Key words cell-free fetal DNA - NIPT - positive predictive value - routine practice - ultrasound
- anomaly scan
Schlüsselwörter zellfreie fötale DNA - NIPT - positiver Vorhersagewert - klinischer Alltag - Ultraschall
- Fehlbildungens-Ultraschall
Introduction
Testing of cell-free DNA (cfDNA), which mainly derives from apoptotic cells of the
trophoblast, has been increasingly adopted into prenatal care during the past decade.
A current meta-analysis confirms an excellent test performance in the detection of
trisomy 21 (T21) with a sensitivity of up to 99.7% and a low false positive rate of
0.04% [1 ]. The accuracy for trisomy 18 (T18) and trisomy 13 (T13) seems only marginally lower
[1 ], [2 ]. However, due to the high costs, it has not been deemed suitable as a first-line
screening method and the question as to how it can be best integrated into medical
care is still under debate.
The current international guidelines recommend cfDNA testing for the trisomies T21/18/13
in combination with a qualified ultrasound (US) examination and stress the importance
of adequate patient education and counselling [3 ], [4 ]. Although the providers of cfDNA-screening tests offer screening for sex chromosome
anomalies (SCAs) and microdeletion syndromes (especially microdeletion 22q11.2) the
test performance is clearly lower and data on validity is scarce. Thus, testing for
SCAs or microdeletions via cfDNA is not advised [3 ], [4 ].
In Germany, there is generally no reimbursement neither for conventional first-trimester
screening nor cfDNA testing. Only recently (09/2019) the authorities decided on limited
coverage for cfDNA testing in individual cases with a high risk for T21, 18 and 13.
However, since cfDNA testing allows virtually risk-free screening at an early stage
of pregnancy and involves only a simple blood draw for the patient, many parents are
willing to meet the costs themselves. Since cfDNA testing is a genetic examination,
it is all the more important that the parents are well informed and properly counselled
about the test performance of cfDNA testing, its advantages and limitations and potential
interpretation of its results.
Whereas initially the accuracy of cfDNA testing was determined in high-risk populations,
it is increasingly used also in populations with a low risk for aneuploidies. Large
studies on the performance of cfDNA tests in clinical routine report a consistently
high specificity and sensitivity [5 ]. However, several reports demonstrate that the actual positive predictive value
(PPV) in clinical routine is lower than expected and describe a relevant proportion
of false positive cfDNA test results [6 ], [7 ], [8 ]. Reasons such as confined placental or true fetal mosaicism and technical or human
errors were identified as reasons for cfDNA test results that are discordant with
the true fetal karyotype [9 ]. Thus, more data from routine practice is required to improve counselling of parents
and enable informed decision-making,
especially if it is taken into consideration that false positive or negative results
may have far-reaching consequences such as unnecessary or late terminations, respectively.
During recent years we observed a considerable number of patients with a positive
cfDNA test result in our referral centre for specialised prenatal care. With a structured
workup of these cases we aim to describe how cfDNA-testing is applied and to provide
PPV estimates in a routine setting in Germany.
Methods
Study design
This is a retrospective study including all patients with a positive cfDNA test result
that were encountered in our unit specialising in prenatal diagnosis between September
2013 and December 2019. Patients are usually referred to our practice in southern
Germany by their regional primary gynaecologist for specialised diagnostics and further
counselling. Referral indications include suspicious clinical findings (e.g. positive
cfDNA test, anomalies detected in routine US scans), high-risk pregnancies or the
patientʼs own request.
Parameters
The PPVs of cfDNA tests for autosomal aneuploidies (T21, T18, T13), sex chromosome
aneuploidies (X0, XXX, XXY, XYY) or a 22q11.2 microdeletion (DiGeorge syndrome) were
defined as the ratio of true positive cases divided by all patients with a positive
test result for the respective anomaly. Confirmatory genetic testing (pre- or postnatally)
was used as a reference standard.
Pregnancy outcomes were “delivered”, “intrauterine death” (IUD) and “termination of
pregnancy” (TOP).
To describe the current utilisation of cfDNA testing in clinical practice, the following
parameters were assessed: maternal characteristics, gestational age (GA) at cfDNA
test, ultrasound (US) examination or first trimester screening (FTS) before cfDNA
test, presence of fetal anomalies, indication for cfDNA test, invasive prenatal testing
method. A cfDNA test was defined as a “screening cfDNA test” if either no US/FTS was
performed before blood sampling or if a US/FTS before blood sampling yielded normal
results.
