_:b412437148 . _:b5892433 . _:b412437149 . _:b5892418 . _:b412437150 . _:b412437151 . _:b412437283 "2"^^ . _:b412437216 . _:b5892434 . _:b412437217 . _:b412437219 . _:b412437243 . _:b412437218 . _:b412437219 . _:b412437282 "2"^^ . _:b412437220 . _:b5892435 . _:b412437221 . _:b412437222 . _:b412437223 . _:b412437224 . _:b5892444 . _:b412437225 . _:b5892435 . _:b412437226 . _:b412437227 . _:b412437228 . _:b5892499 . _:b5892445 . _:b412437283 . _:b412437229 . _:b5892434 . _:b412437230 . _:b412437231 . _:b412437232 . _:b5892446 . _:b412437257 . _:b412437233 . _:b412437234 . _:b412437230 . _:b412437235 . _:b5892483 "In agreement with previous studies (Hartmann et al, 2004; Rand et al, 2005; Reis et al, 2005; Sihto et al, >>2005<<; Wen et al, 2006; McLendon et al, 2008; Parsons et al, 2008), no PDGFRA-activating mutations were found." . _:b412437236 . _:b5892480 "In agreement with previous studies (Hartmann et al, >>2004<<; Rand et al, 2005; Reis et al, 2005; Sihto et al, 2005; Wen et al, 2006; McLendon et al, 2008; Parsons et al, 2008), no PDGFRA-activating mutations were found." . _:b5892447 . . _:b412437237 . _:b412437238 . _:b412437239 . _:b412437240 . _:b5892440 . _:b412437241 . _:b5892455 "Despite the positive response, none of the new targeted therapies has shown significant clinical activity as a single agent in phase II studies (Brandsma and van den Bent, >>2007<<; Raymond et al, 2008)." . . _:b412437242 . _:b412437243 . _:b412437244 . _:b5892490 "PDGFRA gene amplification analysis revealed PDGFRA amplification in 21.1% (12 out of 57) of gliomas, a frequency similar to that described in previous studies (Fleming et al, >>1992<<; Kumabe et al, 1992; Smith et al, 2000; Alonso et al, 2005; Arjona et al, 2005; Puputti et al, 2006; Beroukhim et al, 2007; McLendon et al, 2008)." . _:b5892441 . _:b412437172 . _:b412437245 . _:b412437246 . _:b412437247 . _:b412437184 . _:b5892442 . _:b412437146 . _:b412437185 . _:b412437186 . _:b412437187 . _:b412437211 . _:b412437188 . _:b5892443 . _:b412437189 . _:b412437190 . _:b412437191 . _:b412437192 . _:b5892452 . _:b412437193 . _:b412437194 . _:b412437195 . _:b412437196 . _:b5892453 . . _:b412437197 . _:b412437198 . _:b412437199 . _:b412437200 . _:b5892454 . _:b412437201 . _:b412437202 . _:b412437188 . _:b412437203 . _:b412437204 . _:b5892455 . _:b5892438 . . _:b412437205 . _:b412437191 . _:b412437206 . _:b412437207 . _:b412437208 . _:b5892448 . _:b412437209 . _:b412437210 . _:b412437211 . _:b412437212 . _:b5892470 "These frequencies are in agreement with previous studies, where approximately 50% for malignant astrocytomas were reported to express this receptor (Nister et al, 1988, 1991; Hermanson et al, 1992; Black et al, >>1996<<; Di Rocco et al, 1998; Ribom et al, 2002; Varela et al, 2004; Liang et al, 2008; Takei et al, 2008; Thorarinsdottir et al, 2008)." . _:b5892449 . . _:b412437213 . _:b412437214 . _:b412437215 . _:b5892468 . _:b5892442 "PDGF and PDGFRs have both been found to be overexpressed in glial tumour cell lines and tumour surgical samples (Nister et al, 1988, 1991; Hermanson et al, 1992; Black et al, >>1996<<; Di Rocco et al, 1998; Varela et al, 2004)." . _:b412437280 . _:b5892450 . _:b412437281 . _:b412437282 . _:b412437283 . _:b412437284 . _:b5892451 . . _:b412437285 . _:b412437286 . _:b412437214 . _:b5892460 . . _:b412437236 . . . . _:b5892461 . _:b412437286 . _:b5892462 . _:b5892485 "In agreement with previous studies (Hartmann et al, 2004; Rand et al, 2005; Reis et al, 2005; Sihto et al, 2005; Wen et al, 2006; McLendon et al, >>2008<<; Parsons et al, 2008), no PDGFRA-activating mutations were found." . . _:b5892413 . _:b412437248 . _:b5892412 "with rabbit polyclonal antibodies raised against human PDGFA (clone N-30, dilution 1:80; Santa Cruz Biotechnology, Santa Cruz, USA), and PDGFRA (dilution 1:175; LabVision Corporation) as previously described (Carvalho et al, >>2005<<; Reis et al, 2005). In brief, deparaffinised and rehydrated sections used to study PDGFA expression were pre-treated by microwaving in 10\u2009mM citrate buffer (pH 6.0) three times for 5\u2009min at 600\u2009W." . _:b5892463 . _:b5892456 . _:b5892415 . . . _:b5892457 . _:b5892414 . _:b5892483 . _:b412437248 . _:b5892458 . _:b412437249 . _:b412437250 . . . _:b412437251 . _:b412437252 . _:b5892459 . _:b412437253 . _:b412437254 . . _:b5892464 . _:b412437255 . _:b412437256 . _:b5892468 . _:b412437257 . _:b412437258 . _:b412437259 . _:b412437260 . _:b5892469 . _:b412437261 . _:b412437262 . _:b412437263 . _:b412437264 . _:b5892470 . _:b412437265 . _:b412437266 . _:b412437267 . _:b412437268 . _:b5892497 . _:b5892471 . _:b412437269 . _:b412437270 . _:b412437178 . _:b412437271 . . _:b412437272 . _:b5892464 . _:b412437273 . . _:b412437274 . _:b412437275 . _:b5892469 . _:b412437276 . _:b5892495 . _:b5892465 . _:b412437277 . _:b412437278 . _:b412437279 . _:b5892466 . . . _:b5892467 . _:b5892473 "previous studies, where approximately 50% for malignant astrocytomas were reported to express this receptor (Nister et al, 1988, 1991; Hermanson et al, 1992; Black et al, 1996; Di Rocco et al, 1998; Ribom et al, 2002; Varela et al, >>2004<<; Liang et al, 2008; Takei et al, 2008; Thorarinsdottir et al, 2008). A previous study showed PDGFRA expression in approximately 50% of gliomas and a correlation with poor prognosis in low-grade gliomas (Varela et al, 2004)." . _:b5892476 . _:b5892477 . _:b412437198 . _:b412437204 . _:b412437232 . _:b5892478 . . _:b5892479 . _:b412437166 . _:b412437274 . _:b5892472 . _:b412437282 . . _:b5892473 . _:b412437201 . . _:b412437256 . _:b5892498 . _:b5892474 . . _:b5892445 . _:b5892475 . _:b5892485 . _:b412437197 . _:b5892484 . . _:b5892485 . . _:b412437242 . _:b5892486 . _:b5892487 . _:b5892480 . _:b5892481 . _:b412437180 . . . _:b412437151 . _:b5892452 "Clinical trials evaluating the efficacy of anti-PDGFRA drugs in patients with glioblastomas are ongoing (Reardon et al, >>2005<<; Wen et al, 2006; Desjardins et al, 2007; Newton, 2007; Raymond et al, 2008)." . _:b5892482 . _:b412437234 . _:b412437261 . . . _:b5892483 . _:b412437159 . _:b5892492 . . _:b5892493 . _:b5892430 "Labelling, hybridisation, washes, image acquisition and data normalisation were carried out as previously described (Arriola et al, 2007, >>2008<<; Huang et al, 2007; Marchi\u00F2 et al, 2008a, 2008b)." . _:b5892494 . _:b5892477 "Overexpression of RTKs in cancer has been shown to be driven by underlying genetic events in a substantial proportion of cases (Gschwind et al, >>2004<<). For instance, KIT overexpression in GISTs is driven by activating KIT mutations (Gomes et al, 2008), whereas HER2 overexpression in breast cancer is driven by HER2 gene amplification (Arriola et al, 2008)." . _:b5892495 . _:b412437238 . _:b412437228 . _:b412437255 . _:b5892488 . . _:b412437208 . . _:b5892489 . _:b5892456 "Despite the positive response, none of the new targeted therapies has shown significant clinical activity as a single agent in phase II studies (Brandsma and van den Bent, 2007; Raymond et al, >>2008<<). Currently, combination of anti-PDGFRA drugs with chemotherapy agents is being evaluated (Reardon et al, 2008). However, the molecular alterations underlying glioma patients\u2019 response to PDGFRA antagonists are unknown. One study analysed" . _:b5892490 . _:b5892463 "Previous studies on PDGFA mRNA expression reported high levels of PDGFA in gliomas (Maxwell et al, 1990; Mapstone, >>1991<<; Hermanson et al, 1992; Di Rocco et al, 1998)." . _:b5892491 . _:b412437193 . . _:b5892500 . _:b5892447 "PDGFRA and PDGFA have been shown to be expressed in tumour cells, whereas PDGFB and PDGFRB have been found in glioma-associated endothelial cells (Hermansson et al, 1988; Hermanson et al, 1992; Plate et al, >>1992<<). Furthermore, studies on the PDGFC and D ligands, also demonstrate their expression in gliomas; however, the clinical and biological significance of their expression remain to be determined (Lokker et al, 2002). Importantly, the function" . _:b5892501 . _:b412437275 . . . _:b5892502 . _:b5892477 . _:b412437141 . _:b5892492 . _:b5892503 . _:b5892496 . _:b412437141 . _:b5892497 . _:b5892441 "PDGF and PDGFRs have both been found to be overexpressed in glial tumour cell lines and tumour surgical samples (Nister et al, 1988, 1991; Hermanson et al, >>1992<<; Black et al, 1996; Di Rocco et al, 1998; Varela et al, 2004)." . _:b5892498 . _:b5892436 . _:b412437143 . _:b5892413 "polyclonal antibodies raised against human PDGFA (clone N-30, dilution 1:80; Santa Cruz Biotechnology, Santa Cruz, USA), and PDGFRA (dilution 1:175; LabVision Corporation) as previously described (Carvalho et al, 2005; Reis et al, >>2005<<). In brief, deparaffinised and rehydrated sections used to study PDGFA expression were pre-treated by microwaving in 10\u2009mM citrate buffer (pH 6.0) three times for 5\u2009min at 600\u2009W." . _:b5892499 . _:b412437142 . _:b5892493 "PDGFRA gene amplification analysis revealed PDGFRA amplification in 21.1% (12 out of 57) of gliomas, a frequency similar to that described in previous studies (Fleming et al, 1992; Kumabe et al, 1992; Smith et al, 2000; Alonso et al, >>2005<<; Arjona et al, 2005; Puputti et al, 2006; Beroukhim et al, 2007; McLendon et al, 2008)." . . _:b412437260 . _:b412437229 . _:b5892441 . . _:b5892446 . . _:b412437152 . _:b5892504 . _:b412437269 . _:b412437149 . _:b5892436 _:b5892500 . _:b5892447 . . _:b5892436 _:b5892501 . _:b412437148 . _:b5892436 _:b5892502 . _:b5892436 _:b5892503 . _:b5892436 _:b5892496 . _:b5892436 _:b5892497 . _:b412437195 . _:b412437151 . _:b5892436 _:b5892498 . _:b5892436 _:b5892499 . _:b412437150 . _:b5892410 . _:b5892436 _:b5892504 . . . . _:b5892484 "In agreement with previous studies (Hartmann et al, 2004; Rand et al, 2005; Reis et al, 2005; Sihto et al, 2005; Wen et al, >>2006<<; McLendon et al, 2008; Parsons et al, 2008), no PDGFRA-activating mutations were found." . _:b412437145 . _:b412437186 . _:b5892436 _:b5892484 . _:b5892468 "These frequencies are in agreement with previous studies, where approximately 50% for malignant astrocytomas were reported to express this receptor (Nister et al, 1988, >>1991<<; Hermanson et al, 1992; Black et al, 1996; Di Rocco et al, 1998; Ribom et al, 2002; Varela et al, 2004; Liang et al, 2008; Takei et al, 2008; Thorarinsdottir et al, 2008)." . _:b5892462 . _:b5892436 _:b5892485 . _:b412437144 . _:b412437279 . _:b5892436 _:b5892486 . _:b5892436 _:b5892487 . _:b5892436 _:b5892480 . _:b412437150 . _:b412437240 . _:b5892436 _:b5892481 . _:b412437147 . _:b5892436 _:b5892482 . _:b5892436 _:b5892483 . . _:b5892460 "to targeted agents is associated with overall PDGFR activation; others point to the putative role of PDGFRB or failed to show any association between PDGFR status and response to imatinib (Gross et al, 2006; H\u00E4gerstrand et al, >>2006<<; Servidei et al, 2006)." . _:b5892436 _:b5892492 . _:b5892436 _:b5892493 . . _:b412437146 . _:b412437161 . _:b5892436 _:b5892494 . _:b5892436 _:b5892495 . _:b5892436 _:b5892488 . _:b5892436 _:b5892489 . _:b412437157 . _:b5892416 "Primer sequences for exons 12 and 18 were previously reported (Reis et al, >>2005<<), and for exon 23 were 5\u2032-GCTCTTCTCTCCCTCCTCCA-3\u2032 (sense) and 5\u2032-TTTCTGAACGGGATCCAGAG-3\u2032 (antisense)." . _:b5892436 _:b5892490 . _:b5892436 _:b5892491 . _:b412437156 . . _:b412437159 . _:b5892442 . . _:b412437158 . _:b412437207 . . . _:b412437153 . _:b412437216 . _:b5892437 . . _:b412437152 . _:b412437155 . _:b412437179 . _:b412437154 . . _:b412437165 . _:b5892498 "Co-amplification of these three oncogenes has already been detected with other methodologies in gliomas (Joensuu et al, >>2005<<; Puputti et al, 2006; Holtkamp et al, 2007)." . _:b412437164 . _:b412437164 . . _:b412437271 . _:b412437259 . _:b412437167 . _:b5892502 . _:b5892443 . _:b412437166 . _:b412437184 . . _:b5892503 "of PDGFA and PDGFRA in glioblastomas, it could be hypothesised that autocrine/paracrine loops may be present in these tumours, corroborating the importance of this signalling pathway in gliomas (Nister et al, 1991; Guha et al, >>1995<<; Fomchenko and Holland, 2007). PDGFRA gene amplification may be the underlying genetic mechanism driving PDGFRA overexpression in gliomas." . . _:b5892418 "Primers and probes for 18S gene were previously described (Gomes et al, >>2007<<), for PDGFRA were as follow:" . _:b412437262 . _:b412437161 . _:b412437160 . . _:b412437163 . . "10.1038%2Fsj.bjc.6605225" . _:b412437162 . . . _:b412437173 . _:b5892467 "These frequencies are in agreement with previous studies, where approximately 50% for malignant astrocytomas were reported to express this receptor (Nister et al, >>1988<<, 1991; Hermanson et al, 1992; Black et al, 1996; Di Rocco et al, 1998; Ribom et al, 2002; Varela et al, 2004; Liang et al, 2008; Takei et al, 2008; Thorarinsdottir et al, 2008)." . _:b412437172 . _:b412437182 . _:b5892486 "In agreement with previous studies (Hartmann et al, 2004; Rand et al, 2005; Reis et al, 2005; Sihto et al, 2005; Wen et al, 2006; McLendon et al, 2008; Parsons et al, >>2008<<), no PDGFRA-activating mutations were found." . _:b412437175 . . _:b5892454 "Clinical trials evaluating the efficacy of anti-PDGFRA drugs in patients with glioblastomas are ongoing (Reardon et al, 2005; Wen et al, 2006; Desjardins et al, 2007; Newton, 2007; Raymond et al, >>2008<<). Despite the positive response, none of the new targeted therapies has shown significant clinical activity as a single agent in phase II studies (Brandsma and van den Bent, 2007; Raymond et al, 2008)." . _:b412437264 . _:b412437174 . . _:b412437169 . _:b412437168 . _:b412437171 . _:b5892415 "23 of the PDGFRA gene was carried out by PCR-single-strand conformational polymorphism (PCR\u2013SSCP) followed by direct DNA sequencing of samples that showed a mobility shift in the PCR\u2013SSCP analysis, as previously described (Reis et al, >>2005<<). Briefly, PCR was carried out in a total volume of 25\u2009\u03BCl, consisting of 1\u20132\u2009\u03BCl of DNA solution, 0.5\u2009\u03BCM of both sense and anti-sense primers, 200\u2009\u03BCM of dNTPs (Fermentas Inc., Glen Burnie, MD, USA), 1.5\u20132\u2009mM of MgCl2 (Bioron GmbH," . . _:b412437170 . _:b5892504 . _:b412437181 . . . _:b412437180 . . _:b412437183 . _:b5892436 "discussion" . _:b412437270 . . _:b412437182 . "PMC0" . . _:b5892412 . _:b412437272 . _:b412437177 . . . _:b412437176 . . . _:b412437179 . . _:b412437178 . _:b412437189 . . _:b5892436 _:b5892468 . _:b5892502 "the concurrent expression of PDGFA and PDGFRA in glioblastomas, it could be hypothesised that autocrine/paracrine loops may be present in these tumours, corroborating the importance of this signalling pathway in gliomas (Nister et al, >>1991<<; Guha et al, 1995; Fomchenko and Holland, 2007). PDGFRA gene amplification may be the underlying genetic mechanism driving PDGFRA overexpression in gliomas." . _:b5892436 _:b5892469 . _:b412437188 . _:b5892436 _:b5892470 . _:b5892436 _:b5892471 . _:b412437268 . _:b5892436 _:b5892464 . _:b5892436 _:b5892465 . _:b412437191 . . _:b5892436 _:b5892466 . _:b5892436 _:b5892467 . _:b412437210 . _:b5892436 _:b5892476 . _:b412437181 . _:b5892436 _:b5892477 . _:b412437190 . _:b412437170 . _:b5892436 _:b5892478 . _:b5892436 _:b5892479 . _:b5892436 _:b5892472 . _:b5892436 _:b5892473 . _:b412437185 . _:b5892436 _:b5892474 . _:b5892436 _:b5892475 . _:b5892436 _:b5892452 . _:b5892436 _:b5892453 . _:b412437184 . _:b5892423 . _:b5892436 _:b5892454 . _:b5892436 _:b5892455 . . _:b5892436 _:b5892448 . _:b5892436 _:b5892449 . _:b412437187 . _:b5892449 . _:b5892436 _:b5892450 . _:b5892436 _:b5892451 . _:b5892436 _:b5892460 . _:b5892436 _:b5892461 . _:b412437186 . _:b5892495 "PDGFRA amplification in 21.1% (12 out of 57) of gliomas, a frequency similar to that described in previous studies (Fleming et al, 1992; Kumabe et al, 1992; Smith et al, 2000; Alonso et al, 2005; Arjona et al, 2005; Puputti et al, >>2006<<; Beroukhim et al, 2007; McLendon et al, 2008). We have further shown by aCGH that the amplicon encompasses a region of 3.6\u2009Mb, which, in addition to PDGFRA, also includes KIT and KDR oncogenes." . _:b5892436 _:b5892462 . _:b5892436 _:b5892463 . _:b5892436 _:b5892456 . . _:b5892436 _:b5892457 . _:b412437197 . _:b5892436 _:b5892458 . . _:b5892436 _:b5892459 . _:b412437250 . _:b5892436 _:b5892437 . _:b5892459 "some suggested that sensitivity to targeted agents is associated with overall PDGFR activation; others point to the putative role of PDGFRB or failed to show any association between PDGFR status and response to imatinib (Gross et al, >>2006<<; H\u00E4gerstrand et al, 2006; Servidei et al, 2006)." . _:b412437196 . _:b5892436 _:b5892438 . . _:b5892436 _:b5892439 . _:b5892458 "One study analysed the prevalence of PDGFRA mutations in patients enrolled in a phase I/II study of imatinib mesylate in recurrent