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

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.

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

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 If negative or decline If positive *for ethnicity-based screening, if both members of the couple are carriers of the same condition 18-20 week fetal morphology scan Refer to Genetics If indicated (e.g. fetal anomalies )

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 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

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?

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?

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?

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)

What is Non-Invasive Prenatal Testing? NIPT measures circulating cell-free DNA (cfDNA) from placenta present in maternal blood ‘fetal’ cfDNA comprises ~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)

How does non-invasive prenatal testing compare to traditional prenatal screening for Down syndrome? Screening test Test info Detection rate / Sensitivity for T211 False Positive Rate for T211 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% 0.1-0.2% ~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 18-20 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 Apr 1 [Epub ahead of print]

Non-Invasive Prenatal Testing (NIPT) Landscape Increasing demand from women Increasing uptake in most (urban) centres 3 separate companies, 3 separate technologies Costs between $795 and $1200 8-10 days for result

Recommendations Offer to all women: Prenatal screening using either FTS, IPS or MSS (SIPS or Quad) Fetal morphology scan at about 18-20 weeks gestation Consider NIPT as an option for women who have a high risk for having a baby with an aneuploidy 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

Consider offering Non-Invasive Prenatal Testing (NIPT) for women who: 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 a fetal nuchal translucency (NT) measurement of 3.5mm or greater  Have had a previous pregnancy or child with aneuploidy

Consider offering Non-Invasive Prenatal Testing (NIPT) for women who: Have other high risk factors: Fetal congenital anomalies on ultrasound highly suggestive of trisomy 13, 18 or 21* Soft markers on ultrasound which are highly suggestive of aneuploidy [Refer to SOGC guidelines, 2005].* Are at risk of carrying a male fetus with an X-linked condition (NIPT would be used for sex determination)* In Ontario, MOHLTC funding applications for the indications above marked by an asterisk (*) must be submitted by a geneticist or maternal fetal medicine (MFM) specialist. In other provinces circumstances may be different. Consult your local genetics centre or MFM specialist.

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

Non-Invasive Prenatal Testing (NIPT) results If the result is negative, this is reassuring Your patient should still be offered: fetal morphology scan at 18-20 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 SOGC - Society of Obstetricians and Gynaecologists of Canada

Non-Invasive Prenatal Testing (NIPT) results If the result is positive: Genetic counselling Confirmation by diagnostic testing No irrevocable obstetrical decisions should be made in pregnancies with abnormal NIPT results without confirmatory invasive testing (CVS or amniocentesis) - SOGC

Benefits of Non-Invasive Prenatal Testing (NIPT) Fewer women having diagnostic tests with associated risk of pregnancy loss 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

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 Failed results (~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 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.

Non-Invasive Prenatal Testing: Discordant results Condition Overall True Positive Overall False Positive Down syndrome 91-93% 6-9% Trisomy 18 60-77% 23-47% Trisomy 13 44-54% 46-56% Sex chromosome abnormality (varies with type e.g. Monosomy X, XXY, XYY) 38-88% 12-100% Table 2 Wang – 3 studies The most common initial NIPT-positive result was trisomy 21 (41 cases), followed by trisomy 18 (25 cases), trisomy 13 (16 cases), sex chromosome aneuploidy (16 cases), trisomy 16 (3 cases), monosomy 21 (2 cases), and 1 case each of triploidy and microdeletion of 22q11.2. Four samples negative for NIPT but positive for ultrasound findings were included. Cheung Monosomy X True POS 8/21 (38) Tru NEG 13/21 (62) XXX or XXY 15/17 (88) 2/17 (12) XYY 0/1 1/1 (100) The positive predictive value (PPV) is proportional to the specificity (true negative) of the test and the prevalence of the disorder Wang et al 2014 Genet in Med 17(3) Cheung et al 2015 N Engl J Med [Epub ahead of print]

Limitations of Non-Invasive Prenatal Testing: Biological factors Vanishing twin Fetal fraction (ff) Mosaicism Maternal BMI  ff at 11-13W in 60kg woman = 11.7% and in 160kg woman 3.9%

Limitations of Non-Invasive Prenatal Testing: Biological factors Fetal fraction (ff) Is the amount of fetal cfDNA divided by total (maternal and fetal) cfDNA Low ff result in test failures (<4%) Is affected by: Maternal BMI Gestational age Multiple gestations Higher overall ff, but lower per fetus, risk of false negative Mosaicism High level of mosaicism vs low level mosaicism Maternal BMI  ff at 11-13W in 60kg woman = 11.7% and in 160kg woman 3.9%

Limitations of Non-Invasive Prenatal Testing: Biological factors Confined placental mosaicism Present in 1-2% of first trimester placentas Trisomy 13 and 18  lower placenta volume Rava RP et al. (2014). Clinical Chemistry, 60, 243-50. Wegrzyn et al. (2005). Ultrasound in Obstetrics & Gynecology, 26, 28-32.

