Cell-Free DNA Screening

Overview

Cell-free DNA (cfDNA) screening has been rapidly introduced into prenatal care since it became clinically available in 2011. cfDNA screening can detect more than 99% of cases of trisomy 21 and also has high sensitivity and specificity for the detection of trisomy 13 and 18 and the common sex chromosome aneuploidies. cfDNA screens for aneuploidy by sequencing cfDNA from the maternal serum, which includes fetal cfDNA, largely derived from the placenta from apoptotic trophoblasts.

Pretest counseling should be performed for all women considering cfDNA screening, and all women with positive tests or test failures should receive posttest counseling. Confirmatory testing with chorionic villus sampling (CVS) or amniocentesis should be recommended to women with positive results as false-positive results occur and may be due to confined placental mosaicism (CPM) as well as other rare biologic causes including an underlying maternal chromosomal abnormality, a vanishing twin and in very rare cases, a maternal tumor or malignancy.

The entire fetal genome is represented in short cell-free fragments in the maternal plasma. Additional potential applications using cfDNA technology include screening for microdeletions, fetal Rh-determination, and detection of single gene disorders.

What Is Cell-Free DNA and How Is It Used for Screening for Fetal Aneuploidy?

cfDNA is increasingly being used to screen for the common fetal aneuploidies because of the higher sensitivity and specificities compared with conventional serum screening. Prenatal cfDNA screening was initially described in 1997 and became clinically available in 2011. The cfDNA in the maternal plasma consists of free-floating small fragments of maternal DNA and placental DNA. Placental cfDNA is primarily derived from apoptotic trophoblasts. It can be detected in maternal blood as early as 4 weeks’ gestation. The fetal fraction, which is the proportion of cfDNA of fetal (placental) origin to maternal cfDNA, exceeds 4% in the majority of pregnant women beginning at about 10 weeks’ gestation. It continues to increase by 0.1% per week through 21 weeks’ gestation and approximately 1% per week after 21 weeks’ gestation. Data suggest that a fetal fraction of at least 4% is needed to obtain a reliable result with cfDNA screening.

cfDNA screening is currently used to detect trisomies 21, 18, and 13; 45 X; and 47 XXX, XXY, and XYY. The American College of Medical Genetics and Genomics (ACMG) has endorsed the acronym NIPS (noninvasive prenatal screening) to emphasize the fact that cfDNA is a screening test and not a diagnostic test. In this chapter, we use the terminology “cell-free DNA” instead of “noninvasive prenatal screening,” as all current screening tests are considered noninvasive.

Placental cfDNA has a very short half-life; therefore, a cfDNA screening result will not be affected by a previous pregnancy. Screening is available after approximately 9–10 weeks of gestation when the fetal fraction is generally above 4%. cfDNA screening may be used either as a primary screening test or as a secondary screening test. Traditional serum screening (first trimester screening or second trimester maternal serum screening) is not recommended after cfDNA screening, and concurrent testing with cfDNA and traditional serum screening is also not recommended.

What Techniques Are Used in Cell-Free DNA Screening?

Several different methods of assaying cfDNA are currently in use for aneuploidy screening using next-generation sequencing technology: whole-genome sequencing (massively parallel sequencing), chromosome-selective (or targeted) sequencing, and single-nucleotide polymorphism (SNP) analysis. Although all the three methods appear to have similar sensitivities and specificities, there are advantages and limitations associated with each method.

Massively parallel sequencing randomly analyzes DNA from the entire genome. It sequences the entire DNA that is extracted from the maternal sample, counts cfDNA fragments, and assigns them to their specific chromosomal origin.

Targeted sequencing, also known as chromosome-specific sequencing, specifically amplifies chromosomal areas that are of interest and subsequently evaluates if there is an excess of one specific chromosome over another. An advantage of this technique is increased efficiency and overall lower cost (because only specific chromosomal areas are sequenced). A microarray-based technology that does not require next-generation sequencing has been used to quantify the relative contributions from specific chromosomes. This approach yielded high sensitivities and specificities that were comparable with next-generation sequencing approaches. This approach may result in additional decreased cost and turnaround time and testing of samples with a lower fetal fraction because of individual hybridization instead of sample multiplexing.

The third method uses a single-nucleotide polymorphism-based approach with analysis of the quantitative contributions of maternal and fetal DNA. The SNP-based approach cannot be used in pregnancies involving egg donors because this approach uses maternal genotypic information. This approach may identify consanguinity or uniparental disomy, in which both copies of a chromosome are inherited from the same parent.

Although the sensitivities and specificities for the detection of aneuploidy are similar for all three approaches, the whole genome sequencing approach is reported to have the lowest failure rate at 1.58%, followed by targeted sequencing at 3.56%, and SNP-based approach at 6.39%. The etiology of these failure rates is multifactorial and varies depending on quality control metrics, measurement of fetal fraction, and patient factors.

