Proximity Extension Assay in Combination with Next-Generation Sequencing for High-throughput Proteome-wide Analysis.

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.

Identification of Candidate Plasma Protein Biomarkers for Cervical Cancer Using the Multiplex Proximity Extension Assay.

Human papillomavirus (HPV) is recommended as the primary test in cervical cancer screening, with co-testing by cytology for HPV-positive women to identify cervical lesions. Cytology has low sensitivity and there is a need to identify biomarkers that could identify dysplasia that are likely to progress to cancer. We searched for plasma proteins that could identify women with cervical cancer using the multiplex proximity extension assay (PEA). The abundance of 100 proteins were measured in plasma collected at the time of diagnosis of patients with invasive cervical cancer and in population controls using the Olink Multiplex panels CVD II, INF I, and ONC II. Eighty proteins showed increased levels in cases compared with controls. We identified a signature of 11 proteins (PTX3, ITGB1BP2, AXIN1, STAMPB, SRC, SIRT2, 4E-BP1, PAPPA, HB-EGF, NEMO and IL27) that distinguished cases and controls with a sensitivity of 0.96 at a specificity of 1.0. This signature was evaluated in a prospective replication cohort with samples collected before, at or after diagnosis and achieved a sensitivity of 0.78 and a specificity 0.56 separating samples collected at the time of diagnosis of invasive cancer from samples collected prior to diagnosis. No difference in abundance was seen between samples collected prior to diagnosis or after treatment as compared with population controls, indicating that this protein signature is mainly informative close to time of diagnosis. Further studies are needed to determine the optimal window in time prior to diagnosis for these biomarker candidates.

A two-step strategy for identification of plasma protein biomarkers for endometrial and ovarian cancer.

BACKGROUND: Over 500,000 women worldwide are diagnosed with ovarian or endometrial cancer each year. We have used a two-step strategy to identify plasma proteins that could be used to improve the diagnosis of women with an indication of gynecologic tumor and in population screening. METHODS: In the discovery step we screened 441 proteins in plasma using the proximity extension assay (PEA) and five Olink Multiplex assays (CVD II, CVD III, INF I, ONC II, NEU I) in women with ovarian cancer (n = 106), endometrial cancer (n = 74), benign ovarian tumors (n = 150) and healthy population controls (n = 399). Based on the discovery analyses a set of 27 proteins were selected and two focused multiplex PEA assays were developed. In a replication step the focused assays were used to study an independent set of cases with ovarian cancer (n = 280), endometrial cancer (n = 228), women with benign ovarian tumors (n = 76) and healthy controls (n = 57). RESULTS: In the discovery step, 27 proteins that showed an association to cancer status were identified. In the replication analyses, the focused assays distinguished benign tumors from ovarian cancer stage III-IV with a sensitivity of 0.88 and specificity of 0.92 (AUC = 0.92). The assays had a significantly higher AUC for distinguishing benign tumors from late stage ovarian cancer than using CA125 and HE4 (p = 9.56e-22). Also, population controls could be distinguished from ovarian cancer stage III-IV with a sensitivity of 0.85 and a specificity of 0.92 (AUC = 0.89). CONCLUSION: The PEA assays represent useful tools for identification of new biomarkers for gynecologic cancers. The selected protein assays could be used to distinguish benign tumors from ovarian and endometrial cancer in women diagnosed with an unknown suspicious pelvic mass. The panels could also be used in population screening, for identification of women in need of specialized gynecologic transvaginal ultrasound examination. FUNDING: The Swedish Cancer Foundation, Vinnova (SWELIFE), The Foundation for Strategic Research (SSF), Assar Gabrielsson Foundation.

Detection of serological biomarkers by proximity extension assay for detection of colorectal neoplasias in symptomatic individuals.

