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Molecular Testing For Brain Tumors

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Vol. 19 • Issue 9 • Page 12

The Molecular Edge

The American Cancer Society estimates that over 20,000 new cases of primary malignant brain tumors are diagnosed each year in the U.S.1 The increasing influence of molecular diagnostics (MDx) has provided information about the genetic changes behind gliomas, and a subset of these changes are proving to have utility as diagnostic, prognostic and/or predictive markers.

Glioma Diagnosis

Oligodendrogliomas are a prime example of the relationship between MDx and glioma diagnosis. Fifty to 80% of oligodendrogliomas harbor a loss of chromosome arms 1p and 19q.2 This co-deletion is thought to be a predictor of response to chemotherapy.3 Further research has raised the possibility that the co-deletion may also be a prognostic factor, as survival studies have demonstrated that oligodendrogliomas with the co-deletion grow more slowly than those with intact chromosome arms.4

Standard methodologies for detecting the co-deletion include fluorescence in situ hybridization (FISH) and PCR-based loss of heterozygosity techniques. For FISH, after appropriate preparation of formalin-fixed paraffin embedded (FFPE) tissue, commercially available probes for both 1p and 19q are applied and the ratio of signal from 1p and 19q is compared to signals from their corresponding centromeres. An equal ratio (e.g., 1:1, 2:2, 4:4) indicates no imbalance while a ratio less than 1 indicates loss of the chromosome arm.

Loss of heterozygosity (LOH) can be determined using polymorphic microsatellites from tumor and normal DNA using a PCR-based technique.5 Tumor and normal DNA is extracted from a patient's FFPE tissue and numerous microsatellites on 1p and 19q amplified via PCR and sequenced. Modern multiplexing techniques allow numerous markers to be amplified together. The gain or loss of alleles is determined by comparing the peak area of each allele for the tumor with that of the normal tissue and determining whether LOH is present.

New Marker

More recently, investigators have discovered a novel prognostic marker, O-6-methylguanine methyltransferase (MGMT), in the most frequent glioma, glioblastoma multiforme (GBM). GBM is a high-grade glioma characterized by enhancement on radiology studies and characteristic histologic findings, including microvascular proliferation and "pseudopalisading" necrosis. The outcome for patients with GBM is dire, with most dying within one to two years of the initial diagnosis.6 The MGMT protein functions by removing alkylating groups introduced by chemotherapy agents. When MGMT activity is down-regulated by methylation of its promoter, its DNA repair activity is decreased, which increases a cell's sensitivity to chemotherapy.

In 2000, research demonstrated that the MGMT methylation was associated with response to chemotherapy in gliomas as well as prolonged overall and disease-free survival.7 Similar findings were soon demonstrated between MGMT methylation status and GBM responsiveness to temozolomide.8

Methylation-specific PCR (MSPCR) takes advantage of the fact that methylation occurs at CpG dinucleotides, with the methyl group added to the cytosine base. Following extraction from tumor tissue, DNA is treated with sodium bisulfite that converts unmethylated cytosines to uracil, leaving methylated cytosines intact. Specific primer pairs can then be used that identify and amplify only the methylated or unmethylated DNA.

Some employ restriction endonucleases with methylation-sensitive enzymes.9 There have been questions as to whether MSPCR represents the best methodology for determining MGMT expression status, as results do not appear to correlate well with MGMT protein expression status.10 Commercial pyrosequencing kits have become available that target select sites within the MGMT promoter, and initial studies have demonstrated agreement between MGMT methylation and protein expression.11

The most recent discoveries have come from large-scale genomic studies sequencing the genomes of brain tumors. The sequencing of over 20,000 genes in 22 glioblastomas demonstrated that over 10% of the cases demonstrated mutations in the IDH1 gene that catalyzes the carboxylation of isocitrate to -ketoglutarate.12 Each tumor demonstrated a mutation at the R132 residue, the majority being G->A changes creating an Arg->His substitution.13 Alterations are also seen in gliomas in IDH1's companion gene, IDH2, all at site R172, which is the analog of the R132 residue in IDH1.14 Further work demonstrated that IDH1 mutations are frequent in secondary glioblastomas and would appear to portend a better diagnosis.14, 15

Dr. Trembath is assistant professor, Department of Pathology and Laboratory Medicine, The University of North Carolina Hospitals, Women and Children's Hospital, Chapel Hill.

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