High resolution oligonucleotide CGH using DNA from archived prostate tissue

Abstract

Background: The current focus on biomarker discovery is a result of an improved understanding of the biological basis for carcinogenesis and advances in technology. Biomarkers can aid in diagnosis, prognosis, treatment selection, and drug development. There is an urgent need for high-resolution tools that perform well using archived tissue for biomarker discovery and tools that can translate into the clinic.

Methods: Oligonucleotide array comparative genomic hybridization (oCGH) was compared to BAC-based aCGH using unamplified total genomic DNA from formalin fixed paraffin-embedded (FFPE) prostate tissue.

Results: The copy number aberrations detected with the BAC and oligonucleotide arrays were highly correlated in cases where the arrays contained probes in similar genomic locations. The oligonucleotide array platform provided more precise mapping due to the higher density of oligonucleotide probes.

Conclusions: These results demonstrate the utility of high-resolution oligonucleotide arrays designed to use genomic DNA for CGH measurements using archived tissue samples for discovery and clinic based assays.

Fig. 1

Genome view of a CGH aberration calls for samples 13P (A) and 33P (B). The upper panel of each set shows the oligonucleotide array dye flip pair results and the lower panel displays the BAC array results, both display log₂ ratio splotted as a function of chromosomal position using Agilent’s CGH Analytics software. For both the oligonucleotide and BAC data plots, aberration calls from ADM-1 (threshold 10) in positive polarity are shown with blue lines, aberration calls in negative polarity (i.e., dye flip) are shown with red lines. The heights of the corresponding shaded rectangles indicate average log₂ ratio in each aberrant interval. To enable easier visualization of the aberrant intervals, log₂ ratios from individual oligonucleotide probes are not shown. For the BAC aCGH plots, log₂ ratios from individual probes are plotted as a function of chromosomal position with copy number gains in red (log₂ ratio >0.25) and losses in green (log₂ ratio <−0.25). Note the good concordance between the oligonucleotide and BAC aberration calls displayed as blue horizontal bars, as well as the consistency between the dye flip pairs displayed as red and blue horizontal bars from the oligonucleotide experiments.

Fig. 2

Detailed view of oligonucleotide and BAC aCGH data on chromosomes 8 and 12. Log₂ ratios are plotted for each probe as a function of chromosomal position. Probes with log₂ ratio >0.25 are shown in red, probes with log₂ ratio <−0.25 are shown in green. Vertical blue lines show the extent of the deleted intervals while the width of the blue rectangles correspond to theaveragelog₂ ratio of the probes inside a given aberration interval. (A) Sample 13P and 33P oligonucleotide and BAC array data plots. Whole arm loss of 8p and gain of 8q were detected with both techniques for both samples. (B) Sample 13P oligonucleotide and BAC array data is plotted in the first two panels, respectively, showing all of chromosome 12. The right two panels show a zoomed-in subregion of chromosome 12 near p 12.3. Gray bars indicate specific genes in this region. A single BAC probe with a log₂ ratio consistent with a loss (circled) agrees with the aberration call in the oligonucleotide data, however the increased density of probes on the oligonucleotide array in this region enables are fined view of the breakpoints flanking the deleted region.

Fig. 3

DU145 fresh and fixed tissue penetrance plot for the frozen and FFPE 244KoCGH data. The frequency of a copy number call at a particular locus is shown for each chromosome, with gains in red and losses in green.

Fig. 4

Genome view of aCGH and oCGH aberration calls using Agilent’s CGH Analytics software. A: Sample 13P 44KoCGH with 1 μg sample input (purple) and oCGH with 500 ng input (blue), and BAC aCGH with 1 μg input (green). Aberration calls from ADM-1 are shown with vertical lines, in each sample’s respective color, next to the ideograms. Gains are depicted with vertical lines to the right and losses to the left of the gray vertical line corresponding to each chromosome. B,C: Additional FFPE samples on oCGH with 500 ng input DNA.41P 44KoCGHinblue and BAC aCGH in purple (panel B) and similarly 34P 244KoCGHin green and BAC aCGH in brown (panel C).

Fig. 5

FFPE prostate biopsy and matched primary oCGH penetrance plot. Both samples were run with 500 ng unamplified DNA on Agilent’s 244K oCGH platform. The frequency of gains and deletions are shown in red and green, respectively, for each chromosome.