Routine Noninvasive Prenatal Testing Research Articles

NanoNIPT: Short-fragment nanopore sequencing of cell-free DNA for non-invasive prenatal testing of fetal aneuploidies and sex chromosome aberrations.

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Performance of whole-genome promoter nucleosome profiling of maternal plasma cell-free DNA for prenatal noninvasive prediction of fetal macrosomia: a retrospective nested case-control study in mainland China

BackgroundFetal macrosomia is common occurrence in pregnancy, which is associated with several adverse prognosis both of maternal and neonatal. While, the accuracy of prediction of fetal macrosomia is poor. The aim of this study was to develop a reliable noninvasive prediction classifier of fetal macrosomia.MethodsA total of 3600 samples of routine noninvasive prenatal testing (NIPT) data at 12+ 0–27+ 6 weeks of gestation, which were subjected to low-coverage whole-genome sequencing of maternal plasma cell-free DNA (cfDNA), were collected from three independent hospitals. We identified set of genes with significant differential coverages by comparing the promoter profiling between macrosomia cases and controls. We selected genes to develop classifier for noninvasive predicting, by using support vector machine (SVM) and logistic regression models, respectively. The performance of each classifier was evaluated by area under the curve (AUC) analysis.ResultsAccording to the available follow-up results, 162 fetal macrosomia pregnancies and 648 matched controls were included. A total of 1086 genes with significantly differential promoter profiling were found between pregnancies with macrosomia and controls (p < 0.05). With the AUC as a reference，the classifier based on SVM (CMA-A2) had the best performance, with an AUC of 0.8256 (95% CI: 0.7927–0.8586).ConclusionsOur study provides that assessing the risk of fetal macrosomia by whole-genome promoter nucleosome profiling of maternal plasma cfDNA based on low-coverage next-generation sequencing is feasible.

Estimation of cell-free fetal DNA fraction from maternal plasma based on linkage disequilibrium information

Cell-free fetal DNA fraction (FF) in maternal plasma is a key parameter affecting the performance of noninvasive prenatal testing (NIPT). Accurate quantitation of FF plays a pivotal role in these tests. However, there are few methods that could determine FF with high accuracy using shallow‐depth whole‐genome sequencing data. In this study, we hypothesized that the actual FF in maternal plasma should be proportional to the discrepancy rate between the observed genotypes and inferred genotypes based on the linkage disequilibrium rule in certain polymorphism sites. Based on this hypothesis, we developed a method named Linkage Disequilibrium information-based cell-free Fetal DNA Fraction (LDFF) to accurately quantify FF in maternal plasma. This method achieves a high performance and outperforms existing methods in the fetal DNA fraction estimation. As LDFF is a gender-independent method and developed on shallow-depth samples, it can be easily incorporated into routine NIPT test and may enhance the current NIPT performance.

Genomic studies of Hodgkin lymphoma using circulating cell-free DNA

The revolutionary finding of cell free DNA (cfDNA) circulating in the bloodstream had a huge impact on the development of non-invasive prenatal testing (NIPT) (obstetrics) and liquid biopsies (oncology). The latter, combined with the sequencing of tumor DNA-containing cfDNA, have been widely applied in cancer research, demonstrating the potential of these techniques to improve prognostication and guide individualized treatment strategies. During routine NIPT analysis of more than 88,000 pregnant women performed in our institution, 14 abnormal genomic profiles suggestive of maternal tumor have been identified. Interestingly, one patient was further diagnosed with classic Hodgkin lymphoma (cHL), a tumor characterized by a low content (&lt; 2%) of neoplastic cells in tumor mass. To examine whether circulating cfDNA can be informative about genomic imbalances in neoplastic Hodgkin/Reed-Sternberg (HRS) cells, we performed a pilot study of nine prospective cHL cases. This study showed that genomic profiles of cfDNA correspond to the profiles of HRS cells. To get further insights into the genome of cHL, a large study on cfDNA from 177 prospective cHL patients was subsequently established. Based on ultra-low pass sequencing of cfDNA from this cohort, we built a comprehensive catalog of genomic abnormalities, as well as their frequencies and patterns. Besides the known recurrent imbalances, such as gain/amplification of 2p16/ REL-BCL11A and 9p24/ JAK2-CD274-PDCDLG2 , novel recurrent abnormalities were identified in cHL. Altogether, we have provided evidence that cHL is characterized by consistent and recurrent genomic imbalances and we have shown the potential of genomic profiling of cfDNA as a novel and non-invasive tool in the diagnosis and follow up of cHL patients.

Comprehensive genome-wide analysis of routine non-invasive test data allows cancer prediction: A single-center retrospective analysis of over 85,000 pregnancies.

