John Broberg

Understanding the dynamics of the human proteome is crucial for developing biomarkers to be used as measurable indicators for disease severity and progression, patient stratification, and drug development. The Proximity Extension Assay (PEA) is a technology that translates protein information into actionable knowledge by linking protein-specific antibodies to DNA-encoded tags. In this report we demonstrate how we have combined the unique PEA technology with an innovative and automated sample preparation and high-throughput sequencing readout enabling parallel measurement of nearly 1500 proteins in 96 samples generating close to 150,000 data points per run. This advancement will have a major impact on the discovery of new biomarkers for disease prediction and prognosis and contribute to the development of the rapidly evolving fields of wellness monitoring and precision medicine.

Ovarian cancer is usually detected at a late stage and the overall 5-year survival is 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 593 proteins in three cohorts of patients with ovarian cancer and benign tumors, using the proximity extension assay (PEA). A combinatorial strategy was developed for identification of different multivariate biomarker signatures. A final model consisting of 11 biomarkers plus age was developed into a multiplex PEA test reporting in absolute concentrations. The final model was evaluated in a fourth independent cohort and has an AUC = 0.94, PPV = 0.92, sensitivity = 0.85 and specificity = 0.93 for detection of ovarian cancer stages I-IV. 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.

Background A number of factors regarding blood collection, handling and storage may affect sample quality. The purpose of this study was to assess the impact on plasma protein profiles by delayed centrifugation and plasma separation and multiple freeze-thaw cycles. Methods Blood samples drawn from 16 healthy individuals were collected into ethylenediaminetetraacetic acid tubes and kept either at 4 °C or 22 °C for 1-36 h prior to centrifugation. Plasma samples prepared 1 h after venipuncture were also subjected to two to eight cycles of freezing at -80 °C and thawing at 22 °C. Multiplex proximity extension assay, an antibody-based protein assay, was used to investigate the influence on plasma proteins. Results Up to 36 h delay before blood centrifugation resulted in significant increases of 16 and 40 out of 139 detectable proteins in samples kept at 4 °C or 22 °C, respectively. Some increases became noticeable after 8 h delay at 4 °C but already after 1 h at 22 °C. For samples stored at 4 °C, epidermal growth factor (EGF), NF-kappa-B essential modulator, SRC, interleukin 16 and CD6 increased the most, whereas the five most significantly increased proteins after storage at 22 °C were CD40 antigen ligand (CD40-L), EGF, platelet-derived growth factor subunit B, C-X-C motif chemokine ligand 5 and matrix metallopeptidase 1 (MMP1). Only matrix metallopeptidase 7 (MMP7) decreased significantly over time and only after storage at 22 °C. No protein levels were found to be significantly affected by up to eight freeze-thaw cycles. Conclusions Plasma should be prepared from blood after a limited precentrifugation delay at a refrigerated temperature. By contrast, the influence by several freeze-thaw cycles on detectable protein levels in plasma was negligible.

Abstract 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.

The in situ proximity ligation assay (in situ PLA) is an immunoassay that enables directvisualisation of single protein targets or protein interactions in cell or tissue samples. This project revolves around designing and introducing several novel multiplexable components tobe used in conjunction with Olink Bioscience's Duolink product line. In this report, a novel in silico approach to DNA oligomer interaction design is presented. Using this in silico method, a multiplexed system of DNA oligomers has been designed andevaluated using in situ PLA and fluorescence microscopy.