Characterization of gene expression profiles associated with glioma progression using oligonucleotide-based microarray analysis and real-time reverse transcription-polymerase chain reaction

Abstract

Diffuse astrocytoma of World Health Organization (WHO) grade II has an inherent tendency to spontaneously progress to anaplastic astrocytoma (WHO grade III) and/or glioblastoma (WHO grade IV). The molecular basis of astrocytoma progression is still poorly understood, in particular with respect to the progression-associated changes at the mRNA level. Therefore, we compared the transcriptional profile of approximately 6800 genes in primary WHO grade II gliomas and corresponding recurrent high-grade (WHO grade III or IV) gliomas from eight patients using oligonucleotide-based microarray analysis. We identified 66 genes whose mRNA levels differed significantly (P < 0.01, > or =2-fold change) between the primary and recurrent tumors. The microarray data were corroborated by real-time reverse transcription-polymerase chain reaction analysis of 12 selected genes, including 7 genes with increased expression and 5 genes with reduced expression on progression. In addition, the expression of these 12 genes was determined in an independent series of 43 astrocytic gliomas (9 diffuse astrocytomas, 10 anaplastic astrocytomas, 17 primary, and 7 secondary glioblastomas). These analyses confirmed that the transcript levels of nine of the selected genes (COL4A2, FOXM1, MGP, TOP2A, CENPF, IGFBP4, VEGFA, ADD3, and CAMK2G) differed significantly in WHO grade II astrocytomas as compared to anaplastic astrocytomas and/or glioblastomas. Thus, we identified and validated a set of interesting candidate genes whose differential expression likely plays a role in astrocytoma progression.

Figure 1.

a and b: Histological features of the primary and recurrent glioma of patient 2 on hematoxylin-eosin stained sections. a: The primary tumor (A128D) was a gemistocytic astrocytoma (WHO grade II). b: The recurrent tumor (GB119D) displayed increased cellularity, high mitotic activity, microvascular proliferation, and pseudopalisading necroses (nec). This tumor was classified as glioblastoma (WHO grade IV). c to e: Examples of molecular genetic results obtained for these tumors (lane 1, A128D; lane 2, GB119D; lane 3, leukocyte DNA from patient 2). c: SSCP analysis of TP53 showed identical aberrant band patterns in A128D and GB119D. DNA sequencing revealed the same somatic TP53 mutation (g.14070C>T; R248W) in both tumors. Shown are the mutant sequence in A128D and the wild-type sequence in the corresponding leukocyte DNA. d: Microsatellite analysis of polymorphic loci from 9p (D9S162 and D9S157) revealed LOH only in GB119D but not in A128D. LOH on 10q was detectable in both tumors, as indicated for the loci D10S541 and D10S212. However, the fraction of tumor cells with LOH on 10q appeared to be higher in GB119D than in A128D (arrowheads point to the alleles lost in the tumor DNA). e: Duplex PCR analysis demonstrated homozygous deletion of CDKN2A, p14^ARF and CDKN2B in GB119D but not in A128D. The sizes of the respective PCR fragments amplified from each of these genes and the reference gene APRT are indicated on the right side.

Figure 2.

Results of principal components and cluster analyses of the microarray expression data obtained for the 8 pairs of primary and recurrent gliomas. a: 1400 probe sets with mean >200 and coefficient of variation (SD/mean) >0.5 were standardized by subtracting the mean and dividing by the SD for each probe-set. The first two principal components are plotted. b: Dendrogram from average clustering using the same 1400 probe sets, transformed by taking the logs of ratios to the mean. Each tumor sample is labeled with the corresponding patient number supplemented by P (primary tumor) or R (recurrent tumor). In b, each tumor is additionally labeled by its tumor number and histological diagnosis (in brackets). The abbreviations used are: AII, diffuse astrocytoma WHO grade II; AAIII, anaplastic astrocytoma WHO grade III; sGBIV, secondary glioblastoma WHO grade IV; OAII, oligoastrocytoma WHO grade II; AOAIII, anaplastic oligoastrocytoma WHO grade III.

Figure 3.

The 70 probe sets (corresponding to 66 genes) that gave a P value of <0.01 for paired t-test comparing the 8 pairs of primary and recurrent tumors and that also had greater than two-fold differences between the means for primary and recurrent tumors. The heat map was made using Treeview. The individual case numbers and diagnoses are given on top of the heat map (the abbreviations correspond to those in Figure 2 ▶ ).