BRAF mutations and Phosphatidylinositol-3-Kinase pathway gene alterations in gastrointestinal stromal tumors (GIST)

Cancer Letters 312 (2011) 43–54

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Spectrum of KIT/PDGFRA/BRAF mutations and Phosphatidylinositol-3-Kinase pathway gene alterations in gastrointestinal stromal tumors (GIST) Marc Daniels a, Irene Lurkin b, Roland Pauli c, Erhard Erbstößer d, Uwe Hildebrandt e, Karsten Hellwig f, Uwe Zschille g, Petra Lüders h, Gabriele Krüger i, Jürgen Knolle j, Bernd Stengel k, Friedrich Prall l, Kay Hertel m, Hartmut Lobeck n, Brigitte Popp o, Franz Theissig p, Peter Wünsch q, Ellen Zwarthoff b, Abbas Agaimy a, Regine Schneider-Stock a,⇑ a

Institute of Pathology, University Erlangen, Germany Department Pathology, Erasmus MC, Rotterdam, The Netherlands Institute of Pathology, Klinikum Brandenburg, Germany d Institute of Pathology, AMEOS Klinikum St. Salvator, Halberstadt, Germany e Institute of Pathology, Klinikum Quedlinburg, Germany f Institute of Pathology, Klinikum Magdeburg, Germany g Institute of Pathology, Krankenhaus Bautzen, Germany h Practice of Pathology, Stendal, Germany i Institute of Pathology, Kreisklinik Aschersleben-Straßfurt gGmbH, Germany j Institute of Pathology, Städtisches Klinikum Dessau, Germany k Institute of Pathology, KMG Klinikum Güstrow GmbH, Germany l Institute of Pathology, University Rostock, Germany m Institute of Pathology, HELIOS Klinikum, Erfurt, Germany n Institute of Pathology, Ernst von Bergmann Klinikum, Potsdam, Germany o Practice of Pathology, Ingolstadt, Germany p Institute of Pathology, Carl-Thiem-Klinikum, Cottbus, Germany q Institute of Pathology, Klinikum Nürnberg, Germany b c

a r t i c l e

i n f o

Article history: Received 19 May 2011 Received in revised form 25 July 2011 Accepted 26 July 2011

Keywords: Gastrointestinal stromal tumor (GIST) KIT Platelet derived growth factor receptor alpha (PDGFRA) BRAF PI3K PIK3CA

a b s t r a c t Pathogenetic pathways of gastrointestinal stromal tumors (GIST) lacking mutations in KIT and PDGFRA (15%) are still poorly studied. Nearly nothing is known about PI3K alterations in GISTs and only a few GISTs with BRAF mutations have been reported. BRAF mutations (V600E) were found in 3/87 tumors (3.5%) concomitantly were wild type for KIT and PDGFRA. No mutations were detected in KRAS, NRAS, and FGFR3. For the first-time we demonstrated a PIK3CA mutation (H1047L) simultaneously occurring with a 15-bp deletion in KIT exon 11 in one tumor. We suggest that BRAF mutations are of pathogenetic significance in wild type GISTs. The PI3K pathway should be assessed in future studies. Ó 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction ⇑ Corresponding author. Address: Department of Pathology, Experimental Tumor Pathology, Friedrich-Alexander University Erlangen-Nürnberg, Universitätsstrasse 22, 91054 Erlangen, Germany. Tel.: +49 9131 8526070; fax: +49 9131 8526197. E-mail address: [email protected] (R. Schneider-Stock). 0304-3835/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2011.07.029

Although rare, gastrointestinal stromal tumors (GIST) are the most common mesenchymal tumors of the gastrointestinal tract [1]. The disease usually affects adults at an age of 50–70 without clear sex predilection [2]. GISTs are

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M. Daniels et al. / Cancer Letters 312 (2011) 43–54

Fig. 1. RPTK = receptor protein tyrosine kinase; FGFR3 = fibroblast growth factor receptor; PIP2 = phosphatidylinositol-4,5-bisphosphate; PIP3 = phosphatidylinositol-3,4,5-triphosphate; PI3K = Phosphatidylinositol 3-kinase; PDK ½ = 30 -phosphoinositide-dependent kinases; PTEN = phosphatase and tensin homologue deleted from chromosome 10; AKT = serine/threonine protein-kinase; mTOR = mammalian target of rapamycin; SHC = SRC-homology-2domain-containing; GRB2 = growth factor receptor-bound protein 2; SOS = son of sevenless; RAS = rat sarcoma; BRAF = serine/threonine-specific kinase; MEK = mitogen activated protein kinase; ERK = extracellular regulated kinase. Activation of receptor tyrosine kinases results in dimerisation of receptors and phosphorylation of tyrosines, which leads to a phosphorylation cascade of the tyrosine residues in multiple downstream signaling molecules and activation of signal transduction pathways including RAS/MAP kinase and PI3K/AKT signaling networks [26]. PI3K pathway activation by growth factors through RPTKs like FGFR3 leads to phosphorylation of PIP2 to PIP3. This process can be reversed by PTEN. The cascade through activation of PDK by PIP3 signals leads to AKT activation resulting in gene expression through mTOR. A second pathway interacts with SHC triggers activation of PI3K’s 110 kDa catalytic subunit (p110) as well as the activation of RAS over GRB2 and SOS and therefore starts the RAS–BRAF–MEK–ERK pathway. In addition, activation of the RAS genes activates the pathway through the interaction of RAS with p110a [26]. BRAF and PI3K are interacting with each other by mutual inhibition.

