Clinical implications of crown-rump length discordance at 11 to 14 weeks in dichorionic twins.

BACKGROUND: Crown-rump length discordance, defined as ≥10% discordance, has been investigated as an early sonographic marker of subsequent growth abnormalities and is associated with an increased risk of fetal loss in twin pregnancies. Previous studies have not investigated the prevalence of fetal aneuploidy or structural anomalies in twins with discordance or the independent association of crown-rump length discordance with adverse perinatal outcomes. Moreover, data are limited on cell-free DNA screening for aneuploidy in dichorionic twins with discordance. OBJECTIVE: This study aimed to evaluate whether crown-rump length discordance in dichorionic twins between 11 and 14 weeks of gestation is associated with a higher risk of aneuploidy, structural anomalies, or adverse perinatal outcomes and to assess the performance of cell-free DNA screening in dichorionic twin pregnancies with crown-rump length discordance. STUDY DESIGN: This was a secondary analysis of a multicenter retrospective cohort study that evaluated the performance of cell-free DNA screening for the common trisomies in twin pregnancies from December 2011 to February 2020. For this secondary analysis, we included live dichorionic pregnancies with crown-rump length measurements between 11 and 14 weeks of gestation. First, we compared twin pregnancies with discordant crown-rump lengths with twin pregnancies with concordant crown-rump lengths and analyzed the prevalence of aneuploidy and fetal structural anomalies in either twin. Second, we compared the prevalence of a composite adverse perinatal outcome, which included preterm birth at <34 weeks of gestation, hypertensive disorders of pregnancy, stillbirth or miscarriage, small-for-gestational-age birthweight, and birthweight discordance. Moreover, we assessed the performance of cell-free DNA screening in pregnancies with and without crown-rump length discordance. Outcomes were compared with multivariable regression to adjust for confounders. RESULTS: Of 987 dichorionic twins, 142 (14%) had crown-rump length discordance. The prevalence of aneuploidy was higher in twins with crown-rump length discordance than in twins with concordance (9.9% vs 3.9%, respectively; adjusted relative risk, 2.7; 95% confidence interval, 1.4-4.9). Similarly, structural anomalies (adjusted relative risk, 2.5; 95% confidence interval, 1.4-4.4]) and composite adverse perinatal outcomes (adjusted relative risk, 1.2; 95% confidence interval, 1.04-1.3) were significantly higher in twins with discordance. A stratified analysis demonstrated that even without other ultrasound markers, there were increased risks of aneuploidy (adjusted relative risk, 3.5; 95% confidence interval, 1.5-8.4) and structural anomalies (adjusted relative risk, 2.7; 95% confidence interval, 1.5-4.8) in twins with CRL discordance. Cell-free DNA screening had high negative predictive values for trisomy 21, trisomy 18, and trisomy 13, regardless of crown-rump length discordance, with 1 false-negative for trisomy 21 in a twin pregnancy with discordance. CONCLUSION: Crown-rump length discordance in dichorionic twins is associated with an increased risk of aneuploidy, structural anomalies, and adverse perinatal outcomes, even without other sonographic abnormalities. Cell-free DNA screening demonstrated high sensitivity and negative predictive values irrespective of crown-rump length discordance; however, 1 false-negative result illustrated that there is a role for diagnostic testing. These data may prove useful in identifying twin pregnancies that may benefit from increased screening and surveillance and are not ascertained by other early sonographic markers.

First-Trimester Cell-Free DNA Fetal Fraction and Birth Weight in Twin Pregnancies.

