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cfDNA screening in clinical practice
María del Mar Gil
1997
Maternal blood
Cell-free DNA in maternal bloodLo YM, Corbetta N, Chamberlain PF, Rai V, Sargent
IL, Redman CW, Wainscoat JS. Presence of fetal
DNA in maternal plasma and serum. Lancet 1997;
350:485-487.
2008
Fan HC, Blumenfeld YJ, Chitkara U, Hudgins L, Quake SR:
Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA
from maternal blood. Proc Natl Acad Sci USA 2008;105:266.
Chiu RW, Chan KC, Gao Y, Lau VY, Zheng W, Leung TY, Foo CH, Xie
B, Tsui NB, Lun FM, Zee BC, Lau TK, Cantor CR, Lo YM: Noninvasive
prenatal diagnosis of fetal chromosomal aneuploidy by massively
parallel genomic sequencing of DNA in maternal plasma. Proc Natl
Acad Sci USA 2008;105:20458.
13 18 214 X Y
The cf DNA fragments are sequenced, their chromosome origin is established and the quantity of each chromosome is determined
0.04%
0.04%Trisomy 21 n=1.963
Trisomy 18 n= 563
0.04%Trisomy 13 n= 119
99.7%
97.9%
99.0%
Gil MM, Accurti V, Santacruz B, Plana MN, Nicolaides KH. Analysis of cell-free DNA in maternal blood in screening for
aneuploidies: updated meta-analysis. Ultrasound Obstet Gynecol 2017;50:302-314.
Meta-analyses:
• Singletons: 35 studies (Jan 2011 to Jan 2017)
• Clinical validation or implementation
• Pregnancy outcome > 85% of study population
cfDNA testingPerformance of screening
- Modelo de implementación: universal vs contingente
- Malformaciones fetales y marcadores de cromosomopatía
- Test sin resultado
- Gestaciones gemelares y gemelo evanescente
- Condiciones a incluir en la solicitud del test
Implementación clínica
Models for clinical implementation
- Contingent after results from 1st line screening- Universal screening
OPTION 1
Models for clinical implementation
Universal screening by cfDNA
CVS
Gil MM, Quezada M, Bregant B, Ferraro M, Nicolaides K. Implementation of maternal blood cell-free DNA testing in early screening for aneuploidies. Ultrasound
Obstet Gynecol 2013; 42: 34-40.
10 weeks:
• Scan to measure the fetus
• Blood for cf DNA test
• Blood for combined test
12 weeks:
•Combined test for trisomies, fetal
defects and pregnancy
complications
• +ve cfDNA test
• Fetal defects
• NT >3.5 mm
cf DNA test
Normal: 2738
Trisomy 21: 32
Trisomy 18: 10
Trisomy 13: 5
cfDNA test Combined test
32 (100%)
9 (90%)
2 (40%)
8 (0.3%)
32 (100%)
10 (100%)
5 (100%)
124 (4.5%)
T21 1 (0.04%)
T18/13 7 (0.26%)
Fetal Medicine Centre n=2,905
Quezada MS, Gil MM, Francisco C, Oròsz G, Nicolaides KH. Screening for trisomies 21, 18 and 13 by cell-free DNA analysis of maternal blood
at 10-11 weeks' gestation and the combined test at 11-13 weeks. Ultrasound Obstet Gynecol 2015;45:36-41.
Models for clinical implementation
Universal screening by cfDNA
Trisomía 21 53%
Trisomía 18 72%
Trisomía 13 88%
Triploidía 34%
Monosomía X 94%
Otras 23%
50% T21 80% T13 20% todas 45% T18
Syngelaki A, Guerra L, Ceccacci I, Efeturk T, Nicolaides KH. Impact of holoprosencephaly, exomphalos, megacystis and high NT in first trimester screening for
chromosomal abnormalities. Ultrasound Obstet Gynecol 2016; doi 10.1002/uog.17283
n = 108,982
Screen +ve rate 1.1%
This group contained 57% of
all aneuploidies
Clinical implementationUniversal screening: Fetal malformations
OPTION 2
cfDNA test
No
CVS
Yes
Fetal malformationsNT > 3.5 mm
11-13 weeks:
• Viability, number of fetuses, dating...