believed to originate from or differentiate similar to the gastrointestinal pacemaker cells, the interstitial cells of Cajal or their precursor stem cells [3,4]. Most of GISTs are defined as KIT (CD117) – positive and KIT-signaling or PDGFRA mutation-driven mesenchymal tumors. GISTs are most common in the stomach and the small bowel, but they are encountered with less frequency in the colon, rectum, esophagus, and rarely in the omentum, and mesentery. Histologically, GISTs vary from spindled to epitheloid and mixed cell tumors [2]. These microscopic features are site-dependent: whereas gastric GISTs show spindle cells as well as epitheloid cell appearance, most small intestinal GISTs are composed of spindle cells. The epitheloid pattern in small bowel GISTs was significantly linked to adverse outcome [2]. Around 85% of GISTs harbor mutations in KIT or PDGFRA [2,5]. These receptors are tyrosine kinase proteins and their mutations lead to ligandindependent dimerization and constitutive activation of the receptor causing an increased cellular proliferation and a decreased apoptosis [6]. The majority of KIT mutations involve the proximal part of exon 11 [7]. Exon 9 is the second most often involved part and concerns in the majority of cases an identical 6-nucleotide duplication encoding A502_Y503 [8]. In a small minority of cases exon 13 and exon 17 are mutated. Mutations in the PDGFRA are found in exons 18, 12, and 14 (in decreasing order of fre-

quency) [9]. Most KIT mutated tumors are sensitive to receptor tyrosine kinase inhibitors e.g. Imatinib mesylate (Gleevec, Novartis, Basel, Switzerland) [10]. However, mutations in exon 17 of KIT and exon 18 of PDGFRA (D842V) are resistant to Imatinib while exon 9 mutations of KIT are less sensitive [11]. The remaining 15% of GISTs are wild type for KIT and PDGFRA and responsible pathogenetic pathways are still unknown. Recent studies reported the involvement of Phosphatidylinositol-3-Kinase (PI3K) mutations in several human cancers [12]. PI3K is activated by receptors with tyrosine kinase activity and plays a crucial role in cell growth, proliferation, and survival [13]. PI3K activation leads to phosphorylation of cell membrane phosphatidylinositol and as a result phosphatidylinositol-3,4-bisphosphate (PIP2) and phosphatidylinositol-3,4,5-triphosphate (PIP3) are generated. PIP2 and PIP3 binding to the serine/threonine kinase Akt leads to its conformational change. This activation of Akt which leads to phosphorylation of several downstream substrates plays a key role in the cell process which is relevant to cancer cells, for example in cell survival, proliferation, and cell growth (Fig. 1). It could be postulated that the dysregulation of the PI3K pathway may play a role in GIST pathogenesis and/or progression. Thus the detection of alterations in the PI3K pathway in GIST would be innovative and could be relevant in tumorigenesis and receptor

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M. Daniels et al. / Cancer Letters 312 (2011) 43–54 Table 1 Tumor/study group. Summary of clinicopathological features of 87 cases. Tumor Sex/age no.

Tumor-site

Size (mm)

Mitosis/ 50 HPF

Histology Risk Miettinen and Lasota [5]

Gene

Mutation Mutation (ex)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49

m/59 m/68 f/47 m/77 m/59 f/43 m/68 f/64 m/65 m/68 m/47 f/62 f/66 f/78 m/78 m/49 m/79 m/78 m/70 f/52 m/71 f/68 f/72 m/38 f/52 f/55 m/68 f/58 f/74 m/72 m/71 f/38 m/72 m/74 m/59 f/82 m/57 m/74 m/61 f/80 m/59 f/39 f/58 f/46 m/66 m/73 f/60 f/61 f/61

Stomach Small intestine Small intestine Small intestine Small intestine Rectum Abdomen Esophagus Stomach Stomach Stomach Stomach Stomach Stomach Stomach Stomach Stomach Stomach Stomach Stomach Stomach Stomach Stomach Stomach Stomach Stomach Stomach Stomach Stomach Stomach Stomach Stomach Stomach Stomach Stomach Stomach Stomach Small intestine Small intestine Small intestine Small intestine Small intestine Small intestine Small intestine Small intestine Small intestine Small intestine Small intestine Small intestine

13 60 <50 55 130 n.a. 90 >50 40 110 60 65 70 n.a. 25 25 130 75 130 280 50 21 70 55 150 65 3 37 50 60 25 71 55 55 50 60 50 50 140 74 80 120 56 25 180 50 130 17 21

5 8 125 2 4 60 150 12 <5 <5 10 1 <5 110 0 16 20 65 1 >100 7 13 10 9 8 15 0 5 <5 5 <5 4 >100 4 10 >10 6 >14 4 12 <5 62 16 2 9 >10 2 >10 1

sp sp ep sp m sp sp m sp sp sp m sp sp sp sp sp m sp sp sp sp sp sp sp sp sp sp sp m sp sp sp sp sp sp sp m m m m m sp sp sp sp sp sp sp

No High High Intermediate High High High High Very low Intermediate High Low Low High Very low Intermediate High High Intermediate High Intermediate Intermediate High High High High No Very low Very low Low Very low Low High Low Intermediate High Intermediate High High High Intermediate High High Low High High High High Low

KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT

9 9 9 9 9 9 9 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11 11

50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66

m/75 f/67 f/68 f/59 f/42 m/77 m/69 m/55 f/79 f/68 f/79 m/68 m/62 f/73 f/48 m/75 m/53