OBJECTIVE: The relationship between fetal fraction and birth weight in twin gestations is poorly understood. This study aimed to investigate the relationship between first-trimester cell-free DNA (cfDNA) fetal fraction and birth weight <10th percentile in twin gestations. STUDY DESIGN: This is a planned secondary analysis of the Twin cfDNA Study, a 17-center retrospective cohort of twin pregnancies screened for aneuploidy using cfDNA in the first trimester from December 2011 to February 2022, excluding those with positive screen results for chromosomal aneuploidy. cfDNA testing was performed by a single laboratory using massively parallel sequencing. Baseline characteristics and birth weight of pregnancies with normal fetal fraction were compared with those with low (<5%) and high (>95%) fetal fraction using univariable analyses and multivariable regression. RESULTS: A total of 1,041 twin pregnancies were included. Chronic hypertension, elevated body mass index, and self-identified Black race were associated with fetal fraction <5th percentile. There was no difference in median fetal fraction between those with birth weight <10th percentile in at least one twin (median [interquartile range (IQR)] fetal fraction: 12.2% [9.8, 14.8] vs. those with normal birth weight (≥10th percentile) in both twins (median [IQR] fetal fraction: 12.3% [9.7, 15.2] for normal birth weight, p = 0.49). There was no association between high or low fetal fraction and birth weight <10th percentile for one (p = 0.45) or both (p = 0.81) twins, and there was no association between high or low fetal fraction and birth weight <5th percentile for one (p = 0.44) or both (p = 0.74) twins. The results were unchanged after adjustment for potential confounders. CONCLUSION: In this large cohort, there was no association between the extremes of cfDNA fetal fraction and birth weight <10th percentile, suggesting that first-trimester fetal fraction may not predict impaired fetal growth in twin gestations. KEY POINTS: · No association between fetal fraction and small for gestational age birth weight in twins.. · Results suggest that fetal fraction does not predict birth weight in twin gestations.. · These results differ from the relationship between fetal fraction and birth weight in singletons..

Cell-free DNA screening for trisomy 21 in twin pregnancy: a large multicenter cohort study.

BACKGROUND: Analysis of cell-free DNA from maternal blood provides effective screening for trisomy 21 in singleton pregnancies. Data on cell-free DNA screening in twin gestations are promising although limited. In previous twin studies, cell-free DNA screening was primarily performed in the second trimester and many studies did not report chorionicity. OBJECTIVE: This study aimed to evaluate the screening performance of cell-free DNA for trisomy 21 in twin pregnancies in a large, diverse cohort. A secondary aim was to evaluate screening performance for trisomy 18 and trisomy 13. STUDY DESIGN: This was a retrospective cohort study of twin pregnancies from 17 centers for which cell-free DNA screening was performed from December 2011 to February 2020 by one laboratory using massively parallel sequencing technology. Medical record review was conducted for all newborns and data on the birth outcome, the presence of any congenital abnormalities, phenotypic appearance at birth, and any chromosomal testing that was undertaken in the antenatal or postnatal period were extracted. Cases with a possible fetal chromosomal abnormality with no genetic test results were reviewed by a committee of maternal-fetal medicine geneticists. Cases with a vanishing twin and inadequate follow-up information were excluded. A minimum of 35 confirmed cases of trisomy 21 was required to capture a sensitivity of at least 90% with a prevalence of at least 1.9% with 80% power. Test characteristics were calculated for each outcome. RESULTS: A total of 1764 samples were sent for twin cell-free DNA screening. Of those, 78 cases with a vanishing twin and 239 cases with inadequate follow-up were excluded, leaving a total of 1447 cases for inclusion in the analysis. The median maternal age was 35 years and the median gestational age at cell-free DNA testing was 12.3 weeks. In total, 81% of the twins were dichorionic. The median fetal fraction was 12.4%. Trisomy 21 was detected in 41 of 42 pregnancies, yielding a detection rate of 97.6% (95% confidence interval, 83.8-99.7). There was 1 false negative and no false positive cases. Trisomy 21 was detected in 38 out of 39 dichorionic twin pregnancies, yielding a detection rate of 97.4% (95% confidence interval, 82.6-99.7). Trisomy 18 was detected in 10 of the 10 affected pregnancies. There was 1 false positive case. Trisomy 13 was detected in 4 of the 5 cases, yielding a detection rate of 80% (95% confidence interval, 11.1-99.2). There was one false negative and no false positive cases. The nonreportable rate was low at 3.9 %. CONCLUSION: Cell-free DNA testing is effective in screening for trisomy 21 in twin gestations from the first trimester of pregnancy. Detection of trisomy 21 was high in dichorionic and monochorionic twins, and the nonreportable result rates were low. This study included high numbers of cases of trisomy 18 and 13 when compared with the current literature. Although screening for these conditions in twins seems to be promising, the numbers were too small to make definitive conclusions regarding the screening efficacy for these conditions. It is possible that cell-free DNA testing performance may differ among laboratories and vary with screening methodologies.

