Chad Bradford (Presenter)
Sera Prognostics, Inc.
Bio: Chad Bradford is experienced in the development and validation of mass spectrometry based analytical methods for protein biomarkers and small molecules, serving the clinical diagnostic and pharmaceutical industries.
Authorship: Chad L. Bradford (1), Robert D. Severinsen (1), Trina Pugmire (1), Tracey C. Fleischer (1), Ashoka D. Polpitiya (1), Jeff S. Flick (1), Angela Fox (1), Max T. Dufford (1), Durlin Hickok (1), J. Jay Boniface (1), Ilia Ichetovkin (1)
(1) Sera Prognostics, Inc.
| Short Abstract Spontaneous preterm delivery, the leading cause of mortality and morbidity in neonates, lacks an adequately performing diagnostic test. Targeted quantitative proteomics approaches allow for interrogating multiple biological pathways in a single assay. 5,501 pregnant women were enrolled in the Proteomic Assessment of Preterm Risk (PAPR) clinical trial. Utilizing subsets of PAPR serum samples and several QC pools, the performance of 147 candidate proteins was determined in development and verification studies. Using two proteins with acceptable performance, a PreTRM™ score for spontaneous preterm delivery risk was validated. |
Long Abstract
Spontaneous preterm delivery, a condition with complex etiological factors, is the leading cause of neonatal mortality and morbidity. Existing tests have inadequate performance especially for women expectant with a first child. A test based solely on clinical observations or a single analyte measurement has not had adequate performance for identification of asymptomatic women at high risk for spontaneous preterm delivery. A validated multi-analyte targeted quantitative proteomics assay would provide a significant improvement to current methodologies. The Proteomic Assessment of Preterm Risk (PAPR) clinical trial enrolled 5,501 pregnant women representative of the diverse U.S. population at 11 IRB approved sites. Maternal serum was collected at enrollment between 17 and 28 weeks gestational age, and patients were followed through for delivery outcome.
PAPR serum samples and quality control serum materials were diluted, filtered and then depleted of high-abundance proteins through the use of an automated immuno-depletion system. Fractions of samples containing proteins of interest were reduced, alkylated, and then digested overnight with porcine trypsin. Digested samples were fortified with a solution of pooled stable isotope standard (SIS) peptides, corresponding to the proteotypic peptide serving as a surrogate analyte for the protein of interest. Samples were split into two equal fractions to allow for repeat analysis. One set of the sample splits were desalted by solid phase extraction, lyophilized and reconstituted before liquid chromatography-mass spectrometry (LC-MS) analysis. The triple quadrupole mass spectrometer, served by a reverse-phase gradient LC, used a dynamic multiple reaction monitoring (MRM) method to monitor for 442 transitions representing 147 proteins. The response from two transitions for each light endogenous peptide analyte were normalized by the corresponding SIS response (same charge state and product ions used). One normalized transition response was used for quantitation, and the other was used for quality control purposes. The measured peptides served as surrogate analytes for biologically significant proteins as well as proteins monitored for process quality control. Original selection of targeted proteins was performed by both hypothesis dependent and independent approaches.
Prior discovery and verification studies involved the blinded analysis of 136 nested case (<37 weeks gestational age) and control samples (>37 weeks gestational age) from the PAPR trial. These studies provided the data needed to establish the performance of the 147 proteins as preterm birth risk biomarkers, as measured through the surrogate peptide analyte. Several peptide analytes were eliminated because of unacceptable analytical performance or analyte instability. The ability of an analyte to distinguish between case and control samples was measured individually using AUROC. Data on acceptable analytes from the discovery and verification studies showed 44 candidate biomarkers that could separate case samples from control samples.
The analytical performance of proteins and surrogate peptide analytes was established during an analytical validation. Performance aspects determined during analytical validation included intra- and inter-batch precision, endogenous interferents, short and long term stability, freeze/thaw stability, carryover, limit of detection, matrix specificity, and assessment of multiple serum processing tube types. An analyte passing all acceptance criteria was considered to have the analytical performance necessary for use as a candidate preterm delivery biomarker.
The 44 candidate biomarkers were separated into groups of up-regulated proteins and down-regulated proteins and combined into reversals (up-regulated protein response/down-regulated protein response). Reversals provided an amplified biological signal and helped to normalize variability in pre-analytical and analytical steps since both proteins were subjected to the source of variability. The predictive performance of each reversal was established by measuring the individual reversal AUROC in overlapping three week gestational age windows. The reversal combination with the highest AUROC was with the up-regulated insulin-type growth factor-binding protein (IBP4) and the down-regulated sex hormone binding globulin (SHBG) measured between 19 and 21 weeks gestation. The performance of this predictor was improved with a pre-pregnancy BMI stratification of >22 and
≤37 kg/m2. Other clinical parameters did not improve predictive performance.
Clinical validation involved the blinded assaying of 18 case samples and 36 control samples matched by gestational age at blood draw. The samples used for clinical validation were independent of the samples used during discovery, verification and analytical validation. The serum samples were processed with the validated analytical method and the resultant IBP4/SHBG reversal data was generated. The blinded dataset was delivered to two independent external statisticians and un-blinded. AUROC and significance testing results were generated. The AUROC was 0.75 (P = 0.02), sensitivity of 0.75, specificity of 0.74, a PPV of 0.152, and a NPV of 0.974. The validated test is currently being offered as the PreTRM™ test by Sera Prognostics CLIA Laboratory.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Sera Prognostics, Inc. |
| Board Member | no | |
| Stock | yes | Sera Prognostics, Inc. |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | yes |