Parameters were described in the total population and in groups with a cfDNA test
positive for autosomal aneuploidies, SCAs or 22q11.2 microdeletion.
Data and measurements
In our practice, all patients routinely receive a detailed 2D US scan at their first
visit. Maternal demographic characteristics, clinical findings and pregnancy outcome
are recorded in a database.
At a CRL ≥ 45 and ≤ 84 mm, a detailed first trimester US examination including measurement
of fetal NT thickness, assessment of the additional ultrasound markers nasal bone
(NB), flow in the ductus venosus (DV) and across the tricuspid valve (TV) was performed.
Additionally, a thorough anatomical assessment was carried out to detect any fetal
defects. At a CRL > 84 mm, a detailed fetal anomaly scan was performed. All US examinations
followed the current guidelines [10 ], [11 ], [12 ], [13 ]. A single operator using a 2- to 6-Mhz multifrequency transabdominal probe (Voluson
E8, GE Medical Systems, Zipf, Austria) conducted all measurements. A transvaginal
probe (6- to 12-MHz multifrequency) was used when the fetus was at risk of a cardiac
anomaly or in order to complete the anatomical survey when the scanning quality was
not acceptable
transabdominally. The examiner was certified for evaluation of the ultrasound
markers by the Fetal Medicine Foundation (FMF), UK.
In our practice FTS was based on MA, NT thickness and the additional US markers DV
and TV. The risk was calculated according to the algorithm of the FMF 2012 with the
program ViewPoint (GE healthcare, Chicago, Illinois, USA). Pregnancies with a risk
for T21 ≥ 1 : 50 were classified as “high-risk FTS”. FTS by the primary gynaecologists
was usually based on MA, NT thickness and maternal levels of serum free β-human chorionic
gonadotropin (β-hCG) and pregnancy-associated plasma protein A (PAPP-A).
Invasive prenatal testing (IPT) was offered to all patients with a positive cfDNA
test following the detailed US examination [14 ], [15 ]. The samples were sent for karyotyping to the Genetikum® Stuttgart.
For this study, we collected the required data from our database and (if necessary)
retrieved information on GA at blood sampling for cfDNA test, the cfDNA test provider,
previous results from US examinations or FTS, pregnancy outcome and results of postnatal
genetic testing from the patientsʼ primary gynaecologist, the patients or the Genetikum
Stuttgart (with the patientsʼ consent). The cfDNA tests used were the Harmony® prenatal test (Roche Inc., San Jose, CA, USA), the PrenaTest® (Eurofins Lifecodexx AG, Konstanz, Germany) and the PreviaTest® (Eluthia GmbH, Gießen, Germany).
Statistics
For the calculation of the true and false positive rates, only patients with a confirmatory
genetic testing result were included in the analysis. The 95-%-confidence intervals
were calculated according to Pearson-Clopper. Data analysis was carried out using
SAS version 9.3.
Ethics
All procedures performed in this study were in accordance with the ethical standards
of the 1964 Helsinki Declaration and its later amendments. The patients gave their
general consent to anonymised data handling before their examinations. As this is
a retrospective analysis of data derived from a routine clinical examination, the
approval of the Ethics Committee was not required.
Results
Patient population
From 09/2013 to 12/2019 we encountered 81 patients with a positive cfDNA test result
in our specialised prenatal practice. Most women (n = 71, 87.7%) were referred to
our practice due to the positive cfDNA test result. In 10 cases (12.3%), the cfDNA
test was initiated in our practice. The median maternal age of the total study population
(n = 81) was 37 (range: 27 – 44) years and the median GA at the first examination
at our practice was 13.6 (range: 11.6 – 26.6) weeks. All pregnancies were singleton
pregnancies. Five (5.2%) fetuses were conceived by in vitro fertilisation (IVF). [Fig. 1 ] provides an overview over the results of cfDNA testing, confirmatory genetic testing
and the pregnancy outcomes of the study population.
Fig. 1 Flow chart depicting patient flow, results of confirmatory testing results and outcomes.
AC: amniocentesis; cfDNA: cell free DNA; CVS: chorionic villus sampling; IUD: intrauterine
death; IPT: invasive prenatal testing; SCA: sex chromosome aneuploidy; TOP: termination
of pregnancy; T: trisomy. * including the fetus with a double positive cfDNA result
(T18 and triple X syndrome).