Small intestine Small intestine Small intestine Small intestine Small intestine Colon Colon Rectum Rectum Retroperitoneum Retroperitoneum Small intestine Stomach Stomach Stomach Stomach Stomach

220 22 70 55 12 12 30 55 biopsy 4 10 80 28 22 38 155 140

>5 <5 n.a. 4 <5 10 <5 5 >5 52 32 6 <5 <5 <5 1 <5

n.a. ep sp sp sp sp sp sp sp sp sp sp ep sp m m sp

High Low Highb Intermediate No High Low High Highb High High High Very low Very low Very low Intermediate Intermediate

KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT KIT PDGFRA PDGFRA PDGFRA PDGFRA PDGFRA

11 11 11 11 11 11 11 11 11 11 11 13 12 12 12 18 18

A502_Y503dup A502_Y503dup A502_Y503dup A502_Y503dup A502_Y503dup A502_Y503dup A502_Y503dup W557_V559delinsC V555_K558del G565_D572del Y553_K558del W557R D572_P573del W557_K558del L576P P577_E583dup W557_K558del K558_E562del + PIK3CA H1047L K550_K558dela E554_V555del P577_K581dup V559G K558_V560delinsNH H580_W582delinsKTIdupP573_W582 W557_K558del W557_K558del P551_V559del W557_K558del Q575_D579dup P573_H580dup P551_V555del M552_W557del V560D L576_D579dup T574_R586dup Q556_E561delinsPS V559A D579del V555_Q556del E554_W557del P551_Q556del Q556_Q575del K550_W557del K550_V555del W557R Y552_W556del V559G E554_K558del E554D, V555_L576delinsrevNCSHLSPQQP W557R W557R W557_K558del W557_P573del I563_P577delinsTI K550_K558dela W557R L576P W557_V559delinsC W557_559delinsPF E554_N566del K642E V561D V561D R585_E587del D842V D842V (continued on next page)

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Table 1 (continued) Tumor Sex/age no.

Tumor-site

Size (mm)

Mitosis/ 50 HPF

Histology Risk Miettinen and Lasota [5]

Gene

Mutation Mutation (ex)

67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87

Stomach Stomach Stomach Stomach Stomach Stomach Esophagus Stomach Small intestine Stomach Stomach Stomach Stomach Stomach Stomach Stomach Small intestine Small intestine Small intestine Colon Colon

40 120 55 75 45 170 28 105 80 15 24 10 350 170 30 220 42 60 biopsy 80 70

<5 7 5 2 3 3 <5 2 <5 1 3 <5 45 20 18 32 >50 2 <5 >10 <5

sp m ep sp sp ep sp sp sp m m sp sp sp sp m ep m ep sp ep

PDGFRA PDGFRA PDGFRA PDGFRA PDGFRA PDGFRA BRAF BRAF BRAF wt wt wt wt wt wt wt wt wt wt wt wt

18 18 18 18 18 18 15 15 15 wt wt wt wt wt wt wt wt wt wt wt wt

m/60 f/71 m/68 m/75 f/57 m/59 f/71 f/68 f/60 f/80 f/78 m/71 m/68 f/65 f/71 m/72 f/51 f/89 m/69 m/59 m/67

Very low High Low Low Very low Intermediate Low Intermediate Intermediate No Very low No High High Intermediate High High High n.a. High High

D842V I843-S847delinsH D842V D842_H845del D842V D842_H845delinsV V600E V600E V600E wt wt wt wt wt wt wt wt wt wt wt wt

Abbreviations: n.a., not available; sp, spindle; ep, epitheloid; m, mixed; wt, wild type; HPF, high-power-fields; ex, exon; del, deletion; dup, duplication; ins, insertion. a +50 splice-acceptorsite 3 bp del CAG. b According to result.

tyrosine kinase inhibitor resistance. Furthermore recent studies have disclosed activating mutations of BRAF in a small subset of GISTs and in other neoplasms [14–17]. BRAF is a serine/threonine protein kinase which is an effector of RAS activation and is therefore involved in the RAS–BRAF–ERK signaling pathway which is important for transcriptional regulation (Fig. 1). The RAS–BRAF–ERK signaling pathway and the PI3K pathway are related to each other by an activation of the catalytic subunit p110 of PI3K through interaction with RAS. Additionally BRAF and PI3K inhibit each other mutually [18]. Moreover recent studies have shown that BRAF and KRAS mutations are generally mutually exclusive [19]. Since understanding the genetic aberrations beyond KIT and PDGFRA may lead to the identification of additional therapeutic targets for GISTs, we investigated 87 tumors for mutations in KIT, PDGFRA, PIK3CA (catalytic subunit of PI3K), BRAF, KRAS, NRAS and FGFR3 [20], which represents the first study encompassing all these interrelated genes of the RAS– BRAF–ERK pathway in addition to analysis for usual tyrosine kinase mutations.

inated in the stomach, 26 (30.0%) in the small intestine, 4 (4.6%) in the colon, 2 (2.3%) in the esophagus, 3 (3.4%) in the rectum and three cases (3.4%) were from unspecified GI sites. The mean size for 83 tumors with known size was 73 mm (range, 3–350 mm). Tumors were assigned to risk assessment categories based on size, mitotic index and location according to Miettinen and Lasota [5]. The clinicopathologic features of the cases are summarized in Table 1.

