Richard P. Rava

Genome-Wide Fetal Aneuploidy Detection by Maternal Plasma DNA Sequencing

OBJECTIVE: To prospectively determine the diagnostic accuracy of massively parallel sequencing to detect whole chromosome fetal aneuploidy from maternal plasma. METHODS: Blood samples were collected in a prospective, blinded study from 2,882 women undergoing prenatal diagnostic procedures at 60 U.S. sites. An independent biostatistician selected all singleton pregnancies with any abnormal karyotype and a balanced number of randomly selected pregnancies with euploid karyotypes. Chromosome classifications were made for each sample by massively parallel sequencing and compared with fetal karyotype. RESULTS: Within an analysis cohort of 532 samples, the following were classified correctly: 89 of 89 trisomy 21 cases (sensitivity 100%, 95% [confidence interval] CI 95.9–100), 35 of 36 trisomy 18 cases (sensitivity 97.2%, 95% CI 85.5–99.9), 11 of 14 trisomy 13 cases (sensitivity 78.6%, 95% CI 49.2–99.9), 232 of 233 females (sensitivity 99.6%, 95% CI 97.6 to more than 99.9), 184 of 184 males (sensitivity 100%, 95% CI 98.0–100), and 15 of 16 monosomy X cases (sensitivity 93.8%, 95% CI 69.8–99.8). There were no false-positive results for autosomal aneuploidies (100% specificity, 95% CI more than 98.5 to 100). In addition, fetuses with mosaicism for trisomy 21 (3/3), trisomy 18 (1/1), and monosomy X (2/7), three cases of translocation trisomy, two cases of other autosomal trisomies (20 and 16), and other sex chromosome aneuploidies (XXX, XXY, and XYY) were classified correctly. CONCLUSION: This prospective study demonstrates the efficacy of massively parallel sequencing of maternal plasma DNA to detect fetal aneuploidy for multiple chromosomes across the genome. The high sensitivity and specificity for the detection of trisomies 21, 18, 13, and monosomy X suggest that massively parallel sequencing can be incorporated into existing aneuploidy screening algorithms to reduce unnecessary invasive procedures. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, www.clinicaltrials.gov, NCT01122524. LEVEL OF EVIDENCE: II

DNA Sequencing versus Standard Prenatal Aneuploidy Screening

BACKGROUND In high-risk pregnant women, noninvasive prenatal testing with the use of massively parallel sequencing of maternal plasma cell-free DNA (cfDNA testing) accurately detects fetal autosomal aneuploidy. Its performance in low-risk women is unclear. METHODS At 21 centers in the United States, we collected blood samples from women with singleton pregnancies who were undergoing standard aneuploidy screening (serum biochemical assays with or without nuchal translucency measurement). We performed massively parallel sequencing in a blinded fashion to determine the chromosome dosage for each sample. The primary end point was a comparison of the false positive rates of detection of fetal trisomies 21 and 18 with the use of standard screening and cfDNA testing. Birth outcomes or karyotypes were the reference standard. RESULTS The primary series included 1914 women (mean age, 29.6 years) with an eligible sample, a singleton fetus without aneuploidy, results from cfDNA testing, and a risk classification based on standard screening. For trisomies 21 and 18, the false positive rates with cfDNA testing were significantly lower than those with standard screening (0.3% vs. 3.6% for trisomy 21, P<0.001; and 0.2% vs. 0.6% for trisomy 18, P=0.03). The use of cfDNA testing detected all cases of aneuploidy (5 for trisomy 21, 2 for trisomy 18, and 1 for trisomy 13; negative predictive value, 100% [95% confidence interval, 99.8 to 100]). The positive predictive values for cfDNA testing versus standard screening were 45.5% versus 4.2% for trisomy 21 and 40.0% versus 8.3% for trisomy 18. CONCLUSIONS In a general obstetrical population, prenatal testing with the use of cfDNA had significantly lower false positive rates and higher positive predictive values for detection of trisomies 21 and 18 than standard screening. (Funded by Illumina; ClinicalTrials.gov number, NCT01663350.).

