Whole-genome sequencing (WGS) of maternal plasma cell-free DNA (cfDNA) can potentially evaluate all 24 chromosomes to identify abnormalities of the placenta, fetus, or pregnant woman. Current bioinformatics algorithms typically only report on chromosomes 21, 18, 13, X, and Y; sequencing results from other chromosomes may be masked. We hypothesized that by systematically analyzing WGS data from all chromosomes, we could identify rare autosomal trisomies (RATs) to improve understanding of feto-placental biology. We analyzed two independent cohorts from clinical laboratories, both of which used a similar quality control parameter, normalized chromosome denominator quality. The entire data set included 89,817 samples. Samples flagged for analysis and classified as abnormal were 328 of 72,932 (0.45%) and 71 of 16,885 (0.42%) in cohorts 1 and 2, respectively. Clinical outcome data were available for 57 of 71 (80%) of abnormal cases in cohort 2. Visual analysis of WGS data demonstrated RATs, copy number variants, and extensive genome-wide imbalances. Trisomies 7, 15, 16, and 22 were the most frequently observed RATs in both cohorts. Cytogenetic or pregnancy outcome data were available in 52 of 60 (87%) of cases with RATs in cohort 2. Cases with RATs detected were associated with miscarriage, true fetal mosaicism, and confirmed or suspected uniparental disomy. Comparing the trisomic fraction with the fetal fraction allowed estimation of possible mosaicism. Analysis and reporting of aneuploidies in all chromosomes can clarify cases in which cfDNA findings on selected "target" chromosomes (21, 18, and 13) are discordant with the fetal karyotype and may identify pregnancies at risk of miscarriage and other complications.

BackgroundNon-invasive prenatal testing (NIPT) identifies fetal aneuploidy by sequencing cell-free DNA in the maternal plasma. Pre-symptomatic maternal malignancies have been incidentally detected during NIPT based on abnormal genomic profiles. This low coverage sequencing approach could have potential for ovarian cancer screening in the non-pregnant population. Our objective was to investigate whether plasma DNA sequencing with a clinical whole genome NIPT platform can detect early- and late-stage high-grade serous ovarian carcinomas (HGSOC).MethodsThis is a case control study of prospectively-collected biobank samples comprising preoperative plasma from 32 women with HGSOC (16 ‘early cancer’ (FIGO I–II) and 16 ‘advanced cancer’ (FIGO III–IV)) and 32 benign controls. Plasma DNA from cases and controls were sequenced using a commercial NIPT platform and chromosome dosage measured.Sequencing data were blindly analyzed with two methods: (1) Subchromosomal changes were called using an open source algorithm WISECONDOR (WIthin-SamplE COpy Number aberration DetectOR). Genomic gains or losses ≥ 15 Mb were prespecified as “screen positive” calls, and mapped to recurrent copy number variations reported in an ovarian cancer genome atlas. (2) Selected whole chromosome gains or losses were reported using the routine NIPT pipeline for fetal aneuploidy.ResultsWe detected 13/32 cancer cases using the subchromosomal analysis (sensitivity 40.6 %, 95 % CI, 23.7–59.4 %), including 6/16 early and 7/16 advanced HGSOC cases. Two of 32 benign controls had subchromosomal gains ≥ 15 Mb (specificity 93.8 %, 95 % CI, 79.2–99.2 %). Twelve of the 13 true positive cancer cases exhibited specific recurrent changes reported in HGSOC tumors. The NIPT pipeline resulted in one “monosomy 18” call from the cancer group, and two “monosomy X” calls in the controls.ConclusionsLow coverage plasma DNA sequencing used for prenatal testing detected 40.6 % of all HGSOC, including 38 % of early stage cases. Our findings demonstrate the potential of a high throughput sequencing platform to screen for early HGSOC in plasma based on characteristic multiple segmental chromosome gains and losses. The performance of this approach may be further improved by refining bioinformatics algorithms and targeting selected cancer copy number variations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12916-016-0667-6) contains supplementary material, which is available to authorized users.

