Genomics-based non-invasive prenatal testing for detection of fetal chromosomal aneuploidy in pregnant women

Abstract

Background: Common fetal aneuploidies include Down syndrome (trisomy 21 or T21), Edward syndrome (trisomy 18 or T18), Patau syndrome (trisomy 13 or T13), Turner syndrome (45,X), Klinefelter syndrome (47,XXY), Triple X syndrome (47,XXX) and 47,XYY syndrome (47,XYY). Prenatal screening for fetal aneuploidies is standard care in many countries, but current biochemical and ultrasound tests have high false negative and false positive rates. The discovery of fetal circulating cell-free DNA (ccfDNA) in maternal blood offers the potential for genomics-based non-invasive prenatal testing (gNIPT) as a more accurate screening method. Two approaches used for gNIPT are massively parallel shotgun sequencing (MPSS) and targeted massively parallel sequencing (TMPS).

Objectives: To evaluate and compare the diagnostic accuracy of MPSS and TMPS for gNIPT as a first-tier test in unselected populations of pregnant women undergoing aneuploidy screening or as a second-tier test in pregnant women considered to be high risk after first-tier screening for common fetal aneuploidies. The gNIPT results were confirmed by a reference standard such as fetal karyotype or neonatal clinical examination.

Search methods: We searched 13 databases (including MEDLINE, Embase and Web of Science) from 1 January 2007 to 12 July 2016 without any language, search filter or publication type restrictions. We also screened reference lists of relevant full-text articles, websites of private prenatal diagnosis companies and conference abstracts.

Selection criteria: Studies could include pregnant women of any age, ethnicity and gestational age with singleton or multifetal pregnancy. The women must have had a screening test for fetal aneuploidy by MPSS or TMPS and a reference standard such as fetal karyotype or medical records from birth.

Data collection and analysis: Two review authors independently carried out study selection, data extraction and quality assessment (using the QUADAS-2 tool). Where possible, hierarchical models or simpler alternatives were used for meta-analysis.

Main results: Sixty-five studies of 86,139 pregnant women (3141 aneuploids and 82,998 euploids) were included. No study was judged to be at low risk of bias across the four domains of the QUADAS-2 tool but applicability concerns were generally low. Of the 65 studies, 42 enrolled pregnant women at high risk, five recruited an unselected population and 18 recruited cohorts with a mix of prior risk of fetal aneuploidy. Among the 65 studies, 44 evaluated MPSS and 21 evaluated TMPS; of these, five studies also compared gNIPT with a traditional screening test (biochemical, ultrasound or both). Forty-six out of 65 studies (71%) reported gNIPT assay failure rate, which ranged between 0% and 25% for MPSS, and between 0.8% and 7.5% for TMPS.In the population of unselected pregnant women, MPSS was evaluated by only one study; the study assessed T21, T18 and T13. TMPS was assessed for T21 in four studies involving unselected cohorts; three of the studies also assessed T18 and 13. In pooled analyses (88 T21 cases, 22 T18 cases, eight T13 cases and 20,649 unaffected pregnancies (non T21, T18 and T13)), the clinical sensitivity (95% confidence interval (CI)) of TMPS was 99.2% (78.2% to 100%), 90.9% (70.0% to 97.7%) and 65.1% (9.16% to 97.2%) for T21, T18 and T13, respectively. The corresponding clinical specificity was above 99.9% for T21, T18 and T13.In high-risk populations, MPSS was assessed for T21, T18, T13 and 45,X in 30, 28, 20 and 12 studies, respectively. In pooled analyses (1048 T21 cases, 332 T18 cases, 128 T13 cases and 15,797 unaffected pregnancies), the clinical sensitivity (95% confidence interval (CI)) of MPSS was 99.7% (98.0% to 100%), 97.8% (92.5% to 99.4%), 95.8% (86.1% to 98.9%) and 91.7% (78.3% to 97.1%) for T21, T18, T13 and 45,X, respectively. The corresponding clinical specificities (95% CI) were 99.9% (99.8% to 100%), 99.9% (99.8% to 100%), 99.8% (99.8% to 99.9%) and 99.6% (98.9% to 99.8%). In this risk group, TMPS was assessed for T21, T18, T13 and 45,X in six, five, two and four studies. In pooled analyses (246 T21 cases, 112 T18 cases, 20 T13 cases and 4282 unaffected pregnancies), the clinical sensitivity (95% CI) of TMPS was 99.2% (96.8% to 99.8%), 98.2% (93.1% to 99.6%), 100% (83.9% to 100%) and 92.4% (84.1% to 96.5%) for T21, T18, T13 and 45,X respectively. The clinical specificities were above 100% for T21, T18 and T13 and 99.8% (98.3% to 100%) for 45,X. Indirect comparisons of MPSS and TMPS for T21, T18 and 45,X showed no statistical difference in clinical sensitivity, clinical specificity or both. Due to limited data, comparative meta-analysis of MPSS and TMPS was not possible for T13.We were unable to perform meta-analyses of gNIPT for 47,XXX, 47,XXY and 47,XYY because there were very few or no studies in one or more risk groups.