BACKGROUND: Although the potential of biomarkers to aid in early detection of colorectal cancer (CRC) is recognized and numerous biomarker candidates have been reported in the literature, to date only few molecular markers have been approved for daily clinical use. METHODS: In order to improve the translation of biomarkers from the bench to clinical practice we initiated a biomarker study focusing on a novel technique, the proximity extension assay, with multiplexing capability and the possible additive effect obtained from biomarker panels. We performed a screening of 74 different biomarkers in plasma derived from a case-control sample set consisting of symptomatic individuals representing CRC patients, patients with adenoma, patients with non-neoplastic large bowel diseases and healthy individuals. RESULTS: After statistical evaluation we found 12 significant indicators of CRC and the receiver operating characteristic (ROC) curve of Carcinoembryonic antigen (CEA), Transferrin Receptor-1 (TFRC), Macrophage migration inhibitory factor (MIF), Osteopontin (OPN/SPP1) and cancer antigen 242 (CA242) showed additive effect. This biomarker panel identified CRC patients with a sensitivity of 56% at 90% specificity and thus the performance is sufficiently high to further investigate this combination of five proteins as serological biomarkers for detection of CRC. Furthermore, when applying the indicators to identify early-stage CRC a combination of CEA, TFRC and CA242 resulted in a ROC curve with an area under the curve of 0.861. CONCLUSIONS: Five plasma protein biomarkers were found to be potential CRC discriminators and three of these were additionally found to be discriminators of early-stage CRC. These explorative data in symptomatic individuals demonstrates the feasibility of the multiplex proximity extension assay for screening of potential serological protein biomarkers and warrants independent analyses in a larger sample cohort, including asymptomatic individuals, to further validate the performances of our CRC biomarker panel.

Plasma levels of the MMP-9:TIMP-1 complex as prognostic biomarker in breast cancer: a retrospective study.

BACKGROUND: Worldwide more than one million women are annually diagnosed with breast cancer. A considerable fraction of these women receive systemic adjuvant therapy; however, some are cured by primary surgery and radiotherapy alone. Prognostic biomarkers guide stratification of patients into different risk groups and hence improve management of breast cancer patients. Plasma levels of Matrix Metalloproteinase-9 (MMP-9) and its natural inhibitor Tissue inhibitor of metalloproteinase-1 (TIMP-1) have previously been associated with poor patient outcome and resistance to certain forms of chemotherapy. To pursue additional prognostic information from MMP-9 and TIMP-1, the level of the MMP-9 and TIMP-1 complex (MMP-9:TIMP-1) was investigated in plasma from breast cancer patients. METHODS: Detection of protein:protein complexes in plasma was performed using a commercially available ELISA kit and, for the first time, the highly sensitive in-solution proximity ligation assay (PLA). We screened plasma from 465 patients with primary breast cancer for prognostic value of the MMP-9:TIMP-1 complex. Both assays were validated and applied for quantification of MMP-9:TIMP-1 concentration. In this retrospective study, we analyzed the association between the concentration of the MMP-9:TIMP-1 complex and clinicopathological data and disease free survival (DFS) in univariate and multivariate survival analyses. RESULTS: Following successful validation both assays were applied for MMP-9:TIMP-1 measurements. Of the clinicopathological parameters, only menopausal status demonstrated significant association with the MMP-9:TIMP-1 complex; P = 0.03 and P = 0.028 for the ELISA and PLA measurements, respectively. We found no correlation between the MMP-9:TIMP-1 protein complex and DFS neither in univariate nor in multivariate survival analyses. CONCLUSIONS: Despite earlier reports linking MMP-9 and TIMP-1 with prognosis in breast cancer patients, we here demonstrate that plasma levels of the MMP-9:TIMP-1 protein complex hold no prognostic information in primary breast cancer as a stand-alone marker. We demonstrate that the highly sensitive in-solution PLA can be employed for measurements of protein:protein complexes in plasma.

Multiplexed homogeneous proximity ligation assays for high-throughput protein biomarker research in serological material.

