; 113120.
21. Joensuu H, Roberts PJ, Sarlomo-Rikala M et al. Effect of
tyrosine kinase inhibitor STI571 in a patient with a metastatic
gastrointestinal stromal tumor. N. Engl. J. Med. 2001; 344;
10521056.
22. Demetri GD. Identification and treatment of chemoresistant
inoperable or metastatic GIST: experience with the selective
tyrosine kinase inhibitor imatinib mesylate (STI571). Eur. J.
Cancer 2002; 38 (Suppl. 5); S52S59.
23. Chen LL, Trent JC, Wu EF et al. A missense mutation in KIT
domain 1 correlates with imatinib resistance in gastrointestinal
stromal tumors. Cancer Res. 2004; 64; 59135919.
24. Debiec-Rychter M, Cools J, Dumez H et al. Mechanisms of
resistence to imatinib mesylate in gastrointestinal stromal
tumors and activity of the PKC412 inhibitor against imatinib-
resistant mutants. Gastroentereology 2005; 128; 270279.
25. Tamborini E, Bonadiman L, Greco A et al. A new mutation in
the KIT ATP pocket causes acquired resistance to imatinib in a
gastrointestinal stromal tumor patient. Gastroenterology 2004;
127; 294299.
26. McLean SR, Gana-Weisz M, Hartzoulakis B et al. Imatinib
binding and cKIT inhibition is abrogated by the cKIT kinase
domain I missense mutation Val654Ala. Mol. Cancer Ther.
2005; 4; 20082015.
27. Heinrich MC, Corless CL, Blanke CD et al. Molecular correlates
of imatinib resistance in gastrointestinal stromal tumors.
J. Clin. Oncol. 2006; 24; 47644774.
28. Miselli F, Casieri P, Negri T et al. C-KIT PDGFRA gene statusalterations possibly related to primary imatinib resistance in
gastrointestinal stromal tumors. Clin. Cancer Res. 2007; 13;
23692377.
29. Heinrich MC, Corless CL, Demetri GD et al. Kinase mutations
and imatinib response in patients with metastatic gastrointes-
tinal stromal tumor. J. Clin. Oncol. 2003; 21; 43424349.
Clinical significance of oncogenic KIT and PDGFRA mutations in GISTs 261
2008 The Authors. Journal compilation 2008 Blackwell Publishing Ltd, Histopathology, 53, 245266.
-
30. Corless CL, Schroeder A, Griffith D et al. PDGFRA mutations in
gastrointestinal stromal tumors: frequency, spectrum and
in vitro sensitivity to imatinib. J. Clin. Oncol. 2005; 23;
53575364.
31. Prenen H, Cools J, Mentens N et al. Efficacy of the kinase
inhibitor SU11248 against gastrointestinal stromal tumor
mutants refractory to imatinib mesylate. Clin. Cancer Res.
2006; 12; 26222627.
32. Heinrich MC, Corless CL, Liegl B et al. Mechanisms of sunitinib
malate (SU) resistance in gastrointestinal stromal tumors
(GISTs). 2007 ASCO Annual Meeting Proceedings Part I.
J. Clin. Oncol. 2007; 25; 10006.
33. Hostein I, Longy M, Gastaldello B et al. Detection of a new
mutation in KIT exon 9 in a gastrointestinal stromal tumor.
Int. J. Cancer 2006; 118; 20892091.
34. Lasota J, Miettinen M. KIT exon 11 deletion-inversions
represent complex mutations in gastrointestinal stromal
tumors. Cancer Genet. Cytogenet. 2007; 175; 6972.
35. Antonescu CR, Besmer P, Guo T et al. Acquired resistance to
imatinib in gastrointestinal stromal tumors occurs through
secondary gene mutation. Clin. Cancer Res. 2005; 11; 4182
4190.
36. Longley BJ, Reguera MJ, Ma Y. Classes of c-KIT activating
mutations: proposed mechanisms of action and implications in
disease classification and therapy. Leuk. Res. 2001; 25; 571
576.
37. Ma Y, Cunningham M, Wang X, Ghosh I, Regan L, Longley
B. Inhibition of spontaneous receptor phosphorylation by
residues in putative alpha-helix in the KIT intracellular
juxtamembrane region. J. Biol. Chem. 1999; 274; 13399
13402.
38. Chan PM, Ilangumaran S, La Rose J, Chakrabartty A, Rottapel
R. Autoinhibition of the kit receptor tyrosine kinase by the
cytosolic juxtamembrane region. Mol. Cell. Biol. 2003; 23;
30673078.
39. Duensing A, Medeiros F, McConarty B et al. Mechanisms of
oncogenic KIT signal transduction in primary gastrointestinal
stromal tumours (GISTs). Oncogene 2004; 23; 39994006.
40. Rubin BP, Heinrich MC, Corless CL. Gastrointestinal stromal
tumour. Lancet 2007; 368; 17311741.
