cfDNA screening in clinical practice · OPTION 1 Models for clinical implementation Universal...

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


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.



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.



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%



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


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


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


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


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!

cfDNA screening in clinical practice · OPTION 1 Models for clinical implementation Universal...

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