Conceived and designed the experiments: PNT. Performed the experiments: AHB SLA KKO. Analyzed the data: AHB SLA KKO CMG A-MM-M PNT. Contributed reagents/materials/analysis tools: KR AKW DP CMG A-MM-M PNT. Wrote the paper: AHB PNT. Pathology review: KR AKW.
The authors have declared that no competing interests exist.
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
Epithelial-stromal tumours of the serous histopathological subtype represent the largest group of epithelial ovarian cancers (EOC) and account for significant morbidity and mortality. Ovarian serous tumours may present as benign, low malignant potential (LMP) or malignant disease. Benign tumours account for up to 60% of ovarian serous tumours, present bilaterally in 20% of cases, and are cured through surgical removal of the disease
Karyotyping and array comparative genomic hybridization (aCGH) studies of benign, LMP and malignant serous tumours indicate an increasing frequency of chromosomal abnormalities, with the most extensive aneuploidy and structural abnormalities occurring in malignant tumours
Defining the genes involved in the etiology of ovarian serous neoplasms would provide a means to further stratify patients for optimal treatment regimens, as well as identify new molecular pathways to explore in the development of biomarkers. This is particularly prescient for LMP cases given that the majority of patients do not succumb to the disease, although most cases are usually subjected to aggressive management. Although studies of DNA ploidy in LMP tumours have been used to stratify patients for aggressive treatment, the overall impact on survival is not clear
In this study we have performed an extensive genetic analysis of benign and LMP ovarian serous tumours to further characterize somatic genetic events associated with the most indolent form of ovarian disease. We performed a targeted LOH analysis of the 3p12-pcen locus of interest generated from our previous analyses of benign, LMP and malignant ovarian carcinomas
LOH of 3p has been reported in up to 20% of benign ovarian serous tumours
To increase the resolution of markers in order to detect LOH events in tumour samples, we applied Illumina's HumanHap300-Duo Genotyping BeadChip, which assays approximately 317,500 SNPs across the human genome, to three benign ovarian tumour samples. As proof of principle, we investigated sample 1781T, a benign ovarian serous tumour that has been shown to exhibit LOH of 3p14-pcen. Samples BOV-1329GT and BOV-2564DT, which did not exhibit evidence of LOH in the present study, were also examined. BeadChip analysis identified a 9.1 Mb run of homozygosity (ROH) at 3p12-p11 in 1781T that did not display a corresponding decrease in the Log R ratio, which would have been consistent with a deletion occurring in this region (
SNP array imaging results for chr3 (
To investigate the possibility that LOH analyses underestimated the frequency of 3p abnormalities in benign and LMP serous tumours, we applied the 610K BeadChip technology to an additional 21 benign ovarian serous cases (32 tumours) and 53 LMP ovarian serous cases (58 tumours), of which 10 benign and 5 LMP cases included samples taken from both the left and the right ovaries. We also included 11 LGOSC cases (12 tumours), for which both bilateral tumours of one patient were arrayed. HGOSCs have already been shown to demonstrate LOH and abnormalities of 3p using genotyping arrays
Using the Genome Viewer module of the BeadStudio software, we visually assessed the data, which was aligned according to genomic position. The B allele frequency and Log R ratio were examined in order to infer allelic imbalance of whole chromosomes or chromosomal arms and intrachromosomal breaks (
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.
Sample | Pathology | Age | Stage | Imbalance of whole chromosome or chromosomal arms | Intrachromosomal breaks |
|
|
|
||
BOV-392 | DT | GT | Benign | 57 | - | - | - | - | - | - |
BOV-846 | DT | GT | Benign | 67 | - | - | - | - | - | - |
BOV-1172 | DT | GT | Benign | 66 | - | - | - | - | - | - |
BOV-1588 | DT | GT | Benign | 56 | - | - | - | - | - | - |
BOV-2314 | DT | GT | Benign | 64 | - | - | - | - | - | - |
BOV-2889 | DT | GT | Benign | 65 | - | - | - | - | - | - |
BOV-3057 | DT | GT | Benign | 52 | - | - | - | - | - | - |
BOV-3097 | DT | GT | Benign | 73 | - | - | - | - | - | - |
BOV-3150 | DT | GT | Benign | 56 | - | - | - | - | - | - |
BOV-3268 | DT | GT | Benign | 48 | - | - | - | - | - | - |
BOV-1329 | GT | Benign | 26 | - | - | - | - | - | - | |
BOV-2564 | DT | Benign | 53 | - | - | - | - | - | - | |
1781 | T | Benign | 66 | - | 3,9 | - | - | - | - | |
BOV-1207 | DT | Benign | 51 | - | - | 13q | - | - | - | |
BOV-1296 | DT | Benign | 71 | - | - | - | - | - | - | |
BOV-1332 | DT | Benign | 67 | - | - | - | - | - | - | |
BOV-1761 | GT | Benign | 63 | - | - | - | - | - | - | |
BOV-2023 | DT | Benign | 70 | - | - | 21q | - | - | - | |
BOV-2328 | DT | Benign | 52 | - | - | - | - | - | - | |
BOV-2331 | GT | Benign | 57 | - | - | - | - | - | - | |
BOV-2418 | GT | Benign | 71 | - | - | - | - | - | - | |
BOV-2506 | DT | Benign | 67 | - | - | - | - | - | - |
Description of chromosomal aberrations and mutations observed in a panel of 32 benign ovarian serous tumours from 22 patients. All chromosomal arms which display an intrachromosomal break or allelic imbalance are shown for each tumour, along with the corresponding mutations. T, tumour; DT, tumour on right ovary; GT, tumour on left ovary; EPT, tumour on omentum.
