Conceived and designed the experiments: NW PH. Performed the experiments: KJ SC. Analyzed the data: NW AB HPY PH. Contributed reagents/materials/analysis tools: NW AB KJ HPY CDB NC UP LR SSB SC PH. Wrote the paper: NW AB HPY CDB SC PH. Contributed genotyping data: NC UP.
LR is employed by Westat, Inc., a company that serves as the PLCO Study Coordinating Center. This relationship does not influence the authors' adherence to the PLoS ONE policies on sharing data and materials. The other authors have declared that no competing interests exist.
A recent ovarian cancer genome-wide association study (GWAS) identified a locus on 9p22 associated with reduced ovarian cancer risk. The single nucleotide polymorphism (SNP) markers localize to the
We analyzed the association of 9p22 SNPs with transvaginal ultrasound (TVU) screening results and CA-125 blood levels from participants without ovarian cancer in the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO); 1,106 women with adequate ultrasound screening results and available genotyping information were included in the study.
We observed a significantly increased risk of abnormal suspicious TVU results for seven SNPs on 9p22, with odds ratios between 1.68 (95% CI: 1.04–2.72) for rs4961501 and 2.10 (95% CI: 1.31–3.38) for rs12379183. Associations were restricted to abnormal suspicious findings at the first TVU screen. We did not observe an association between 9p22 SNPs and CA-125 levels.
Our findings suggest that 9p22 SNPs, which were found to be associated with decreased risk of ovarian cancer in a recent GWAS, are associated with sonographically detectable ovarian abnormalities. Our results corroborate the relevance of the 9p22 locus for ovarian biology. Further studies are required to understand the complex relationship between screening abnormalities and ovarian carcinogenesis and to evaluate whether this locus can influence the risk stratification of ovarian cancer screening.
Ovarian cancer is the 8th most common cancer and the 5th leading cause of cancer death among women in the US
The use of TVU is hampered by false-positive findings, resulting in unnecessary surgical procedures with possible complications
A recent genome wide association study identified the first ovarian cancer susceptibility locus with genome-wide significance
We included all 1,106 women with TVU data and genome-wide scan data covering the 9p22 region from the PLCO screening arm (total n = 39,115 of whom n = 34,261 had not had prior oophorectomy). Each of the 10 screening centers obtained local Institutional Review Board approval to carry out the trial. NCI Institutional Review Board Approval was obtained to conduct genotyping among women who agreed to participate in genetic studies. We included only women with adequate TVU results and genotyping information for at least one of the SNPs on 9p22 previously found to be associated with ovarian cancer
Variable | Baseline Abnormal TVU | Incident Abnormal TVU | Other TVU | Baseline vs. Incident/Otherx2 p-value | Baseline/Incident vs. Otherx2 p-value |
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55–59 | 12 (24.0) | 7 (15.2) | 246 (24.4) | ||
60–64 | 15 (30.0) | 13 (28.3) | 321 (31.8) | ||
65–69 | 14 (28.0) | 14 (30.4) | 280 (27.7) | ||
70+ | 9 (18.0) | 12 (26.1) | 163 (16.1) | 0.99 | 0.43 |
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Caucasian | 43 (86.0) | 37 (80.4) | 912 (90.3) | ||
Non-Caucasian | 7 (14.0) | 9 (19.6) | 98 (9.7) | 0.38 | 0.03 |
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No | 45 (90.0) | 42 (91.3) | 912 (90.4) | ||
Yes | 5 (10.0) | 4 (8.7) | 97 (9.6) | 0.92 | 0.94 |
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No | 44 (88.0) | 36 (78.3) | 850 (84.6) | ||
Yes, female relative | 6 (12.0) | 8 (17.4) | 138 (13.7) | ||
Yes, male relative | 0 | 1 (2.2) | 2 (0.2) | ||
Possibly | 0 | 1 (2.2) | 15 (1.5) | 0.78 | 0.48 |