• Screening for defects
• Screening for pregnancy complications
Kagan KO, Sroka F, Sonek J, Abele H, Lüthgens K, Schmid M, Wagner P, Brucker S, Wallwiener D, Hoopmann M First-trimester risk assessment based on ultrasound and
cell-free DNA vs combined screening: a randomized controlled trial Ultrasound Obstet Gynecol 2018; 51: 437-444.
Models for clinical implementation
Universal screening by cfDNA
Kagan KO, Sroka F, Sonek J, Abele H, Lüthgens K, Schmid M, Wagner P, Brucker S, Wallwiener D, Hoopmann M First-trimester risk
assessment based on ultrasound and cell-free DNA vs combined screening: a randomized controlled trial Ultrasound Obstet Gynecol 2018; 51:
437-444.
cfDNA test Combined
test688
0%
688
17 (2.5%)
N
FPR
10 (1.5%) 0%FailedRandomise to cfDNA test or combined test
Yes 2%
Fetal defectsNT > 3.5 mm
CVS
Ultrasound at 11-13 SG
No 98%
Models for clinical implementation
Universal screening by cfDNA
Clinical implemetationContingent screening: only to high-risk
Clinical implementation within the NHS
High-risk
>1 in 100 (3%)
First - line screening by the combined test (11-13 w)
Low-risk
<1 in 100 (97%)
Invasive test Nothing else
Nothing else
cfDNA test
National Screening Committee
Objectives:- Decrease invasive testing- Cost neutral- Maintain DR at about 85%
Clinical implemetationContingent screening:intermediate-risk
Santorum M, Wright D, Syngelaki A, Karagioti N, Nicolaides KH. Accuracy of first trimester combined test in screening for trisomies 21, 18 and 13. Ultrasound Obstet Gynecol
2016; doi 10.1002/uog.17283
Dete
cti
on
ra
te (%
)
0
10
20
30
40
50
60
70
80
90
100
Screen positive rate (%)
0 10 20
1 in 2000
1 in 100
4 12
1 in 500
98%
88%
96%
70%1 in 10
1st trimester combined screening at 11-13 weeks
(maternal age, NT, serum free ß-hCG & PAPP-A)
High risk Low risk
Invasive test
Intermediate risk
Nothing else
cfDNA test
+ -
1 in 10 1 in 500
Kings’ College & Medway Maritime hospitals
October 2013 – January 2018
Singleton pregnancies n>40,000
Study supported
by Ariosa - Roche
Management of “no results”
Fetal fractionIs it important?
Fetal fraction
Ch
rom
oso
me 2
1 Z
-sco
re
-3
0
3
6
9
12
15
18
21
0 5 10 15 20 25 30 35
24
Wright D, Wright A, Nicolaides KH: A unified approach to risk assessment for fetal aneuploidies. Ultrasound Obstet Gynecol 2015; 45: 48-54
40% FF
Units of chromosome 21
20% FF 5% FF 2% FF
Galeva S, Gil MM, Konstantinidou L, Akolekar R, Nicolaides KH. First-trimester screening for trisomies by cfDNA testing of maternal
blood in singleton and twin pregnancies: factors affecting test failure. Ultrasound Obstet Gynecol 2019; doi: 10.1002/uog.20290
Singletons
No result: 3%
Result after repeat testing
No result: 1%
MC twins
No result: 5%
No result: 0%
DC twins
No result: 12%
No result: 5%
Increased risk for test failure
- Increasing maternal weight
- Nulliparity
- Black and South Asian race
- IVF conception (x3.8)
- Dichorionic twins (x1.7)
- Early gestational age
- Low PAPP-A and free ß-hCG
Harmony test at 10-14 w
- KCH, MMH, FMC
- Singletons n=23,495
- Twins n=928
No result from cfDNA test
Clinical implementationManagement of “no results”
Singletons(n=10,619)
25
20
15
10
5
0Normal
n=10,393
T21
n=160
T18
n=50
T13
n=16
Feta
l fr
acti
on
(%
)
Revello R, Sarno L, Ispas A, Akolekar R, Nicolaides KH.