Sample | Initial Pathology | Revised Pathology | Age | Stage | Imbalance of whole chromosome or chromosomal arms | Intrachromosomal breaks |
|
|
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Other | ||
TOV-335 | DT | LMP | 44 | IIIB | 5, 8, 12, 15, 20 | - | c.35G>T; p.Gly12Val | - | - | |||
TOV-929(B) | T | LMP | 54 | IIIA | 12, 18 | - | c.35G>T; p.Gly12Val | - | - | |||
TOV-978 | T | LMP | 34 | IA | 8, 12, 20 | 6p, 13q | c.35G>A; p.Gly12Asp | - | - | |||
TOV-1068 | T | LMP | 58 | IIIC | 3,5, 7, 8, 12, 18 | - | c.35G>A; p.Gly12Asp | - | - | |||
TOV-1215 | GT | LMP | 27 | IIIA | 12p, 19 | 1p, 6q, 22q | c.34G>C; p.Gly12Arg | - | - | |||
TOV-2262 | DT | LMP | 62 | IB | 1q, 8, 12 | 1p | c.35G>T; p.Gly12Val | - | - | |||
TOV-3922 | GT | LMP | 65 | IA | 2, 3, 6, 7, 9, 12, 20 | - | c.35G>A; p.Gly12Asp | - | - | |||
TOV-1228 | GT | LMP | 42 | IIIC | - | 7p, 16q | c.35G>T; p.Gly12Val | - | - | |||
TOV-4105 | GT | LMP | 66 | IA | - | 8q, 12p | c.35G>T; p.Gly12Val | - | - | |||
TOV-3492 | DT | LMP | 41 | IIC | - | - | c.35G>T; p.Gly12Val | - | - | |||
TOV-3882 | DT | LMP | 49 | IIIA | - | - | c.35G>A; p.Gly12Asp | - | - | |||
N3426 | DT | LMP | 60 | - | - | c.35G>T; p.Gly12Val | - | - | ||||
TOV-1010 | DT | LMP | 31 | IB | 7, 8 | 21q | - | c.1799T>A; p.Val600Glu | - | |||
TOV-1010 | GT | LMP | 31 | IB | - | - | - | - | - | |||
TOV-4269 | DT | LMP | 21 | 7p | 7q | - | c.1799T>A; p.Val600Glu | - | ||||
TOV-696 | GT | LMP | 41 | IIIC | - | 12p, 15q, 17q | - | c.1799T>A; p.Val600Glu | - | |||
TOV-920 | DT | GT | LMP | 45 | IIIA | - | 3q, 22q | - | c.1799T>A; p.Val600Glu | - | ||
TOV-991 | DT | LMP | 67 | IB | - | - | - | c.1397G>T; p.Gly466Val | - | |||
TOV-991 | GT | LMP | 67 | IB | - | 11p | - | c.1397G>T; p.Gly466Val | - | |||
TOV-2173 | T | LMP | 49 | IA | - | 7q | - | c.1799T>A; p.Val600Glu | - | |||
TOV-3165 | GT | LMP | 35 | IA | - | 2p | - | c.1799T>A; p.Val600Glu | - | |||
TOV-933 | DT | LMP | 77 | IA | - | - | - | c.1799T>A; p.Val600Glu | - | |||
TOV-984 | DT | LMP | 34 | IB | - | - | - | c.1799T>A; p.Val600Glu | - | |||
TOV-1267 | DT | LMP | 44 | IIIA | - | - | - | c.1799T>A; p.Val600Glu | - | |||
TOV-1300 | GT | LMP | 47 | IIIC | - | - | - | c.1799T>A; p.Val600Glu | - | |||
TOV-3094 | GT | LMP | 29 | IIIC | - | - | - | c.1799T>A; p.Val600Glu | - | |||
TOV-3973 | GT | LMP | 62 | IA | - | - | - | c.1799T>A; p.Val600Glu | - | |||
TOV-4262 | GT | LMP | 26 | IIIC | - | - | - | c.1799T>A; p.Val600Glu | - | |||
TOV-1685 | GT | LMP | LMP | 26 | IA | 5p, 13q, 17p | 1p, 1q, 2p, 2q, 3p, 3q, 4p, 4q, 5q, 6p, 6q, 7p, 7q, 8p, 8q, 9p, 9q, 10q, 14q, 16p, 17q, 18q, 19p, 21q, 22q, Xp, Xq | - | - | c.1024delC; p.Arg342na; putative stop aa344 | ||
TOV-942 | GT | LMP | LMP | 58 | IC | 8p, 17p, 20q, X | 3p, 4p, 6p, 9p, 9q, 10q, 12p, 12q, 13q, 17q, 19p, 20p, 22q | - | - | - | ||
TOV-4054 | DT | LMP | 50 | IC | - | 6q | - | - | - | |||
TOV-4054 | GT | LMP | 50 | IC | 11, 12 | 6q | - | - | - | |||
TOV-206 | DT | LMP | 28 | IIIC | 12 | - | - | - | - | |||
TOV-845 | T | LMP | 50 | IIIC | 1q | 1p | - | - | - | |||
TOV-1694 | DT | LMP | 71 | IIIA | 17q | - | - | - | - | |||
TOV-1775 | DT | GT | LMP | 49 | IIIA | 1q, 16 | - | - | - | - | ||
