Analysis of Plasma Cell-Free DNA by Ultradeep Sequencing in Patients With Stages I to III Colorectal Cancer.

IMPORTANCE: Novel sensitive methods for detection and monitoring of residual disease can improve postoperative risk stratification with implications for patient selection for adjuvant chemotherapy (ACT), ACT duration, intensity of radiologic surveillance, and, ultimately, outcome for patients with colorectal cancer (CRC). OBJECTIVE: To investigate the association of circulating tumor DNA (ctDNA) with recurrence using longitudinal data from ultradeep sequencing of plasma cell-free DNA in patients with CRC before and after surgery, during and after ACT, and during surveillance. DESIGN, SETTING, AND PARTICIPANTS: In this prospective, multicenter cohort study, ctDNA was quantified in the preoperative and postoperative settings of stages I to III CRC by personalized multiplex, polymerase chain reaction-based, next-generation sequencing. The study enrolled 130 patients at the surgical departments of Aarhus University Hospital, Randers Hospital, and Herning Hospital in Denmark from May 1, 2014, to January 31, 2017. Plasma samples (n = 829) were collected before surgery, postoperatively at day 30, and every third month for up to 3 years. MAIN OUTCOMES AND MEASURES: Outcomes were ctDNA measurement, clinical recurrence, and recurrence-free survival. RESULTS: A total of 130 patients with stages I to III CRC (mean [SD] age, 67.9 [10.1] years; 74 [56.9%] male) were enrolled in the study; 5 patients discontinued participation, leaving 125 patients for analysis. Preoperatively, ctDNA was detectable in 108 of 122 patients (88.5%). After definitive treatment, longitudinal ctDNA analysis identified 14 of 16 relapses (87.5%). At postoperative day 30, ctDNA-positive patients were 7 times more likely to relapse than ctDNA-negative patients (hazard ratio [HR], 7.2; 95% CI, 2.7-19.0; P < .001). Similarly, shortly after ACT ctDNA-positive patients were 17 times (HR, 17.5; 95% CI, 5.4-56.5; P < .001) more likely to relapse. All 7 patients who were ctDNA positive after ACT experienced relapse. Monitoring during and after ACT indicated that 3 of the 10 ctDNA-positive patients (30.0%) were cleared by ACT. During surveillance after definitive therapy, ctDNA-positive patients were more than 40 times more likely to experience disease recurrence than ctDNA-negative patients (HR, 43.5; 95% CI, 9.8-193.5 P < .001). In all multivariate analyses, ctDNA status was independently associated with relapse after adjusting for known clinicopathologic risk factors. Serial ctDNA analyses revealed disease recurrence up to 16.5 months ahead of standard-of-care radiologic imaging (mean, 8.7 months; range, 0.8-16.5 months). Actionable mutations were identified in 81.8% of the ctDNA-positive relapse samples. CONCLUSIONS AND RELEVANCE: Circulating tumor DNA analysis can potentially change the postoperative management of CRC by enabling risk stratification, ACT monitoring, and early relapse detection.

Early Detection of Metastatic Relapse and Monitoring of Therapeutic Efficacy by Ultra-Deep Sequencing of Plasma Cell-Free DNA in Patients With Urothelial Bladder Carcinoma.

PURPOSE: Novel sensitive methods for early detection of relapse and for monitoring therapeutic efficacy may have a huge impact on risk stratification, treatment, and ultimately outcome for patients with bladder cancer. We addressed the prognostic and predictive impact of ultra-deep sequencing of cell-free DNA in patients before and after cystectomy and during chemotherapy. PATIENTS AND METHODS: We included 68 patients with localized advanced bladder cancer. Patient-specific somatic mutations, identified by whole-exome sequencing, were used to assess circulating tumor DNA (ctDNA) by ultra-deep sequencing (median, 105,000×) of plasma DNA. Plasma samples (n = 656) were procured at diagnosis, during chemotherapy, before cystectomy, and during surveillance. Expression profiling was performed for tumor subtype and immune signature analyses. RESULTS: Presence of ctDNA was highly prognostic at diagnosis before chemotherapy (hazard ratio, 29.1; P = .001). After cystectomy, ctDNA analysis correctly identified all patients with metastatic relapse during disease monitoring (100% sensitivity, 98% specificity). A median lead time over radiographic imaging of 96 days was observed. In addition, for high-risk patients (ctDNA positive before or during treatment), the dynamics of ctDNA during chemotherapy was associated with disease recurrence (P = .023), whereas pathologic downstaging was not. Analysis of tumor-centric biomarkers showed that mutational processes (signature 5) were associated with pathologic downstaging (P = .024); however, no significant correlation for tumor subtypes, DNA damage response mutations, and other biomarkers was observed. Our results suggest that ctDNA analysis is better associated with treatment efficacy compared with other available methods. CONCLUSION: ctDNA assessment for early risk stratification, therapy monitoring, and early relapse detection in bladder cancer is feasible and provides a basis for clinical studies that evaluate early therapeutic interventions.