Optimal Detection of Fetal Chromosomal Abnormalities by Massively Parallel DNA Sequencing of Cell-Free Fetal DNA from Maternal Blood

BACKGROUND Massively parallel DNA sequencing of cell-free fetal DNA from maternal blood can detect fetal chromosomal abnormalities. Although existing algorithms focus on the detection of fetal trisomy 21 (T21), these same algorithms have difficulty detecting trisomy 18 (T18). METHODS Blood samples were collected from 1014 patients at 13 US clinic locations before they underwent an invasive prenatal procedure. All samples were processed to plasma, and the DNA extracted from 119 samples underwent massively parallel DNA sequencing. Fifty-three sequenced samples came from women with an abnormal fetal karyotype. To minimize the intra- and interrun sequencing variation, we developed an optimized algorithm by using normalized chromosome values (NCVs) from the sequencing data on a training set of 71 samples with 26 abnormal karyotypes. The classification process was then evaluated on an independent test set of 48 samples with 27 abnormal karyotypes. RESULTS Mapped sites for chromosomes of interest in the sequencing data from the training set were normalized individually by calculating the ratio of the number of sites on the specified chromosome to the number of sites observed on an optimized normalizing chromosome (or chromosome set). Threshold values for trisomy or sex chromosome classification were then established for all chromosomes of interest, and a classification schema was defined. Sequencing of the independent test set led to 100% correct classification of T21 (13 of 13) and T18 (8 of 8) samples. Other chromosomal abnormalities were also identified. CONCLUSION Massively parallel sequencing is capable of detecting multiple fetal chromosomal abnormalities from maternal plasma when an optimized algorithm is used.

Noninvasive Detection of Fetal Subchromosome Abnormalities via Deep Sequencing of Maternal Plasma

The purpose of this study was to determine the deep sequencing and analytic conditions needed to detect fetal subchromosome abnormalities across the genome from a maternal blood sample. Cell-free (cf) DNA was isolated from the plasma of 11 pregnant women carrying fetuses with subchromosomal duplications and deletions, translocations, mosaicism, and trisomy 20 diagnosed by metaphase karyotype. Massively parallel sequencing (MPS) was performed with 25-mer tags at approximately 10(9) tags per sample and mapped to reference human genome assembly hg19. Tags were counted and normalized to fixed genome bin sizes of 1 Mb or 100 kb to detect statistically distinct copy-number changes compared to the reference. All seven cases of microdeletions, duplications, translocations, and the trisomy 20 were detected blindly by MPS, including a microdeletion as small as 300 kb. In two of these cases in which the metaphase karyotype showed additional material of unknown origin, MPS identified both the translocation breakpoint and the chromosomal origin of the additional material. In the four mosaic cases, the subchromosomal abnormality was not demonstrated by MPS. This work shows that in nonmosaic cases, it is possible to obtain a fetal molecular karyotype by MPS of maternal plasma cfDNA that is equivalent to a chromosome microarray and in some cases is better than a metaphase karyotype. This approach combines the advantage of enhanced fetal genomic resolution with the improved safety of a noninvasive maternal blood test.

Initial clinical laboratory experience in noninvasive prenatal testing for fetal aneuploidy from maternal plasma DNA samples

The aim of this study is to report the experience of noninvasive prenatal DNA testing using massively parallel sequencing in an accredited clinical laboratory.

Circulating Fetal Cell-Free DNA Fractions Differ in Autosomal Aneuploidies and Monosomy X

BACKGROUND Noninvasive prenatal testing based on massively parallel sequencing (MPS) of cell-free DNA in maternal plasma has become rapidly integrated into clinical practice for detecting fetal chromosomal aneuploidy. We directly determined the fetal fraction (FF) from results obtained with MPS tag counting and examined the relationships of FF to such biological parameters as fetal karyotype and maternal demographics. METHODS FF was determined from samples previously collected for the MELISSA (Maternal Blood Is Source to Accurately Diagnose Fetal Aneuploidy) study. Samples were resequenced, analyzed blindly, and aligned to the human genome (assembly hg19). FF was calculated in pregnancies with male or aneuploid fetuses by means of an equation that incorporated the ratio of the tags in these samples to those of a euploid training set. RESULTS The mean (SD) FF from euploid male pregnancies was 0.126 (0.052) (n = 160). Weak but statistically significant correlations were found between FF and the maternal body mass index (r(2) = 0.18; P = 2.3 × 10(-8)) and between FF and gestational age (r(2) = 0.02; P = 0.047). No relationship with maternal ethnicity or age was observed. Mean FF values for trisomies 21 (n = 90), 18 (n = 38), and 13 (n = 16) and for monosomy X (n = 20) were 0.135 (0.051), 0.089 (0.039), 0.090 (0.062), and 0.106 (0.045), respectively. CONCLUSIONS MPS tag-count data can be used to determine FF directly and accurately. Compared with male euploid fetuses, the FF is higher in maternal plasma when the fetus has trisomy 21 and is lower when the fetus has trisomy 18, 13, or monosomy X. The different biologies of these aneuploidies have practical implications for the determination of cutoff values, which in turn will affect the diagnostic sensitivity and specificity of the test.

