Our CQ-PCR detected 1p and/or 19q deletions in majority of classical oligodendroglioma samples (81% of OG and 47% of AO), but in minority of OA (13%). These results data consistent with those generally reported for LOH and FISH assays studying these tumor types.3,4,23
In addition, 15 out 18 samples showed concordant results between FISH and CQ-PCR. Considering the small tumor sample size and limited marker loci used in our study, such a high concordance is remarkable, and suggests that our CQ-PCR has the potential to be used to define 1p/19q deletion status independent of FISH and LOH analysis. CQ-PCR does not require paired blood samples, strong reliance on technical considerations such as technician consistency and experience, and is much less costly than either LOH or FISH. Thus we could use this technology to effectively screen all available tumor DNA samples to determine 1p/19q deletion status which will aid in building multivariate prognosis models for OT. The prognostic and predictive values of CQ-PCR-derived 1p/19q status will be determined based on a larger sample size with a large enough event number.
Our CQ-PCR finding of no 1p deletions in GBMs is consistent with results reported with LOH and FISH. However, we did detect PLAUR deletion in19q in 57% of the GBM samples, which has not been previously recognized. In contrast to GBM samples, none of the 8 OA samples had deletion of PLAUR. The identification of gain at 1p marker genes CAMTA1, E2F2 and NOTCH2 and 19 q marker gene PLAUR was not the focus of this study; however, in many OT and GBM samples, gains were detected by CQ-PCR that were not evident by FISH. Their biological implications and prognostic/predictive values on survival and treatment response are yet to be determined.
There have being prior explorations on obtaining 1p/19q status based solely on tumor DNA. High throughput genomic DNA microarray-based technology known as comparative genomic hybridization is able to detect 1p/19q deletions in gliomas, in addition to providing information on DNA CNV in the entire genome.28,29
It requires well trained personnel to not only perform the assay, but also process the data; thus incurring high cost in detecting a few known marker loci. Other PCR-based assays have also emerged including quantitative microsatellite analysis (QUMA) 30,31
and multiplex ligation-dependent probe amplification (MLPA).32
QUMA uses PCR amplification of microsatellite loci that contain (CA)n repeats to determine CNV of genes of interest, and has also been validated with FISH technology on detection of 1p and 19q loss in oligodendroglioma.31
However use of primers to amplify microsatellite DNA in quantitative PCR can be noisy, as multiple lengths of PCR product can be produced from cells even with homozygous loci, and amplification efficiency of different microsatellite markers can be different, thus the comparability between different platforms would be challenged. MLPA-based detection of 1p/19q has the advantage of detecting copy number changes of up to 45 loci in one relatively simple PCR based assay but it is semi quantitative and has yet to be widely adopted.32
Our real-time CQ-PCR utilizes multi-gene recombinant standard DNA to determine the absolute ratio of DNA copy numbers between the marker genes and multiple reference genes in tumor specimens. Both marker and reference genes were selected for their locations of interest and single-copy in genome with PCR primers designed to avoid amplification of corresponding pseudogenes in genome. It has advantages offered by QUMA in that all loci are informative, paired normal tissue from the same patient is not required, and even gain can be distinguished from loss. In comparison to QUMA, which requires accurate input of tumor DNA quantity, result from CQ-PCR is an absolute ratio of two gene’s DNA quantity. Thus the result is independent of DNA input quantity and the accuracy is ensured by the specificity of PCR primers.
Our results showed that CQ-PCR is a highly sensitive test; 100 copy numbers of a specific gene were able to be robustly quantified in highly diluted DNA samples (0.5 ng/μL). In addition to determine loss (here 1p/19q), gain of gene copy numbers, such as EGFR was also be detected by CQ-PCR (our unpublished data).
Inherited with PCR-based molecular assay, this technique has the disadvantages that it will not detect events without causing an alteration of DNA copy number, such as chromosomal translations, and it relies on the stability of reference genes. The former concern is not an issue for detecting deletion of 1p/19q, while the later has been considered by selecting multiple reference genes residing in relatively stable areas of genome in specific cancer types, which information is based on whole genome comparative analyses. It still needs to be empirically validated based on a large number of tumor samples. In this study, we have tested four candidate reference genes in total 58 gliomas, with ERC2, SPOCK1, and/or SPAG16 giving similar results for the marker gene on CNV, while not when normalized with PGCP, a reference gene selected based on stable area of chromosome (data not shown). In this study, we have taken average ratios to ERC2, SPOCK1, and/or SPAG16 to determine deletion status of 1p/19q marker genes. The average (medium) of the SDs from different reference ratios for the four marker genes is 10%–15% (7%–9%), which verified our proper use of the reference genes.
In conclusion, CQ-PCR technology reported here could be used to effectively screen all available tumor DNA samples to get 1p/19q deletion status. Our prior studies of glioma prognosis have revealed a prognostic model for OT with high predictive accuracy that explained 63% survival variation with significant likelihood ratio P
-value = 0.0076.33
It is yet to be determined if the CQ-PCR-detected 1p/19q deletion has prognostic value, and furthermore if it improves the multivariable OT models we have established with combined clinical and gene expression variables.