Assessment and Clinical Utility of a Non-Next-Generation Sequencing-Based Non-Invasive Prenatal Testing Technology

Abstract

Background: Rolling-circle replication (RCR) is a novel technology that has not been applied to cell-free DNA (cfDNA) testing until recently. Given the cost and simplicity advantages of this technology compared to other platforms currently used in cfDNA analysis, an assessment of RCR in clinical laboratories was performed. Here, we present the first validation study from clinical laboratories utilizing RCR technology. Methods: 831 samples from spontaneously pregnant women carrying a singleton fetus, and 25 synthetic samples, were analyzed for the fetal risk of trisomy 21 (T21), trisomy 18 (T18) and trisomy 13 (T13), by three laboratories on three continents. All the screen-positive pregnancies were provided post-test genetic counseling and confirmatory diagnostic invasive testing (e.g., amniocentesis). The screen-negative pregnancies were routinely evaluated at birth for fetal aneuploidies, using newborn examinations, and any suspected aneuploidies would have been offered diagnostic testing or confirmed with karyotyping. Results: The study found rolling-circle replication to be a highly viable technology for the clinical assessment of fetal aneuploidies, with 100% sensitivity for T21 (95% CI: 82.35–100.00%); 100.00% sensitivity for T18 (71.51–100.00%); and 100.00% sensitivity for T13 analyses (66.37–100.00%). The specificities were >99% for each trisomy (99.7% (99.01–99.97%) for T21; 99.5% (98.62–99.85%) for T18; 99.7% (99.03–99.97%) for T13), along with a first-pass no-call rate of 0.93%. Conclusions: The study showed that using a rolling-circle replication-based cfDNA system for the evaluation of the common aneuploidies would provide greater accuracy and clinical utility compared to conventional biochemical screening, and it would provide comparable results to other reported cfDNA methodologies.

A Critical Evaluation of Validation and Clinical Experience Studies in Non-Invasive Prenatal Testing for Trisomies 21, 18, and 13 and Monosomy X

“…Figure S1: Forest Plots of all Data. References [ 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 , 45 , 46 , 47 , 48 , 49 , 50 , 51 , 52 , 53 , 54 , 55 , 56 , 57 , 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 , …”

Non‐Invasive prenatal testing with rolling circle amplification: Real‐world clinical experience in a non‐molecular laboratory

“…However, an argument can be made that these samples could be considered as true additional FNs. To account for that possibility, if we compared the performance of the system with these FN excluded; with these FN included; and with how the system has performed based on a summary of the eight previously published Vanadis‐based studies, 10 , 11 , 12 , 13 , 17 , 18 , 19 , 20 it would show a consistent 99.8% specificity and sensitivities of 96.7%, 90.6%, and 99.5% respectively.…”

“… 10 , 11 While NGS‐based NIPT testing has been suggested to be this first‐line screen, some drawbacks have prevented its widespread implementation, including the cost and complexity of NGS analysis, which requires complex bioinformatic analysis, high run costs, and extensive hands‐on time. 12 With the lower degree of complexity and substantially automated workflow associated with the Vanadis system, this methodology provided a solution for our clinicians and patients. Our laboratory experienced a reduced start‐up time and decreased costs, by eliminating the need for building and developing dedicated pre‐ and post‐PCR rooms and complicated bioinformatics servers.…”

“… a Performance metrics as reported in literature and including our data. 10 , 11 , 12 , 13 , 17 , 18 , 19 , 20 b Performance metrics as reported in literature. 21 , 39 , 40 , 41 …”

“… 10 , 11 The Vanadis® system (PerkinElmer, Waltham, MA, USA) screens chromosomes by converting chromosome‐unique fragments into digitally quantifiable objects through rolling circle replication. 10 , 11 , 12 With this methodology, target fragments from a chromosome of interest are directly captured using specific probes before being converted into circular DNA. This DNA is then amplified via rolling circle replication to form a single rolling circle product (RCP), which is fluorescently labeled for counting by the imaging system.…”