Bead-based ELISA for validation of ovarian cancer early detection markers

University of Washington Seattle, Seattle, Washington, United States
Clinical Cancer Research (Impact Factor: 8.72). 04/2006; 12(7 Pt 1):2117-24. DOI: 10.1158/1078-0432.CCR-05-2007
Source: PubMed


Efforts to validate ovarian cancer early detection biomarkers with immunoassays are challenged by the limited specimen volumes available. We sought to develop a specimen-efficient assay to measure CA125 in serum, assess its reproducibility, validity, and performance, and test its potential for multiplexing and combining with human epididymis protein 4 (HE4), a promising novel ovarian cancer marker.
Four pairs of commercially available anti-CA125 antibodies and one pair of anti-HE4 antibodies were evaluated for accuracy in measuring known concentrations of antigen on a bead-based platform. The two best pairs were further assessed for reproducibility, validity, and the ability to discriminate between blinded serum samples obtained from ovarian cancer cases (n = 66) and women without ovarian cancer (n = 125).
Suitability for use in a bead-based assay varied across CA125 antibody pairs. Two CA125 bead-based assays were highly reproducible (overall correlations between replicates >/= 0.95; coefficients of variation < 0.2) and strongly correlated with the research standard CA125II RIA (correlations >/= 0.9). Their ability to distinguish ovarian cancer cases from non-cases based on receiver operating characteristic analyses (area under the curve, AUC, of 0.85 and 0.84) was close to that of the CA125II RIA (AUC, 0.87). The HE4 bead-based assay showed lower reproducibility but yielded an AUC of 0.89 in receiver operating characteristics analysis. Multiplexing was not possible but a composite marker including CA125 and HE4 achieved an AUC of 0.91.
Optimization procedures yielded two bead-based assays for CA125 that perform comparably to the standard CA125II RIA, which could be combined with an HE4 bead-based assay to improve diagnostic performance, and requires only 15 muL of sample each.

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    • "Several groups have investigated HE4 in a possible screening setting and report good ROC AUC separating women with either early or late stage epithelial ovarian cancer from healthy controls [10,16,22,23]. HE4 is a biomarker that increases progressively with disease and is markedly lower in healthy women than in women with benign ovarian cysts [12]. "
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    ABSTRACT: Women presenting with a large or complex ovarian cyst are referred to extensive surgical staging to ensure the correct diagnosis and treatment of a possible epithelial ovarian cancer. We hypothesized that measurement of the biomarkers HE4 and CA-125 preoperatively would improve the assignment of these patients to the correct level of care. Patients diagnosed with a cystic ovarian mass and scheduled for an operation at our center of excellence for ovarian cancer surgery from 2001 to 2010 were prospectively included (n=394) and plasma was collected consecutively. Cut-off for HE4 was calculated at 75% specificity (85 pM and 71.8 pM for post and premenopausal women). For CA-125, 35 U/mL cut-off was used. The study population included women with malignant (n=114), borderline (n=45), and benign (n=215) ovarian tumors. Receiver operator characteristic (ROC) area under the curve (AUC) in the benign versus malignant cohorts was 86.8% for CA-125 and 84.4% for HE4. Negative predictive value was 91.7% when at least one of the biomarkers was positive, with only early stage epithelial ovarian cancer showing false negative results. Sensitivity at set specificity (75%) was 87% for risk of ovarian malignancy algorithm (ROMA) in the postmenopausal cohort (cut-off point, 26.0%) and 81% in the premenopausal cohort (cut-off point, 17.3%). ROC AUC in the benign versus stage I epithelial ovarian cancer was only 72% for HE4 and 76% for CA-125. In our study, population HE4 did not outperform CA-125. Based on our data a prospective trial with patients already diagnosed with an ovarian cyst may be conducted.
    Journal of Gynecologic Oncology 12/2011; 22(4):244-52. DOI:10.3802/jgo.2011.22.4.244 · 2.49 Impact Factor
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    • "Normally expressed on the surface of mesothelial cells lining the body cavities as the name suggests, mesothelin is a protein that has shown to be a helpful diagnostic marker of several cancers such as mesotheliomas, ovarian, and pancreatic cancers, where it is overexpressed [30]. This marker has very high specificity as well as sensitivity for patients with ovarian cancer [29]. "
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    ABSTRACT: This paper reviews current screening techniques as well as novel biomarkers and their potential role in early detection of ovarian cancer. Ovarian cancer is one of the most common reproductive cancers and has the highest mortality rate amongst gynecologic cancers. Because most ovarian cancer diagnoses occur in the late stages of the disease, five-year survival rates fall below 20%. To improve survival rates and to lower mortality rates for ovarian cancer, improved detection at early stages of the disease is needed. Current screening approaches include tumor markers, ultrasound, or a combination. Efforts are underway to discover new biomarkers of ovarian cancer in order to surmount the obstacles in early-stage diagnosis. Among serum protein markers, HE4 and mesothelin can augment CA125 detection providing higher sensitivity and specificity due to the presence of these proteins in early-stage ovarian cancer. Detection testing that includes methylation of the MCJ gene and increased expression of vascular endothelial growth factor is correlated to poor prognosis and may predict patient survival outcome. Detection testing of biomarkers with long-term stability and combination panels of markers, will likely lead to effective screening strategies with high specificity and sensitivity for early detection of ovarian cancer.
    Journal of Oncology 04/2011; 2011(1687-8450):475983. DOI:10.1155/2011/475983
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    • "When the ROC–AUCs of the different tumour markers were compared, HE4 and CA125 performed similarly, except for the post-menopausal patients in whom CA125 performed better. This similar performance of HE4 and CA125 was also noted in other studies (Hellstrom et al, 2003; Scholler et al, 2006; Palmer et al, 2008; Montagnana et al, 2009; Andersen et al, 2010). Combining HE4 and CA125 in the ROMA improved HE4 but not CA125 performance, regardless of menopausal status. "
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    ABSTRACT: Recently, a Risk of Ovarian Malignancy Algorithm (ROMA) utilising human epididymis secretory protein 4 (HE4) and CA125 successfully classified patients as presenting a high or low risk for epithelial ovarian cancer (EOC). We validated this algorithm in an independent prospective study. Women with a pelvic mass, who were scheduled to have surgery, were enrolled in a prospective study. Preoperative serum levels of HE4 and CA125 were measured in 389 patients. The performance of each of the markers, as well as that of ROMA, was analysed. When all malignant tumours were included, ROMA (receiver operator characteristic (ROC)-area under curve (AUC)=0.898) and HE4 (ROC-AUC)=0.857) did not perform significantly better than CA125 alone (ROC-AUC=0.877). Using a cutoff for ROMA of 12.5% for pre-menopausal patients, the test had a sensitivity of 67.5% and a specificity of 87.9%. With a cutoff of 14.4% for post-menopausal patients, the test had a sensitivity of 90.8% and a specificity of 66.3%. For EOC vs benign disease, the ROC-AUC of ROMA increased to 0.913 and for invasive EOC vs benign disease to 0.957. This independent validation study demonstrated similar performance indices to those recently published. However, in this study, HE4 and ROMA did not increase the detection of malignant disease compared with CA125 alone. Although the initial reports were promising, measurement of HE4 serum levels does not contribute to the diagnosis of ovarian cancer.
    British Journal of Cancer 02/2011; 104(5):863-70. DOI:10.1038/sj.bjc.6606092 · 4.84 Impact Factor
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