A high throughput protein biomarker discovery tool has been developed based on multiplexed proximity ligation assays in a homogeneous format in the sense of no washing steps. The platform consists of four 24-plex panels profiling 74 putative biomarkers with sub-pm sensitivity each consuming only 1 µl of human plasma sample. The system uses either matched monoclonal antibody pairs or the more readily available single batches of affinity purified polyclonal antibodies to generate the target specific reagents by covalently linking with unique nucleic acid sequences. These paired sequences are united by DNA ligation upon simultaneous target binding forming a PCR amplicon. Multiplex proximity ligation assays thereby converts multiple target analytes into real-time PCR amplicons that are individually quantified using microfluidic high capacity qPCR in nano liter volumes. The assay shows excellent specificity, even in multiplex, by its dual recognition feature, its proximity requirement, and most importantly by using unique sequence specific reporter fragments on both antibody-based probes. To illustrate the potential of this protein detection technology, a pilot biomarker research project was performed using biobanked plasma samples for the detection of colorectal cancer using a multivariate signature.

Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood.

Convenient and well-performing protein detection methods for a wide range of targets are in great demand for biomedical research and future diagnostics. Assays without the need for washing steps while still unaffected when analyzing complex biological samples are difficult to develop. Herein, we report a well-characterized nucleic acid proximity-based assay using antibodies, called Proximity Extension Assay (PEA), showing good performance in plasma samples. Target-specific antibody pairs are linked to DNA strands that upon simultaneous binding to the target analyte create a real-time PCR amplicon in a proximity-dependent manner enabled by the action of a DNA polymerase. 3'Exonuclease-capable polymerases were found to be clearly superior in sensitivity over non-3'exonuclease ones. A PEA was set up for IL-8 and GDNF in a user-friendly, homogenous assay displaying femtomolar detection sensitivity, good recovery in human plasma, high specificity and up to 5-log dynamic range in 1 µL samples. Furthermore, we have illustrated the use of a macro-molecular crowding matrix in combination with this homogeneous assay to drive target binding for low-affinity antibodies, thereby improving the sensitivity and increasing affinity reagent availability by lowering assay development dependency on high-affinity antibodies. Assay performance was also confirmed for a multiplex version of PEA.

Development and validation of a quantitative Proximity Extension Assay instrument with 21 proteins associated with cardiovascular risk (CVD-21).

BACKGROUND: Treatment of cardiovascular diseases (CVD) is a substantial burden to healthcare systems worldwide. New tools are needed to improve precision of treatment by optimizing the balance between efficacy, safety, and cost. We developed a high-throughput multi-marker decision support instrument which simultaneously quantifies proteins associated with CVD. METHODS AND FINDINGS: Candidate proteins independently associated with different clinical outcomes were selected from clinical studies by the screening of 368 circulating biomarkers. We then custom-designed a quantitative PEA-panel with 21 proteins (CVD-21) by including recombinant antigens as calibrator samples for normalization and absolute quantification of the proteins. The utility of the CVD-21 tool was evaluated in plasma samples from a case-control cohort of 4224 patients with chronic coronary syndrome (CCS) using multivariable Cox regression analyses and machine learning techniques. The assays in the CVD-21 tool gave good precision and high sensitivity with lower level of determination (LOD) between 0.03-0.7 pg/ml for five of the biomarkers. The dynamic range for the assays was sufficient to accurately quantify the biomarkers in the validation study except for troponin I, which in the modeling was replaced by high-sensitive cardiac troponin T (hs-TnT). We created seven different multimarker models, including a reference model with NT-proBNP, hs-TnT, GDF-15, IL-6, and cystatin C and one model with only clinical variables, for the comparison of the discriminative value of the CVD-21 tool. All models with biomarkers including hs-TnT provided similar discrimination for all outcomes, e.g. c-index between 0.68-0.86 and outperformed models using only clinical variables. Most important prognostic biomarkers were MMP-12, U-PAR, REN, VEGF-D, FGF-23, TFF3, ADM, and SCF. CONCLUSIONS: The CVD-21 tool is the very first instrument which with PEA simultaneously quantifies 21 proteins with associations to different CVD. Novel pathophysiologic and prognostic information beyond that of established biomarkers were identified by a number of proteins.