Background Noninvasive prenatal tests (NIPTs) detect fetal chromosomal anomalies with high clinical sensitivity and specificity. We examined the performance of a paired-end sequencing-based NIPT in the detection of genome-wide fetal chromosomal anomalies including common trisomies, sex chromosomal aneuploidies (SCA), rare autosomal aneuploidies (RAAs), and partial deletions/duplications ≥7 Mb. Methods Frozen plasma samples from pregnant women were tested using the VeriSeq NIPT Solution v2 assay. All samples were previously tested with a laboratory-developed NIPT and had known clinical outcomes. Individuals performing the sequencing were blinded to clinical outcome data. Clinical sensitivity and specificity were determined for basic (chromosomes 21, 18, 13, X, and Y) and genome-wide screening modes. Results Of 2335 samples that underwent genome-wide analysis, 28 did not meet QC requirements, resulting in a first-pass assay failure rate of 1.2%. Basic screening analysis, excluding known mosaics, correctly classified 130/130 trisomy 21 samples (sensitivity >99.9%, 95% confidence interval [CI] 97.1%–100%), 41/41 trisomy 18 samples (sensitivity >99.9%, 95% CI 91.4%–100%), and 26/26 trisomy 13 samples (sensitivity >99.9%, 95% CI 87.1%–100%) with 6 false-positive results; specificities ≥99.90% were reported for all 3 trisomies. Concordance for SCAs ranged from 90.5%–100%. Genome-wide screening analysis including known mosaics correctly classified 27/28 RAAs and 20/27 partial deletions/duplications with a specificity of 99.80% for both anomalies, and an overall genome-wide specificity for all anomalies of 99.34%. Conclusions The VeriSeq NIPT Solution v2 assay enables accurate identification of fetal aneuploidy, allowing detection of genome-wide fetal chromosomal anomalies with high clinical sensitivities and specificities and a low assay failure rate. Clinical Trial Notification [CTN] identification number [ID]: CT-2018-CTN-01585-1 v1, Protocol: NIPT T05 002.

Noninvasive prenatal testing (NIPT) is a screening test for fetal chromosomal aneuploidy using cell-free DNA derived from maternal blood. It has been rapidly accepted into obstetric practice because of its application from 10-weeks' gestation, and its high sensitivity and specificity. NIPT results can be influenced by several factors including placental or maternal mosaicism and co-twin demise; cell-free DNA from a maternal origin can also complicate interpretation, with evidence that NIPT can detect previously unsuspected malignancies. This study aimed to develop management guidelines for women with NIPT results suspicious of maternal malignancy. The Peter MacCallum Cancer Center's experience of seven cases where abnormal NIPT results led to investigation for maternal malignancy between 2016 and 2019 were reviewed, along with the published literature. Six of the seven women (86%) referred for investigation were diagnosed with advanced malignancies, including colorectal cancer, breast cancer, melanoma, and Hodgkin lymphoma. Based on our single-center experience, as well as the available literature, guidelines for the investigation of women with NIPT results suspicious of malignancy are proposed, including utilization of fluorodeoxyglucose positron emission tomography-computed tomography, which had a high concordance with other investigations and diagnoses. These guidelines include maternal and fetal investigations, as well as consideration of the complex medical, psychologic, social, and ethical needs of these patients and their families.

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False-negative cell-free DNA (cfDNA) screening results involving Down syndrome are rare, but have high clinical impact on patients and their healthcare providers. Understanding the biology behind these results may allow for improved diagnostic follow-up and counseling. In 5 different centers offering cfDNA prenatal screening, 9 false-negative results were documented in 646 confirmed cases of trisomy 21; a false-negative rate of 1.4% (95% CI, 0.7-2.6). False-negative results included 4 cases of classical trisomy 21 and 5 cases with a de novo 21q;21q rearrangement. Two out of five rearrangements had molecular studies and were confirmed as isochromosomes. When combined with reports from the cfDNA screening literature, 8 out of 29 (28%) Down syndrome cases with a false-negative "non-invasive prenatal test" (NIPT) were associated with a 21q;21q rearrangement, compared with 2% reported in live born children with Down syndrome. In our laboratory series, evidence for placental or fetal mosaicism was present in 3 out of 3 true-positive cases involving a 21q;21q rearrangement and was confirmed in one false-negative case where placental material was available for study. Isochromosome 21q rearrangements are thus overrepresented among false-negative cfDNA screening results involving Down syndrome. Postzygotic isochromosome formation leading to placental mosaicism provides a biological cause for the increased prevalence of these rearrangements among false-negative cases. For clinical practice, a low trisomic fraction (z-score or equivalent measure) relative to the fetal fraction suggests placental mosaicism. Care should be taken as these cases may not reflect confined placental mosaicism, but rather full trisomy in the presence of a placenta containing normal cells.