2. Materials and methods

2.3. DNA isolation

2.1. Patients and tissue samples

For the extraction of genomic DNA, 2–5 sections of about 5 lm of the tumor tissue were used. Genomic DNA purification from tissue was initiated by deparaffinization. Genomic DNA was prepared by NucleoSpin Tissue kit according to the protocol of the manufacturer (MACHEREY–NAGEL, Düren, Germany). After lysis with proteinase K at a concentration of 0.8–1.0 U per digest at 55 °C over night, the obtained DNA could be used for polymerase chain reaction (PCR).

Eighty-seven formalin-fixed and paraffin-embedded tumor samples were extracted from 87 patients between 2002 and 2009. All tumors were primary GISTs that were not subjected to receptor tyrosine kinase inhibitor therapy prior to surgery. The mean age of all patients was 64.9 years (range, 38–89 years). 42 (48.3%) patients were female and 45 (51.7%) were male. 49 tumors (56.3%) orig-

2.2. Immunohistochemistry Paraffin sections of formalin-fixed tissue were used for conventional hematoxylin and eosin staining and immunohistochemistry which was performed by polymer kit purchased from Zytomed Systems Ltd. (Berlin, Germany) according to the manufacturer’s instructions. The following antibodies were used: CD 117 (anti-Human c-kit protooncogene product, polyclonal, 1:200, DakoCytomation, Hamburg, Germany) and CD34 (BI-3C5, 1:200, Zytomed, Berlin, Germany).

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Table 2 PCR primers used for detection of mutations of KIT or PDGFRA or BRAF and primers used for multiplex amplification of HRAS, KRAS, NRAS, PIK3CA and FGFR3. Primer

Exon

Sequence (50 > 30 )

Length (bp)

Annealing temperature (°C)

Cycles

KIT KIT KIT KIT KIT KIT KIT KIT

9F 9R 11F 11R 13F 13R 17F 17R

CTA GAG TAA GCC AGG GCT TTT GTT CCT AAA CAT CCC CTT AAA TTG GAT T GTG CTC TAA TGA CTG AGA C TAC CCA AAA AGG TGA CAT GG TTT TGC TAA AAT GCA TGT TTC CA TAA AAG GCA GCT TGG ACA CG TTA AAT GGT TTT CTT TTC TCC TCC AA CAG GAC TGT CAA GCA GAG AAT GG

269

62 62 57 57 58 58 64 64

36 36 36 36 36 36 36 36

PDGFRA PDGFRA PDGFRA PDGFRA PDGFRA PDGFRA

12F 12R 14F 14R 18F 18R

TGG TGC ACT GGG ACT TTG GTA AAA GGG AGT CTT GGG AGG TTA CC GTA GCT CAG CTG GAC TGA TA CAC ATG TGT CCA GTG AA CAG GGG TGA TGC TAT ATC AGC TAC A GTC CAG TGT GGG AAG TGT GGA C

222

62 62 50 50 62 62

36 36 36 36 37 37

BRAF BRAF

15F 15R

TCT TCA TGA AGA CCT CAC AGT CCA GAC AAC TGT TCA AAC TGA

96

55 55

35 35

HRAS HRAS HRAS HRAS

1F 1R 2F 2R

CAG GAG ACC CTG TAG GAG G TCG TCC ACA AAA TGG TTC TG GGA GAC GTG CCT GTT GGA GGT GGA TGT CCT CAA AAG AC

139

55 55 55 55

35 35 35 35

KRAS KRAS KRAS KRAS

1F 1R 2F 2R

GGC CTG CTG AAA ATG ACT G GGT CCT GCA CCA GTA ATA TG CCA GAC TGT GTT TCT CCC TT CAC AAA GAA AGC CCT CCC CA

163

55 55 55 55

35 35 35 35

NRAS NRAS NRAS NRAS

1F 1R 2F 2R

GGT GTG AAA TGA CTG AGT AC GGG CCT CAC CTC TAT GGT G GGT GAA ACC TGT TTG TTG GA ATA CAC AGA GGA AGC CTT CG

128

55 55 55 55

35 35 35 35

PIK3CA PIK3CA PIK3CA PIK3CA

9F 9R 20F 20R

AGT AAC AGA CTA GCT AGA GA ATT TTA GCA CTT ACC TGT GAC GAC CCT AGC CTT AGA TAA AAC GTG GAA GAT CCA ATC CAT TT

139

55 55 55 55

35 35 35 35

FGFR3 FGFR3 FGFR3 FGFR3 FGFR3 FGFR3

7F 7R 10F 10R 15F 15R

AGT GGC GGT GGT GGT GAG GGA G GCA CCG CCG TCT GGT TGG CAA CGC CCA TGT CTT TGC AG AGG CGG CAG AGC GTC ACA G GAC CGA GGA CAA CGT GAT G GTG TGG GAA GGC GGT GTT G

115

55 55 55 55 55 55

35 35 35 35 35 35

199 168 188

197 214

140

155

103

109

138 160

Abbreviations: F, forward; R, reverse; bp, base pair.

2.4. Analysis of KIT and PDGFRA mutations KIT exons 9, 11, 13, and 17 and PDGFRA exons 12, 14, and 18 were screened for mutations by using the PCR amplification. The PCR reaction was carried out in a final volume of 25 ll under the following conditions: 12.5 ll of QIAGEN Multiplex PCR Master Mix (QIAGEN GmbH, Hilden, Germany) with HotStarTaq DNA Polymerase and a unique PCR buffer, 7.5 ll distilled water, and 1.5 ll of forward and reverse primers (10 lM) each. In every set of amplifications negative controls were included. The used primer sets, annealing temperatures, and number of cycles are displayed in Table 2. With the help of a multicapillary electrophoresis system QIAxcel System (Qiagen GmbH, Hilden, Germany) amplification products were controlled for correct amplification. The amplified DNA fragments were purified and sequenced on an ABI PRISM 310 Genetic Analyzer (Applied Biosystems, Foster City, California, USA) using the BigDye Termination kit (Applied Biosystems).