41. Nishida T, Hirota S, Taniguchi M et al. Familial gastrointestinal
stromal tumours with germline mutation of the KIT gene. Nat.
Genet. 1998; 19; 323324.
42. Isozaki K, Terris B, Belghiti J et al. Germline-activating
mutation in the kinase domain on KIT gene in familial
gastrointestinal stromal tumors. Am. J. Pathol. 2001; 157;
15811585.
43. Beghini A, Tibiletti MG, Roversi G et al. Germline mutation in
the juxtamembrane domain of the kit gene in a family with
gastrointestinal stromal tumors and urticaria pigmentosa.
Cancer 2001; 92; 657662.
44. Maeyama H, Hidaka E, Ota H et al. Familial gastrointestinal
stromal tumor with hyperpigmentation: association with a
germline mutation of the c-kit gene. Gastroenterology 2001;
120; 210215.
45. Hirota S, Nishida T, Isozaki K et al. Familial gastrointestinal
stromal tumors associated with dysphagia and novel type
germline mutation of KIT gene. Gastroenterology 2002; 122;
14931499.
46. Chompret A, Kannengiesser C, Barrois M et al. PDGFRA
germline mutation in a family with multiple cases of gastro-
intestinal stromal tumor. Gastroenterology 2004; 126; 318
321.
47. Robson ME, Glogowski E, Sommer G et al. Pleomorphic
characteristics of a germ-line KIT mutation in a large kindred
with gastrointestinal stromal tumors, hyperpigmentation, and
dysphagia. Clin. Cancer Res. 2004; 10; 12501254.
48. Carballo M, Roig I, Aquilar F et al. Novel c-KIT germline
mutation in a family with gastrointestinal stromal tumors and
cutaneous hyperpigmantation. Am. J. Med. Genet. A 2005;
132; 361364.
49. Li FP, Fletcher JA, Heinrich MC et al. Familial gastrointestinal
stromal tumor syndrome: phenotypic and molecular features in
a kindred. J. Clin. Oncol. 2005; 23; 27352743.
50. Tarn C, Merkel E, Canutescu AA et al. Analysis of KIT
mutations in sporadic and familial gastrointestinal stromal
tumors: therapeutic implications through protein modeling.
Clin. Cancer Res. 2005; 11; 36683677.
51. ORiain C, Corless CL, Heinrich MC et al. Gastrointestinal
stromal tumors: insights from a new familial GIST kindred with
unusual genetic and pathologic features. Am. J. Surg. Pathol.
2005; 29; 16801683.
52. Hartmann K, Wardelmann E, Ma Y et al. Novel germline
mutation of KIT associated with familial gastrointestinal
stromal tumors and mastocytosis. Gastroenterology 2005;
129; 10421046.
53. Lasota J, Miettinen M. A new familial GIST identified. Am. J.
Surg. Pathol. 2006; 30; 1342.
54. Hirota S, Okazaki T, Kitamura Y et al. Cause of familial and
multiple gastrointestinal autonomic nerve tumors with hyper-
plasia of interstitial cells of Cajal is germline mutation of the
c-kit gene. Am. J. Surg. Pathol. 2000; 24; 326327.
55. Kang DY, Park CK, Choi JS et al. Multiple gastrointestinal
stromal tumors: clinicopathologic and genetic analysis of 12
patients. Am. J. Surg. Pathol. 2007; 31; 224232.
56. Graham J, Debiec-Rychter M, Corless CL et al. Imatinib in the
management of multiple gastrointestinal stromal tumors asso-
ciated with a germline KIT K642E mutation. Arch. Pathol. Lab.
Med. 2007; 131; 13931396.
57. Sommer G, Agosti V, Ehlers I et al. Gastrointestinal stromal
tumors in a mouse model by targeted mutation of the Kit
receptor tyrosine kinase. Proc. Natl Acad. Sci. USA 2003; 100;
67066711.
58. Rubin BP, Antonescu CR, Scott-Browne JP et al. A knock-in
mouse model of gastrointestinal stromal tumor harboring Kit
K641E. Cancer Res. 2005; 65; 66316639.
59. Nakahara M, Isozaki K, Hirota S et al. A novel gain-of-function
mutation of c-kit gene in gastrointestinal stromal tumors.
Gastroenterology 1998; 115; 10901095.
60. Tuveson DA, Willis NA, Jacks T et al. STI571 inactivation of
the gastrointestinal stromal c-KIT oncoprotein: biological and
clinical implications. Oncogene 2001; 20; 50545058.
61. Nakatani H, Kobayashi M, Jin T et al. STI571 (Glivec) inhibits
the interaction between c-KIT and heat shock protein 90 of the
gastrointestinal stromal tumor cell line, GIST-T1. Cancer Sci.
2005; 96; 116119.