Non-invasive prenatal testing (NIPT) for fetal aneuploidies by massively parallel sequencing has transformed the approach to prenatal care. The presence of circulating fetal cell-free DNA (cfDNA) in maternal plasma, which is derived primarily from the placenta, is the basis of this test.1 Studies comparing NIPT to serum or combined screening have shown that NIPT outperforms these screening options in trisomy detection and has significantly lower false positive rates. 2-4 Biological causes of false positive NIPT results may include maternal mosaicism (primarily sex chromosome mosaicism), confined placental mosaicism, co-twin demise, maternal malignancy and maternal copy number variation. 2,5 Here, we present a case of a nonstandard NIPT result involving a large and pathogenic partial duplication of chromosome 18q first indicated by NIPT. Subsequent prenatal diagnosis confirmed the presence of the duplication in the fetus, which was inherited from the phenotypically normal mother who carried the same chromosomal imbalance, but in mosaic form. A review of the NIPT data indicates that maternal mosaicism for this large segmental imbalance is sufficient to cause a false positive NIPT result in subsequent pregnancies, in the absence of the imbalance being transmitted to the fetus. Therefore, maternal mosaicism for large segmental copy number abnormalities may be yet another rare cause of false positive NIPT results, which further highlights the need for confirmatory invasive testing whenever an abnormal NIPT result is returned.The patient was a 38-year-old G3P2 woman who underwent first trimester combined screening. Risks for trisomy 18 and trisomy 21 were calculated at 1:13,100 and 1:1,690, respectively. No further investigations for Down syndrome or trisomy 18 were recommended on the basis of these low risk results. A fetal anomaly ultrasound scan at 20 weeks of gestation identified an isolated aberrant right subclavian artery. The sonologist counselled a low risk for chromosome abnormality 6 but informed the patient of the option of NIPT and amniocentesis. The patient was not referred for formal genetic counselling and pursued NIPT independently.Maternal venous blood was collected for NIPT at 20 + 6 weeks of gestation, and testing was performed by Natera Panorama ™ test, which is a targeted, multiplexed, single nucleotide polymorphism (SNP)-based NIPT assay. 7 The NIPT returned an unexpected finding with no result for chromosome 18 because of an 'atypical finding outside the current scope of the test'. The atypical finding was a suspicion of a partial duplication of chromosome 18. To help direct follow-up testing, a request was made for genomic or cytogenetic breakpoint information but was declined on the basis that the information being sought was outside the current scope of the test. Genetic counselling was arranged, and the patient elected amniocentesis at 22 weeks of gestation, at which time a small ventricular septal defect on ultrasound was also noted. As no positional information for the suspected duplication...

Objectives: To evaluate the performance of cell-free DNA (cfDNA) screening for common fetal aneuploidies, choice of prenatal procedure, and chromosome conditions identified during pregnancy after low-risk cfDNA screening.Method: A single-center prenatal cfDNA screening test was employed to detect trisomies 21, 18, and 13 (T21, T18, T13) and sex chromosome aneuploidies (SCAs).Test performance, choice of prenatal procedure, and cytogenetic results in pregnancies with low-risk cfDNA screening were reviewed.Results: CfDNA screening of 38,289 consecutive samples identified 720 (1.9%) pregnancies at increased risk for aneuploidy. Positive predictive values (PPVs) for high-risk singleton pregnancies were 98.5% (T21), 92.5% (T18) and 55.2% (T13).PPVs for SCAs ranged from 30.6% to 95.2%. Most women elected chorionic villus sampling for prenatal diagnosis of T21, T18 and T13; amniocentesis and/or postnatal testing were commonly chosen for SCAs. Cytogenetic tests from 616 screennegative pregnancies identified 64 cases (12.7%) with chromosome conditions not detected by cfDNA screening, including triploidy (n = 30) and pathogenic and likely pathogenic copy number variants (n = 34). A further 15 (0.04%) false-negative common aneuploidy results were identified.Conclusions: CfDNA screening was highly accurate for detecting fetal aneuploidy in this general-risk obstetric population. Fetal ultrasound and prenatal diagnostic testing were important in identifying chromosome conditions in pregnancies screened as low-risk. Key pointsWhat's already known about this topic? � Prenatal cell-free DNA (cfDNA) screening for common autosomal trisomies has high sensitivity and specificity. Fewer data are available for sex chromosome aneuploidies (SCAs), which show greater variability in screening performance.