2.5. Analysis of PIK3CA, KRAS, NRAS, FGFR3, and BRAF mutations Three multiplex PCRs were performed for (1) BRAF exon 15 and KRAS exons 2 and 3, (2) PIK3CA exons 9 and 20 and NRAS exons 2 and 3 and (3) FGFR3 exons 7, 10 and 15 as described previously [21]. Primer sequences are given in Table 2. Multiplex PCRs were performed in a volume of 15 ll, containing 1  PCR buffer, 1.5 mM MgCl2, 0.5 units Go-Taq DNA polymerase (Promega, Madison, WI), 0.17 mM dNTP’s (Roche, Basel, Switzerland), 0.3–1 lM primers (Invitrogen, Carlsbad, CA), 5% glycerol (Fluka, Buchs SG, Switzerland) and 5 ng genomic DNA. Thermal cycling conditions were: 5 min at 95 °C, 35 cycles at 95 °C for 45 s, 55 °C for 45 s and 72 °C for 45 s, followed by 10 min at 72 °C. The PCR products were treated with two units Exonuclease I (ExoI) and three units Shrimp Alkaline Phosphatase (SAP) (USB, Cleveland, Ohio USA). This was followed by a single nucleotide probe extension assay using a SNaPshot Multiplex kit (Applied Biosystems, Foster

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Fig. 2. Amino-acid sequences in exon 11 of KIT in 53 sporadic GISTs. Deletions are indicated by dashes (-) and red color, substitutions are indicated by the one letter code of the substituted amino-acid and green color, insertions by the one letter code of the inserted amino-acid and blue color, and duplications by the one letter code of the duplicated amino-acids and violet color. Complex mutations such as deletion-insertions are indicated by dashes (-) and the inserted amino-acid (-/AA) marked by the corresponding colors. On the right side, case numbers are given.

City, CA) and probes designed to anneal to either the forward or the reverse strand of a PCR product adjacent to the mutation site of interest. These probes were fitted with T tails of different length at their 50 ends to allow separa-

tion of the extension products by size. The mutation detection reactions were performed in a volume of 10 ll, containing 1 ll SAP/ExoI treated PCR product, 2.5 ll SNaPshot Multiplex Ready Reaction mix, 1  Big Dye

M. Daniels et al. / Cancer Letters 312 (2011) 43–54

49

Fig. 3. Amino-acid sequences in exon 18 of PDGFRA in 8 sporadic GISTs. Deletions are indicated by dashes (-) and red color, substitutions are indicated by the one letter code of the substituted amino-acid and green color, and insertions by the one letter code of the inserted amino-acid and blue color. Complex mutations such as deletion-insertions are indicated by dashes (-) and the inserted amino-acid (-/AA) marked by the corresponding colors. On the right side, case numbers are given.

sequencing buffer and 1 ll probe mix. Thermal cycler conditions were: 35 cycles of 10 s at 96 °C and 40 s at 58.5 °C. The products were treated with 1 unit SAP at 37 °C for 60 min and 72 °C for 15 min and analyzed on an automatic sequencer (ABI PRISM 3130 XL Genetic Analyzer, Applied Biosystems) with the fluorescent label on the incorporated ddNTP indicating the presence or absence of a mutation. For analysis of the data Genescan Analysis Software version 3.7 (Applied Biosystems) was used. 2.6. Statistical analysis Statistical analysis was performed using Fisher’s or v2 exact test in cross tables and one-way ANOVA (for comparisons of means) to evaluate the differences between mutation (data type, localization, risk, or mitosis) and clinico-pathologic factors. All statistical tests were two sided. Differences with p-values < 0.05 were considered statistically significant. All calculations were performed using SPSS version 19.0.0 software package (SPSS, Inc., Chicago, IL, USA).

Exon 13 was mutated in only one case (1.2%) with a substitution of E642 for K642 which is an encoding part of the tyrosine kinase 1, the ATP binding-domain of KIT.

3.1.2. Correlation with clinico-pathological findings Exon 11 deletions and substitutions occurred in different localizations and affected tumors showed a spectrum of spindled or epitheloid phenotypes. All duplications in exon 11 (n = 6) were associated with gastric localization and are predominantly spindle cell tumors (5/6; 83.3%). All six duplications clustered in 30 Kit exon 11. None of the six tumors with exon 11 duplications was classified as high risk (p = 0.041) which is in accordance with Miettinen and Lasota [5]. Generally, tumors with KIT exon 11 deletions were more frequently classified as high risk ones according to Miettinen et al. (23/36; 63.9%) than tumors with exon 11 single nucleotide substitutions 5/11 (45.5%). Four of 6 (66.7%) exon 9 mutated tumors with known primary location were located in the small intestine (p = 0.032) and predominantly showed spindle cell type (5/7; 71.4%). 83.3% of exon 9 mutants were intestinal tumors (four small and one large bowel GISTs). Furthermore GISTs with KIT exon 9 duplications were associated with high risk group in the classification system published by Miettinen and Lasota [5] (5/7; 71.4%).

