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 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 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


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.



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.



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.



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.



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.



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).



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).



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.



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



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.



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).



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.

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