TOV-3028 | GT | LMP | 74 | IA | 16q | 1p | - | - | - | |||
TOV-306 | DT | LMP | 62 | IIIC | - | 15q, 22q | - | - | - | |||
N945 | T | LMP | 64 | IIB | - | 6q | - | - | - | |||
TOV-1101 | GT | LMP | 52 | IIIC | - | 19q | - | - | - | |||
TOV-1319(A) | T | LMP | 80 | IA | - | 22q | - | - | - | |||
TOV-2395 | DT | LMP | 33 | IA | - | 2q | - | - | - | |||
TOV-3546 | DT | LMP | 51 | IIIC | - | 1p | - | - | - | |||
TOV-107 | GT | LMP | 63 | IA | - | - | - | - | - | |||
TOV-838 | GT | LMP | 38 | IB | - | - | - | - | - | |||
TOV-916 | T | LMP | 65 | IIIA | - | - | - | - | - | |||
TOV-1112 | T | LMP | 69 | - | - | - | - | - | ||||
TOV-1157 | T | LMP | 61 | IA | - | - | - | - | - | |||
TOV-1533 | GT | LMP | 60 | IA | - | - | - | - | - | |||
TOV-1607 | T | LMP | 58 | IB | - | - | - | - | - | |||
TOV-1615 | DT | LMP | 62 | IA | - | - | - | - | - | |||
TOV-1915 | T | LMP | 61 | IA | - | - | - | - | - | |||
TOV-2005 | DT | LMP | 54 | I | - | - | - | - | - | |||
TOV-2563 | DT | LMP | 50 | IB | - | - | - | - | - | |||
TOV-3423 | DT | LMP | 40 | IIB | - | - | - | - | - | |||
TOV-3703 | GT | LMP | 69 | IA | - | - | - | - | - |
Description of chromosomal aberrations and mutations observed in a panel of 58 LMP serous tumours from 53 patients. Two tumours displaying a high level of chromosomal instability were reevaluated by a gynecologic pathologist (TOV-1685GT and TOV-942GT). All chromosomal arms which display an intrachromosomal break or allelic imbalance are shown for each tumour, along with the corresponding mutations. T, tumour; DT, tumour on right ovary; GT, tumour on left ovary; EPT, tumour on omentum.
Sample | Initial Pathology | Revised Pathology | Age | Stage | Imbalance of whole chromosome or chromosomal arms | Intrachromosomal breaks |
|
|
|
||
832 | T | LGOSC | 37 | IIIC | - | 1p, 2q, 5q, 6p, 6q, 19q | c.35G>A; p.Gly12Asp | - | - | ||
682 | T | LGOSC | 40 | IIIC | - | - | - | c.1799T>A; p.Val600Glu | - | ||
TOV-854 | DT | GT | LGOSC | 61 | IIIC | 4, 5, 7, 8, 11, 13, 15, 20, X | - | - | c.1799T>A; p.Val600Glu | - | |
TOV-947 | DT | LGOSC | LMP | 53 | IA | 4, 14, 17p, 18p, 21, X | 1p, 1q, 2p, 2q, 3p, 3q, 5q, 6p, 6q, 7p, 7q, 8q, 9p, 9q, 10q, 11p, 12q, 13q, 15q, 16q, 17q, 18q, 19p, 19q, 20q, 22q | - | - | c.725G>A; p.Cys242Tyr | |
TOV-553 | EPT | LGOSC | HGSOC | 48 | IIIC | 16p, 17q | 1p, 1q, 2p, 2q, 3p, 3q, 4p, 4q, 5p, 5q, 6p, 6q, 7p, 7q, 8p, 8q, 9p, 9q, 10p, 10q, 11p, 11q, 12p, 12q, 13q, 14q, 15q, 16q, 17p, 18p, 18q, 19p, 19q, 20p, 20q, 21q, 22q, Xp, Xq | - | - | c.659A>G; p.Tyr220Cys | |
TOV-81 | DT | LGOSC | non invasive implant | 66 | IIIC | 17p, 18p, Xq | 1p, 1q, 2p, 2q, 3p, 3q, 4p, 4q, 5p, 5q, 6p, 6q, 7p, 7q, 8p, 8q, 9p, 9q, 10p, 10q, 11p, 11q, 12p, 12q, 13q, 14q, 15q, 16p, 16q, 17q, 18q, 19p, 20p, 20q, 22q, Xp | - | - | c.818G>A; p.Arg273His | |
TOV-490 | T | LGOSC | HGSOC | 71 | IIIC | 1q, 2q, 3q, 4, 5p, 5q, 6p, 7p, 9q, 10p, 14, 16p, 16q, 17, 18q, 20, | 1p, 2p, 3p, 6q, 7q, 9p, 10q, 12p, 12q, 13q, 18p, 19p, 19q, 21q, 22q, Xp, Xq | - | - | c.382delC; p.Pro128na; putative stop aa169 | |
TOV-812 | EPT | LGOSC | Metastatic serous | 70 | IIIC | 6q, 9, 16, 17, 18, 22q, X | 1p, 1q, 4q, 11p, 19p | - | - | c.455_456insC; p.Pro152na; putative stop aa180 | |