3.2. PDGFRA mutations 3. Results Data for risk stratification (size, site and mitotic count) were available for 86 tumors. According to the risk classification system published by Miettinen and Lasota [5], five patients (5.8%) were classified at no risk, 11 (12.8%) at very low risk, 12 (14.0%) at low risk, 16 (18.6%) at intermediate risk, and 42 (48.8%) at high risk. KIT or PDGFRA mutations were detected in 72/87 (82.8%) and were found to be mutually exclusive. 3.1. KIT mutations 3.1.1. Mutation spectrum and frequency of KIT mutations Of the 87 tumors 61 (70.1%) had KIT gene mutations: 53 (60.9%) in exon 11 (Fig. 2), 7 (8.1%) in exon 9, and 1 (1.2%) in exon 13. None had a mutation in exon 17. All deletions were in-frame deletions and they tend to cluster in 50 Kit exon 11 (Fig. 2). Mutations of exon 11 included nucleotide deletions in 36/53 cases (67.9%). Among these deletion mutations 8/ 36 (22.2%) were complex mutations (deletion combined with insertion and/or duplication). Single point mutations were diagnosed in 11/53 cases (20.8%), duplications in 6/53 cases (11.3%). Isolated insertions were not detected in any of the cases. In 40/53 cases (75.5%) mutations of exon 11 clustered in the hot-spot region between codons 550 and 560; of them, 6/53 mutations (11.3%) were localized in the known ‘‘hot-spot’’ region (W557_K558). Different size deletions in 50 Kit can affect KIT intron 10exon-11 splice-acceptor sites. In our tumor group we detected 2/53 (3.8%) of this mutation which was formerly described by Lasota and Miettinen [22]. This mutation obviously forms a novel intraexonic pre mRNA 30 splice acceptor site and consistently leads to K550_K558del. All 7 exon 9 mutations were structurally identical tandem duplications with insertion of six nucleotides (GCCTAT) encoding A502_Y503.

3.2.1. Mutation spectrum and frequency of PDGFRA mutations Among the tumors without KIT mutations (n = 26), 11 tumors (42.3%) showed a PDGFRA mutation. These made up 12.6% of the whole tumor group. 8/87 tumors (9.2%) had mutations in exon 18 (Fig. 3) and 3/87 cases (3.4%) had exon 12 mutations. 5/8 (62.5%) of the exon 18 mutations represented a substitution of V842 for D842. Exon 18 mutations tend to cluster between codons 840 and 848 (8/8; 100%) (Fig. 3). Two of the 3 exon 12 mutations represented a V561D mutation at the protein level.

3.2.2. Correlation of PDGFRA mutations with clinico-pathological Findings Both exon 18 and exon 12 mutations were strongly associated with gastric localization (p = 0.032; 11/11; 100%). Additionally there is a tendency for exon 18 mutations to occur in male patients (6/8; 75%). Furthermore, PDGFRA mutants have a low mitotic index (all of 3 exon 12 mutations and 7/8 of exon 18 mutants; with a mean mitotic index of 4/ 50 HPF). Mean mitotic rates were 51/50 HPFs, 17/50 HPFs, and 4/50 HPFs for GISTs with mutations in exon 9, exon 11 and PDGFRA exon 18 respectively. KIT and PDGFRA wild type genotypes were found in 15/87 cases (17.2%). They were associated with spindle cell morphology in 8/15 cases (53.3%) and showed a highly variable mitotic activity (range, 1 to >50/50 HPFs).

3.3. NRAS, KRAS, and FGFR3 mutations All tested tumors were wild type for NRAS (data available for 83 tumors) and KRAS (data available for 78 tumors). Furthermore 82 tumors were tested for FGFR3 gene mutations; none showed a mutation in this gene.

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M. Daniels et al. / Cancer Letters 312 (2011) 43–54 3.5. Spectrum and frequency of the PIK3CA mutation A PIK3CA mutation (substitution H1047L) was detected in one tumor (Fig. 4). A second DNA-preparation has been done to verify the mutant signal. The PIK3CA mutation occurred together with an KIT exon 11 deletion K558_E562del. The patient (no. 18; Table 1) was a 78 year old man. The tumor was localized in the stomach, had a size of 75 mm and showed mixed cell morphology (Fig. 5) and a mitotic activity of 65/50 HPFs. Therefore it is classified as a high risk GIST (Table 1) [5]. A strong positivity for CD 117, CD 34, and smooth muscle actin was proven. S-100 protein was negative.

4. Discussion

Fig. 4. Mutations in the BRAF and PIK3CA genes. (a) Wild type pattern for/BRAF/codons 600 and 601 (no. 12). (b–d) The next to the wild type A nucleotide a T (thymine) is visible indicative of mutation V600E (No. 73, No. 74, No. 75). (e) Wild type pattern for PIK3CA codons 542, 545 (two peaks) and 1047 (no. 61). (f) A small percentage of tumor cells have a mutation A > T leading to H1047L (no. 25).

3.4. Spectrum and frequency of BRAF mutations BRAF mutations were found in 3/15 (20%) wild type GIST. All three cases had a substitution in the so called ‘‘hot spot’’ region V600E (Fig. 4). All BRAF mutations are associated with female gender and spindle cell morphology (Fig. 5). The three patients had an age of 60, 68, and 71 years, respectively. Mean size of these three tumors was 71 mm (range, 28–105 mm). They were localized in esophagus, stomach and small intestine. BRAF mutants significantly had a lower mitotic activity (p < 0.001; mean, 3/50 HPFs) compared to KIT mutated tumors (21/50 HPFs). In one case metastasis has occurred. There was no association with a specific risk group (Table 3). All three tumors were positive for CD117 and CD34 and were negative for smooth muscle actin and S-100 protein.