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No | 47 (94.0) | 41 (89.1) | 949 (94.4) | ||
Yes, immediate family | 3 (6.0) | 3 (6.5) | 40 (4.0) | ||
Possibly | 0 | 2 (4.4) | 16 (1.6) | 0.53 | 0.53 |
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Ever | 33 (66.0) | 30 (65.2) | 617 (61.1) | ||
Never | 17 (34.0) | 16 (34.8) | 388 (38.5) | ||
Unknown | 0 | 0 | 4 (0.4) | 0.74 | 0.59 |
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Never | 23 (46.0) | 26 (56.5) | 511 (50.7) | ||
Ever | 27 (54.0) | 20 (43.5) | 496 (49.3) | 0.49 | 0.96 |
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Never | 13 (26.0) | 13 (28.3) | 324 (32.1) | ||
Current | 15 (30.0) | 16 (34.8) | 310 (30.7) | ||
Former | 22 (44.0) | 17 (37.0) | 376 (37.2) | 0.57 | 0.6 |
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No | 35 (79.5) | 37 (84.1) | 867 (90.4) | ||
Yes | 9 (20.5) | 7 (15.9) | 92 (9.6) |
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0.01 |
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<10 | 0 | 0 | 12 (1.2) | ||
10–11 | 8 (16.0) | 12 (26.1) | 167 (16.6) | ||
12–13 | 27 (54.0) | 27 (58.7) | 572 (56.8) | ||
14–15 | 12 (24.0) | 5 (10.9) | 215 (21.4) | ||
16+ | 3 (6.0) | 2 (4.4) | 41 (4.1) | 0.86 | 0.57 |
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Control | 22 (44.0) | 26 (56.5) | 520 (51.5) | ||
Case | 28 (56.0) | 20 (43.5) | 490 (48.5) | 0.29 | 0.78 |
Bladder | 4 | 6 | 84 | ||
Breast | 4 | 1 | 32 | ||
Colon | 4 | 1 | 79 | ||
Lung | 14 | 9 | 249 | ||
Pancreas | 2 | 3 | 46 | ||
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TVU = transvaginal ultrasound; Family hx = family history; PMH = Post-menopausal hormone; OC = oral contraceptive; GWAS = genome-wide association study.
Genotyping was performed at the National Cancer Institute Core Genotyping Facility and Fred Hutchinson Cancer Research Center on the HumanHap550 Infinium II and Human 610-Quad chips (Illumina, San Diego, CA). Genotyping quality control followed standard procedures at the Core Genotyping Facility
TVU results were dichotomized into abnormal suspicious vs. normal and abnormal not suspicious. SNP associations were studied in relation to three outcomes: The TVU result at the first screen performed, the most severe TVU result of all screens, and the most severe incident TVU result (i.e. after excluding exams with abnormal screening results at the first screen). We used additive models defining the minor allele as risk allele to study the association of 9p22 SNPs with abnormal suspicious TVU results restricted to Caucasian individuals. We ran crude models and models adjusted for age as a continuous variable. For sensitivity analyses, we re-ran the models excluding individuals who were genotyped at the Fred Hutchinson Cancer Center but did not see any effect related to the site of genotyping. A sensitivity analysis restricted to control women only did not change the direction of the results. We used the Bonferroni correction as a conservative adjustment for multiple comparisons (n = 10), lowering the significance threshold to 0.005. Next, we studied the risk of abnormal TVU results associated with combinations of 0–2, 3–5, and 6–8 minor alleles from the four most significantly associated SNPs (rs10756819, rs12379183, rs3814113, and rs7861573). In addition, we combined the two least correlated SNPs (rs7861573 and rs10810666,
The LD-plot was generated with Haploview based on r2 of the 10 SNPs on 9p22 in 992 Caucasian women with genotyping information and transvaginal ultrasound results available.
Overall, 96 of the 1106 women included in this analysis had abnormal TVU screening results (8.7%) at any screening visit. In 50 women, abnormal TVU results were found at the first screening visit in PLCO (baseline abnormal TVU result), in the remaining 46 women, the abnormal TVU result was reported after an initially normal finding (incident abnormal TVU result).