635 | T | LGOSC | 48 | IIIC | 1p, 1q, 4, 9, 22q, X | 8p, 17p | - | - | - | ||
TOV-1284 | T | LGOSC | 39 | IIIC | - | - | - | - | - | ||
TOV-1949 | T | LGOSC | 52 | IIIC | 8q | 3q, 5p, 7p | - | - | - |
Description of chromosomal aberrations and mutations observed in a panel of 12 LGOSC samples from 11 patients. Five tumours displaying a high level of chromosomal instability and
Bilateral tumours from 16 samples in this study were genotyped. None of the 10 paired bilateral benign tumours exhibited any evidence of genomic anomalies. Of the five paired LMP samples examined, one or both tumour samples exhibited evidence of chromosomal abnormalities. Some of these cases exhibited identical (cases TOV-1775 and TOV-920) or similar (case TOV-4054) abnormalities, suggesting the possibility of common clonal origins in these cases, as has been proposed for malignant ovarian cancers
Homozygous deletions may be inferred by identifying markers associated with a downward deviation of the Log R ratio and the absence of allele frequency scores. Null alleles resulting from somatic homozygous deletions are of particular interest, as they may affect the function of tumour suppressor genes. Furthermore, breaks occurring adjacent to cancer-associated genes may affect their regulation.
Chr | Sample | Pathology | Flanking SNPs | Genomic Location | Cytoband | Maximum Size | # Markers within Deletion | Nearest Upstream Gene | Genes included in deletion | Full Gene Name | Nearest Downstream Gene | High Grade Amplifications | Homozygous Deletions |
|
BOV-2506DT | Benign | rs1983365;rs1175861 | 4,198,741–4,224,068 | 2p25.3 | 25,328 bp | 6/7 |
|
|
- |
|
2 | 22 |
|
TOV-933DT | LMP | rs869817;rs17546105 | 12,016,999–12,031,541 | 2p25.1 | 14,543 bp | 4 |
|
|
- |
|
3 | 0 |
|
BOV-1172DT/GT | Benign | rs17864582;rs1028145 | 51,922,567–51,928,042 | 2p16.3 | 5,476 bp | 8 |
|
|
mRNA AK127244 |
|
1 | 0 |
|
TOV-916T | LMP | rs1011572;rs12328023 | 53,551,600–53,561,575 | 2p16.2 | 9,976 bp | 3 |
|
|
- |
|
3 | 0 |
|
TOV-845T | LMP | rs12615852;rs12469535 | 242,974,521–243,044,147 | 2q37.3 | 69,627 bp | 8 |
|
|
hypothetical LOC728323 | Telomere | 0 | 8 |
|
|
mRNA AK125674 | |||||||||||
|
BOV-3097DT/GT | Benign | rs28625386;rs7693378 | 70,323,237–70,636,709 | 4q13.2 | 104,491 bp | 6/7 |
|
|
UDP glucuronosyltransferase 2 family, polypeptide B28 |
|
0 | 44 |
|
TOV-854 DT/GT | LGSOC | rs1507939;rs950206 | 116,165,372–116,180,555 | 4q26 | 15,184 bp | 4 |
|
|
- |
|
1 | 2 |
|
TOV1112T | LMP | rs10518388;rs2013332 | 122,280,335–122,291,522 | 4q27 | 11,188 bp | 5 |
|
|
pyroglutamylated RFamide peptide receptor |
|
0 | 10 |
|
BOV-1588DT/GT | Benign | rs7692005;rs4692824 | 171,253,046–171,288,473 | 4q33 | 35,428 bp | 3 |
|
|
- |
|
0 | 0 |
|
TOV-306DT | LMP | rs4374757;rs4571472 | 15,719,350–15,721,360 | 5p15.1 | 2,011 bp | 4 |
|
|
F-box and leucine-rich repeat protein 7 |
|
13 | 6 |
|
BOV-3057DT/GT | Benign | rs4704943;rs12187915 | 155,470,446–155,500,631 | 5q33.2 | 30,186 bp | 3 |
|
|
sarcoglycan, delta |
|
0 | 3 |
|
TOV-3165GT | LMP | rs9350099;rs926274 | 19,039,776–19,051,695 | 6p22.3 | 11,920 bp | 4 |