malignant gliomas; however, no activating mutations were observed (Wen et al, >>2006<<). The in vitro studies using PDGFR-targeted drugs (e.g., imatinib) have provided conflicting information about the molecular underpinning of sensitivity to those agents. Although some suggested that sensitivity to targeted agents is" . _:b412437199 . . _:b5892436 _:b5892444 . _:b5892464 "Previous studies on PDGFA mRNA expression reported high levels of PDGFA in gliomas (Maxwell et al, 1990; Mapstone, 1991; Hermanson et al, >>1992<<; Di Rocco et al, 1998)." . _:b5892436 _:b5892445 . _:b5892431 "Labelling, hybridisation, washes, image acquisition and data normalisation were carried out as previously described (Arriola et al, 2007, 2008; Huang et al, >>2007<<; Marchi\u00F2 et al, 2008a, 2008b)." . _:b412437198 . _:b5892436 _:b5892446 . _:b412437153 . _:b5892436 _:b5892447 . . _:b5892436 _:b5892440 . _:b5892436 _:b5892441 . _:b412437193 . _:b5892436 _:b5892442 . _:b5892436 _:b5892443 . _:b412437194 . _:b412437192 . . _:b412437195 . _:b5892470 . . _:b5892414 "As previously described (Reis et al, >>2005<<), both the distribution and intense immunoreactivity were semi-quantitatively scored by JML and ALF independently with the observers blinded to the clinical information and results of the other molecular tests as follows:" . . _:b5892429 . _:b412437252 . _:b412437194 . . _:b412437165 . _:b5892426 . _:b412437205 . _:b5892465 . _:b412437204 . _:b5892479 "For instance, KIT overexpression in GISTs is driven by activating KIT mutations (Gomes et al, 2008), whereas HER2 overexpression in breast cancer is driven by HER2 gene amplification (Arriola et al, >>2008<<). Here, we investigated the prevalence of PDGFRA-activating mutations and gene amplification in gliomas. In agreement with previous studies (Hartmann et al, 2004; Rand et al, 2005; Reis et al, 2005; Sihto et al, 2005; Wen et al, 2006;" . _:b412437207 . _:b412437162 . . _:b412437202 . _:b5892458 . _:b412437206 . _:b412437201 . _:b5892475 . _:b412437205 . _:b5892499 "Co-amplification of these three oncogenes has already been detected with other methodologies in gliomas (Joensuu et al, 2005; Puputti et al, >>2006<<; Holtkamp et al, 2007)." . _:b412437200 . _:b412437155 . _:b5892489 "gov/projects/SNP/; Carvalho et al, 2005; Reis et al, 2005; Wen et al, >>2006<<). The impact of these genetic polymorphisms in PDGFRA function remains to be elucidated." . . _:b412437226 . _:b412437206 . _:b412437203 . _:b5892473 . . _:b5892471 . . . _:b412437202 . _:b412437213 . . _:b5892493 . _:b412437212 . . . _:b5892453 . . _:b412437215 . _:b412437183 . _:b412437214 . _:b412437209 . _:b412437253 . _:b5892410 _:b5892412 . . _:b5892410 _:b5892413 . _:b412437208 . _:b5892461 . _:b5892410 _:b5892414 . _:b5892410 _:b5892415 . . . _:b5892500 . _:b5892481 . _:b412437148 . _:b412437211 . _:b5892410 _:b5892411 . _:b5892411 "Cases were classified according to the WHO criteria (Louis et al, >>2007<<). This series (Table 1) includes 83 astrocytic, 68 oligodendroglial and 9 oligoastrocytic tumours." . _:b412437210 . _:b5892433 . . _:b412437221 . _:b5892419 "The in-house probe was generated, biotin-labelled and used in hybridisations as previously described (Lambros et al, >>2006<<; Gomes et al, 2007)." . . . _:b5892410 _:b5892428 . _:b5892410 _:b5892429 . _:b412437220 . _:b5892410 _:b5892430 . . _:b5892410 _:b5892431 . _:b5892410 _:b5892424 . _:b5892410 _:b5892425 . _:b412437223 . _:b5892410 _:b5892426 . _:b412437143 "10"^^ . _:b5892410 _:b5892427 . _:b5892410 _:b5892420 . _:b5892410 _:b5892421 . _:b412437222 . _:b5892410 _:b5892422 . _:b5892410 _:b5892423 . _:b412437142 "11"^^ . _:b5892410 _:b5892416 . _:b5892410 _:b5892417 . _:b412437217 . _:b5892410 _:b5892418 . _:b5892410 _:b5892419 . _:b412437216 . _:b5892474 . . . _:b412437219 . _:b412437266 . _:b412437218 . _:b412437265 . _:b412437141 "14"^^ . _:b5892471 "These frequencies are in agreement with previous studies, where approximately 50% for malignant astrocytomas were reported to express this receptor (Nister et al, 1988, 1991; Hermanson et al, 1992; Black et al, 1996; Di Rocco et al, >>1998<<; Ribom et al, 2002; Varela et al, 2004; Liang et al, 2008; Takei et al, 2008; Thorarinsdottir et al, 2008)." . _:b5892410 _:b5892432 . _:b5892466 "We have previously shown that 100% of gliosarcomas express PDGFA (Reis et al, >>2005<<). PDGFRA expression was detected in 29.6% of gliomas, and more frequently expressed in 45\u201360% of malignant astrocytic tumours. These frequencies are in agreement with previous studies, where approximately 50% for malignant astrocytomas" . _:b5892410 _:b5892433 . _:b5892410 _:b5892434 . _:b412437229 . _:b5892410 _:b5892435 . _:b5892449 "been suggested that activation of PDGFRA signalling, directly or indirectly through creating a favourable microenvironment niche, can contribute to the transformation of neural stem/progenitor cells into glioma tumours (Jackson et al, >>2006<<; Fomchenko and Holland, 2007; Siebzehnrubl et al, 2008)." . _:b412437228 . _:b5892448 . . _:b412437231 . _:b412437150 "6"^^ . _:b412437230 . _:b412437151 "6"^^ . _:b5892490 . _:b412437225 . . _:b412437221 . _:b412437148 "6"^^ . _:b412437224 . _:b5892459 . . _:b412437149 "6"^^ . _:b412437227 . _:b5892432 "Labelling, hybridisation, washes, image acquisition and data normalisation were carried out as previously described (Arriola et al, 2007, 2008; Huang et al, 2007; Marchi\u00F2 et al, 2008a, 2008b). Polymorphic BACs identified in an analysis of 50 male/female and female/female hybridisations were filtered out. This left a final dataset of 13711 clones with unambiguous mapping