Cell-free DNA testing for trisomies: management of failed
result. Ultrasound Obstet Gynecol 2016; 47: 698-704.
“No result” from cf DNA test
Detailed ultrasound scan
AbnormalNormal
Invasive test
Repeat cfDNA test
AbnormalNormal “No result”
No IT
Prior risk T21
Low High
MC twins: same as in singletons
DC twins: the placental products of the normal fetus may mask the abnormality of the co-twin.
- Measure total fetal fraction and set cut-offfor failure at higher level than singletons
- Measure fetal fraction separately for eachfetus and set cut-off at level of the one with the lowest fraction
- Do not measure fetal fraction.
Trisomies in twins:
Screening by cfDNA test
FPRDR
0.04%
0.04%Trisomy 21 n=1.963
Trisomy 18 n= 563
0.04%Trisomy 13 n= 119
99.7%
97.9%
99.0%
Gil MM, Accurti V, Santacruz B, Plana MN, Nicolaides KH. Analysis of cell-free DNA in maternal blood in screening for aneuploidies: updated meta-analysis.
Ultrasound Obstet Gynecol 2017;50:302-314.
Gil MM, Galeva S, Jani J, Konstantinidou L, Akolekar R, Nicolaides KH. Screening for trisomies by cfDNA testing of maternal blood in twin pregnancy:
update of the FMF results and meta-analysis. Ultrasound Obstet Gynecol 2019; in press
Singleton pregnancies
FPRDR
0.03%
0.05%Trisomy 21 n=56
Trisomy 18 n=18
0.19%Trisomy 13 n= 3
98.2%
88.9%
66.7%
Twin pregnancies
cfDNA test in twins
Performance of screening
Meta-analyses:
• Singletons: 35 studies (Jan 2011 to Jan 2017)
• Twins: 8 studies (Jan 2011 to Jan 2019)
• Clinical validation or implementation
• Pregnancy outcome > 85% of study population
0.23% of all pregnancies
Responsible of 0.27-0.36% of false positives
Discordant results at least 15 weeks later
Gestational age (weeks)
Fetal demise
cfDNA test
6 8 10 12 14 16 18 20 22
cfDNA test in twins
Vanishing twin
Curnow KJ, Wilkins-Haug L, Ryan A, Kırkızlar E, Stosic M, Hall MP, Sigurjonsson S, Demko Z, Rabinowitz M, Gross SJ. Detection of triploid, molar,and vanishing twin
pregnancies by a single-nucleotide polymorphism-based noninvasive prenatal test. Am J Obstet Gynecol 2015; 212:79.e1-9.
Thurik FF, Ait Soussan A, Bossers B, Woortmeijer H, Veldhuisen B, Page-Christiaens GC, de Haas M, van der Schoot CE. Analysis of false-positive results of fetal RHD typing in
a national screening program reveals vanishing twins as potential cause for discrepancy. Prenat Diagn 2015; 35: 754–760.
Niles KM, Murji A, Chitayat D. Prolongued duration of persistent cell free DNA from a vanishing twin. Ultrasound Obstet Gynecol 2018; doi: 10.1002/uog.19004.
T21, T18, T13Sex chromos.