Optimizing Detection of Kidney Transplant Injury by Assessment of Donor-Derived Cell-Free DNA via Massively Multiplex PCR.

Standard noninvasive methods for detecting renal allograft rejection and injury have poor sensitivity and specificity. Plasma donor-derived cell-free DNA (dd-cfDNA) has been reported to accurately detect allograft rejection and injury in transplant recipients and shown to discriminate rejection from stable organ function in kidney transplant recipients. This study used a novel single nucleotide polymorphism (SNP)-based massively multiplexed PCR (mmPCR) methodology to measure dd-cfDNA in various types of renal transplant recipients for the detection of allograft rejection/injury without prior knowledge of donor genotypes. A total of 300 plasma samples (217 biopsy-matched: 38 with active rejection (AR), 72 borderline rejection (BL), 82 with stable allografts (STA), and 25 with other injury (OI)) were collected from 193 unique renal transplant patients; dd- cfDNA was processed by mmPCR targeting 13,392 SNPs. Median dd-cfDNA was significantly higher in samples with biopsy-proven AR (2.3%) versus BL (0.6%), OI (0.7%), and STA (0.4%) (p < 0.0001 all comparisons). The SNP-based dd-cfDNA assay discriminated active from non-rejection status with an area under the curve (AUC) of 0.87, 88.7% sensitivity (95% CI, 77.7⁻99.8%) and 72.6% specificity (95% CI, 65.4⁻79.8%) at a prespecified cutoff (>1% dd-cfDNA). Of 13 patients with AR findings at a routine protocol biopsy six-months post transplantation, 12 (92%) were detected positive by dd-cfDNA. This SNP-based dd-cfDNA assay detected allograft rejection with superior performance compared with the current standard of care. These data support the feasibility of using this assay to detect disease prior to renal failure and optimize patient management in the case of allograft injury.

Tripolar chromosome segregation drives the association between maternal genotype at variants spanning PLK4 and aneuploidy in human preimplantation embryos.

Aneuploidy is prevalent in human embryos and is the leading cause of pregnancy loss. Many aneuploidies arise during oogenesis, increasing with maternal age. Superimposed on these meiotic aneuploidies are frequent errors occurring during early mitotic divisions, contributing to widespread chromosomal mosaicism. Here we reanalyzed a published dataset comprising preimplantation genetic testing for aneuploidy in 24 653 blastomere biopsies from day-3 cleavage-stage embryos, as well as 17 051 trophectoderm biopsies from day-5 blastocysts. We focused on complex abnormalities that affected multiple chromosomes simultaneously, seeking insights into their formation. In addition to well-described patterns such as triploidy and haploidy, we identified 4.7% of blastomeres possessing characteristic hypodiploid karyotypes. We inferred this signature to have arisen from tripolar chromosome segregation in normally fertilized diploid zygotes or their descendant diploid cells. This could occur via segregation on a tripolar mitotic spindle or by rapid sequential bipolar mitoses without an intervening S-phase. Both models are consistent with time-lapse data from an intersecting set of 77 cleavage-stage embryos, which were enriched for the tripolar signature among embryos exhibiting abnormal cleavage. The tripolar signature was strongly associated with common maternal genetic variants spanning the centrosomal regulator PLK4, driving the association we previously reported with overall mitotic errors. Our findings are consistent with the known capacity of PLK4 to induce tripolar mitosis or precocious M-phase upon dysregulation. Together, our data support tripolar chromosome segregation as a key mechanism generating complex aneuploidy in cleavage-stage embryos and implicate maternal genotype at a quantitative trait locus spanning PLK4 as a factor influencing its occurrence.

Incidence of the 22q11.2 deletion in a large cohort of miscarriage samples.