Cell-free DNA screening in twin pregnancies: A more accurate and reliable screening tool.

OBJECTIVE: Outcome data from cell-free DNA (cfDNA) screening in twin gestations are limited. This study adds an appreciable number of confirmed outcomes to the literature, and assesses performance of cfDNA screening in twins over a 4.5-year period at one large clinical laboratory. METHOD: Prenatal cytogenetic and SNP microarray results were cross-referenced with cfDNA results for twin pregnancies, yielding 422 matched cases. Using diagnostic results as truth, performance of cfDNA screening in this population was assessed. RESULTS: Of the 422 twin pregnancies with both cfDNA and diagnostic results, 3 specimens failed amniocyte analysis, and 48 samples (11.5%) were nonreportable from the initial cfDNA draw. Analysis of the 371 reportable samples demonstrated a collective sensitivity of 98.7% and specificity of 93.2% for trisomies 21/18/13. Positive predictive values (PPVs) in this study population, which is enriched for aneuploidy, were 78.7%, 84.6%, and 66.7% for trisomy 21, 18, and 13, respectively. CONCLUSION: CfDNA screening in a cohort of twin pregnancies with matched diagnostic results showed superior performance compared to traditional serum biochemical screening in twins. This study adds to a growing body of evidence suggesting that cfDNA is an accurate and reliable screening tool for the major trisomies in twin pregnancies.

Clinical validation of a noninvasive prenatal test for genomewide detection of fetal copy number variants.

BACKGROUND: Current cell-free DNA assessment of fetal chromosomes does not analyze and report on all chromosomes. Hence, a significant proportion of fetal chromosomal abnormalities are not detectable by current noninvasive methods. Here we report the clinical validation of a novel noninvasive prenatal test (NIPT) designed to detect genomewide gains and losses of chromosomal material ≥7 Mb and losses associated with specific deletions <7 Mb. OBJECTIVE: The objective of this study is to provide a clinical validation of the sensitivity and specificity of a novel NIPT for detection of genomewide abnormalities. STUDY DESIGN: This retrospective, blinded study included maternal plasma collected from 1222 study subjects with pregnancies at increased risk for fetal chromosomal abnormalities that were assessed for trisomy 21 (T21), trisomy 18 (T18), trisomy 13 (T13), sex chromosome aneuploidies (SCAs), fetal sex, genomewide copy number variants (CNVs) ≥7 Mb, and select deletions <7 Mb. Performance was assessed by comparing test results with findings from G-band karyotyping, microarray data, or high coverage sequencing. RESULTS: Clinical sensitivity within this study was determined to be 100% for T21 (95% confidence interval [CI], 94.6-100%), T18 (95% CI, 84.4-100%), T13 (95% CI, 74.7-100%), and SCAs (95% CI, 84-100%), and 97.7% for genomewide CNVs (95% CI, 86.2-99.9%). Clinical specificity within this study was determined to be 100% for T21 (95% CI, 99.6-100%), T18 (95% CI, 99.6-100%), and T13 (95% CI, 99.6-100%), and 99.9% for SCAs and CNVs (95% CI, 99.4-100% for both). Fetal sex classification had an accuracy of 99.6% (95% CI, 98.9-99.8%). CONCLUSION: This study has demonstrated that genomewide NIPT for fetal chromosomal abnormalities can provide high resolution, sensitive, and specific detection of a wide range of subchromosomal and whole chromosomal abnormalities that were previously only detectable by invasive karyotype analysis. In some instances, this NIPT also provided additional clarification about the origin of genetic material that had not been identified by invasive karyotype analysis.