Test results and utilisation of cfDNA testing
The most frequent positive cfDNA test result was T21 (n = 40; 49.4%), followed by
T18 (n = 8; 9.9%) and T13 (n = 7; 8.6%). In 18 cases (22.2%), the cfDNA test predicted
a SCA (X0: n = 6 [7.4%], XXX: n = 6 [7.4%], XXY: n = 5 [6.2%]; XYY: n = 1 [1.2%]).
One patient had a double positive cfDNA test for T18 and triple X syndrome. Seven
patients (8.6%) had a positive cfDNA test for a 22q11.2 deletion.
Blood sampling for cfDNA testing mainly took place in the first trimester of pregnancy
(88.9%) and 70.4% of the pregnant women were ≥ 35 years old.
In 85.2% of the cases, cfDNA testing was used as a screening method, i.e. before blood
sampling no US or FTS had been performed or it had yielded a normal result. Only 11.1%
of the cfDNA test were initiated due to an abnormal US result, whereas 63.0% of the
patients had not received a first-trimester anomaly scan before. [Table 1 ] describes the characteristics of the positive cfDNA test cases stratified for autosomal
aneuploidies, SCAs and 22q11.2 deletion.
Table 1 Utilisation of cfDNA-testing.
All
n = 81
T21
n = 40
T18*
n = 9
T13
n = 7
SCA*
n = 19
22q12.2
n = 7
* including the fetus with a double positive cfDNA test result (T18 and triple X
syndrome)
** GA at blood sampling for cfDNA test
# cfDNA test was classified as “screening cfDNA test” if no US/FTS was performed before
blood sampling or a US/FTS before blood sampling was normal.
AC: amniocentesis; cfDNA: cell-free fetal DNA; CVS: chorionic villus sampling; GA:
gestational age; FTS: first trimester screening
Maternal age
24 (29.6)
10 (25.0)
3 (33.3)
3 (42.9)
7 (36.8)
2 (28.6)
57 (70.4)
30 (75.0)
6 (66.7)
4 (57.1)
12 (63.2)
5 (71.4)
Median GA at cfDNA test**
12 + 3 (10 + 1 – 27 + 4)
12 + 3 (10 + 1 – 27 + 4)
12 + 4 (10 + 1 – 21 + 6)
12 + 4 (11 + 0 – 13 + 0)
12 + 2 (10 + 1 – 13 + 5)
12 + 4 (12 + 1 – 13 + 0)
72 (88.9)
33 (82.5)
7 (77.8)
7 (100.0)
19 (100.0)
7 (100.0)
9 (11.1)
7 (17.5)
2 (22.2)
0
0
0
Screening cfDNA test#
69 (85.2%)
30 (75.0)
8 (88.9)
6 (85.7)
19 (100.0)
7 (100.0)
Fetal anomaly scan before cfDNA testing
51 (63.0)
26 (65.0)
7 (77.8)
5 (71.4)
11 (57.9)
3 (42.9)
20 (24.7)
7 (17.5)
1 (11.1)
1 (14.3)
8 (42.1)
4 (57.1)
9 (11.1)
7 (17.5)
1 (11.1)
3 (42.9)
0
0
FTS before cfDNA testing
62 (76.5)
29 (72.5)
7 (77.8)
5 (71.4)
15 (78.9)
7 (100.0)
13 (16.1)
6 (15.0)
2 (22.2)
1 (14.3)
4 (21.1)
0
6 (7.4)
5 (12.5)
0
1 (14.3)
0
0
cfDNA testing performed at
71 (87.7)
37 (92.5)
7 (77.8)
6 (85.7)
15 (78.9)
7 (100.0)
10 (12.3)
3 (7.5)
2 (22.2)
1 (14.3)
4 (21.1)
0
Invasive prenatal testing
40 (49.4)
28 (70.0)
5 (55.6)
2 (28.6)
2 (10.5)
4 (57.1)
18 (22.2)
5 (12.5)
3 (33.3)
4 (57.1)
6 (31.6)
0
2 (2.5)
1 (2.5)
1 (1.1)
0
0
0
21 (25.9)
6 (15.0)
0
1 (14.3)
11 (57.9)
3 (42.9)
In the total population, the median percentage of fetal cfDNA was 8.7% and in all
cases, it was above the threshold of 4% (range: 4.3 – 23.4%). The blood samples were
mainly sent to the local cfDNA test provider Cenata GmbH (Tübingen, Germany) using
the Harmony prenatal test (n = 72; 88.9%). Eight patients (9.9%) chose the PrenaTest
and one (1.2%) the PreviaTest.