Limitations of Non-Invasive Prenatal Testing: Control and Standards Takoudes and Hamar (2014) made headlines when blood samples from 2 non-pregnant women were sent to 5 American commercial labs offering NIPT 3/5 labs reported normal result (no aneuploidy) 2/5 labs unable to report due to low fetal fraction Some companies differ on their assertions that ff influences accuracy of NIPT testing

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 Geneticseducation.ca has more information, links to companies and sample completed forms Refer for genetic counselling when appropriate Company websites: Harmony Prenatal Test™ by Ariosa Diagnostics through Gamma-Dynacare Panorama™ by Natera through Lifelabs/CML Verifi® Prenatal Test by Verinata through Mount Sinai Services Inc. or Medcan Clinic Incidentals e.g. maternal aneuploidy, maternal malignancy Other companies offering NIPT: Sequenom Counsyl Nukleo in Quebec http://www.cliniqueovo.com/en/prenatal/prenatal-screening/fetal-dna-in-maternal-blood/

Ordering Non-Invasive Prenatal Testing (NIPT) in Ontario www.geneticseducation.ca Educational Resources > GECKO on the run > NIPT

Expansion of non-invasive prenatal testing In October 2013 one company expanded their NIPT test to include screening for microdeletion syndromes (e.g. 22q deletion/DiGeorge and trisomies 16 and 22), and in spring 2014 others followed suit Rationale: Cumulatively common disorders The combined at-birth incidence of the 5 commonly offered microdeletion syndromes is approximately 1 in 1,000 Incidence is independent of maternal age Appear to have high sensitivity BUT no clinical validation studies, no guidelines, rare conditions decrease PPV and increase FPR No MOHLTC approval Incidence = 1 in 4,000 to 1 in 50,000

Case 1 A 36 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?  

Yes But consider: She is eligible for NIPT with MOH funding 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

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?

Yes Advanced maternal age (greater than 40 years at EDB) is an appropriate indication for NIPT Covered by MOH funding Better screen than IPS Earlier result Decreased chance with NIPT that patient would receive screen positive result

Case 3 A 29 year old patient had an 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?

Genetic counselling is recommended Patient likely to be offered: Chromosomal microarray Genetic testing for other single gene conditions Level II ultrasound Fetal echocardiogram

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 Not an all purpose genetic test, only gives info on specific chromosomes, and so not indicated in all circumstances

Prenatal Screening Summary Offer all pregnant women, regardless of age: PN screening for fetal aneuploidy (trisomy 13, 18, 21) through FTS, IPS, SIPS or Quad screening Second trimester ultrasound for dating, assessment of fetal anatomy and detection of multiples Maternal age should not be used as the basis for recommending invasive testing when non-invasive PN screening is available Prenatal testing menu continues to evolve and expand with new screening and diagnostic tests More, complex options may add to patient’s decisional conflict

Don’t forget Take a family history to identify familial and/or ethnicity-specific disorders and screen accordingly Consider consanguinity and screen and test accordingly Refer or consult genetics when in doubt

Resources NIPT GECKO on the run GEC-KO website: Genetics Education www.geneticseducation.ca Society of Obstetricians and Gynaecologists of Canada (SOGC) national clinical guidelines in prenatal genetics visit http://sogc.org/clinical-practice-guidelines/ and scroll down to Genetics. Recent Reviews: Cuckle H, Benn P, Pergament E. Cell-free DNA screening for fetal aneuploidy as a clinical service Clin Biochem. 2015; [Epub ahead of print] Bianchi D and Wilkins-Haug L. Integration of non-invasive DNA testing for aneuploidy into prenatal care: What has happened since the rubber met the road? Clin Chem 2014; 60:1