Most GISTs require oncogenic activation of the KIT or PDGFRA receptor tyrosine kinase proteins, and the genomic mechanisms of oncogene activation are heterogeneous. Notably, the kinase mutation type correlates with both tumor biology and imatinib response. For example, GISTs with KIT exon 11 mutations are typically gastric and have excellent imatinib response, whereas those with KIT exon 9 mutations generally arise in the small bowel and are less responsive to imatinib. To identify genes that might contribute to these biological differences, we carried out gene expression profiling of 26 GISTs with known KIT and PDGFRA mutational status. Expression differences were then evaluated further by RNA in situ hybridization, immunohistochemistry, and immunoblotting. Unsupervised hierarchical clustering grouped tumors with similar mutations together, but the distinction between the different groups was not absolute. Differentially expressed genes included ezrin, p70S6K, and PKCs, which are known to have key roles in KIT or PDGFRA signaling, and which might therefore contribute to the distinctive clinicopathological features in GISTs with different mutation types. These gene products could serve as highly selective therapeutic targets in GISTs containing the KIT or PDGFRA mutational types with which they are associated.
Activating mutations in the KIT gene, which encodes a transmembrane receptor tyrosine kinase (RTK), are present in the majority of GISTs (Hirota et al., 1998; Hirota et al., 2001; Kinoshita et al., 2003). Most GISTs (67%) are characterized by mutations in exon 11 of KIT, which causes ligand-independent activation of its tyrosine kinase function. Mutations in KIT exon 9 are present in about 11% of GISTs (Lux et al., 2000; Antonescu et al., 2003). More rarely mutations are found in exon 13 or 17 (Rubin et al., 2001; Kinoshita et al., 2003). Although most GISTs have activating KIT mutations, a subset is KIT wild type (Hirota et al., 1998). Heinrich et al. (2003a) showed that approximately 35% of these GISTs have intragenic activating mutations in the related receptor tyrosine kinase, platelet-derived growth factor receptor alpha (PDGFRA), and this has been confirmed by Hirota et al. (2003). The clinical relevance of different mutations in GIST became apparent in two recent studies, where KIT exon 11 mutants were highly sensitive to imatinib, whereas GISTs with KIT exon 9 mutations – and those without demonstrable KIT and PDGFRA mutations – often failed to respond (Heinrich et al., 2003b, Debiec-Rychter et al., 2004a).
The various kinase mutations in GIST also correlate with certain clinicopathological features. For example, GISTs with KIT exon 9 mutations generally arise in the small bowel (Antonescu et al., 2003), and GISTs with PDGFRA mutations often lack KIT expression, and contain epithelioid cells (Heinrich et al., 2003a; Debiec-Rychter et al., 2004b).
KIT and PDGFRA mutations are mutually exclusive oncogenic mechanisms in GISTs, and the biological sequelae of these alternate mechanisms appear to be similar (Heinrich et al., 2003a). To understand the genes that are associated with the activation of these two kinases in GIST tumorigenesis, we have examined the gene expression profiles of 26 tumors with known mutations in KIT (exons 9, 11, or 17) and PDGFRA (exon 12 or 18) genes using 42 000 spot cDNA microarrays. We confirmed the differences in expression profile by using Western blots on a separate set of GISTs. In addition, we used immunohistochemistry (IHC) and in situ hybridization (ISH) on a tissue microarray with a different group of 127 GISTs representing various KIT and PDGFRA mutants and KIT and PDGFRA wild-type GISTs.
