Abstract:BackgroundCurrently, there are no FDA approved screening tools for detecting early stage ovarian cancer in the general population. Development of a biomarker-based assay for early detection would significantly improve the survival of ovarian cancer patients.
MethodsWe used a multiplex approach to identify protein biomarkers for detecting early stage ovarian cancer. This new technology (Proseek® Multiplex Oncology Plates) can simultaneously measure the expression of 92 proteins in serum based on a proximity ext… Show more
“…In agreement with our previous study on the Oncology Iv2 plate (13) and others (25,28), we found high levels of both MK and IL6 in the sera of patients with ovarian cancer. FR-alpha, KLK11, and NECT4 also showed a significant increase in their levels of expression in the sera of ovarian cancer patients as previously reported (24,26,27).…”
Section: Discussionsupporting
confidence: 93%
“…The Proseek platform also includes three "Interplate controls" for data normalization between plates and three "Negative controls" to establish background levels. One microliter of serum from each of the samples was mixed with the Oncology II reagents following the manufacturer's protocol, then processed in combination with the Fluidigm BioMark HD high-throughput PCR instrument at the University of Minnesota Genomics Center as previously described (13). Data generated from the plates were analyzed, including normalization and linearization, per manufacturer's protocol.…”
Section: Sample Processingmentioning
confidence: 99%
“…Recently, we tested a small sample set (20 cases each of healthy, benign ovarian, early stage serous ovarian cancer, and late-stage serous ovarian cancer) on the Proseek Multiplex Oncology Iv2 panel to determine its feasibility as a means to identify candidate biomarkers for early stage serous ovarian cancer (13). We demonstrated that the Proseek technology provides similar results to conventional clinical assays for CA125, and can identify new candidate biomarkers that improve the sensitivity and specificity of CA125 alone.…”
The best known ovarian cancer biomarker, CA125, is neither adequately sensitive nor specific for screening the general population. By using a combination of proteins for screening, it may be possible to increase the sensitivity and specificity over CA125 alone. In this study, we used Proseek Multiplex Oncology II plates to simultaneously measure the expression of 92 cancer-related proteins in serum using proximity extension assays. This technology combines the sensitivity of the PCR with the specificity of antibodybased detection methods, allowing multiplex biomarker detection and high-throughput quantification. We analyzed 1 mL of sera from each of 61 women with ovarian cancer and compared the values obtained with those from 88 age-matched healthy women. Principle component analysis and unsupervised hier-archical clustering separated the ovarian cancer patients from the healthy, with minimal misclassification. Data from the Proseek plates for CA125 levels exhibited a strong correlation with clinical values for CA125. We identified 52 proteins that differed significantly (P < 0.006) between ovarian cancer and healthy samples, several of which are novel serum biomarkers for ovarian cancer. In total, 40 proteins had an estimated area under the ROC curve of 0.70 or greater, suggesting their potential to serve as biomarkers for ovarian cancer. CA125 alone achieved a sensitivity of 93.4% at a specificity of 98%. By adding the Oncology II values for five proteins to CA125 in a multiprotein classifier, we increased the assay sensitivity to 98.4% at a specificity of 98%, thereby improving the sensitivity and specificity of CA125 alone.
