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LifeCodexx AG

PrenaTest
Setting standards in NIPT in Europe

Dr. Wera Hofmann


CSO LifeCodexx AG
Belgrade, 2015-02-27

PrenaTest - The NIPT leader in Europe


Quality Made in Germany
LifeCodexx AG as a German leading company in the field of molecular diagnostics has been setting
standards in Europe with PrenaTest since 2012:
First product to European market in Aug. 2012
First CE-certified test
PrenaTest detects fetal trisomies 21, 18 and 13 with a sensitivity of 99% and a false-positive rate of
0.1% for singleton and multiple pregnancies
PrenaTest successfully validated for gonosomal aneuploidies
Lowest no call rate in industry: 0.6%
Clear result no risk score
First company with guaranteed turnaround time < 1 week (PrenaTest Express)
Over 20.000 samples successfully analyzed since launch
Proprietary QuantYfeX assay for quantification of the fetal DNA amount at the beginning of the
analysis: fast call back if a new blood sample is needed
Positive reimbursement decision in Germany (Erprobungsregelung) following LifeCodexx AG
request, positive decision expected in Switzerland soon

What is NIPT?

Non-invasive prenatal testing (NIPT) - new option in prenatal care

According to International Society for Prenatal Diagnosis (ISPD) NIPT is an advanced screening
method for women at increased risk of common fetal aneuploidies
Numerous publications with first experiences in the clinical application

Scientific Basis of NIPT cell free fetal DNA (cffDNA)

1997: detection of cffDNA fragments in maternal plasma


Detection: starting week 4
Fetal fraction:
2 40 %
Stability:
< 2 hours
Characteristics:
short fragments, 80 % < 200 bp
released from trophoblast cells
(placenta) by apoptosis / necrosis

* Lo et al., Lancet 1997; 350:485-7

Technical Basis of NIPT Next Generation Sequencing (NGS)


Since 2008:
Massively Parallel (Shotgun) Sequencing (MPS) for NIPT
-Very high throughput, short reads (36bp)
-15 million reads / sample
-16 plex; 12( samples per run (HiSeq2500)

Random MPS is the best validated method with the vast majority of NIPT data

ER Mardis. Nature 470, 198-203 (2011) doi:10.1038/nature09796

PraenaTest Process Overview

Regulatory environment for next generation sequencing (NGS)


based aneuploidy tests in Europe
In-Vitro Diagnostics Directive 98/79/EG* of the European
Union, national laws:
The software must be CE marked and monitored by a
Notified Body
The Legal Manufacturer must ensure the
application
of a full quality assurance system
(EN ISO 13485)
PraenaTest is the first NIPT performed in Europe in
accordance with these regulatory requirements.

* List B, Appendix II, IVD Products

Method Overview
1. Blood collection
2. Plasma preparation
3. Extraction of cell free DNA
4. Quantification of fetal DNA
(QuantYfeX)
5. Preparation of a genomic
library
6. next generation
sequencing

Streck cell-free DNA BCT labelled with


sample barcode, patient name and patient birthdate

Method Overview
1. Blood collection
2. Plasma preparation
3. Extraction of cell free DNA
4. Quantification of fetal DNA
(QuantYfeX)
5. Preparation of a genomic
library
6. next generation
sequencing

Minimum of plasma volume isolated: 3,5 ml

Method Overview
1. Blood collection
2. Plasma preparation
3. Extraction of cell free DNA
4. Quantification of fetal DNA
(QuantYfeX)
5. Preparation of a genomic
library
6. next generation
sequencing

11

Method Overview
1. Blood collection
2. Plasma preparation

QuantYfeX
Proprietary qPCR-Assay

3. Extraction of cell free DNA


4. Quantification of fetal DNA
(QuantYfeX)
5. Preparation of a genomic
library

Minimum 4% cffDNA for


singleton and
8% cffDNA for multiple
pregnancies

6. next generation
sequencing
4. Add on: determination of
fetal sex

Method Overview
1. Blood collection
2. Plasma preparation
3. Extraction of cell free DNA
4. Quantification of fetal DNA
(QuantYfeX)
5. Preparation of a genomic
library
6. next generation
sequencing

13

Method Overview
1. Blood collection
2. Plasma preparation

Illumina Sequencing
Technology:
HiSeq2500
Single-reads

3. Extraction of cell free DNA


4. Quantification of fetal DNA
(QuantYfeX)
5. Preparation of a genomic
library

PraenaTest
express

6. next generation
sequencing

Rapid run (7h)