Massively parallel sequencing of maternal plasma DNA in 113 cases of fetal nuchal cystic hygroma.

OBJECTIVE: To estimate the accuracy and potential clinical effect of using massively parallel sequencing of maternal plasma DNA to detect fetal aneuploidy in a cohort of pregnant women carrying fetuses with nuchal cystic hygroma. METHODS: The MatErnal BLood IS Source to Accurately diagnose fetal aneuploidy (MELISSA) study database was queried to identify eligible patients carrying fetuses with cystic hygroma (n=113) based on clinical ultrasonographic examination reports near enrollment. Archived plasma samples were newly sequenced and normalized chromosome values were determined. Aneuploidy classifications for chromosomes 21, 18, 13, and X were made using the massively parallel sequencing data by laboratory personnel blinded to fetal karyotype and compared for analysis. RESULTS: Sixty-nine of 113 (61%) patients had fetuses with abnormal karyotypes, including trisomy 21 (n=30), monosomy X (n=21), trisomy 18 (n=10), trisomy 13 (n=4), and other (n=4). There were 44 euploid cases; none was called positive for aneuploidy. The massively parallel sequencing detection rates were as follows: T21: 30 of 30, T18: 10 of 10, T13: three of four, and monosomy X: 20 of 21, including two complex mosaic cases. Overall, using massively parallel sequencing results of the four studied chromosomes, 107 of 113 (95%, 95% confidence interval [CI] 88.8–98.0) cases were accurately called by massively parallel sequencing, including 63 of 65 (97%, 95% CI 89.3–99.6) of cases of whole chromosome aneuploidy. CONCLUSION: Massively parallel sequencing provides an accurate way of detecting the most prevalent aneuploidies associated with cystic hygroma. Massively parallel sequencing could advance prenatal care by providing alternative point-of-care noninvasive testing for pregnant women who either decline or do not have access to an invasive procedure. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov, www.clinicaltrials.gov, NCT01122524. LEVEL OF EVIDENCE: II

A new era in noninvasive prenatal testing.

To the Editor: In their Perspective article, Morain et al. (Aug. 8 issue)1 say that commercial companies have not met reasonable standards for demonstrating clinical validity. This is not correct. In five independent cohorts, researchers have examined 463 pregnancies with fetuses with Down’s syndrome, 187 with trisomy 18, and 37 with trisomy 13. To maximize confidence in sensitivity estimates, all were high-risk pregnancies (in a general population, more than 250,000 pregnancies would have had to be studied). Five professional organizations, including the American Congress of Obstetricians and Gynecologists,2 recommend offering such testing for “high-risk” pregnancies. The Perspective article also implies that a positive predictive value (PPV) of 62% might be inadequate for decision making. Using the authors’ numbers, we modeled the performance of cell-free fetal DNA (cfDNA) testing in 200,000 pregnancies at a risk level of 1 in 200. We assumed that the cfDNA test had a sensitivity of 99.9% and a specificity of 99.7%. Of the pregnant women tested, only 1596 (0.8%) would be candidates for diagnostic studies, and 999 of 1000 cases would be detected. More than 198,000 women would have a negative screen, which would result in approximately 660 fewer procedure-related fetal losses. Suggesting that testing is unreliable because it is not approved by the Food and Drug Administration (FDA) may discourage its valid use in avoiding unnecessary invasive diagnostic testing.499 developing world, might help to encourage companies to spend resources on breakthrough innovation, rather than on minor modifications and lawyer fees. But whatever its implications for innovation, this much is clear: poor people around the world need better access to affordable drugs, and this decision will help to provide it. Disclosure forms provided by the author are available with the full text of this article at NEJM.org.