Allan T. Bombard

DNA sequencing of maternal plasma to detect Down syndrome: An international clinical validation study

Purpose: Prenatal screening for Down syndrome has improved, but the number of resulting invasive diagnostic procedures remains problematic. Measurement of circulating cell-free DNA in maternal plasma might offer improvement.Methods: A blinded, nested case-control study was designed within a cohort of 4664 pregnancies at high risk for Down syndrome. Fetal karyotyping was compared with an internally validated, laboratory-developed test based on next-generation sequencing in 212 Down syndrome and 1484 matched euploid pregnancies. None had been previously tested. Primary testing occurred at a CLIA-certified commercial laboratory, with cross validation by a CLIA-certified university laboratory.Results: Down syndrome detection rate was 98.6% (209/212), the false-positive rate was 0.20% (3/1471), and the testing failed in 13 pregnancies (0.8%); all were euploid. Before unblinding, the primary testing laboratory also reported multiple alternative interpretations. Adjusting chromosome 21 counts for guanine cytosine base content had the largest impact on improving performance.Conclusion: When applied to high-risk pregnancies, measuring maternal plasma DNA detects nearly all cases of Down syndrome at a very low false-positive rate. This method can substantially reduce the need for invasive diagnostic procedures and attendant procedure-related fetal losses. Although implementation issues need to be addressed, the evidence supports introducing this testing on a clinical basis.

Noninvasive detection of fetal trisomy 21 by sequencing of DNA in maternal blood: a study in a clinical setting

OBJECTIVE We sought to evaluate a multiplexed massively parallel shotgun sequencing assay for noninvasive trisomy 21 detection using circulating cell-free fetal DNA. STUDY DESIGN Sample multiplexing and cost-optimized reagents were evaluated as improvements to a noninvasive fetal trisomy 21 detection assay. A total of 480 plasma samples from high-risk pregnant women were employed. RESULTS In all, 480 prospectively collected samples were obtained from our third-party storage site; 13 of these were removed due to insufficient quantity or quality. Eighteen samples failed prespecified assay quality control parameters. In all, 449 samples remained: 39 trisomy 21 samples were correctly classified; 1 sample was misclassified as trisomy 21. The overall classification showed 100% sensitivity (95% confidence interval, 89-100%) and 99.7% specificity (95% confidence interval, 98.5-99.9%). CONCLUSION Extending the scope of previous reports, this study demonstrates that plasma DNA sequencing is a viable method for noninvasive detection of fetal trisomy 21 and warrants clinical validation in a larger multicenter study.

DNA sequencing of maternal plasma reliably identifies trisomy 18 and trisomy 13 as well as Down syndrome: an international collaborative study.

Purpose:To determine whether maternal plasma cell–free DNA sequencing can effectively identify trisomy 18 and 13.Methods:Sixty-two pregnancies with trisomy 18 and 12 with trisomy 13 were selected from a cohort of 4,664 pregnancies along with matched euploid controls (including 212 additional Down syndrome and matched controls already reported), and their samples tested using a laboratory-developed, next-generation sequencing test. Interpretation of the results for chromosome 18 and 13 included adjustment for CG content bias.Results:Among the 99.1% of samples interpreted (1,971/1,988), observed trisomy 18 and 13 detection rates were 100% (59/59) and 91.7% (11/12) at false-positive rates of 0.28% and 0.97%, respectively. Among the 17 samples without an interpretation, three were trisomy 18. If z-score cutoffs for trisomy 18 and 13 were raised slightly, the overall false-positive rates for the three aneuploidies could be as low as 0.1% (2/1,688) at an overall detection rate of 98.9% (280/283) for common aneuploidies. An independent academic laboratory confirmed performance in a subset.Conclusion:Among high-risk pregnancies, sequencing circulating cell–free DNA detects nearly all cases of Down syndrome, trisomy 18, and trisomy 13, at a low false-positive rate. This can potentially reduce invasive diagnostic procedures and related fetal losses by 95%. Evidence supports clinical testing for these aneuploidies.Genet Med 2012:14(3):296–305

DNA sequencing of maternal plasma to identify Down syndrome and other trisomies in multiple gestations

Studies on prenatal testing for Down syndrome (trisomy 21), trisomy 18, and trisomy 13 by massively parallel shotgun sequencing (MPSS) of circulating cell free DNA have been, for the most part, limited to singleton pregnancies. If MPSS testing is offered clinically, it is important to know if these trisomies will also be identified in multiple pregnancies.

