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Molecular and Biochemical Analyses Of Human Cancer Biology Molecular and Biochemical Analyses of Platelet-Derived Growth Factor Receptor (PDGFR) B, PDGFRA, and KIT Receptors in Chordomas ElenaTamborini,1Francesca Miselli,1Tiziana Negri,1M. Stefania Lagonigro,1Samantha Staurengo,1 Gian Paolo Dagrada,1Silvia Stacchiotti,2 Elisa Pastore,1Alessandro Gronchi,2 Federica Perrone,1 Antonino Carbone,1Marco A. Pierotti,3 Paolo G. Casali,2 and Silvana Pilotti1 Abstract Purpose: We have previously shown the presence of an activated platelet-derived growth factor (PDGF) receptor (PDGFR) B and its ligand PDGFB in a limited number of patients with clinical and radiological responses to imatinib mesylate treatment.This article describes the results of comprehensive molecular/biochemical analyses of the three receptors targeted by the drug (PDGFRB, PDGFRA, and KIT) in a series of 31chordoma patients. Experimental Design: The presence and activation status of PDGFRB, PDGFRA, and KIT receptors were investigated by means of immunoprecipitation and Western blot analyses complemented by immunohistochemistry, their expression level was analyzed by means of real-time PCR, and the occurrence of activating point mutations was investigated by means of cDNA sequencing. The PDGFB, PDGFA, and stem cell factor cognate ligands were investigated by reverse transcription-PCR, and gene status was assessed by fluorescence in situ hybridization. Results: The results show that PDGFRB was highly expressed and phosphorylated, whereas PDGFRA and KIT were less expressed but phosphorylated and thus activated. These findings, together with the absence of gain-of-function mutations and the presence of the cognate ligands, strongly support the hypothesis that the activation mechanism is the autocrine/paracrine loop. No role seems to be played by gene amplification. Conclusions: In the light of our findings, the clinical benefit observed in chordoma patients treated with imatinib seems to be attributable to the switching off of all three receptors. Chordomas are rare, low-grade neoplasms originating from most frequent event is the deletion of 1p, the location of the remnants of the fetal nothocord that mainly affect the sacrum, potential cancer susceptibility region of locus 1p36.31-1p36.13. skull base region and, in a minority of cases, the cervical and The results of combined loss of heterozygosity and reverse thoracolumbar vertebrae (1). Three histologic variants with transcription-PCR analyses have suggested that several genes, different morphologies have been described: the conventional or including CASP9, EPH2A, and DVL1, may play an oncosup- classic type, the chondroid type, and the dedifferentiated type. pressing role and be involved in chordoma development (2). Classic cytogenetic studies define these tumors as complex Finally, losses of 1p and 3p and gains of chromosomes 7, 20, sarcomas because they are generally characterized by complex 5q, and 12q have been reported based on comparative genomic karyotypes. The analyses published thus far have found that the hybridization studies (3). In relation to cell cycle–controlling genes, an immunophe- notype-based study has found an alteration in the G1-S checkpoint, principally restricted to p53 overexpression (4). Authors’ Affiliations: 1Experimental Molecular Pathology, Department of Very little is known about receptor tyrosine kinase (RTK) Pathology, 2Department of Medical/Surgical Oncology, and 3Scientific Director, activation, but their RTK expression could offer useful alternative Fondazione IRCCS, Istituto Nazionale dei Tumori, Milan, Italy therapeutic targets because, as it is well known that chordomas Received 6/30/06; revised 9/13/06; accepted 9/21/06. Grant support: Associazione Italiana per la Ricerca sul Cancro 2004 and Italian do not respond to radiotherapy or conventional chemotherapy, Ministry of Health (Ricerca Finalizzata 2004). the standard treatment is surgery. It has in fact been recently The costs of publication of this article were defrayed in part by the payment of page found that Met and epidermal growth factor receptor are highly advertisement charges. This article must therefore be hereby marked in accordance expressed especially in primary chordomas (5). with 18 U.S.C. Section 1734 solely to indicate this fact. Note: E. Tamborini and F. Miselli contributed equally to this work. P.G. Casali and We have previously shown the presence of an activated S. Pilotti are senior coauthors. platelet-derived growth factor (PDGF) receptor (PDGFR) B and Requests for reprints: Silvana Pilotti, Unit of Experimental Molecular Pathology, its PDGFB ligand in some chordomas and reported that an Department of Pathology, Istituto Nazionale per lo Studio e la Cura deiTumori,Via G. albeit limited number of patients