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Multiplex targeted proteomic assay for biomarker detection in plasma: a pancreatic cancer biomarker case study.

ABSTRACT: Biomarkers are most frequently proteins that are measured in the blood. Their development largely relies on antibody creation to test the protein candidate performance in blood samples of diseased versus nondiseased patients. The creation of such antibody assays has been a bottleneck in biomarker progress due to the cost, extensive time, and effort required to complete the task. Targeted proteomics is an emerging technology that is playing an increasingly important role to facilitate disease biomarker development. In this study, we applied a SRM-based targeted proteomics platform to directly detect candidate biomarker proteins in plasma to evaluate their clinical utility for pancreatic cancer detection. The characterization of these protein candidates used a clinically well-characterized cohort that included plasma samples from patients with pancreatic cancer, chronic pancreatitis, and healthy age-matched controls. Three of the five candidate proteins, including gelsolin, lumican, and tissue inhibitor of metalloproteinase 1, demonstrated an AUC value greater than 0.75 in distinguishing pancreatic cancer from the controls. In addition, we provide an analysis of the reproducibility, accuracy, and robustness of the SRM-based proteomics platform. This information addresses important technical issues that could aid in the adoption of the targeted proteomics platform for practical clinical utility.

SUBMITTER: Pan S

PROVIDER: S-EPMC3292708 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Multiplex targeted proteomic assay for biomarker detection in plasma: a pancreatic cancer biomarker case study.

Pan Sheng S Chen Ru R Brand Randall E RE Hawley Sarah S Tamura Yasuko Y Gafken Philip R PR Milless Brian P BP Goodlett David R DR Rush John J Brentnall Teresa A TA

Journal of proteome research 20120208 3

Biomarkers are most frequently proteins that are measured in the blood. Their development largely relies on antibody creation to test the protein candidate performance in blood samples of diseased versus nondiseased patients. The creation of such antibody assays has been a bottleneck in biomarker progress due to the cost, extensive time, and effort required to complete the task. Targeted proteomics is an emerging technology that is playing an increasingly important role to facilitate disease bio ...[more]

PMID: 22316387

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Project description:Molecular diagnostic testing of KRAS and BRAF mutations has become critical in the management of colorectal cancer (CRC) patients. Some progress has been made in liquid biopsy detection of mutations in circulating tumor DNA (ctDNA), which is a fraction of circulating cell-free DNA (cfDNA), but slow analysis for DNA sequencing methods has limited rapid diagnostics. Other methods such as quantitative PCR and more recently, droplet digital PCR (ddPCR), have limitations in multiplexed capacity and the need for expensive specialized equipment. Hence, a robust, rapid and facile strategy is needed for detecting multiple ctDNA mutations to improve the management of CRC patients. To address this significant problem, herein, we propose a new application of multiplex PCR/SERS (surface-enhanced Raman scattering) assay for the detection of ctDNA in CRC, in a fast and non-invasive manner to diagnose and stratify patients for effective treatment. Methods: To discriminate ctDNA mutations from wild-type cfDNA, allele-specific primers were designed for the amplification of three clinically important DNA point mutations in CRC including KRAS G12V, KRAS G13D and BRAF V600E. Surface-enhanced Raman scattering (SERS) nanotags were labelled with a short and specific sequence of oligonucleotide, which can hybridize with the corresponding PCR amplicons. The PCR/SERS assay was implemented by firstly amplifying the multiple mutations, followed by binding with multicolor SERS nanotags specific to each mutation, and subsequent enrichment with magnetic beads. The mutation status was evaluated using a portable Raman spectrometer where the fingerprint spectral peaks of the corresponding SERS nanotags indicate the presence of the mutant targets. The method was then applied to detect ctDNA from CRC patients under a blinded test, the results were further validated by ddPCR. Results: The PCR/SERS strategy showed high specificity and sensitivity for genotyping CRC cell lines and plasma ctDNA, where as few as 0.1% mutant alleles could be detected from a background of abundant wild-type cfDNA. The blinded test using 9 samples from advanced CRC patients by PCR/SERS assay was validated with ddPCR and showed good consistency with pathology testing results. Conclusions: With ddPCR-like sensitivity yet at the convenience of standard PCR, the proposed assay shows great potential in sensitive detection of multiple ctDNA mutations for clinical decision-making.

Dataset Information

Multiplex targeted proteomic assay for biomarker detection in plasma: a pancreatic cancer biomarker case study.

Publications

Multiplex targeted proteomic assay for biomarker detection in plasma: a pancreatic cancer biomarker case study.

Similar Datasets

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