62. Tarn C, Skorobogatko YV, Taguchi T, Eisenberg B, von Mehren
M, Godwin AK. Therapeutic effect of imatinib in gastrointes-
tinal stromal tumors: AKT signaling dependent and indepen-
dent mechanisms. Cancer Res. 2006; 66; 54775486.
63. Bauer S, Duensing A, Demetri GD et al. KIT oncogenic
signaling mechanisms in imatinib-resistant gastrointestinal
stromal tumor: PI3-kinase AKT is a crucial survival pathway.Oncogene 2007; 26; 75607568.
64. Sciot R, Debiec-Rychter M. GIST under imatinib therapy.
Semin. Diagn. Pathol. 2006; 23; 8490.
262 J Lasota and M Miettinen
2008 The Authors. Journal compilation 2008 Blackwell Publishing Ltd, Histopathology, 53, 245266.
-
65. de Raedt T, Cools J, Debiec-Rychter M et al. Intestinal neuro-
fibromatosis is a subtype of familial GIST and results from
dominant activating mutation in PDGFRA. Gastroenterology
2006; 131; 19071912.
66. Pasini B, Matyakhina L, Bei T et al. Multiple gastrointestinal
stromal tumors caused by platelet-derived growth factor
receptor a gene mutations: a case associated with a germlineV561D defect. J. Clin. Endocrin. Metab. 2007; 92; 3728
3732.
67. Corless CL, Fletcher JA, Heinrich MC. Biology of gastrointes-
tinal stromal tumors. J. Clin. Oncol. 2004; 22; 38133825.
68. Lasota J, Jasinski M, Sarlomo-Rikala M et al. Mutations in
exon 11 of c-kit occur preferentially in malignant versus
benign gastrointestinal stromal tumors and do not occur in
leiomyomas or leiomyosarcomas. Am. J. Pathol. 1999; 154;
5360.
69. Taniguchi M, Nishida T, Hirota S et al. Effect of c-kit mutation
on prognosis of gastrointestinal stromal tumors. Cancer Res.
1999; 59; 42974300.
70. Sakurai S, Fukasawa T, Chong JM et al. C-kit gene abnormal-
ities in gastrointestinal stromal tumors (tumors of interstitial
cells of Cajal). Jpn. J. Cancer Res. 1999; 90; 13211328.
71. Rubin BP, Singer S, Tsao C et al. KIT activation is a ubiquitous
feature of gastrointestinal stromal tumors. Cancer Res. 2001;
61; 81188121.
72. Wardelmann E, Neidt I, Bierhoff E et al. c-kit mutations in
gastrointestinal stromal tumors occur preferentially in the
spindle rather than in the epithelioid cell variant. Mod. Pathol.
2002; 15; 125136.
73. Antonescu CR, Sommer G, Sarran L et al. Association of KIT
exon 9 mutation with nongastric primary site and aggressive
behavior: KIT mutation analysis and clinical correlates of 120
gastrointestinal stromal tumors. Clin. Cancer Res. 2003; 9;
33293337.
74. Corless CL, McGreevey L, Town A et al. KIT gene deletion at the
intron 10-exon 11 boundry in GI stromal tumors. J. Mol. Diag.
2004; 6; 366370.
75. Chen LL, Sabripour M, Wu EF et al. A mutation-created novel
intra-exonic pre-mRNA splice site causes constitutive activa-
tion of KIT in human gastrointestinal stromal tumors. Oncogene
2005; 24; 42714280.
76. Andersson J, Sjogren H, Meis-Kindblom JM et al. The complex-
ity of KIT gene mutations and chromosome rearrangements
and their clinical correlation in gastrointestinal stromal (pace-
maker cell) tumors. Am. J. Pathol. 2002; 160; 1522.
77. Nakagomi N, Hirota S. Juxtamembrane-type c-kit gene muta-
tion found in aggressive systematic mastocytosis induces
imatinib-resistant constitutive KIT activation. Lab. Invest.
2007; 87; 365371.
78. Lux ML, Rubin BP, Biase TL et al. KIT extracellular and kinase
domain mutations in gastrointestinal stromal tumors. Am. J.
Pathol. 2000; 156; 791795.
79. Lasota J, Corless CL, Heinrich MC et al. Clinicopathologic profile
of gastrointestinal stromal tumors (GISTs) with primary KIT
exon 13 or exon 17 mutations. A multi-center study on 54
cases. Mod. Pathol. 2008; Epub ahead of print.
80. He HY, Xiang YN, Zhong HH et al. c-kit and PDGFRS
mutations in 60 cases of gastrointestinal tumors (GISTs).
Beijing Da Xue Xue Bao 2005; 37; 320324.
81. Cho S, Kitadai Y, Yoshida S et al. Deletion of the KIT gene is
associated with liver metastasis and poor prognosis in patients
with gastrointestinal stromal tumor in the stomach. Int. J.
Oncol. 2006; 28; 13611367.