Developing a Prospective Gestational Lyme Disease Study.

Lyme disease in pregnancy is understudied. The few available reports of Borrelia infection during pregnancy collecting clinical outcomes, with or without confirmed fetal infection both in utero and neonatal, are limited to case reports and small series. Population-based studies are not available. We propose a prospective study of Borrelia infection during pregnancy based in obstetrical practices in both endemic and nonendemic areas, with long term follow-up of pregnancy outcomes and development assessment of offspring infected or exposed to Borrelia in utero using current serological, microscopic, culture, and molecular techniques. In addition to detection of Borrelia burgdorferi sensu stricto, additional Borrelia species and other pathogens known to be transmitted by ticks will be tested. Serial biospecimens including maternal and cord blood, maternal peripheral blood mononuclear cells and urine, and, when clinically indicated, amniotic fluid, chorionic villi, intrauterine cord blood, will be collected with clinical data, imaging, and for infections treatment medications. Offspring will be followed until age 5 years with annual developmental assessments to assess pregnancy outcomes. The study will require parallel development of a biorepository with strategies for management, data security and data sharing. A public-private partnership will be required to support the study.

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

OBJECTIVE: 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. METHOD: Massively parallel sequencing was performed on ccf DNA isolated from the plasma of 1564 pregnant women with known fetal karyotype. A classification algorithm for SCA detection was constructed and trained on this cohort. Another study of 411 maternal samples from women with blinded-to-laboratory fetal karyotypes was then performed to determine the accuracy of the classification algorithm. RESULTS: In the training cohort, the new algorithm had a detection rate (DR) of 100% (95%CI: 82.3%, 100%), a false positive rate (FPR) of 0.1% (95%CI: 0%, 0.3%), and nonreportable rate of 6% (95%CI: 4.9%, 7.4%) for SCA determination. The blinded validation yielded similar results: DR of 96.2% (95%CI: 78.4%, 99.8%), FPR of 0.3% (95%CI: 0%, 1.8%), and nonreportable rate of 5% (95%CI: 3.2%, 7.7%) for SCA determination CONCLUSION: Noninvasive prenatal identification of the most common sex chromosome aneuploidies is possible using ccf DNA and massively parallel sequencing with a high DR and a low FPR.

Laboratory performance of genome-wide cfDNA for copy number variants as compared to prenatal microarray.

BACKGROUND: Noninvasive prenatal testing (NIPT) allows for screening of fetal aneuploidy and copy number variants (CNVs) from cell-free DNA (cfDNA) in maternal plasma. Professional societies have not yet embraced NIPT for fetal CNVs, citing a need for additional performance data. A clinically available genome-wide cfDNA test screens for fetal aneuploidy and CNVs larger than 7 megabases (Mb). RESULTS: This study reviews 701 pregnancies with "high risk" indications for fetal aneuploidy which underwent both genome-wide cfDNA and prenatal microarray. For aneuploidies and CNVs considered 'in-scope' for the cfDNA test (CNVs ≥ 7 Mb and select microdeletions), sensitivity and specificity was 93.8% and 97.3% respectively, with positive and negative predictive values of 63.8% and 99.7% as compared to microarray. When including 'out-of-scope' CNVs on array as false negatives, the sensitivity of cfDNA falls to 48.3%. If only pathogenic out-of-scope CNVs are treated as false negatives, the sensitivity is 63.8%. Of the out-of-scope CNVs identified by array smaller than 7 Mb, 50% were classified as variants of uncertain significance (VUS), with an overall VUS rate in the study of 2.29%. CONCLUSIONS: While microarray provides the most robust assessment of fetal CNVs, this study suggests that genome-wide cfDNA can reliably screen for large CNVs in a high-risk cohort. Informed consent and adequate pretest counseling are essential to ensuring patients understand the benefits and limitations of all prenatal testing and screening options.