BACKGROUND: The 22q11.2 deletion syndrome is the most common microdeletion syndrome in livebirths, but data regarding its incidence in other populations is limited and also include ascertainment bias. This study was designed to determine the incidence of the 22q11.2 deletion in miscarriage samples sent for clinical molecular cytogenetic testing. RESULTS: Twenty-six thousand one hundred one fresh product of conception (POC) samples were sent to a CLIA- certified, CAP-accredited laboratory from April 2010--May 2016 for molecular cytogenetic miscarriage testing using a single-nucleotide polymorphism (SNP)-based microarray platform. A retrospective review determined the incidence of the 22q11.2 deletion in this sample set. Fetal results were obtained in 22,451 (86%) cases, of which, 15 (0.07%) had a microdeletion in the 22q11.2 region (incidence, 1/1497). Of those, 12 (80%) cases were found in samples that were normal at the resolution of traditional karyotyping (i.e., had no chromosome abnormalities above 10 Mb in size) and three (20%) cases had additional findings (Trisomy 15, Trisomy 16, XXY). Ten (67%) cases with a 22q11.2 deletion had the common ~3 Mb deletion; the remaining 5 cases had deletions ranging in size from 0.65 to 1.5 Mb. A majority (12/15) of cases had a deletion on the maternally inherited chromosome. No significant relationship between maternal age and presence of a fetal 22q11.2 deletion was observed. CONCLUSIONS: The observed incidence of 1/1497 for the 22q11.2 deletion in miscarriage samples is higher than the reported general population prevalence (1/4000-1/6000). Further research is needed to determine whether the 22q11.2 deletion is a causal factor for miscarriage.

Validation of an Enhanced Version of a Single-Nucleotide Polymorphism-Based Noninvasive Prenatal Test for Detection of Fetal Aneuploidies.

OBJECTIVE: To validate an updated version (Version 2) of a single-nucleotide polymorphism (SNP)-based noninvasive prenatal test (NIPT) and to determine the likelihood of success when testing for fetal aneuploidies following a redraw. METHODS: Version 2 was analytically validated using 587 plasma samples with known genotype (184 trisomy 21, 37 trisomy 18, 15 trisomy 13, 9 monosomy X, 4 triploidy and 338 euploid). Sensitivity, specificity and no-call rate were calculated, and a fetal-fraction adjustment was applied to enable projection of these values in a commercial distribution. Likelihood of success of a second blood draw was computed based on fetal fraction and maternal weight from the first draw. RESULTS: Validation of this methodology yielded high sensitivities (≥99.4%) and specificities (100%) for all conditions tested with an observed no-call rate of 2.3%. The no-call threshold for sample calling was reduced to 2.8% fetal fraction. The redraw success rate was driven by higher initial fetal fractions and lower maternal weights, with the fetal fraction being the more significant variable. CONCLUSIONS: The enhanced version of this SNP-based NIPT method showed a reduced no-call rate and a reduced fetal-fraction threshold for sample calling in comparison to the earlier version, while maintaining high sensitivity and specificity.

Detection of Clonal and Subclonal Copy-Number Variants in Cell-Free DNA from Patients with Breast Cancer Using a Massively Multiplexed PCR Methodology.

We demonstrate proof-of-concept for the use of massively multiplexed PCR and next-generation sequencing (mmPCR-NGS) to identify both clonal and subclonal copy-number variants (CNVs) in circulating tumor DNA. This is the first report of a targeted methodology for detection of CNVs in plasma. Using an in vitro model of cell-free DNA, we show that mmPCR-NGS can accurately detect CNVs with average allelic imbalances as low as 0.5%, an improvement over previously reported whole-genome sequencing approaches. Our method revealed differences in the spectrum of CNVs detected in tumor tissue subsections and matching plasma samples from 11 patients with stage II breast cancer. Moreover, we showed that liquid biopsies are able to detect subclonal mutations that may be missed in tumor tissue biopsies. We anticipate that this mmPCR-NGS methodology will have broad applicability for the characterization, diagnosis, and therapeutic monitoring of CNV-enriched cancers, such as breast, ovarian, and lung cancer.

Evidence of Selection against Complex Mitotic-Origin Aneuploidy during Preimplantation Development.