82. Cho S, Kitadai Y, Yoshida S et al. Genetic and pathologic
characteristics of gastrointestinal stromal tumors in extra-
gastric lesions. Int. J. Mol. Med. 2006; 18; 1067
1071.
83. Kinoshita K, Hirota S, Isozaki K et al. Characterization of
tyrosine kinase I domain c-kit gene mutation Asn655Lys newly
found in primary jejunal gastrointestinal stromal tumor. Am. J.
Gastroenterol. 2007; 102; 11341136.
84. Debiec-Rychter M, Sciot R, Le Cesne A et al. KIT mutations and
dose selection for imatinib in patients with advanced gastro-
intestinal stromal tumors. Eur. J. Cancer 2006; 42; 1093
1103.
85. Hongyo T, Li T, Syaifudin M et al. Specific c-kit mutations in
sinonasal natural killer T-cell lymphoma in China and Japan.Cancer Res. 2000; 60; 23452347.
86. Tian Q, Frierson HF Jr, Krystal GW et al. Activating c-kit gene
mutations in human germ cell tumors. Am. J. Pathol. 1999;
154; 16431647.
87. Przygodzki RM, Hubbs AE, Zhao F-Q et al. Primary mediastinal
seminomas: evidence of single and multiple KIT mutations.
Lab. Invest. 2002; 82; 13691375.
88. Kemmer K, Corless CL, Fletcher JA et al. KIT mutations are
common in testicular seminomas. Am. J. Pathol. 2004; 164;
305313.
89. Longley BJ, Tyrrell L, Lu S-Z et al. Somatic c-KIT activating
mutation in urticaria pigmentosa and aggressive mastocytosis:
establishment of clonality in a human mast cell neoplasm.
Nat. Genet. 1996; 12; 312314.
90. Martin J, Poveda J, Llombart-Bosch A et al. Deletions
affecting codons 557-558 of the c-KIT gene indicate a poor
prognosis in patients with completely resected gastrointesti-
nal stromal tumors: a study by the Spanish Group for
Sarcoma Research (GEIS). J. Clin. Oncol. 2005; 23; 6190
6198.
91. Moskaluk CA, Tian Q, Marshall CR et al. Mutations of c-kit JM
domain are found in a minority of human gastrointestinal
stromal tumors. Oncogene 1999; 18; 18971902.
92. Lasota J, Wozniak A, Sarlomo-Rikala M et al. Mutations in
exons 9 and 13 of KIT gene are rare events in gastrointestinal
stromal tumors. A study of two hundred cases. Am. J. Pathol.
2000; 157; 10911095.
93. Hirota S, Nishida T, Isozaki K et al. Gain-of-function mutation
at the extracellular domain of KIT in gastrointestinal stromal
tumours. J. Pathol. 2001; 193; 505510.
94. Sakurai S, Oguni S, Hironaka M et al. Mutations in c-kit gene
exons 9 and 13 in gastrointestinal stromal tumors among
Japanese. Jpn. J. Cancer Res. 2001; 92; 494498.
95. Subramanian S, West R, Corless CL et al. Gastrointestinal
stromal tumors (GISTs) wit KIT and PDGFRA mutations have
distinct gene expression profiles. Oncogene 2004; 23; 7780
7790.
96. Wardelmann E, Hrychyk A, Markelbach-Bruse S et al. Associ-
ation of platelet-derived growth factor receptor a mutationswith gastric primary site and epithelioid or mixed cell
morphology in gastrointestinal stromal tumors. J. Mol. Diagn.
2004; 6; 197204.
97. Andersson J, Bumming P, Meis-Kindblom JM et al. Gastro-
intestinal stromal tumors with KIT exon 11 deletions are
associated with poor prognosis. Gastroenterology 2006; 130;
15731581.
98. Kim TW, Lee H, Kang Y-K et al. Prognostic significance of
c-kit mutation in localized gastrointestinal stromal tumors.
Clin. Cancer Res. 2004; 10; 30763081.
Clinical significance of oncogenic KIT and PDGFRA mutations in GISTs 263
2008 The Authors. Journal compilation 2008 Blackwell Publishing Ltd, Histopathology, 53, 245266.
-
99. Kang HJ, Nam SW, Kim H et al. Correlation of KIT and platelet-
derived growth factor receptor a mutations with gene activa-tion and expression profiles in gastrointestinal stromal tumors.
Oncogene 2005; 24; 10661074.
100. Choi YR, Kim H, Kang HJ et al. Overexpression of high mobility
group box 1 in gastrointestinal stromal tumors with KIT
mutation. Cancer Res. 2003; 63; 21882193.
101. Feng F, Liu XH, Xie Q et al. Expression and mutation of c-kit
gene in gastrointestinal stromal tumors. World J. Gastroenterol.
2003; 9; 25482551.
102. Hou YY, Tan YS, Sun MH et al. C-kit gene mutation in
gastrointestinal stromal tumors. World J. Gastroenterol. 2004;
10; 13101314.