Authors' conclusions: These results show that MPSS and TMPS perform similarly in terms of clinical sensitivity and specificity for the detection of fetal T31, T18, T13 and sex chromosome aneuploidy (SCA). However, no study compared the two approaches head-to-head in the same cohort of patients. The accuracy of gNIPT as a prenatal screening test has been mainly evaluated as a second-tier screening test to identify pregnancies at very low risk of fetal aneuploidies (T21, T18 and T13), thus avoiding invasive procedures. Genomics-based non-invasive prenatal testing methods appear to be sensitive and highly specific for detection of fetal trisomies 21, 18 and 13 in high-risk populations. There is paucity of data on the accuracy of gNIPT as a first-tier aneuploidy screening test in a population of unselected pregnant women. With respect to the replacement of invasive tests, the performance of gNIPT observed in this review is not sufficient to replace current invasive diagnostic tests.We conclude that given the current data on the performance of gNIPT, invasive fetal karyotyping is still the required diagnostic approach to confirm the presence of a chromosomal abnormality prior to making irreversible decisions relative to the pregnancy outcome. However, most of the gNIPT studies were prone to bias, especially in terms of the selection of participants.

Conflict of interest statement

Mylene Badeau: none known.

Jonatan Blais: none known.

Yves Giguère (and some other members of the review team ‐ FR, SL, FL) are investigators in a Research Project funded under the auspices of Genome Canada and the Canadian Institutes for Health Research (both non‐for‐profit organisations funded by the Canadian government) but that call for some mandatory in‐kind contributions from other partners. This Research Project thus receives in‐kind funding from private corporations which either offer commercial NIPT tests (Ariosa Diagnostics Inc, San Jose, CA) or offer reagents and/or equipment that can be used to perform NIPT assays (Life Technologies Inc, NY, USA; Illumina, San Diego, CA, USA; QIAGEN, Hilden, GER; Perkin Elmer, Waltham, MASS, USA). This funding is at arms length from the scientific components of the research project. The present review is not funded by this Project. Some of the authors may eventually publish results from clinical trials that could be considered in this review. None of the four authors involved in gNIPT studies will take part in the selection of studies, nor in any decisions/analyses related to their own studies. All authors declared no other conflict of interest

Sylvie Langlois (and some other members of the review team ‐ FR, YG, FL) are investigators in a Research Project funded under the auspices of Genome Canada and the Canadian Institutes for Health Research (both non‐for‐profit organisations funded by the Canadian government) but that call for some mandatory in‐kind contributions from other partners. This Research Project thus receives in‐kind funding from private corporations which either offer commercial NIPT tests (Ariosa Diagnostics Inc, San Jose, CA) or offer reagents and/or equipment that can be used to perform NIPT assays (Life Technologies Inc, NY, USA; Illumina, San Diego, CA, USA; QIAGEN, Hilden, GER; Perkin Elmer, Waltham, MASS, USA). This funding is at arms length from the scientific components of the research project. The present review is not funded by this Project. Some of the authors (FR, SL, YG and FL) may eventually publish results from clinical trials that could be considered in this review. None of these four authors involved in gNIPT studies will take part in the selection of studies, nor in any decisions/analyses related to their own studies. All authors declared no other conflict of interest. This review was supported by a Canadian Institutes of Health Research (CIHR) Knowledge Synthesis Grant: Fall 2014 Competition (2014‐11‐17) awarded to AFT, FR, FL, and SL. CIHR in no way influenced the results or conclusions of this review.

France Légaré (and some other members of the review team ‐ FR, YG, SL) are investigators in a Research Project funded under the auspices of Genome Canada and the Canadian Institutes for Health Research (both non‐for‐profit organisations funded by the Canadian government) but that call for some mandatory in‐kind contributions from other partners. This Research Project thus receives in‐kind funding from private corporations which either offer commercial NIPT tests (Ariosa Diagnostics Inc, San Jose, CA) or offer reagents and/or equipment that can be used to perform NIPT assays (Life Technologies Inc, NY, USA; Illumina, San Diego, CA, USA; QIAGEN, Hilden, GER; Perkin Elmer, Waltham, MASS, USA). This funding is at arms length from the scientific components of the research project. The present review is not funded by this Project. Some of the authors (FR, SL, YG and FL) may eventually publish results from clinical trials that could be considered in this review. None of these four authors involved in gNIPT studies will take part in the selection of studies, nor in any decisions/analyses related to their own studies. All authors declared no other conflict of interest. This review was supported by a Canadian Institutes of Health Research (CIHR) Knowledge Synthesis Grant: Fall 2014 Competition (2014‐11‐17) awarded to AFT, FR, FL, and SL. CIHR in no way influenced the results or conclusions of this review.