Assessing CAR T-Cell Therapy Response Using Genome-Wide Sequencing of Cell-Free DNA in Patients With B-Cell Lymphomas.

Methods that enable monitoring of therapeutic efficacy of autologous chimeric antigen receptor (CAR) T-cell therapy will be clinically useful. The aim of this study is to demonstrate the feasibility of blood-derived cell-free DNA (cfDNA) to predict CAR T-cell therapy response in patients with refractory B-cell lymphomas. Whole blood was collected before and throughout CAR T-cell therapy until day 154. Low-coverage (∼0.4×), genome-wide cfDNA sequencing, similar to that established for noninvasive prenatal testing, was performed. The genomic instability number (GIN) was used to quantify plasma copy number alteration level. Twelve patients were enrolled. Seven (58%) patients achieved a complete response (CR); 2 (25%), a partial response. Median progression-free survival was 99 days; median overall survival was not reached (median follow-up, 247 days). Altogether, 127 blood samples were analyzed (median, 10 samples/patient [range 8-13]). All 5 patients who remained in CR at the time of last measurement had GIN <170 (threshold). Two patients who attained CR, but later relapsed, and all but one patient who had best response other than CR had last GIN measurement of >170. In 5 of 6 patients with relapsed or progressive disease, increasing GIN was observed before the diagnosis by imaging. The abundance of CAR T-cell construct (absolute number of construct copies relative to the number of human genome equivalents) also showed a trend to correlate with outcome (day 10, P = .052). These data describe a proof-of-concept for the use of multiple liquid biopsy technologies to monitor therapeutic response in B-cell lymphoma patients receiving CAR T-cell therapy.

Application of mosaicism ratio to multifetal gestations.

Mosaicism ratio, or MR, is a laboratory metric that can be calculated using massively parallel sequencing data from cell-free DNA (cfDNA) screening. MR compares the amount of cfDNA present from a particular chromosome or chromosomal region to the overall fetal fraction of the specimen. In singleton gestations, MR may be used to refine the positive predictive value of an abnormal cfDNA screening result by identifying cases that could be impacted by various biological factors, such as placental mosaicism or prior co-twin demise. The current study was designed to examine the behavior of mosaicism ratio (MR) in multifetal gestations. Multifetal cfDNA specimens with positive results for trisomies 21, 18, or 13 and confirmed diagnostic outcomes were compiled to examine MR of the aneuploid chromosome based on the number of affected fetuses/placentas. A second multifetal cohort was assembled to analyze the MR of the Y chromosome in cases with at least one male fetus. For aneuploid cases, the average MR of affected singletons (used as a biological proxy for two affected twins) was significantly higher than the average MR for twins in which one fetus was affected. The average MR of the aneuploid chromosome for one affected twin was 52%, 42%, and 48% of that of singleton gestations for trisomy 21, 18, and 13 cases, respectively. MR cutoffs of 0.7 for trisomy 21, and 0.5 for trisomies 18 and 13 may help predict whether one versus both twins are affected with aneuploidy when clinical concern arises. For male cases, the Y MR of XX/XY gestations was 48% of the Y MR for XY/XY gestations. Using a Y MR cutoff of 0.8 allowed determination of XX/XY versus XY/XY gestations with 92.3-94.9% accuracy. Based on the data presented, MR may have utility in the analysis and interpretation of cfDNA data from multifetal gestations.

Early, On-Treatment Levels and Dynamic Changes of Genomic Instability in Circulating Tumor DNA Predict Response to Treatment and Outcome in Metastatic Breast Cancer Patients.