103. Lasota J, Dansonka-Mieszkowska A, Stachura T et al. Gastro-
intestinal stromal tumors with internal tandem duplications in
3 end of KIT juxtamembrane domain occur predominantlyin stomach and generally seem to have a favorable course.
Mod. Pathol. 2003; 16; 12571264.
104. Haller F, Gunawan B, von Heydebreck A et al. Prognostic role
of E2F1 and members of the CDKN2A network in gastro-
intestinal stromal tumors. Clin. Cancer Res. 2005; 11; 6589
6597.
105. Daum O, Grossmann P, Vanecek T et al. Diagnostic morpho-
logical features of PDGFRA-mutated gastrointestinal stromal
tumors: molecular genetic and histologic analysis of 60 cases of
gastric gastrointestinal stromal tumors. Ann. Diagn. Pathol.
2007; 11; 2733.
106. Penzel R, Aulmann S, Moock M et al. The location of KIT and
PDGFRA gene mutations in gastrointestinal stromal tumours is
site and phenotype associated. J. Clin. Pathol. 2005; 58; 634
639.
107. London CA, Galli SJ, Yuuki T et al. Spontaneous canine mast
cell tumors express tandem duplications in the proto-oncogene
c-kit. Exp. Hematol. 1999; 27; 689697.
108. Ma Y, Longley BJ, Wang X et al. Clustering of activating
mutations in c-KITs juxtamembrane coding region in canine
mast cell neoplasms. J. Invest. Dermatol. 1999; 112; 165
170.
109. Corbacioglu S, Kilic M, Westhoff MA et al. Newly identified c-kit
receptor tyrosine kinase ITD in childhood AML induces ligand
independent growth and is responsive to a synergistic effect of
imatinib and rapamycin. Blood 2006; 108; 35043513.
110. Lasota J, Dansonka-Mieszkowska A, Sobin LH et al. A great
majority of GISTs with PDGFRA mutations represents gastric
tumors of low or no malignant potential. Lab. Invest. 2004; 84;
874883.
111. Pauls K, Merkelbach-Bruse S, Thal D et al. PDGFRa- and c-kit-mutated gastrointestinal stromal tumours (GISTs) are charac-
terized by distinctive histological and immunohistochemical
features. Histopathology 2005; 46; 166175.
112. Wasag B, Debiec-Rychter M, Pauwels P et al. Differential
expression of KIT PDGFRA mutant isoforms in epithelioid andmixed variants of gastrointestinal stromal tumors depends
predominantly on the tumor site. Mod. Pathol. 2004; 17; 889
894.
113. Sakurai S, Hasegawa T, Sakuma Y et al. Myxoid epithelioid
gastrointestinal stromal tumor (GIST) with mast cell infiltra-
tions: a subtype of GIST with mutations of platelet-derived
growth factor receptor alfa gene. Human Pathol. 2004; 35;
12231230.
114. Haller F, Happel N, Schulten H-J et al. Site-dependent differen-
tial KIT and PDGFRA expression in gastric and intestinal
stromal tumors. Mod. Pathol. 2007; 20; 11031111.
115. Holden JA, Willmore-Payne C, Coppola D et al. High-resolution
melting amplicon analysis as a method to detect c-kit and
platelet-derived growth factor receptor a activating mutationsin gastrointestinal stromal tumors. Am. J. Clin. Pathol. 2007;
128; 230238.
116. Medeiros F, Corless CL, Duensing A et al. KIT-negative gastro-
intestinal stromal tumors. Proof of concept and therapeutic
implications. Am. J. Surg. Pathol. 2004; 28; 889894.
117. Hirota S, Ohashi A, Nishida T et al. Gain-of-function mutations
of platelet-derived growth factor receptor alpha gene in
gastrointestinal stromal tumors. Gastroenterology 2003; 125;
660667.
118. Lasota J, Stachura J, Miettinen M. GISTs with PDGFRA exon 14
mutations represent subset of clinically favorable gastric
tumors with epithelioid morphology. Lab. Invest. 2006; 86;
94100.
119. Miettinen M, Sarlomo-Rikala M, Sobin LH et al. Esophageal
stromal tumors: a clinicopathologic, immunohistochemical,
and molecular genetic study of 17 cases and comparison with
esophageal leiomyomas and leiomyosarcomas. Am. J. Surg.
Pathol. 2000; 24; 211222.
120. Miettinen M, Sobin LH, Lasota J. Gastrointestinal stromal
tumors (GISTs) of the stomacha clinicopathologic, immu-
nohistochemical and molecular genetic study of 1756 cases
with long-term follow-up. Am. J. Surg. Pathol. 2005; 29;
5268.
