1. Non-Invasive Prenatal Testing (NIPT)
Developed by Dr. Judith Allanson, Ms. Shawna Morrison and Dr. June Carroll
Last updated November 2015

2. Disclaimer
This presentation is for educational purposes only and should not be used as a substitute for clinical judgement. GEC-KO aims to aid the practicing clinician by providing informed opinions regarding genetic services that have been developed in a rigorous and evidence-based manner. Physicians must use their own clinical judgement in addition to published articles and the information presented herein. GEC-KO assumes no responsibility or liability resulting from the use of information contained herein.

3. Objectives Following this session the learner will be able to:
Refer to their local genetics centre and/or order genetic testing appropriately regarding non-invasive prenatal testing (NIPT)
Discuss and address patient concerns regarding NIPT
Find high quality genomics educational resources appropriate for primary care

4. Typical Prenatal Testing Algorithm
Offer PN screening to all pregnant women
FTS/IPS/SIPS
NIPT for AMA and for women willing to pay
Family history
Ethnicity-based screening
AMA – Advanced Maternal Age,
If negative or decline
If positive*
*for ethnicity-based screening, if both members of the couple are carriers of the same condition
SOGC Nov 2011 age 40 = AMA
Family Hx Q: hx of genetic conditions, ID/DD, congenital anomalies, infertility, consanguinity
18-20 week fetal morphology scan
Refer to Genetics
If indicated (e.g. fetal anomalies )

5. Prenatal Testing Algorithm for Women at Increased Risk
Indication
Advanced maternal age, multiple soft markers on ultrasound, ultrasound anomaly, positive prenatal screen, etc.
Genetic counselling with testing options
No further testing
Invasive Testing
(diagnostic)
Screening Test
e.g. NIPT
If positive
If negative
QF-PCR
Detects common aneuploidies: Down syndrome, Trisomy 18, Trisomy 13 and sex chromosome aneuploidies
Depending on indication:
No further testing
Consider additional testing
Recommend that all women be offered prenatal screening
CCMG/SOGC Guidelines Jan 2011 Use of a DNA Method, QF-PCR, in the Prenatal Diagnosis of Fetal Aneuploidies –
SEE PRENATAL CHROMOSOMAL MICROARRAY EDUCATION MODULE FOR MORE
2.4% CMA will pick up abnormality for any indication
6.4% if u/s abnormality
~1% VUS
If positive
Karyotype
If negative
No further testing
Additional Testing
e.g. Chromosomal microarray

6. Case 1
A 32 year old woman has had a positive Integrated Prenatal Screening Test (IPS) result for Down syndrome. She is about 17 weeks gestation.
Is NIPT a good option?

7. Case 2
A 40 year old G1 woman is about 9 weeks gestation. She is in your office to discuss prenatal testing options in this pregnancy conceived by IVF.
Is NIPT a good option?

8. Case 3
A 29 year old patient had a nuchal translucency (NT) of 4.4mm at 12+5 weeks gestation
You offered NIPT and she accepted
NIPT results were normal. She is now 14+2 weeks gestation
What are the next steps?

9. What is Non-Invasive Prenatal Testing?
Screening test to prenatally detect Down syndrome and other aneuploidies (extra or missing chromosomes)
trisomy 21, 18, 13
trisomy of sex chromosomes (XXX, XXY, XYY)
Turner syndrome (monosomy X)
triploidy (extra copy of all chromosomes)
Aneuploidy: refers to an abnormal chromosome number (extra or missing)

10. What is Non-Invasive Prenatal Testing?
NIPT measures circulating cell-free DNA (cfDNA) from placenta present in maternal blood
‘fetal’ cfDNA comprises ~3-10% of DNA in maternal blood
Increases with gestational age
Companies offering NIPT use various technologies to analyze cfDNA and determine chromosome quantities
Performed on maternal blood sample
As early as 9 weeks gestation (company specific)
Prior U/S preferable– viability, accurate GA, exclude multiples
Technologies:
detect higher relative amounts of DNA from an aneuploidy fetus by comparing quantity to a reference chromosome, determining if there is a normal, higher or lower than expected quantity of particular DNA sequences
amplify and sequence chromosomes of interest using single-nucleotide polymorphisms (SNPs)

