Non-Invasive Prenatal Testing: Increasing Detection of Down’s

Syndrome to >99%

Dr Shian Miller Obstetrician & Gynaecologist

BSc (Hon), MBBS, FRANZCOG

Prenatal screening �  Prenatal screening & diagnosis aims to detect

conditions before a fetus is born � Can be non-invasive: maternal serum

screening, ultrasound � Or can be invasive: amniocentesis, CVS,

fetal blood sampling � Genetic testing of fetuses previously

required invasive techniques – now Non-Invasive Prenatal Testing (NIPT) can indirectly sample fetal DNA from maternal blood

Overview

� Biology of NIPT – cfDNA � Clinical applications of NIPT � Use of NIPT for aneuploidy screening � Discordant results � NIPT in practice � Currently available NIPT options � The future of NIPT

Cell-free DNA in maternal blood �  In 1997, Lo et al published the discovery of

circulating cell-free fetal DNA in the maternal serum

�  Presence of fetal DNA in maternal plasma and serum. YM Dennis Lo et al. The Lancet 1997; 350: 485-87

�  Fragments of DNA are released from placental cells through apopotosis into the maternal circulation

�  Generally, cell-free DNA (cfDNA) in maternal blood is 90% from maternal bone marrow and 10% from placenta

�  Placental cfDNA is detectable before placental circulation is developed – present in useful amounts from 8-9 weeks gestation

Placental cell-free DNA

�  Increased levels with abnormal placentation – placenta accreta, pre-eclampsia – and Trisomy 21 (more apoptosis)

� Decreased levels in obese women – thought to be due to apoptosis of adipose tissue increasing maternal fraction

Clearance of placental cfDNA

� As the DNA fragments are not contained within a cell, they are unstable and have a short half-life of 4-30 minutes

� Placental cfDNA is undetectable in maternal serum within hours of delivery

� Thus, placental cfDNA detected during a pregnancy is considered to be representative of the current fetus

Non-Invasive Prenatal Test (NIPT)

� NIPT involves collecting maternal blood and analysing it for placental cfDNA

� DNA fragments need to be multiplied millions of times in a short period of time

� Although cfDNA discovered in 1997, not until the technology became commercially available in 2005, could NIPT be realised

� The technology was ‘Massively Parallel Sequencing’ or Next Generation Sequencing – complex molecular biology

Overview of NIPT process

Sequencing cfDNA �  The simplified explanation is: �  The cfDNA is multiplied millions of times

(both maternal and placental/fetal) �  The relative quantity of each chromosome is

compared to a reference genome �  Then look for over-representation of a

chromosome �  Eg. Excess chromosome 21 compared to

other chromosomes suggest Down’s syndrome

Excess Chromosome

Not just chromosomes � Using targeted sequencing, can also detect

single gene mutations and microdeletions

Clinical applications of NIPT � Aneuploidy, typically Trisomy 21 (Down’s),

Trisomy 18 (Edward’s), Trisomy 13 (Patau’s) and XY abnormalities

� Main reason for screening these trisomies is that, unlike other trisomies, these often progress to a live birth

� Gender determination, particularly for history of X-linked disorders

�  Single gene disorders – cystic fibrosis, myotonic dystrophy

�  Fetal RhD typing to direct management of pregnant women with Anti-D antibodies

Use of NIPT in aneuploidy screening

�  Since introduction in Australia in late 2012, more than 2000 women have used NIPT for aneuploidy screening

� Generally targeted to high-risk women but appears to be as effective in a low-risk population

� Currently, the most appropriate implementation strategy for NIPT in the Australian context is still to be determined

Current prenatal aneuploidy screening

�  In Australia, high uptake of combined first trimester screening (CFTS)

�  Involves a blood test for bHCG and PAPP-A � Also an ultrasound scan at 11-13+6 weeks � Apart from aneuploidy, screening also

beneficial for: ◦  Correct dating of pregnancy ◦  Anatomy assessment ◦  Increased NT is a marker for other abnormalities ◦  Biochemistry also predicts pre-eclampsia, IUGR ◦  Information about multiple pregnancies

NIPT compared to CFTS

CFTS NIPT Sensitivity 90% >99% Specificity 95% >99% False positive

3-5% <0.5%

False negative

10% <1%

PPV 4% 45%

NIPT advantages � High negative predictive value – reduces

need for invasive testing � High detection rate of >99% for Trisomy

21 and >90% for Trisomy 18 & 13 � Only a maternal blood test � No risk to the fetus � Gender determination � Available from 10 weeks gestation

onwards – useful for those who have missed CFTS

NIPT disadvantages � Anatomical abnormalities not detected –

cfDNA even with anencephaly �  Still considered a screening test – if a

result is ‘positive’, still need invasive testing (CVS/amniocentesis) to confirm

� Does not target atypical chromosome abnormalities – account for 30% of abnormal karyotypes