Gene expression profiling, unsupervised hierarchical clustering
As summarized in Table 1, the 26 GIST cases included a spectrum of mutations in KIT and PDGFRA, as well as three cases that were wild type for all examined exons in both these genes. Clinical information and IHC staining profiles of the cases are shown in Table 1. Gene selection for cDNA gene array analysis was based on signal-to-noise ratio and the degree of variation in expression across the samples (see Materials and methods) and yielded 1875 genes (Figure 1a). By using unsupervised hierarchical clustering most tumors were grouped together according to mutation type (Figure 1b). Three of four GISTs with a mutation in KIT exon 9 clustered together with one wild-type case as branch 1 (Figure 1b). All GIST cases with a mutation within PDGFRA clustered on branch 2, together with two wild-type cases and two cases with mutations in KIT exon 11. Finally, eight of 10 GISTs with a mutation in KIT exon 11 clustered on branch 3, together with two cases with mutations in KIT exons 9 and 17, respectively. Thus, by using an unsupervised clustering method we could see a general separation of GIST cases based on their kinase mutation status.
Inspection of the clustered gene array data showed that the KIT gene was relatively highly expressed in most of the KIT exons 11 and 9 mutant GISTs, but was expressed at lower levels in most of the PDGFRA mutant GISTs. Expression of the PDGFRA gene was seen predominantly in the PDGFRA mutant GIST cases (Figure 1b). However, four GISTs with KIT exon 11 mutations also showed relatively high expression of PDGFRA. These findings are similar to those previously reported using IHC and in situ hybridization (ISH) on an independent set of 127 GIST cases with known mutational status (West et al., 2004). None of the KIT exon 9 mutants showed expression of PDGFRA (Figure 1b). The wild-type GISTs that clustered along with PDGFRA mutant GISTs showed expression of PDGFRA, while the wild-type GIST that clustered along with KIT exon 9 mutants did not. CD34 was highly expressed in most of the KIT exon 11 mutants and present at much lower levels in KIT exon 9 and PDGFRA mutants (Figure 1b).
Gene expression profiling, SAM analysis
We subsequently analyzed the expression data by significance analysis of microarrays (SAM) (Tusher et al., 2001) to identify and rank order the genes that differentiate GISTs based on KIT or PDGFRA mutations. Three SAM analyses were performed. First, we considered the KIT exon 11 mutants as a group distinct from KIT exon 9, PDGFRA mutants and wild-type GISTs. Second, we analyzed genes that separated KIT exon 9 mutants from the KIT exon 11, PDGFRA and wild-type cases. Finally, we investigated the genes specifically expressed in PDGFRA mutants. The highest-ranking genes identified in these three separate SAM analyses are shown in Tables 2a–c respectively. The complete SAM list of genes for each GIST mutant group is given in the supplemental data at http://microarray-pubs.stanford.edu/tma_portal/GISTmutations/.
Confirmation of gene array findings
We used a tissue microarray (TMA) of 127 GISTs with known KIT and PDGFRA mutational status to confirm the expression profiles for genes selected from SAM analysis using antibodies and ISH probes. We tested MAP1B by ISH and found that positive signals were more common among KIT exon 9 mutant cases (71.4%) than among KIT exon 11 and wild-type GISTs (22.4 and 46.2%, respectively) (Figure 2). None of the PDGFRA mutant GISTs was positive for MAP1B. These findings are comparable with the gene array data. A detailed table (Supplementary Table 1) that shows the site of origin and mutation type of the 127 GIST cases on the TMA is shown at the accompanying website.
From our gene expression data we selected DLK1 as a candidate marker for PDGFRA GIST mutants. The DLK1 ISH probe was predominantly positive in PDGFRA mutants (57.1%), but in contrast to the gene array findings, 42.9% of KIT exon 9 mutants were also positive for this probe, while fewer exon 11 cases (17.3%) reacted (Figure 2). Interestingly, the DLK1 gene is located on the chromosome 14q. This part of the chromosome is often lost in GIST (Breiner et al., 2000; Gunawan et al., 2004). This raises the possibility that chromosome 14 loss could be associated with KIT exon 11 mutants, and thus explain the low level of DLK1 expression in this subset of GIST. However, Heinrich et al. (2003a) showed that cytogenetic profiles were the same in PDGFRA-mutant and KIT-mutant GISTs: each of four PDGFRA-mutants had a 14q deletion. As shown previously (West et al., 2004), a PDGFRA ISH probe stained the majority (80%) of PDGFRA GIST mutants (Figure 2), confirming the gene array findings (Figure 1b).