11. How does non-invasive prenatal testing compare to traditional prenatal screening for Down syndrome?
Screening test
Test info
Detection rate / Sensitivity for T211,4
False Positive Rate for T211,4
Positive predictive value for T212,3
FTS
MA, NT, PAPP-A, beta-hCG
80-85%
3-9%
~4%
IPS
MA, NT, PAPP-A, AFP, uE3, hCG
85-90%
2-4%
Quad/MSS
MA, AFP, uE3, total hCG, inhibin
75-85%
5-10%
SIPS
MA, PAPP-A, AFP, beta-hCG/total hCG, uE3, inhibin
80-90%
2-7%
NIPT
+/-MA, cfDNA
>99%
<0.1%
~80.9% for all populations (high and low risk women)
Norton 2015 – PPV for NIPT for T21 was 50% in low risk women and 76.0% in women <35y
Recommendations: any prenatal screen offered to women who present in T1 should have DR of 75% and no more than 3% FPR, if presenting in T2 DR of 75% with no more than 5% FPR
Open Neural Tube Defect (ONTD): Maternal Serum – alphafetoprotein (MSAFP) in T2 + comprehensive U/S screen at weeks
U/S for delayed ossification of fetal nasal bone – T1 (11-14 weeks) or T2
T1 DR 69% FPR depending on maternal ethnicity (9% Afro-Caribbeans, 5% Asians, 2.2% Caucasian)
1 Prenatal Screening Ontario
2 Bianchi et al 2014 N Engl J Med 370:9
3 Norton et al 2015 N Engl J Med 372:17
4 Gil et al 2015 Ultrasound Obstet Gynecol 45

12. How does non-invasive prenatal testing perform for other common aneuploidies?
Aneuploidy
Detection Rate
False Positive Rate
Trisomy 18
96.3%
0.13%
Trisomy 13
91.0%
Monosomy X (Turner syndrome)
90.3%
0.14%
Other sex chromosome aneuploidies
93.0%
Comment on PPV
Gil et al 2015 Ultrasound Obstet Gynecol 45

13. Non-Invasive Prenatal Testing (NIPT) Landscape
Offered at genetics centres all over Canada
To all vs. high risk varies
Increasing demand and uptake by women
3 separate companies, 3 separate technologies (available in Canada)
Costs about 500$
8-10 days for result

14. Recommendations Offer to all women:
Conventional prenatal screening using either FTS, IPS or MSS (SIPS or Quad)
Fetal morphology scan at about weeks gestation
Consider NIPT as an option for women who have a high risk for having a baby with an aneuploidy
ACOG 2015 Given the performance of conventional screening methods and the limitations of cell-free DNA, conventional screening methods remain the most appropriate choice for first-line screening for most women in the general obstetric population. Traditional serum analyte screening methods allow for higher detection rates of these other chromosome abnormalities as well as the risk of other adverse pregnancy outcomes. For example, a positive integrated screening test result may indirectly identify a fetus with an unbalanced rearrangement of a chromosome other than trisomies 13, 18, or 21. One study of women with abnormal traditional screening test results who had diagnostic testing estimated that up to 17% of clinically significant chromosomal abnormalities would not be detectable with most of the current cell-free DNA techniques.
Offer to everyone?
Society of Obstetricians and Gynaecologists of Canada, American College of Obstetricians and Gynecologists, International Society for Prenatal Diagnosis, National Society of Genetic Counselors, Society for Maternal-Fetal Medicine

15. Women who have a high risk for having a baby with an aneuploidy
Are of advanced maternal age, defined as 40 years of age or older at estimated date of birth
Have an abnormal multiple marker screen i.e. FTS/IPS/MSS
Have had a previous pregnancy or child with aneuploidy

16. Women who have a high risk for having a baby with an aneuploidy
In consultation with genetics or maternal-fetal-medicine, consider NIPT for women where:
Fetal nuchal translucency (NT) measures 3.5mm or greater
Fetal congenital anomalies on ultrasound are highly suggestive of trisomy 13, 18 or 21
Soft markers on ultrasound are highly suggestive of aneuploidy [Refer to SOGC guidelines, 2005].
There is a risk of carrying a male fetus with an X-linked condition (NIPT would be used for sex determination)