Whole-chromosome imbalances affect over half of early human embryos and are the leading cause of pregnancy loss. While these errors frequently arise in oocyte meiosis, many such whole-chromosome abnormalities affecting cleavage-stage embryos are the result of chromosome missegregation occurring during the initial mitotic cell divisions. The first wave of zygotic genome activation at the 4-8 cell stage results in the arrest of a large proportion of embryos, the vast majority of which contain whole-chromosome abnormalities. Thus, the full spectrum of meiotic and mitotic errors can only be detected by sampling after the initial cell divisions, but prior to this selective filter. Here, we apply 24-chromosome preimplantation genetic screening (PGS) to 28,052 single-cell day-3 blastomere biopsies and 18,387 multi-cell day-5 trophectoderm biopsies from 6,366 in vitro fertilization (IVF) cycles. We precisely characterize the rates and patterns of whole-chromosome abnormalities at each developmental stage and distinguish errors of meiotic and mitotic origin without embryo disaggregation, based on informative chromosomal signatures. We show that mitotic errors frequently involve multiple chromosome losses that are not biased toward maternal or paternal homologs. This outcome is characteristic of spindle abnormalities and chaotic cell division detected in previous studies. In contrast to meiotic errors, our data also show that mitotic errors are not significantly associated with maternal age. PGS patients referred due to previous IVF failure had elevated rates of mitotic error, while patients referred due to recurrent pregnancy loss had elevated rates of meiotic error, controlling for maternal age. These results support the conclusion that mitotic error is the predominant mechanism contributing to pregnancy losses occurring prior to blastocyst formation. This high-resolution view of the full spectrum of whole-chromosome abnormalities affecting early embryos provides insight into the cytogenetic mechanisms underlying their formation and the consequences for human fertility.

Whole genome prediction for preimplantation genetic diagnosis.

BACKGROUND: Preimplantation genetic diagnosis (PGD) enables profiling of embryos for genetic disorders prior to implantation. The majority of PGD testing is restricted in the scope of variants assayed or by the availability of extended family members. While recent advances in single cell sequencing show promise, they remain limited by bias in DNA amplification and the rapid turnaround time (<36 h) required for fresh embryo transfer. Here, we describe and validate a method for inferring the inherited whole genome sequence of an embryo for preimplantation genetic diagnosis (PGD). METHODS: We combine haplotype-resolved, parental genome sequencing with rapid embryo genotyping to predict the whole genome sequence of a day-5 human embryo in a couple at risk of transmitting alpha-thalassemia. RESULTS: Inheritance was predicted at approximately 3 million paternally and/or maternally heterozygous sites with greater than 99% accuracy. Furthermore, we successfully phase and predict the transmission of an HBA1/HBA2 deletion from each parent. CONCLUSIONS: Our results suggest that preimplantation whole genome prediction may facilitate the comprehensive diagnosis of diseases with a known genetic basis in embryos.

Common variants spanning PLK4 are associated with mitotic-origin aneuploidy in human embryos.

Aneuploidy, the inheritance of an atypical chromosome complement, is common in early human development and is the primary cause of pregnancy loss. By screening day-3 embryos during in vitro fertilization cycles, we identified an association between aneuploidy of putative mitotic origin and linked genetic variants on chromosome 4 of maternal genomes. This associated region contains a candidate gene, Polo-like kinase 4 (PLK4), that plays a well-characterized role in centriole duplication and has the ability to alter mitotic fidelity upon minor dysregulation. Mothers with the high-risk genotypes contributed fewer embryos for testing at day 5, suggesting that their embryos are less likely to survive to blastocyst formation. The associated region coincides with a signature of a selective sweep in ancient humans, suggesting that the causal variant was either the target of selection or hitchhiked to substantial frequency.

Detection of triploid, molar, and vanishing twin pregnancies by a single-nucleotide polymorphism-based noninvasive prenatal test.