Performance of cfDNA testing in routine practice
To assess the performance of cfDNA testing in routine practice, only the 73 fetuses
(74 positive cfDNA test results) with a confirmatory genetic testing (pre- or postnatally)
were included in the analysis.
In 26 out of 74 cases (35.1%) the positive cfDNA test result was discordant with the
diagnostic cytogenetic testing result ([Table 2 ]). cfDNA testing for T21 yielded the highest predictive value: 38 out of 40 cases
with a positive cfDNA test for T21 were confirmed, yielding a PPV of 95.0%. However,
for T18 and T13 the PPV was only 55.6% (T18) and 28.6% (T13), respectively. Only 23.1%
of the predicted SCAs were confirmed and no fetus was diagnosed with DiGeorge syndrome
([Table 2 ]).
Table 2 Prediction values of cfDNA testing.
cfDNA test positive, n
Diagnostic testing positive, n
Diagnostic testing negative, n
PPV, % (95% CI)
FPR, % (95% CI)
* including the fetus with a double positive cfDNA test result (T18 and triple X syndrome)
cfDNA: cell free DNA, PPV: positive predictive value, FPR: false positive rate; TPR:
true positive rate; SCA: sex chromosome aneuploidy
Trisomy 21
40
38
2
95.0 (83.1 – 99.4)
5.0 (0.1 – 16.9)
Trisomy 18
9*
5
4
55.6 (21.2 – 86.3)
44.4 (13.7 – 78.8)
Trisomy 13
7
2
5
28.6 (3.7 – 71.0)
71.4 (29.0 – 96.3)
SCA
13*
3
10
23.1 (5.5 – 57.2)
76.9 (46.2 – 95.0)
5
1
4
20 (n. a.)
80 (n. a.)
5*
1
4
20 (n. a.)
80 (n. a.)
1
1
100 (n. a.)
1
1
DiGeorge syndrome
5
5
100 (7.8 – 100)
Structured workup of cases
For a structured workup, we divided the population into four groups based on the cfDNA
test result:
autosomal aneuploidy,
double positive test,
SCA and
22q11.2 deletion.
1. Cases with a positive cfDNA test for an autosomal aneuploidy
Fifty-five fetuses had a positive cfDNA test result for an autosomal aneuploidy. Since
our US examination routine differs in the first and second trimester of pregnancy,
we further separated the group in fetuses that had a CRL ≥ 45 and ≤ 84 mm (n = 38)
and those with a CRL > 84 mm (n = 17) at the first visit in our practice (which could
be before or after the cfDNA test).
First visit in first trimester: 38 (69.1%) fetuses had a CRL ≥ 45 and ≤ 84 mm at the first visit in our practice
and thus received a detailed first trimester ultrasound examination including measurement
of NT and the additional markers NB, DV, TV as well as a thorough anatomical assessment.
Thirty women had an increased risk for an autosomal aneuploidy based on US/FTS results:
in 28 fetuses, we detected an US anomaly ([Table 3 ]) and 2 cases had a high-risk FTS due to abnormal biochemical markers only. Of those,
27 women opted for invasive prenatal testing (25 CVS, 2 AC). In all these cases, the
cfDNA test results were confirmed by karyotyping (T21: n = 25; T18: n = 3; T13: n = 2).
25 pregnancies were terminated, one IUD occurred (T21) and one child with T21 was
delivered. Three women that had fetuses displaying an enlarged NT, nasal bone hypoplasia
and other US anomalies did not undergo IPT. One fetus with a screening cfDNA test
positive for T21 died spontaneously in utero. In the other two cases, the expectant
parents had first obtained the US result indicating a high risk for T21 and chose
the cfDNA test instead of IPT for confirmation. Both children with T21 were delivered.
Table 3 US anomalies in fetuses with a positive cfDNA test result for T21, T18 or T13.
