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Study Suggests Cell-Free DNA Approach Can Outperform Standard Screens for Fetal Aneuploidy

NEW YORK (GenomeWeb News) – A new multi-center study performed in the US suggests fetal aneuploidy screening based on cell-free DNA sequencing from pregnant women's blood finds fewer false-positive cases than standard serum screening methods in a general population of pregnant women.

As they reported in the New England Journal of Medicine last night, researchers based at Tufts Medical Center, Illumina, and elsewhere did blinded sequencing on DNA in maternal blood samples collected from thousands of pregnant women at centers in the US. From chromosome dosage profiles revealed by sequence read coverage, they detected all of the chromosome 21, 18, or 13 trisomies present in the samples — fetal aneuploidies that were verified by birth outcomes or chromosomal karyotyping.

Notably, the team reported that the sequencing-based method also led to far fewer false-positive aneuploidy predictions than did standard screening approaches. The latter biochemical methods focus on serum biochemical markers such as pregnancy-related proteins and hormones and are sometimes used in conjunction with first-trimester ultrasound measurements.

The study was meant as a "real-world comparison" between cell-free DNA-based aneuploidy screening and the serum biochemical assays currently used for such purposes, co-author Richard Rava, Illumina's vice president of R&D for reproductive and genetic health, told GenomeWeb Daily News.

Illumina is one of several firms, including Natera, Ariosa Diagnostics, and Sequenom, that are selling sequencing-based cell-free DNA tests for detecting fetal aneuploidy.

Based on findings from the study, he argued that the cell-free DNA screening strategy "really merits consideration as a primary [fetal aneuploidy] screen for the entire obstetrical population," Rava said. "If it was instituted for that, it would really reduce the number of diagnostic procedures … because you would not be sending as many women with false-positive on to invasive procedures."

Prenatal tests based on sequencing of the cell-free DNA found in expectant mothers' blood have entered the clinical sphere in the US over the past few years on the heels of studies verifying the accuracy and validity of such approaches, the researchers noted. But most research has focused on women deemed to be at higher-than-usual risk of carrying a fetus affected by aneuploidy.

"High-risk is generally defined as women over 35 or women who have had a positive biochemical screen or, sometimes, a biochemical screen with ultrasound," Rava said.

"The question that people have begun asking as this [cell-free DNA sequencing] test becomes more pervasive is, 'How does the test operate in a general obstetric population, where we don't separate low-risk and high-risk," he added.

For their new study, the investigators focused on that more general population, noting that some clinicians have already started replacing standard biochemical screening for aneuploidy with cell-free DNA screens.

As part of the Comparison of Aneuploidy Risk Evaluations (CARE) study, Rava and his co-authors compared cell-free DNA based testing to conventional serum screening for aneuploidy in a prospective, blinded manner at 21 medical centers in the US over six months, from the summer of 2012 to the following January.

The 2,052 pregnant women enrolled were all at least 18 years old (the average maternal age was 29.6 years old) and carrying a single fetus that had reached at least eight weeks gestational age.

Blood samples from the pregnant women were shipped to Illumina's Redwood City, Calif. research lab, which the company took over when it acquired Verinata Health last year. Lab personnel blinded to the study questions at hand prepared the samples and sequenced cell-free DNA from 2,042 women with eligible blood samples using Illumina's HiSeq 2000.

From there, investigators used established sequence mapping and read tag methods to estimate the fraction of fetal DNA in the pool of circulating cell-free DNA in these samples.

Such approaches also made it possible to see situations in which chromosome dosages jumped for chromosomes 21, 18, or 13, as they do when an extra copy of one of the chromosomes is present in a fetus.

The team ultimately detected eight aneuploidies from fetal DNA sequencing: five instances of chromosome 21 trisomy (Down syndrome), two chromosome 18 trisomies (Edwards syndrome), and one fetus with trisomy 13 (Patau syndrome). Eighteen of the 2,042 samples did not yield information either way.

Samples from most of the same women were also assessed using standard blood-based biochemical screens that have been established for finding markers associated with aneuploidy during the first and second trimesters.

Both approaches detected authentic aneuploidies, the researchers reported, though the circulating fetal DNA-based approach had enhanced specificity over serum-based biochemical screening strategies.

For the 1,914 women successfully screened using both approaches who were available for follow-up during the group's primary analysis, the cell-free DNA test had false-positive rates of 0.3 percent and 0.2 percent when predicting trisomy 21 and trisomy 18, respectively.

In contrast, the false-positive rates for conventional serum screenings were 3.6 percent in the case of trisomy 21 and 0.6 percent of chromosome 18 trisomies.

The team also performed a secondary analysis using data for nearly 900 women who'd been screened for fetal trisomy 13, a fetal aneuploidy that's screened for less frequently than the chromosome 21 and 18 trisomies. There, the cell-free circulating DNA approach generated one false-positive prediction compared to six serum screening-based false-positives.

False positives at the screening stage are problematic, Rava explained, because it may prompt women and their doctors to unnecessarily pursue invasive aneuploidy testing methods such as amniocentesis or chorionic villus sampling that are costly and carry their own set of risks.

Those involved in developing commercial versions of the circulating fetal DNA-based screening tests for aneuploidy hope to see professional societies such as the International Society for Prenatal Diagnosis or the American College of Obstetrics and Gynecology take into account findings from the current analysis and similar studies done in the future as they offer recommendations about the feasibility of using cell-free DNA in screens of pregnant women regardless of their perceived risk levels.

"Over the past year, [the societies] have all recommended [cell-free DNA screening] for the high-risk population and they're looking for more data in the general obstetrical population to decide whether they should be recommending it, just as they did for biochemical screens," Rava said.

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