Real-Time Quantitative PCR Analysis of Gene Dosages Reveals Gene Amplification in Low-Grade Oligodendrogliomas

M. Eva Alonso, PhD; M. Josefa Bello, PhD; Dolores Arjona, PhD; Victor Martinez-Glez, MD; Jose M. de Campos, MD, PhD; Alberto Isla, MD, PhD; M. Elena Kusak, MD; FAs Vaquero, MD, PhD; Manuel Gutierrez, MD, PhD; Jose L. Sarasa, MD, PhD; Juan A. Rey, PhD

Disclosures

Am J Clin Pathol. 2005;123(6):900-906. 

In This Article

Abstract and Introduction

Proto-oncogene amplification is an important alteration that is present in about 45% to 50% of high-grade human gliomas. We studied this mechanism in 8 genes (cyclin-dependent kinase-4 [ CDK4 ], MDM2 , MDM4 , renin-angiotensin system-1, ELF3 , GAC1 , human epidermal growth factor receptor-2, and platelet-derived growth factor receptor-A gene) in a series of 40 oligodendrogliomas (World Health Organization (WHO) grade II, 21; WHO grade III, 13; and WHO grade II-III oligoastrocytomas, 6) using real-time quantitative polymerase chain reaction. Amplification of at least 1 of these genes was detected in 58% of samples (23/40). By histopathologic grade, 67% of grade II oligodendrogliomas (14/21), 46% of grade III anaplastic oligodendrogliomas (6/13), and 50% of mixed oligoastrocytomas (3/6) were positive for amplification of at least 1 gene. CDK4 , MDM2 , and GAC1 were the most frequently involved genes (12/40 [30%], 12/40 [30%], and 13/40 [33%], respectively). Our findings demonstrate gene amplification in low-grade samples indicating that it is an important alteration in the early steps of oligodendroglioma development and, therefore, might be considered a molecular mechanism leading to malignant progression toward anaplastic forms.

Oligodendrogliomas are primary tumors of the central nervous system that occur predominantly in the cerebral hemispheres and are more frequent in the fifth and sixth decades of life.[1] They account for 5% to 18% of all gliomas and include pure oligodendrogliomas, corresponding primarily to World Health Organization (WHO) grades II and III (anaplastic) of malignancy, and mixed oligoastrocytomas.[1] Chromosomal, genetic, and epigenetic aberrations in oligodendrogliomas involved with their development and progression have been identified and include allelic losses at 1p and 19q,[2,3] gene mutations of PTEN and p16ink4A ,[4,5] and aberrant promoter methylation of certain tumor-related genes.[6,7] Oncogene amplification is involved in the development of many solid tumors in humans and is a reflection of the genetic instability of tumor cells; proto-oncogene amplification, therefore, has been associated with tumor stage and progression in human gliomas.[8] This genetic alteration induces amplification of different tumor-related genes and leads to altered expression of proteins that have important roles in the regulation of cell proliferation.[8]

Some genes with potential oncogenic features located at 12q13-14 are amplified and their proteins overexpressed in malignant gliomas.[9] These genes are CDK4 (cyclin-dependent kinase-4 involved in the cell cycle regulation), SAS (associated with growth alterations and frequently coamplified with CDK4 ), and MDM2 (oncogene regulator of TP53 ).[9,10] Because individual malignant gliomas show CDK4/SAS amplification but no MDM2 amplification or vice versa, the possibility exists of a common amplification target gene located between CDK4 and MDM2. These amplification data between CDK4/SAS and MDM2 provide additional evidence that these genes would represent 2 independent targets for amplification in high-grade gliomas.[9,10,11]

MDM4, also known as MDMX, located at 1q32, encodes for a p53-binding protein that shares structural similarities with Mdm2 and has been proposed as a negative regulator of p53 function.[12] Other genes at 1q32 that can be coamplified with MDM4 in high-grade gliomas are GAC1 (also overexpressed in these brain tumors), REN1 (renin-angiotensin system gene implicated in tumoral cell supply), and ELF3 (a transcription factor with a role in cellular differentiation).[13] The epidermal growth factor receptor ( EGFR ) gene frequently is amplified in oligodendrogliomas and, together with the other 3 members of this gene family, has an important role in the development of other tumor types.[14] For example, amplification of human epidermal growth factor receptor-2 ( HER-2 ) is one of the first consistent genetic alterations found in breast cancer, but few studies have so far been performed on HER-2 amplification in gliomas.[15] Another important growth factor is the platelet-derived growth factor (PDGF); its signaling activation system has been involved in the development and malignant progression of gliomas.[16] Overexpression of PDGF system components, particularly the α subtype receptor (PDGFRA), is common in glial tumors, primarily those with oligodendroglial differentiation and highly anaplastic features.[17]

All of these findings suggest that overexpression of the aforementioned genes might be related to the gene amplification process. Because data are scarce on this subject, we studied a series of 40 oligodendrogliomas by quantitative polymerase chain reaction (PCR) of 8 genes located at 12q13-14 ( CDK4 and MDM2 ), 1q32 ( MDM4, GAC1, REN1, and ELF3 ), 17q21 ( HER-2 ), and 4q11 ( PDGFRA ) to determine the role that their amplification might have in the development of oligodendroglioma.

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