BackgroundImplausible false positive results in non-invasive prenatal testing (NIPT) have been occasionally associated with the detection of occult maternal malignancies. Hence, there is a need for approaches allowing accurate prediction of whether the NIPT result is pointing to an underlying malignancy, as well as for organized programs ensuring efficient downstream clinical management of these cases.MethodsUsing a data set of 88,294 NIPT performed at University Hospital Leuven (Belgium) between November 2013 and March 2020, we retrospectively evaluated the positive predictive value (PPV) of our NIPT approach for cancer detection. In this approach, whole-genome cell-free DNA (cfDNA) data from NIPT were scrutinized for the presence of (sub)chromosomal copy number alterations (CNAs) predictive for a malignancy, using an unbiased NIPT analysis pipeline coined GIPSeq. For suspected cases, the presence of a maternal cancer was evaluated via subsequent multidisciplinary clinical follow-up examinations. The cancer-specificity of the identified CNAs in cfDNA was assessed through genetic analyses of a tumor biopsy.FindingsFifteen women without a cancer history were identified with a GIPSeq result suggestive of a malignant process. Their cfDNA profiles showed either genome-wide aberrations or a single trisomy 8. Upon clinical examinations, a solid or hematological cancer was identified in 4 and 7 cases, respectively. Three women were identified as having a clonal mosaicism. For one case no underlying condition was found. These numbers add to a PPV of 73%. Based on this experience, we presented a multidisciplinary care path for efficient clinical management of these cases.InterpretationThe presented approach for analysing NIPT results has a high PPV, yet unknown sensitivity, for detecting asymptomatic malignancies upon routine NIPT. Given the complexity of diagnosing a pregnant woman with cancer, clinical follow-up should occur in a well-designed multidisciplinary setting, such as via the care model that we presented here.FundingThis work was supported by Research Foundation Flanders and KU Leuven funding.

Breast Cancer Detection and Treatment Monitoring Using a Noninvasive Prenatal Testing Platform: Utility in Pregnant and Nonpregnant Populations.

Numerous publications have reported the incidental detection of occult malignancies upon routine noninvasive prenatal testing (NIPT). However, these studies were not designed to evaluate the NIPT performance for cancer detection. We investigated the sensitivity of a genome-wide NIPT pipeline, called GIPSeq, for detecting cancer-specific copy number alterations (CNAs) in plasma tumor DNA (ctDNA) of patients with breast cancer. To assess whether a pregnancy itself, with fetal cell-free DNA (cfDNA) in the maternal circulation, might influence the detection of ctDNA, results were compared in pregnant (n = 25) and nonpregnant (n = 25) cancer patients. Furthermore, the ability of GIPSeq to monitor treatment response was assessed. Overall GIPSeq sensitivity for detecting cancer-specific CNAs in plasma cfDNA was 26%. Fifteen percent of detected cases were asymptomatic at the time of blood sampling. GIPSeq sensitivity mainly depended on the tumor stage. Also, triple negative breast cancers (TNBC) were more frequently identified compared to hormone-positive or HER2-enriched tumors. This might be due to the presence of high-level gains and losses of cfDNA or high ctDNA loads in plasma of TNBC. Although higher GIPSeq sensitivity was noted in pregnant (36%) than in nonpregnant women (16%), the limited sample size prohibits a definite conclusion. Finally, GIPSeq profiling of cfDNA during therapy allowed monitoring of early treatment response. The results underscore the potential of NIPT-based tests, analyzing CNAs in plasma cfDNA in a genome-wide and unbiased fashion for breast cancer detection, cancer subtyping and treatment monitoring in a pregnant and nonpregnant target population.

Clinical value of routine non-invasive prenatal testing for the screening of non-target chromosomal abnormalities

To assess the value of non-invasive prenatal testing (NIPT) for the identification of sex chromosome aneuploidies (SCAs), copy number variants (CNVs) and rare autosomal trisomies (RATs). A total of 11 429 women with singleton pregnancy in Ningbo area were screened by NIPT. 106 women were subjected to invasive prenatal diagnosis due to high risk of chromosomal abnormalities other than 21, 18 and 13 aneuploidies. All cases were followed up for pregnancy outcome and postnatal status. Sixty-six women were signaled by NIPT for fetal SCAs, among whom 54 were willing to undergo prenatal diagnosis. Eighteen cases of fetal SCAs were verified as true positives and 4 were suspected positives, which yielded a positive predictive value (PPV) of 33.3%. Half of the women decided to continue their pregnancy. Forty women were signaled by NIPT for fetal CNVs, among which 32 underwent prenatal diagnosis. 19 cases of fetal CNVs were verified as true positives and 3 cases were suspected positives, which yielded a PPV of 46.8%. All women with pathological or possibly pathological CNVs decided to terminate their pregnancies. Thirty-one women were signaled for with fetal RATs. Two fetuses were confirmed to harbor mosaicism trisomies by prenatal diagnosis, and 1 case was suspected to be positive, which yielded a PPV of 9.7%. All of the three women have decided to terminate their pregnancy. In addition to aneuploidies of target chromosomes, NIPT also has important value for the detection of SCAs and CNVs. The results can help to further reduce birth defects. Nevertheless, in view of its low PPV, pregnant women with positive result still need appropriate genetic counseling and prenatal diagnosis to avoid unnecessary induced labor.

