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Vincenzo Cirigliano

Molecular Genetics Synlab, Barcelona

vincenzo.cirigliano@synlab.es

cfDNA Based Prenatal Screening for Common Aneuploidies

0%

20%

40%

60%

80%

100%

Maternal age AFP only Quad MarkerScreen

FirstTrimester

Screen

FullIntegrated

Screen

Detection Rate for Down Syndrome*

1970’s 1980’s 1990’s 2000

*ACOG practice bulletin no. 77, Obstet Gynecol 2007;109:217-27.

Evolution of Prenatal Screening

FPR=5.0%

Cell-free DNA in Maternal Blood

• Released through apoptosis

Fetal cfDNA from the Cytotrophoblast

• Fetal DNA reliably detected from

7w gestation

• Cleared by the kidney disappears

within hours postpartum

• Maternal Plasma contains both

fetal and maternal cfDNA

1-20% of cfDNA is of fetal origin

• Fetal cfDNA is shorter than maternal

(140-160b.p.)

Chromosome 21 fragments

Reference chromosome

Fetal cfDNA

Maternal cfDNA

Detecting Fetal Trisomy by cfDNA Analysis

Fetal cfDNA

Maternal cfDNA

Extra fragments derived

from fetal trisomy 21

Chromosome 21 fragments

Reference chromosome

Detecting Fetal Trisomy by cfDNA Analysis

Alignment of Fragments

Fragment Count

Fetal Fraction

Expected ratio for Trisomy

4% 1.02

10% 1.05

20% 1.10

40% 1.20

Higher Fetal Fraction Easier Detection Fetal cfDNA

Maternal cfDNA

Detecting Fetal Trisomy by cfDNA Analysis

Chromosome 21 fragments

Reference chromosome

Massively Parallel Shotgun Sequencing (MPSS)

• MPSS is a random sampling of cfDNA fragments

• An arbitrary z-score value is used as a cut-off for trisomy

Palomaki et al.. Genet Med. 2011 Nov;13(11): 913-20.

N=1696

• “Unclassified” zone for values between 2.5-4

• true and false positives in this zone

Bianchi, DW, et al.. Obstet Gynecol. 2012 May;119(5): 890-901.

MPSS Unclassified Values

Chr 21, 18, 13, X & Y cfDNA

Other Chr cfDNA

Unmapped cfDNA

cfDNA in blood

MPSS (shotgun)

Selected Tags

Directed

Random analysis of cfDNA

Targeted vs WGS

Sensitivity is only driven by the number of useful counts (Sequencing Depth)

Specificity increases with the Fetal Fraction (FF)

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

FF 8% FF 2%

24

Fetal fraction

Ch

rom

oso

me

21

Z-s

co

re

-3

0

3

6

9

12

15

18

21

0 5 10 15 20 25 30 35

FF 8% FF 2%

24

a) b)

Fracción fetal

Z-s

core

para

Cro

mosm

a 2

1

FF 2% FF 8%

Cut-off at 4% FF for reportable cases

Aneuploidy detection by MPSS

Wright et al. 2014 UOG 45(1), 48–54

Detection rate FPR

Trisomy 21 99.7% 0.04%

Trisomy 18 97.9% 0.04%

Trisomy 13 99.0% 0.04%

cfDNA and Aneuploidy Screening

Gil et al, Ultrasound Obstet Gynecol 2017; 50: 302–314

cfDNA not always reflects fetal genotype (placental /maternal mosaicism, vanishing twin…)

Trisomy 18 and 13 – discordance

Explaining “false positive” and “false negative” NIPT results

*Kalousek DK et al., Am J Hum Genet. 1989 Mar;44(3): 338-43 .

cfDNA originates from placenta ● May be similar to “Direct prep” of chorionic villi

Chromosomal makeup of placenta and fetus can be different

Occurs more frequently with chromosomes 13 and 18

● T13 and T18 (Kalousek et al*) — Only 30% of trophoblast showed trisomy when other fetal tissue