17. Non-Invasive Prenatal Testing (NIPT) results
Results will be reported in various ways and may be worded as:
positive or negative
aneuploidy detected, no aneuploidy detected or aneuploidy suspected/borderline value
high risk or low risk
Labs that report the chance of aneuploidy commonly use “>99%” as indicative of high risk and “<1/10,000” as indicative of low risk, intermediate results are also occasionally reported

18. Non-Invasive Prenatal Testing (NIPT) results
Negative result: Low risk
Your patient should still be offered:
Fetal morphology scan at weeks’ gestation
Referral for genetic and/or maternal fetal medicine consultation, which may be indicated for additional counselling and testing, depending on the reason your patient qualified for NIPT (e.g. increased NT)
As per SOGC guidelines, MS-AFP should only be offered to pregnant women with a pre-pregnant body mass index ≥ 35 kg/m2 or when geographical or clinical access factors limit timely and good quality ultrasound screening

19. Non-Invasive Prenatal Testing (NIPT) results
Positive result: High risk
Genetic counselling
Confirmation by diagnostic testing
Consider the positive predictive value (PPV)
No irrevocable obstetrical decisions should be made in pregnancies based on abnormal NIPT results alone without confirmatory invasive testing (CVS or amniocentesis) - SOGC
Reasons for false positive: placental mosaicism, a vanishing twin or a maternal tumor

20. NIPT and PPV

21. NIPT and PPV www.perinatalquality.org/Vendors/NSGC/NIPT/
But even for those at a very-high a priori risk of 1:5, PPV does not exceed 99% (Dondorp et al 2015 EJHG)

22. Confined Placental Mosaicism
Possible explanation for a false positive result
Present in 1-2% of first trimester placentas
Important concept
Trisomy 13 and 18  lower placenta volume
Rava RP et al. (2014). Clinical Chemistry, 60,
Wegrzyn et al. (2005). Ultrasound in Obstetrics & Gynecology, 26,

23. Benefits of Non-Invasive Prenatal Testing (NIPT)
Fewer women having diagnostic tests with associated risk of pregnancy loss
Increased access
Early test result (drawn at ≥ 9-10 weeks at earliest)
No risk of miscarriage
Detects the most common chromosomal aneuploidies
Higher detection rates and lower false positive rates than IPS or MSS
Negative predictive values are greater than 99% for all patient populations who receive a test result

24. Limitations of Non-Invasive Prenatal Testing (NIPT)
NIPT cannot:
Detect chromosome differences other than aneuploidy of chromosomes 13, 18, 21, X and Y
some companies are now adding screening for other trisomies and certain microdeletion syndromes
Completely rule out aneuploidy
Detect single gene conditions
Detect congenital anomalies
Possibility of no result (~6%)
1/2- 2/3 can be successfully resolved with redraw at later gestation
False positives and false negatives
Twins and IVF pregnancies
No result: . According to recent review (Cuckle, 2015) There are three circumstances when no result can happen: (1) a poor sample or quality assessment failure; (2) a low FF; or (3) an ‘uninterpretable’ result, regarded by the laboratory as too close to the cut-off 
? About reason for FPR can be addressed in Q&A – low ff, confined placental mosaicism, maternal aneuploidy, vanishing twin
Twins- lower sensitivity in discordant twins – lower ff per twin
ISPD (f) Multiple gestational pregnancies: Testing has also been extended to twin pregnancies. Provided the cfDNA test is interpretable, performance in twins concordant for aneuploidy is expected to be equivalent to that for singletons. A meta-analysis of published studies found sensitivity in discordant twins was similar to that found for singletons.31 The specificity for all twins was also similar to singletons. However, when fetal fraction is measured for each fetus and the lowest value used to decide on interpretability, the failure rate will be higher than in singletons.
ACOG 2015: Regardless of the method, the accuracy of screening for aneuploidy is limited in multiple gestations. With any method based on maternal blood (serum analytes or DNA), only a single composite result for the entire gestation is provided, with no ability to distinguish a differential risk between fetuses. The data regarding the performance of cell-free DNA screening in twin gestations are limited. Although preliminary findings suggest that this screening is accurate, larger prospective studies and published data are needed before this method can be recommended for multiple gestations. Cell-free DNA screening is not recommended for women with multiple gestations. There are no available data on higher-order multiples.
IVF – limited data, may have higher fail rate
Lambert-Messerlian et al., (2014) have compared the performance of NIPT for autosomal aneuploidy in pregnancies achieved by assisted reproductive technologies (ART) versus controls. There was no significant difference in cell-free DNA fetal fraction between the two groups. However, they did find that z-scores for trisomy 21 and trisomy 18 were lower in the ART group. They conclude that the findings need to be confirmed before any adjustment is made to testing and reporting protocols. It is also possible that ART pregnancies are more likely to include a vanishing twin and residual placental tissue. If these findings are confirmed, it may be useful to make minor adjustments to the z-scores or their interpretation in pregnancies conceived via ART.