BACKGROUND: Circulating tumor DNA (ctDNA) offers high sensitivity and specificity in metastatic cancer. However, many ctDNA assays rely on specific mutations in recurrent genes or require the sequencing of tumor tissue, difficult to do in a metastatic disease. The purpose of this study was to define the predictive and prognostic values of the whole-genome sequencing (WGS) of ctDNA in metastatic breast cancer (MBC). METHODS: Plasma from 25 patients with MBC were taken at the baseline, prior to treatment (T0), one week (T1) and two weeks (T2) after treatment initiation and subjected to low-pass WGS. DNA copy number changes were used to calculate a Genomic Instability Number (GIN). A minimum predefined GIN value of 170 indicated detectable ctDNA. GIN values were correlated with the treatment response at three and six months by Response Evaluation Criteria in Solid Tumours assessed by imaging (RECIST) criteria and with overall survival (OS). RESULTS: GIN values were detectable (>170) in 64% of patients at the baseline and were significantly prognostic (41 vs. 18 months OS for nondetectable vs. detectable GIN). Detectable GIN values at T1 and T2 were significantly associated with poor OS. Declines in GIN at T1 and T2 of > 50% compared to the baseline were associated with three-month response and, in the case of T1, with OS. On the other hand, a rise in GIN at T2 was associated with a poor response at three months. CONCLUSIONS: Very early measurements using WGS of cell-free DNA (cfDNA) from the plasma of MBC patients provided a tumor biopsy-free approach to ctDNA measurement that was both predictive of the early tumor response at three months and prognostic.

Genome-wide Sequencing of Cell-free DNA Enables Detection of Copy-number Alterations in Patients with Cancer Where Tissue Biopsy is Not Feasible.

When tissue biopsy is not medically prudent or tissue is insufficient for molecular testing, alternative methods are needed. Because cell-free DNA (cfDNA) has been shown to provide a representative surrogate for tumor tissue, we sought to evaluate its utility in this clinical scenario. cfDNA was isolated from the plasma of patients and assayed with low-coverage (∼0.3×), genome-wide sequencing. Copy-number alterations (CNA) were identified and characterized using analytic methods originally developed for noninvasive prenatal testing (NIPT) and quantified using the genomic instability number (GIN), a metric that reflects the quantity and magnitude of CNAs across the genome. The technical variability of the GIN was first evaluated in an independent cohort comprising genome-wide sequencing results from 27,754 women who consented to have their samples used for research and whose NIPT results yielded no detected CNAs to establish a detection threshold. Subsequently, cfDNA sequencing data from 96 patients with known cancers but for whom a tissue biopsy could not be obtained are presented. An elevated GIN was detected in 35% of patients and detection rates varied by tumor origin. Collectively, CNAs covered 96.6% of all autosomes. Survival was significantly reduced in patients with an elevated GIN relative to those without. Overall, these data provide a proof of concept for the use of low-coverage, genome-wide sequencing of cfDNA from patients with cancer to obtain relevant molecular information in instances where tissue is difficult to access. These data may ultimately serve as an informative complement to other molecular tests.

Genome-Wide Sequencing of Cell-Free DNA Identifies Copy-Number Alterations That Can Be Used for Monitoring Response to Immunotherapy in Cancer Patients.

Inhibitors of the PD-1/PD-L1/CTLA-4 immune checkpoint pathway have revolutionized cancer treatment. Indeed, some patients with advanced, refractory malignancies achieve durable responses; however, only a subset of patients benefit, necessitating new biomarkers to predict outcome. Interrogating cell-free DNA (cfDNA) isolated from plasma (liquid biopsy) provides a promising method for monitoring response. We describe the use of low-coverage, genome-wide sequencing of cfDNA, validated extensively for noninvasive prenatal testing, to detect tumor-specific copy-number alterations, and the development of a new metric-the genome instability number (GIN)-to monitor response to these drugs. We demonstrate how the GIN can be used to discriminate clinical response from progression, differentiate progression from pseudoprogression, and identify hyperprogressive disease. Finally, we provide evidence for delayed kinetics in responses to checkpoint inhibitors relative to molecularly targeted therapies. Overall, these data demonstrate a proof of concept for using this method for monitoring treatment outcome in patients with cancer receiving immunotherapy.