Improving lithium dose prediction using population pharmacokinetics and pharmacogenomics: a cohort genome-wide association study in Sweden.

BACKGROUND: Lithium is the most effective treatment for bipolar disorder, resulting in strong suicide prevention effects. The therapeutic range of lithium, however, is narrow and treatment initiation requires individual titration to address inter-individual variability. We aimed to improve lithium dose prediction using clinical and genomic data. METHODS: We performed a population pharmacokinetic study followed by a genome-wide association study (GWAS), including two clinical Swedish cohorts. Participants in cohort 1 were from specialised outpatient clinics at Huddinge Hospital, in Stockholm, Sweden, and participants in cohort 2 were identified using the Swedish National Quality Registry for Bipolar disorder (BipoläR). Patients who received a lithium dose corresponding to at least one tablet of lithium sulphate (6 mmol) per day and had clinically relevant plasma concentrations of lithium were included in the study. Data on age, sex, bodyweight, height, creatinine concentration, estimated glomerular filtration rate (eGFR), lithium preparation, number of tablets of lithium per day, serum lithium concentration, and medications affecting kidney function (C09 antihypertensives, C03 [except C03D] sodium-retaining diuretics, and non-steroidal anti-inflammatory drugs) were obtained retrospectively for several timepoints when possible from electronic health records, BipoläR, and the Swedish prescription registry. The median time between timepoints was 1·07 years for cohort 1 and 1·09 years for cohort 2. The primary outcome of interest was the natural logarithm of total body clearance for lithium (CLLi) associated with the clinical variables. The residual effects after accounting for age and sex, representing the individual-level effects (CLLi,age/sex), were used as the dependent variable in a GWAS. FINDINGS: 2357 patients who were administered lithium (1423 women [60·4%] and 934 men [39·6%]; mean age 53·6 years [range 17-89], mainly of European descent) were included and 5627 data points were obtained. Age (variance explained [R2]: R2cohort1=0·41 and R2cohort2=0·31; both p<0·0001), sex (R2cohort1=0·0063 [p=0·045] and R2cohort2=0·026 [p<0·0001]), eGFR (R2cohort1=0·38 and R2cohort2=0·20; both p<0·0001), comedication with diuretics (R2cohort1=0·0058 [p=0·014] and R2cohort2=0·0026 [p<0·0001]), and agents acting on the renin-aldosterone-angiotensin system (R2cohort1=0·028 and R2cohort2=0·015; both p<0·0001) were clinical predictors of CLLi. Notably, an association between CLLi and serum lithium was observed, with a lower CLLi being associated with higher serum lithium (R2cohort1=0·13 and R2cohort2=0·15; both p<0·0001). In a GWAS of CLLi,age/sex, one locus was associated with a change in CLLi (rs583503; ß=-0·053 [95% CI -0·071 to -0·034]; p<0·00000005). We also found enrichment of the associations with genes expressed in the medulla (p=0·0014, corrected FDR=0·04) and cortex of the kidney (p=0·0015, corrected FDR=0·04), as well as associations with polygenic risk scores for eGFR (p value threshold: 0·05, p=0·01), body-mass index (p value threshold: 0·05, p=0·00025), and blood urea nitrogen (p value threshold: 0·001, p=0·00043). The model based on six clinical predictors explained 61·4% of the variance in CLLi in cohort 1 and 49·8% in cohort 2. Adding genetic markers did not lead to major improvement of the models: within the subsample of genotyped individuals, the variance explained only increased from 59·32% to 59·36% in cohort 1 and from 49·21% to 50·03% in cohort 2 when including rs583503 and the four first principal components. INTERPRETATION: Our model predictors could be used clinically to better guide lithium dosage, shortening the time to reach therapeutic concentrations, thus improving care. Identification of the first genomic locus and PRS to be associated with CLLi introduces the opportunity of individualised medicine in lithium treatment. FUNDING: Stanley Medical Research Institute, Swedish Research Council, Swedish Foundation for Strategic Research, Swedish Brain Foundation, Swedish Research Council, Söderström-Königska Foundation, Bror Gadelius Minnesfond, Swedish Mental Health Fund, Karolinska Institutet and Hospital.