121. Miettinen M, Kopczynski J, Maklouf HR et al. Gastrointestinal
stromal tumors, intramural leiomyomas and leiomyosarcomas
in the duodenuma clinicopathologic, immunohistochemical
and molecular genetic study of 167 cases. Am. J. Surg. Pathol.
2003; 27; 625641.
122. Miettinen M, Makhlouf H, Sobin LH et al. Gastrointestinal
stromal tumors (GISTs) of the jejunum and ileum: a clinico-
pathologic, immunohistochemical and molecular genetic study
of 906 cases before imatinib with long-term follow-up. Am. J.
Surg. Pathol. 2006; 30; 477489.
123. Miettinen M, Sarlomo-Rikala M, Sobin LH et al. Gastrointes-
tinal stromal tumors and leiomyosarcomas in the colon: a
clinicopathologic, immunohistochemical and molecular genet-
ic study of 44 cases. Am. J. Surg. Pathol. 2000; 24; 1339
1352.
124. Miettinen M, Furlong M, Sarlomo-Rikala M et al. Gastrointes-
tinal stromal tumors, intramural leiomyomas, and leiomyo-
sarcomas in the rectum and anus. A clinicopathologic,
immunohistochemical and molecular genetic study of 144
cases. Am. J. Surg. Pathol. 2001; 25; 11211133.
125. Lasota J, Kopczynski J, Sarlomo-Rikala M et al. KIT 1530ins6
mutation defines a subset of predominantly malignant gastro-
intestinal stromal tumors of intestinal origin. Hum. Pathol.
2003; 34; 13061312.
126. Tzen CY, Wang MN, Mau BL. Spectrum and prognostication of
KIT and PDGFRA mutation in gastrointestinal stromal tumors.
Eur. J. Surg. Oncol. 2007; Epub May 29.
127. Todoroki T, Sano T, Sakurai S et al. Primary omental gastro-
intestinal stromal tumor (GIST). World J. Surg. Oncol. 2007; 5;
66.
128. Miettinen M, Lasota J, Sobin LH. Gastrointestinal stromal
tumors of the stomach in children and young adults. Am. J.
Surg. Pathol. 2005; 29; 19.
129. Prakash S, Sarran L, Socci N et al. Gastrointestinal stromal
tumors in children and young adults: a clinicopathologic,
molecular, and genomic study of 15 cases and review of the
literature. J. Pediatr. Hematol. Oncol. 2005; 27; 179187.
264 J Lasota and M Miettinen
2008 The Authors. Journal compilation 2008 Blackwell Publishing Ltd, Histopathology, 53, 245266.
-
130. OSullivan MJ, McCabe A, Gillett P et al. Multiple gastric
stromal tumors in a child without syndromic association lacks
common KIT or PDGFRalpha mutations. Pediatr. Dev. Pathol.
2005; 8; 685689.
131. Diment J, Tamborini E, Casali P et al. Carney triad: case report
and molecular analysis of gastric tumor. Hum. Pathol. 2005;
36; 112116.
132. Perry CG, Young WF Jr, McWhinney SR et al. Functioning
paraganglioma and gastrointestinal stromal tumor of the
jejunum in three women: syndrome or coincidence. Am. J.
Surg. Pathol. 2006; 30; 4249.
133. Bumming P, Nilsson B, Sorensen J et al. Use of 2-tracer PET to
diagnose gastrointestinal stromal tumour and pheochromo-
cytoma in patients with Carney triad and neurofibromatosis
type 1. Scand. J. Gastroenterol. 2005; 41; 626630.
134. Kuroiwa M, Hiwatari M, Hirato J et al. Advanced-stage
gastrointestinal stromal tumor treated with imatinib in a
12-year-old girl with unique mutation of PDGFRA. J. Pediatr.
Surg. 2005; 40; 17981801.
135. Price VE, Zielenska M, Chilton-MacNeill S et al. Clinical
and molecular characteristics of pediatric gastrointestinal
stromal tumors (GISTs). Pediatr. Blood Cancer 2005; 45; 2024.
136. Kinoshita K, Hirota S, Isozaki K et al. Absence of c-kit gene
mutations in gastrointestinal stromal tumours from neuro-
fibromatosis type 1 patients. J. Pathol. 2004; 202; 8085.
137. Andersson J, Sihto H, Meis-Kindblom JM et al. NF1-associated
gastrointestinal stromal tumors have unique clinical, pheno-
typic, and genotypic characteristics. Am. J. Surg. Pathol. 2005;
29; 11701176.
138. Takazawa Y, Sakurai S, Sakuma Y et al. Gastrointestinal
stromal tumors of neurofibromatosis type I (von Reckling-
hausens disease). Am. J. Surg. Pathol. 2005; 29; 755763.
139. Miettinen M, Fetsch JF, Sobin LH et al. Gastrointestinal stromal
tumors in patients with neurofibromatosis 1: a clinicopatho-
logic and molecular genetic study of 45 cases. Am. J. Surg.
Pathol. 2006; 30; 9096.