20 samples

High output
run 48h
128 samples

Bioinformatic analysis in the case of a fetal trisomy 21

Method Bioinformatic analysis of the sequencing data

Sequencing and Alignment

Quantification

Alignment of the fragments to


the respective chromo-some by
comparison with a human
reference genome in the public
data base

Counting of the sequences per


chromosome, calculation of
percentage
(e.g. % chromosome 21)

Method Bioinformatic analysis of the sequencing data


Quantification

Examples:
Karyotype

% Chr
13

% Chr
18

% Chr
21

46XX / 46XY*

3.61 %

2.97 %

1.27 %

47XX+13 / 47XY+13

3.65 %

2.97 %

1.27 %

47XX+18 / 47XY+18

3.61 %

3.02 %

1.27 %

47XX+21 / 47XY+21

3.61 %

2.97 %

1.32 %

* Median calculations of chromosomal percentages of more than 400 samples having normal karyotype

Method z-score Calculation

Example for a frequency distribution of z-scores


The z-score represents the distance
between the value measured for a given
sample and the median of the reference set
in terms of the median absolute deviation
(MAD)
z-score of 1 = exactly 1 deviation
z-score of 3 = 3 deviations
z-score calculation:

z-score

3 T21 positive
< 3 T21 negative

PraenaTest result report - negative

PraenaTest result report - positive

PraenaTest Test performance

PrenaTest clinically validated for singleton pregnancies


Two independent studies performed:
European Validation Study (EVS 2012)
Samples from women with high risk pregnancies provided by Sequenom (SCS 2013)
Results published in peer-reviewed journals
Stumm et al. Prenatal Diagnosis 2012
Stumm et al. Prenatal Diagnosis 2014

EVS 2012

SCS 2013

total

Correct
classified
(total)

466/468*

340/340

806/808

Trisomy 13 (n)

5/5

3/3

8/8

Trisomy 18 (n)

8/8

6/6

14/14

Trisomy 21 (n)

40/41

34/34

74/75

Total detection
rate

98.11%

100%

98.97%

False-positive
rate

1/414
0.24%

0/297
0%

1/711
0.14%

Within the EVS, one result was false-negative for trisomy 21 and one result was false-positive for trisomy 18 (a sample with an
aberration of chromosome 10).

as well as for multiple pregnancies


Launch: Feb. 6th, 2014
Generally to be applied after IVF, egg donation and other infertility treatments
New performance qualification of the PrenaTest validated by own R&D study and a joint study with
samples kindly provided by collaboration partner Sequenom
All positive T21 cases correctly classified (1 monochorionic, concordant; 5 dichorionic,
discordant)

Referenz

T21

T18

T13

Sehnert et al. 2011

Leung et al. 2013

Lau et al. 2013

12

Canick et al. 2013

27

Gil et al. 2013

275

9*

1
* one T21 not detected

PraenaTest Validation
Groemminger et al. 2014

62**

** 60 twins and 2 tiplets; 6 T21 cases in twins

Gonosomal aneuplodies recently validated as well


Launch: June 30th, 2014
Offered as extra option to interested pregnant women

PrenaTest is independently confirmed in clinical application

Since PrenaTest introduction:

Generally increased applications of prenatal diagnosis methods (+3,6%) after FTS, primarily in
an intermediate risk (1:300 1:50) cohort (+10,7%)

Decreasing number of invasive procedures by 67,4%

High test accuracy for PrenaTest confirmed in diagnostic routine


(2014-12-31*)

# of successfully reported samples:

17,527 (since launch)

Insuspicious results:

97.8% (n= 17,134)*

Affected cases:
Trisomy 13:
Trisomy 18:
Trisomie 21:

0.12% (n= 19 / 16,310)*


0.37% (n= 61 / 16,310)
1.68% (n= 295 / 17,527)

Turner Syndrome:
Klinefelter Syndrome:
47XYY Syndrome:
TripleX Syndrome:

Twins:

0.24% (n= 5 / 2,105)*


0.24% (n= 5 / 2,105)
0.14% (n= 3 / 2,105)
0.24% (n= 5 / 2,105)
3.75% (n= 396)

Discordant results :
False positive: 1 x T13, 16 x T18, 5 x T21, 1x Klinefelter Syndrome
False negative: 1 x T13, 1x T18, 1x T21
General PrenaTest performance parameter:
Overall false positive rate (FPR): 0.13%
Overall detection rate: 98.7%
Failure rate due to low cffDNA fraction in first blood samples: 1.2%
Overall no call rate: 0.6%

Patient profile Age of women


More than 45% of women are older than 35 years

Patient profile In which week of pregnancy is the PrenaTest done?