High expression levels for CD34 RNA were found by gene array analysis almost exclusively in KIT exon 11 mutation cases. By IHC, CD34 staining on the GIST TMA was mostly restricted to KIT exon 11 mutants (51.3%), although it was weakly positive in one of eight scorable KIT exon 9 mutant cores on the TMA (12.5%) and 41% of wild-type GISTs. None of the PDGFRA mutant GISTs showed staining for CD34 (Figure 2). For comparison, we included our previously published ISH and IHC results for KIT, PDGFRA and DOG1 markers in Figure 2 (West et al., 2004). Representative cores from the TMA showing staining for DLK1, MAP1B and CD34 are shown in Figure 3, with digital images of all TMA stains available on the accompanying website http://microarray-pubs.stanford.edu/tma_portal/GISTmutations/.
We also used Western blot analysis to validate gene microarray data, focusing on a representative set of genes (CD34, EPHA4, ezrin/VIL2, p70S6K/RPS6KA1, PRKCQ, and PRKCA) differentially expressed among GISTs based on expression profiling, the availability of reagents, and their known relevance in receptor tyrosine kinase signaling. The total cell extracts from 11 GISTs (four KIT exon 9 mutants, four KIT exon 11 mutants and three PDGFRA GIST mutants) were used for this analysis; this set of tumors was distinct from that used for expression profiling and was also different from that represented on the TMA.
Ezrin/VIL2 is of clinical interest as it is involved in both cell motility and cell spreading signaling pathways and has been shown to play a role in metastasis (Louvet-Vallee, 2000; Bretscher et al., 2002; Khanna et al., 2004; Yu et al., 2004). Western blot studies with ezrin/VIL2 antibody showed that the protein is expressed at detectable levels in all the GIST samples tested; however, the PDGFRA mutants showed lower levels of ezrin/VIL2 compared with KIT exon 9 and 11 mutants (Figure 4). This correlates well with the gene expression data (Figure 1b).
Protein kinase C (PKC) proteins regulate KIT signaling through inhibitory phosphorylation of KIT interkinase domain serine residues, and through effects on downstream signaling intermediates (Blume-Jensen et al., 1994; Taylor and Metcalfe 2000). In addition, KIT activation generates critical PKC cofactors, including diacylglycerol, through phospholipase C gamma and PI3K pathways (Plo et al., 2001) Strong expression of protein kinase C alpha (PRKCA) was demonstrated by Western blotting in one PDGFRA mutant GIST (Figure 4). This finding partially confirms the gene array, where we found PRKCA to be expressed predominantly in PDGFRA mutant cases (Figure 1b). Western blot analysis for another PKC family member, protein kinase C theta (PRKCQ), revealed that the KIT exon 9 and 11 mutant GISTs expressed higher levels of this protein as compared to PDGFRA mutants (Figure 4). PRKCQ has been implicated previously as a potential diagnostic marker in GIST (Nielsen et al., 2002). In the present study, we found PRKCQ RNA expression lower in the PDGFRA-mutant GISTs (see spot image of the data in Supplementary Figure 1 on the accompanying website) although not sufficiently so as to result in inclusion in the initial gene selection data set.
EPHA4 is a member of the ephrin family of receptor tyrosine kinases, and has not previously been implicated in GIST tyrosine kinase signaling mechanisms. In agreement with the gene array studies, the Western blot analysis showed strong EPHA4 expression in each of four KIT exon 9 mutant GISTs, but in only one of four KIT exon 11 mutant GISTs, and in none of the PDGFRA mutant GISTs (Figure 4).
