Detection of 1p and 19q loss in oligodendroglioma by quantitative microsatellite analysis, a real-time quantitative polymerase chain reaction assay

Abstract

The combined loss of chromosomes 1p and 19q has recently emerged as a genetic predictor of chemosensitivity in anaplastic oligodendrogliomas. Here, we describe a strategy that uses a novel method of real-time quantitative polymerase chain reaction, quantitative microsatellite analysis (QuMA), for the molecular analysis of 1p and 19q loss in oligodendrogliomas and oligoastrocytomas in archival routinely processed paraffin material. QuMA is performed on the ABI 7700 and based on amplifications of microsatellite loci that contain (CA)n repeats where the repeat itself is the target for hybridization by the fluorescently labeled probe. This single probe can therefore be used to determine copy number of microsatellite loci spread throughout the human genome. In genomic DNA prepared from paraffin-embedded brain tumor specimens, QuMA detected combined loss of 1p and 19q in 64% (21 of 32) of oligodendrogliomas and 67% (6 of 9) of oligoastrocytomas. We validate the use of QuMA as a reliable method to detect copy number by showing concordance between QuMA and fluorescence in situ hybridization at 37 of 45 chromosomal arms tested. These results indicate that QuMA is an accurate, high-throughput assay for the detection of copy number at multiple loci; as many as 31 loci of an individual tumor can be analyzed on a 96-well plate in a single 2-hour run. In addition, it has advantages over standard allelic imbalance/loss of heterozygosity assays in that all loci are potentially informative, paired normal tissue is not required, and gain can be distinguished from loss. QuMA may therefore be a powerful molecular tool to expedite the genotypic analysis of human gliomas in a clinical setting for diagnostic/prognostic purposes.

Figure 1.

TaqMan chemistry and strategy for QuMA. A: Fluorescence is detected when the 5′-label of the probe, or reporter (R), is liberated from the 3′-quenching signal (Q) through the exonucleolytic activity of Taq polymerase. The probe for QuMA hybridizes to the CA repeat of microsatellite sequences. B: Schematic illustration of QuMA experiment comparing tumor DNA with loss (short-dashed lines) to normal DNA (long-dashed lines). The curves for the reference locus for two DNAs are superimposed and for simplicity are shown as a single curve (solid line). Comparison of each microsatellite curve with its appropriate reference is represented as the δCt, where δCt = Ct (microsatellite) − Ct (reference), and Ct is cycle number at threshold. The relative copy number (tumor to normal DNA) is determined from the δδCt, where δδCt = δCt (tumor) − δCt (normal). The copy number in the tumor DNA is equal to 2 × 2^−δδCt.

Figure 2.

PCR efficiency of paraffin-embedded DNA. A: DNA from normal paraffin-embedded tissue was serially diluted starting at 50 ng, and a microsatellite locus from chromosome 1 was amplified. The diluted DNAs reach threshold as predicted from the fundamental equation for PCR, X_n = X₀(1+E)ⁿ, or X_n = X₀2ⁿ when efficiency (E) is 100%. For example, the cycle difference at threshold for the 20 ng and 5 ng quantities is 2 reflecting the fourfold difference in starting DNA amounts. B: Plot of the log of input DNA quantities versus Ct. The equation for this line is derived from X_n = X₀(1+E)ⁿ when n = Ct, cycle at threshold fluorescence. The efficiency of PCR can be calculated from the slope of the line, E = 10^−1/slope − 1. Because the PCR efficiency is near 100% for all of our primer sets, the fundamental equation for PCR [X_n = X₀(1+E)ⁿ] can be assumed to be X_n = X₀2ⁿ. In the experiment shown, the data points have a correlation of 0.9996 with E = 96% demonstrating that amplification of paraffin-embedded DNA over this range of input DNA closely adheres to the fundamental equation for PCR.

Figure 3.

Copy number of loci in tumors as determined by QuMA. Forty-one cases of oligodendroglioma or oligoastrocytoma have been ordered by the extent of 1p loss beginning with tumors where all five 1p loci have been deleted. Rows represent individual cases, and copy numbers for genomic loci (position indicated in cM) have been arranged in columns. Pathological diagnosis is designated as: O, oligodendroglioma; AO, anaplastic oligodendroglioma; OA, oligoastrocytoma; AOA, anaplastic oligoastrocytoma. Gain or loss is assigned to a copy number by using a tolerance interval as previously described. For these data, a calculated copy number <1.58 was scored as a loss, whereas >2.53 was scored as a gain. Calculated numbers are as indicated in the boxes, and dark-gray boxes represent losses, white boxes normal, and light gray boxes indicate gains. Boxes without numbers represent loci not determined. Locations of the FISH probes relative to 1p and 19q markers are also indicated at the top of the diagram.

Figure 4.

Histology and molecular results from FISH and QuMA for oligodendroglioma. A: Histology of a typical oligodendroglioma (case 8758) showing round nuclei, perinuclear halos, and chicken-wire vasculature. B: FISH showing loss of 1p36 (red signal) relative to 1q24 (green signal). C: Delay of one cycle in a tumor DNA relative to a normal DNA at D1S468. PCR is performed in triplicate for both the microsatellite and reference loci for each DNA so that each curve actually represents three curves. Because the reference curves (six curves total) for the normal and tumor DNA reach threshold at precisely the same cycle, this experiment directly illustrates 1p loss through the delay of one cycle at the threshold in a tumor DNA relative to a normal diploid DNA.

Figure 5.

QuMA and FISH results compared. FISH was performed on a subset of specimens from Figure 3 ▶ for comparison to QuMA results. Paraffin-embedded tumor sections were hybridized with probes for regions of loss on 1p and 19q. Hybridizations also included control probes from opposing arms, 1q and 19p, respectively, to create a ratio of total signals counted in 300 to 500 nuclei. Results are tabulated as nml for retention (two copies), del for loss (one copy), and gain for more than two copies. Chromosomal arms that were discordant between the two methods are shaded in the QuMA results.

Figure 6.

Correlation of 1p/19q status with histology. Photomicrographs (original magnification, ×400) of H&E-stained paraffin sections from three oligodendrogliomas (A–C) and a single oligoastrocytoma (D and E) are shown. A: Case 5457: round nuclei and perinuclear halos highlight this classic oligodendroglioma with 1p/19q loss. B: Case 2470: an anaplastic oligodendroglioma that does not display classic oligodendroglial morphology, but showing 1p/19q loss. C: Case 16223: glioma with some features of oligodendroglioma (round, regular nuclei) where 1p/19q has remained intact. D and E: Case 2286: oligoastrocytoma with 1p/19q loss that exhibits distinct areas of oligodendroglial (D) and astrocytic (E) morphology.

Figure 7.

1p loss detected by QuMA maps to a common region of deletion previously characterized in gliomas. QuMA detected partial deletions (loss of more than one consecutive marker) in four oligodendrogliomas (8503, 307, 7876, 10896). All of these tumors have lost the marker D1S214. The FISH probe that is centromeric to D1S468 was retained in tumor 8503 limiting the overlapping region of loss from the probe region to the marker D1S2736. Tumor 14670 with an isolated loss at D1S468 was included to illustrate that retention of the FISH probe in both 14670 and 8503 raises the possibility that deletions in these tumors both end within the region complementary to the probe.