Possible from gestational week 9+0 onwards; used only by 2%
The highest peak at week 13 corresponds to the time of the nuchal translucency measurements

Patient profile Indications for the application of NIPT


Increased maternal age
Increased risk for aneuploidy based on first trimester screening
The third important reason for doing NIPT is listed as other medical reasons, mainly representing
women with anxiety and a strong psychological need for assurance, even in the absence of
indicators for a fetal trisomy.

PraenaTest New insights

Limitations of NIPT - Discordant results between NIPT and IPT


Test material originates from trophoblast cells, thus fetoplacental discrepancies have to be taken into account
No conclusions can be drawn regarding structural chromosomal aberrations
Mosaics are not recognizable or cannot fully be ascertained quantitatively

depends on degree of mosaicism and amount of cffDNA

Consider confined placental mosaics

NIPT is much more sensitive in detecting CPM then CVS. by Rava et al. 2013
More precisely: investigate cell-free placental DNA

Vanishing twins

False negative results

! 100 % can never be achieved in clinical practice


! All positive cases must be confirmed by karyotyping
False positive results

PrenaTest - recent successful case studies


Case report 1
Turkey: Fetus payraceus
Singleton pregnancy; gestational week17+2
positive z-score of chr. 21: 13.5
cffDNA 20.7% (QFX) and 9.2 (rMPS/Y reads)

Invasive prenatal testing:


AC: 46,XY

Confirmation: started as a multiple pregnancy (ICSI); deceased fet still recognizable


Before birth: New blood test (week 38 +2) with a negative z-score of chr 21: -0.3
After birth:
Cultered cells of placental tissue of the fetus payraceus
karyotype 47,XX,+21
Grmminger et al. J. Clin. Med. 2014

in week 17 +2 (US examination)


cffDNA fraction 21.7%

Case report 2
Hungary: Vanishing Twin
Singleton pregnancy
Borderline z-score of chr. 21: 3.4
cffDNA fraction 13.44%

Gender not clearly determined using QFX; 3% based on Y reads for chr. Y
Interpretation: consider possibility of a vanishing twin with trisomy 21

Confirmation: started as a multiple pregnancy (using ART); Sex of the living fetus is female
Grmminger et al. J. Clin. Med. 2014

Correlation between z-score chr. 21 and cffDNA

Case report 3
Germany: Confined Placental Mosaicism (CPM)
positive z-score of chr. 18: 4.6
cffDNA fraction 10.1%

Invasive prenatal testing:


AC: 46,XY
Placenta tissue/FISH: 80% T18 mosaicism

Frequency of CPM about 1-2%; Artan et al. [1995] reported 4.8%

Case report 4
Germany: T21 mosaicism
Examined as part of R&D
Karyotyp using CVS:
47,XX,+21[10]/46,XX [ 5]
Confirmed:
positive z-score of chr. 21 is 7.4
cffDNA 16.68%

In clinical study:
T21 mosaicism 47,XY,+21[6]/46,XY[34]
z-score: 0.5
cffDNA 7%

Case report 5
Germany: T18 deletion
z-score of chr. 18: -6.3
inconspicuous for trisomy 18
Indication for (partial) deletion
of chr.18

Invasive prenatal testing:


Prenatal quicktest with markers for chr. 13, 18, 21, and sex chromosomes: diploid signal constellation
Array-CGH: 18q22.1q23(63,909,745-78,010,032)x1 dn; 14 Mb-deletion with at least 12 genes involved
Termination of pregnancy

Case report 6
Germany: Triple X
z-scores of chr. 13/18/21 < -3

z-score for chr. X: 8.49

Additional findings: additional chr X; fetal Klinefelter-Syndrom/maternales Triple X


Confirmation: maternal TripleX
When prevalence of 1:1000 of Triple X = clear limitation in the detection of fetal gonosomal aneuploidies