How Effective Is Cell-Free DNA in Screening for Fetal Aneuploidy?

cfDNA screening has a higher sensitivity and lower false-positive rate than conventional screening modalities (see Chapter 8 ) in detecting fetal aneuploidy. Clinical studies have demonstrated that the detection rates and false-positive rates for fetal aneuploidy do not differ with maternal age. The performance characteristics of cfDNA screening for fetal aneuploidy are summarized in Table 9.1 . A metaanalysis that included data from 35 studies reported cfDNA screening detection rates of 99.7% for trisomy 21, 97.9% for trisomy 18, and 99.0% for trisomy 13. The detection rates for sex chromosome aneuploidies were greater than 95% ( Table 9.1 ). Of note, it is important to recognize that the cases of fetal aneuploidy in women who did not receive a cfDNA test result were not included in the calculation of the detection rates. As expected, the positive predictive values (PPVs) for trisomy 21, 18, and 13 are higher in older women as the prevalence of these conditions increases with advancing maternal age.

What Is the Accuracy of Screening for Sex Chromosome Aneuploidies?

The performance characteristics are presented in Table 9.1 . Although the overall false-positive and false-negative rates for the detection of sex chromosome aneuploidies are very low, the PPV appears to be limited. Bianchi et al. reported a low frequency (0.26%–1.05%) of false-positive results for sex chromosome aneuploidy in a cohort of approximately 18,000 women who had cfDNA testing. Although clinical follow-up was only obtained for 44 of the 148 cases predicted to have monosomy X, 35 (79.5%) of the cases were false positives. Among 38 screen positive cases of XXX, 12 of 13 (92.3%) with clinical follow-up were false positive. Of the 12 false-positive cases, 2 were due to a maternal XXX cell line. Among six XYY cases, two had follow-up. One was confirmed as XYY and the other as XXY. Of the 12 XXY cases, 2 had follow-up and were confirmed to be true positives. These findings raise concerns about the limitations of cfDNA in screening for sex chromosome aneuploidy and highlight the importance of pretest counseling and confirmatory diagnostic testing following a screen-positive cfDNA test result. False-negative cases are likely to remain largely undetected because of the absence of fetal and neonatal findings in the most common sex chromosome aneuploidies. More information about performance characteristics of cfDNA for detection of sex chromosome aneuploidies will likely be forthcoming from future large prospective studies.

What Are the Potential Advantages and Disadvantages Associated With Cell-Free DNA Screening for Fetal Aneuploidy Compared With Conventional Screening Tests?

cfDNA is the most sensitive screening option for trisomy 13, 18, and 21. The false-positive rates associated with cfDNA screening are lower compared with conventional screening. Conventional screening tests (first trimester screening, second trimester screening, or stepwise sequential screening) have detection rates of 80%–95% with false-positive rates of 5%. This could potentially lead to an increased number of diagnostic procedures in women who have conventional screening compared with cfDNA screening.

In addition, the PPV (the likelihood that a woman with a positive test has an affected fetus) is considerably higher with cfDNA screening compared with conventional screening. In a study of approximately 1900 women with a mean age of 29.6 years, the PPVs for the detection of trisomy 21 were 45.5% with cfDNA screening and 4.2% with conventional screening.

An additional potential benefit of cfDNA is the option for earlier genetic screening results compared with some traditional screening methods. This allows for definitive diagnostic testing at an earlier gestational age.

cfDNA screening does not include screening for open neural tube defects. Patients should be offered MSAFP level at approximately 16–18 weeks’ gestation unless the fetal anatomy is adequately evaluated with a second trimester ultrasound by a provider with experience in the detection of neural tube defects, ventral wall defects, and other fetal disorders.

A study of a cohort of 1.3 million women who had traditional aneuploidy screening through the California State screening program showed that 506 (17%) of the 2993 women with a positive aneuploidy screen and an abnormal result on invasive diagnostic testing had chromosomal abnormalities that would not be detectable with cfDNA screening. The abnormalities characterized as undetectable by cfDNA screening included mosaic cases of trisomy 21, trisomy 18 and trisomy 13, mosaic sex chromosome aneuploidies, other trisomies (nonmosaic and mosaic), balanced rearrangements, unbalanced rearrangements, insertions and deletions, triploidy, tetraploidy, and cases with extra structurally abnormal chromosomes. There were 45 cases with CPM. Of note, the clinical outcomes associated with these abnormalities ranged from mild to having a significant disability. Some of the undetected abnormalities including the triploidy cases and the tetraploidy case would likely have resulted in spontaneous pregnancy loss, and the fetuses with balanced translocations would likely have been phenotypically normal. Of note, although cfDNA screening currently does not report on the detection of rare autosomal trisomies, it is possible to detect these abnormalities using this technology. However, with present methods, traditional multiple marker screening is a broader screen for the detection of fetal anomalies compared with the high precision of cfDNA screening. Large-scale prospective studies are needed to evaluate the clinical validity and utility of reporting information on the rare autosomal trisomies.

A decision analytic model comparing clinical outcomes, quality-adjusted life-years and costs associated with different strategies for Down syndrome testing using cost estimates from 2015 determined that cfDNA is the optimal and most cost-effective screen for women of 40 years and older. The authors assessed the outcomes of six testing strategies incorporating diagnostic testing with chromosomal microarray, multiple marker screening, cfDNA screening, and nuchal translucency screening alone, in combination, or in sequence. Multiple marker screening with optional follow-up diagnostic testing was the most effective (highest quality-adjusted life-years) and least expensive strategy at ages of 20–38 years.

What Are Other Potential Uses for Cell-Free DNA in Prenatal Testing?