treated with imatinib Venezian 1, 20133 Milan, Italy. Phone: 39-02-23902260; Fax: 39-02-23902877; mesylate (Gleevec, Novartis Pharma AG, Basel, Switzerland), E-mail: [email protected]. F 2006 American Association for Cancer Research. an inhibitor of some RTKs, including KIT, PDGFRA, and doi:10.1158/1078-0432.CCR-06-1584 PDGFRB, all showed clinical and radiological response ClinCancerRes2006;12(23)December1,2006 6920 www.aacrjournals.org Downloaded from clincancerres.aacrjournals.org on September 25, 2021. © 2006 American Association for Cancer Research. RTKs Expression and Activation in Chordoma improvements and experienced an improvement in their quality of life (6). Table 1. Clinical features of chordoma patients In this study, we extended our analyses to all of the Case Sex Age Site Sample Material Type receptors targeted by imatinib mesylate in a series of 31 type patients with conventional chordomas by investigating the presence and activation status of PDGFRB, PDGFRA, and KIT 1 M 61 Sacrum Primary Frozen Surgical specimen 2 M 25 Sacrum Primary Frozen Surgical specimen by means of immunoprecipitation and Western blot analyses 3 M 58 Sacrum Primary Frozen Biopsy complemented by immunohistochemistry. Their expression 4F 64Vertebrae Primary Frozen Surgical specimen level was analyzed by real-time PCR, the presence of activating 5 F 65 Vertebrae Primary Frozen Surgical specimen point mutations was investigated by means of cDNA 6 F 66 Sacrum Recurrent Frozen Biopsy PDGFB, PDGFA, 7 M 52 Sacrum Primary Frozen Biopsy sequencing, and the presence of the and 8 M 61 Sacrum Primary Frozen Biopsy stem cell factor (SCF) cognate ligands was investigated by 9 F 73 Sacrum Primary Frozen Surgical specimen means of reverse transcription-PCR. As gene amplification may 10 M 57 Clivus Primary Frozen Biopsy also be one of the mechanisms responsible for receptor 11 F 28 Clivus Primary Frozen Surgical specimen activation, gene status was assessed by means of fluorescence 12 F 60 Vertebrae Primary Frozen Biopsy in situ 13 M 58 Sacrum Primary Frozen Surgical specimen hybridization (FISH). 14M 56 Sacrum Primary Frozen Surgical specimen The results showed that PDGFRB, PDGFRA, and KIT were all 15 M 70 Sacrum Recurrent Frozen Biopsy expressed and phosphorylated even if with different level of 16 M 24Clivus Recurrent Frozen Surgical specimen expression as shown also by quantitative analysis on their 17 M 41 Sacrum Recurrent Frozen Surgical specimen 18 M 49 Sacrum Primary Frozen Surgical specimen mRNAs. Together with the absence of gain-of-function muta- 19 F 47 Sacrum Recurrent Frozen Surgical specimen tions and the presence of the cognate ligands, these findings 20 M 66 Sacrum Primary Frozen Surgical specimen strongly support that the activation mechanism is the auto- 21 M 77 Sacrum Primary Frozen Surgical specimen crine/paracrine loop. Gene amplification did not seem to play 22 M 59 Sacrum Primary Frozen Surgical specimen any role. 23 M 66 Sacrum Primary Frozen Surgical specimen 24F 65 Sacrum Recurrent Frozen Surgical specimen 25 F 60 Sacrum Primary Frozen Surgical specimen 26 M 75 Sacrum Primary Frozen Surgical specimen Materials and Methods 27 F 41 Sacrum Recurrent Frozen Biopsy 28 M 69 Vertebrae Primary Frozen Surgical specimen Patients and materials 29 F 59 Sacrum Primary Paraffin — We studied 31 chordomas affecting 19 males and 12 females with a 30 M 51 Vertebrae Recurrent Paraffin — median age of 59 years (23 primary tumors and 8 recurrences: 3 involving 31 F 66 Sacrum Primary Paraffin — the clivus, 5 the vertebrae, and 23 the sacrum) by analyzing 20 frozen surgical specimens (15 in-house and 5 consultation cases), 8 frozen small biopsy specimens (6 in-house and 2 consultation cases), and 3 RNA extraction, reverse-transcription PCR, sequencing specimens in paraffin-embedded blocks (all consultation cases). All of analysis, and ligand assessment the specimens used for the molecular/biochemical analyses had been Total RNA was extracted from freshly frozen tissue and reverse excised before imatinib treatment. Eleven of the patients had undergone transcribed as described previously (7). The obtained cDNA was standard chemotherapy and/or radiotherapy (Table 1). Written in- amplified using specific primer pairs for PDGFRA, PDGFRB, and c-KIT formed consent was obtained from all the patients. genes as listed in a previous publication (9). The same cDNAs were used All the biochemical and molecular analyses were done on tissue to detect the presence of PDGFA, PDGFB, and SCF ligands as already sections carefully evaluated by the pathologist in terms of presence of reported (9). normal tissues or necrosis. Fluorescence in situ hybridization Biochemical analysis Cytologic specimens and touch imprints. The slides were placed Positive and negative controls.
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