information according to the March 2006 build" . _:b412437146 "7"^^ . _:b412437226 . _:b412437147 "6"^^ . _:b412437237 . _:b412437190 . _:b412437144 "9"^^ . _:b412437236 . _:b5892444 . _:b412437145 "7"^^ . . _:b412437239 . _:b412437157 "5"^^ . _:b412437238 . _:b412437156 "5"^^ . _:b412437233 . . _:b412437159 "4"^^ . _:b412437232 . _:b412437158 "4"^^ . _:b412437235 . _:b412437187 . _:b412437153 "5"^^ . _:b412437234 . . _:b412437152 "5"^^ . _:b412437245 . . _:b5892494 . _:b412437155 "5"^^ . _:b5892421 "Amplification was defined as >5 signals per nucleus in more than 50% of tumour cells, or when large gene copy clusters were seen (Lambros et al, >>2006<<; Gomes et al, 2007)." . _:b412437244 . _:b5892478 . _:b412437224 . _:b412437154 "5"^^ . _:b412437247 . _:b412437165 "3"^^ . _:b5892488 . _:b412437246 . _:b412437164 "3"^^ . _:b412437163 . _:b412437280 . _:b412437241 . _:b412437167 "3"^^ . _:b412437174 . _:b412437169 . _:b412437240 . _:b5892491 . _:b412437166 "3"^^ . . _:b412437215 . _:b412437243 . _:b412437212 . . _:b412437161 "4"^^ . _:b412437242 . _:b5892450 "of PDGFRA signalling, directly or indirectly through creating a favourable microenvironment niche, can contribute to the transformation of neural stem/progenitor cells into glioma tumours (Jackson et al, 2006; Fomchenko and Holland, >>2007<<; Siebzehnrubl et al, 2008)." . _:b412437158 . . _:b5892425 "This type of BAC array platform has been shown to be as robust as high-density oligonucleotide arrays (Coe et al, 2007; Gunnarsson et al, >>2008<<) and its actual resolution is approximately 100\u2009kb for >98% of the genome (Arriola et al, 2007, 2008; Marchi\u00F2 et al, 2008a)." . _:b412437160 "4"^^ . . . . _:b412437163 "4"^^ . . _:b5892448 "Furthermore, studies on the PDGFC and D ligands, also demonstrate their expression in gliomas; however, the clinical and biological significance of their expression remain to be determined (Lokker et al, >>2002<<). Importantly, the function of PDGF signalling in gliomagenesis has been enlightened by its potential role in cancer stem cell hypothesis of gliomagenesis. It has been suggested that activation of PDGFRA signalling, directly or indirectly" . _:b412437162 "4"^^ . _:b5892492 "PDGFRA gene amplification analysis revealed PDGFRA amplification in 21.1% (12 out of 57) of gliomas, a frequency similar to that described in previous studies (Fleming et al, 1992; Kumabe et al, 1992; Smith et al, >>2000<<; Alonso et al, 2005; Arjona et al, 2005; Puputti et al, 2006; Beroukhim et al, 2007; McLendon et al, 2008)." . _:b5892481 "In agreement with previous studies (Hartmann et al, 2004; Rand et al, >>2005<<; Reis et al, 2005; Sihto et al, 2005; Wen et al, 2006; McLendon et al, 2008; Parsons et al, 2008), no PDGFRA-activating mutations were found." . . _:b412437173 "3"^^ . _:b412437172 "3"^^ . _:b412437192 . . _:b5892417 "showing a mobility shift in the PCR\u2013SSCP analysis different from the normal pattern were directly sequenced (Stab Vida, Investigation and Services in Biological Sciences Lda, Oeiras, Portugal) as previously described (Gomes et al, >>2007<<). All positive cases were confirmed twice with a new and independent PCR amplification, followed by direct sequencing." . _:b412437175 "3"^^ . _:b5892482 . _:b5892482 "In agreement with previous studies (Hartmann et al, 2004; Rand et al, 2005; Reis et al, >>2005<<; Sihto et al, 2005; Wen et al, 2006; McLendon et al, 2008; Parsons et al, 2008), no PDGFRA-activating mutations were found." . _:b412437174 "3"^^ . _:b5892452 . _:b412437169 "3"^^ . . _:b412437223 . _:b412437168 "3"^^ . . _:b412437285 . _:b412437171 "3"^^ . _:b5892424 "This type of BAC array platform has been shown to be as robust as high-density oligonucleotide arrays (Coe et al, >>2007<<; Gunnarsson et al, 2008) and its actual resolution is approximately 100\u2009kb for >98% of the genome (Arriola et al, 2007, 2008; Marchi\u00F2 et al, 2008a)." . _:b412437170 "3"^^ . . . . . _:b5892476 "However, in a report of 40 patients with grade II astrocytomas and oligoastrocytomas, there was an association between high PDGFRA expression and a favourable patient outcome (Ribom et al, >>2002<<). Recently, Liang et al, (2008) in a paediatric high-grade glioma series failed to find any significant impact of PDGFRA expression on survival. In our series, PDGFRA expression was not correlated with patients\u2019 survival. Interestingly," . _:b412437181 "3"^^ . _:b412437180 "3"^^ . . . _:b412437183 "3"^^ . _:b412437217 . _:b5892422 "Amplification was defined as >5 signals per nucleus in more than 50% of tumour cells, or when large gene copy clusters were seen (Lambros et al, 2006; Gomes et al, >>2007<<). CISH hybridisations were evaluated with observers blinded to the clinical information and results of immunohistochemical and QPCR analysis." . _:b412437182 "3"^^ . . _:b412437177 "3"^^ . . _:b412437176 "3"^^ . _:b412437203 . _:b412437179 "3"^^ . _:b412437149 . . _:b412437178 "3"^^ . . . _:b5892455 . _:b412437189 "3"^^ . _:b412437188 "3"^^ . _:b412437231 . _:b412437191 "3"^^ . _:b412437157 . . _:b412437190 "3"^^ . _:b412437222 . _:b412437185 "3"^^ . _:b412437277 . _:b412437184 "3"^^ . . . _:b412437187 "3"^^ . _:b412437186 "3"^^ . . _:b412437235 . _:b412437197 "2"^^ . _:b5892426 "This type of BAC array platform has been shown to be as robust as high-density oligonucleotide arrays (Coe et al, 2007; Gunnarsson et al, 2008) and its actual resolution is approximately 100\u2009kb for >98% of the genome (Arriola et al, >>2007<<, 2008; Marchi\u00F2 et al, 2008a). Labelling, hybridisation, washes, image acquisition and data normalisation were