Two SNPs, rs10756819 (OR = 1.48, p = 0.025) and rs12379183 (OR = 1.46, p = 0.046), showed a significant association with an abnormal TVU result at any time during follow up, while two other SNPs, rs3814113 and rs7861573, showed marginally significant results. None of these associations was significant after adjusting for multiple comparisons. After restricting to prevalent abnormal TVU findings as outcome only, the effect increased substantially: All ten SNPs on 9p22 showed increased ORs for suspicious TVU results at the first screen (
Worst TVU result (n = 992) | First TVU result (n = 992) | Incident TVU result (n = 949) | |||||||||||||
SNP | Case/Control | OR | Lower CL | Upper CL | P-value | Case/Control | OR | Lower CL | Upper CL | P-value | Case/Control | OR | Lower CL | Upper CL | P-value |
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80/912 | 1.48 | 1.05 | 2.08 |
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43/949 | 2.01 | 1.28 | 3.14 |
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37/912 | 1.01 | 0.61 | 1.68 | 0.9763 |
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80/910 | 1.27 | 0.86 | 1.89 | 0.2275 | 43/947 | 1.72 | 1.05 | 2.82 |
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37/910 | 0.84 | 0.45 | 1.59 | 0.5982 |
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80/911 | 1.22 | 0.79 | 1.89 | 0.3684 | 43/948 | 1.73 | 1.02 | 2.94 |
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37/911 | 0.73 | 0.34 | 1.54 | 0.4024 |
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79/909 | 1.46 | 1.01 | 2.13 |
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42/946 | 2.10 | 1.31 | 3.38 |
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37/909 | 0.90 | 0.50 | 1.64 | 0.7363 |
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79/911 | 1.21 | 0.78 | 1.87 | 0.3884 | 43/947 | 1.68 | 0.99 | 2.85 | 0.0527 | 36/911 | 0.74 | 0.35 | 1.55 | 0.4184 |
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75/790 | 1.10 | 0.78 | 1.56 | 0.5870 | 41/824 | 1.30 | 0.82 | 2.05 | 0.2602 | 34/790 | 0.90 | 0.54 | 1.50 | 0.6934 |
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78/911 | 1.12 | 0.80 | 1.58 | 0.5030 | 42/947 | 1.42 | 0.91 | 2.23 | 0.1241 | 36/911 | 0.85 | 0.51 | 1.41 | 0.5263 |
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79/912 | 1.39 | 0.98 | 1.97 | 0.0652 | 42/949 | 1.93 | 1.22 | 3.06 |
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37/912 | 0.93 | 0.55 | 1.57 | 0.7849 |
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80/910 | 1.25 | 0.85 | 1.82 | 0.2563 | 43/947 | 1.68 | 1.04 | 2.72 |
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37/910 | 0.84 | 0.47 | 1.52 | 0.5711 |
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79/908 | 1.42 | 0.97 | 2.07 | 0.0694 | 42/945 | 1.99 | 1.23 | 3.21 |
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37/908 | 0.90 | 0.50 | 1.64 | 0.7414 |
Per allele odds ratios obtained with an additive model restricted to the Caucasian population for the association of 9p22 SNPs with abnormal screening results are shown. Cases are women with suspicious screening results; controls are women with normal or non-suspicious screening results. Worst TVU results indicate abnormal TVU results at any screen during the 4-year follow-up. First TVU results indicate abnormal TVU results at the first screen a woman participated in. Incident TVU results are abnormal results among women that were normal or non-suspicious at the first screening. An asterisk indicates p-values lower than 0.005, the significance level after conservative Bonferroni correction.
SNP combination | Case/Control | OR | Lower CL | Upper CL | P-value |
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43/949 | 1.951 | 1.259 | 3.022 | 0.0028 |
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43/949 | 1.616 | 1.141 | 2.290 | 0.0069 |
Per allele odds ratios obtained with an additive model restricted to the Caucasian population for the association of combinations of 9p22 SNPs with abnormal screening results are shown. First, combinations of the four most strongly associated SNPs were analyzed. Three groups were created based on the number of minor alleles: 0–2 alleles present, 3–5 alleles present, 6–8 alleles present. Next, the two least correlated SNPs were combined. For the two-SNP combination, homozygote major alleles were considered low risk, while heterozygous alleles and homozygous minor allele genotypes were considered high risk. Three groups were created as follows: low risk by both SNPs, high risk by either one of the SNPs, and high risk by both SNPs.
We explored the association of 9p22 SNPs with TVU characteristics and CA-125 levels. For the rs12379183 SNP that showed the strongest effect in this analysis, we observed a trend of increasing ovarian volume measured at first ultrasound associated with risk alleles in all age groups. For the rs3814113 SNP that was most strongly associated with ovarian cancer risk
SNP | Genotype | Age | N | First volume median cm3 (IQR) | Maximal volume median cm3 (IQR) |
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AA |
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55–59 | 122 | 1.2 (1.6) | 1.6 (2.3) | ||
60–64 | 169 | 1.2 (1.8) | 1.6 (2.3) | ||
65–69 | 141 | 1.2 (1.3) | 1.6 (1.6) | ||
70–74 | 86 | 1.1 (1.6) | 1.65 (2.4) | ||
AG |
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55–59 | 71 | 1.5 (1.7) | 1.8 (1.4) | ||
60–64 | 93 | 1.4 (2.2) | 2.1 (2.7) | ||
65–69 | 77 | 1.4 (1.6 | 1.6 (2.1) | ||
70–74 | 50 | 1 (1.2) | 1.35 (1.3) | ||
GG |
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55–59 | 8 | 1.95 (1.2) | 1.95 (1.85) | ||
60–64 | 12 | 1.7 (1.35) | 2.25 (3.85) | ||
65–69 | 11 | 1.6 (1.7) | 2.4 (3.0) | ||
70–74 | 5 | 1.2 (0.6) | 1.4 (0.4) | ||
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TT |
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55–59 | 97 | 1.3 (1.8) | 1.7 (2.3) | ||
60–64 | 125 | 1.2 (1.7) | 1.6 (2.1) | ||
65–69 | 109 | 1.2 (1.2) | 1.5 (1.4) | ||
70–74 | 62 | 1.1 (1.7) | 1.45 (2.1) | ||
TC |
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55–59 | 94 | 1.4 (1.6) | 1.7 (1.6) | ||
60–64 | 125 | 1.4 (2.2) | 2.1 (2.9) | ||
65–69 | 92 | 1.4 (1.65) | 1.6 (1.8) | ||
70–74 | 68 | 1.1 (1.25) | 1.7 (1.95) | ||
CC |
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55–59 | 11 | 2 (1.8) | 2.1 (3.1) | ||
60–64 | 26 | 1.2 (1) | 1.85 (2.2) | ||
65–69 | 29 | 1.3 (1.7) | 2.1 (3.1) | ||
70–74 | 10 | 0.9 (0.5) | 1.05 (0.7) |
Median ovarian volume and interquartile range at the first visit and median of the highest measured volume per woman is shown stratified by genotypes and age groups.