25. Non-Invasive Prenatal Testing (NIPT) and counselling
Pre- and post-test counselling is important
Invasive testing following positive results
Conditions tested for in addition to T21
Incidental findings
Consult genetics if unsure
has more information, links to companies and sample completed forms
Refer for genetic counselling when appropriate
Incidentals e.g. maternal aneuploidy, maternal malignancy
Other companies offering NIPT:
Sequenom
Counsyl
Nukleo in Quebec

26. Ordering Non-Invasive Prenatal Testing (NIPT)
Educational Resources > GECKO on the run > NIPT
Harmony Prenatal Test™ by Ariosa Diagnostics through Dynacare
Panorama™ by Natera through Lifelabs Genetics
Verifi® Prenatal Test by Verinata through Mount Sinai Services Inc. and Medcan Clinic in Toronto

27. Expansion of non-invasive prenatal testing
In October 2013 one company expanded their NIPT test menu to include screening for microdeletion syndromes (e.g. 22q deletion/DiGeorge) and trisomies associated with early pregnancy loss (e.g. trisomy 16) and in spring 2014 others followed suit
Incidence = 1 in 4,000 to 1 in 50,000
ACOG 2015 Routine cell-free DNA screening for microdeletion syndromes should not be performed.
Microdeletion syndromes occur sporadically or are due to other genetic mechanisms.
Screening for these microdeletions has not been validated in clinical studies, and the true sensitivity and specificity of this screening test is uncertain.
ISPD
about the prenatal incidence of many of these conditions – impacts PPV
For many disorders there are other/alternative molecular mechanisms (e.g. imprinting defect, nested deletions) and not all cases are detectable and this information should be included in estimated detection rates
22q.org – Teddy Bohan, 3y

28. Expansion of non-invasive prenatal testing
Arguments for NIPT with microdeletions now
Clinically relevant microdeletions and duplications occur in 1-1.7% of all structurally normal pregnancies
Incidence is independent of maternal age
Cumulatively these disorders are common
The combined at-birth incidence of the 5 commonly offered microdeletion syndromes is approximately 1 in 1,000
Less women are expected to have invasive procedures and be offered microarray, so these conditions should be added to NIPT
. 30yo woman, wants every test available, willing to pay - NIPT in general obstetrical population looks like it performs well and better than conventional screening - Professional societies do not recommend this for the general obstestrical population, but may be reasonable.  Addiing microdeletions will increase the FPR and decrease the PPV and so increasing the likelihood of onvasive procedure yo woman, first trimester ultrasound shows NT4.5mm, declines all testing, echo at 20W u/s shows tetralogy of fallot % associated with 22qdel, declines invasive testing but would like NIPT, maybe reasonable in consultation with genetics