We also assessed the protein expression for p70S6K/RPS6KA1, a protein that is involved in the PI3K/AKT signaling pathway. Expression of p70S6K/RPS6KA1 was confined to KIT exon 9 and 11 mutants (Figure 4). However, not all the KIT mutants expressed this protein, a situation similar to that noted on the gene arrays (Figure 1b). CD34 was expressed strongly in two of four KIT exon 11 mutants and was not detected in the KIT exon 9 mutant and PDGFRA mutant GISTs. These data corroborate the TMA demonstrations of CD34 immunohistochemical staining in KIT exon 11 mutant GISTs.
GISTs are the most common mesenchymal neoplasms of the intestinal tract. Most GISTs express the KIT receptor, a type III receptor tyrosine kinase (RTK) (Hirota et al., 1998; Tuveson et al., 2001). The majority of these tumors are sensitive to imatinib (Demetri et al., 2002). When phosphorylated, the KIT receptor stimulates intracellular signaling, resulting in activation of cell proliferation, differentiation and survival pathways during embryogenesis and in the postnatal organism (Besmer, 1991; Besmer, 1997; Sanders et al., 1999). Gain-of-function mutations in KIT have been identified in most GISTs leading to constitutive ligand-independent activation of KIT tyrosine kinase (Hirota et al., 1998; Nakahara et al., 1998). Recently, it was reported that a subset of GISTs that have no mutation in KIT carry a mutation in PDGFRA, another type III RTK. While most KIT mutant isoforms respond to imatinib, only a small subset of PDGFRA mutant isoforms respond (Heinrich et al., 2003b).
Several aspects of GIST biology have been reported to be similar in tumors with KIT and PDGFRA mutations (Heinrich et al., 2003a). These include mechanisms of molecular cytogenetic progression and activation of PI3K/AKT and MAPK signaling pathways. However, there are characteristic clinicopathological differences in GISTs with different KIT and PDGFRA mutation types, with one example being the proclivity for small bowel primaries in GISTs with KIT exon 9 mutations. Therefore, we hypothesized that cDNA array profiles might reveal distinctive gene expression patterns accounting for the somewhat different clinicopathological features in GISTs with various kinase mutation types. We addressed this hypothesis by characterizing the cDNA microarray gene expression signatures for 26 GISTs, including 23 with KIT or PDGFRA mutations and three which lacked mutations. Unsupervised hierarchical clustering revealed a noticeable grouping of KIT mutants separate from the PDGFRA mutants. In addition, the two main mutational types in KIT (exon 11 and exon 9) grouped on separate branches of the dendrogram. The gene array analysis data were confirmed by ISH, IHC, and Western blot analyses of independent sets of GIST cases with a variety of mutations. The genes that were chosen to help confirm the gene array data were selected based on the availability of reagents and/or the role that they are known to play in receptor tyrosine kinase pathway signaling. We found good confirmation of the expression profiles for the genes ezrin/VIL2, PRKCQ, EPHA4, DLK1, MAP1B, and p70S6K/RPS6KA1 and a partial confirmation for genes PRKCA and CD34.
Expression analysis of genes in RTK signaling pathway
Signaling pathways that play key roles downstream of type III RTKs include the PI3K/AKT and RAS/MAPK pathways (Porter and Vaillancourt, 1998; Cantley, 2002; Hahn and Weinberg, 2002). AKT3 (v-akt murine thymoma viral oncogene homolog 3; protein kinase B, gamma) was highly expressed in most of the KIT exon 11 and 9 mutants but in lower amounts in PDGFRA mutants (Figure 1b). The AKT3 protein product belongs to the serine/threonine protein kinase family and is involved in cell proliferation, differentiation, apoptosis and tumorigenesis. AKT3 is activated by growth factors via PI3K (phosphatidyl inositol 3-kinase). Interestingly, there was a higher level of expression of the AKT/PI3K pathway genes in KIT mutants as compared to PDGFRA mutants. This suggests that GISTs with different mutations have subtle differences in signal transduction networks that might account for their various clinicopathologic differences.
