prenatalsafe® karyo · prenatalsafe® karyo analyzes every chromosome in the genome. unlike any...

PrenatalSAFE® Karyo test is the most technologically advanced genome-wide NIPT

Through cfDNA analysis from maternal plasma, PrenatalSAFE®Karyo detects:

Aneuploidies

structural chromosomal aberrations(deletions or duplications) across the fetal genome

providing karyotype-level insight.

What is

PrenatalSafe® Karyo analyzes every chromosome in the genome.

Unlike any other noninvasive prenatal test available to date, it offers a level of information previously only available from a fetal karyotype analysis, performed with invasive prenatal diagnosis procedures (amniocentesis and CVS)

the next level in noninvasive prenatal testing

Maternal blood sample

Maternal cfDNA Fetal cfDNA

NIPT uses cell-free DNA (cfDNA)

Whole-genome sequencingtechnology

Fetal FractiondeterminationcfDNA

extraction

DNA Sequencescounting

2 levels of genome-wide screening

It can also test for 9 common microdeletion syndromes

Microdeletion Syndrome Prevalence

DiGeorge syndrome (22q11.2) 1 in 4,0001p36 deletion syndrome 1 in 4,000 to 1 in 10,000Angelman syndrome (15q11.2) 1 in 12,000Prader-Willi syndrome (15q11.2) 1 in 10,000 to 1 in 25,000Cri du Chat syndrome (5p15.3) 1 in 20,000 to 1 in 50,000Wolf-Hirschhorn syndrome (4p16.3) 1 in 50,000Langer-Giedion syndrome (8q24) 1/200.000Jacobsen syndrome (11q23) 1/100.000Smith-Magenis syndrome (17p11.2) 1/15.000 - 1/25.000

Detection rate

Detection rate

PrenatalSAFE® Karyo Plus test identifies 95.5% of chromosomal anomalies prenatally detected and 99.1% of those anomalies observed at birth, achieving a level of detection rate very closed to the traditional fetal karyotyping (96.9%), obtained with invasive, prenatal diagnostic techniques.

Conventional Prenatal Screening vsDetection Rates for Trisomy 21

ACOG Practice Bulletin No. 77, January 2007

False Positive Rate (FPR): 5%

Integrated Screen

Quadruple Screen

1st Trimester Blood Screen

+ NTSerum

Integrated

Fetal Karyotyping vs Traditional Karyotyping

Analyzes every chromosome

Detects chromosomalgains or losses >7 Mb

Detects chromosomegains or losses >10 Mb

Requires an invasive procedure

Detects unbalanced translocations

Detects segmental deletions/duplications

Detects mosaic trisomies

Detects marker chromosomes

Detects microdeletion syndromes

Detects triploidy

Considered diagnostic

Pre-clinical Validation performance

Fiorentino et al., EJHG conference 2016; ISPD conference 2016

Performance Clinical Cases

Performance Standard protocol

PPV: Positive Predictive Value; NPV: Negative Predictive Value; SCA: Sex Chromosomes Aneuploidy

Follow-up March 2016

Performance FAST protocol

PPV: Positive Predictive Value; NPV: Negative Predictive Value; SCA: Sex Chromosomes Aneuploidy

Follow-up March 2016

Published validation studies

Which Patients Should Be Offered?

This test is intended for patients at 10 weeks or greater gestation who meet any of the following criteria:

Maternal age-related risks (e35 years)

Positive results on maternal serum screening

Abnormal ultrasound finding(s)

Prior pregnancy with aneuploidy

Parental translocation

Low risk pregnancies

Patients wanting early, accurate testing and are at average risk of aneuploidy

The test is suitable for both singleand twin pregnancies.

It can be performed in patients whose pregnancies have been achieved by IVF techniques, including pregnancies with egg donation.

pregnancy characteristics

Free services supplied

Case study

Case Study 1

31 y.o. patient

5

Negative Result

Fetal abnormal ultrasound findings(cerebellar ipoplasia; Ventriculomegaly )

Reassessment of the NIPT data with

Amniocentesis

20^ week gestation

12^ weeks gestation

del5p15.33p13.2

Chr. 5Chr. 5

PrenatalSafe® KaryoArray-CGH

cfDNAAmniotic Fluid

34 Mb

Detection of a deletion 5p confirmed by array-CGH

32 y.o. patient

5

Multiple fetal abnormal ultrasound findings

Amniocentesis

13^ weeks gestation

Negative Result

21^ weeks gestation

Reassessment of the NIPT data with

Case Study 2

Del18p11.32-p11.31

Chr. 18

PrenatalSafe® KaryoArray-CGH

3.8 Mb

Del18q21.32-q2321.3 Mb

Identification of 2 deletions (18p / 18q) confirmed by array-CGH

Chr. 18

cfDNAAmniotic Fluid

32 y.o patientCarrier of a reciprocal translocation 46,XX,t(7;9)(p15;q22)

