Detection of 1p19q deletion by real-time comparative quantitative PCR

Abstract

1p/19q (1p and/or 19q) deletions are prognostic factors in oligodendroglial tumors (OT) and predict better survival after both chemotherapy and radiotherapy. While studying 1p/19q status as a potential variable within multivariate prognosis models for OT, we have frequently encountered unknown 1p/19q status within our glioma sample database due to lack of paired blood samples for loss of heterozygosity (LOH) assay and/or failure to perform fluorescence in situ hybridization (FISH). We realized that a 1p and 19q deletion assay that could be reliably performed solely on tumor DNA samples would allow us to fill in these molecular biology data "holes". We built recombinant DNA with fragments of the selected "marker" genes in 1p (E2F2, NOTCH2), and 19q (PLAUR) and "reference" genes (ERC2, SPOCK1, and SPAG16 ) and used it as quantification standard in real-time PCR to gain absolute ratios of marker/reference gene copy numbers in tumor DNA samples, thus called comparative quantitative PCR (CQ-PCR). Using CQ-PCR, we identified 1p and/ or 19q deletions in majority of pure low-grade oligodenroglioma (OG) tumors (17/21, 81%), a large portion of anaplastic oligodendroglioma (AO) tumors (6/15, 47%), but rarely found in mixed oligoastrcytomas (OA) tumors (1/8, 13%). These data are consistent with results of LOH and FISH assays generally reported for these tumor types. In addition, 15 out 18 samples showed concordant results between FISH and CQ-PCR. We conclude that CQ-PCR is a potential means to gain 1p/19q deletion information, which prognostic and predictive values of CQ-PCR-derived 1p/19q status will be determined in a future study.

Keywords: 1p19q deletion; glioblastoma multiforme; glioma; oligodendroglial tumors; real-time comparative quantitative PCR.

Figure 1

Real-time CQ-PCR detection of gender information based on human DNA samples. Bar and line height are mean (SD) ratios of DNA copy numbers of ERC2 gene in 3p14.3 (open bar) and NLGN4Y gene in Yq11.221 (filled bar) to three other genes in 8q22.2 (PGCP), 2q34 (SPAG16), 5q31.2 (SPOCK1), in a male and a female blood DNA.

Figure 2

Comparison of gene copy number variation between paired DNA samples from patient’s blood and tumor. Bar and line height are mean (SD) ratios of DNA copy numbers of E2F2, NOTCH2, and PLAUR to two reference genes (ERC2 and SPOCK1), quantified by real-time CQ-PCR.