Carmen Lindsay: none known.

Leon Nshimyumukiza: none known.

François Rousseau (and some other members of the review team ‐ SL, YG, FL) are investigators in a Research Project funded under the auspices of Genome Canada and the Canadian Institutes for Health Research (both non‐for‐profit organisations funded by the Canadian government) but that call for some mandatory in‐kind contributions from other partners. This Research Project thus receives in‐kind funding from private corporations which either offer commercial NIPT tests (Ariosa Diagnostics Inc, San Jose, CA) or offer reagents and/or equipment that can be used to perform NIPT assays (Life Technologies Inc, NY, USA; Illumina, San Diego, CA, USA; QIAGEN, Hilden, GER; Perkin Elmer, Waltham, MASS, USA). This funding is at arms length from the scientific components of the research project. The present review is not funded by this Project. Some of the authors (FR, SL, YG and FL) may eventually publish results from clinical trials that could be considered in this review. None of these four authors involved in gNIPT studies will take part in the selection of studies, nor in any decisions/analyses related to their own studies. All authors declared no other conflict of interest. This review was supported by a Canadian Institutes of Health Research (CIHR) Knowledge Synthesis Grant: Fall 2014 Competition (2014‐11‐17) awarded to AFT, FR, FL, and SL. CIHR in no way influenced the results or conclusions of this review.

Yemisi Takwoingi: The University of Birmingham received consultancy fees from Universite Laval for statistical analysis.

Alexis F Turgeon: This review was supported by a Canadian Institutes of Health Research (CIHR) Knowledge Synthesis Grant: Fall 2014 Competition (2014‐11‐17) awarded to AFT, FR, FL, and SL. CIHR in no way influenced the results or conclusions of this review.

William Witteman: none known.

Figures

1

Difference between massively parallel shotgun sequencing (MPSS) and targeted massively parallel sequencing (TMPS). Genomics‐based non‐invasive prenatal testing (gNIPT) aims to count the number of copies of DNA fragments from the chromosomes of interest (chromosome 21 (Chrom. 21) in this example) present in circulating cell‐free DNA (ccfDNA) from a pregnant woman, relative to a reference set of chromosomes (Ref. Chrom.). DNA fragments circulating in maternal blood in the case of a euploid (left) and aneuploid (right) pregnancy are illustrated (top). MPSS produces a large number of sequence reads from all chromosomes while TMPS generates a larger proportion of reads from the chromosomes of interest (bottom). In both methods, sequence reads can be used to detect a slight excess of fetal genomic material coming from the chromosome of interest. Figure was created by FR.

2

Current clinical pathway and three proposed uses of genomics‐based non‐invasive prenatal testing (gNIPT). Currently (on the left), pregnant women can have a prenatal screening test consisting of biomarkers or ultrasound, or both. For high‐risk pregnant women, an invasive diagnostic test (karyotyping) is offered. In the present review, we propose 3 different clinical pathways. First, gNIPT could be used as a triage test, to decide which pregnant women should receive further testing. Second, gNIPT could be used to replace current prenatal screening tests. Finally, gNIPT could be used to replace current invasive diagnostic tests (if diagnostic performance permits). At any point in a clinical pathway, a pregnant woman may decide not to proceed with other tests (not shown in the figure). Figure was designed by CL, JB, MB and YT.

3

PRISMA flow diagram for selection of studies from January 2007 to October 2016. #: number, DTA: diagnostic test accuracy, NTIS: The National Technical Information Service and WHO ICTRP: World Health Organization International Clinical Trials Registry Platform.

4

Risk of bias and applicability concerns summary: review authors' judgements about each domain for each of the studies included for massively parallel shotgun sequencing (MPSS).

5

Risk of bias and applicability concerns summary: review authors' judgements about each domain for each study included for targeted massively parallel sequencing (TMPS).

6

Risk of bias and applicability concerns (all tests included): review authors' judgements about each domains presented as percentages across included studies. MPSS: massively parallel shotgun sequencing, TMPS: targeted massively parallel sequencing.

7

Forest plot of MPSS and TMPS for T21 in unselected pregnant women undergoing aneuploidy screening. FN: false negative, FP: false positive, MPSS: massively parallel shotgun sequencing, TMPS: targeted massively parallel sequencing, TN: true negative and TP: true positive.