Noninvasive prenatal detection of sex chromosomal aneuploidies by sequencing circulating cell-free DNA from maternal plasma

Whole‐genome sequencing of circulating cell free (ccf) DNA from maternal plasma has enabled noninvasive prenatal testing for common autosomal aneuploidies. The purpose of this study was to extend the detection to include common sex chromosome aneuploidies (SCAs): [47,XXX], [45,X], [47,XXY], and [47,XYY] syndromes.

Non-Invasive Prenatal Chromosomal Aneuploidy Testing - Clinical Experience: 100,000 Clinical Samples

Objective As the first laboratory to offer massively parallel sequencing-based noninvasive prenatal testing (NIPT) for fetal aneuploidies, Sequenom Laboratories has been able to collect the largest clinical population experience data to date, including >100,000 clinical samples from all 50 U.S. states and 13 other countries. The objective of this study is to give a robust clinical picture of the current laboratory performance of the MaterniT21 PLUS LDT. Study Design The study includes plasma samples collected from patients with high-risk pregnancies in our CLIA–licensed, CAP-accredited laboratory between August 2012 to June 2013. Samples were assessed for trisomies 13, 18, 21 and for the presence of chromosome Y-specific DNA. Sample data and ad hoc outcome information provided by the clinician was compiled and reviewed to determine the characteristics of this patient population, as well as estimate the assay performance in a clinical setting. Results NIPT patients most commonly undergo testing at an average of 15 weeks, 3 days gestation; and average 35.1 years of age. The average turnaround time is 4.54 business days and an overall 1.3% not reportable rate. The positivity rate for Trisomy 21 was 1.51%, followed by 0.45% and 0.21% rate for Trisomies 18 and 13, respectively. NIPT positivity rates are similar to previous large clinical studies of aneuploidy in women of maternal age ≥35 undergoing amniocentesis. In this population 3519 patients had multifetal gestations (3.5%) with 2.61% yielding a positive NIPT result. Conclusion NIPT has been commercially offered for just over 2 years and the clinical use by patients and clinicians has increased significantly. The risks associated with invasive testing have been substantially reduced by providing another assessment of aneuploidy status in high-risk patients. The accuracy and NIPT assay positivity rate are as predicted by clinical validations and the test demonstrates improvement in the current standard of care.

Circulating cell free DNA testing: are some test failures informative?

The proportion of circulating cell free DNA derived from the feto‐placental unit (fetal fraction or FF) correlates with test success and interpretation reliability. Some fetal disorders are associated with systematically lower FF, sometimes resulting in noninformative results.

Fetal sex determination using circulating cell-free fetal DNA (ccffDNA) at 11 to 13 weeks of gestation.

To examine the performance of a mass spectrometry‐based detection platform using three Y‐chromosome sequences for fetal sex determination from circulating cell‐free fetal DNA (ccffDNA) in maternal blood in the first trimester of pregnancy.

Factors affecting levels of circulating cell‐free fetal DNA in maternal plasma and their implications for noninvasive prenatal testing

Sufficient fetal DNA in a maternal plasma sample is required for accurate aneuploidy detection via noninvasive prenatal testing, thus highlighting a need to understand the factors affecting fetal fraction.

Procedure-related fetal losses in transplacental versus nontransplacental genetic amniocentesis

OBJECTIVE We hypothesize that loss rates after amniocentesis do not differ in transplacental and nontransplacental taps performed by experienced operators. STUDY DESIGN Subjects were 1000 women undergoing second-trimester amniocentesis: 745 were referred for maternal age; 132 for positive maternal serum alpha-fetoprotein screens, 41 indicating a risk for fetal neural tube defect, 91 indicating a risk for fetal chromosome abnormality; and 123 were referred for other reasons. All procedures were videotaped. The placenta was anterior in 518 cases; in 306 of these the needle went through the placenta. All pregnancies were prospectively evaluated through delivery. RESULTS There were 13 losses among the 1000 procedures (1.3%). The transplacental losses occurred from 4 to 71 days after procedure, median 26.5 days; the nontransplacental losses from 12 days after procedure to term, median 25 days. The loss rate was essentially similar in the two categories: six transplacental (1.96%) and seven nontransplacental (1%) (relative risk 1.52 [95% confidence limits 0.84 to 2.75], p = 0.23). If the three patients with elevated maternal serum alpha-fetoprotein values were excluded from data analysis, the loss rates in the two groups were virtually identical (relative risk 0.98 [95% confidence limits 0.38 to 2.54], p = 1.0000). CONCLUSION Transplacental amniocentesis does not appear to increase the fetal loss rate in the hands of experienced surgeons. Moreover, in view of the time span between amniocentesis and loss in both groups, a procedural cause seems questionable.