�  Some types of NIPT not suitable for twins � Cost – no Medicare rebate

Discordant results �  Low false positive rate of 0.2% � Causes include: ◦  Placental mosaicism ◦  Co-twin demise ◦ Organ transplant eg kidney transplant; male donor ◦  Bone marrow transplant ◦  False negative 45XO – cryptic mosaic – fetus XO,

placenta XX ◦ Maternal chromosomal abnormality ◦  Increasing maternal age – white cells can ‘lose an X’

and become ‘XO’ – somatic mosaicism ◦  Laboratory error ◦ Occult cancer – sheds chromosomes chaotically

Multiple aneuploidy in occult cancer

No result from NIPT

� Test failure rate <5% � Obese women have a significantly

reduced fetal fraction of cfDNA �  For a maternal weight of 160kg, 50% of

samples will have fetal fraction <4% �  If sample taken too early in the pregnancy,

may have false negative results for Y chromosome or Rhesus status

NIPT for Twin pregnancies

� Currently there is limited experience with performance of NIPT for twins

� Results to date are promising � Lower sensitivity for detecting aneuploidy �  Fetal fraction is not double that of a

singleton but less than that – may affect test performance

NIPT in practice �  NIPT can be performed from 10 weeks gestation �  Currently no controls or guidelines for use �  Women have taken it up in an ad hoc way �  As it involves only a maternal blood test and poses

no risk to the fetus, very attractive to mothers �  Previously prohibitively expensive but now easily

obtained at under $500 (becoming comparable to CFTS ~$300)

�  Ease of access may mean implications of test results may not be considered adequately

�  Risk of commercialisation – marketing direct to consumer

Models for the use of NIPT in aneuploidy screening

1.  CFTS – if high risk >1:300, offer NIPT or invasive testing

2.  CFTS – tiered management (contingent) ◦  Very low risk <1:1000 – no further testing ◦  Low risk 1:1000 to 1:300 – offered no

further testing or NIPT ◦  High risk >1:300 – offered NIPT or invasive

testing ◦  Very high risk >1:10 – invasive testing

Other models for using NIPT

3.  Routine NIPT at 10 weeks gestation then ultrasound at 12 weeks (for dating, anatomy assessment) – if no result, recommend CFTS

4.  Routine NIPT, ultrasound, and biochemistry

5.  NIPT only for high risk women: maternal age >35, previous history of aneuploidy, known parental balanced translocation

Ethical considerations with NIPT � Gender determination used for gender

selection � Use in paternity testing � Discovery of previously unknown maternal

genetic disorder, eg Turner mosaicism �  Screening non-specifically for genetic

alterations may lead to findings of uncertain significance

�  Termination decision based on NIPT result only (trend in the USA)

Current availability of NIPT in Australia

�  Previously required shipping samples overseas in a Streck tube (contains a preservative that allows DNA to remain stable even in ambient temperatures)

� Now, laboratories available in Australia �  Turnaround time for overseas labs 7-10 days

– Australian labs 3-4 days � Available at major pathology labs, obstetric

ultrasound places, fertility specialists and private obstetricians

� Different labs & technology but very similar NPV and sensitivity and specificity

GPs will be the front-line �  Increasing public awareness of NIPT � Consultations regarding NIPT are likely to

be initiated by pregnant women themselves

� GPs will be the first point of contact � GPs should be able to provide counseling

and guidance to inform appropriate decision making

� Likely, GPs will order NIPT for patients before contact with obstetrician/hospital

CASE ONE

� 19yo G1P0 at 10 weeks gestation � Planned pregnancy; excited � No significant history

CASE TWO

� 40yo G1P0 at 10 weeks gestation � Planned pregnancy; excited �  Fit and well, no significant history

CASE THREE

� 30yo G3P2 at 10 weeks gestation �  First child had Down’s syndrome � Parental karyotypes normal � No other history of note

CASE FOUR

� 28yo G1P0 at 10 weeks gestation � Known maternal balanced translocation

of chromosome 21 � No other history of note

The future of NIPT �  Still some doubt about the place of NIPT

in prenatal screening in Australia � The USA has taken it up enthusiastically –

uptake of CFTS has always been poorer � Prices will continue to fall � Professional organisations are under

pressure to establish guidelines for use � NIPT is very likely to take a central role

in the future of Australian prenatal screening