Sato et al. (2000) showed that AKT dephosphorylation resulted from increased activity of the PP2A family of protein phosphatases. PP2A family members also control cell proliferation signals by regulating phosphorylation of the mitogen-activated protein kinase (MAPK) cascade, and appear to be linked to carcinogenesis (Kohno et al., 1999; Silverstein et al., 2002). PPP2R3A, which encodes a regulatory subunit of PP2A, is specifically expressed in a subset of GISTs with KIT exon 11 mutants. Another PP2A regulatory subunit gene, protein phosphatase PPP2R1B, is expressed specifically in most of the PDGFRA mutant cases. The PPP2R1B gene encodes the beta isoform of the A subunit of PP2A and has been identified as a possible human tumor suppressor gene (Wang et al., 1998; Schonthal, 2001). Our data suggest that expression of specific PP2A family subunits may be associated with the kinase mutation status of GISTs. The differences in expression levels for PP2A family proteins in KIT exon 11 and PDGFRA mutants may contribute to differences in AKT/PI3K pathway signaling. These observations are of potential clinical relevance, because therapeutic synergies with KIT and PDGFRA inhibition might be obtained by inhibition of downstream PI3K/AKT/mTOR mechanisms. Our preliminary evidence, as discussed below, suggests that the mTOR/p70S6K axis might be more relevant in KIT mutant GISTs than in those with PDGFRA mutations, given that p70S6K expression is lower in the latter group.
A subset of PDGFRA mutants showed high levels of MAP2K1 (MEK), another activator of the MAPK gene family. Furthermore, MAPK target genes such as COL1A1, EGR1, ENPP2, JUN and FOS were expressed predominantly in the PDGFRA GIST mutants (Figure 1b). However, variable levels of MEK were also found in tumors with mutation in KIT and MEK expression alone cannot explain the high levels of MAPK target genes seen in PDGFRA mutant GISTs. At the protein level, Western blot studies have shown no differences in p42/44 MAPK in primary GISTs with PDGFRA mutations and with different KIT mutations (Heinrich et al., 2003a; Duensing et al., 2004).
PDGFRA mutants were associated with a large number of genes that are involved in T-cell receptor signaling (see complete SAM gene list for PDGFRA mutants in the accompanying website). Histologic examination of the PDGFRA cases showed a trend towards a higher rate of pleomorphism and many cases did indeed contain lymphocytes, either diffusely or in aggregates. However, the significance of the elevated levels of T-cell genes in our PDGFRA cases remains to be determined.
Gene expression and Western blot data confirmed the differential expression of ezrin/VIL2 between the KIT and PDGFRA mutants, with high levels of expression found in KIT exon 11 and a subset of KIT exon 9 mutants. Ezrin presents a functional link between the plasma membrane and the cortical actin cytoskeleton of the cell and also participates in essential signal transduction pathways including cell survival, motility, invasion and adherence. (Louvet-Vallee, 2000; Bretscher et al., 2002; Khanna et al., 2004). Ezrin is a crucial regulator of metastasis in rhabdomyosarcoma and high levels of expression are significantly correlated with clinical stage (Yu et al., 2004). A recent study showed that ezrin was highly expressed in malignant GISTs as compared to benign cases (Koon et al., 2004). Further studies will be needed to determine whether ezrin has a similar predictive role in KIT and PDGFRA mutant GISTs. In a further comparison of genes highly expressed, COL8A1 and CENP-F are two genes present in both data sets.
By both Western blot and gene array analysis, p70S6K/RPS6KA1 was found in most KIT exon 11 and 9 mutants, but not in PDGFRA mutants. p70S6K/RPS6KA1 belongs to the RSK family of genes that is implicated in activation of the mitogen-activated kinase (MAPK) cascade, and stimulation of cell proliferation and differentiation (Frodin and Gammeltoft, 1999). The RSKs are known to phosphorylate the transcription factor CREB, and activated CREB promotes cell survival (Frodin and Gammeltoft, 1999). In addition, p70S6K plays a key role in the PI3K/AKT pathway, downstream of mTOR (Brazil et al., 2002; Cantley, 2002). As shown above, the PI3K/AKT pathway appears to be more highly expressed in KIT exon 11 and 9 mutants than in PDGFRA mutants. This suggests that p70S6K/RPS6KA1 may be a better target for therapeutic intervention in KIT mutant rather than PDGFRA mutant GISTs.