140. Nemoto H, Tate G, Schirinzi A et al. Novel NF1 gene mutation
in a Japanese patient with neurofibromatosis type 1 and a
gastrointestinal stromal tumor. J. Gastroenterol. 2006; 41;
378382.
141. Yantiss R, Rosenberg AE, Sarran L et al. Multiple gastro-
intestinal stromal tumors in type I neurofibromatosis: a
pathologic and molecular study. Mod. Pathol. 2005; 18; 475
484.
142. Haller F, Schulten H-J, Armbrust T et al. Multicentric sporadic
gastrointestinal stromal tumors (GISTs) of the stomach with
distinct clonal origin: differential diagnosis to familial and
syndromal GIST variants and peritoneal metastasis. Am. J.
Surg. Pathol. 2006; 31; 933937.
143. Bumming P, Andersson J, Meis-Kindblom JM et al. Neoadju-
vant, adjuvant and palliative treatment of gastrointestinal
stromal tumours (GIST) with imatinib: a centre-based study of
17 patients. Cancer Res. UK 2003; 89; 460464.
144. Steigen SE, Eide TJ, Wasag B et al. Mutations in gastrointestinal
stromal tumorsa population-based study from Northern
Norway. APMIS 2007; 115; 289298.
145. Ernst SI, Hubbs AE, Przygodzki RM et al. KIT mutation
portends poor prognosis in gastrointestinal stromal smoothmuscle tumors. Lab. Invest. 1998; 78; 16331636.
146. Corless CL, McGreevey L, Haley A et al. KIT mutations are
common in incidental gastrointestinal stromal tumors one
centimeter or less in size. Am. J. Pathol. 2002; 160; 1567
1572.
147. Wardelmann E, Losen I, Hans V et al. Deletion of Trp-557 and
Lys-558 in the juxtamembrane domain of the c-kit protoonco-
gene is associated with metastatic behavior of gastrointestinal
stromal tumors. Int. J. Cancer 2003; 106; 887895.
148. Kikuchi H, Yamashita K, Kawabata T et al. Immunohisto-
chemical and genetic features of gastric and metastatic liver
gastrointestinal stromal tumors: sequential analyses. Cancer
Sci. 2006; 97; 127132.
149. Lasota J, Jerzak vel Dobosz A, Wasag B et al. Presence of
homozygous KIT exon 11 mutations is strongly associated with
malignant clinical behavior in gastrointestinal stromal tumors.
Lab. Invest. 2007; 87; 10291041.
150. Van Glabbeke M, Verweij J, Casali PG et al. Initial and late
resistance to imatinib in advanced gastrointestinal stromal
tumors are predicted by different prognostic factors: a Euro-
pean Organization for Research and Treatment of Cancer-
Italian Sarcoma Group-Australasian Gastrointestinal Trials
Group study. J. Clin. Oncol. 2005; 23; 57955804.
151. Wardelmann E, Thomas N, Merkelbach-Bruse S et al. Acquired
resistance to imatinib in gastrointestinal stromal tumours
caused by multiple KIT mutations. Lancet Oncol. 2005; 6; 249
251.
152. Wardelmann E, Merkelbach-Bruse S, Pauls K et al. Polyclonal
evolution of multiple secondary KIT mutations in gastrointes-
tinal stromal tumors under treatment with imatinib mesylate.
Clin. Cancer Res. 2006; 12; 17431749.
153. Haller F, Detken S, Schulten HJ et al. Surgical management
after neoadjuvant imatinib therapy in gastrointestinal stromal
tumours (GISTs) with respect to imatinib resistance caused by
secondary KIT mutations. Ann. Surg. Oncol. 2007; 14; 526
532.
154. Grabellus F, Ebeling P, Worm K et al. Double resistance to
imatinib and AMG 706 caused by multiple acquired KIT exon
17 mutations in gastrointestinal stromal tumour. Gut 2007;
56; 10251026.
155. Loughrey MB, Waring PM, Dobrovic A et al. Polyclonal
resistance in gastrointestinal stromal tumor treated with
sequential kinase inhibitors. Clin. Cancer Res. 2006; 12;
62056206.
156. Wakai T, Kanda T, Hirota S et al. Late resistance to imatinib
therapy in a metastatic gastrointestinal stromal tumour is
associated with second KIT mutation. Br. J. Cancer 2004; 90;
20592061.
157. Grimpen F, Yip D, McArthur G et al. Resistance to imatinib,
low-grade FDG-avidity on PET, and acquiered KIT exon 17
mutation in gastrointestinal stromal tumour. Lancet Oncol.
2005; 6; 724727.
158. Bertucci F, Goncalves A, Monges G et al. Acquired resistance to
imatinib and secondary KIT exon 13 mutation in gastrointes-
tinal stromal tumour. Oncol. Rep. 2006; 97; 97101.