100% for trisomy 13 and 18 (viable)

● Case report of discordance between NIPT and fetus for trisomy 13

— NIPT: “positive” for T13

— CVS: mosaic 47,XY,+13[10]/46,XY[ 12]

— Amnio normal, fetus normal

— Placental biopsies = 2/4 mosaic trisomy 13

**Hall AL, Drendel HM, Verbrugge JL, Reese AM, Schumacher KL, Griffith CB, Weaver DD, Abernathy MP, Litton CG, Vance GH, Genetics in Medicine ( 2013)

Detection rate FPR

Trisomy 21 99.7% 0.04%

Trisomy 18 97.9% 0.04%

Trisomy 13 99.0% 0.04%

cfDNA and Aneuploidy Screening

It’s a screening test

Gil et al, Ultrasound Obstet Gynecol 2017; 50: 302–314

0%

20%

40%

60%

80%

100%

Maternal age AFP only Quad MarkerScreen

FirstTrimester

Screen

FullIntegrated

Screen

Detection Rate for Down Syndrome* FPR < 0.1%

Cell-free

DNA

analysis

1970’s 1980’s 1990’s 2000 *ACOG practice bulletin no. 77, Obstet Gynecol 2007;109:217-27.

FPR=5.0%

Evolution of Prenatal Screening

0

50

100

150

200

250

300

350

2012 2013

ANXIETY

RISK

N 314

N 96

142

29

Invasive Procedures

Pedregosa et. al. 2015 Progresos de Obstetricia y Ginecología 01;58(3).

18

Paired-end Sequencing More information, maintained accuracy

CGCTAGAAG

ATTTCCGCGATCTTCCCGTTCGACTGCAGACCTTCAGCGCGCATATATCGCTAGCATACCGTTATAC

GAAGTCGCG

Human Genome

Alignment Better accuracy

2 x 36 bp

Fragment Size determination

Fragment length

Count:

Paired End Sequencing More information, maintained accuracy

Improved Allignment, Increased Depth and Resolution

2xMPSS

Size Comparison – Maternal and Fetal Fragments

Lo et al. Sci Transl Med 2010 Vol 2 Issue 61 61ra91

Maternal and fetal cfDNA size distribution

Fragment size distribution

Cromosoma de interés

Cromosoma(s) de referencia

Cromosoma(s) de referencia

Cromosoma de interés

a) b)

Cromosoma de interés

Cromosoma(s) de referencia

Cromosoma(s) de referencia

Cromosoma de interés

a) b)

Cromosoma de interés

Cromosoma(s) de referencia

Cromosoma(s) de referencia

Cromosoma de interés

a) b)

Short Fragments Long Fragments

Standard methodology: counting statistics to determine if more than the expected number of reads are mapped to the target chromosome

New methodology: fragment size statistics, determine if the fraction of short fragments was higher on the target chromosome.

Paired-end MPSS: new analysis method

Cromosoma de interés

Cromosoma(s) de referencia

Cromosoma(s) de referencia

Cromosoma de interés

a) b)

Cromosoma de interés

Cromosoma(s) de referencia

Cromosoma(s) de referencia

Cromosoma de interés

a) b)

Cromosoma de interés

Cromosoma(s) de referencia

Cromosoma(s) de referencia

Cromosoma de interés

a) b)

Cromosoma de interés

Cromosoma(s) de referencia

Cromosoma(s) de referencia

Cromosoma de interés

a) b)

Paired-end MPSS: new analysis method

New methodology: fragment size statistics, determine if the fraction of short fragments was higher on the target chromosome.