Circulating Cell-Free DNA to Determine the Fetal RHD Status in All Three Trimesters of Pregnancy.

OBJECTIVE: To estimate the accuracy of a new assay to determine the fetal RHD status using circulating cell-free DNA. METHODS: This was a prospective, observational study. Maternal blood samples were collected in each trimester of pregnancy in 520 nonalloimmunized RhD-negative patients. Plasma samples were analyzed for circulating cell-free DNA using the SensiGENE RHD test, which used primers for exons 4 and 7 as previously described and incorporated a new primer design for exon 5 of the RHD gene. Neonatal serology for RhD typing using cord blood at birth was undertaken and results were stored in a separate clinical database. After unblinding the data, results of the DNA analysis were compared with the neonatal serology. RESULTS: Inconclusive results secondary to the presence of the RHD pseudogene or an RHD variant were noted in 5.6%, 5.7%, and 6.1% of the first-, second-, and third-trimester samples, respectively. The incidence of false-positive rates for RhD (an RhD-negative fetus with an RHD-positive result) was 1.54% (95% confidence interval [CI] 0.42-5.44%), 1.53% (CI 0.42-5.40%), and 0.82% (CI 0.04-4.50%), respectively. There was only one false-negative diagnosis (an RhD-positive fetus with an RHD-negative result), which occurred in the first trimester (0.32%; 95% CI 0.08-1.78%). Genotyping for mismatches across repeated samples revealed that this error was related to mislabeling of samples from two patients collected on the same day at one of the collection sites. Overall test results were in agreement across all three trimesters (P>.99). CONCLUSION: Circulating cell-free DNA can accurately predict the fetal RhD status in all three trimesters of pregnancy.

High-throughput massively parallel sequencing for fetal aneuploidy detection from maternal plasma.

BACKGROUND: Circulating cell-free (ccf) fetal DNA comprises 3-20% of all the cell-free DNA present in maternal plasma. Numerous research and clinical studies have described the analysis of ccf DNA using next generation sequencing for the detection of fetal aneuploidies with high sensitivity and specificity. We sought to extend the utility of this approach by assessing semi-automated library preparation, higher sample multiplexing during sequencing, and improved bioinformatic tools to enable a higher throughput, more efficient assay while maintaining or improving clinical performance. METHODS: Whole blood (10mL) was collected from pregnant female donors and plasma separated using centrifugation. Ccf DNA was extracted using column-based methods. Libraries were prepared using an optimized semi-automated library preparation method and sequenced on an Illumina HiSeq2000 sequencer in a 12-plex format. Z-scores were calculated for affected chromosomes using a robust method after normalization and genomic segment filtering. Classification was based upon a standard normal transformed cutoff value of z = 3 for chromosome 21 and z = 3.95 for chromosomes 18 and 13. RESULTS: Two parallel assay development studies using a total of more than 1900 ccf DNA samples were performed to evaluate the technical feasibility of automating library preparation and increasing the sample multiplexing level. These processes were subsequently combined and a study of 1587 samples was completed to verify the stability of the process-optimized assay. Finally, an unblinded clinical evaluation of 1269 euploid and aneuploid samples utilizing this high-throughput assay coupled to improved bioinformatic procedures was performed. We were able to correctly detect all aneuploid cases with extremely low false positive rates of 0.09%, <0.01%, and 0.08% for trisomies 21, 18, and 13, respectively. CONCLUSIONS: These data suggest that the developed laboratory methods in concert with improved bioinformatic approaches enable higher sample throughput while maintaining high classification accuracy.