Genome-wide cell-free DNA screening: a focus on copy-number variants

Purpose Of 86,902 prenatal genome-wide cell-free DNA (cfDNA) screening tests, 4,121 were positive for a chromosome abnormality. This study examines 490 cases screen-positive for one or more subchromosomal copy-number variants (CNV) from genome-wide cfDNA screening. Methods Cases positive for one or more subchromosomal CNV from genome-wide cfDNA screening and diagnostic outcomes were compiled. Diagnostic testing trends were analyzed, positive predictive values (PPVs) were calculated, and the type of chromosomal abnormalities ultimately confirmed by diagnostic testing were described. Results CNVs were identified in 0.56% of screened specimens. Of the 490 cases screen-positive for one or more CNV, diagnostic outcomes were available for 244 cases (50%). The overall PPV among the cases with diagnostic outcomes was 74.2% (95% CI: 68.1–79.5%) and 71.8% (95% CI: 65.5–77.4%) for “fetal-only” events. Overall, isolated CNVs showed a lower PPV of 61.0% (95% CI: 52.5–68.8%) compared to complex CNVs at 93.9% (95% CI: 86.6–97.5%). Isolated deletions/duplications and unbalanced structural rearrangements were the most common diagnostic outcomes when isolated and complex CNVs were identified by cfDNA screening, respectively. Conclusion Genome-wide cfDNA screening identifies chromosomal abnormalities beyond the scope of traditional cfDNA screening, and the overall PPV associated with subchromosomal CNVs in cases with diagnostic outcomes was >70%.

Three years of clinical experience with a genome-wide cfDNA screening test for aneuploidies and copy-number variants

Purpose Pregnant women have unprecedented choices for prenatal screening and testing. Cell-free DNA (cfDNA) offers the option to screen for aneuploidy of all chromosomes and genome-wide copy-number variants (CNVs), expanding screening beyond the common trisomies (“traditional” cfDNA). We sought to review the utilization trends and clinical performance characteristics of a commercially available genome-wide cfDNA test, with a subset having available diagnostic testing outcomes. Methods Retrospective analysis of 55,517 samples submitted for genome-wide cfDNA screening at a commercial laboratory, assessing indications, demographics, results, and performance. The cohort was broken into three “testing years”’ to compare trends. Results Indications shifted over time, with a decrease in referrals for ultrasound findings (22.0% to 12.0%) and an increase in no known high-risk indication (3.0% to 16.6%). Of the positive results, 25% would be missed with traditional cfDNA screening. High sensitivity and specificity were observed with a positive predictive value (PPV) of 72.6% for genome-wide CNVs and 22.4% for rare autosomal trisomies (RATs). Conclusion A broader patient population is utilizing genome-wide cfDNA, yet positivity rates and the contribution of genome-wide events have remained stable at approximately 5% and 25%, respectively. Test performance in a real-world clinical population shows high PPVs in those CNVs tested, with diagnostic outcomes in over 40% of positive cases.

Application of mosaicism ratio to multifetal gestations

Mosaicism ratio, or MR, is a laboratory metric that can be calculated using massively parallel sequencing data from cell-free DNA (cfDNA) screening. MR compares the amount of cfDNA present from a particular chromosome or chromosomal region to the overall fetal fraction of the specimen. In singleton gestations, MR may be used to refine the positive predictive value of an abnormal cfDNA screening result by identifying cases that could be impacted by various biological factors, such as placental mosaicism or prior co-twin demise. The current study was designed to examine the behavior of mosaicism ratio (MR) in multifetal gestations. Multifetal cfDNA specimens with positive results for trisomies 21, 18, or 13 and confirmed diagnostic outcomes were compiled to examine MR of the aneuploid chromosome based on the number of affected fetuses/placentas. A second multifetal cohort was assembled to analyze the MR of the Y chromosome in cases with at least one male fetus. For aneuploid cases, the average MR of affected singletons (used as a biological proxy for two affected twins) was significantly higher than the average MR for twins in which one fetus was affected. The average MR of the aneuploid chromosome for one affected twin was 52%, 42%, and 48% of that of singleton gestations for trisomy 21, 18, and 13 cases, respectively. MR cutoffs of 0.7 for trisomy 21, and 0.5 for trisomies 18 and 13 may help predict whether one versus both twins are affected with aneuploidy when clinical concern arises. For male cases, the Y MR of XX/XY gestations was 48% of the Y MR for XY/XY gestations. Using a Y MR cutoff of 0.8 allowed determination of XX/XY versus XY/XY gestations with 92.3–94.9% accuracy. Based on the data presented, MR may have utility in the analysis and interpretation of cfDNA data from multifetal gestations.

Prenatal screening for trisomy 21: a comparative performance and cost analysis of different screening strategies

Background Prenatal screening for chromosome aneuploidies have constantly been evolving, especially with the introduction of cell-free fetal DNA (cfDNA) screening in the most recent years. This study compares the performance, costs and timing of test results of three cfDNA screening implementation strategies: contingent, reflex and primary. Methods We modelled enhanced first trimester screening (eFTS) as the first-tier test in contingent or reflex strategies. cfDNA test was performed contingent on or reflex from eFTS results. A comparison was made between cfDNA screening using sequencing technology and Rolling Circle Amplification (RCA)/imaging solution. All model assumptions were based on results from previous publications or information from the Ontario prenatal screening population. Results At an eFTS risk cut-off of ≥1/1000, contingent and reflex cfDNA screening have the same detection rate (DR) (94%) for trisomy 21. Reflex cfDNA screening using RCA/Imaging solution provided the lowest false positive rate and cost. The number of women requiring genetic counselling and diagnostic testing was significantly reduced and women received their cfDNA screening result 9 days sooner compared with the contingent model. While primary cfDNA screening improved the trisomy 21 DR by 3–5%, it was more costly and more women required diagnostic testing. Conclusion Reflex cfDNA screening is the most cost-effective prenatal screening strategy. It can improve the efficiency of prenatal aneuploidy screening by reducing the number of patient visits and providing more timely results.