OBJECTIVE: We sought to determine the ability of single-nucleotide polymorphism-based noninvasive prenatal testing (NIPT) to identify triploid, unrecognized twin, and vanishing twin pregnancies. STUDY DESIGN: The study included 30,795 consecutive reported clinical cases received for NIPT for fetal whole-chromosome aneuploidies; known multiple gestations were excluded. Cell-free DNA was isolated from maternal blood samples, amplified via 19,488-plex polymerase chain reaction, and sequenced. Sequencing results were analyzed to determine fetal chromosome copy number and to identify the presence of additional fetal haplotypes. RESULTS: Additional fetal haplotypes, indicative of fetal triploidy, vanishing twin, or undetected twin pregnancy, were identified in 130 (0.42%) cases. Clinical confirmation (karyotype for singleton pregnancies, ultrasound for multifetal pregnancies) was available for 58.5% (76/130) of cases. Of the 76 cases with confirmation, 42.1% were vanishing twin, 48.7% were viable twin, 5.3% were diandric triploids, and 3.9% were nontriploid pregnancies that lacked evidence of co-twin demise. One pregnancy had other indications suggesting triploidy but lacked karyotype confirmation. Of the 5 vanishing twin cases with a known date of demise, 100% of losses occurred in the first trimester; up to 8 weeks elapsed between loss and detection by NIPT. CONCLUSION: This single-nucleotide polymorphism-based NIPT successfully identified vanished twin, previously unrecognized twin, and triploid pregnancies. As vanishing twins are more likely to be aneuploid, and undetected residual cell-free DNA could bias NIPT results, the ability of this method to identify additional fetal haplotypes is expected to result in fewer false-positive calls and prevent incorrect fetal sex calls.

Expanding the scope of noninvasive prenatal testing: detection of fetal microdeletion syndromes.

OBJECTIVE: The purpose of this study was to estimate the performance of a single-nucleotide polymorphism (SNP)-based noninvasive prenatal test for 5 microdeletion syndromes. STUDY DESIGN: Four hundred sixty-nine samples (358 plasma samples from pregnant women, 111 artificial plasma mixtures) were amplified with the use of a massively multiplexed polymerase chain reaction, sequenced, and analyzed with the use of the Next-generation Aneuploidy Test Using SNPs algorithm for the presence or absence of deletions of 22q11.2, 1p36, distal 5p, and the Prader-Willi/Angelman region. RESULTS: Detection rates were 97.8% for a 22q11.2 deletion (45/46) and 100% for Prader-Willi (15/15), Angelman (21/21), 1p36 deletion (1/1), and cri-du-chat syndromes (24/24). False-positive rates were 0.76% for 22q11.2 deletion syndrome (3/397) and 0.24% for cri-du-chat syndrome (1/419). No false positives occurred for Prader-Willi (0/428), Angelman (0/442), or 1p36 deletion syndromes (0/422). CONCLUSION: SNP-based noninvasive prenatal microdeletion screening is highly accurate. Because clinically relevant microdeletions and duplications occur in >1% of pregnancies, regardless of maternal age, noninvasive screening for the general pregnant population should be considered.

Clinical experience and follow-up with large scale single-nucleotide polymorphism-based noninvasive prenatal aneuploidy testing.

OBJECTIVE: We sought to report on laboratory and clinical experience following 6 months of clinical implementation of a single-nucleotide polymorphism-based noninvasive prenatal aneuploidy test in high- and low-risk women. STUDY DESIGN: All samples received from March through September 2013 and drawn ≥9 weeks' gestation were included. Samples that passed quality control were analyzed for trisomy 21, trisomy 18, trisomy 13, and monosomy X. Results were reported as high or low risk for fetal aneuploidy for each interrogated chromosome. Relationships between fetal fraction and gestational age and maternal weight were analyzed. Follow-up on outcome was sought for a subset of high-risk cases. False-negative results were reported voluntarily by providers. Positive predictive value (PPV) was calculated from cases with an available prenatal or postnatal karyotype or clinical evaluation at birth. RESULTS: Samples were received from 31,030 patients, 30,705 met study criteria, and 28,739 passed quality-control metrics and received a report detailing aneuploidy risk. Fetal fraction correlated positively with gestational age, and negatively with maternal weight. In all, 507 patients received a high-risk result for any of the 4 tested conditions (324 trisomy 21, 82 trisomy 18, 41 trisomy 13, 61 monosomy X; including 1 double aneuploidy case). Within the 17,885 cases included in follow-up analysis, 356 were high risk, and outcome information revealed 184 (51.7%) true positives, 38 (10.7%) false positives, 19 (5.3%) with ultrasound findings suggestive of aneuploidy, 36 (10.1%) spontaneous abortions without karyotype confirmation, 22 (6.2%) terminations without karyotype confirmation, and 57 (16.0%) lost to follow-up. This yielded an 82.9% PPV for all aneuploidies, and a 90.9% PPV for trisomy 21. The overall PPV for women aged ≥35 years was similar to the PPV for women aged <35 years. Two patients were reported as false negatives. CONCLUSION: The data from this large-scale report on clinical application of a commercially available noninvasive prenatal test suggest that the clinical performance of this single-nucleotide polymorphism-based noninvasive prenatal test in a mixed high- and low-risk population is consistent with performance in validation studies.