carried out as previously described (Arriola et al, 2007, 2008; Huang et al, 2007; Marchi\u00F2 et al, 2008a, 2008b)." . _:b5892480 . _:b412437196 "2"^^ . _:b5892478 "For instance, KIT overexpression in GISTs is driven by activating KIT mutations (Gomes et al, >>2008<<), whereas HER2 overexpression in breast cancer is driven by HER2 gene amplification (Arriola et al, 2008)." . _:b412437220 . _:b412437199 "2"^^ . _:b5892427 "type of BAC array platform has been shown to be as robust as high-density oligonucleotide arrays (Coe et al, 2007; Gunnarsson et al, 2008) and its actual resolution is approximately 100\u2009kb for >98% of the genome (Arriola et al, 2007, >>2008<<; Marchi\u00F2 et al, 2008a). Labelling, hybridisation, washes, image acquisition and data normalisation were carried out as previously described (Arriola et al, 2007, 2008; Huang et al, 2007; Marchi\u00F2 et al, 2008a, 2008b)." . . _:b412437198 "2"^^ . _:b412437193 "2"^^ . . _:b412437177 . . _:b412437192 "2"^^ . _:b5892463 . _:b412437195 "2"^^ . _:b412437246 . _:b412437194 "2"^^ . . _:b5892443 "PDGF and PDGFRs have both been found to be overexpressed in glial tumour cell lines and tumour surgical samples (Nister et al, 1988, 1991; Hermanson et al, 1992; Black et al, 1996; Di Rocco et al, >>1998<<; Varela et al, 2004)." . _:b412437205 "2"^^ . _:b412437204 "2"^^ . . _:b412437207 "2"^^ . . . _:b5892424 . _:b412437206 "2"^^ . _:b412437201 "2"^^ . _:b412437200 "2"^^ . _:b5892411 . . _:b412437200 . . _:b412437203 "2"^^ . _:b5892431 . . _:b412437202 "2"^^ . _:b5892487 "gov/projects/SNP/; Carvalho et al, >>2005<<; Reis et al, 2005; Wen et al, 2006). The impact of these genetic polymorphisms in PDGFRA function remains to be elucidated." . _:b412437213 "2"^^ . _:b5892496 "in 21.1% (12 out of 57) of gliomas, a frequency similar to that described in previous studies (Fleming et al, 1992; Kumabe et al, 1992; Smith et al, 2000; Alonso et al, 2005; Arjona et al, 2005; Puputti et al, 2006; Beroukhim et al, >>2007<<; McLendon et al, 2008). We have further shown by aCGH that the amplicon encompasses a region of 3.6\u2009Mb, which, in addition to PDGFRA, also includes KIT and KDR oncogenes." . _:b412437212 "2"^^ . _:b412437278 . _:b412437143 . _:b5892444 "PDGF and PDGFRs have both been found to be overexpressed in glial tumour cell lines and tumour surgical samples (Nister et al, 1988, 1991; Hermanson et al, 1992; Black et al, 1996; Di Rocco et al, 1998; Varela et al, >>2004<<). PDGFRA and PDGFA have been shown to be expressed in tumour cells, whereas PDGFB and PDGFRB have been found in glioma-associated endothelial cells (Hermansson et al, 1988; Hermanson et al, 1992; Plate et al, 1992). Furthermore, studies" . . _:b5892497 "57) of gliomas, a frequency similar to that described in previous studies (Fleming et al, 1992; Kumabe et al, 1992; Smith et al, 2000; Alonso et al, 2005; Arjona et al, 2005; Puputti et al, 2006; Beroukhim et al, 2007; McLendon et al, >>2008<<). We have further shown by aCGH that the amplicon encompasses a region of 3.6\u2009Mb, which, in addition to PDGFRA, also includes KIT and KDR oncogenes." . _:b412437215 "2"^^ . _:b412437214 "2"^^ . _:b412437209 "2"^^ . _:b412437218 . _:b412437208 "2"^^ . . _:b412437284 . _:b5892438 "The PDGF pathway is one of the most consistently altered cellular signalling system in glial tumourigenesis (Shih and Holland, 2006; Fomchenko and Holland, >>2007<<). PDGF and PDGFRs have both been found to be overexpressed in glial tumour cell lines and tumour surgical samples (Nister et al, 1988, 1991; Hermanson et al, 1992; Black et al, 1996; Di Rocco et al, 1998; Varela et al, 2004)." . . _:b5892476 . _:b412437211 "2"^^ . _:b412437147 . _:b412437210 "2"^^ . . . _:b412437221 "2"^^ . _:b412437220 "2"^^ . . . . _:b5892472 . . _:b412437233 . _:b412437223 "2"^^ . _:b412437222 "2"^^ . _:b412437217 "2"^^ . _:b412437263 . _:b5892494 "analysis revealed PDGFRA amplification in 21.1% (12 out of 57) of gliomas, a frequency similar to that described in previous studies (Fleming et al, 1992; Kumabe et al, 1992; Smith et al, 2000; Alonso et al, 2005; Arjona et al, >>2005<<; Puputti et al, 2006; Beroukhim et al, 2007; McLendon et al, 2008). We have further shown by aCGH that the amplicon encompasses a region of 3.6\u2009Mb, which, in addition to PDGFRA, also includes KIT and KDR oncogenes." . _:b412437216 "2"^^ . _:b5892446 "PDGFRA and PDGFA have been shown to be expressed in tumour cells, whereas PDGFB and PDGFRB have been found in glioma-associated endothelial cells (Hermansson et al, 1988; Hermanson et al, >>1992<<; Plate et al, 1992)." . . _:b412437219 "2"^^ . _:b412437281 . _:b412437185 . . _:b5892451 "or indirectly through creating a favourable microenvironment niche, can contribute to the transformation of neural stem/progenitor cells into glioma tumours (Jackson et al, 2006; Fomchenko and Holland, 2007; Siebzehnrubl et al, >>2008<<)." . _:b412437218 "2"^^ . _:b412437145 . . _:b412437251 . . _:b412437229 "2"^^ . . _:b5892435 "as a smoothed Log2 ratio of between 0.12 and 0.40, corresponding to approximately 3\u20135 copies of the locus, whereas gene amplification was defined as having a Log2 ratio >0.40, corresponding to more than 5 copies (Arriola et al, >>2008<<). These figures were obtained by comparison with interphase FISH data for markers at different chromosomal locations (Reis-Filho et al, 2008)." . _:b412437228 "2"^^ . _:b412437231 "2"^^ . . _:b412437230 "2"^^ . . _:b412437196 . _:b412437225 "2"^^ . _:b412437267 . _:b412437171 . _:b412437258 . . _:b412437224 "2"^^ . _:b5892437 "The PDGF pathway is one of the most consistently altered