During submission of this manuscript, a paper was published that indicated that there are differences in gene expression related to location within the GI tract (Antonescu et al., 2004). This may reflect some of the differences that we see between our KIT exon 9 and KIT exon 11 mutants, as several exon 9 cases arise from the small bowel, and all KIT exon 11 mutants from the stomach. However, almost all the PDGFRA mutants and all the KIT exon 11 mutants used for gene arrays originate from the stomach. We find significant differences in gene expression between these two groups and these differences thus appear to be caused by genotype rather than by location of the tumors. The heatmap in Figure 1a shows that there are significant numbers of genes that are differentially expressed between KIT exon 11 and PDGFRA mutants, more than those seen between KIT exon 11 and KIT exon 9 mutants.
Distinct gene expression profiles can distinguish most GISTs with mutations in different kinases and also among those with different types of mutations in the KIT gene. Nevertheless, our data show no absolute distinction between KIT and PDGFRA mutant pathways, and previous studies (Heinrich et al., 2003a) have shown that both KIT and PDGFRA mutant GISTs feature functional activation of the PI3K/AKT and MAPK pathways. Although gene expression studies in larger numbers of GISTs might reveal additional correlates with kinase mutation type, it is likely that a considerable overlap of expressed genes – as seen in this study – reflects the close biologic relationships between GISTs, irrespective of their kinase oncogene mechanisms. Nevertheless, genes such as ezrin, p70S6K and PRKCQ are differentially expressed in KIT vs PDGFRA mutant GISTs. These findings suggest that various signaling proteins might be more effective therapeutic targets in some GISTs than in others, depending on their kinase mutation type.
Materials and methods
Gene expression profiling on microarrays
In total, 26 fresh frozen GIST tumor tissue samples were used for gene array analysis. The institutional review board at Stanford University approved the study. The mutation status of each case was determined by denaturing HPLC and direct sequencing as described previously (Corless et al., 2002). The microarrays used in the study contain a total of about 42 000 cDNAs representing about 28 000 genes or ESTs printed on polylysine coated glass slides by the Stanford Functional Genomics Facility (http://www.microarray.org/). Preparation and details of microarray construction were described previously (Perou et al., 2000). Briefly, tissue was homogenized in Trizol reagent (Invitrogen, Carlsbad, CA, USA) and total RNA was extracted. Preparation of Cy-3-dUTP labeled cDNA from reference RNA and Cy-5-dUTP labeled cDNA tumor specimen total RNA, microarray hybridization and washing of arrays was performed as described by Perou et al. (2000). Microarrays were scanned on a GenePix 4000 microarray scanner (Axon Instruments, Foster City, CA, USA) and fluorescence ratios (tumor/reference) were calculated using GenePix software. The raw data and the image files are available from the Stanford Microarray Database (http://smd.stanford.edu/ and accompanying website http://microarray-pubs.stanford.edu/tma_portal/GISTmutations/). Control and empty spots on the arrays were not included for the analysis, as well as those spots flagged as bad spots due to technical errors. Only cDNA spots with a ratio of signal over background of at least 1.8 in either the Cy3 or Cy5 channel were included; genes with 80% good data were included and those genes were filtered based on those where expression levels differed by at least four-fold in at least three arrays. Using these criteria, 1875 genes passed the filtering criteria and were used for further analysis. Unsupervised hierarchical clustering analysis (Eisen et al., 1998) and significance analysis of microarrays (SAM) (Tusher et al., 2001) were then performed as described previously (Nielsen et al., 2002).