29. Microdeletion syndrome
Incidence (at birth)
Clinical features
Babies born with this syndrome often have:
22q11.2 deletion syndrome/ DiGeorge
1 in 4,000-2,000
heart defects, immune system problems, and mild-to moderate intellectual disability. They may also have kidney problems, feeding problems, and/or seizures.
1p36 deletion syndrome
1 in 10,000-5,000
weak muscle tone, heart and other birth defects, intellectual disabilities, and behavior problems. About half will have seizures.
Angelman syndrome
1 in 12,000
delayed milestones (like sitting, crawling and walking), seizures, and problems with balance and walking. They also have severe intellectual disability and most do not develop speech.
Prader-Willi syndrome
1 in 25, ,000
low muscle tone and problems with feeding and gaining weight. They also have intellectual disability. As children and adults, they have rapid weight gain and often develop obesity related medical problems.
Cri-du-chat syndrome (5p-)
1 in 50, ,000
low birth weight, small head size, and decreased muscle tone. Feeding and breathing difficulties are also common. They have moderate-to-severe intellectual disability.
Cumulative incidence of 1 in 1000
22q - 85% of ind. will have typical full deletion, 15% will have ‘nested’ deletions within the region
PWS - Approximately 70% of individuals with PWS have a deletion on one number 15 chromosome involving bands 15q11.2-q13 (paternal del)
Angelman – about 65-70% caused by 5- to 7-Mb deletions in 15q11.2-q13( maternal)

30. Expansion of non-invasive prenatal testing
Arguments against NIPT with microdeletions now
There are no published clinical validation studies, only proof of concept
There are no guidelines – not considered standard of care
Because these are low-prevalence disorders, false positive results are inevitable and the positive predictive value will be low
Undermine benefit of NIPT reducing the number of women undergoing invasive testing
Uncertainty about the prenatal prevalence
Informed consent
Familiarity with rare conditions
Familiarity with testing limitations
E.g. 22q - 85% of individuals with 22qdeletion will have typical full deletion screened for by NIPT while 15% will have ‘nested’ deletions within the region; ~ 65-70% of Angelman syndrome is caused by (maternal) deletions at 15q11.2 screened for by NIPT while others will have various other genetic mechanisms
Provinces are not currently funding
It is recognized that validating test detection rates and false-positive rates for additional rare disorders is problematic because alternative comparative screening methods do not exist and test samples from affected pregnancies may be unavailable (ISPD)
. 30yo woman, wants every test available, willing to pay - NIPT in general obstetrical population looks like it performs well and better than conventional screening - Professional societies do not recommend this for the general obstestrical population, but may be reasonable.  Addiing microdeletions will increase the FPR and decrease the PPV and so increasing the likelihood of onvasive procedure yo woman, first trimester ultrasound shows NT4.5mm, declines all testing, echo at 20W u/s shows tetralogy of fallot % associated with 22qdel, declines invasive testing but would like NIPT, maybe reasonable in consultation with genetics

31. How does NIPT perform with microdeletions?
If the original purpose of NIPT was to decrease the number of women having invasive testing, adding relatively rare disorders and consequently increasing the false positive rate – more women could end up having invasive procedures unnecessarily

32. Back to case 1: IPS positive, 17W GA
Is NIPT a good option?
Yes
She is at high risk to have a baby with aneuploidy
May be eligible for provincial funding where applicable
But consider:
In the event of an abnormal result, is termination of pregnancy an option for the couple?
More rapid result from amniocentesis, consider GA
If NIPT is positive, guidelines recommend confirmatory diagnostic testing by amniocentesis – delays timing for diagnosis
What is her IPS risk?
1 in 2 versus 1 in 120

33. Back to case 2: 40yo G1 Yes Is NIPT a good option?
Advanced maternal age (greater than 40 years at EDB) is an appropriate indication for NIPT
Covered by some provincial health plans
Better screen than IPS
Earlier result
Decreased chance with NIPT that patient would receive screen positive result 9W
IVF

34. Back to case 3: NT 4.4mm, low risk NIPT
What are the next steps?
Genetic counselling is recommended
Patient likely to be offered:
Chromosomal microarray
Genetic testing for other single gene conditions
Level II ultrasound
Fetal echocardiogram

35. Non-Invasive Prenatal Testing Pearls
Consider offering NIPT to high risk women
Consider NIPT as a screen of higher sensitivity than current screening if your patient is willing to pay for the test
Not a diagnostic test, diagnostic testing should follow a positive result
Not an all purpose genetic test, only gives info on specific chromosomes, and so not indicated in all circumstances

36. Resources
See for more details and how to connect to your local genetics centre and the GECKO on the run resource for more information
For a recent review on NIPT see Cuckle H, Benn P, Pergament E. Cell-free DNA screening for fetal aneuploidy as a clinical service. Clin Biochem 2015; [Epub ahead of print]