The next generation of glioma biomarkers: MGMT methylation, BRAF fusions and IDH1 mutations

Abstract

For some, glioma biomarkers have been expected to solve common diagnostic problems in routine neuropathology service caused by insufficient material, technical shortcomings or lack of experience. Further, biomarkers should predict patient outcome and direct optimal therapy for the individual patient. Unfortunately, current biomarkers still fall somewhat short of these grand expectations. While there has been some progress, it has generally been slow and in small steps. In this review, the newest set of glioma biomarkers: O(6) -methylguanine-DNA methyltransferase (MGMT) methylation, BRAF fusion and IDH1 mutation are discussed. MGMT methylation is well established as a prognostic/predictive marker for glioblastoma; however, technical questions regarding testing remain, it is not currently utilized widely in guiding patient management, and it has proven to be of no assistance in diagnostics. In contrast, BRAF fusion and IDH1 mutation analyses promise to be very helpful for classifying and grading gliomas, while their potential predictive value has yet to be established.

Figure 1

Genomic area covering the promoter, CpG island and exon 1 of O⁶‐methylguanine‐DNA methyltransferase (MGMT). Genomic data based on University of California Santa Cruz (UCSC) Genome Browser February 2009 assembly (http://genome.ucsc.edu/), chromosome 10 genomic contig GL000100.1 (GenBank)—a web‐based service offered by the NIH, MGMT sequence: NM_002412.3 (GenBank), CpG island predicted by UCSC Genome Bioinformatics position chr10: 131 264 949–131 265 710. Whole promoter DNA sequence according to (35). Minimal promoter region, enhancer region, upstream highly methylated region and downstream highly methylated region according to (65). CpG sites with best correlation between methylation status and MGMT expression according to (25). Methylation‐specific PCR (MS‐PCR) primers according to 23, 39, 99. Pyrosequencing region according to (62). HhaI cleaving sites used for methylation‐specific multiplex ligation dependent probe amplification (MS‐MLPA) according to (47). Real‐time‐MS‐PC (RT‐MS‐PCR) region according to (92).

Figure 2

Bar plot showing different results for MGMT promoter hypermethylation analysis by pyrosequencing according to (62) . T: amount of thymidine in %. C: amount of cytosine in %. CpG 1—5: the different CpG sites. Control: a cytosine not followed by guanine that always becomes converted to 100% uracil/thymidine when DNA bisulfite treatment was successful. A. Nonambiguous pattern indicating methylation of all five CpG sites between 17% and 53%. B. Nonambiguous pattern indicating no methylation of all five CpG sites. C. Methylation of only CpG site no. 2 with 26%.

Figure 4

Detection of KIAA1549/BRAF fusion by 5′ rapid amplification of cDNA ends (RACE) analysis. Sequence traces consequently demonstrating a fusion between KIAA1549 exon 15: BRAF exon 11, KIAA1549 exon 16: BRAF exon 9 and KIAA1549 exon 15: BRAF exon 9.

Figure 3

Detection of KIAA1549/BRAF fusion by fluorescence in situ hybridization (FISH) analysis. Nucleus of pilocytic astrocytoma carrying tandem duplication of BRAF (red signals) and KIAA1549 (green signals) resulting in a fusion of these loci (yellow signal).

Figure 5

Binding of antibody H09 to tumor cells in infiltrating edge of oligodendroglioma.