The pathogenetic pathways underlying wild type GISTs are still unknown. GISTs lacking a detectable kinase mutation had a lower overall response to therapy with receptor tyrosine kinase inhibitors than tumors with exon 11 or 9 mutations and consequently these patients had a significantly shorter overall survival. Therefore it is of great value to analyze the molecular pathogenesis, histopathologic spectrum, and biological potential of these KIT and PDGFRA wild type tumors. Recently, it has been suggested that BRAF or NRAS might play a role in wild type GIST pathogenesis [15]. BRAF, an effector of RAS protein, and NRAS are commonly mutated in cancer such as endometrial carcinoma [16] and represent the most frequent genetic events in malignant melanoma whereas a subset of these melanomas also harbor KIT mutations [15]. Furthermore the Phosphatidylinositol-3-Kinase (PI3K)/Akt pathway is involved in the pathogenesis of several human cancers [12,23–27]. In breast carcinoma the deregulation of the PI3K/Akt pathway contributes to a more aggressive phenotype with poor outcome in prognostically favorable node negative tumors [28]. Based on the key role of the PI3K/ Akt pathway in proliferation we postulated its possible contribution to the pathogenesis of wild type GIST. Therefore we investigated 87 tumors for mutations in KIT, PDGFRA, BRAF, PIK3CA, KRAS, NRAS and FGFR3 (Fig. 1). To our knowledge this is the first study encompassing all these interrelated genes of the RAS–BRAF–ERK pathway in addition to analysis for usual tyrosine kinase mutations in GIST. In general, our results are consistent with previous studies regarding the frequency, type and localization of KIT and PDGFRA mutations. In our tumor group KIT mutations were the most frequent mutations followed by PDGFRA mutations. Most KIT mutations involve exon 11 the proximal part of KIT clustering between codons K550 and E561 [6,29–31]. In our study, 5/6 (88.3%) exon 11 deletions with loss of W557 and K558, the most common simple deletion in GISTs, were classified as high risk in the risk classification by Miettinen et al. [5,32–34]. We confirmed recent findings that internal tandem duplications of 1 upon more than 20 codons occur preferentially in the distal part of KIT exon 11 and are associated with gastric localization [35,36]. For exon 9 (duplication A502_Y503) mutations we verified recent published data that most of them seemed to be associated with small intestinal origin [8,37] and pursued a malignant course [35,38]. In our study exon 13 was mutated in only one case (1.2%) classified as high risk. These results are in accordance with two large GIST series

M. Daniels et al. / Cancer Letters 312 (2011) 43–54

51

Fig. 5. Representative images of gastrointestinal stromal tumors (GIST) harboring different kinase mutations. KIT mutants displayed spindled histology (a) and diffusely expressed CD117 (b). On the other hand, PDGFRA-mutated GISTs (c) were predominantly epitheloid with weak to absent CD117 immunostaining (d, note strongly staining mast cells). BRAF-mutated GIST showed a spindled histomorphology with variable sclerosis (e) and strong CD117 expression (not shown). The single tumor with KIT exon 11 mutation and concurrent PIK3CA mutation revealed varied histological pattern with focally prominent whorls (f) in addition to sarcomatous spindled (g) and round cell/epitheloid areas with brisk mitotic activity (h). (i) CD117 immunostaining (same case) displayed both membranous (upper left field) and dot-like Golgi-pattern (lower right).

[8,37]. Only a few GISTs with mutations involving exon 17 have been reported [39]. We did not detect any exon 17 mutation in our tumor group. Substitution of V842 for D842 is the most common PDGFRA mutation [2]. PDGFRA mutations tended to cluster between codons 840_848 in exon 18 (8/8; 100%), confirming recent studies [9,40–43]. We showed that 2/3 (66.7%) single nucleotide substitutions identified in PDGFRA exon 12 resulted in V561D which is the most common reported mutation in exon 12 [9,22,40,41]. It has been reported that PDGFRA mutations occur almost exclusively in GISTs of stomach [40,41,43,44] and have low mitotic rate [5] as shown in our study. Moreover we confirmed that exon 11 mutants had a significantly higher mitotic activity (mean 17/50 HPF) than exon 18 mutants (4/50 HPF) showing that Kit exon 11 mutations are more common in large, mitotically active tumors [30,31]. To date there are only three reports on BRAF mutations in GIST. The study of Agaram et al. [15] detected BRAF exon 15 (V600E) missense mutations in 3 of 61 (4%) KIT/PDGFRA

wild type GISTs. These BRAF mutations occurred in middleaged females with small bowel localization, spindle cell morphology, and high risk of malignancy. NRAS mutations were not identified. Agaimy et al. [14] analyzed 69 GISTs and detected BRAF mutations in 2/28 (7%) wild type GIST. They did not find mutations in exon 2 of the KRAS gene. The two BRAF-mutated tumors originated from male patients and different sites (stomach and small intestine) and were mitotically inactive spindle cell GISTs. Hostein et al. [17] screened 70 KIT/PDGFRA wild type GISTs (among total of 524 GISTs; 13.4%) of the cases. They detected BRAF mutations in 9 out of the 70 cases (13%); 7/9 (78%) BRAFmutant patients were males. These GISTs were more commonly located in the small intestine (6/9; 67%), but also in the stomach 2/9 (22%) and in the peritoneum 1/9 (11%). The three BRAF-mutant GISTs identified in our study were associated with female gender and spindle cell morphology (Fig. 5). All our BRAF mutants had a low mitotic activity (mean activity 3/50 HPF) and showed varied localization (esophagus, stomach and small intestine).