Lithium and the Interplay Between Telomeres and Mitochondria in Bipolar Disorder.

Bipolar disorder is a severe psychiatric disorder which affects more than 1% of the world's population and is a leading cause of disability among young people. For the past 50 years, lithium has been the drug of choice for maintenance treatment of bipolar disorder due to its potent ability to prevent both manic and depressive episodes as well as suicide. However, though lithium has been associated with a multitude of effects within different cellular pathways and biological systems, its specific mechanism of action in stabilizing mood remains largely elusive. Mitochondrial dysfunction and telomere shortening have been implicated in both the pathophysiology of bipolar disorder and as targets of lithium treatment. Interestingly, it has in recent years become clear that these phenomena are intimately linked, partly through reactive oxygen species signaling and the subcellular translocation and non-canonical actions of telomerase reverse transcriptase. In this review, we integrate the current understanding of mitochondrial dysfunction, oxidative stress and telomere shortening in bipolar disorder with documented effects of lithium. Moreover, we propose that lithium's mechanism of action is intimately connected with the interdependent regulation of mitochondrial bioenergetics and telomere maintenance.

Expression of telomerase reverse transcriptase positively correlates with duration of lithium treatment in bipolar disorder.

Bipolar disorder (BD) may be associated with accelerated cellular aging. However, previous studies on telomere length (TL), an important biomarker of cellular aging, have yielded mixed results in BD. We aimed to evaluate the hypothesis that BD is associated with telomere shortening and whether this is counteracted by long-term lithium treatment. We also sought to determine whether long-term lithium treatment is associated with increased expression of telomerase reverse transcriptase (TERT), the catalytic subunit of telomerase. We determined TL and TERT expression in 100 BD I patients and 100 healthy controls. We also genotyped three single nucleotide polymorphisms associated with TL. TERT expression was significantly increased in BD I patients currently on lithium treatment. TERT expression was also significantly positively correlated with duration of lithium treatment in patients treated for 24 months or more. However, we did not find any significant effect of lithium treatment on TL. Neither did we find significant differences in TL between BD patients and controls. We suggest that long-term lithium treatment is associated with an increase in the expression of TERT. We hypothesize that an increase in TERT expression may contribute to lithium's mood stabilizing and neuroprotective properties by improving mitochondrial function and decreasing oxidative stress.

High throughput proteomics identifies a high-accuracy 11 plasma protein biomarker signature for ovarian cancer.

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.

Simultaneous Multiplexed Measurement of RNA and Proteins in Single Cells.

Significant advances have been made in methods to analyze genomes and transcriptomes of single cells, but to fully define cell states, proteins must also be accessed as central actors defining a cell's phenotype. Methods currently used to analyze endogenous protein expression in single cells are limited in specificity, throughput, or multiplex capability. Here, we present an approach to simultaneously and specifically interrogate large sets of protein and RNA targets in lysates from individual cells, enabling investigations of cell functions and responses. We applied our method to investigate the effects of BMP4, an experimental therapeutic agent, on early-passage glioblastoma cell cultures. We uncovered significant heterogeneity in responses to treatment at levels of RNA and protein, with a subset of cells reacting in a distinct manner to BMP4. Moreover, we found overall poor correlation between protein and RNA at the level of single cells, with proteins more accurately defining responses to treatment.

Multiplexed, targeted profiling of single-cell proteomes and transcriptomes in a single reaction.

