non-invasive prenatal testing: increasing detection of...
TRANSCRIPT
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
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
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
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 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