ARSA: aberrant right subclavian artery; AVSD: atrioventricular septal defect; CRL:
crown rump length; DV: ductus venosus; LV: left ventricle; NT: nuchal translucency;
TR: tricuspid regurgitation; US: ultrasound; VSD: ventricular septal defect
Fetuses receiving a detailed US in first trimester (CRL ≥ 45 and ≤ 84 mm) (n = 38)
Confirmed T21
n = 25
Confirmed T18
n = 4
Confirmed T13
n = 2
False positive*
n = 7
Any US anomaly
23 (88,5%)
3 (75%)
2 (100%)
0
15 (65.2%)
1
20 (80.0%)
0
1
12 (48.0)
2 (50.0%)
12 (48.0)
1 (25.0%)
1
6 (24.0)
2 (50.0%)
1
White spot LV Holoprosencephaly
Singular umbilical artery
Fetal tachycardia
Cleft lip and palate
Early fetal retardation
Singular umbilical artery
Microcephaly
Polydactyly
Micrognathia
Lateral neck cyst
Fetuses receiving a detailed US in second trimester (≥ 84 mm) (n = 17)
Confirmed T21
n = 13
T18
n = 1
T13
n = 0
False positive
n = 3
Any US anomaly
11 (84.6%)
1 (100.0)
0
9 (69.2%)
Nuchal oedema
Lateral neck cysts
Nasal bone hypoplasia
0
3 (23.1%)
AVSD (3)
Small muscular VSD with left to right shunt
Small LV and large intra-atrial aneurysm
White Spot in LV (5)
TR (1)
Short femur (1)
Reverse flow in DV (3)
Mild hydronephrosis (1)
White spot liver (1)
ARSA (1)
There were 8 first-trimester fetuses with a positive test result for an autosomal
aneuploidy which did not detect an US anomaly (T21: n = 1, T18: n = 3, T13: n = 4).
Seven women opted for IPT (CVS: n = 2; AC: n = 4; both: n = 1). One fetus was confirmed
to have T18 following AC and the pregnancy was terminated. In the other 6 cases, the
cfDNA test result turned out to be false positive. Among those, there was one case
with placental trisomy 18 mosaicism detected by long-term CVS culture. Karyotyping
following AC in week 17 + 0 affirmed euploidy.
A 31-year-old patient referred for counselling due to a positive cfDNA test for T13
(GA 11 + 1 weeks) decided against IPT, since no anomalies were detected at the detailed
first trimester scan at a GA 13 + 4. A healthy child was delivered.
Taken together, in the group without anomalies in first trimester US examination (or
no high-risk FTS) all but one of the cases with a positive cfDNA test were discordant
with the genetic result. One case with T18 that was otherwise unsuspicious was identified.
First visit in second trimester: 17 (30.9%) pregnancies with a cfDNA test positive for an autosomal aneuploidy were
already in the second or third trimester at the first visit in our practice and received
a thorough anatomical assessment only. Fetal anomalies were detected in 12 fetuses
([Table 3 ]). Among those were 8 women who had a screening cfDNA test predicting T21 and were
referred to our practice for IPT. T21 was confirmed in all cases (CVS: n = 5; AC:
n = 2; both: n = 1) and the pregnancies were terminated.
A 35-year-old woman was referred for detailed second trimester US screening at a GA
21 + 6. We identified a heart anomaly and initiated cfDNA test, which resulted in
T18 ([Table 3 ]). The couple opted for AC in week 26 + 2. T18 was confirmed and the pregnancy was
terminated.
There were 3 cases with typical ultrasound anomalies pointing towards a high risk
for T21 that were detected in the second trimester of pregnancy (GA 22 + 2; 23 + 0;
26 + 6, respectively). All had nasal bone hypoplasia, two had white spots in the left
ventricle and one had an atrioventricular septal defect (AVSD) ([Table 3 ]). cfDNA test was performed following the detailed anomaly scan whereas the couples
refrained from IPT. All children were delivered and T21 postnatally confirmed.
In 5 fetuses that were seen in the second trimester of pregnancy (GA 14 + 1 – 18 + 0),
no anomalies were detected. The women had obtained a positive result in a cfDNA test
ordered for primary screening and were referred to our practice for IPT (T21: n = 3;
T18: n = 1; T13: n = 1). Two cases of T21 were confirmed by CVS and both pregnancies
were terminated. In the other three cases, AC revealed a normal karyotype and the
children were delivered.