159. Koyama T, Nimura H, Kobayashi K et al. Reccurent gastro-
intestinal stromal tumor (GIST) of the stomach associated with
a novel c-kit mutation after imatinib treatment. Gastric. Cancer
2006; 9; 235239.
160. Tamborini E, Gabanti E, Lagonigro SM et al. KIT Val654Alareceptor detected in one imatinib-resistant GIST patient. Cancer
Res. 2004; 65; 1115.
161. Faivre S, Delbaldo C, Vera K et al. Safety, pharmacokinetic, and
antitumor activity of SU11248, a novel oral multitarget
tyrosine kinase inhibitor, in patients with cancer. J. Clin. Oncol.
2006; 24; 2535.
162. Joensuu H. Second line therapies for the treatment of gastro-
intestinal stromal tumor. Curr. Opin. Oncol. 2007; 19; 353358.
Clinical significance of oncogenic KIT and PDGFRA mutations in GISTs 265
2008 The Authors. Journal compilation 2008 Blackwell Publishing Ltd, Histopathology, 53, 245266.
-
163. Maki RG. Recent advances in therapy for gastrointestinal
stromal tumors. Curr. Oncol. Rep. 2007; 9; 165169.
164. Heinrich MC, Maki RG, Corless CL et al. Sunitinib (SU) response
in imatinib-resistant (IM-R) GIST correlates with KIT and
PDGFRA mutation status. ASCO Annual Meeting Proceedings
Part I. J. Clin. Oncol. 2006; 24; 9502.
165. Schittenhelm MM, Shiraga S, Schroeder A et al. Dasatinib
(BMS-354825), a dual SRC ABL kinase inhibitor, inhibitsthe kinase activity of wild-type, juxtamembrane, and activation
loop mutant KIT isoforms associated with human malignan-
cies. Cancer Res. 2006; 66; 473481.
166. Guo T, Agaram NP, Wong GC et al. Sorafenib inhibits the
imatinib-resistant KIT T670I gatekeeper mutation in gastro-
intestinal stromal tumor. Clin. Cancer Res. 2007; 13; 4874
4881.
167. von Mehren M. Beyond imatinib: second generation c-KIT
inhibitors for the management of gastrointestinal stromal
tumors. Clin. Colorectal. Cancer 2006; 6 (Suppl. 1); S30S34.
168. Sambol EB, Ambrosini G, Geha RC et al. Flavopiridol targets
c-KIT transcription and induces apoptosis in gastrointestinal
stromal tumor cells. Cancer Res. 2006; 60; 58585866.
169. Emmerson P, Maynard J, Jones S et al. Characterizing muta-
tions in samples with low-level mosaicism by collection and
analysis of DHPLC fractionated heteroduplexes. Hum. Mutat.
2003; 21; 112115.
170. Metaxa-Mariatou V, Papadopoulos S, Papadopoulos E et al.
Molecular analysis of GISTs: evaluation of sequencing and
dHPLC. DNA Cell Biol. 2004; 23; 777782.
171. Lasota J, Wasag B, Steigen SE et al. Improved detection of KIT
exon 11 duplications in formalin fixed paraffin embedded
gastrointestinal stromal tumors (GISTs). J. Mol. Diagn. 2007; 9;
8994.
172. Emile JF, Theou N, Tabone S et al. Clinicopathologic, pheno-
typic, and genotypic characteristics of gastrointestinal mesen-
chymal tumors. Clin. Gastroenterol. Hepatol. 2004; 2; 597605.
173. Vu HA, Xinh PT, Kikushima M et al. A recurrent duodenal
gastrointestinal stromal tumor with a frameshift mutation
resulting in a stop codon in KIT exon 13. Genes Chromosomes
Cancer 2005; 42; 179183.
174. Williams C, Ponten F, Moberg C et al. A high frequency of
sequence alterations is due to formalin fixation of archival
specimens. Am. J. Pathol. 1999; 155; 14671471.
175. Crosier PS, Ricciardi ST, Hall LR et al. Expression of isoforms of
the human receptor tyrosine kinase c-kit in leukemic cell lines
and acute myeloid leukemia. Blood 1993; 82; 11511158.
176. Zhu WM, Dong WF, Minden M. Alternative splicing creates
two forms of the human kit protein. Leuk. Lymphoma 1994; 12;
441447.
177. Lasota J, Kopczynski J, Majidi M et al. Apparent KIT Ser715
deletion in GISTs mRNA is not detectable in genomic DNA and
represents a previously known splice variant of KIT transcript.
Am. J. Pathol. 2002; 161; 739741.
178. Yamamoto H, Oda Y, Kawaguchi K et al. c-kit and PDGFRA
mutations in extragastrointestinal stromal tumor (gastrointes-
tinal stromal tumor of the soft tissue). Am. J. Surg. Pathol.
2004; 28; 479488.
179. Sihto H, Sarlomo-Rikala M, Tynninen O