|
|
- |
|
2 | 0 |
|
TOV-306DT | LMP | rs9469655;rs2495975 | 33,935,903–33,944,014 | 6p21.31 | 8,112 bp | 4/5 |
|
|
- |
|
1 | 0 |
|
TOV-1949T | LGSOC | rs2749135;rs11155845 | 101,491,425–101,507,345 | 6q16.3 | 15,921 bp | 3 |
|
|
- |
|
0 | 0 |
|
TOV4054DT | LMP | rs492132;rs12194183 | 117,643,433–117,885,959 | 6q22.1 | 242,527 bp | 70/74 |
|
|
c-ros oncogene 1, receptor tyrosine kinase |
|
0 | 1 |
|
|
discoidin, CUB and LCCL domain containing 1 | |||||||||||
|
|
golgi-associated PDZ and coiled-coil motif containing | |||||||||||
|
TOV-490T | LGSOC | rs9374781;rs606955 | 119,444,784–119,457,914 | 6q22.31 | 13,131 bp | 3 |
|
|
family with sequence similarity 184, member A |
|
1 | 0 |
|
TOV-490T | LGSOC | rs6900527;rs7761698 | 149,437,584–149,442,245 | 6q25.1 | 4,662 bp | 3 |
|
|
- |
|
0 | 0 |
|
BOV2328DT | Benign | rs10085387;rs11763921 | 97,389,030–97,404,277 | 7q21.3 | 15,248 bp | 3 |
|
|
- |
|
6 | 4 |
|
TOV-490T | LGSOC | rs10739110;rs7865244 | 6,697,128–6,715,730 | 9p24.1 | 18,603 bp | 3 |
|
|
- |
|
4 | 0 |
|
TOV-490T | LGSOC | rs10960291;rs10511574 | 11,827,014–11,951,204 | 9p23 | 124,191 bp | 38/39 |
|
|
- |
|
5 | 10 |
|
TOV-490T | LGSOC | rs8181148;rs4977974 | 25,282,007–25,294,701 | 9p21.3 | 12,695 bp | 3 |
|
|
- |
|
3 | 28 |
|
635T | LGSOC | rs4149303;rs2065412 | 107,594,515–107,598,740 | 9q31.1 | 4,226 bp | 3 |
|
|
ATP-binding cassette, sub-family A (ABC1), member 1 |
|
1 | 0 |
|
TOV4262GT | LMP | rs11788366;rs12237388 | 119,262,628–119,287,555 | 9q33.1 | 24,928 bp | 3 |
|
|
astrotactin 2 |
|
0 | 0 |
|
TOV-553EPT | LGSOC | rs7971309;rs1863552 | 108,423,276–108,459,275 | 12q23.3 | 36,000 bp | 3 |
|
|
- |
|
1 | 0 |
|
BOV-2314DT/GT | Benign | rs7998352;rs1928393 | 34,134,809–34,149,388 | 13q13.2 | 14,580 bp | 3 |
|
|
StAR-related lipid transfer (START) domain containing 13 |
|
2 | 0 |
|
TOV-490T | LGSOC | rs1198316;rs1211304 | 50,370,205–50,381,016 | 13q14.2 | 10,812 bp | 3 |
|
|
- |
|
2 | 1 |
|
TOV-1228GT | LMP | rs9546330;rs2669264 | 83,787,475–83,793,975 | 13q31.1 | 6,501 BP |
|
|
- |
|
3 | 1 | |
|
BOV-392DT/GT | Benign | rs7987913;rs9517112 | 98,527,866–98,536,863 | 13q32.2 | 8,998 bp | 4 |
|
|
- |
|
5 | 0 |
|
TOV4262GT | LMP | rs183112;rs1861320 | 55,527,682–55,541,040 | 16q12.2 | 13,359 bp | 3 |
|
|
Matrix metalloproteinase 2 |
|
0 | 0 |
|
TOV-490T | LGSOC | rs8075188;rs917344 | 69,387,158–69,399,273 | 17q24.3 | 12,116 bp | 3 |
|
|
- |
|
3 | 0 |
|
TOV4262GT | LMP | rs644016;rs11662635 | 55,164,547–55,170,274 | 18q21.31 | 5,728 bp | 3 |
|
|
- |
|
0 | 0 |
|
BOV-3150DT/GT | Benign | rs6511105;rs7254995 | 20,612,645–20,728,777 | 19p12 | 116,133 bp | 15 |
|
|
mRNA AF338193 |
|
2 | 19 |
|
|
zinc finger protein 737 | |||||||||||
|
TOV-696GT | LMP | rs12110;rs4805110 | 35,660,508–35,669,071 | 19q13.12 | 8,564 bp | 3 |
|
|
FXYD domain containing ion transport regulator 5 |
|
3 | 0 |
|
BOV-3150GT | Benign | cnvi0010506;rs3969184 | 26,248,774–28,118,678 | 20p11.1-q11.1 | 1,869,905 bp | 5/6 | centromere |
|
- |
|
1 | 0 |
|
TOV-490T | LGSOC | rs5748755;rs4819923 | 17,426,401–17,433,210 | 22q11.1 | 6,810 bp | 3 |
|
|
- |
|
1 | 0 |
|
