Molecular Diagnosis of Oligodendroglioma in Paraffin Sections

Abstract

SUMMARY:Distinction of oligodendrogliomas from other gliomas is clinically important, but the histologic diagnosis of oligodendroglioma has been a difficult and notoriously subjective task. Testing for loss of heterozygosity (LOH) on chromosomal arms 1p and 19q, the genetic signature of oligodendroglioma, has emerged as a useful, objective adjunct to the traditional histologic evaluation. However, LOH testing of glioma specimens has not yet been widely implemented, presumably because of a lack of a practical LOH assay. We describe a 1p and 19q LOH assay suitable for routine diagnostics. In contrast to traditional microsatellite-based LOH analysis, we show that detection of LOH is usually possible even without normal tissue or blood from the same patient. A small area of tumor on a single paraffin section is sufficient for the assay. The assay protocol consists of a one-step DNA extraction, multiplex PCR for microsatellites on 1p and 19q, and capillary electrophoresis of the PCR products. LOH is detected by analysis of the allelic patterns and by integration of data from multiple highly polymorphic microsatellites. In a validation study on 19 gliomas, the results were concordant with results obtained by established methods and correlated well with histologic diagnoses. Because only a paraffin section is required, the pathologist can perform both the traditional histopathologic evaluation and this supporting molecular assay from the material at hand. (Lab Invest 2003, 83:419 -428).

Multiplex Ligation-Dependent Probe Amplification

“…The most commonly applied techniques, LOH, 10,16 fluorescent in situ hybridization (FISH), 17,18 or quantitative microsatellite analysis, 19,20 specifically analyze the chromosomal region 1p36 to identify OTs with a favorable prognosis and therapy response. The clinical value of such tests has been clearly proven.…”

A Novel Multiplexing, Polymerase Chain Reaction-Based Assay for the Analysis of Chromosome 18q Status in Colorectal Cancer

“…PCR multiplexing allows a faster analysis of different microsatellite markers in a single assay. 17,18 Because reliability of LOH-multiplexed assays has been controversial when using DNA obtained from paraffin-embedded tissues, 19 we performed reproducibility experiments to determine the robustness of this technique, demonstrating its high reliability for LOH anal- GeneScan electropherograms of two different microsatellites using three decreasing DNA concentrations. For each informative marker, the major peak of each allele selected for LOH analysis, consistent with highest one, is pointed by an arrow.…”

Genomic Changes in Gliomas Detected Using Single Nucleotide Polymorphism Array in Formalin-Fixed, Paraffin-Embedded Tissue

“…Corresponding tissue from two to five unstained, 10-m-thick tissue sections was removed using Pinpoint reagents (ZymoResearch, Orange, CA) as previously described. 3,15 Genomic DNA was extracted from the sample with use of a QIAmp DNA Kit (Qiagen, Valencia, CA) and quantified by OD at 260 nm using the NanoDrop ND-1000 (NanoDrop Technologies, Inc., Wilmington, DE).…”

“…3 Briefly, fluorescentlabeled PCR products were detected by capillary electrophoresis with use of the ABI 3130 Genetic Analyzer and GeneMapper software version 4 (Applied Biosystems, Carlsbad, CA). Interpretation was made by the molecular pathologist (C.D.G., J.R.E.)…”

“…3 Both assays are limited in that the probes or primers interrogate only small portions of the chromosomes, and thus small deletions can be missed or misinterpreted as involving the whole arm. Additionally, STR loci are not always informative; that is, some may be germline homozygous.…”