52

M. Daniels et al. / Cancer Letters 312 (2011) 43–54

Table 3 Clinicopathological and molecular features of GISTs with primary V600E BRAF mutations from the current and previous studies (n = 17). No.

Reference

Sex/ age

Tumor-site

Size (mm)

Mitosis/ 50 HPF

Histology

Risk Miettinen and Lasota [5]

KIT exon 9, 11, 13, 17/PDGFRA exon 12, 18

BRAF exon 15

KRAS exon 2

NRAS; FGFR3; PIK3CA exons 2, 3; 7, 10, 15; 9, 20

1

Patient no. 73, current Patient no. 74 , current Patient no. 75, current Agaimy et al. [14] Agaimy et al. [14] Agaram et al. [15] Agaram et al. [15] Agaram et al. [15] Hostein et al. [17] Hostein et al. [17] Hostein et al. [17] Hostein et al. [17] Hostein et al. [17] Hostein et al. [17] Hostein et al. [17] Hostein et al. [17] Hostein et al. [17]

f/71

Esophagus

28

<5

sp

Low

wt

V600E

wt

wt

f/68

Stomach

105

2

sp

Intermediate

wt

V600E

wt

wt

f/60

Small intestine

80

<5

sp

Intermediate

wt

V600E

wt

wt

m/ 71 m/ 81 f/52

Stomach

40

0

sp

Very low

wt

V600E

wt

n.a.

Small intestine Small intestine Small intestine Small intestine Small intestine Small intestine Stomach

40

0

sp

Low

wt

V600E

wt

n.a.

100

90

sp

High

wt

V600E

n.a.

n.a.

100

5

sp

Intermediate

wt

V600E

n.a.

n.a.

90

50

sp

High

wt

V600E

n.a.

n.a.

200

6

sp

High

wt

V600E

n.a.

n.a.

25

5

sp + ep

Low

wt

V600E

n.a.

n.a.

n.a.

n.a.

sp

n.a.

wt

V600E

n.a.

n.a.

Stomach

30

1

sp

Very low

wt

V600E

n.a.

n.a.

Small intestine Small intestine Small intestine Small intestine Peritoneum

25

10

sp

High

wt

V600E

n.a.

n.a.

25

1

sp + ep

Low

wt

V600E

n.a.

n.a.

25

6

sp

High

wt

V600E

n.a.

n.a.

25

3

sp

Low

wt

V600E

n.a.

n.a.

28

50

ep

High

wt

V600E

n.a.

n.a.

2

3

4 5 6 7 8 9 10 11 12 13 14 15 16 17

f/55 f/49 m/ 53 m/ 38 m/ 63 m/ 78 f/51 m/ 58 m/ 58 m/ 41 f/50

Abbreviations: f, female; m, male; n.a., not analyzed; sp, spindle; ep, epitheloid; HPF, high-power-fields; wt, wild type.

Our study confirms recent data that BRAF mutations and KIT/PDGFRA mutations seem to be mutually exclusive oncogenic events [14,17]. Table 3 gives an overview of previously reported BRAF mutations in GIST. No clinicopathologic data such as gender, tumor size, mitotic count, risk, or age are specific for V600E BRAF-mutant GISTs. Nevertheless there seems to exist a relation between the occurrence of BRAF mutation and spindle cell morphology 16/17 (94%) and small intestine origin 11/17 (65%). One 15 bp deletion in KIT exon 11 (K558_E562del) was detected in a GIST that harbored an additional PIK3CA mutation (Substitution H1047L). This tumor was wild type for NRAS/KRAS/FGFR3/BRAF and showed a high mitotic activity (65/50 HPFs) and a large size (75 mm) indicating a high risk tumor [5]. The coexistence of a PIK3CA mutation besides a KIT mutation in this tumor suggests that the PIK3CA mutation probably represents a secondary event correlating with tumor progression. Indeed, in some cancer types PIK3CA mutations have been associated with invasiveness and a worse prognosis [45]. In our case a 3 mm liver metastasis was detected at time of primary sur-

gery and verified histologically. Of note, the patient received no chemotherapy or receptor tyrosine kinase inhibitor therapy prior to surgery indicating that the PIK3CA mutation is a spontaneous event unrelated to prior therapy. The patient was lost to follow up 1 year after surgery, because of severe co-morbidities. In summary this study confirms a wide spectrum of common kinase mutations in GISTs and underlines the need for a more extended genetic testing in individual cases especially with the background that KIT and PDGFRA wild type GISTs show worse response to receptor tyrosine kinase inhibitor treatment. We confirmed that BRAF mutations play a role in a subset of KIT/PDGFRA wild type GISTs. Our study is the first one to document a PIK3CA mutation in GIST. Future studies on larger tumor groups are needed to explore the impact of BRAF and PIK3CA mutations for therapy with receptor tyrosine kinase inhibitors in KIT wild type tumors. Possibly this new molecular group of GIST patients may benefit from selective BRAF inhibitors or PI3K pathway inhibitors such as p110a and mTOR inhibitors.

M. Daniels et al. / Cancer Letters 312 (2011) 43–54

Conflict of interest Prof. Dr. rer. nat. Regine Schneider-Stock and PD Dr. med. Abbas Agaimy have a project grant support from Novartis.

[17]

[18]

Acknowledgment [19]

Marc Daniels was supported by Interdisciplinary Center of Clinical Research, Erlangen. Grant Number: IZKF grant A20.

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