TOV-490T | LGSOC | rs5963931;rs5918139 | 41,064,184–41,116,530 | Xp11.4 | 52,347 bp | 3 |
|
|
ubiquitin specific peptidase 9, X-linked |
|
0 | 2 |
Deletions are mapped on the Human Feb. 2009 (GRCh37/hg19) assembly of the human genome, except for the deletions flanked by non-SNP markers, which are mapped to the Human March 2006 (NCBI36/hg18) assembly. Flanking SNPs refers to the SNP markers flanking the homozygously deleted SNPs, and represent the largest possible size of the deletion. The genes located within and directly upstream and downstream from the hypothesized deleted regions are indicated. The genes that contain exons which may fall in the region of deletion are bolded. Genes found to be differentially expressed in Bonome
Given the large ROH overlapping the 3p12-p11 region in the benign tumour sample 1781T, we investigated whether ROHs of this interval were also observed in other samples. This analysis was restricted to the benign and LMP samples, as they exhibited low levels of generalized genomic instability. We examined ROHs larger than 5 Mb, as previous studies have shown that smaller ROHs, particularly those less than 1.5 Mb, may be common occurrences
Sample | Pathlogy | Chr | Location (MB) | Size (bp) | |||
BOV-1588 | DT | GT | Benign | 1 | 160.7–194.5 | 33,841,667 | 6654 |
1 | 196.2–205 | 8,752,119 | 2007 | ||||
2 | 5.0–23.0 | 18,066,407 | 4085 | ||||
2 | 235.5–242.7 | 7,160,696 | 1656 | ||||
3 | 128.9–173.1 | 44,169,552 | 8290 | ||||
7 | 145.8–157.6 | 11,483,243 | 2810 | ||||
11 | 89.1–94.6 | 5,530,828 | 1005 | ||||
12 | 51.8–62.6 | 10,847,129 | 1827 | ||||
13 | 24.6–43.6 | 18,960,867 | 4658 | ||||
15 | 29.2–34.3 | 5,117,368 | 1316 | ||||
21 | 18.7–27.5 | 8,753,964 | 2144 | ||||
22 | 14.4–36.3 | 21,832,954 | 5490 | ||||
22 | 41.2–49.6 | 8,361,420 | 2645 | ||||
X | 19.4–29.4 | 10,007,381 | 1267 | ||||
BOV-1172 | DT | GT | Benign | 1 | 155.7–161.8 | 6,055,835 | 1491 |
BOV-2506 | DT | Benign | 13 | 49.3–60.9 | 11,593,401 | 2076 | |
N1781 | T | Benign | 3 | 78.4–87.4 | 9,078,581 | 1206 | |
N3426-RT | DT | LMP | 3 | 190.2–196.4 | 6,176,699 | 1173 | |
6 | 39.0–82.4 | 43,394,086 | 8120 | ||||
10 | 12.7–25.6 | 12,881,505 | 3541 | ||||
X | 48.0–68.4 | 20,413,402 | 1608 | ||||
TOV-206 | DT | LMP | 1 | 37.9–55.6 | 17,698,088 | 2995 | |
15 | 57.6–66.7 | 9,077,633 | 2004 | ||||
TOV-916 | T | LMP | 2 | 169.9–206.4 | 36,453,242 | 6293 | |
3 | 190.0–196.7 | 7,768,872 | 1594 | ||||
TOV-1694 | DT | LMP | 3 | 71.7–128.3 | 56,621,515 | 9502 | |
15 | 40.1–51.5 | 11,391,842 | 2027 | ||||
TOV-3882 | DT | LMP | 3 | 2.6–8.4 | 5,795,529 | 1963 | |
3 | 65.3–71.7 | 6,417,920 | 1437 | ||||
TOV-4105(A) | GT | LMP | 3 | 0–5.1 | 5,082,955 | 1947 | |
3 | 177.1–186.6 | 9,530,536 | 1788 | ||||
TOV-1267 | DT | LMP | X | 71.9–77.6 | 5,692,402 | 314 | |
TOV-107 | GT | LMP | X | 55.1–67.3 | 12,153,847 | 538 | |
TOV-1775 | DT | GT | LMP | 8 | 24.5–29.7 | 5,229,945 | 1429 |
TOV-3165 | GT | LMP | 17 | 32.3–48.1 | 15,814,993 | 2498 | |
TOV-933 | DT | LMP | 3 | 77.2–101.8 | 24,659,990 | 3046 |
SNPs in the region refers to the number of polymorphic SNP markers located within the ROH. Only benign and LMP cases were examined in this analysis.