2. Case with a double positive cfDNA test
One 30-year old patient was referred to our practice for amniocentesis at a GA 15 + 1
due to a double positive cfDNA test result for T18 and triple X syndrome. The fetus
received a detailed US and appeared normal without any suspicious findings. Confirmatory
genetic testing following AC revealed a normal karyotype and a healthy child was delivered.
3. Cases with positive cfDNA test results for SCAs
There were 18 fetuses that had a positive cfDNA test for a SCA. In all cases, cfDNA
test was used as a primary screening method in the first trimester of pregnancy (GA
10 + 1 to 13 + 5 weeks). None of the fetuses displayed anomalies in detailed US scan
in our practice (GA 12 + 2 to 21 + 0 weeks). Only 7 (38.9%) of the parents opted for
IPT (2 CVS, 5 AC). Postnatal testing results were available for 5 children and 6 had
no confirmatory genetic testing at all. All children were delivered and 3 had a confirmed
SCA (XXX, X0 and XXY).
4. Cases with a positive cfDNA test results for a microdeletion
All 7 cases that had a cfDNA test predicting a 22q11.2 deletion were primary screening
tests ordered by the patientsʼ primary gynaecologists at a GA between 12 + 1 and 12 + 7
weeks. The patients were referred to our practice for IPT and/or further counselling.
At a detailed US examination in our practice (GA 13 + 4 to 15 + 2 weeks), all fetuses
appeared normal. Four couples opted for CVS with a negative result. Three decided
against IPT, one of those had postnatal testing. There was no case with a confirmed
DiGeorge syndrome.
Number of positive cfDNA test per year
The number of patients with a positive cfDNA test result in our practice strongly
increased from one case in 2013 to 27 cases in 2019 ([Fig. 2 ]). We encountered more than half of the cases (56.8%) in the last two years (2018/2019).
Furthermore, positive cfDNA test results for 22q11.2 deletion occurred only in 2018
and 2019.
Fig. 2 Number of cases with a positive cfDNA result per year. cfDNA: cell free DNA; SCA:
sex chromosome aneuploidy.
Discussion
This study retrospectively describes 81 cases with a cfDNA test predicting T21, 18
or 13, a SCA or a 22q11.2 microdeletion in a referral practice specialising in prenatal
diagnosis in Germany. The PPV of cfDNA testing for T21 was 95%. In contrast, only
55.6 and 28.6% of the cases with a positive cfDNA test for T18 and T13, respectively,
were confirmed. About 75% of the test results predicting a SCA turned out to be false
positive. No fetus was ultimately diagnosed with DiGeorge syndrome. Furthermore, our
data reveals that adherence to the guideline recommendations on utilisation of cfDNA
test by the primary obstetric providers tends to be low. Whereas national and international
guidelines recommend cfDNA testing only following or in conjunction with a qualified
US examination [3 ], [4 ], almost ⅔ of the patients had not received a fetal anomaly scan before cfDNA test.
Twenty-six patients had a cfDNA test positive
for a SCA or a microdeletion, although testing for SCAs and microdeletions is
not recommended due to insufficient clinical evidence [3 ], [4 ]. Most of those occurred during the previous two years. Since we only included patients
with a positive cfDNA test, we can merely speculate about the total number of cfDNA
tests ordered for SCA or DiGeorge syndrome testing in our region.
The limitations of this study are its retrospective design that includes only patients
with a positive cfDNA test result in our practice. Thus, we cannot evaluate the specificity,
sensitivity or negative predictive value of cfDNA test. Although the cohort is rather
large for a single referral centre the small sample allows only descriptive statistical
analysis on the individual aneuploidies. Nevertheless, this study is based on a comprehensive
database with known pregnancy outcome for all cases and covers the entire time period
since cfDNA test is available in Germany. Moreover, it represents a population with
a mixed aneuploidy risk typically seen in routine clinical practice.
It has been reported that the test quality of cfDNA tests in terms of specificity
and sensitivity remains high in real-world conditions [5 ]. However, these parameters do not include prevalence. On the other hand, in the
light of the excellent test quality of cfDNA test, the PPV, which largely depends
on prevalence (and thus maternal age) of the tested condition, is often neglected
[16 ]. Our study, which describes PPV estimates for cfDNA test in routine conditions in
Germany, is in line with other real-world studies from the US, Sweden and China [6 ], [8 ], [17 ], [18 ]: The studies report PPVs for the individual autosomal aneuploidies ranging from
T21: 83 to 94%; T18: 64 to 76% and T13: 44 to 75% [6 ], [8 ], [17 ], [18 ]. Two studies report PPVs of approximately 40% for a combination of SCAs [8 ], [17 ]. And three out of the four studies report only false positive cfDNA test results
for 22q11.2 microdeletion [6 ], [8 ], [17 ]. Only the study of Petersen et al., which includes the largest cohort with 712 patients,
reports a PPV of 21% for 22q11.2 microdeletion [18 ].