aneuploidiesMicrodeletions
Genome wide
assessment
Single gene
disorders
Clinical relevance
Prevalence
Evidence on performance
Degree of performance
Simplicity of counseling
Content of cfDNA test
Thank you
Conclusions
• Universal screening: better after the 12 weeks’ scan
• Contingent model: intermediate risks without malformations
• If there is no result: reassessment and consider re-draw
• Never in vanishing twins or triplets, recommended in twins
Ultrasound examination will help us interpret cfDNA
test results and choose the best management option
www.fetalmedicine.org
The Fetal Medicine
Foundation
18th World Congress in Fetal Medicine
Alicante 25-29 June 2019
Expandir las patologías estudiadas
Preocupaciones clínicas
Cobertura test ADN-lc
de defectos fetales:
8-10%
Defectos
estructurales (no
cardiacos):
42.8%
Anomalías
cardiacas
congénitas:
21.4%
Otras anomalías
cromosómicas: 4.6%
VNC: 21.4%Trisomía 21: 1.8%
Trisomía 18: 0.5%
Trisomía 13: 0.2%
Aneuploidías cromosomas
sexuales: 3.6%
Monosomía X: 3.8%
Triploidía: 0.07%
Cobertura test ADN-lc de
anomalias cromosómicas:
55.5%
Norton M., SMFM, 2015
Expandir las patologías estudiadas
Triploidía
T. Diándrica: 4 Correcto 4
T. Digínica: 4 ‘Baja FF’ 3
Fallo 1
Normal: 48 Correcto 44
Fallo 4
Feta
l fr
acti
on
(%
)
0
5
10
15
20
25
30
35
40
50 60 70 80 90 100 110 120
Maternal weight (kg)
0.5th centile
Nicolaides KH, Syngelaki A, Gil MM, Quezada MS, Zinevich Y. Prenatal detection of fetal triploidy from cell-free DNA testing in maternal blood. Fetal Diagn Ther 2014; 35: 212-
217.
Posible con SNPs
No resultado si FFNormal si no FF
Expandir las patologías estudiadas
Anomalías cromosomas sexuales
• Síndrome de Turner letal: fácilmente detectable por ecografía
• La mayoría de casos son leves y sin discapacidad intelectual
• 50% de las aneuploidías de los cromosomas sexuales son mosaicos
• Alta incidencia de mosaicismos maternos
• El rendimiento del cribado es incierto
- Tasa de “no resultados” es mayor que para trisomías
- Tasa de detección menor (alrededor del 90%) que para T21 (>99%)
- Tasa de falsos positivos mayor (alrededor del 0,3%) que para T21
(<0,1%)
- Valor predictivo positivo menor que para trisomía 21
• Manejo de un resultado positivo: necesidad de amniocentesis
Expandir las patologías estudiadas
Anomalías cromosomas sexuales: triple X
• Anomalía cromosómica más común en las mujeres
• Aproximadamente 1 de cada 1.000 nacimientos femeninos
• Sólo un 10% de las personas con XXX está diagnosticadas
• Mayoría leves o asintomáticos
Expandir las patologías estudiadas
Anomalías subcromosómicas
Microdeleciones 1/nac. 3>Mb
22q11.2 deletion 2-4.000 85%
Angelman / Prader Willi
(deleción 15q11.2-q13)20.000 70%
1p36 del 5.000 85%
Cri-du-chat (deleción 5p) 50.000 99% Edad materna (años)
Riesgo
T21
Di George – hallazgos comunes:
75% deficiencias inmunológicas
50% hipocalcemia
30% problemas alimenticios
35% anomalías renales
75% defectos cardiacos
95% déficit intelectual
25% esquizofrenia en el adulto
- Tasas de detección*: 60-99%- Tasa total de falsos positivos: 1%- Valor predictivo positivo para deleción 22q11.2: 5%- Proporción de microdeleciones significativas incluidas: 10%
* reportado por las compañías, pero no incluídas aquellas de <3 Mb
Yaron Y et al. Current status of testing for microdeletion syndromes and rare autosomal trisomies using cell-free DNA technology. Obstet Gynecol 2015;126:1095
Wapner RJ et al. Expanding the scope of noninvasive prenatal testing: detection of fetal microdeletion syndromes. Am J Obstet Gynecol 2015;212:332.e1-9.
• Modelo universal: mejor tras ecografía semana 12
• Modelo contingente: riesgos intermedios sin malformaciones
• Si un test no da resultado: reevaluar y considerar repetir
• Nunca en gemelo evanescente ni triples, sí en gemelares
• Condiciones a incluir: Trisomías 21, 18 y 13 (+/- sexo fetal)
Conclusiones
La ecografía, incluídos los marcadores, nos ayudan a
interpretar resultados y decidir el siguiente paso
- Modelo de implementación: universal vs contingente
- Malformaciones fetales y marcadores de cromosomopatía
- Test sin resultado
- Gestaciones gemelares y gemelo evanescente
- Condiciones a incluir en la solicitud del test
¿Lo hemos respondido todo?
¡Gracias!