Mutations of
In general, the LMP and LGOSC cases with somatic
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
A gynecologic pathologist independently reviewed the LMP and LGOSC samples that were found to harbor
Genotyping data were analyzed by GenoCNA to evaluate various states of copy number variations that include allelic content occurring within each group of benign and LMP samples. The LGOSCs were not analyzed, given the small number of cases within the group and the fact that a number of cases were later designated by histopathology as not LGOSC.
As noted in
GenoCNA graphs showing gain (red) and loss (blue) in 20 serous benign tumour samples (
Discrete CNVs and somatic gains and losses of whole chromosomes and chromosomal arms are reflected in the GenoCNA analyses of the LMP tumours (
The ROH in the 3p12-p11 interval, along with the allelic imbalance of chr3 observed in the benign tumour sample 1781T, is interesting in light of recent research in our group suggesting the possibility of tumour suppressor gene(s) in this interval
Gene (RefSeq) | Genomic Location | Coding Location | 1781T | Ref. NCBI | Ref. Celera | Codon Change | Amino Acid Change | Function | dbSNP | HapMap CEU Frequency |
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g.79067965G>A | c.-610G>A |
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G | A | 5′UTR | rs1550930 |
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(NM_133631.3) | g.78796078G>T | c.1346-28G>T |
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G | G | intronic | rs2304503 |
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g.78737962G>A | c.1892-40G>A |
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G | G | intronic | rs967454 |
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g.78717508C>T | c.2477-56C>T |
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C | C | intronic | rs2255164 |
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g.78711350A>G | c.2813-86A>G |
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A | A | intronic | rs9864412 |
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g.78700779G>T | c.3658+103G>T |
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G | T | intronic | rs3925684 |
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g.78680578A>G | c.4328-123A>G |
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A | A | intronic | rs6548592 |
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g.78676467T>C | c.4721+4T>C |
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T | G | intronic | rs7636043 |
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g.78676422C>T | c.4721+49C>T |
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C | T | intronic | rs7614084 |
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g.78666765A>G | c.5128+20A>G |
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A | G | intronic | rs9839790 |
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g.78663956C>T | c.5129-6C>T |
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C | C | intronic | rs1027832 |
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g.81810749C>T | c.-90C>T |
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C | C | 5′UTR | ||||
(NM_000158.3) | g.81810703delG | c.-44delG |
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- | - | 5′UTR | rs11391701 |
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g.81810516G>T | c.143+10G>T |
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G | G | intronic | rs9820490 |
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g.81720221A>G | c.514-117A>G |
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A | A | intronic | rs9863136 |
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g.81643167A>G | c.1000A>G |
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A | G | ATT>GTT |
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non-synonymous | rs2172397 |
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g.81630214C>T | c.1730+102C>T |
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G | G | intronic | rs9870056 |
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g.81548210insTTC | c.2335+51insTTC |
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- | - | intronic | rs34988523 |
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No sequence variants were observed in
BeadChip analysis of 1781T demonstrated extensive allelic imbalance of chr3 and chr9. Chromosome 3 harbours
The inferred 242.5 kb homozygous deletion observed at 6q22.1 in LMP tumour TOV-4054DT stood out in part because it is much larger than the size of the average homozygous deletion (28.3 kb) observed in the present study (
Analysis of the
Although LOH of 3p has been reported in benign serous tumours at frequencies of up to 20%
It is possible that an excess of contaminating stromal cells may have obscured chromosomal anomalies in a subset of the samples analyzed. Previous studies using LOH analysis or CGH have observed chromosomal abnormalities without enriching for tumour cells, as chromosomal anomalies present in even 40% of cells can be detected by SNP array analyses
It is interesting that the 9.1 Mb ROH at 3p12 observed in sample 1781T overlaps a tumour suppressor region identified by our group using a functional complementation study involving the transfer of chr3 fragments into an EOC cell line
Case BOV-1588 exhibited the most extensive ROHs, as approximately 212 Mb of the genome (7.1%) occurred in ROHs longer than 5 Mb. These ROHs were confirmed to be germline in this patient. As the offspring of first cousins are expected to have about 6.25% genomic autozygosity, it is possible that the extensive ROHs observed in BOV-1588 were the consequence of a consanguineous mating. Upon further review of the medical history of this case, it was revealed that the patient has schizophrenia, a condition that has recently been associated with ROHs