RS12379183 | RS10756819 | RS3814113 | ||||||||
AA (n = 16) | AB/BB (n = 25) | p-value | AA (n = 10) | AB/BB (n = 32) | p-value | AA (n = 11) | AB/BB (n = 30) | p-value | ||
Number of cysts | Mean | 1.69 | 1.23 | 0.21 | 2.22 | 1.18 |
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1.82 | 1.19 | 0.08 |
SE | 0.38 | 0.15 | 0.64 | 0.12 | 0.46 | 0.13 | ||||
Cyst diameter (cm) | Mean | 3.87 | 3.74 | 0.81 | 3.56 | 3.86 | 0.48 | 3.83 | 3.76 | 0.92 |
SE | 0.24 | 0.46 | 0.36 | 0.36 | 0.39 | 0.38 | ||||
Cyst volume (cm3) | Mean | 35.38 | 60.78 | 0.21 | 29.36 | 56.92 | 0.07 | 38.03 | 55.5 | 0.34 |
SE | 5.3 | 18.85 | 7.46 | 14.79 | 9.14 | 15.72 |
A = major allele; B = minor allele. SE = standard error. T-test p-values are shown.
A large consortial GWAS effort recently identified several SNPs on 9p22 that are associated with ovarian cancer risk
In our study of women without ovarian cancer, we observed a significantly increased risk of abnormal suspicious TVU results for several SNPs on 9p22 that have been found to be associated with reduced ovarian cancer risk
We explored the association of 9p22 genotypes with morphologic characteristics recorded during TVU in women with abnormal TVU screening results. Although numbers were limited, we observed that women carrying minor 9p22 alleles had ultrasound features corresponding to complex ovarian cysts
Most importantly, our findings require independent confirmation, which is challenging, as there are only few resources that provide both TVU screening information and genetic data from a population-based study. If confirmed, our data suggest that some genes potentially protective against ovarian cancer actually are associated with suspicious TVU findings such as increased ovarian volume or complex cysts that gradually arise over decades and are detected at the first TVU screen.
The biology of ovarian cancer development is not well understood. It has been suggested that incessant ovulation, associated with repeated disruption and micro-traumas of the ovarian surface epithelium, may lead to initial transformation
Ovarian abnormalities associated with SNPs at the 9p22 locus may protect against cancer development by interfering with these carcinogenic mechanisms, e.g. by reducing the number of lifetime ovulations or by modulating the exposure of ovarian tissue to endogenous or exogenous hormones. Ovarian cysts may impede implantation of early transformed cell clones derived from the Fallopian tube. Furthermore, although we did not see any evidence in PLCO, we cannot exclude that the reduced ovarian cancer risk associated with these SNPs is related to more frequent oophorectomies following suspicious TVU results, rather than to a direct biological mechanism.
If the 9p22 locus is associated with false positive ovarian cancer screening results, genotyping might have influence on the interpretation of TVU results.
A recent study demonstrated that cancer-related SNPs may influence prostate cancer risk estimates related to prostate specific antigen levels
The authors thank Dr. Philip Prorok, Division of Cancer Prevention, National Cancer Institute, the Screening Center investigators and staff of the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial, Mr. Tom Riley and staff, Information Management Services, Inc., Ms. Barbara O'Brien and staff, Westat, Inc., Mr. Tim Sheehy and staff, DNA Extraction and Staging Laboratory, SAIC-Frederick, Inc, and Ms. Jackie King and staff, BioReliance, Inc. Most importantly, we acknowledge the study participants for their contributions to making this study possible.