cellular signalling system in glial tumourigenesis (Shih and Holland, >>2006<<; Fomchenko and Holland, 2007)." . _:b5892460 . _:b5892504 "glioblastomas, it could be hypothesised that autocrine/paracrine loops may be present in these tumours, corroborating the importance of this signalling pathway in gliomas (Nister et al, 1991; Guha et al, 1995; Fomchenko and Holland, >>2007<<). PDGFRA gene amplification may be the underlying genetic mechanism driving PDGFRA overexpression in gliomas." . . _:b412437227 "2"^^ . _:b412437226 "2"^^ . _:b5892454 . _:b5892439 . _:b5892423 "The aCGH platform used for this study was constructed at the Breakthrough Breast Cancer Research Centre and comprises >16\u2009000 BAC clones tiled across the genome (Arriola et al, >>2007<<). This type of BAC array platform has been shown to be as robust as high-density oligonucleotide arrays (Coe et al, 2007; Gunnarsson et al, 2008) and its actual resolution is approximately 100\u2009kb for >98% of the genome (Arriola et al," . . _:b412437237 "2"^^ . . _:b5892453 "Clinical trials evaluating the efficacy of anti-PDGFRA drugs in patients with glioblastomas are ongoing (Reardon et al, 2005; Wen et al, >>2006<<; Desjardins et al, 2007; Newton, 2007; Raymond et al, 2008)." . _:b5892475 "A previous study showed PDGFRA expression in approximately 50% of gliomas and a correlation with poor prognosis in low-grade gliomas (Varela et al, >>2004<<). However, in a report of 40 patients with grade II astrocytomas and oligoastrocytomas, there was an association between high PDGFRA expression and a favourable patient outcome (Ribom et al, 2002). Recently, Liang et al, (2008) in a" . _:b412437236 "2"^^ . _:b412437144 . _:b5892474 "were reported to express this receptor (Nister et al, 1988, 1991; Hermanson et al, 1992; Black et al, 1996; Di Rocco et al, 1998; Ribom et al, 2002; Varela et al, 2004; Liang et al, 2008; Takei et al, 2008; Thorarinsdottir et al, >>2008<<). A previous study showed PDGFRA expression in approximately 50% of gliomas and a correlation with poor prognosis in low-grade gliomas (Varela et al, 2004)." . _:b5892500 "Co-amplification of these three oncogenes has already been detected with other methodologies in gliomas (Joensuu et al, 2005; Puputti et al, 2006; Holtkamp et al, >>2007<<). A statistically significant association between PDGFRA amplification and overexpression was found only in diffuse astrocytomas (grade II). Given that PDGFRA overexpression appears to be an early event in gliomagenesis (Hermanson et al," . _:b412437239 "2"^^ . _:b412437238 "2"^^ . _:b412437233 "2"^^ . . _:b412437154 . _:b412437232 "2"^^ . _:b5892456 . _:b412437235 "2"^^ . _:b5892432 . _:b5892440 "PDGF and PDGFRs have both been found to be overexpressed in glial tumour cell lines and tumour surgical samples (Nister et al, 1988, >>1991<<; Hermanson et al, 1992; Black et al, 1996; Di Rocco et al, 1998; Varela et al, 2004)." . . _:b412437234 "2"^^ . _:b5892501 . _:b5892496 . . _:b412437245 "2"^^ . . . _:b5892433 "Labelling, hybridisation, washes, image acquisition and data normalisation were carried out as previously described (Arriola et al, 2007, 2008; Huang et al, 2007; Marchi\u00F2 et al, 2008a, 2008b). Polymorphic BACs identified in an analysis of 50 male/female and female/female hybridisations were filtered out. This left a final dataset of 13711 clones with unambiguous mapping information according to the March 2006 build (hg18) of" . _:b412437244 "2"^^ . _:b412437249 . . _:b412437247 "2"^^ . _:b412437239 . _:b412437189 . _:b412437246 "2"^^ . _:b412437241 "2"^^ . _:b412437253 . _:b412437273 . _:b412437240 "2"^^ . _:b412437252 . _:b5892469 "These frequencies are in agreement with previous studies, where approximately 50% for malignant astrocytomas were reported to express this receptor (Nister et al, 1988, 1991; Hermanson et al, >>1992<<; Black et al, 1996; Di Rocco et al, 1998; Ribom et al, 2002; Varela et al, 2004; Liang et al, 2008; Takei et al, 2008; Thorarinsdottir et al, 2008)." . _:b412437243 "2"^^ . _:b412437255 . . . _:b412437242 "2"^^ . _:b5892501 "Given that PDGFRA overexpression appears to be an early event in gliomagenesis (Hermanson et al, >>1996<<), our results provide support to the contention that gene amplification may be one of the underlying mechanisms at this stage." . _:b412437254 . _:b5892450 . . . _:b412437253 "2"^^ . _:b412437249 . . . _:b5892465 "Previous studies on PDGFA mRNA expression reported high levels of PDGFA in gliomas (Maxwell et al, 1990; Mapstone, 1991; Hermanson et al, 1992; Di Rocco et al, >>1998<<). It should be noted, however, that there is a paucity of data on prevalence of PDGFA protein expression in primary glioma specimens. We have previously shown that 100% of gliosarcomas express PDGFA (Reis et al, 2005). PDGFRA expression" . _:b412437252 "2"^^ . _:b412437248 . _:b412437156 . _:b412437255 "2"^^ . _:b412437251 . _:b5892487 . . _:b412437254 "2"^^ . _:b412437250 . _:b5892434 "of the normal ratios obtained from the filtered clones mapping to chromosomes 1\u201322, assessed in multiple hybridisations between DNA extracted from a pool of male and female blood donors as previously described (Arriola et al, >>2008<<; Reis-Filho et al, 2008) (Log2 ratio of \u00B10.08)." . . _:b412437249 "2"^^ . _:b412437261 . _:b5892419 . _:b5892479 . _:b5892457 . _:b412437248 "2"^^ . _:b412437260 . _:b412437225 . _:b412437244 . _:b412437251 "2"^^ . _:b412437263 . _:b412437213 . _:b412437250 "2"^^ . _:b412437245 . _:b412437262 . _:b5892429 "Labelling, hybridisation, washes, image acquisition and data normalisation were carried out as previously