Abnormal Resultdetected fetal karyotype with an unbalanced translocation

Villocentesis

Chr. 7

Chr. 9

Results confirmed

Case Study 3

11^ weeks gestation

PrenatalSafe® KaryoArray-CGHcfDNACVS

PrenatalSafe® KaryoArray-CGHCVS

Dup7p22.3p21.2 15 Mb Dup9p24.3-q31.1 107.5 Mb

cfDNA

Detected a fetal karyotype with an unbalanced translocation confirmed by array-CGH

35 y.o patient

Amniocentesis(traditional karyotyping)

Negative Result(Fetale karyotype 46, XY)

(Suspected DiGeorge syndrome)

Identified deletion 22q11.2 (compatible with DiGeorge syndrome)

Amniocentesis

Case Study 4

13^ weeks gestation

20^ weeks gestation

Fetal abnormal ultrasound findings

PrenatalSafe® KaryoArray-CGH

cfDNA

del22q11.21DiGeorge Syndrome

Plus

4 Mb

Chr.22

Amniotic Fluid

Identification of 22q11.2 deletionconfirmed by Array-CGH

33 y.o. patient(She was not aware to be carrier of a chromosomal translocation)

Abnormal Resultdetected fetal karyotype with an unbalanced translocation

Paziente portatrice di traslocazione reciproca

46,XX,t(13;20)(q33;q13.3)

Chr. 13 Chr. 20

Case Study 5

12^ weeks gestation

Villocentesis

Results confirmed

(cfDNA)

Del13q33.1q34 11.4 Mb Dup20q13.33 1.9 Mb

Array-CGH(CVS)

Chr. 13 Chr. 20

(cfDNA)Array-CGH

(CVS)PrenatalSafe® KaryoPrenatalSafe® Karyo

Detected a fetal karyotype with an unbalanced translocation confirmed by array-CGH

40 y.o patient

Abnormal Resultdetected fetal karyotype with duplication 18p

Villocentesis

Results confirmed

Case Study 6

11^ weeks gestation

Chr. 18

Detection of a duplication 18p confirmed by array-CGH

38 y.o patient

Abnormal Resultdetected fetal karyotype with duplication 11p

Amniocentesis

Results confirmed

Case Study 7

10^ weeks gestation

Detection of a duplication 11p confirmed by array-CGH

Chr. 11

PrenatalSafe® KaryoArray-CGH

cfDNAAmniotic

Fluid

Dup11p15.4p15.1

16 Mb

Dup11p15.4p15.1

16 Mb

35 y.o patient

Abnormal Resultdetected fetal karyotype with deletion Xp

Amniocentesis

Results confirmed

Case Study 8

12^ weeks gestation

16^ weeks gestation

PrenatalSafe® KaryoArray-CGH

cfDNAAmniotic Fluid

delXp22.33p11.1

58.5 Mb

delXp22.33p11.1

58.5 Mb

Detection of a deletion Xpconfirmed by array-CGH

40 y.o. patient

NIPT(5 chromosomes screening)

Trisomy 21 not detectedMaternal CNV detected

causing the false positive result

Positive ResultHigh risk for Trisomy 21

Test performed by a different lab

Case Study 9

13^ weeks gestation

Lower the risk of false positive results determined by maternal CNVs

PrenatalSafe® KaryocfDNA

Array-CGHMaternal blood

Chudova et al., 2016 NEJM

Lower the risk of false positive results determined by maternal CNVs

Strom et al., 2017 NEJM

Lower the risk of false positive results determined by maternal CNVs

42 y.o patient

Abnormal Resultdetected fetal karyotype with Trisomy 9

Amniocentesis

Results confirmed

Case Study 10

14^ weeks gestation

16^ weeks gestation

Trisomy 9 mosaic (20%)

Chr. 9

46,XX[80]/47,XX,+9[20]

Detection of Trisomy 9 mosaic confirmed by traditional karyotyping

PrenatalSafe® Karyo Traditional Karyotyping

40 y.o patient

Abnormal Resultdetected fetal karyotype with Trisomy 22

Amniocentesis

Results confirmed

Case Study 11

10^ weeks gestation

16^ weeks gestation

Trisomy 22 mosaic (16%)

Chr.22

46,XY[42]/47,XY,+22[8]

