Glioma test array for use with formalin-fixed, paraffin-embedded tissue: array comparative genomic hybridization correlates with loss of heterozygosity and fluorescence in situ hybridization

Abstract

Array-based comparative genomic hybridization (aCGH) is a powerful, high-throughput tool for whole genome analysis. Until recently, aCGH could only be reproducibly performed on frozen tissue samples and with significant tissue amounts. For brain tumors however, paraffin-embedded tissue blocks from small stereotactic biopsies may be the only tissue routinely available. The development of methods to analyze formalin-fixed, paraffin-embedded (FFPE) material therefore has the potential to impact molecular diagnosis in a significant way. To this end, we constructed a BAC array representing chromosomes 1, 7, 19, and X because 1p/19q deletion and EGFR gene amplification provide clinically relevant information for glioma diagnosis. We also optimized a two-step labeling procedure using an amine-modified nucleotide for generating aCGH probes. Using this approach, we analyzed a series of 28 FFPE oligodendroglial tumors for alterations of chromosomes 1, 7, and 19. We also independently assayed these tumors for 1p/19q deletion by fluorescence in situ hybridization and by loss of heterozygosity analyses. The concordance between aCGH, standard loss of heterozygosity and fluorescence in situ hybridization was nearly 100% for the chromosomes analyzed. These results suggest that aCGH could offer an improved molecular diagnostic approach for gliomas because of its ability to detect clinically relevant molecular alterations in small FFPE specimens.

Figure 1

Array validation. aCGH was performed using male and female lymphocyte DNA from healthy donors. Male DNA was labeled with Cy3-dCTP and female DNA was labeled with Cy5-dCTP by random priming. Hybridization was performed mismatching the sex chromosome. Log2 ratios along the autosomes appear normal and along the X chromosome appear deleted, indicating the sex mismatch for the X chromosome. Ratio profiles from five different control hybridizations are shown. Arrows indicate centromeres of 1 and 19.

Figure 2

Comparison of labeling procedures. DNA from oligodendroglioma blocks was isolated as described in the Materials and Methods section. a and b: A tumor without 1p/19q loss, with a labeled with random priming and b labeled with amino-allyl-dUTP; sex is mismatched. c and d: A tumor with 1p/19q loss, with c labeled with random priming and d labeled with amino-allyl-dUTP; sex is mismatched. In both cases, the amino-allyl-dUTP-labeling procedure produced smoother ratio profiles compared to randomly primed labeled DNA. Arrows indicate centromeres of 1 and 19.

Figure 3

Comparison of frozen versus FFPE DNA in two oligodendrogliomas. Array CGH was performed using frozen DNA (a and b) labeled by random priming. 1p and 19q loss was clearly visible in both samples. Sex chromosomes were mismatched as an internal hybridization control. FFPE DNA from the same tumors labeled by amino-allyl-dUTP clearly detected the losses, as shown in c and d. Arrows indicate centromeres of 1 and 19.

Figure 4

Comparison of aCGH, FISH, and LOH in representative oligodendroglial samples. a: aCGH of xT-3608 shows normal 1p and 19q copy number. aCGH result was confirmed by FISH and LOH using markers described in the Materials and Methods. b: aCGH of xT-3533 shows loss of 1p and 19q. aCGH result was confirmed by FISH and LOH. c: aCGH of xT-3643 shows increase in copy number for chromosome 1. FISH analysis on tissue section shows increase in copy number for both 1p and 1q probes. d: aCGH of xT-3508 shows amplification of the EGFR gene. Three additional clones from 7p mapping in close proximity to the EGFR gene and five clones from 7q were included in the array. None of the other chromosome 7 clones were increased in copy number or amplified. aCGH result was confirmed by FISH on tumor section. The sex chromosomes were mismatched for all hybridizations. Red “lollipop” denotes the BAC clone used for FISH and green arrows denote positions of markers used for LOH analysis relative to the BAC used for FISH. Downward arrows indicate centromeres of 1 and 19.