Observed and Modeled Positive Predictive Values Using Cell-free DNA Testing for Fetal Trisomy in a Clinical Laboratory Population.

INTRODUCTION: The objective of this study was to explore different approaches to communicating the positive predictive value (PPV) of cell-free DNA screening for fetal trisomy. METHODS: PPV was established for 4 maternal age-groups (<30, 30-34, 35-39, and >39 years) from clinical laboratory data and compared to the modeled PPV from an online calculator. In women under 35, PPV was compared between 2 subsets, high risk and low risk, classified based on the diagnosis codes that were provided to the laboratory. RESULTS: In 503 high-probability trisomy 21 results, the observed PPVs in the 4 age-groups were 97.0% (<30), 98.9% (30-34), 99.5% (35-39), and 96.3% (>39), all higher than those from the calculator, which ranged from 53 to 95%. Likewise, PPVs were 77.4-97.0% observed versus 16-78% modeled in 131 trisomy 18 cases and 30.4-80.0% observed versus 6-61% modeled in 80 trisomy 13 cases. In women under 35, PPV for the trisomies combined was 90.4% in the higher-risk group compared to 79.7% in the lower-risk group. CONCLUSION: Modeling PPV based on maternal age will provide an underestimate in a clinical population. Although the PPV is higher for the samples with higher-risk diagnosis codes, the information that accompanies clinical samples is too general to model PPV for a specific patient.

Clinical performance of non-invasive prenatal testing (NIPT) using targeted cell-free DNA analysis in maternal plasma with microarrays or next generation sequencing (NGS) is consistent across multiple controlled clinical studies.

OBJECTIVE: To evaluate the clinical performance of non-invasive prenatal testing for trisomy 21, 18, and 13 using targeted cell-free DNA (cfDNA) analysis. METHODS: Targeted cfDNA analysis using DANSR™ and FORTE™ with microarray quantitation was used to evaluate the risk of trisomy 21, 18, and 13 in blinded samples from 799 singleton, twin, natural, and IVF pregnancies. Subjects either had fetal chromosome evaluation by karyotype, FISH, QF-PCR, or karyotype for newborns with suspected aneuploidy at birth. The results of targeted cfDNA analysis were compared to clinical genetic testing outcomes to assess clinical performance. RESULTS: Targeted cfDNA analysis with microarray quantification identified 107/108 trisomy 21 cases (99.1%), 29/30 trisomy 18 cases (96.7%), and 12/12 trisomy 13 cases (100%). The specificity was 100% for all three trisomies. Combining this data with all published clinical performance studies using DANSR/FORTE methodology for greater than 23 000 pregnancies, the sensitivity of targeted cfDNA analysis was calculated to be greater than 99% for trisomy 21, 97% for trisomy 18, and 94% for trisomy 13. Specificity for each trisomy was greater than 99.9%. CONCLUSION: Targeted cfDNA analysis demonstrates consistently high sensitivity and extremely low false positive rates for common autosomal trisomies in pregnancy across quantitation platforms.

Fraction of cell-free fetal DNA in the maternal serum as a predictor of abnormal placental invasion-a pilot study.

OBJECTIVE: Pilot studies have suggested the amount of cell-free fetal DNA (cffDNA) in the maternal serum is increased in pregnancies complicated by placenta accreta. Our objective was to determine if levels of cffDNA can predict invasive placentation. METHODS: We enrolled women with antenatally suspected placenta accreta compared with gestational age-matched cases of placenta previa and women with prior cesarean deliveries (CDs) and normal placentation. The fetal fraction of cffDNA was quantified using DANSR™ as compared with total DNA. Patient characteristics were compared between the three groups using ANOVA, and linear regression was used to compare the fraction of cffDNA between pathologically confirmed cases of placenta accreta, placenta previa and normal placentation. RESULTS: Twenty women were enrolled, (seven cases of placenta accreta, six cases of placenta previa and seven cases of normal placentation with prior CD). The groups did not differ by maternal weight, placental weight, number of prior CD or years from prior CD. The mean fraction of cffDNA did not differ significantly by group when controlling for the aforementioned factors (accreta = 19.1%, previa = 27.2%, prior CD = 28.9%, p = 0.26), nor did the median (accreta = 17.0%, previa = 30.1%, prior CD = 22.7%). CONCLUSIONS: We could not confirm diagnostic benefit. Further investigation of other biomarkers including placental mRNA is warranted.

Gestational age and maternal weight effects on fetal cell-free DNA in maternal plasma.

OBJECTIVE: To determine the effects of gestational age and maternal weight on percent fetal cell-free DNA (cfDNA) in maternal plasma and the change in fetal cfDNA amounts within the same patient over time. METHODS: The cfDNA was extracted from maternal plasma from 22 384 singleton pregnancies of at least 10 weeks gestation undergoing the Harmony(TM) Prenatal Test. The Harmony Prenatal Test determined fetal percentage via directed analysis of cfDNA. RESULTS: At 10 weeks 0 days to 10 weeks 6 days gestation, the median percent fetal cfDNA was 10.2%. Between 10 and 21 weeks gestation, percent fetal increased 0.1% per week (p < 0.0001), and 2% of pregnancies were below 4% fetal cfDNA. Beyond 21 weeks gestation, fetal cfDNA increased 1% per week (p < 0.0001). Fetal cfDNA percentage was proportional to gestational age and inversely proportional to maternal weight (p = 0.0016). Of 135 samples that were redrawn because of insufficient fetal cfDNA of the initial sample, 76 (56%) had greater than 4% fetal cfDNA in the sample from the second draw. CONCLUSION: Fetal cfDNA increases with gestation, decreases with increasing maternal weight, and generally improves upon a blood redraw when the first attempt has insufficient fetal cfDNA.

Non-invasive prenatal testing with cell-free DNA: US physician attitudes toward implementation in clinical practice.

OBJECTIVE: The aim of this study was to assess awareness, potential adoption, and current utilization of non-invasive prenatal testing (NIPT) analysis for common fetal aneuploidies among obstetricians. METHODS: A 36-item web-based survey was designed to assess the current practice of fetal aneuploidy screening and knowledge and utilization of NIPT for fetal trisomy. Practicing obstetricians in the United States were invited via email to participate in the survey. RESULTS: Of the 101 obstetricians that completed the survey (27% academic-based, 73% private practice), 97% offer screening to high-risk patients and 91% offer screening to average-risk patients. With regard to current screening tests, the top three advantages were as follows: recommendation by professional societies, no risk to the pregnancy, and long history/experience with the test, whereas the top three limitations were as follows: patient anxiety, risks of follow-up invasive testing, and high false positives. NIPT had been used by 32% of respondents and 22% were familiar with NIPT and the associated clinical data. The majority of physicians predicted that they would offer NIPT to high-risk women (86.1%) and average-risk women (76.2%) within 12 months. CONCLUSION: Obstetricians plan to increase their utilization of NIPT and expect that the majority of both high-risk and average-risk patients will be offered NIPT as an option.