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Despite intensive effort, the target genes on 1p and 19q have not been identified. However, molecular testing for 1p/19q deletion can be performed by loss of heterozygosity (LOH) analysis (currently by polymerase chain reaction [PCR] analysis of microsatellite loci or by high-throughput analysis of single nucleotide polymorphisms) and by fluorescence in situ hybridization (FISH) with locus-specific bacterial artificial chromosome (BAC) probes. FISH can be performed on two paraffin-embedded sections and does not require microdissection, which is an advantage over the other non-in situ methods. An advantage of FISH is that 1p/19q deletion can be focal,29 and normal cells are frequently present in gliomas. FISH can directly evaluate lesions and tumor cells of interest. An advantage of LOH analysis is that it can detect mitotic recombination (which is not detected by FISH). However, this LOH mechanism has been shown to be rare in gliomas.22

FISH detection of 1p and 19q deletions has been described.22,25 Figure 27-2 illustrates typical 1p/19q FISH results for two anaplastic oligodendrogliomas, one with and one without 1p/19q deletion. Approximately 60% of the nuclei in the tumor with 1p and 19q deletion contain one 1p36 or 19q13.3 probe signal and two 1q24 or 19p13 control probe signals (Figure 27-2a). The remaining cells with two signals for each probe are likely contaminating normal glial, neuronal, or endothelial cells or tumor cells that lack deletion. The mean 1p/1q and 19q/19p signal ratios per nucleus are 0.62 and 0.71, respectively. Normal value studies and an evaluation of a large series of tumors by multiple methods indicate that ratios less than 0.80 are associated with 1p or 19q deletion. By comparison, approximately 60% of the nuclei in the tumor without 1p and 19q deletion contain two signals for all four probes (Figure 27-2b). The mean 1p/1q and 19q/19p signal ratios per nucleus are 1.04 and 1.01, respectively. The nuclei with one signal for each probe are likely a result of truncation of signals by paraffin sectioning. The nuclei with three or more signals are likely a result of nuclear overlap.

When used with appropriate control probes (e.g., the BAC probes on 1q42 and 19p13), FISH is able to detect 1p and 19q deletions in polyploid or aneuploid tumors. In addition, trisomy 19 is detected by this FISH method, an alteration long known to be associated with high-grade astrocytic tumors.30 Based on normal value studies, we do not report chromosome 1 or 19 aneusomy until the proportion of nuclei with three or more signals for each probe exceeds 30% of the nuclei evaluated.

Quantitative analysis of microsatellite alleles (QuMA) also has been shown to detect 1p and 19q deletions (and trisomy 19).31 Both QuMA and current LOH analysis methods have the capacity for automation and rapid throughput. However, both require microdissection for accurate deletion detection, and this process increases turnaround time.

For similar reasons, FISH is the optimal method for the detection of chromosome 10 loss and EGFR amplification. In a careful method-comparison study, semiquantitative PCR analysis and Southern blot analysis did not detect small foci of EGFR amplification compared to FISH.8 The most accurate means to detect the EGFRvIII variant in gliomas is by immunohistochemistry with specific anti-bodies.32 The other EGFR variants can be detected by DNA sequencing of tumor specimens.

TP53 and PTEN mutation detection is best performed by high-throughput DNA sequencing/alteration methods (see chapter 2). The Mayo Clinic/NCCTG study included full sequencing of the PTEN gene and exons 5 to 8 of the TP53 gene.8

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