Performance of a universally offered prenatal screening program incorporating cfDNA in Ontario, Canada: a descriptive population-based cohort study of 280,000 pregnancies.

Objectives: To measure the population-based performance and impact of Ontario, Canada's modified-contingent prenatal screening system for the detection of trisomies 21 (T21) and 18 (T18). Design: A retrospective, descriptive cohort study examining routinely collected data from BORN Ontario, which captures linkable population data for prenatal and neonatal health encounters across a variety of settings (e.g., laboratories, birthing hospitals and midwifery practice groups). Setting: A province-wide and publicly funded prenatal screening program in Ontario, Canada offering cfDNA screening for those at increased risk of having a pregnancy with T21 or T18. Participants: 373,682 singleton pregnancies with an estimated due date between September 1 2016 and March 31, 2019 who were offered publicly funded prenatal screening. Main outcome measures: Prenatal detection of T21 or T18, ascertained by cytogenetic results. Performance was assessed by calculating sensitivity, specificity, positive predictive value and negative predictive value against confirmatory diagnostic cytogenetic results and birth outcomes. The secondary objective was to determine the impact of contingent cfDNA screening by measuring uptake and the proportion of T21 screen-positive pregnancies undergoing subsequent cfDNA screening and invasive prenatal diagnostic testing (PND). Results: 69% of pregnancies in Ontario underwent prenatal screening for T21/T18. The modified-contingent screen sensitivity was 89.9% for T21 and 80.5% for T18. The modified-contingent screen-positive rate was 1.6% for T21 and 0.2% for T18. The cfDNA screening test failure rate was 2.2% (final result including multiple attempts). The PND rate among pregnancies screened was 2.4%. Conclusions: This study is the largest evaluation of population-based performance of a publicly funded cfDNA prenatal screening system. We demonstrated a robust screening system with high sensitivity and low PND consistent with smaller validation studies.

Clinical and Economic Evaluation after Adopting Contingent Cell-Free DNA Screening for Fetal Trisomies in South Spain

<b><i>Introduction:</i></b> Contingent cell-free (cf) DNA screening on the basis of the first-trimester combined test (FCT) results has emerged as a cost-effective strategy for screening of trisomy 21 (T21). <b><i>Objectives:</i></b> To assess performance, patients’ uptake, and cost of contingent cfDNA screening and to compare them with those of the established FCT. <b><i>Methods:</i></b> This is a prospective cohort study including all singleton pregnancies attending to their FCT for screening of T21 at 2 university hospitals in South Spain. When the FCT risk was ≥1:50, there were major fetal malformations, or the nuchal translucency was ≥3.5 mm, women were recommended invasive testing (IT); if the risk was between 1:50 and 1:270, women were recommended cfDNA testing; and for risks bellow 1:270, no further testing was recommended. Detection rate (DR), false-positive rate (FPR), patients’ uptake, and associated costs were evaluated. <b><i>Results:</i></b> We analyzed 10,541 women, including 46 T21 cases. DR of our contingent strategy was 89.1% (41/46) at 1.4% (146/10,541) FPR. Uptake of cfDNA testing was 91.2% (340/373), and overall IT rate was 2.0%. The total cost of our strategy was €1,462,895.7, similar to €1,446,525.7 had cfDNA testing not been available. <b><i>Conclusions:</i></b> Contingent cfDNA screening shows high DR, low IT rate, and high uptake at a similar cost than traditional screening.

Low Fetal Fraction and Birth Weight in Women with Negative First-Trimester Cell-Free DNA Screening

Objective To determine the association between low fetal fraction and birth weight among women with a negative cell-free DNA (cfDNA) result for common aneuploidies in the first trimester. Study Design This is a retrospective cohort of women who delivered a singleton between July 2016 and June 2018 at a single institution and had normal cfDNA testing in the first trimester. The primary variable of interest was “low fetal fraction,” which was defined as fetal fractions less than 5th percentile among all fetal fractions in the cohort (fetal fraction < 5.34%). The primary outcomes were birth weight ≤ 5th and ≤ 10th percentiles. Multivariable logistic regressions assessed for the association between low fetal fraction and birth weight. Results A total of 7,478 women delivered a singleton at ≥24 weeks' gestation, of which 2,387 (32%) underwent genetic screening through cfDNA; the majority were in the first trimester (n = 2,052 [86%]). 2,035 met the inclusion criteria. Birth weight ≤ 5th percentile was significantly higher in the low fetal fraction group (6.9 vs. 3.2%; p = 0.04). A low fetal fraction was associated with higher odds of an infant with a low birth weight: adjusted odds ratio (aOR) of 2.32 (95% CI 1.15–4.67) for birth weight ≤ 10th percentile (p = 0.02) and aOR of 3.73 (95% CI 1.40–9.03) for birth weight ≤ 5th percentile (p = 0.004). Conclusion Low fetal fractions of ≤ 5th percentile were associated with an increased risk of birth weights ≤ 5th and ≤ 10th percentiles in women with negative cfDNA screening in the first trimester. Future work is needed to further investigate this relationship and to determine the potential clinical implications, such as third-trimester screening for growth restriction in women with low fetal fractions and negative cfDNA screening results.