described (Arriola et al, >>2007<<, 2008; Huang et al, 2007; Marchi\u00F2 et al, 2008a, 2008b)." . . _:b412437261 "2"^^ . . _:b5892425 . _:b5892412 . _:b412437257 . _:b5892445 "PDGFRA and PDGFA have been shown to be expressed in tumour cells, whereas PDGFB and PDGFRB have been found in glioma-associated endothelial cells (Hermansson et al, >>1988<<; Hermanson et al, 1992; Plate et al, 1992)." . _:b412437199 . _:b412437260 "2"^^ . . _:b5892413 . _:b412437256 . . _:b5892466 . _:b412437263 "2"^^ . _:b5892488 "gov/projects/SNP/; Carvalho et al, 2005; Reis et al, >>2005<<; Wen et al, 2006). The impact of these genetic polymorphisms in PDGFRA function remains to be elucidated." . _:b412437259 . _:b5892428 . _:b5892421 . _:b5892414 . . _:b5892489 . _:b5892472 "are in agreement with previous studies, where approximately 50% for malignant astrocytomas were reported to express this receptor (Nister et al, 1988, 1991; Hermanson et al, 1992; Black et al, 1996; Di Rocco et al, 1998; Ribom et al, >>2002<<; Varela et al, 2004; Liang et al, 2008; Takei et al, 2008; Thorarinsdottir et al, 2008)." . _:b412437262 "2"^^ . _:b412437258 . _:b5892467 . _:b5892415 . . _:b412437173 . _:b412437257 "2"^^ . _:b412437269 . _:b5892461 "is associated with overall PDGFR activation; others point to the putative role of PDGFRB or failed to show any association between PDGFR status and response to imatinib (Gross et al, 2006; H\u00E4gerstrand et al, 2006; Servidei et al, >>2006<<)." . _:b412437256 "2"^^ . _:b412437268 . _:b5892484 . _:b412437259 "2"^^ . _:b412437271 . _:b5892410 . _:b412437167 . . _:b412437258 "2"^^ . _:b412437270 . _:b5892411 . _:b412437160 . _:b412437209 . . _:b412437269 "2"^^ . _:b412437265 . _:b5892420 . . _:b5892427 . . . _:b412437268 "2"^^ . _:b412437264 . _:b5892421 . . _:b412437271 "2"^^ . _:b412437267 . _:b5892422 . _:b5892457 "Currently, combination of anti-PDGFRA drugs with chemotherapy agents is being evaluated (Reardon et al, >>2008<<). However, the molecular alterations underlying glioma patients\u2019 response to PDGFRA antagonists are unknown. One study analysed the prevalence of PDGFRA mutations in patients enrolled in a phase I/II study of imatinib mesylate in" . _:b412437270 "2"^^ . . _:b5892423 . _:b412437266 . _:b412437168 . _:b412437237 . _:b412437265 "2"^^ . . _:b5892416 . _:b412437277 . _:b412437247 . _:b5892491 "PDGFRA gene amplification analysis revealed PDGFRA amplification in 21.1% (12 out of 57) of gliomas, a frequency similar to that described in previous studies (Fleming et al, 1992; Kumabe et al, >>1992<<; Smith et al, 2000; Alonso et al, 2005; Arjona et al, 2005; Puputti et al, 2006; Beroukhim et al, 2007; McLendon et al, 2008)." . _:b412437264 "2"^^ . _:b5892428 "has been shown to be as robust as high-density oligonucleotide arrays (Coe et al, 2007; Gunnarsson et al, 2008) and its actual resolution is approximately 100\u2009kb for >98% of the genome (Arriola et al, 2007, 2008; Marchi\u00F2 et al, 2008a). Labelling, hybridisation, washes, image acquisition and data normalisation were carried out as previously described (Arriola et al, 2007, 2008; Huang et al, 2007; Marchi\u00F2 et al, 2008a, 2008b)." . _:b412437276 . _:b5892420 "The in-house probe was generated, biotin-labelled and used in hybridisations as previously described (Lambros et al, 2006; Gomes et al, >>2007<<). Briefly, heat pre-treatment of deparaffinised sections were incubated for 15\u2009min at 98\u2009\u00B0C in CISH pre-treatment buffer (SPOT-light tissue pre-treatment kit, Zymed Laboratories, San Francisco, CA, USA) and digested with pepsin for 6\u2009min" . _:b5892417 . . _:b5892430 . _:b412437267 "2"^^ . _:b412437152 . _:b5892418 . _:b412437279 . _:b412437153 . _:b412437154 . _:b412437155 . _:b5892486 . _:b412437266 "2"^^ . . _:b412437156 . _:b5892419 . _:b412437278 . _:b412437157 . _:b5892416 . _:b412437158 . . _:b412437159 . _:b412437277 "2"^^ . . _:b412437160 . _:b5892439 "PDGF and PDGFRs have both been found to be overexpressed in glial tumour cell lines and tumour surgical samples (Nister et al, >>1988<<, 1991; Hermanson et al, 1992; Black et al, 1996; Di Rocco et al, 1998; Varela et al, 2004)." . _:b5892428 . _:b412437273 . _:b412437161 . _:b412437162 . _:b412437163 . _:b412437276 "2"^^ . _:b412437164 . _:b5892429 . _:b412437272 . _:b412437165 . _:b412437166 . . _:b412437167 . _:b412437279 "2"^^ . _:b412437168 . _:b5892430 . _:b412437275 . _:b412437169 . _:b412437170 . . _:b412437278 "2"^^ . . _:b412437171 . _:b412437176 . _:b5892431 . _:b412437274 . _:b412437172 . _:b412437173 . _:b412437174 . _:b412437175 . _:b412437273 "2"^^ . _:b412437176 . _:b5892424 . _:b412437285 . _:b5892410 "materials and methods" . _:b412437177 . _:b412437178 . _:b412437179 . _:b412437272 "2"^^ . _:b412437180 . _:b5892425 . _:b412437284 . _:b412437181 . _:b412437276 . _:b412437182 . _:b5892462 "Previous studies on PDGFA mRNA expression reported high levels of PDGFA in gliomas (Maxwell et al, >>1990<<; Mapstone, 1991; Hermanson et al, 1992; Di Rocco et al, 1998)." . _:b5892451 . _:b412437183 . _:b412437275 "2"^^ . _:b5892426 . . _:b412437274 "2"^^ . _:b412437241 . _:b5892503 . _:b5892427 . _:b412437286 . _:b5892420 . . _:b412437285 "2"^^ . _:b412437281 . _:b5892436 . . _:b412437175 . _:b412437284 "2"^^ . . _:b5892437 . _:b412437280 . . _:b5892422 . _:b5892440 . . _:b5892438 . _:b412437283 . _:b5892417 . . _:b412437227 . _:b412437286 "2"^^ . _:b412437254 . _:b412437282 . _:b5892439 . _:b412437141 . _:b412437142 . _:b412437143 . _:b412437281 "2"^^ . . _:b412437142 . _:b5892432 . _:b412437144 . _:b412437145 . . _:b412437146 . _:b412437147 . _:b412437280 "2"^^ .