ISH and IHC
A tissue microarray containing 127 cases of GISTs with known mutation status was constructed as described previously (West et al., 2004). There was one core for each tumor, and all of the GISTs on this TMA were analyzed for mutations in exons 9, 11, 13 and 17 of the KIT gene using a combination of denaturing HPLC and direct sequencing, as previously described (Corless et al., 2002; Heinrich et al., 2003a). KIT wild-type tumors included on the array were also screened for mutations in exons 12 and 18 of the PDGFRA gene. ISH of TMA sections was performed as previously described (West et al., 2004). The primer sequences used for the amplification of probes are for DLK1, MAP1B, and PDGFRA are given in Supplementary Table 2 (http://microarray-pubs.stanford.edu/tma_portal/GISTmutations/).
We performed IHC with CD34 antibody (Becton Dickinson, Franklin Lakes, NJ, USA) as previously described (Linn et al., 2003; West et al., 2004). The IHC data for DOG1 and KIT, and the ISH data for PDGFRA in Figure 2 were previously published (West et al., 2004).
Scoring of IHC and ISH
For IHC scoring of the TMA, a score of ‘−2’ was given for absent or insignificant staining defined as less than 5% tumor cells with staining. Data fields for unscorable cores were left empty. A score of ‘1’ was given for hybridization signal in greater than 5% of tumor cells or less than 50% of tumor cells. A score of ‘2’ was given for hybridization signal in greater than 50% tumor cells. Nontumor cells and cells of unknown origin were not scored. Cores scored as ‘1’ or ‘2’ were scored as positive in Figure 2. For ISH, only a prominent dot-like stain was counted towards the scoring. A score of ‘−2’ was given for absent or insignificant staining defined as less than 1 dot-like stain per observed nucleus. Data fields for unscorable cores were left empty. A score of ‘1’ was given for greater than 1 dot-like stain per tumor cell. A score of ‘2’ was given for multiple dot-like stains the tumor cells. Cores scored as ‘1’ or ‘2’ were scored as positive in Figure 2. All stained cores were reviewed by two observers (MvdR, RW). To aid in the analysis of numerous tissue cores stained by ISH, digital images were collected using the BLISS instrument (Bacuslabs, Lombard IL; USA http://bacuslabs.com). Scoring results were combined using Deconvoluter and represented in Treeview (Liu et al., 2002). All 809 digital images taken for IHC and ISH experiments are available at the accompanying website (http://microarray-pubs.stanford.edu/tma_portal/GISTmutations/). Digital images for PDGFRA, KIT (CD117), DOG1 ISH and KIT (CD117), DOG1 immunostaining were previously published (West et al., 2004) and are also available at http://microarray-pubs.stanford.edu/tma_portal/dog1/.
Whole-cell lysates from tumor specimens were prepared by using lysis buffer (1% NP-40, 50 mM Tris-HCl pH 8.0, 100 mM sodium fluoride, 30 mM sodium pyrophosphate, 2 mM sodium molybdate, 5 mM EDTA, 2 mM sodium orthovanadate) containing protease inhibitors (10 μg/ml aprotinin, 10 ug/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride). Protein concentrations were determined with Bio-Rad protein assay (Bio-Rad Laboratories Hercules, CA, USA). Electrophoresis and Western blotting were performed as described previously (Rubin et al., 2001). The hybridization signals were detected by chemiluminescence (ECL, Amersham Pharmacia Biotech) and were captured and quantitated using a FUJI LAS1000-plus chemiluminescence imaging system.
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SS was supported by a fellowship from the Laboratory of Surgical Pathology, Department of Pathology, Stanford University Medical Center. This work was supported by NIH Grant CA85129.
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Subramanian, S., West, R., Corless, C. et al. Gastrointestinal stromal tumors (GISTs) with KIT and PDGFRA mutations have distinct gene expression profiles. Oncogene 23, 7780–7790 (2004) doi:10.1038/sj.onc.1208056
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