Chromosome 3 anomalies investigated by genome wide SNP analysis of benign, low malignant potential and low grade ovarian serous tumours

Abstract

Ovarian carcinomas exhibit extensive heterogeneity, and their etiology remains unknown. Histological and genetic evidence has led to the proposal that low grade ovarian serous carcinomas (LGOSC) have a different etiology than high grade carcinomas (HGOSC), arising from serous tumours of low malignant potential (LMP). Common regions of chromosome (chr) 3 loss have been observed in all types of serous ovarian tumours, including benign, suggesting that these regions contain genes important in the development of all ovarian serous carcinomas. A high-density genome-wide genotyping bead array technology, which assayed >600,000 markers, was applied to a panel of serous benign and LMP tumours and a small set of LGOSC, to characterize somatic events associated with the most indolent forms of ovarian disease. The genomic patterns inferred were related to TP53, KRAS and BRAF mutations. An increasing frequency of genomic anomalies was observed with pathology of disease: 3/22 (13.6%) benign cases, 40/53 (75.5%) LMP cases and 10/11 (90.9%) LGOSC cases. Low frequencies of chr3 anomalies occurred in all tumour types. Runs of homozygosity were most commonly observed on chr3, with the 3p12-p11 candidate tumour suppressor region the most frequently homozygous region in the genome. An LMP harboured a homozygous deletion on chr6 which created a GOPC-ROS1 fusion gene, previously reported as oncogenic in other cancer types. Somatic TP53, KRAS and BRAF mutations were not observed in benign tumours. KRAS-mutation positive LMP cases displayed significantly more chromosomal aberrations than BRAF-mutation positive or KRAS and BRAF mutation negative cases. Gain of 12p, which harbours the KRAS gene, was particularly evident. A pathology review reclassified all TP53-mutation positive LGOSC cases, some of which acquired a HGOSC status. Taken together, our results support the view that LGOSC could arise from serous benign and LMP tumours, but does not exclude the possibility that HGOSC may derive from LMP tumours.

Figure 1. SNP array imaging results for chr3 and chr9 of the benign serous tumour 1781T.

SNP array imaging results for chr3 (A, C, E) and chr9 (B, D, F) of the benign serous tumour 1781T, using Illumina's HumanHap300-Duo Genotyping BeadChip (A and B) and Illumina's Human610-Quad Genotyping BeadChip (C–F). Two different DNA preparations were used with the HumanHap610-Quad Genotyping BeadChip. The top plot of each figure shows the B allele frequency (BAF) for each SNP marker aligned to its chromosomal position. In heterozygous diploid cells, alleles are present in AA, AB or BB pairs. The B Allele frequencies for these possible allele pairs are 0, 0.5 or 1, respectively. Any deviation from this ratio indicates a chromosomal aberration. In one DNA preparation, the double row in the BAF plot indicates allelic imbalance of SNP markers across the entire chromosome (C and D). A 9.1 Mb ROH is observed on chr3 and is highlighted in blue. No markers are located in the centromeric region of either chromosome, as noted by a lack of markers in both the B allele frequency and Log R ratio (LRR) plots. The bottom plot of each figure contains the Log R ratio, which provides an indication of the copy number for each SNP marker aligned to its chromosomal position. Note the absence of a drop in the Log R ratio in the highlighted ROH.

Figure 2. Example of intrachromosomal breaks and allelic imbalance in an LMP tumour.

SNP array imaging results for chr1 of LMP sample TOV-845T. Several intrachromosomal breaks are denoted by arrows on 1p, and are visualized by breaks in the continuity of both the B allele frequency and Log R ratio plots. Note the Log R ratio indicates loss of copy number for most of the 1p arm, with gains of copy number near the centromere. The double row in the BAF plot observed on 1q indicates allelic imbalance of SNP markers across the entire chromosomal arm. Note the Log R ratio for the 1q arm averages above 0, indicating a gain of copy number.

Figure 3. High level amplification of a 1.59 Mb region containing KRAS in an LMP sample.

SNP array imaging results for chr12 of LMP sample TOV-942GT. A high-grade amplification of a discrete 1.59 Mb region (arrow) containing the proto-oncogene KRAS is observed (A). Depiction of the amplified region that contains 12 genes, including KRAS (arrow) (B).

Figure 4. GenoCNA graphs showing gain and loss in serous benign tumour samples and serous LMP samples.

GenoCNA graphs showing gain (red) and loss (blue) in 20 serous benign tumour samples (A) and 53 serous LMP samples (B). Peaks describing gain in >30% of samples represent repetitive regions around centromeres and/or telomeres. Peaks describing loss in >30% of samples represent common CNVs that often display loss of copy number. Somatic gains and losses of chromosomes are visible in the GenoCNA graph of the LMP samples, such as loss of 1p and gain of 1q, gain of chr7, chr8 and chr12.

Figure 5. Analysis of the GOPC-ROS1 fusion gene in an ovarian LMP sample.

Analysis of the GOPC-ROS1 fusion gene. Plots representing the B allele frequency and the Log R ratio on chr6 in TOV-4054GT (left) and TOV-4054DT (right) (A). A homozygous deletion is present at 6q22.2 in TOV-4054DT (circled), as observed by a Log R ratio ≤−2 and associated loss of B allele frequency organization. The genomic region located within the 242.5 kb homozygous deletion includes coding exons of the genes ROS1, DCBLD1 and GOPC, as visualized by the UCSC Genome Browser (B). RT-PCR analysis of the GOPC-ROS1 fusion gene (C). The fusion gene is highly expressed in TOV-4054DT and lowly expressed in TOV-4054GT. The EOC cell line OV-90neo^r was used as a negative control. Sequencing of the GOPC-ROS1 fusion cDNA indicates that exon 7 of GOPC is fused in-frame to exon 35 of ROS1 (D).