PREFACE: In silico pipeline for accurate cell‐free fetal DNA fraction prediction

ObjectiveDuring routine noninvasive prenatal testing (NIPT), cell‐free fetal DNA fraction is ideally derived from shallow‐depth whole‐genome sequencing data, preventing the need for additional experimental assays. The fraction of aligned reads to chromosome Y enables proper quantification for male fetuses, unlike for females, where advanced predictive procedures are required. This study introduces PREdict FetAl ComponEnt (PREFACE), a novel bioinformatics pipeline to establish fetal fraction in a gender‐independent manner.MethodsPREFACE combines the strengths of principal component analysis and neural networks to model copy number profiles.ResultsFor sets of roughly 1100 male NIPT samples, a cross‐validated Pearson correlation of 0.9 between predictions and fetal fractions according to Y chromosomal read counts was noted. PREFACE enables training with both male and unlabeled female fetuses. Using our complete cohort (nfemale = 2468, nmale = 2723), the correlation metric reached 0.94.ConclusionsAllowing individual institutions to generate optimized models sidelines between‐laboratory bias, as PREFACE enables user‐friendly training with a limited amount of retrospective data. In addition, our software provides the fetal fraction based on the copy number state of chromosome X. We show that these measures can predict mixed multiple pregnancies, sex chromosomal aneuploidies, and the source of observed aberrations.

Noninvasive prenatal testing for fetal subchromosomal copy number variations and chromosomal aneuploidy by low-pass whole-genome sequencing.

BackgroundExpanding noninvasive prenatal testing (NIPT) to include the detection of fetal subchromosomal copy number variations (CNVs) significantly decreased the sensitivity and specificity. Developing analytic pipeline to achieve high performance in the noninvasive detection of CNVs will largely contribute to the application of CNVs screening in clinical practice.MethodsWe developed the Noninvasively Prenatal Subchromosomal Copy number variation Detection (NIPSCCD) method based on low‐pass whole‐genome sequencing, and evaluated its efficacy in detecting fetal CNVs and chromosomal aneuploidies with 20,003 pregnant women.ResultsTotally, NIPSCCD identified 36 CNVs, including 29 CNVs consistent and 7 CNVs inconsistent with amniocytes tests. Additionally, seven fetal CNVs identified by amniocytes testing were undetected by NIPSCCD. The sensitivities for detecting CNVs > 10 Mb, 5 Mb–10 Mb, and CNVs < 5 Mb were 91.67%, 100.00%, and 68.42%, respectively. Moreover, NIPSCCD identified 103/ true positive trisomy 21/18/13 cases and 21 false positives, producing an overall 100.00% sensitivity and 99.89% specificity.ConclusionNIPSCCD showed a good performance in detecting fetal subchromosomal CNVs, especially for CNVs >10 Mb, and can be incorporated into the routine NIPT chromosomal aneuploidies screening with high sensitivity and specificity.

Moving Towards Routine Non-Invasive Prenatal Testing (NIPT): Challenges Related to Women’s Autonomy

Women’s reproductive autonomy, and its translation into informed free choice regarding prenatal testing, is a dominant concept in the bioethical discourse concerning prenatal testing. This discourse is based on the premise that access to information regarding the pregnancy promotes autonomous decision-making. However, studies show that the offer of prenatal screening as a routine part of pregnancy care is not supported, to a large degree, by appropriate informed consent mechanisms. This means that the implementation of the concept of autonomy ought to be reconsidered. On the backdrop of these challenges, the introduction of Non-Invasive Prenatal Testing (NIPT) may present new challenges. The main advantages of NIPT, compared with earlier technologies, (namely the early availability of results, the non-invasive and risk-free nature of the test, as well as its increased accuracy) may exacerbate threats to women’s autonomy. Four of the most likely future uses of NIPT – first-tier screening, diagnosis, expanded targeted use, and whole-genome sequencing – will continue to challenge patients’ autonomy. To shed light on these developments, this paper presents some longstanding ethical concerns regarding prenatal screening, examines what makes NIPT different from earlier screening technologies, and charts the possible future courses of NIPT, while anticipating the ethical and social implications of the various signposts potentially encountered, particularly as they relate to reproductive autonomy.

Size-selective separation and overall-amplification of cell-free fetal DNA fragments using PCR-based enrichment

This study aimed to establish a method for the selective amplification of cell-free fetal DNA (cffDNA) in maternal plasma and preserve the integrity of DNA fragments during amplification, thereby providing a sufficient amount of cffDNA to meet the requirement of routine non-invasive prenatal testing. We amplified DNA molecules in a one-reaction system without considering their particular sequences and lengths (overall amplification) by using PCR-based enrichment. We then modified PCR conditions to verify the effect of denaturation temperature on DNA amplification on various lengths of DNA (selective overall amplification). Finally, we used an optimum temperature range to amplify cffDNA selectively. Amplification results were validated by electrophoresis and real-time quantitative PCR. Our PCR-based enrichment efficiently amplified all DNA fragments with differing lengths within a single reaction system, as well as preserving the integrity of the DNA fragments. cffDNA was significantly amplified along with the selective amplification of small fragment maternal plasma DNA in an appropriate range of denaturation temperatures. We have established a PCR-based method for the simultaneous enrichment and amplification of cffDNA in order to meet the requirements of high cffDNA quantity for routine non-invasive prenatal testing.