New! Effects of Low Molecular Weight Heparin medication in


pregnant women opting for PraenaTest
Low molecular weight heparin (LMWH) is often used as
prophylaxis against venous thrombosis during such
pregnancies.
In pregnant women on LMWH medication the cell-free
plasma DNA contains, in comparison to an average
plasma sample, a higher proportion of small DNA
fragments with an unusually high Guanosin-Cytosin
(GC)-content.
This biases NIPT-results so that they cannot be
interpreted correctly not reported by anybody
before!
The disturbing effect of LMWH can be cleared away by
taking the blood sample for NIPT right before the next
application of LWMW when the plasma level is lowest.
manuscript submitted in NEJM

39

Summary - NIPT - PrenaTest


NIPT is a new, but rapidly evolving field in the area of prenatal care
PraenaTest is increasingly established in prenatal diagnosis with very high test accuracy and very low
failure rate
The first NIPT for fetal trisomies 21, 18 and 13 available in Europe, which is in accordance with the In-Vitro
Diagnostics Directive 98/79/EG of the European Union
No stand alone test but complements present non-invasive prenatal methods
NIPT is capable of detecting common fetal aneuploidies with almost similar accuracy as chorion villus
sampling (CVS) and amniocentesis (AC), but without any risk for miscarriage
The number of invasive tests can be reduced substantially, procedure-related fetal losses can be avoided
Limits of NIPT method are better described; further clarification is necessary in a strong collaboration
between gynecologists specialized in ultrasonography and geneticists
Considering additional findings as incidental findings; high importance of genetic counseling

Thank you very much.

Backup

QuantYfeX Quality Control for PraenaTest


Specific regions of the human genome are modified by methylation
and differ in their pattern from mother to child (Differential methylation).
the DNA sequence is identical,
but: maternal DNA is not methylated,
fetal DNA is methylated.

Specific enzymes digest only non-methylated DNA methylated DNA remains intact.

specific, hypomethylated DNA region of maternal origin


specific, hypermethylated DNA region of fetal origin (target)
specific reference DNA region of both fetal and maternal origin
reference-specific VIC-labelled flourescent probe
target-specific FAM-labelled flourescent probe
Grmminger et al. J. Clin. Med. 2014, 3(3), 679-692;

http://www.mdpi.com/2077-0383/3/3/679

based on qPCR platform


Quantitative PCR of two regions in the genome:

a)One region which is not digested by the used enzyme,


b)One region which is digested depending on the methylation.
PCR signal for a) represents the mixture of maternal and fetal DNA (100%)
PCR signal for b) represents only the fetal proportion (x %)

a)

b)

LightCycler 480 System

QuantYfeX principle of cffDNA assay


Assay Design multiplex fluorescent probe quantitative real-time PCR assay
Insensitive marker (total DNA)
fetal DNA
maternal DNA
no restriction sites

sensitive marker

(fetal DNA)
fetal DNA
maternal DNA
methylation sensitive restriction sites

QuantYfeX assay principle for gender determination (1/2)


Detection of Y-chromosomal region
Y-chromosomal sequences origination from male fetuses are detectable.

DNA-sample of a pregnancy with a male fetus

DNA-sample of a pregnancy with a female fetus

autosomal und gonosomal DNA of maternal and fetal origin


Y-chromosomal DNA of fetal origin

QuantYfeX assay principle for gender determination (2/2)


Gender determination

Gender determination

Target gene: SRY

(detection of male specific DNA sequences)

male fetal DNA


No respective
maternal DNA region existing

QuantYfeX principle of cffDNA assay


Assay Design multiplex fluorescent probe quantitative real-time PCR assay
Insensitive marker (total DNA)
fetal DNA
maternal DNA
no restriction sites

sensitive marker

(fetal DNA)
fetal DNA
maternal DNA
methylation sensitive restriction sites

QuantYfeX assay principle for gender determination (1/2)


Detection of Y-chromosomal region
Y-chromosomal sequences origination from male fetuses are detectable.

DNA-sample of a pregnancy with a male fetus

DNA-sample of a pregnancy with a female fetus

autosomal und gonosomal DNA of maternal and fetal origin


Y-chromosomal DNA of fetal origin

QuantYfeX assay principle for gender determination (2/2)


Gender determination

Gender determination

Target gene: SRY

(detection of male specific DNA sequences)

male fetal DNA


No respective
maternal DNA region existing

Method Library Preparation/Pooling


automated protocol:
32-48 samples/batch
vs. manual protocol:
8-12 samples/batch

https://www.neb.com/products/e6040-nebnext-dna-library-prep-master-mix-set-for-illumina