Genomic imbalance in products of conception: single-nucleotide polymorphism chromosomal microarray analysis.

OBJECTIVE: To report the full cohort of identifiable anomalies, regardless of known clinical significance, in a large-scale cohort of postmiscarriage products-of-conception samples analyzed using a high-resolution single-nucleotide polymorphism (SNP)-based microarray platform. High-resolution chromosomal microarray analysis allows for the identification of visible and submicroscopic cytogenomic imbalances; the specific use of SNPs permits detection of maternal cell contamination, triploidy, and uniparental disomy. METHODS: Miscarriage specimens were sent to a single laboratory for cytogenomic analysis. Chromosomal microarray analysis was performed using a SNP-based genotyping microarray platform. Results were evaluated at the cytogenetic and microscopic (greater than 10 Mb) and submicroscopic (less than 10 Mb) levels. Maternal cell contamination was assessed using information derived from fetal and maternal SNPs. RESULTS: Results were obtained on 2,389 of 2,392 specimens (99.9%) that were less than 20 weeks of gestation. Maternal cell contamination was identified in 528 (22.0%) specimens. The remaining 1,861 specimens were considered to be of true fetal origin. Of these, 1,106 (59.4%) showed classical cytogenetic abnormalities: aneuploidy accounted for 945 (85.4%), triploidy for 114 (10.3%), and structural anomalies or tetraploidy for the remaining 47 (4.2%). Of the 755 (40.6%) cases considered normal at the cytogenetic level, SNP chromosomal microarray analysis revealed a clinically significant copy number change or whole-genome uniparental disomy in 12 (1.6%) and three (0.4%) cases, respectively. CONCLUSION: Chromosomal microarray analysis of products-of-conception specimens yields a high diagnostic return. Using SNPs extends the scope of detectable genomic abnormalities and facilitates reporting "true" fetal results. This supports the use of SNP chromosomal microarray analysis for cytogenomic evaluation of miscarriage specimens when clinically indicated. LEVEL OF EVIDENCE: III.

Single-nucleotide polymorphism-based noninvasive prenatal screening in a high-risk and low-risk cohort.

OBJECTIVE: To estimate performance of a single-nucleotide polymorphism-based noninvasive prenatal screen for fetal aneuploidy in high-risk and low-risk populations on single venopuncture. METHODS: One thousand sixty-four maternal blood samples from 7 weeks of gestation and beyond were included; 1,051 were within specifications and 518 (49.3%) were low risk. Cell-free DNA was amplified, sequenced, and analyzed using the Next-generation Aneuploidy Test Using SNPs algorithm. Samples were called as trisomies 21, 18, 13, or monosomy X, or euploid, and male or female. RESULTS: Nine hundred sixty-six samples (91.9%) successfully generated a cell-free DNA result. Among these, sensitivity was 100% for trisomy 21 (58/58, confidence interval [CI] 93.8-100%), trisomy 13 (12/12, CI 73.5-100%), and fetal sex (358/358 female, CI 99.0-100%; 418/418 male, CI 99.1-100%), 96.0% for trisomy 18 (24/25, CI 79.7-99.9%), and 90% for monosomy X (9/10, CI 55.5-99.8%). Specificity for trisomies 21 and 13 was 100% (905/905, CI 99.6-100%; and 953/953, CI 99.6-100%, respectively) and for trisomy 18 and monosomy X was 99.9% (938/939, CI 99.4-100%; and 953/954, CI 99.4-100%, respectively). However, 16% (20/125) of aneuploid samples did not return a result; 50% (10/20) had a fetal fraction below the 1.5th percentile of euploid pregnancies. Aneuploidy rate was significantly higher in these samples (P<.001, odds ratio 9.2, CI 4.4-19.0). Sensitivity and specificity did not differ in low-risk and high-risk populations. CONCLUSIONS: This noninvasive prenatal screen performed with high sensitivity and specificity in high-risk and low-risk cohorts. Aneuploid samples were significantly more likely to not return a result; the number of aneuploidy samples was especially increased among samples with low fetal fraction. This underscores the importance of redraws or, in rare cases, invasive procedures based on low fetal fraction. LEVEL OF EVIDENCE: II.