The chromosomal abnormalities observed in 58 LMP samples from 53 cases mirror those previously reported in the literature, where 1p and 22q are subject to losses, and chr12 and chr8 display increases in copy number
The genetic spectrum of abnormalities observed in our small set of LGOSC cases is also consistent with independent reports, particularly when factoring in an independent review of the histopathology of cases. All five LGOSC cases that harboured a somatic
Few unique homozygous deletions were inferred in the samples analyzed, and none overlapped regions containing known tumour suppressor genes. It is interesting that 28 genes reported as differentially expressed in transcriptome studies of LMP samples are located directly adjacent to or within homozygous deletions identified in our SNP analyses of LMP samples
A 242.5 kb homozygous deletion at 6q22.1 was observed in the LMP tumour sample TOV-4054DT. Molecular genetic characterization suggests that this resulted in the creation of a transcriptionally active
The fusion gene occurred in a
Our results support the hypothesis that LGOSCs are derived from LMP ovarian serous tumours. Interestingly, chromosomal aberrations, but not genetic mutations, were observed in benign serous tumours. It is possible that acquisition of a mutation, such as
Tumour samples and peripheral blood lymphocytes were collected with informed consent from participants undergoing surgeries performed at the Centre hospitalier de l'Université de Montréal-Hôpital Notre-Dame or from surgeries performed at the McGill University Health Centre – Montreal General Hospital. The study is in compliance with the Helsinki declaration, and has been granted ethical approval by the respective Research Ethics Boards of Centre hospitalier de l'Université de Montréal-Hôpital Notre-Dame and The McGill University Health Centre. Clinical features such as disease stage, and tumour characteristics such as grade and histopathological subtype, were assigned by a gynecologist-oncologist and gynecologic-pathologist, respectively, according to the criteria established by the International Federation of Gynecology and Obstetrics (
EOC cell lines were derived from a stage IIIc/low grade papillary serous adenocarcinoma (TOV-81D), a stage III/high grade clear cell carcinoma (TOV-21G), a stage IIIc/high grade endometrioid carcinoma (TOV-112D), the ascites fluid of a stage IIIc/high grade adenocarcinoma (OV-90), a stage IIIc/high grade serous carcinoma (TOV-2223G), and both the tumour and the ascites fluid of a stage IIIc/high grade serous tumour (TOV-1946 and OV-1946), all from chemotherapy-naïve patients, as described
DNA was extracted from EOC cell lines, fresh frozen tumour specimens and peripheral blood lymphocytes as described previously
Total RNA was extracted with TRIzol™ reagent (Invitrogen Canada Inc., Burlington, ON) from the OV-90neor cell line grown to 80% confluency in 100 mm Petri dishes, or from fresh frozen TOV-4054DT/GT tumours as described previously
LOH analysis was performed using polymorphic microsatellite repeat markers representing various 3p loci:
Mutation analysis of tumour DNA samples was designed to detect variants in the protein coding exons 2 to 11 of
Promoter hypermethylation of
Genome-wide chromosomal anomalies in three benign ovarian tumours were inferred using the Infinium™ genotyping technology with Illumina's HumanHap300-Duo Genotyping BeadChip (Illumina, San Diego, CA, USA), which assays >317,500 SNPs. Genotyping of 32 benign ovarian serous tumours (including the 3 tumours assayed on the 300K BeadChip), 58 serous LMP tumours and 12 LGOSCs was performed using Illumina's Human610-Quad Genotyping BeadChip (Illumina, San Diego, CA, USA). This BeadChip assays 620,901 markers, where over 560,000 are SNPs with an average spacing of 4.7 kb per marker (median spacing is 2.7 kb). Both genotyping, using 750 ng of DNA from frozen tumours, and scanning, using the BeadArray™ Reader, were performed at the McGill University and Genome Quebec Innovation Centre (
Genotyping analysis was performed using the Genome Viewer module in BeadStudio Data Analysis software v2.2.22 (Illumina, San Diego, CA, USA.). The software aligns genotyping data for each marker with genomic map coordinates based on March 2006 NCBI36/hg18 (Build 36.1) assembly of the human reference sequence (genome.ucsc.edu/cgi-bin/hgGateway). An image file was created for inferring genomic rearrangements based on the allele frequency and copy number (Log R ratios) for each marker assayed. LOH was inferred by B allele frequency, where values that deviate from 0.5 (less than 0.4 and greater than 0.6) indicate allelic imbalance when reviewed for a series of adjacently mapped markers. Breakpoints were inferred based on deviation of allele frequencies relative to those of adjacently mapped markers. Log R ratios deviating from 0 suggest copy gain or loss. Homozygous deletions were inferred based on Log R ratios ≤−2 for at least three adjacently mapped markers, and sizes were estimated based on the location of nearest flanking markers with Log R ratios above −2. Regions suggesting extensive homozygosity (or runs of homozygosity; ROH), spanning intervals >5 Mb were inferred from heterozygous SNP markers. ROHs were required to have an average frequency of 1 SNP per 10 kb, and a heterozygous call for a marker was allowed if it was flanked by at least 100 SNP markers with homozygous scores
The distribution of mutations in
Normalized SNP intensity files were also analyzed by GenoCNA
Expression of the
(XLS)
(XLS)
(XLS)
We thank Manon de Ladurantaye for her helpful expertise. Research was conducted at The Research Institute of the McGill University Health Centre which receives support from the Fonds de la recherche du Québec - Santé (FRQS). Clinical specimens were provided by the Banque de tissus et de données of the Réseau de recherche sur le cancer of the FRQS affiliated with the Canadian Tumour Repository Network (CRTNet).