Combine FF, counts and frag size statistics for final scoring (t-score)

Short fragments analysis boosts resolution improving counting statistics

Increased specificity / confident analysis at low FF

V. Cirigliano et al UOG 2017 doi:10.1002/uog.173

The Fetal Medicine

Foundation

Trisomy 21 n = 47

Trisomy 18 n = 30

Trisomy 13 n = 10

Stored plasma samples 1mL from 1000 singleton pregnancies at 11-13 wks

* Failed result n=12 (1.2%)

K. H. Nicolaides, Neobona Presentation, Madrid Feb. 2016

V. Cirigliano et al UOG 2017 doi:10.1002/uog.173

Total cfDNA Failed

Normal 1609 1592 17

Trisomy 21 66 63 3

Trisomy 18 34 33 0

Trisomy 13 13 13 0

45, X 5 5 0

47, XXY 3 3 0

Total 1730 1709 20

Results of testing 1730 plasma samples

117 aneuploidies identified, 6 cases with FF between 0.8 and 3%.

Performance evaluation study

-10

-5

0

5

10

15

20

25

30

35

40

0.0000 0.0500 0.1000 0.1500 0.2000 0.2500 0.3000 0.3500 0.4000FF

NCV

NCV vs FF

V. Cirigliano et al UOG 2017 doi:10.1002/uog.173

-100

-80

-60

-40

-20

0

20

40

60

80

100

0.0000 0.0500 0.1000 0.1500 0.2000 0.2500 0.3000 0.3500 0.4000FF

T18 Score

T-score vs FF

Cut off at 4% FF no longer needed V. Cirigliano et al UOG 2017 doi:10.1002/uog.173

98% of Trisomies and 53% SCA confirmed by fetal karyotype

All pregnancies to term

1stT Screening /

Streck BCT Blood

Paired-end MPSS: Clinical Application

23749 GA > 10w - 728 Twins

Test performed within 5 Days from sampling 1ml Plasma TAT 2-5 Days All samples tested for common trisomies SCAs included in 54% of cases

27%

41%

14%

4% 2%

13%

MaternalAge

Anxiety ScreeningPotitive

Ultrasound History Others

Indications

Paired-end MPSS: Clinical Application

23749 Samples

No result 430 (1.7%)

Not suitable (n=81)

Redraw 406

No result 27 (6.5%)

Normal n= 22767 (98%)

Trisomy 21-18-13 n= 471

XY Chr. n= 11021 (47%)

No Result 0.2%

SCA n= 36

Tested 23668 (730 Twins)

Result 23238 (98.3%)

Result 379 (93.5%)

Result in 99.8%

XXY+18/21 n=3

Paired-end MPSS: Clinical Application

FPR 0.04% *1 T21, 3 T13 miscarried before confirmation

Results of screening for autosomal trisomies

T21* 363 1 5 99.7% 0.02%

T18 72 - 2 100% 0.01%

T13* 37 - 3 100% 0.01%

Low Risk 23238

FPR 0.05%

45,X 20 9 3 0.05%

47,XXY 9 5 0 0.00%

47,XXX 5 3 0 0.00%

47,XYY 2 2 0 0.00%

Mat 3

Total 36 19 1

Results of screening for XY aneuploidies

- 11021 consecutive clinical cases - Invasive procedures in 53% high risk results - All pregnancies to term

T21 14 - 2 100% 0.1%

T18 13 - - 100% 0.0%

T13 7 - - 100% 0.0%

Low Risk 1109

Autosomal trisomies at Low FF

FF<4% n= 1362 (5.5%) 84% reported, 24 Twins (48 Redraw) 20% of total T13 and T18

• Paired-end MPSS already costs effective

• New generation algorithm (Tscore) High DR, Low FPR

• Improved efficiency, no fixed lower limit for FF to report

• All Trisomies with FF <4% correctly scored

• Including low FF didn’t impact DR while only increasing FPR

by 0.005%

• Increased T13 and T18 prevalence at low FF

• Extends cfDNA screening to a larger proportion of pregnancies

Conclusions

Dept. of Molecular Genetics Synlab, Barcelona

Thanks

Vincenzo Cirigliano

Molecular Genetics Synlab, Barcelona

vincenzo.cirigliano@synlab.es