Non-invasive prenatal detection of trisomy 13 using a single nucleotide polymorphism- and informatics-based approach.

PURPOSE: To determine how a single nucleotide polymorphism (SNP)- and informatics-based non-invasive prenatal aneuploidy test performs in detecting trisomy 13. METHODS: Seventeen trisomy 13 and 51 age-matched euploid samples, randomly selected from a larger cohort, were analyzed. Cell-free DNA was isolated from maternal plasma, amplified in a single multiplex polymerase chain reaction assay that interrogated 19,488 SNPs covering chromosomes 13, 18, 21, X, and Y, and sequenced. Analysis and copy number identification involved a Bayesian-based maximum likelihood statistical method that generated chromosome- and sample-specific calculated accuracies. RESULTS: Of the samples that passed a stringent DNA quality threshold (94.1%), the algorithm correctly identified 15/15 trisomy 13 and 49/49 euploid samples, for 320/320 correct copy number calls. CONCLUSIONS: This informatics- and SNP-based method accurately detects trisomy 13-affected fetuses non-invasively and with high calculated accuracy.

Reliability of 46,XX results on miscarriage specimens: a review of 1,222 first-trimester miscarriage specimens.

OBJECTIVE: To examine the rate of maternal contamination in miscarriage specimens. DESIGN: Retrospective review of 1,222 miscarriage specimens submitted for chromosome testing with detection of maternal cell contamination (MCC). SETTING: Referral centers requesting genetic testing of miscarriage specimens at a single reference laboratory. PATIENT(S): Women with pregnancy loss who desire complete chromosome analysis of the pregnancy tissue. INTERVENTION(S): Analysis of miscarriage specimens using single-nucleotide polymorphism (SNP) microarray technology with bioinformatics program to detect maternal cell contamination. MAIN OUTCOME MEASURE(S): Chromosome content of miscarriages and incidence of 46,XX results due to MCC. RESULT(S): Of the 1,222 samples analyzed, 592 had numeric chromosomal abnormalities, and 630 were normal 46,XX or 46,XY (456 and 187, respectively). In 269 of the 46,XX specimens, MCC with no embryonic component was found. With the exclusion of maternal 46,XX results, the chromosomal abnormality rate increased from 48% to 62%, and the ratio for XX to XY results dropped from 2.6 to 1.0. CONCLUSION(S): Over half of the normal 46,XX results in miscarriage specimens were due to MCC. The use of SNPs in MCC testing allows for precise identification of chromosomal abnormalities in miscarriage as well as MCC, improving the accuracy of products of conception testing.

SNP-based non-invasive prenatal testing detects sex chromosome aneuploidies with high accuracy.

OBJECTIVE: This study aimed to develop a single-nucleotide polymorphism-based and informatics-based non-invasive prenatal test that detects sex chromosome aneuploidies early in pregnancy. METHODS: Sixteen aneuploid samples, including thirteen 45,X, two 47,XXY, and one 47,XYY, along with 185 euploid controls, were analyzed. Cell-free DNA was isolated from maternal plasma, amplified in a single multiplex polymerase chain reaction assay that targeted 19,488 polymorphic loci covering chromosomes 13, 18, 21, X, and Y, and sequenced. Sequencing results were analyzed using a Bayesian-based maximum likelihood statistical method to determine copy number of interrogated chromosomes, calculating sample-specific accuracies. RESULTS: Of the samples that passed a stringent quality control metric (93%), the algorithm correctly identified copy number at all five chromosomes in all but one of the 187 samples, for 934/935 correct calls as early as 9.4 weeks of gestation. We detected 45,X with 91.7% sensitivity (CI: 61.5-99.8%) and 100% specificity (CI: 97.9-100%), and 47,XXY and 47,XYY. The average calculated accuracy was 99.78%. CONCLUSION: This method non-invasively detected 45,X, 47,XXY, and 47,XYY fetuses from cell-free DNA isolated from maternal plasma with high calculated accuracies and thus offers a non-invasive method with the potential to function as a routine screen allowing for early prenatal detection of rarely diagnosed yet commonly occurring sex aneuploidies.