8

Forest plot of MPSS and TMPS for T21 in pregnant women selected at high risk of fetal aneuploidy. FN: false negative, FP: false positive, MPSS: massively parallel shotgun sequencing, TMPS: targeted massively parallel sequencing, TN: true negative and TP: true positive.

9

Forest plot of MPSS and TMPS for T18 in pregnant women selected at high risk of fetal aneuploidy. FN: false negative, FP: false positive, MPSS: massively parallel shotgun sequencing, TMPS: targeted massively parallel sequencing, TN: true negative and TP: true positive.

10

Forest plot of MPSS and TMPS for T13 in pregnant women selected at high risk of fetal aneuploidy. FN: false negative, FP: false positive, MPSS: massively parallel shotgun sequencing, TMPS: targeted massively parallel sequencing, TN: true negative and TP: true positive.

11

Forest plot of MPSS and TMPS for 45,X in pregnant women selected at high risk of fetal aneuploidy. FN: false negative, FP: false positive, MPSS: massively parallel shotgun sequencing, TMPS: targeted massively parallel sequencing, TN: true negative and TP: true positive.

12

Forest plot of MPSS and TMPS for 47,XXX, 47,XXY and 47,XYY in pregnant women selected at high risk of fetal aneuploidy. FN: false negative, FP: false positive, MPSS: massively parallel shotgun sequencing, TMPS: targeted massively parallel sequencing, TN: true negative and TP: true positive.

13

Forest plot of MPSS and TMPS for autosomes (T21, T18 and T13 combined) in unselected pregnant women undergoing aneuploidy screening. FN: false negative, FP: false positive, MPSS: massively parallel shotgun sequencing, TMPS: targeted massively parallel sequencing, TN: true negative and TP: true positive.

14

Forest plot of MPSS and TMPS for autosomes (T21, T18 and T13) in pregnant women selected at high risk of fetal aneuploidy. FN: false negative, FP: false positive, MPSS: massively parallel shotgun sequencing, TMPS: targeted massively parallel sequencing, TN: true negative and TP: true positive.

15

Forest plot of MPSS and TMPS for SCA (45,X, 47,XXX, 47,XXY and 47,XYY combined) in pregnant women selected at high risk of fetal aneuploidy. FN: false negative, FP: false positive, MPSS: massively parallel shotgun sequencing, TMPS: targeted massively parallel sequencing, TN: true negative and TP: true positive.

16

Forest plot of traditional screening tests for T21, T18 and T13 in unselected pregnant women undergoing aneuploidy screening. FN: false negative, FP: false positive, MPSS: massively parallel shotgun sequencing, TMPS: targeted massively parallel sequencing, TN: true negative and TP: true positive.

17

Forest plot of traditional screening tests for autosomes (T21, T18 and T13 combined) in unselected pregnant women undergoing aneuploidy screening. FN: false negative, FP: false positive, TN: true negative and TP: true positive.

18

Forest plot of comparative studies of TMPS and traditional screening tests for autosomes (T21, T18 and T13 combined) in unselected pregnant women undergoing aneuploidy screening. FN: false negative, FP: false positive, TN: true negative and TP: true positive.

19

Forest plot of traditional screening tests for autosomes (T21, T18 and T13 combined) in pregnant women with mixed prior risk of fetal aneuploidy. FN: false negative, FP: false positive, MPSS: massively parallel shotgun sequencing, TMPS: targeted massively parallel sequencing, TN: true negative and TP: true positive.

20

Forest plot of MPSS and TMPS for autosomes (T21, T18 and T13 combined) in pregnant women with mixed prior risk of fetal aneuploidy. FN: false negative, FP: false positive, MPSS: massively parallel shotgun sequencing, TMPS: targeted massively parallel sequencing, TN: true negative and TP: true positive.

21

Forest plot of MPSS and TMPS for T21, T18 or T13 in pregnant women with mixed prior risk of fetal aneuploidy. FN: false negative, FP: false positive, MPSS: massively parallel shotgun sequencing, TMPS: targeted massively parallel sequencing, TN: true negative and TP: true positive.

22

Forest plot of MPSS and TMPS for SCA (45,X, 47,XXX, 47,XXY and 47,XYY combined) in pregnant women with mixed prior risk of fetal aneuploidy. FN: false negative, FP: false positive, MPSS: massively parallel shotgun sequencing, TMPS: targeted massively parallel sequencing, TN: true negative and TP: true positive.

23

Forest plot of MPSS and TMPS for 45,X, 47,XXY or 47,XYY in pregnant women with mixed prior risk of fetal aneuploidy. FN: false negative, FP: false positive, MPSS: massively parallel shotgun sequencing, TMPS: targeted massively parallel sequencing, TN: true negative and TP: true positive.