9p21 locus analysis in high‐risk gastrointestinal stromal tumors characterized for c‐kit and platelet‐derived growth factor receptor α gene alterations

Abstract

BACKGROUNDGastrointestinal stromal tumors (GISTs) are noncomplex sarcomas that often are due to c‐kit‐activating and platelet‐derived growth factor receptor α gene (PDGFRα)‐activating mutations and perturbations of their related signaling pathways. Molecular and cytogenetic findings have indicated correlations between tumor progression and high‐risk GISTs with c‐kit mutations, the overexpression of genes such as ezrin, and losses at 9p. In particular, it was reported recently that malignant GISTs showed alterations in the p16INK4a gene located at the 9p21 locus.METHODSTo assess the involvement of p14ARF and p15INK4b in addition to p16INK4a in GISTs, the authors undertook a molecular and cytogenetic study of the 9p21 locus. A series of 22 pre‐Gleevec era, cryopreserved, high‐risk GISTs that were characterized well in terms of KIT and PDGFRα receptors were investigated for mRNA expression, homozygous deletions, mutations, and promoter methylation of locus 9p21, in some instances complemented by fluorescent in situ hybridization studies.RESULTSThe results indicated the loss of p16INK4a mRNA expression in 41% of the GISTs, mainly due to the homozygous deletion of both the p16INK4a gene and the p14ARF gene (24%). No mutations were found, and promoter methylation (detected by means of methylation‐specific polymerase chain reaction analysis in 27% of tumors) was restricted mainly to the p15INK4b gene (20%). It is noteworthy that, in all of the methylated GISTs, the epigenetic promoter alteration was coupled with mRNA expression.CONCLUSIONSAlterations in the 9p21 locus were found cumulatively in 54% of the tumors in the current series and were represented mainly by the loss of tumor suppressor gene expression. The p16INK4a deletion, which always was coupled with p14ARF gene loss, seemed to be the most common 9p21 inactivation mechanism. Cancer 2005. © 2005 American Cancer Society.

Gastrointestinal stromal tumors: what do we know now?

“…97 A significant fraction of malignant GISTs show inactivation of the tumor suppressor gene CDKN2A (which encodes the cell cycle regulatory protein p16 INK4A ) through chromosome 9p21 deletion, either biallelic or in combination with mutation or promoter methylation. [98][99][100][101] TP53 mutations and decreased p53 immunostaining also correlate with a poor prognosis. [102][103][104] Likewise, amplifications of MDM2 and CCND1 (cyclin D1), although uncommon in GISTs, are associated with malignancy.…”

Imatinib response in two GIST patients carrying two hitherto functionally uncharacterized PDGFRA mutations: An imaging, biochemical and molecular modeling study

“…The immunohistochemical analyses were made using 2-lm, formalin-fixed and paraffin-embedded tumoral sections of the surgical specimens and biopsy, using antibodies against CD117, PDGFRA, Ki-67 and desmin, as described previously, 5,6 and DOG1 1:100 diluted (Novocastra, Newcastle upon Tyne, United Kingdom).…”

“…Stem cell factor (SCF) detection by RT-PCR was performed as already described. 5 Biochemical analysis. Biochemical analysis for KIT and PDGFRA expression and activation were performed as described elsewhere.…”

Evidence for activation of KIT, PDGFRα, and PDGFRβ receptors in the Ewing sarcoma family of tumors

“…10 Human SCF, PDGFA and PDGFB were amplified as described elsewhere. 11 All receptor sequence reactions were carried out using an automated sequencing system (377 DNA sequencer, ABI PRISM PE; Applied Biosystems, Foster City, CA) according to standard protocols.…”

“…11 Briefly, the samples were immunoperoxidase-phenotyped using antibodies against PDGFRa (sc-338, Santa Cruz Biotechnology, Santa Cruz, CA; diluted 1:200), PDGFRb (sc-339 Santa Cruz Biotechnology; diluted 1:100), and CD117 (A4502, Dako, Carpinteria CA; diluted 1:50 epitope 963-976) with heatinduced epitope retrieval. The slides were developed using 3,3 0 diaminobenzidine.…”