Similar to our study, these studies analyse fairly small cohorts, which are likely
to differ in their age distribution, their risk profile and the indications for cfDNA
test. This may account for the differing PPV-estimates and renders it impossible to
make a direct comparison of the actual values. However, all studies clearly demonstrate
that, in a typical population in routine practice, positive cfDNA test results must
be carefully interpreted and appropriate counselling of the women concerned is of
major importance.
A particularly worrying observation in this study is that almost two thirds of the
patients did not receive a detailed US examination before cfDNA test. This might indicate
that cfDNA testing is increasingly regarded as a replacement for a first trimester
US anomaly screen and that the pregnant women are not aware that US can additionally
detect anomalies unrelated to aneuploidies. On the other hand, about 90% of the fetuses
with a confirmed autosomal aneuploidy displayed fetal anomalies amenable to first
trimester US. In particular, fetuses with T13 and T18 are usually detectable by US
in early pregnancy. Thus, the case with T18 with an inconspicuous US was rather unusual
in our experience. Interestingly, a recent publication has shown that the time of
intrauterine diagnosis of trisomies 21, 13 and 18 has not changed in the past 10 years
[19 ].
It has been shown that the implementation of cfDNA test as a screening method for
T21 in a high-risk population decreases the rate of invasive diagnostics [20 ]. This positive effect might however be undermined by an indiscriminate usage of
cfDNA test. Since cfDNA testing is a screening method, invasive prenatal testing is
required to confirm a positive result. In our study, approximately half of the patients
with a cfDNA test positive for a SCA or 22q11.2 microdeletion underwent an invasive
procedure – they would most probably not have done this if they had ordered the cfDNA
test only for T21 without additional options and had obtained a negative result. Furthermore,
about 25% of our study population was younger than 35 years old. Due to the low prevalence
in women < 35 years, the PPVs for T18 and T13 are predicted to be lower than 35 and
18%, respectively [16 ]. Thus, broad usage of cfDNA test testing for
T13 and T18 in low-risk pregnancies will be bound to result in false positive
cases entailing unnecessary invasive procedures.
On the other hand – as described in several cases in our study – cfDNA test can be
a valuable option for parents where there is a high probability that their fetus has
T21 due to typical US findings, but who want to have the child delivered and do not
want to take the risk of IPT.
Although it is required by the German Genetic Diagnostics Act that gynaecologists
must obtain a supplementary qualification for “subject-related genetic councelling”
and are obliged to provide appropriate counselling to the patients [3 ], it is uncertain if the cfDNA test providers verify those requirements. In our experience,
counselling prior to cfDNA-testing is often insufficient. Thus, a comprehensive education
programme for primary obstetric providers and responsible counselling for pregnant
women may help to increase sensible usage of cfDNA testing.
Primary gynaecologists must be aware that even a high specificity of cfDNA test for
a certain aneuploidy of > 99% does not mean that less than 1% of the women undergoing
the test will obtain a false positive result. Instead, they must consider the PPV
and be aware that it largely depends on prevalence and thus age of the individual
patient.
Adequate patient education about the various options of prenatal screening and their
informative value is vital and decisions should not be driven by economic/financial
aspects. Parents must understand that the cfDNA test cannot predict whether their
child is healthy but can only exclude specific conditions. They should not be left
alone to make their decision solely based on the information supplied by the cfDNA
test providers (e.g. brochures, web pages), since these argue mainly in terms of specificity
and sensitivity but neglect the PPV. Instead, they deserve the explanation that a
positive test result has a certain probability to be false positive. And last but
not least, parents should be encouraged to think about potential consequences of a
positive test result, before actually undergoing the test.
Conclusions
This study demonstrates that in routine practice the PPVs for cfDNA testing for aneuploidies
other than trisomy 21 are low. Often, cfDNA testing is performed without an accompanying
ultrasound examination. Thus, utilisation of cfDNA testing in routine practice should
be optimised.