“…In agreement with our previous study on the Oncology Iv2 plate (13) and others (25,28), we found high levels of both MK and IL6 in the sera of patients with ovarian cancer. FR-alpha, KLK11, and NECT4 also showed a significant increase in their levels of expression in the sera of ovarian cancer patients as previously reported (24,26,27).…”
Section: Discussionsupporting
confidence: 93%
“…The Proseek platform also includes three "Interplate controls" for data normalization between plates and three "Negative controls" to establish background levels. One microliter of serum from each of the samples was mixed with the Oncology II reagents following the manufacturer's protocol, then processed in combination with the Fluidigm BioMark HD high-throughput PCR instrument at the University of Minnesota Genomics Center as previously described (13). Data generated from the plates were analyzed, including normalization and linearization, per manufacturer's protocol.…”
Section: Sample Processingmentioning
confidence: 99%
“…Recently, we tested a small sample set (20 cases each of healthy, benign ovarian, early stage serous ovarian cancer, and late-stage serous ovarian cancer) on the Proseek Multiplex Oncology Iv2 panel to determine its feasibility as a means to identify candidate biomarkers for early stage serous ovarian cancer (13). We demonstrated that the Proseek technology provides similar results to conventional clinical assays for CA125, and can identify new candidate biomarkers that improve the sensitivity and specificity of CA125 alone.…”
The best known ovarian cancer biomarker, CA125, is neither adequately sensitive nor specific for screening the general population. By using a combination of proteins for screening, it may be possible to increase the sensitivity and specificity over CA125 alone. In this study, we used Proseek Multiplex Oncology II plates to simultaneously measure the expression of 92 cancer-related proteins in serum using proximity extension assays. This technology combines the sensitivity of the PCR with the specificity of antibodybased detection methods, allowing multiplex biomarker detection and high-throughput quantification. We analyzed 1 mL of sera from each of 61 women with ovarian cancer and compared the values obtained with those from 88 age-matched healthy women. Principle component analysis and unsupervised hier-archical clustering separated the ovarian cancer patients from the healthy, with minimal misclassification. Data from the Proseek plates for CA125 levels exhibited a strong correlation with clinical values for CA125. We identified 52 proteins that differed significantly (P < 0.006) between ovarian cancer and healthy samples, several of which are novel serum biomarkers for ovarian cancer. In total, 40 proteins had an estimated area under the ROC curve of 0.70 or greater, suggesting their potential to serve as biomarkers for ovarian cancer. CA125 alone achieved a sensitivity of 93.4% at a specificity of 98%. By adding the Oncology II values for five proteins to CA125 in a multiprotein classifier, we increased the assay sensitivity to 98.4% at a specificity of 98%, thereby improving the sensitivity and specificity of CA125 alone.
“…Our top-ranked model had a sensitivity of 0.85 and specificity of 0.91 under the same conditions. Similar to the results of these previous studies 8,9 , the performance of our models in the test-proportion of the discovery data is very good, with some models showing perfect classification. We also evaluated the selected models in two replication cohorts and found the performance similar, but somewhat lower than in the discovery set.…”
Section: Discussionsupporting
confidence: 87%
“…We then performed model selection as before based solely on the discovery data (benign tumours versus ovarian cancer stages III-IV) and identified a model consisted of 8 proteins. We finally added three proteins (WFDC2, KRT19 and FR-alpha) based on their previous association with ovarian cancer stages I-II in our modelling, or in previous literature 9,12,13 . The 11-protein panel consisted of MUCIN-16, SPINT1, TACSTD2, CLEC6A, ICOSLG, MSMB, PROK1, CDH3, WFDC2, KRT19 and FR-alpha.…”
Section: Proof-of-concept Model For Practical Usementioning
Ovarian cancer is usually detected at a late stage with the 5-year survival at only 30-40%.Additional means for early detection and improved diagnosis are acutely needed. To search for novel biomarkers, we compared circulating plasma levels of 981 proteins in patients with ovarian cancer and benign tumours, using the proximity extension assay. A novel combinatorial strategy was developed for identification of multivariate biomarker signatures, resulting in 484 mutually exclusive models out of which 448 did not contain the present biomarker MUCIN-16. The top-ranking model consisted of 14 proteins and had a AUC=0.95, PPV=1.0, sensitivity=0.99 and specificity=1.0 for detection of stage III-IV ovarian cancer in the discovery data, and an AUC=0.89, PPV=0.93, sensitivity=0.89 and specificity=0.95 in the replication data. The novel plasma protein signature could be used to improve the diagnosis of women with adnexal ovarian mass or in screening to identify women that should be referred to specialized examination.
Effective extension of mass spectrometry-based proteomics to single cells remains challenging. Herein we combined microfluidic nanodroplet technology with tandem mass tag (TMT) isobaric labeling to significantly improve analysis throughput and proteome coverage for single mammalian cells. Isobaric labeling facilitated multiplex analysis of single cell-sized protein quantities to a depth of ~1,600 proteins with median CV of 10.9% and correlation coefficient of 0.98. To demonstrate in-depth high throughput single cell analysis, the platform was applied to measure protein expression in 72 single cells from three murine cell populations (epithelial, immune, and endothelial cells) in <2 days instrument time with over 2,300 proteins identified. Principal component analysis grouped the single cells into three distinct populations based on protein expression with each population characterized by well-known cell-type specific markers. Our platform enables high throughput and unbiased characterization of single cell heterogeneity at the proteome level.
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