We present a scalable, integrated strategy for coupled protein and RNA detection from single cells. Our approach leverages the DNA polymerase activity of reverse transcriptase to simultaneously perform proximity extension assays and complementary DNA synthesis in the same reaction. Using the Fluidigm C1™ system, we profile the transcriptomic and proteomic response of a human breast adenocarcinoma cell line to a chemical perturbation, benchmarking against in situ hybridizations and immunofluorescence staining, as well as recombinant proteins, ERCC Spike-Ins, and population lysate dilutions. Through supervised and unsupervised analyses, we demonstrate synergies enabled by simultaneous measurement of single-cell protein and RNA abundances. Collectively, our generalizable approach highlights the potential for molecular metadata to inform highly-multiplexed single-cell analyses.

Homogenous 96-plex PEA immunoassay exhibiting high sensitivity, specificity, and excellent scalability.

Medical research is developing an ever greater need for comprehensive high-quality data generation to realize the promises of personalized health care based on molecular biomarkers. The nucleic acid proximity-based methods proximity ligation and proximity extension assays have, with their dual reporters, shown potential to relieve the shortcomings of antibodies and their inherent cross-reactivity in multiplex protein quantification applications. The aim of the present study was to develop a robust 96-plex immunoassay based on the proximity extension assay (PEA) for improved high throughput detection of protein biomarkers. This was enabled by: (1) a modified design leading to a reduced number of pipetting steps compared to the existing PEA protocol, as well as improved intra-assay precision; (2) a new enzymatic system that uses a hyper-thermostabile enzyme, Pwo, for uniting the two probes allowing for room temperature addition of all reagents and improved the sensitivity; (3) introduction of an inter-plate control and a new normalization procedure leading to improved inter-assay precision (reproducibility). The multiplex proximity extension assay was found to perform well in complex samples, such as serum and plasma, and also in xenografted mice and resuspended dried blood spots, consuming only 1 µL sample per test. All-in-all, the development of the current multiplex technique is a step toward robust high throughput protein marker discovery and research.

Growth in pre-pubertal children with myelomeningocele (MMC) on growth hormone (GH): the KIGS experience.

PURPOSE: To analyse the auxological data of children with myelomeningocele (MMC) on growth hormone (GH) therapy whose growth data was documented within KIGS (Pfizer International Growth Database). Longitudinal growth data of a sub-group of pre-pubertal children were studied after a treatment period of 3 years. PATIENTS AND METHODS: Eighty patients (38 m, 42 f) with MMC with a median chronological age (CA) of 11.6 years (at latest visit) on GH were registered in the KIGS database. In 52 patients, GH deficiency was documented. GH therapy started with a median dose of 0.23 mg kg(-1) per week. The 3-year longitudinal growth was analysed in 21 patients (13 m, 8 f; median CA 9.2 years, latest visit), all of whom were pre-pubertal at start and during GH therapy. RESULTS: GH therapy started at 7.5 years with a dose of 0.23 mg kg(-1) per week. Birth length SDS (-0.51) and mid-parental height SDS (+0.07) were in the normal range. BMI SDS at start was +0.24, at latest visit -0.03. After a median treatment duration of 3.0 years (latest visit), height SDS improved from -2.97 (start of GH) to -2.01. The sub-group of pre-pubertal MMC patients started GH therapy (dose 0.22 mg kg(-1) per week) at 6.2 years. Growth velocity (GV) SDS increased significantly (at start: -1.77; 1 year: +2.60, 2 years: +2.25, 3 years: +1.24), thus height SDS improved from -3.25 at start to -1.87 at 36 months. BMI SDS was in the normal range and remained unchanged during GH therapy. No major side effects of GH were recorded. CONCLUSION: GH had positive effects on height SDS in MMC patients. The analysis of the longitudinal growth data of pre-pubertal MMC patients showed a significant increase in GV SDS and improvement of height SDS.