PrenatalSafe® Karyo Traditional Karyotyping

Detection of Trisomy 22 mosaic confirmed by traditional karyotyping

31 y.o patient

Abnormal Resultdetected fetal karyotype with Trisomy 7

Amniocentesis

Results confirmed

Case Study 12

12^ weeks gestation

16^ weeks gestation

Trisomy 7 mosaic (4%)

Chr. 7

46,XX[48]/47,XX,+7[2]

PrenatalSafe® Karyo Traditional Karyotyping

Detection of Trisomy 7 mosaic confirmed by traditional karyotyping

Prospective study

Fiorentino et al., submitted

Background Conventional cfDNA-based NIPT approaches focus on detection of common

trisomies and sex-chromosome aneuploidies.

This leaves a gap of approximately 17% of clinically relevant chromosomal/subchromosomal abnormalities that would go undetected.

Previous studies have shown the potential of extending conventional NIPT to detect fetal microdeletion syndromes from maternal plasma (Peters et al., 2011; Srinivasan et al., 2013).

Other studies showed how genome-wide cfDNA testing can contribute in lowering the incidence of false positive results generated by maternal copy number variants (Snyder et al., 2015; Chudova et al., 2016).

At present, there are limited data available on the potential of genome-wide screening to detect rare autosomal trisomies and structural chromosome anomalies in a general population of pregnant women.

Aim of the study

From December 2015 through May 2016, genome-wide cell-free fetal DNA(cfDNA) testing was offered to pregnant women undergoing conventionalcfDNA-based non-invasive prenatal testing (NIPT) for common fetalaneuploidy.

We aimed to compare the performance of the two test in a general obstetricalpopulation.

Demographic and pregnancy characteristics

Characteristics n

No. of eligible patients 12.114

Maternal age-yr- Mean ±SD 35.3±4.1

- Min-max 20-58

Gestational age at sample collection -wk- Mean ±SD 12.3±2.1

- Min-max 10-29

Indications for NIPT

- Parental Anxiety 3804 (31.4%)- Advanced maternal agea 4446 (36.7%)- Positive prenatal screen 1199 (9.9%)- Fetal ultrasound abnormality 472 (3.9%)- Prior pregnancy with fetal aneuploidy 157 (1.3%)- More than one indication 2035 (16.8%)

Results of samples tested

Total

No. of patients analyzed 12.114

Samples with a call - no. (%) 12.078 (99.7)

Total cancellations - no. (%) 182 (1.5)

- Samples with low FF - no. (%) 145 (1.2)

- Samples with assay failure - no. (%) 36 (0.3)

Samples with a conclusive result - no. (%) 11.932 (98.5)

Chromosomally abnormal results

- Genome-wide cfDNA screening - no. (%) 196 (1.6)

- Conventional cfDNA screening - no. (%) 166 (1.4)

Pregnancies confirmed as chromosomally abnormal - no. (%)

- Genome-wide cfDNA screening - no. (%) 169 (1.4)

- Conventional cfDNA screening - no. (%) 151 (1.3)

Clinically relevant chromosomal abnormalities classes detected by genome-wide cfDNA analysis

Types of chromosome anomalies detected by conventional and genome-wide cfDNA testing

^ Clinically relevant chromosomal abnormalities, not detected by conventional cfDNA screening, potentially resulting in the birth of babies with chromosomal anomalies, have been considered as false negative.

§ A P-value of less than 0.05 was considered to indicate statistical significance (**).

Performance of conventional cfDNA screening vs. genome-wide analysis

Conventional cfDNA

screening

Genome-wide cfDNAscreening

P-value§

No. of pregnancies assessed 11.932 11.932Clinical relevant chromosomal abnormalities detected - no. (%)

166 196

Pregnancies confirmed as chromosomally abnormal - no. (%)

151 169

False Positive 15 27

False Negative 12* 0

True Positive 151 169

True Negative 11.754 11.736

Sensitivity 92.64% 100.00% <0.001

Specificity 99.87% 99.77% 0.064

Positive Predictive Value (PPV) 90.96% 86.22% 0.161

Negative Predictive Value (NPV) 99.90% 100.00% <0.001

Performance of the Genome-wide cfDNA screening

Conclusion

The clinical utility of expanding NIPT to cover the entire genome iscontroversial, especially in low-risk pregnancies. In fact, it pertains a risk ofoverdiagnosis with a higher number of false positives because ofchromosomal rearrangements which are confined to the placenta, potentiallyleading to an increase in unnecessary invasive testing.

The results of this study demonstrate that a high specificity may bemaintained while extending the screen to all chromosomal abnormalities.

Thank you for your attention

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